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Expression of Interleukin-6, c- Fos , and zif268 mRNAs in Rat Ischemic Cortex

January 1995
Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism 15(1):166-171

January 1995
15(1):166-171

DOI:10.1038/jcbfm.1995.18

Authors:

Xinkang Wang

Johnson & Johnson

Peter R Young

BioHighland Consulting

Frank C Barone

State University of New York Downstate Medical Center

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The expression of interleukin-6 (IL-6) mRNA in the focal ischemic rat cortex was studied by means of Northern hybridization. IL-6 mRNA was induced after permanent occlusion of the middle cerebral artery, reached a significant level at 3 h, and peaked at 12 h, i.e., ~ 10-fold increase in the ischemic zone compared with the nonischemic cortex or sham-operated controls. The increased IL-6 mRNA was elevated for at least 24 h. Low levels of IL-6 mRNA were detected in sham-operated rats or in the contralateral nonischemic cortex. The expression of c-fos and zif268 mRNAs, two early response genes, was rapid (increased by 1 h postischemia) and transient (returned to basal levels by 24 and 12 h, respectively), clearly having different kinetic patterns from that of IL-6 mRNA. The early response kinetic pattern of c-fos and zif268 mRNAs in focal ischemia suggests their transcriptional regulatory roles in response to ischemic insult, while the delayed induction pattern of IL-6 mRNA suggests a role for this pleiotropic cytokine in the inflammatory response to the focal ischemic damage of the brain.Keywords: c-fos; Focal brain ischemia; Inflammation; Interleukin-6; Stroke; zif268

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   

          

      

 

      

  

        

 

        

   

    

         

      

      

       



      

        

 

       

        

     

       

     

        

   

      

        

      

        

        

     

    

  

      

        

      

        

    

         

       

     

    

  

        



        

    

  

        

       

      

 

      

        

     



      

       

  

      

     

    

        

      

      

      

       

       

           

        

       

        

      



      

       

   

    

      

    

   

 

     

           

      

      

    

        

     

       

      

       

         

     

 

       

     

        

       

     

        

     

 

       

  

     

    

       

       

    

     

    

      

      

    

       

       

    

       

    

        

       

          

      

          

     

         

      

     

    

     

            

         

 

     

       

        

         

      

     

      

   

        

    

       

  

    

    

   

        

      

      

         

   

          

  

     

 

 

       





  

   

      

     

      

      

   

     

        

         

    

     

 

        

    

      

       

         

      

     

        

       

 

         

     

    

   

 

 

 

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          

       



                 



                

   

      

  

     

      

       

          

        

        

       

       

      

       



       

   



 

     

      

      

  

      

   

       

      

       

     

      

         

        

       

       

      

       

        



       

    











  

 

      

   



     

 

      

 



       

       

 

 

       



       

       



   



      

       

      

    

  





    

   

    

        

    

        

      

     

        

          

       

         

       

      

       

       

    

        

     

      

   

     



 





     



       

         

     

       

       

        

  

 

       

       

    

      

    

       

       

   

           

        

      

         

      

       

     

   

     

   

       

       

      

 

           

   

         

 

         

         

 

      

          

       

       

        

       

       

         

    

        

       

       

        



        

     

      

 

        



       

       

       

 

       

        

        

 

    

        

 

     

       

        

       



   

          

    

    

  

      

           

     

     

        

        



          

  

 

          

    

      

      

  

          

   

   

         

     

    

       

        

   

 

         

     

       

   

        

    

  

          

        

     



           

        

       

      



      

           

     

   

         

 

        

      



         



         

   

          

  

       

   

  



         

       

       

 

       

         

      

       

         

       



          

       

   

   

           

       

      

        

  

      

          

         

    

      

      

       

        

   

       

       

     

       

    

   

         

        

       



        

      

          

  

         

      

   

         

        

 

          

    

     

         

         

  

    

       

   

      

     

   

 

       

        

        

 

      

       

        

         

  

       

       



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IL-6 in traumatic brain injury: A Janus-faced player in damage and repair

Article

May 2023
J NEUROTRAUM

Traumatic brain injury (TBI) is a common and often devastating illness, with wide-ranging public health implications. In addition to the primary injury, victims of TBI are at risk for secondary neurological injury by numerous mechanisms. Current treatments are limited and do not target the profound immune response associated with injury. This immune response reflects a convergence of peripheral and CNS-resident immune cells whose interaction is mediated in part by a disruption in the blood brain barrier. The diverse family of cytokines helps to govern this communication and among these, IL-6 is a notable player in the immune response to acute neurological injury. It is also a well-established pharmacological target in a variety of other disease contexts. In TBI, elevated IL-6 levels are associated with worse outcomes, but its role in the response to injury is double-edged. IL-6 promotes neurogenesis and wound healing in animal models of TBI, but it may also contribute to disruptions in the blood brain barrier and the progression of cerebral edema. Here, we review IL-6 biology in the context of TBI, with an eye to clarifying its controversial role and understanding its potential as a target for modulating the immune response in this disease.

Association of Platelet-to-Lymphocyte Ratio with Stroke-Associated Pneumonia in Acute Ischemic Stroke

Article

Full-text available

Mar 2022

A common consequence of acute ischemic stroke (AIS), stroke-associated pneumonia (SAP), might result in a poor prognosis after stroke. Based on the critical position of inflammation in SAP, this study aimed to explore the correlation between platelet-to-lymphocyte ratio (PLR) and the occurrence of SAP. We included 295 patients with acute ischemic stroke, 40 with SAP, and 255 without SAP. The area under the receiver operating characteristic curve was used to determine the diagnostic value of SAP risk factors using binary logistic regression analysis. The comparison between the two groups showed that age, the baseline National Institutes of Health Stroke Scale (NIHSS) score, and the proportion of dysphagia, atrial fibrillation, and total anterior circulation infarct were higher, and the proportion of lacunar circulation infarct was lower in the SAP group (P < 0.001). In terms of laboratory data, the SAP group had considerably greater neutrophil counts and PLR, while the non-SAP group (P < 0.001) had significantly lower lymphocyte counts and triglycerides. Binary logistic regression analysis revealed that older age (aOR = 1.062, 95% CI: 1.023–1.102, P = 0.002), atrial fibrillation (aOR = 3.585, 95% CI: 1.605–8.007, P = 0.019), and PLR (aOR = 1.003, 95% CI: 1.001–1.006, P = 0.020) were independent risk factors associated with SAP after adjusting for potential confounders. The sensitivity and specificity of PLR with a cutoff value of 152.22 (AUC: 0.663, 95% CI: 0.606–0.717, P = 0.0006) were 57.5% and 70.6%, respectively. This study showed that high PLR is an associated factor for SAP in AIS patients. Increased systemic inflammation is linked to SAP in ischemic stroke. Inflammatory biomarkers that are easily accessible may aid in the diagnosis of high-risk SAP patients.

Role of cellular prion protein in splenic CD4+ T cell differentiation in cerebral ischaemic/reperfusion

Article

Full-text available

Sep 2021

Objective Cellular prion protein (PrPC), the primary form of prion diseases pathogen, has received increasing attention for its protective effect against ischaemic stroke. Little is known about its role in peripheral immune responses after cerebral ischaemia/reperfusion (I/R) injury. This study is to detect the variation of splenic CD4⁺ T lymphocytes differentiation and the concentration of inflammatory cytokines after murine cerebral I/R injury in the context of PRNP expression as well as its influence on the ischaemic neuronal apoptosis. Methods We established the cerebral ischaemic murine model of different PRNP genotypes. We detected the percentage of splenic CD4⁺PrPC+ T cells of PRNP wild-type mice and the ratio of splenic Th1/2/17 lymphocytes of mice of different PRNP expression. The relevant inflammatory cytokines were then measured. Oxygen glucose deprivation/reperfusion (OGD/R) HT22 mouse hippocampal neurons were co-cultured with the T-cell-conditioned medium harvested from the spleen of modelled mice and then the neuronal apoptosis was detected. Results CD4⁺ PrPC+ T lymphocytes in wild-type mice elevated after MCAO/R. PRNP expression deficiency led to an elevation of Th1/17 phenotypes and the promotion of pro-inflammatory cytokines, while PRNP overexpression led to the elevation of Th2 phenotype and upregulation of anti-inflammatory cytokines. In addition, PrPC-overexpressed CD4⁺T cells weakened the apoptosis of OGD/R HT-22 murine hippocampal neurons caused by MCAO/R CD4⁺ T-cell-conditioned medium, while PrPC deficiency enhanced apoptosis. Interpretation PrPC works as a neuron protector in the CNS when I/R injury occurs and affects the peripheral immune responses and defends against stroke-induced neuronal apoptosis.

Methylprednisolone Treatment in Brain Death-Induced Lung Inflammation–A Dose Comparative Study in Rats

Article

Full-text available

Feb 2021

Background: The process of brain death (BD) leads to a pro-inflammatory state of the donor lung, which deteriorates its quality. In an attempt to preserve lung quality, methylprednisolone is widely recommended in donor lung management. However, clinical treatment doses vary and the dose-effect relation of methylprednisolone on BD-induced lung inflammation remains unknown. The aim of this study was to investigate the effect of three different doses methylprednisolone on the BD-induced inflammatory response. Methods: BD was induced in rats by inflation of a Fogarty balloon catheter in the epidural space. After 60 min of BD, saline or methylprednisolone (low dose (5 mg/kg), intermediate dose (12.5 mg/kg) or high dose (22.5 mg/kg)) was administered intravenously. The lungs were procured and processed after 4 h of BD. Inflammatory gene expressions were analyzed by RT-qPCR and influx of neutrophils and macrophages were quantified with immunohistochemical staining. Results: Methylprednisolone treatment reduced neutrophil chemotaxis as demonstrated by lower IL-8-like CINC-1 and E-selectin levels, which was most evident in rats treated with intermediate and high doses methylprednisolone. Macrophage chemotaxis was attenuated in all methylprednisolone treated rats, as corroborated by lower MCP-1 levels compared to saline treated rats. Thereby, all doses methylprednisolone reduced TNF-α, IL-6 and IL-1β tissue levels. In addition, intermediate and high doses methylprednisolone induced a protective anti-inflammatory response, as reflected by upregulated IL-10 expression when compared to saline treated brain-dead rats. Conclusion: We showed that intermediate and high doses methylprednisolone share most potential to target BD-induced lung inflammation in rats. Considering possible side effects of high doses methylprednisolone, we conclude from this study that an intermediate dose of 12.5 mg/kg methylprednisolone is the optimal treatment dose for BD-induced lung inflammation in rats, which reduces the pro-inflammatory state and additionally promotes a protective, anti-inflammatory response.

Uric Acid Neuroprotection Associated to IL-6/STAT3 Signaling Pathway Activation in Rat Ischemic Stroke

Article

Full-text available

Jan 2021
MOL NEUROBIOL

Despite the promising neuroprotective effects of uric acid (UA) in acute ischemic stroke, the seemingly pleiotropic underlying mechanisms are not completely understood. Recent evidence points to transcription factors as UA targets. To gain insight into the UA mechanism of action, we investigated its effects on pertinent biomarkers for the most relevant features of ischemic stroke pathophysiology: (1) oxidative stress (antioxidant enzyme mRNAs and MDA), (2) neuroinflammation (cytokine and Socs3 mRNAs, STAT3, NF-κB p65, and reactive microglia), (3) brain swelling (Vegfa, Mmp9, and Timp1 mRNAs), and (4) apoptotic cell death (Bcl-2, Bax, caspase-3, and TUNEL-positive cells). Adult male Wistar rats underwent intraluminal filament transient middle cerebral artery occlusion (tMCAO) and received UA (16 mg/kg) or vehicle (Locke's buffer) i.v. at 20 min reperfusion. The outcome measures were neurofunctional deficit, infarct, and edema. UA treatment reduced cortical infarct and brain edema, as well as neurofunctional impairment. In brain cortex, increased UA: (1) reduced tMCAO-induced increases in Vegfa and Mmp9/Timp1 ratio expressions; (2) induced Sod2 and Cat expressions and reduced MDA levels; (3) induced Il6 expression, upregulated STAT3 and NF-κB p65 phosphorylation, induced Socs3 expression, and inhibited microglia activation; and (4) ameliorated the Bax/Bcl-2 ratio and induced a reduction in caspase-3 cleavage as well as in TUNEL-positive cell counts. In conclusion, the mechanism for morphological and functional neuroprotection by UA in ischemic stroke is multifaceted, since it is associated to activation of the IL-6/STAT3 pathway, attenuation of edematogenic VEGF-A/MMP-9 signaling, and modulation of relevant mediators of oxidative stress, neuroinflammation, and apoptotic cell death.

Expression of TNF and TNF Receptors (p55 and p75) in the Rat Brain after Focal Cerebral Ischemia

Article

Nov 1997

Cerebral ischemia induces a rapid and dramatic up-regulation of tumor necrosis factor (TNF) protein and mRNA, but the cellular sources of TNF in the ischemic brain have not been defined. The diverse activities of TNF are mediated via ligand interaction with two distinct receptors, p55 and p75, which activate separate intracellular signal transduction pathways, leading to distinct biological effects. Since the effects of cerebral ischemia on TNF receptor (TNFR) expression are unknown, we examined the cellular localization and protein expression of TNF and its two receptors in the rat cerebral cortex in response to permanent middle cerebral artery (MCA) occlusion. The results indicate that focal. cerebral ischemia up-regulates expression of TNF and both TNFRs within the ischemic cortex. The most abundant type of TNF immunoreactivity (IR) was a punctate and filamentous pattern of transected cellular processes; however, cell bodies of neurons, astrocytes, and microglia, as well as infiltrating polymorphonuclear (PMN) leukocytes also showed TNF IR. Brain vasculature displayed TNF IR not only within endothelial cells but also in the perivascular space. MCA occlusion induced significant up-regulation of TNF receptors, with p55 IR appearing within 6 hr, significantly before the appearance of p75 IR at 24 hr after the onset of ischemia. Since p55 has been implicated in transducing cytotoxic signalling of TNF, these results support the proposed injurious role of excessive TNF produced during the acute response to cerebral ischemia.

Ischemia Reperfusion Injury Induced Blood Brain Barrier Dysfunction and the Involved Molecular Mechanism

Article

Full-text available

Apr 2023
NEUROCHEM RES

Stroke is characterized by the abrupt failure of blood flow to a specific brain region, resulting in insufficient supply of oxygen and glucose to the ischemic tissues. Timely reperfusion of blood flow can rescue dying tissue but can also lead to secondary damage to both the infarcted tissues and the blood–brain barrier, known as ischemia/reperfusion injury. Both primary and secondary damage result in biphasic opening of the blood–brain barrier, leading to blood–brain barrier dysfunction and vasogenic edema. Importantly, blood–brain barrier dysfunction, inflammation, and microglial activation are critical factors that worsen stroke outcomes. Activated microglia secrete numerous cytokines, chemokines, and inflammatory factors during neuroinflammation, contributing to the second opening of the blood–brain barrier and worsening the outcome of ischemic stroke. TNF-α, IL-1β, IL-6, and other microglia-derived molecules have been shown to be involved in the breakdown of blood–brain barrier. Additionally, other non-microglia-derived molecules such as RNA, HSPs, and transporter proteins also participate in the blood–brain barrier breakdown process after ischemic stroke, either in the primary damage stage directly influencing tight junction proteins and endothelial cells, or in the secondary damage stage participating in the following neuroinflammation. This review summarizes the cellular and molecular components of the blood–brain barrier and concludes the association of microglia-derived and non-microglia-derived molecules with blood–brain barrier dysfunction and its underlying mechanisms.

Interleukin-6 as a Potential Predictor of Neurologic Outcomes in Cardiac Arrest Survivors Who Underwent Target Temperature Management

Article

Oct 2020
J EMERG MED

Background Serum interleukin-6 (IL-6) is a cytokine released in response to an inflammatory stimulus or tissue injury. IL-6 levels are known to increase in patients with brain injury. Objective We investigated the neurologic outcomes associated with serum IL-6 levels in out-of-hospital cardiac arrest (OHCA) survivors who underwent target temperature management (TTM). Methods This was a prospective single-center observational study from October 2018 to November 2019 in a cohort of 45 patients. Serum inflammatory markers (IL-6, C-reactive protein, white blood cells) were determined in samples obtained immediately and at 24, 48, and 72 h after the return of spontaneous circulation (ROSC). Poor neurologic outcome, defined as Cerebral Performance Category 3–5 at 3 months after cardiac arrest, was the primary outcome. Results Among 45 patients enrolled in this study, 25 (55.6%) patients showed a poor neurologic outcome. IL-6 levels were significantly higher in the poor neurologic outcome group immediately (IL-60) after ROSC. The area under the curve (AUC) value of IL-60 was the highest among those of serum IL-6, CRP, and WBC at each time point. The IL-6 levels for predicting poor neurologic outcome had a sensitivity of 75.0%, with 80% specificity at IL-60. The AUC of IL-60 was 0.810 (95% confidence interval 0.664–0.913), with a cutoff value of 346.7 pg mL⁻¹. Conclusions Serum IL-6 level immediately after ROSC was a highly specific and sensitive marker for the 3-month poor neurologic outcome, and may be a useful early predictive marker of neurologic outcome in OHCA survivors treated with TTM.

Quantification of gene expression in post-mortem human brain

Thesis

Jan 2004

Paul Preece

Quantitative human mRNA data are derived from post-mortem or biopsied tissue. Confounding factors, RNA degradation, poor replication and a large variance are often cited, however, as objections to the data's reliability. At issue is whether post-mortem mRNA represents an ordered system and to what degree non-specific factors contribute to the measurements. I developed statistical methods and validated them by measuring 25 mRNA transcripts in an animal model of ischaemia. In the process I discovered novel increases for 3 genes in rats with ischaemic damage: leukaemia inhibitory factor, nestin and galanin mRNA. Additionally, I discovered that reference genes known as "housekeepers"' do not always act as steady-state controls and that the precise value of a test gene response varies according to the baseline choice of reference gene. Once optimised, I applied the analytical methods to human post-mortem brains. I used TaqMan™ real-time RT-PCR to measure 13 mRNAs in 513 cortical samples taken from 90 Alzheimer's disease and 81 control brains. Despite a high variance and confounding factors such as brain pH, I found strong geometric relations between the mRNA transcripts up to and beyond 100 hours autopsy delay. Where a postmortem brain had a high/low level of one mRNA, the same brain invariably had a high/low level of other mRNAs; correlated order is present and provides a means of isolating any mRNA change due specifically to disease. I measured mRNA levels of β-Secretase (BACE), GSK 3 and the isoforms of APP/APRP in the AD and control brains. After adjustment for age of death, brain pH, and gender, there was no change in the mRNA levels for either BACE or GSK 3α mRNA (p = 0.354 and p = 0.054 respectively). There was a change, however, in the ratio of KPI+ to KPI- mRNA isoforms of APP/APRP. Three separate probes, designed only to recognise KPI+ mRNA, each gave increases of between 28 and 50% in AD brains relative to controls (p = 0.002). There was no change in the mRNA levels of KPI-(APP 695) (p = 0.898). Therefore, whilst I KPI- mRNA levels remained level between AD and control brains, the KPI+ species were seen to increase specifically in the AD brains.

Cytokine profile in dynamics of acute period of ischemic stroke

Article

Feb 2010

Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal

Article

Full-text available

Jan 1987

A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.

Tumor necrosis factor-alpha expression in ischemic neurons

Article

Full-text available

Jan 1994

Increase in IL-6, IL-1 and TNF levels in rat brain following traumatic lesion

Article

Full-text available

Feb 1993
J NEUROIMMUNOL

The effects of fluid percussion trauma on brain interleukin (IL)-6, IL-1 and tumor necrosis factor-α (TNF-α) levels have been studied. In the cortex and hippocampus of control and sham-operated rats, the levels of these cytokines were very low (below 4 units/mg protein) and constant. IL-6 and IL-1 levels in the ipsilateral cortex increase rapidly following trauma to reach a maximum of 350 and 16 units/mg protein, respectively, 8 h after the lesion, remained elevated until 18 h and decreased thereafter to basal values. TNF-α levels were maximally elevated (12 units/mg protein) at 3 h and 8 h and returned to basal values by 18 h. Qualitatively similar changes, but with 25–80-fold smaller amplitude, were seen in the contralateral cortex and in the ipsi- and contralateral hippocampus. The levels of IL-6 in the plasma of sham-operated and lesioned rats were only slightly elevated, whereas IL-1 and TNF-α were undetectable. Histological studies of brain tissue at early stages after trauma demonstrated an acute hemorrhage associated with neutrophil invasion. The administration of Ro5 4864 (0.5 mg/kg i.p.), a specific ligand of p (peripheral-type benzodiazepine) binding sites, did not result in any significant effect on the levels of IL-6, IL-1 or TNF-α in the brain of control or sham-operated animals. However, when administered 24 h before or 15 min after trauma, this benzodiazepine enhanced the increase of these cytokines by 2–4-fold in the ipsilateral cortex. The global effect of Ro5 4864 on IL-6 levels was of greater amplitude than those on IL-1 or TNF-α. Ro5 4864 had no effect on IL-6 levels in the plasma of lesioned rats. The activation of p sites by specific benzodiazepine ligands might, by increasing the trauma-induced production of these cytokines, modulate the inflammatory reaction at the site of brain injury.

Human cysteine-rich protein. A member of the LIM/double-finger family displaying coordinate serum induction with c-myc

Article

Full-text available

Jun 1992

We previously reported the structure of the placentally derived human cysteine-rich (h crp) cDNA and demonstrated that it encodes a highly conserved and widely distributed zinc finger-like protein. We now report that the expression of both the mouse and human crp genes is induced as a primary response to serum in quiescent Balb/c 3T3 cells and in human fibroblasts. The profile of this primary response is remarkably parallel to that of c-myc in the Balb/c 3T3 cell line. The structure of the 23.2-kilobase h crp gene demonstrates that it is a member of a gene superfamily encoding proteins sharing a highly characteristic 52-amino acid "LIM/double-finger" motif. The evolutionarily conserved structure of cysteine-rich protein, its structural similarity to a number of developmentally critical proteins, its distinctive tissue distribution, and its primary response to early events in the cell cycle suggest that crp plays an important role in cell function.

Increased plasma interleukin-6 levels in head injury patients

Article

Jan 1990

Induction of Interleukin1? mRNA in Rat Brain After Transient Forebrain Ischemia

Article

Feb 1992
J NEUROCHEM

: The expression of interleukin-1β (IL-1β) mRNA in the cerebral cortex, hippocampus, striatum, and thalamus of rats was studied after transient forebrain ischemia. IL-1β mRNA was not detected in all these regions of sham-operated control rats. IL-1β mRNA was induced after transient forebrain ischemia and reached a detectable level in all regions examined 15 min after the start of recirculation. The induction of IL-1β mRNA had a few peaks, that is, peaks were observed at 30 and 240 min in the four regions examined, and another peak was observed at 90 min in the striatum. One day after the start of recirculation, IL-1β mRNA levels were markedly decreased, but even 7 days after that, IL-1β mRNA was found at very low levels in all regions examined. The amounts of c-fos and β-actin mRNAs on the same blots were also examined. The induction of c-fos mRNA was transient and had only one peak in all regions examined, whereas the levels of β-actin mRNA in these regions were fairly constant throughout the recirculation period. Thus, we provide the first evidence for a characteristic expression of IL-1β mRNA in several brain regions after transient forebrain ischemia.

Construction and identification of cDNA clones for mouse ribosomal proteins: Application for the study of r-protein gene expression

Article

Aug 1980
GENE

A set of recombinant DNAs with inserts of mRNA sequences coding for various mouse ribosomal proteins (r- proteins) were isolated. The recombinants were selected from a library of cDNA clones representing the small (⩽12S) poly(A)+ mRNA of mouse L cells, which is relatively enriched for the r-protein mRNAs. Selection consisted of testing the various recombinant plasmids for their ability to hybridize selectively with an mRNA that directs the synthesis of a recognizable r-protein in a cell-free translational system. Translation products were subjected to a preliminary screen by electrophoresis on one-dimensional Triton X-100/urea slab gels and then more definitively identified by the two-dimensional gel analyses conventionally used for ribosomal proteins. The set of recombinants represents the mRNAs specifying ribosomal proteins S16, L7, L13, L18, L19, L30, L32/33 and probably L10.

RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination

Article

Nov 1977

RNA molecular weight measurements were carried out by gel electrophoresis under four different denaturing conditions including 99% formamide, 10 mM methyl mercury, 2.2 M formaldehyde, and 6 M urea at pH 3.8. Electrophoresis at a series of gel concentrations and at least two different voltage gradients resulted in some RNA species exhibiting apparent molecular weights that vary with both gel concentration and voltage gradient. Three different deviations from the requirement for hydrodynamically equivalent conformations were observed: (1) deformation of the random coil structure of very large RNAs at moderately high gel concentrations and voltage gradients resulting, in extreme cases, in a molecular weight independent migration of RNA molecules; (2) incomplete denaturation of RNA molecules with very GC rich helical regions; and (3) varying charge/mass ratio due to differential protonation at pH 3.8. Reliable molecular weight measurements of RNA molecules as large as 4.0 × 106 containing GC rich helical regions could only be made on dilute (0.5-1.0%) agarose gels after reaction with either 2.2 M formaldehyde or 10 mM methyl mercury hydroxide. A theoretical justification for the use of the empirical log molecular weight-mobility relation is presented. It is also demonstrated that the gel electrophoretic behavior of a homologous series of random coils can be approximated by that of a series of spheres with radii proportional to the square root of radius of gyration of a random coil. Consequently, molecular weight determinations of denatured RNAs, especially those obtained by extrapolation, are more reliable if the square root of the molecular weight is plotted vs. log mobility.

Peripheral and central regulation of IL-6 gene expression in endotoxin-treated rats

Article

Oct 1992

The distribution of IL-6 mRNA in the rat brain was studied by in situ hybridization using 35S-labelled oligonucleotides. The mRNA encoding IL-6 was found in hippocampus, hypothalamus, cerebellum as well as in other brain areas. Microscopic analyses revealed both neuronal and glial cell localization of the mRNA. Subsequently, Northern blot analyses were performed with RNA isolated from spleen, adrenals, pituitary gland, hypothalamus, hippocampus and cerebellum of rats injected intraperitoneally with a non toxic dose of LPS. A rapid induction of the IL-6 mRNA was observed in the peripheral organs, whereas no change in IL-6 transcripts could be measured in the brain. It is concluded that the local synthesis and release of IL-6 in pituitary and adrenal gland might be involved in the activation of the HPA axis following an endotoxin challenge.

Role of monoamine systems in activation of zif268 by cocaine

Article

Oct 1992

Rapid activation of transcription factor genes is thought to play a key role in stimulus-induced neuronal plasticity. To help understand the genomic response that may underlie long-term effects of cocaine and amphetamine, we have investigated the effect of these agents on Zif268, a transcription regulatory factor that is expressed at high levels in brain neurons. Like c-fos, zif268 is markedly activated in striatum by cocaine and amphetamine. This response appears to involve the dopamine system, since it is abolished by SCH23390, a selective D1 dopamine receptor antagonist, or by 6-hydroxydopamine lesions. To assess the role of other monoamine systems in regulating the expression of these transcription factors, we have examined the effects of selective monoamine uptake blockers as well as agents that lesion the norepinephrine and serotonin systems. These studies indicate that, in addition to the dopamine system, the norepinephrine and serotonin systems also play prominent roles in the activation of zif268 and c-fos by cocaine and amphetamine.

Expression of Interleukin-6, c- Fos , and zif268 mRNAs in Rat Ischemic Cortex

Abstract

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