Decrease In Messenger RNA Levels Research Articles

Opposing Roles of Sphingosine 1-Phosphate Receptors 1 and 2 in Fat Deposition and Glucose Tolerance in Obese Male Mice.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that regulates fundamental cellular processes such as proliferation, migration, apoptosis, and differentiation through 5 cognate G protein-coupled receptors (S1P1-S1P5). We previously demonstrated that blockade of S1P2 signaling in S1P2-deficient mice attenuates high-fat diet-induced adipocyte hypertrophy and glucose intolerance and an S1P2-specific antagonist JTE-013 inhibits, whereas an S1P1/S1P3 dual antagonist (VPC23019) activates, adipogenic differentiation of preadipocytes. Based on those observations, this study examined whether an S1P1-specific agonist, SEW-2871, VPC23019, or their combination acts on obesity and glucose intolerance in leptin-deficient ob/ob mice. The oral administration of SEW-2871 or JTE-013 induced significant reductions in body/epididymal fat weight gains and epididymal/inguinal fat adipocyte sizes and improved glucose intolerance and adipocyte inflammation in ob/ob mice but not in their control C57BL/6J mice. Both SEW-2871 and JTE-013 decreased messenger RNA levels of tumor necrosis factor-α and CD11c, whereas they increased those of CD206 and adiponectin in the epididymal fats isolated from ob/ob mice with no changes in the levels of peroxisome proliferator activated receptor γ and its regulated genes. By contrast, VPC23019 did not cause any such alterations but counteracted with all those SEW-2871 actions in these mice. In conclusion, the S1P1 agonist SEW-2871 acted like the S1P2 antagonist JTE-013 to reduce body/epididymal fats and improve glucose tolerance in obese mice. Therefore, this study raises the possibility that endogenous S1P could promote obesity/type 2 diabetes through the S1P2, whereas exogenous S1P could act against them through the S1P1.

Reprogramming the piRNA pathway for multiplexed and transgenerational gene silencing in C. elegans.

Single-guide RNAs can target exogenous CRISPR-Cas proteins to unique DNA locations, enabling genetic tools that are efficient, specific and scalable. Here we show that short synthetic guide Piwi-interacting RNAs (piRNAs) (21-nucleotide sg-piRNAs) expressed from extrachromosomal transgenes can, analogously, reprogram the endogenous piRNA pathway for gene-specific silencing in the hermaphrodite germline, sperm and embryos of Caenorhabditis elegans. piRNA-mediated interference ('piRNAi') is more efficient than RNAi and can be multiplexed, and auxin-mediated degradation of the piRNA-specific Argonaute PRG-1 allows conditional gene silencing. Target-specific silencing results in decreased messenger RNA levels, amplification of secondary small interfering RNAs and repressive chromatin modifications. Short (300 base pairs) piRNAi transgenes amplified from arrayed oligonucleotide pools also induce silencing, potentially making piRNAi highly scalable. We show that piRNAi can induce transgenerational epigenetic silencing of two endogenous genes (him-5 and him-8). Silencing is inherited for four to six generations after target-specific sg-piRNAs are lost, whereas depleting PRG-1 leads to essentially permanent epigenetic silencing.

Neuronal deletion of Gtf2i, associated with Williams syndrome, causes behavioral and myelin alterations rescuable by a remyelinating drug.

Williams syndrome (WS), caused by a heterozygous microdeletion on chromosome 7q11.23, is a neurodevelopmental disorder characterized by hypersociability and neurocognitive abnormalities. Of the deleted genes, general transcription factor IIi (Gtf2i) has been linked to hypersociability in WS, although the underlying mechanisms are poorly understood. We show that selective deletion of Gtf2i in the excitatory neurons of the forebrain caused neuroanatomical defects, fine motor deficits, increased sociability and anxiety. Unexpectedly, 70% of the genes with significantly decreased messenger RNA levels in the mutant mouse cortex are involved in myelination, and mutant mice had reduced mature oligodendrocyte cell numbers, reduced myelin thickness and impaired axonal conductivity. Restoring myelination properties with clemastine or increasing axonal conductivity rescued the behavioral deficits. The frontal cortex from patients with WS similarly showed reduced myelin thickness, mature oligodendrocyte cell numbers and mRNA levels of myelination-related genes. Our study provides molecular and cellular evidence for myelination deficits in WS linked to neuronal deletion of Gtf2i.

In Utero Exposure to a High-Fat Diet Programs Hepatic Hypermethylation and Gene Dysregulation and Development of Metabolic Syndrome in Male Mice.

Exposure to a high-fat (HF) diet in utero is associated with increased incidence of cardiovascular disease, diabetes, and metabolic syndrome later in life. However, the molecular basis of this enhanced susceptibility for metabolic disease is poorly understood. Gene expression microarray and genome-wide DNA methylation analyses of mouse liver revealed that exposure to a maternal HF milieu activated genes of immune response, inflammation, and hepatic dysfunction. DNA methylation analysis revealed 3360 differentially methylated loci, most of which (76%) were hypermethylated and distributed preferentially to hotspots on chromosomes 4 [atherosclerosis susceptibility quantitative trait loci (QTLs) 1] and 18 (insulin-dependent susceptibility QTLs 21). Interestingly, we found six differentially methylated genes within these hotspot QTLs associated with metabolic disease that maintain altered gene expression into adulthood (Arhgef19, Epha2, Zbtb17/Miz-1, Camta1 downregulated; and Ccdc11 and Txnl4a upregulated). Most of the hypermethylated genes in these hotspots are associated with cardiovascular system development and function. There were 140 differentially methylated genes that showed a 1.5-fold increase or decrease in messenger RNA levels. Many of these genes play a role in cell signaling pathways associated with metabolic disease. Of these, metalloproteinase 9, whose dysregulation plays a key role in diabetes, obesity, and cardiovascular disease, was upregulated 1.75-fold and hypermethylated in the gene body. In summary, exposure to a maternal HF diet causes DNA hypermethylation, which is associated with long-term gene expression changes in the liver of exposed offspring, potentially contributing to programmed development of metabolic disease later in life.

Human amniotic mesenchymal stromal cell transplantation improves endometrial regeneration in rodent models of intrauterine adhesions

Background aimsIntrauterine adhesion (IUA) is a common uterine cavity disease characterized by the unsatisfactory regeneration of damaged endometria. Recently, stem cell transplantation has been proposed to promote the recovery process. Here we investigated whether human amniotic mesenchymal stromal cells (hAMSCs), a valuable resource for transplantation therapy, could improve endometrial regeneration in rodent IUA models. MethodsForty female Sprague-Dawley rats were randomly assigned to five groups: normal, sham-operated, mechanical injury, hAMSC transplantation, and negative control group. One week after intervention and transplantation, histological analyses were performed, and immunofluorescent and immunohistochemical expression of cell-specific markers and messenger RNA expression of cytokines were measured. ResultsThicker endometria, increased gland numbers and fewer fibrotic areas were found in the hAMSC transplantation group compared with the mechanical injury group. Engraftment of hAMSCs was detected by the presence of anti-human nuclear antigen–positive cells in the endometrial glands of the transplantation uteri. Transplantation of hAMSCs significantly decreased messenger RNA levels of pro-inflammatory cytokines (tumor necrosis factor-α and interleukin-1β), and increased those of anti-inflammatory cytokines (basic fibroblast growth factor, and interleukin-6) compared with the injured uterine horns. Immunohistochemical expression of endometrial epithelial cells was revealed in specimens after hAMSC transplantation, whereas it was absent in the mechanically injured uteri. ConclusionshAMSC transplantation promotes endometrial regeneration after injury in IUA rat models, possibly due to immunomodulatory properties. These cells provide a more easily accessible source of stem cells for future research into the impact of cell transplantation on damaged endometria.

Chronic Cannabinoid Receptor 2 Activation Reverses Paclitaxel Neuropathy Without Tolerance or Cannabinoid Receptor 1–Dependent Withdrawal

BackgroundMixed cannabinoid receptor 1 and 2 (CB1 and CB2) agonists such as Δ9-tetrahydrocannabinol (Δ9-THC) can produce tolerance, physical withdrawal, and unwanted CB1-mediated central nervous system side effects. Whether repeated systemic administration of a CB2-preferring agonist engages CB1 receptors or produces CB1-mediated side effects is unknown. MethodsWe evaluated antiallodynic efficacy, possible tolerance, and cannabimimetic side effects of repeated dosing with a CB2-preferring agonist AM1710 in a model of chemotherapy-induced neuropathy produced by paclitaxel using CB1 knockout (CB1KO), CB2 knockout (CB2KO), and wild-type (WT) mice. Comparisons were made with the prototypic classic cannabinoid Δ9-THC. We also explored the site and possible mechanism of action of AM1710. ResultsPaclitaxel-induced mechanical and cold allodynia developed to an equivalent degree in CB1KO, CB2KO, and WT mice. Both AM1710 and Δ9-THC suppressed established paclitaxel-induced allodynia in WT mice. In contrast to Δ9-THC, chronic administration of AM1710 did not engage CB1 activity or produce antinociceptive tolerance, CB1-mediated cannabinoid withdrawal, hypothermia, or motor dysfunction. Antiallodynic efficacy of systemic administration of AM1710 was absent in CB2KO mice and WT mice receiving the CB2 antagonist AM630, administered either systemically or intrathecally. Intrathecal administration of AM1710 also attenuated paclitaxel-induced allodynia in WT mice, but not CB2KO mice, implicating a possible role for spinal CB2 receptors in AM1710 antiallodynic efficacy. Finally, both acute and chronic administration of AM1710 decreased messenger RNA levels of tumor necrosis factor-α and monocyte chemoattractant protein 1 in lumbar spinal cord of paclitaxel-treated WT mice. ConclusionsOur results highlight the potential of prolonged use of CB2 agonists for managing chemotherapy-induced allodynia with a favorable therapeutic ratio marked by sustained efficacy and absence of tolerance, physical withdrawal, or CB1-mediated side effects.

Caffeine prevents experimental liver fibrosis by blocking the expression of TGF-β

There is a growing body of evidence that caffeine exerts beneficial effects on the liver; however, the molecular mechanisms by which caffeine exerts beneficial effects on the liver are poorly defined. The aim of the present study was to examine the efficacy of caffeine in preventing thioacetamide (TAA)-induced cirrhosis in rats. Cirrhosis was induced by chronic TAA administration and the effects of coadministration of caffeine for 8 weeks were evaluated, including control groups. The administration of TAA induced liver cirrhosis, which was inhibited by caffeine. Caffeine prevents elevation of liver enzymes. Liver histopathology and hydroxyproline levels were significantly lower in the rats treated with TAA plus caffeine compared with TAA only. Caffeine shows antioxidant properties by restoring the redox equilibrium [lipid peroxidation and glutathione peroxidase (GPx) levels]. Western blot assays showed blockade of the expression of transforming growth factor-β and its downstream inductor connective tissue growth factor. Similarly, caffeine decreases messenger RNA levels of these profibrogenic proteins. In addition, caffeine inhibits hepatic stellate cells because of blockade of the expression of α-smooth muscle actin; in the western blot assay, we also found low levels of mRNA of collagen α1. Zymography assays showed that caffeine had an effect on the activity of matrix metalloproteinases 2 and 9, but no effect on the expression of tissue inhibitor of metalloproteinases-1, using RT-PCR. Our results show that caffeine prevents experimental cirrhosis; the mechanisms of action are associated with its antioxidant properties and mainly by its ability to block the elevation of the profibrogenic cytokine transforming growth factor-β, which may be associated with attenuation of the inflammatory and fibrotic processes.

Bupivacaine—the deadly friend of intervertebral disc cells?

Background context Bupivacaine is commonly used as an adjunct during provocative discography and is administered intradiscally in patients with discogenic back pain. Recent studies demonstrated that bupivacaine is cytotoxic for articular chondrocytes in vitro at clinically used concentrations (0.25%–0.5%). Purpose To analyze a concentration-dependent effect of bupivacaine on cell viability and gene expression of human intervertebral disc (IVD) cells in an in vitro model. Study design In vitro cell culture study. Patient sample Disc cells were isolated from human disc biopsies from 11 patients undergoing surgery because of degenerative disc disease or disc herniation. Outcome measures Cell viability and gene expression after exposure to bupivacaine. Methods Human IVD cells were treated with different concentrations of bupivacaine for 2 (n=5) or 18 hours (n=5) and analyzed for cell viability and proliferation (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay). Additionally, cells were prestimulated with interleukin-1 beta (IL-1β) (5 ng/mL) to increase the levels of proinflammatory cytokines and matrix-degrading enzymes and thereafter treated with 0.75 mmol bupivacaine (as determined in the cell viability test) for 2 (n=5) or 18 hours (n=5). Prestimulated cells with or without bupivacaine treatment were analyzed for gene expression of IL-1β, IL-6, IL-8, tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), matrix metalloproteinase-3 (MMP3), MMP9, MMP13, and a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) using real-time reverse transcription-polymerase chain reaction. Statistical analysis was performed by using the Mann-Whitney U test with a significance level of p<.05. Results After 18 hours, bupivacaine exhibited either a cytotoxic or a proliferative effect on human IVD cells, depending on the concentration. Similar but lower effects could be observed already after 2 hours. With a concentration of 0.75 mmol (proliferative effect), bupivacaine significantly decreased messenger RNA levels of TNF-α, COX-2, MMP13, and ADAMTS4 after 18 hours. In contrast, expression of IL-6, IL-8, and MMP9 did not differ; expression of IL-1β and MMP3 was stimulated with 0.75 mmol. After 2 hours, we observed a reduction in the expression of COX-2, MMP3, MMP13, and ADAMTS4, without any effect regarding IL-1β. Conclusions Application of bupivacaine in clinically relevant concentrations was toxic for IVD cells in vitro. A low concentration stimulated cell proliferation and reduced gene expression of certain matrix-degrading enzymes and proinflammatory cytokines. If these results can be corroborated in tissue explant models or animal studies, caution regarding provocative discography with bupivacaine is prompted.

Decreased Expression of the Voltage-Dependent Anion Channel in Differentiated PC-12 and SH-SY5Y Cells Following Low-Level Pb Exposure

Lead (Pb) has been shown to disrupt cellular energy metabolism, which may underlie the learning deficits and cognitive dysfunctions associated with environmental Pb exposure. The voltage-dependent anion channel (VDAC) plays a central role in regulating energy metabolism in neurons by maintaining cellular ATP levels and regulating calcium buffering, and studies have shown that VDAC expression is associated with learning in mice. In this study, we examined the effect of 5 and 10microM Pb on VDAC expression in vitro in order to determine whether Pb alters VDAC expression levels in neuronal cell lines. PC-12 and SH-SY5Y cells were used since they differentiate to resemble primary neuronal cells. VDAC expression levels were significantly decreased 48 h after exposure to Pb in both cell lines. In contrast, exposure to 24 h of hypoxia failed to produce a decrease in VDAC, suggesting that decreased VDAC expression is not a general cellular stress response but is a result of Pb exposure. This decreased VDAC expression was also correlated with a corresponding decrease in cellular ATP levels. Real-time reverse transcription-polymerase chain reaction demonstrated a significant decrease in messenger RNA levels for the VDAC1 isoform, indicating that Pb reduces transcription of VDAC1. These results demonstrate that exposure to 5 and 10microM Pb reduces VDAC transcription and expression and is associated with reduced cellular ATP levels.

Noncollagenous bone matrix proteins as a part of calcific aortic valve disease regulation

Clinically, calcific aortic valve disease is a progressive continuum from obstructive fibro(sclero)tic valve thickening to aortic stenosis. Recent evidence suggests that, in addition to nonbone miscellaneous mineralization, calcified valves present distinct signs of active bone remodeling; and in this context, noncollagenous bone-associated proteins are assumed to have a critical role. The expression of 5 bone matrix proteins-bone morphogenetic protein-2 and -4, bone sialoprotein II, osteopontin, and osteoprotegerin-was examined by reverse transcriptase polymerase chain reaction (n = 31) and immunolabeling (n = 83) in the clinical continuum from healthy pliable valves to heavily calcified ones. As a known structural pathologic sign, the extent of neovascularization was also examined. We observed progressive increase in the gene expression of osteopontin (7.4-fold elevation, P < .001) and bone sialoprotein II (5.8-fold elevation, P < .05), and also 1.7-fold elevation (P < .05) in osteoprotegerin gene expression during the disease course. These findings were congruent with that of immunohistochemical analysis. Surprisingly, bone morphogenetic protein-2 and -4 showed a comparable significant decrease in messenger RNA levels in calcified valves (P < .01 and P < .05, respectively). Our results support the view that aortic valve calcification is an actively regulated process. Furthermore, the results suggest that the expression of pro- and anticalcific noncollagenous bone-associated matrix proteins is altered during the disease continuum and that this imbalance may contribute to the pathology of calcific aortic valve disease.

Selenite reactivates silenced genes by modifying DNA methylation and histones in prostate cancer cells

DNA hypermethylation is a common epigenetic alteration in human prostate cancer and is considered to contribute to development of this disease. Accumulating data suggest that dietary factors may alter cancer risk by modifications of epigenetic processes in the cell. The present study was designed to investigate whether selenium (Se) would alter epigenetic events to regulate methylation-silenced genes in human prostate cancer cells. DNA methylation, histone modifications and gene expression were studied in LNCaP cells after selenite treatment using polymerase chain reaction, western blot analysis, chromatin immunoprecipitation assay and enzymatic activity assay. Our study shows that selenite treatment caused partial promoter DNA demethylation and reexpression of the pi-class glutathione-S-transferase (GSTP1) in LNCaP cells in a dose- and time-dependent manner. Selenite treatment decreased messenger RNA levels of DNA methyltransferases (DNMTs) 1 and 3A and protein levels of DNMT1. Selenite also decreased histone deacetylase activity and increased levels of acetylated lysine 9 on histone H3 (H3-Lys 9), but decreased levels of methylated H3-Lys 9. Selenite treatment reduced levels of DNMT1 and methylated H3-Lys 9 associated with the GSTP1 promoter, but increased levels of acetylated H3-Lys 9 associated with this promoter. Additionally, selenite treatment decreased general DNA methylation and caused partial promoter demethylation and reexpression of the tumor suppressor adenomatous polyposis coli and cellular stress response 1, a gene involving tumor growth and metastasis. Our study demonstrates that Se can epigenetically modulate DNA and histones to activate methylation-silenced genes. These epigenetic modifications may contribute to cancer prevention by Se.

HLA‐G14‐bp polymorphism at exon 8 in Amerindian populations from the Brazilian Amazon

HLA-G (human leukocyte antigen-G) plays an important role in the modulation of the maternal immune system during pregnancy. HLA-G alleles presenting a 14-bp insertion at exon 8 have been associated with decreased messenger RNA levels, preeclampsia, and miscarriages. This suggests that natural selection may exert a strong influence against the insertion allele in isolated populations. DNA samples from 384 Amazonian Indians spread across seven isolated tribes were evaluated for the 14-bp polymorphism. The insertion frequency (38.54%) was somewhat low. The Ewens-Watterson's neutrality test showed a slight trend toward balancing selection operating at this locus but no correlation between the 14-bp locus and fertility data was found. To sum up, no definitive evidence was obtained indicating that the 14-bp frequencies in Amerindians depart from neutrality.

Polymorphisms in the tumor necrosis factor alpha and heat shock protein HSP70-2 genes and preterm premature rupture of the membranes in multifetal gestations

To determine the relationship between preterm premature rupture of the membranes (PPROM) and polymorphisms in genes involved in pro-inflammatory immunity (TNF) and cell viability under conditions of stress (hsp70) in multifetal gestations. Buccal swabs from 101 mother-neonate pairs of multifetal pregnancies were tested for nucleotide polymorphisms at position 308 of the TNF gene and position 1267 of the hsp70-2 gene. Pregnancy outcome data were subsequently obtained. Carriage of allele 2 of the TNF gene (TNF∗2) by the firstborn was associated with PPROM. Fetus A carried TNF∗2 in 37.0% of cases with PPROM as opposed to 9.0% without PPROM (P = .002; OR = 5.98, 95% CI = 1.7-22.1). Conversely, the first fetus was TNF∗1-homozygous in 91.0% of cases without PPROM as opposed to 63.0% with PPROM (P = .002; OR = .17, 95% CI = .1-.6). Homozygous carriage of hsp70 allele 2 (HSP70∗2) by the firstborn was also associated with PPROM. Fetus A was HSP70∗2 homozygous in 23.1% of PPROM cases as opposed to 6.3% without PPROM (P = .06; OR = 4.43, 95% CI = .97-21.3). There was no relation between TNF∗2 or HSP70∗2 carriage by the second fetus or the mother and PPROM. In pregnancies with PPROM, the firstborn was positive for both TNF∗2 and HSP70∗2 homozygosity in 15.4% as opposed to 0% of cases without PPROM (P = .006; OR = 24.32, 95% CI = 1.3-469.6). The rate of PPROM in multifetal pregnancies is related to carriage of the TNF∗2 allele and/or HSP70∗2 homozygosity in the firstborn. TNF∗2 is associated with increased TNF production as compared to TNF∗1. HSP70∗2 homozygosity is associated with decreased messenger RNA levels compared to HSP70∗1 homozygotes and heterozygotes. Hyper-TNF production by the fetus nearest the chorioamnion in response to stressful stimuli, coupled with a decreased level of protection against cell death due to reduced hsp70 production, may increase susceptibility to PPROM.

Helicobacter pylori impairs DNA mismatch repair in gastric epithelial cells

Background & Aims:Helicobacter pylori infection is a major gastric cancer risk factor. H. pylori gastritis occurs more frequently in individuals with microsatellite instability–positive than those with microsatellite instability–negative gastric cancers, raising the possibility that H. pylori infection affects DNA mismatch repair (MMR). The aim of this study was to determine the effect of H. pylori on the expression of DNA MMR proteins and RNA in gastric epithelial cells. Methods: Gastric cancer cell lines were cocultured with H. pylori, bacterial extracts, and Campylobacter jejuni or Escherichia coli. MutS (hMSH2 and hMSH6) and MutL (hMLH1, hPMS2, and hPMS1) DNA MMR protein and RNA levels were determined. Results: All cell lines examined showed decreased levels of MutS and MutL DNA MMR proteins in a dose-dependent manner after coculture with H. pylori strains. The reduction in DNA MMR protein levels was caused by heat-sensitive H. pylori products. The levels of DNA MMR proteins were affected by C. jejuni but not by E. coli. RNA levels of hMSH2 and hMSH6 were also reduced after exposure to H. pylori.Conclusions:H. pylori infection of gastric epithelial cells leads to a decrease in DNA MMR proteins that is at least in part related to an H. pylori–induced decrease in messenger RNA levels of repair genes. These data suggest that H. pylori infection might lead to a deficiency of DNA MMR in gastric epithelial cells that may increase the risk of mutation accumulation in gastric mucosa cells and the risk of gastric cancer during chronic H. pylori infection.GASTROENTEROLOGY 2002;123:542-553

Verrucous carcinoma of the foot associated with human papillomavirus type 16

Background: We present 2 patients with verrucous carcinoma (VC) of the foot, a malignancy of unknown origin. Objective: Molecular studies from the VCs were undertaken to determine the presence, type, and physical state of human papillomavirus (HPV) as well as the expression levels of certain oncogenes and antioncogenes. Methods: Synthetic consensus and type-specific primers were used to determine the HPV type from both VCs via polymerase chain reaction (PCR). Verification of fragments was accomplished by means of specific isotope-labeled oligonucleotide probes. The physical state of HPV DNA was determined by two-dimensional gel electrophoresis. Quantitative oncogene and antioncogene expression studies were performed with the use of reverse transcriptase PCR. Results: HPV type 16 was identified in episomal and integrated forms in both tumors. Expression studies revealed increased messenger RNA levels of c-Ki-ras oncogene and the p53 antioncogene and decreased messenger RNA levels of the Rb antioncogene in both VCs. Conclusion: Episomal and integrated forms of HPV-16 DNA were found in VCs of the foot, along with alterations of c-Ki-ras, p53, and Rb genes. (J Am Acad Dermatol 2001;45:49-55.)

Homolateral cerebrocortical changes in neuropeptide and receptor expression after minimal cortical infarction.

A cortical infarct of 2 mm diameter was obtained in the parietal cortex after a craniotomy, disruption of the dura mater and topical application of 3 M KCl. It has been shown previously that the presence of a small cortical infarct induces an increase in immediate early gene messenger RNA expression followed by an increase in neuropeptide and glutamic acid decarboxylase messenger RNA expression. Glutamate, acting at N-methyl-D-aspartate receptors, is held responsible for these changes, since they are blocked by pretreatment with dizocilpine. In the present study, we have analysed the consequences of the dramatic changes in messenger RNA expression on the level of immediate early gene products c-fos and zif 268, and on that of neuropeptides by using immunohistochemistry. After just 1 h, an increase in c-fos- and zif 268-like immunoreactivity is observed in the entire cortical hemisphere homolateral to the infarct, and is no longer detected after 6 h. An increase in cholecystokinin octapeptide-, substance P-, neuropeptide Y- and somatostatin-like immunoreactivity is observed in the entire cortical hemisphere homolateral to the infarct after three days, and is no longer detected after 30 days. To investigate if these dramatic increases in neuropeptide immunoreactivities may have functional consequences, we studied the level of cholecystokinin receptors by autoradiographic binding using [125I]cholecystokinin-8S and in situ hybridization for the detection of cholecystokinin-b receptor messenger RNA. A decrease in cholecystokinin binding sites and cholecystokinin-b receptor messenger RNA is observed in the entire cortical hemisphere homolateral to the infarct after three days, and is no longer detected after nine days. This study shows that a topical stimulation has diffuse effects, reaching regions far from the site of the lesion, and some of them are still strongly present after nine days. The increase in neuropeptide messenger RNAs is followed by an increase in the protein products of these genes, which may modify the neurotransmission. As a corollary to this, a decrease in cholecystokinin binding sites occurs. This may have further consequences on signal transduction pathways. This decrease in cholecystokinin binding sites is associated with a decrease in the cholecystokinin-b receptor messenger RNA, and this is the first example of a decrease in messenger RNA levels in this experimental model.

Hypoxemia up-regulates interleukin-8 stimulated phagocytosis of polymorphonuclear leukocytes by differential regulation of CD32w and CD35 messenger RNA expression*

The purpose of these studies was to investigate the effect of hypoxemia on interleukin-8 (IL-8) regulation of phagocytosis and surface opsonic receptor expression of whole blood polymorphonuclear leukocytes (PMNs). Whole blood PMNs were rendered hypoxemic (PvO2 less than 15 mm Hg) or normoxic (PvO2 equals 60 to 80 mm Hg) and incubated with IL-8 (50 ng/ml) before the sequential addition of serum-opsonized fluorescent microspheres (emission wavelength, 560 nm) and fluorescein isothiocyanate-conjugated mouse anti-human CD32w or CD35 antibodies (emission wavelength, 513 nm). Concomitant two color flow cytometric analysis was then performed to measure the mean channel fluorescence (MCF) and the percentage of positive PMNs. Steady-state messenger RNA levels for CD32w and CD35 were quantitated by means of Northern blot analysis by using total RNA extracted from purified PMNs from each study group. Hypoxemia increased IL-8 stimulated PMN phagocytosis (percentage of positive PMNs, 38 +/- 1.5 versus 27 +/- 2.0, MCF, 5491 +/- 182 versus 4060 +/- 121 for hypoxemia plus IL-8 versus normoxia plus IL-8, respectively; p < 0.05). Hypoxemia increased IL-8 stimulated PMN expression of CD32w and CD35 (MCF CD32w, 791 +/- 105 versus 336 +/- 81; MCF CD35, 542 +/- 87 versus 254 +/- 41; p < 0.05). Under normoxic conditions IL-8 decreased messenger RNA levels for both CD32w and CD35, but under hypoxemic conditions IL-8 increased messenger RNA levels only for CD32w. Hypoxemia directly regulates IL-8 control of PMNs by increasing phagocytosis, receptor expression, and messenger RNA for these receptors. Studies investigating cytokine regulation of PMN function need to take into account oxygen tension when establishing the fundamental effects of cytokines on PMN physiology.

Suppression of interleukin-1 beta and tumour necrosis factor-alpha biosynthesis by cadmium in in vitro activated human peripheral blood mononuclear cells.

Cadmium is a highly toxic element responsible for acute and chronic toxicity in man. There is evidence that cadmium induces pathophysiological effects by modulating components of the immune system. Cytokines are being increasingly recognized as essential mediators of normal and pathologic immune responses. Cadmium at concentrations varying from 1.0 x 10(-4) to 3.3 x 10(-6) M inhibited the phytohemagglutinin induced production of interleukin-1 beta and tumour necrosis factor-alpha, in in vitro activated human peripheral blood mononuclear cells. The messenger RNA levels of interleukin-1 beta and tumour necrosis factor-alpha were examined during a 24-h culture period, at different time points. The decreased messenger RNA levels at the time points of the maximum expression of interleukin-1 beta and tumour necrosis factor-alpha indicate that cadmium suppresses their production at the transcriptional level.