Alterations In Steady-state Levels Research Articles

Transposable element sequence fragments incorporated into coding and noncoding transcripts modulate the transcriptome of human pluripotent stem cells.

Transposable elements (TEs) occupy nearly 40% of mammalian genomes and, whilst most are fragmentary and no longer capable of transposition, they can nevertheless contribute to cell function. TEs within genes transcribed by RNA polymerase II can be copied as parts of primary transcripts; however, their full contribution to mature transcript sequences remains unresolved. Here, using long and short read (LR and SR) RNA sequencing data, we show that 26% of coding and 65% of noncoding transcripts in human pluripotent stem cells (hPSCs) contain TE-derived sequences. Different TE families are incorporated into RNAs in unique patterns, with consequences to transcript structure and function. The presence of TE sequences within a transcript is correlated with TE-type specific changes in its subcellular distribution, alterations in steady-state levels and half-life, and differential association with RNA Binding Proteins (RBPs). We identify hPSC-specific incorporation of endogenous retroviruses (ERVs) and LINE:L1 into protein-coding mRNAs, which generate TE sequence-derived peptides. Finally, single cell RNA-seq reveals that hPSCs express ERV-containing transcripts, whilst differentiating subpopulations lack ERVs and express SINE and LINE-containing transcripts. Overall, our comprehensive analysis demonstrates that the incorporation of TE sequences into the RNAs of hPSCs is more widespread and has a greater impact than previously appreciated.

A novel heterozygous OPA3 mutation located in the mitochondrial target sequence results in altered steady-state levels and fragmented mitochondrial network

BackgroundMutations in OPA3 have been reported in patients with autosomal dominant optic atrophy plus cataract and Costeff syndrome. Here, we report the results of a comprehensive study on OPA3 mutations,...

A New Player in Environmentally Induced Oxidative Stress: Polychlorinated Biphenyl Congener, 3,3′-Dichlorobiphenyl (PCB11)

Recent analysis of air samples from Chicago and Lake Michigan areas observed a ubiquitous airborne polychlorinated biphenyl (PCB) congener, 3,3'-dichlorobiphenyl (PCB11). Our analysis of serum samples also revealed the existence of hydroxylated metabolites of PCB11 in human blood. Because PCBs and PCB metabolites have been suggested to induce oxidative stress, this study sought to determine whether environmental exposure to PCB11 and its 4-hydroxyl metabolite could induce alterations in steady-state levels of reactive oxygen species (ROS) and cytotoxicity in immortalized human prostate epithelial cells (RWPE-1). This study also examines if antioxidants could protect the cells from PCB11-induced cytotoxicity. Exponentially growing RWPE-1 cells were exposed to PCB11 and its metabolite, 3,3'-dichlorobiphenyl-4-ol (4-OH-PCB11), as well as an airborne PCB mixture resembling the Chicago ambient air congener profile, every day for 5 days. Results showed that 4-OH-PCB11 could significantly induce cell growth suppression and decrease the viability and plating efficiency of RWPE-1 cells. 4-OH-PCB11 also significantly increased steady-state levels of intracellular superoxide, O₂•⁻), as well as hydroperoxides. Finally, treatment with the combination of polyethylene glycol-conjugated CuZn superoxide dismutase and catalase added 1h after 4-OH-PCB11 exposures, significantly protected RWPE-1 cells from PCB toxicity. The results strongly support the hypothesis that exposure to a hydroxylated metabolite of PCB11 can inhibit cell proliferation and cause cytotoxicity by increasing steady-state levels of ROS. Furthermore, antioxidant treatments following PCBs exposure could significantly mitigate the PCB-induced cytotoxicity in exponentially growing human prostate epithelial cells.

An iTRAQ Proteomics Screen Reveals the Effects of the MDM2 Binding Ligand Nutlin-3 on Cellular Proteostasis

Mouse double minute 2 (MDM2) participates in protein synthesis, folding, and ubiquitin-mediated degradation and is therefore a proteostasic hub protein. The MDM2 interactome contains over 100 proteins, yet stratification of dominant MDM2-interacting proteins has not been achieved. 8-plex iTRAQ (nanoLC-MS/MS) of MCF7 cells treated with the MDM2-binding ligand Nutlin-3 identified the most abundant cellular protein changes over early time points; 1,323 unique proteins were identified including 35 with altered steady-state levels within 2 h of Nutlin-3 treatment, identifying a core group of MDM2 related proteins. Six of these proteins were previously identified MDM2 interactors, and the effects of Nutlin-3 on the MDM2-nucleophosmin interaction (NPM) was further validated. This revealed that Nutlin-3 mediates the in vivo conversion of NPM from an oligomer to a monomer as an MDM2-dependent phenomenon, with Nutlin-3 stimulating MDM2 binding to a peptide motif derived from the oligomerization interface of NPM. These data form the first proteomic screen of Nutlin-3 in cells whereby we (i) identify the most abundant MDM2-interacting proteins whose steady-state levels change early after Nutlin-3 treatment; (ii) identify the first protein apart from p53, nucleophosmin (NPM), whose interaction with MDM2 can be stimulated allosterically by Nutlin-3; and (iii) raise the possibility that Nutlin-3 might act as a general agonist of other MDM2 protein-protein interactions.

Genetic analysis of Parkinson's disease-linked leucine-rich repeat kinase 2

Mutations in LRRK2 (leucine-rich repeat kinase 2) are the most common genetic cause of PD (Parkinson's disease). To investigate how mutations in LRRK2 cause PD, we generated LRRK2 mutant mice either lacking its expression or expressing the R1441C mutant form. Homozygous R1441C knockin mice exhibit no dopaminergic neurodegeneration or alterations in steady-state levels of striatal dopamine, but they show impaired dopamine neurotransmission, as was evident from reductions in amphetamine-induced locomotor activity and stimulated catecholamine release in cultured chromaffin cells as well as impaired dopamine D2 receptor-mediated functions. Whereas LRRK2-/- brains are normal, LRRK2-/- kidneys at 20 months of age develop striking accumulation and aggregation of α-synuclein and ubiquitinated proteins, impairment of the autophagy-lysosomal pathway, and increases in apoptotic cell death, inflammatory responses and oxidative damage. Our further analysis of LRRK2-/- kidneys at multiple ages revealed unique age-dependent biphasic alterations of the autophagic activity, which is unchanged at 1 month of age, enhanced at 7 months, but reduced at 20 months. Levels of α-synuclein and protein carbonyls, a general oxidative damage marker, are also decreased in LRRK2-/- kidneys at 7 months of age. Interestingly, this biphasic alteration is associated with increased levels of lysosomal proteins and proteases as well as progressive accumulation of autolysosomes and lipofuscin granules. We conclude that pathogenic mutations in LRRK2 impair the nigrostriatal dopaminergic pathway, and LRRK2 plays an essential role in the dynamic regulation of autophagy function in vivo.

Translational regulation of Anopheles gambiae mRNAs in the midgut during Plasmodium falciparum infection

BackgroundMalaria is caused by Plasmodium parasites, which are transmitted via the bites of infected Anopheline mosquitoes. Midgut invasion is a major bottleneck for Plasmodium development inside the mosquito vectors. Malaria parasites in the midgut are surrounded by a hostile environment rich in digestive enzymes, while a rapidly responding immune system recognizes Plasmodium ookinetes and recruits killing factors from the midgut and surrounding tissues, dramatically reducing the population of invading ookinetes before they can successfully traverse the midgut epithelium. Understanding molecular details of the parasite-vector interactions requires precise measurement of nascent protein synthesis in the mosquito during Plasmodium infection. Current expression profiling primarily monitors alterations in steady-state levels of mRNA, but does not address the equally critical issue of whether the proteins encoded by the mRNAs are actually synthesized.ResultsIn this study, we used sucrose density gradient centrifugation to isolate actively translating Anopheles gambiae mRNAs based upon their association with polyribosomes (polysomes). The proportion of individual gene transcripts associated with polysomes, which is determined by RNA deep sequencing, reflects mRNA translational status. This approach led to identification of 1017 mosquito transcripts that were primarily regulated at the translational level after ingestion of Plasmodium falciparum-infected blood. Caspar, a negative regulator of the NF-kappaB transcription factor Rel2, appears to be substantially activated at the translational levels during Plasmodium infection. In addition, transcripts of Dcr1, Dcr2 and Drosha, which are involved in small RNA biosynthesis, exhibited enhanced associations with polysomes after P. falciparum challenge. This observation suggests that mosquito microRNAs may play an important role in reactions against Plasmodium invasion.ConclusionsWe analyzed both total cellular mRNAs and mRNAs that are associated with polysomes to simultaneously monitor transcriptomes and nascent protein synthesis in the mosquito. This approach provides more accurate information regarding the rate of protein synthesis, and identifies some mosquito factors that might have gone unrecognized because expression of these proteins is regulated mainly at the translational level rather than at the transcriptional level after mosquitoes ingest a Plasmodium-infected blood meal.

Tryptophan-derived metabolites are required for antifungal defense in the Arabidopsis mlo2 mutant.

Arabidopsis (Arabidopsis thaliana) genes MILDEW RESISTANCE LOCUS O2 (MLO2), MLO6, and MLO12 exhibit unequal genetic redundancy with respect to the modulation of defense responses against powdery mildew fungi and the control of developmental phenotypes such as premature leaf decay. We show that early chlorosis and necrosis of rosette leaves in mlo2 mlo6 mlo12 mutants reflects an authentic but untimely leaf senescence program. Comparative transcriptional profiling revealed that transcripts of several genes encoding tryptophan biosynthetic and metabolic enzymes hyperaccumulate during vegetative development in the mlo2 mlo6 mlo12 mutant. Elevated expression levels of these genes correlate with altered steady-state levels of several indolic metabolites, including the phytoalexin camalexin and indolic glucosinolates, during development in the mlo2 single mutant and the mlo2 mlo6 mlo12 triple mutant. Results of genetic epistasis analysis suggest a decisive role for indolic metabolites in mlo2-conditioned antifungal defense against both biotrophic powdery mildews and a camalexin-sensitive strain of the necrotrophic fungus Botrytis cinerea. The wound- and pathogen-responsive callose synthase POWDERY MILDEW RESISTANCE4/GLUCAN SYNTHASE-LIKE5 was found to be responsible for the spontaneous callose deposits in mlo2 mutant plants but dispensable for mlo2-conditioned penetration resistance. Our data strengthen the notion that powdery mildew resistance of mlo2 genotypes is based on the same defense execution machinery as innate antifungal immune responses that restrict the invasion of nonadapted fungal pathogens.

R1441C mutation in LRRK2 impairs dopaminergic neurotransmission in mice.

Dominantly inherited mutations in leucine-rich repeat kinase 2 (LRRK2) are a common genetic cause of Parkinson's disease (PD). The importance of the R1441 residue in the pathogenesis is highlighted by the identification of three distinct missense mutations. To investigate the pathogenic mechanism underlying LRRK2 dysfunction, we generated a knockin (KI) mouse in which the R1441C mutation is expressed under the control of the endogenous regulatory elements. Homozygous R1441C KI mice appear grossly normal and exhibit no dopaminergic (DA) neurodegeneration or alterations in steady-state levels of striatal dopamine up to 2 years of age. However, these KI mice show reductions in amphetamine (AMPH)-induced locomotor activity and stimulated catecholamine release in cultured chromaffin cells. The introduction of the R1441C mutation also impairs dopamine D2 receptor function, as suggested by decreased responses of KI mice in locomotor activity to the inhibitory effect of a D2 receptor agonist, quinpirole. Furthermore, the firing of nigral neurons in R1441C KI mice show reduced sensitivity to suppression induced by quinpirole, dopamine, or AMPH. Together, our data suggest that the R1441C mutation in LRRK2 impairs stimulated dopamine neurotransmission and D2 receptor function, which may represent pathogenic precursors preceding dopaminergic degeneration in PD brains.

Genetic variation in the CHRNA5 gene affects mRNA levels and is associated with risk for alcohol dependence

Alcohol dependence frequently co-occurs with cigarette smoking, another common addictive behavior. Evidence from genetic studies demonstrates that alcohol dependence and smoking cluster in families and have shared genetic vulnerability. Recently a candidate gene study in nicotine dependent cases and nondependent smoking controls reported strong associations between a missense mutation (rs16969968) in exon 5 of the CHRNA5 gene and a variant in the 3'-UTR of the CHRNA3 gene and nicotine dependence. In this study we performed a comprehensive association analysis of the CHRNA5-CHRNA3-CHRNB4 gene cluster in the Collaborative Study on the Genetics of Alcoholism (COGA) families to investigate the role of genetic variants in risk for alcohol dependence. Using the family-based association test, we observed that a different group of polymorphisms, spanning CHRNA5-CHRNA3, demonstrate association with alcohol dependence defined by Diagnostic and Statistical Manual of Mental Disorders, 4th edn (DSM-IV) criteria. Using logistic regression we replicated this finding in an independent case-control series from the family study of cocaine dependence. These variants show low linkage disequilibrium with the SNPs previously reported to be associated with nicotine dependence and therefore represent an independent observation. Functional studies in human brain reveal that the variants associated with alcohol dependence are also associated with altered steady-state levels of CHRNA5 mRNA.

Insulin Receptor Substrate-1 Is an Important Mediator of Ovarian Cancer Cell Growth Suppression by All-trans Retinoic Acid

There is a need to identify more effective drugs for the treatment of ovarian cancer as it is the leading cause of death among gynecologic tumors. All-trans retinoic acid (ATRA), a natural retinoid, arrests the growth of CA-OV3 ovarian carcinoma cells in G(0)-G(1). Because the insulin-like growth factor-I receptor has been implicated in the proliferation of various tumors, we investigated its potential role in the suppression of ovarian cancer cell growth by ATRA. Our studies revealed that insulin receptor substrate-1 (IRS-1) protein levels decrease in CA-OV3 cells on ATRA treatment, whereas no differences in IRS-1 levels were seen in the ATRA-resistant SK-OV3 cells. Moreover, CA-OV3 clones overexpressing IRS-1 were growth inhibited less by ATRA, whereas SK-OV3 clones in which levels of IRS-1 were reduced by expression of antisense IRS-1 became sensitive to growth inhibition by ATRA treatment. Studies to determine the mechanism by which ATRA reduced IRS-1 expression showed that ATRA altered steady-state levels of IRS-1 mRNA and the stability of IRS-1 protein. Finally, the role of IRS-1 as a potential molecular target of ATRA in ovarian tumors was assessed by immunohistochemistry in an ovarian cancer tissue array. Compared with normal ovary, the majority of malignant epithelial ovarian tumors overexpressed IRS-1. Thus, there seems to be a correlation between IRS-1 expression and malignancy in ovarian tumors. Our results suggest that IRS-1 is in fact an important growth-regulatory molecule that can be a potential effective target for chemotherapeutic intervention with growth-suppressive agents, including retinoids.

Human microvascular endothelial synthesis of interleukin‐8 during in vitro ischemia and reperfusion

These studies were undertaken to evaluate human microvascular endothelial cell (MEC) synthesis of interleukin-8 (IL-8), a potent neutrophil chemoattractant, under in vitro conditions of ischemia and reperfusion. IL-8 and other related CXC chemokines are believed to mediate tissue injury in a variety of pathologic conditions in humans. MEC grown on microcarrier beads were exposed to 3 or 6 h of in vitro ischemia followed by 2 h of reperfusion. Conditioned medium, MEC protein, and total RNA extracts were assayed for IL-8 using an ELISA. During ischemia alone, MEC increased intracellular, but not extracellular levels of IL-8 secretion. In contrast, reperfusion markedly stimulated both intracellular and extracellular IL-8 secretion. Neither 3 h of ischemia alone or followed by reperfusion altered steady-state levels of IL-8 mRNA when compared to pre-ischemic levels. In contrast, after 6 h of ischemia alone and ischemia followed by reperfusion, IL-8 mRNA was increased eight- and sixfold, respectively, when compared to pre-ischemic levels. These studies demonstrate an inverse relationship between the rate of IL-8 protein secretion and the steady-state levels of IL-8 mRNA during ischemia and reperfusion. During ischemia and reperfusion both the increase in cell-associated IL-8 protein and the release of IL-8 into the medium is dependent on de novo protein synthesis rather than the intracellular accumulation of IL-8. These experiments indicate that post-ischemic modulation of IL-8 release and synthesis following ischemia reperfusion will require strategies directed towards inhibition of IL-8 transcription and in depth knowledge of the mechanisms regulating IL-8 secretion.

Persistence of Chlamydia trachomatis is induced by ciprofloxacin and ofloxacin in vitro.

An in vitro cell culture model was used to investigate the long-term effect of ciprofloxacin and ofloxacin on infection with Chlamydia trachomatis. Standard in vitro susceptibility testing clearly indicated successful suppression of chlamydial growth. To mimic better in vivo infection conditions, extended treatment with the drugs was started after infection in vitro had been well established. Incubation of such established chlamydial cultures with ciprofloxacin and ofloxacin not only failed to eradicate the organism from host cells, but rather induced a state of chlamydial persistence. This state was characterized by the presence of nonculturable, but fully viable, bacteria and the development of aberrant inclusions. In addition chlamydia exhibited altered steady-state levels of key chlamydial antigens, with significantly reduced major outer membrane protein and near constant hsp60 levels. Resumption of overt chlamydial growth occurred after withdrawal of ciprofloxacin, confirming the viability of persisting chlamydia. In vitro ciprofloxacin results are consistent with clinical data, thereby providing an explanation for treatment failures of ciprofloxacin. Parallel in vitro studies with ofloxacin suggest a better correlation between clinical and laboratory-defined efficacy, although the clinical studies on which this assessment is based did not include monitoring of chlamydial persistence. The data presented here clearly demonstrate that under at least some circumstances, standard determination of MICs and minimal bactericidal concentrations for C. trachomatis allows no more than a simple definition of whether an antibiotic has some anti chlamydial activity; however, such testing is not always sufficient to verify that the antibiotic will eliminate the organism in vivo.

Birth insult increases amphetamine-induced behavioral responses in the adult rat

We have previously reported that an apparently uncomplicated Caesarean section birth produces long-term alterations in steady-state levels of dopamine in the central nervous system of the rat. In addition, adult rats that had been born by Caesarean section, either with or without acute global anoxia, showed markedly greater dopamine release from the nucleus accumbens in response to repeated stress, in comparison to vaginally born controls. The aim of the present study was to test whether these birth complications also result in long-term changes in behavior mediated by dopamine systems. For this, we investigated effects of a low dose (0.5 mg/kg) of amphetamine on activity levels in three-month-old rats that had been born vaginally (control), by rapid Caesarean section, or by Caesarean section with 15 min of global anoxia. Amphetamine induced a significantly greater increase in locomotor activity in animals born by Caesarean section or by Caesarean section+15 min anoxia, in comparison to the drug's effects in vaginally born controls. Behavioral responses were further analysed from video recordings of the animals' behavior. In confirmation of automated activity counts, both animals born by Caesarean section and by Caesarean section+15 min anoxia showed a significant increase in the duration and frequency of moving and a decrease in the duration and frequency of standing, in comparison to vaginally born controls. Animals delivered by Caesarean section showed a significant increase in the duration of sniffing and a decrease in the duration and frequency of grooming when compared to vaginally born controls. Animals delivered by Caesarean section+15 min anoxia showed a significant increase in the duration and frequency of rearing, in comparison to controls. The pattern of behavioral changes observed indicates that, as adults, animals born by Caesarean section and by Caesarean section with added global anoxia both show heightened behavioral responses to amphetamine, in comparison to vaginally born animals. These findings highlight the sensitivity of dopamine pathways to variations in birth procedure and add experimental support to epidemiological evidence implicating birth complications in the pathophysiology of disorders involving central dopaminergic neurons, such as schizophrenia.

His-65 in the proton-sucrose symporter is an essential amino acid whose modification with site-directed mutagenesis increases transport activity.

The proton-sucrose symporter that mediates phloem loading is a key component of assimilate partitioning in many higher plants. Previous biochemical investigations showed that a diethyl pyrocarbonate-sensitive histidine residue is at or near the substrate-binding site of the symporter. Among the proton-sucrose symporters cloned to date, only the histidine residue at position 65 of AtSUC1 from Arabidopsis thaliana is conserved across species. To test whether His-65 is involved in the transport reaction, we have used site-directed mutagenesis and functional expression in yeast to determine the significance of this residue in the reaction mechanism. Symporters with mutations at His-65 exhibited a range of activities; for example, the H65C mutant resulted in the complete loss of transport capacity, whereas H65Q was almost as active as wild type. Surprisingly, the H65K and H65R symporters transport sucrose at significantly higher rates (increased Vmax) than the wild-type symporter, suggesting His-65 may be associated with a rate-limiting step in the transport reaction. RNA gel blot and protein blot analyses showed that, with the exception of H65C, the variation in transport activity was not because of alterations in steady-state levels of mRNA or symporter protein. Significantly, those symporters with substitutions of His-65 that remained transport competent were no longer sensitive to inactivation by diethyl pyrocarbonate, demonstrating that this is the inhibitor-sensitive histidine residue. Taken together with our previous results, these data show that His-65 is involved in sucrose binding, and increased rates of transport implicate this region of the protein in the transport reaction.

Alterations in Catecholamine Turnover in Specific Regions of the Rat Brain Following Acute Exposure to Nitrous Oxide

The effects of nitrous oxide (N 2O) on steady-state concentrations and turnover rates of catecholamines in the olfactory bulb, hypothalamus, brain stem, hippocampus, striatum, thalamus, cerebral cortex, and spinal cord were determined in rats. Animals were exposed for 2 h to either 60% N 2O or air. Immediately following exposure, all animals were injected intraperitoneally with alpha-methylparatyrosine (αMPT), a competitive inhibitor of tyrosine hydroxylase, and sacrificed at 0, 30, or 90 min postinjection. Brain catecholamine concentrations were determined using high-performance liquid chromatography coupled with electrochemical detection (HPLC-EC). Results indicate that N 2O exposure significantly elevates steady-state concentrations of norepinephrine (NE) in the hypothalamus and striatum yet decreases amine levels in the brain stem region. Steady-state levels of dopamine (DA) were not significantly altered in any region of the CNS by N 2O exposure. Acute exposure to N 2O also resulted in significant decreases in the turnover rate of NE in the brain stem, yet it increased turnover of this amine in the olfactory bulb, hypothalamus, and striatum. Acute exposure to N 2O resulted in a decreased turnover rate of DA in the hippocampus and striatum. In contrast, N 2O appears to increase DA turnover in the olfactory bulb. These results indicate that acute exposure to N 2O in rats causes region-specific alterations in steady-state levels and turnover rates of DA and NE within the central nervous system.

Temporal Alterations in Basement Membrane Components in the Pulmonary Vasculature of the Chronically Hypoxic Rat: Impact of Hypoxia and Recovery

The hypoxic model of pulmonary hypertension was used to examine temporal alterations in the deposition of the basement membrane (BM) and components of fibronectin, laminin, and Type IV collagen within vascular, airway, and gas exchange compartments of the lung. Because hypoxic pulmonary hypertension is a reversible model of hypertension, changes in fibronectin and laminin synthesis/deposition in the recovering lung were also examined. Long-term hypoxic exposure produced decreases in body weight, increased right ventricular and lung dry weights and elevations in pulmonary arterial pressure. Immunohistochemical analysis revealed consistent and progressive increases in the deposition of fibronectin and laminin, but not type IV collagen, in the subendothelial and medial BMs of large and small pulmonary arteries, but not in airways or lung parenchyma. These changes were observed by day 4 of hypoxia and were most prominent in the conducting vasculature. Northern analysis showed a biphasic pattern of alterations in steady-state levels of BM component mRNA in hypoxic rats with early reductions at days 4 and 7 followed by increases at day 12. Recovery from 12 days of hypoxia resulted in regression of pulmonary hypertension and right ventricular hypertrophy but not increased lung weight. Immunohistochemical analysis of fibronectin, laminin, and type IV collagen levels in the vasculature showed a temporal regression to levels that were not remarkably different from time-matched controls at day 30 of recovery. Northern analysis of lungs from hypoxic-recovery rats revealed increased steady-state levels of mRNA for fibronectin, laminin, and type IV collagen at all time points. These data indicate that long-term hypoxic exposure elicits marked alterations in the synthetic capacity and deposition of the important cell attachment BM glycoproteins fibronectin and laminin. In addition, recovery from hypoxia appears to be characterized by a lack of increased fibronectin and laminin levels in the conducting vasculature, suggesting a marked and rapid reorganization of the vascular BMs on both hypoxic exposure and recovery from hypoxia.

Temporal alterations in basement membrane components in the pulmonary vasculature of the chronically hypoxic rat: impact of hypoxia and recovery.

The hypoxic model of pulmonary hypertension was used to examine temporal alterations in the deposition of the basement membrane (BM) and components of fibronectin, laminin, and Type IV collagen within vascular, airway, and gas exchange compartments of the lung. Because hypoxic pulmonary hypertension is a reversible model of hypertension, changes in fibronectin and laminin synthesis/deposition in the recovering lung were also examined. Long-term hypoxic exposure produced decreases in body weight, increased right ventricular and lung dry weights and elevations in pulmonary arterial pressure. Immunohistochemical analysis revealed consistent and progressive increases in the deposition of fibronectin and laminin, but not type IV collagen, in the subendothelial and medial BMs of large and small pulmonary arteries, but not in airways or lung parenchyma. These changes were observed by day 4 of hypoxia and were most prominent in the conducting vasculature. Northern analysis showed a biphasic pattern of alterations in steady-state levels of BM component mRNA in hypoxic rats with early reductions at days 4 and 7 followed by increases at day 12. Recovery from 12 days of hypoxia resulted in regression of pulmonary hypertension and right ventricular hypertrophy but not increased lung weight. Immunohistochemical analysis of fibronectin, laminin, and type IV collagen levels in the vasculature showed a temporal regression to levels that were not remarkably different from time-matched controls at day 30 of recovery. Northern analysis of lungs from hypoxic-recovery rats revealed increased steady-state levels of mRNA for fibronectin, laminin, and type IV collagen at all time points. These data indicate that long-term hypoxic exposure elicits marked alterations in the synthetic capacity and deposition of the important cell attachment BM glycoproteins fibronectin and laminin. In addition, recovery from hypoxia appears to be characterized by a lack of increased fibronectin and laminin levels in the conducting vasculature, suggesting a marked and rapid reorganization of the vascular BMs on both hypoxic exposure and recovery from hypoxia.

Effects of chronic ethanol administration on the endocytosis of cytokines by rat hepatocytes.

The effects of chronic ethanol administration on the endocytosis of three representative cytokines were investigated in isolated rat hepatocytes. When hepatocytes were isolated from rats that were fed an ethanol liquid diet for 12 to 13 weeks, these cells exhibited a decreased ability to internalize and degrade transforming growth factor-alpha, tumor necrosis factor-alpha and interleukin-6, compared with hepatocytes from the pair-fed controls. This impaired endocytosis of all three cytokines was accompanied by significant decreases in the amount of hepatocyte surface-bound cytokine. Changes in cytokine binding to surface receptors and reduced rates of receptor-cytokine complex internalization into the cells seem to be major contributors to defective endocytosis in hepatocytes from the ethanol-fed rats. Impaired hepatocyte endocytosis could lead to altered steady-state levels of cytokines in the liver and modified physiological responses to cytokines. These changes could affect homeostasis among the various cell types in the liver and could contribute to liver dysfunction and injury.

Temporal Alterations in Specific Basement Membrane Components in Lungs from Monocrotaline-treated Rats

The present study utilized the monocrotaline (MCT) model of pulmonary hypertension in rats to examine temporal alterations in steady-state levels of basement membrane (BM) component mRNA and deposition of protein using Northern analysis and immunohistochemistry, respectively. MCT (60 mg/kg, subcutaneous) produced sustained increases in lung dry tissue mass by 7 days, right ventricular mass by 14 days, and pulmonary arterial pressure by 21 days after administration. mRNA levels specific for laminin (LM) were elevated as early as 1 day after MCT treatment, while mRNA for all BM components examined except type IV collagen were increased in lungs from MCT-treated rats by day 4. Differences in LM, perlecan (PN), and type IV collagen-specific mRNAs from lung tissue between MCT-treated and control rats disappeared by day 14. In contrast, fibronectin (FN) mRNA remained elevated in lung tissue from MCT-treated rats from day 4 onward. Increases in immunolocalizable FN and LM in the vasculature, and PN and type IV collagen in gas exchange areas, were observed 4 days after MCT treatment compared with controls. These changes generally became more pronounced by 21 days after MCT administration, at which time the parenchyma of MCT-treated rats also demonstrated increases in immunolocalizable FN, LM, and BM-chondroitin sulfate proteoglycan (BM-CSPG). The pulmonary vasculature additionally showed increases in type IV collagen, PN, and BM-CSPG in MCT-treated rats compared with controls by 21 days. These observations suggest that the accumulation of specific BM components in the pulmonary vasculature and parenchyma may contribute to the pathogenesis and maintenance of MCT-induced hypertensive pulmonary vascular disease.

Alterations in the levels of expression of specific cellular genes in adenovirus-infected and -transformed cells

There is increasing evidence that changes in the transcriptional program of cellular genes in virus-transformed cells can contribute to virus transformation. It is, therefore, important to study altered expression patterns of cellular genes in adenovirus-infected and -transformed cells. We have used 40 different cellular genes or gene segments as hybridization probes to analyze the cytoplasmic RNA from adenovirus type 2 (Ad2)-infected KB cells, from Ad5-transformed human cells (293) or from several Ad2- or adenovirus type 12 (Ad12)-transformed hamster cell lines. Many of the genes probed were not expressed in human or hamster cells. Transcription of the ADPRT and the heat shock protein 70 genes was increased in Ad2-infected KB cells and in 293 cells. In Ad2-infected KB cells, c- myc gene transcription was decreased. In 293 cells and in three adenovirus-transformed hamster cell lines (T637, BHK21-Ad2E1A-E1B, and BHK21-Ad2 HindIII-G), the transcription of the c- jun gene was increased, whereas c- myc transcription was decreased in the latter two cell lines. The data presented here demonstrate that, among 40 different mammalian gene probes, alterations in steady state levels of RNA were detected for five of these genes. These results suggest major alterations in transcription patterns in adenovirus-infected and -transformed cells.