Comparison of two measures of human immunodeficiency virus (HIV) type 1 load in HIV risk groups.

Levels of viral burden were compared across risk group and gender populations among 485 human immunodeficiency virus type 1 (HIV-1)-infected participants consisting of 190 male injection drug users (IDUs), 92 female IDUs, and 203 homosexual men. Viral burden was quantified by a microculture technique to determine cell-associated infectious units per 10(6) peripheral blood mononuclear cells (IUPM) and by reverse transcriptase PCR (Amplicor) to determine plasma HIV RNA levels. Adjusting for CD4(+) cell count, females had a lower infectious HIV load than all males combined (0. 33 log10 lower; P = 0.004), and homosexual men had a 0.29 log10 higher infectious viral load than all IDUs combined (P = 0.001). For HIV RNA levels, females had lower levels than males (0.19 log10 lower; P = 0.04), but no differences were observed by risk group. After controlling for percent CD4(+) cells, no differences were found by risk group for either assay, but females still had a 0.25 log10 lower infectious viral load than males (P = 0.04) and a viral RNA load similar to that of males (P = 0.25). The correlation between infectious viral load and HIV RNA load was 0.58 overall, which did not differ by gender or risk group. Our data suggest that differences in viral load may exist by gender and that any differences observed by risk group are driven predominantly by gender or percent CD4(+) cell differences. These data also confirm a moderate correlation between cell-associated infectious viral load and plasma HIV RNA load, which appears to be similar by gender and across risk groups.

Ectopic expression of metallothionein-III causes pancreatic acinar cell necrosis in transgenic mice.

Mice express four distinct metallothioneins (MTs) that have similar metal-binding properties. MT-I and MT-II are expressed coordinately in most organs, whereas MT-III is expressed predominantly in a subset of neurons and MT-IV is expressed in certain stratified epithelia. The restricted expression of MT-III suggests that it may severe a specialized function. To test this hypothesis, transgenic mice were generated that express MT-III in the wider expression domain of MT-I. Similar transgenic lines expressing extra MT-I under the same regulation were generated as controls for the effect of over-expression of MT. Transgenic mice that express MT-III ectopically frequently die at 2-3 months of age. The pancreata of moribund mice were abnormally small and histological examination, at various ages, revealed a progressive degeneration of the acinar cells. At early stages multifocal acinar cell eosinophilia and swollen nuclei were seen and this pathology progressed to multifocal acinar cell necrosis and fibrosis. The terminal stages were characterized by a loss of the acinar compartment, leaving the islets embedded in a fibrotic remnant. Other organs of these mice were grossly and histologically normal. All organs examined from mice expressing excess MT-I were unremarkable even though expression of either MT-I or MT-III transgenes resulted in similar accumulations of zinc and copper in the pancreata. This study indicates that pancreatic acinar cells are unusually sensitive to chronic expression of MT-III. The mechanism by which MT-III disrupts pancreatic function is unclear, but the results provide further evidence that MT isoforms exhibit distinct properties and probably serve distinct biological functions.

Inhibition of cholesterol synthesis by squalene synthase inhibitors does not induce myotoxicity in vitro.

The cholesterol-lowering HMG CoA reductase inhibitors (HMGRI), pravastatin and lovastatin, have been associated with skeletal myopathy in humans and in rats. In a previous in vitro study, HMGRI-induced changes in neonatal rat skeletal muscle cells were characterized by reversible inhibition of protein synthesis and loss of differentiated myotubes at concentrations markedly lower than those inducing enzyme leakage. Myotoxicity was determined to be directly related to inhibition of HMG CoA reductase, since mevalonate, the immediate product of HMG CoA reductase metabolism, abrogated the drug-induced changes. Farnesol, geranylgeraniol, and squalene are metabolites of mevalonate. Squalene, formed from farnesol by squalene synthase, is the first metabolite solely committed to cholesterol synthesis. In contrast, geranylgeraniol, formed by the addition of an isoprene group to farnesol, is the first metabolite uncommitted to cholesterol synthesis. The objective of the present study was to determine the role of inhibition of cholesterol synthesis in HMGRI-induced in vitro myotoxicity. HMGRI-treated neonatal rat skeletal muscle cultures were supplemented with farnesol and geranylgeraniol, and in another study, muscle cultures were exposed to two squalene synthase inhibitors (SSI), BMS-187745 and its prodrug ester, BMS-188494. Endpoints evaluated for both studies included protein synthesis ([3H]leucine incorporation), total cellular protein (a measure of cell loss), intra- and extracellular lactate dehydrogenase activity (a measure of membrane integrity), cholesterol biosynthesis ([14C]acetate incorporation), and morphology. HMG CoA reductase inhibitor-induced morphologic changes and inhibition of protein synthesis were significantly ameliorated by supplementation with farnesol and geranylgeraniol. In contrast to HMGRI-induced in vitro myotoxicity, SSI induced an irreversible, minimal cytotoxicity at close to maximum soluble concentrations. These results indicate that depletion of metabolites of geranylgeranyl pyrophosphate, and not inhibition of cholesterol synthesis, is the primary cause of HMG CoA reductase-induced myotoxicity.

HMG CoA reductase inhibitor-induced myotoxicity: pravastatin and lovastatin inhibit the geranylgeranylation of low-molecular-weight proteins in neonatal rat muscle cell culture.

In previous studies, inhibition of cholesterol synthesis by HMG CoA reductase inhibitors (HMGRI) was associated with myotoxicity in cultures of neonatal rat skeletal myotubes, and rhabdomyolysis in rats, rabbits, and humans in vivo. In vitro myotoxicity was directly related to HMGRI-induced depletion of mevalonate, farnesol, and geranylgeraniol, since supplementation with these intermediate metabolites abrogated the toxicity. Both farnesol and geranylgeraniol are required for the posttranslational modification, or isoprenylation, of essential regulatory proteins in mammalian cells. The objective of the present study was to measure changes in protein isoprenylation in cultured neonatal rat skeletal muscle cells exposed for 24 hr to increasing concentrations of pravastatin or lovastatin. Proteins were labeled with [3H]mevalonate, [3H]farnesyl pyrophosphate (FPP), or [3H]geranylgeranyl pyrophosphate (GGPP), and then separated by SDS-PAGE and quantitated by scintillation counting and densitometry of autoradiographs. Mevalonate and FPP labeling of the majority of proteins increased in a concentration-dependent manner, even at concentrations greater than 2 microM lovastatin and 25 microM pravastatin that completely inhibited cholesterol synthesis. In contrast, mevalonate and FPP labeling of three protein bands with molecular weights of 26.6, 27.7, and 28.9 kDa was markedly inhibited at concentrations higher than 1 microM lovastatin and 400 microM pravastatin, which inhibited protein synthesis and disrupted myotube morphology after longer exposures in a previous study. In contrast, these proteins were equally well labeled by GGPP at all HMGRI concentrations tested, suggesting that isoprenylation of the 26.9-, 27.8-, and 28.9-kDa proteins requires geranylgeraniol. The results of this study indicate that HMGRI-induced myotoxicity is most likely related to reduced posttranslational modification of specific regulatory proteins by geranylgeraniol.

Expression of human metallothionein-III in transgenic mice.

Transgenic mice that express human metallothionein-III (hMT-III) were generated. Human MT-III mRNA expression was prominent in brain, resulting in a 9-fold elevation of MT-III mRNA in cortex, a 3-5-fold elevation in hippocampus, thalamus, brainstem, and olfactory bulb, and a 1.4-fold elevation in cerebellum. Human MT-III protein was detected biochemically and accounted for a 3.4-fold increase in total brain MT. The concentration of zinc (but not copper) was elevated in those brain regions that expressed the most hMT-III mRNA. The histochemically reactive pool of zinc, as measured by Timm's stain or TS-Q histofluorescence, was not appreciably altered. No changes in brain weight, morphology or histology have been noted; the mice breed normally and appear to have normal behavior.

In vitro myotoxicity of the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, pravastatin, lovastatin, and simvastatin, using neonatal rat skeletal myocytes.

Pravastatin, lovastatin, and simvastatin, drugs which lower cholesterol by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, have been linked to skeletal myopathies in humans and rats. The myotoxicity of these three drugs was compared, after 48 hr exposure, in cultures of primary neonatal rat skeletal myotubes. Measurements included HMG CoA reductase activity ([14C]acetate incorporation into cholesterol), indicators of membrane damage (CPK, LDH, and AST), cell viability (mitochondrial dehydrogenase metabolism of MTT), protein synthesis ([3H]leucine incorporation), and energy status (ATP). All three drugs inhibited cholesterol synthesis to the same extent in rat hepatocytes (IC50s approximately 0.07 microM). Lovastatin- and simvastatin-induced inhibition of cholesterol synthesis in myotubes was unchanged compared to that of hepatocytes, but pravastatin was 85-fold less potent (IC50 = 5.9 microM). Protein synthesis and ATP levels were the most sensitive indicators of toxicity. Pravastatin (IC50 = 759 microM) was > 100-fold less inhibitory of protein synthesis than lovastatin (IC50 = 5.4 microM) or simvastatin (IC50 = 1.9 microM). Addition of mevalonic acid (the immediate product of the HMG CoA reductase reaction), as 100 microM mevalonic acid lactone, reversed the toxicity of all three drugs. Removal of serum for 24-72 hr did not alter the toxicity of any of the drugs compared to cultures containing 10% serum, suggesting that differences in protein binding did not account for the differences in toxicity of the drugs. These results indicate that pravastatin is less myotoxic than lovastatin or simvastatin in this in vitro system using neonatal rat skeletal muscle cells, and this differential toxicity is correlated with the selective decrease in inhibition of HMG CoA reductase by pravastatin in nonhepatic tissues.

Metallothionein III is expressed in neurons that sequester zinc in synaptic vesicles.

MT-III, a brain-specific member of the metallothionein gene family, binds zinc and may facilitate the storage of zinc in neurons. The distribution of MT-III mRNA within the adult brain was determined by solution and in situ hybridization and compared to that of MT-I mRNA. MT-III mRNA is particularly abundant within the cerebral cortex, hippocampus, amygdala, and nuclei at base of the cerebellum. Transgenic mice generated using 11.5 kb of the mouse MT-III 5' flanking region fused to the E. coli lacZ gene express beta-galactosidase in many of the same regions identified by in situ hybridization. MT-III mRNA was present in readily identifiable neurons within the olfactory bulb, hippocampus, and cerebellum, and beta-galactosidase activity was localized to neurons throughout the brain, but not to glia, as determined by costaining with X-Gal and neural- and glia-specific antibodies. There is marked correspondence between the neurons that are rich in MT-III mRNA and those neurons that store zinc in their terminal vesicles. MT-III is found complexed with zinc in vivo and its expression in cultured cells leads to the intracellular accumulation of zinc and enhanced histochemical detection of zinc. These results are discussed in light of the possibility that MT-III may participate in the utilization of zinc as a neuromodulator.

Leukemia inhibitory factor induces neurotransmitter switching in transgenic mice.

Leukemia inhibitory factor (LIF) is a cytokine growth factor that induces rat sympathetic neurons to switch their neurotransmitter phenotype from noradrenergic to cholinergic in vitro. To test whether LIF can influence neuronal differentiation in vivo, we generated transgenic mice that expressed LIF in pancreatic islets under the control of the insulin promoter and evaluated the neurotransmitter phenotype of the pancreatic sympathetic innervation. We also used the insulin promoter to coexpress nerve growth factor in the islets, which greatly increased the density of sympathetic innervation and facilitated analysis of the effects of LIF. Our data demonstrate that tyrosine hydroxylase and catecholamines declined and choline acetyltransferase increased in response to LIF. We conclude that LIF can induce neurotransmitter switching of sympathetic neurons in vivo.

Targeted disruption of metallothionein I and II genes increases sensitivity to cadmium.

We inactivated the mouse metallothionein (MT)-I and MT-II genes in embryonic stem cells and generated mice homozygous for these mutant alleles. These mice were viable and reproduced normally when reared under normal laboratory conditions. They were, however, more susceptible to hepatic poisoning by cadmium. This proves that these widely expressed MTs are not essential for development but that they do protect against cadmium toxicity. These mice provide a means for testing other proposed functions of MT in vivo.

Insulin-like growth factor I receptors and IGF-I actions in neuronal cultures from the brain.

Neurons in primary culture have been used to study IGF-I receptors and IGF-I-induced cellular actions in the brain. Intact neurons in culture specifically bind [125I]IGF with high affinity. The potency for the competition of [125I]IGF-I binding was IGF-I > IGF-II > insulin. A curvilinear Scatchard plot represented high-affinity (0.15 nM) and low-affinity (3 nM) binding sites with a Bmax of 142 fmol and 618 fmol/mg protein, respectively. These binding sites are predominantly localized on neurites with relatively few sites on the cell soma. IGF-I induced synthesis of protein(s) in the M(r) range of 48,000-50,000 with pI values of 6.1-6.4. These observations show that IGF-I receptor mediates induction of specific proteins and suggest that these proteins may be involved in the neurotrophic activity of IGF-I in the brain.

Characterization of rabbit primary proximal tubule kidney cell cultures grown on Millicell-HA membrane filters.

With the aim of developing a kidney cell culture system that can be used to assess renal toxicity in vivo, freshly isolated rabbit proximal tubules were plated on Millipore cellulose filters mounted in plastic inserts (Millicell-HA). DNA synthesis peaked on day 6 of culture and cells reached confluency by days 12-14. The integrity of the monolayer was confirmed by exclusion of [(14)C]inulin and cell viability demonstrated by linearity of protein synthesis over a 24-hr period. In confluent cultures, the organic anion, [(14)C]p-aminohippuric acid (PAH) and cation [(14)C]tetraethylammonium bromide (TEA) were shown to be transported from the basolateral to the apical side at a rate 5-6 times greater than that from the apical to basolateral side during the first 60 min of exposure. Probenecid decreased PAH transport by 60% and N-methylnicotinamide and quinine inhibited TEA transport by 40 and 56%, respectively. Uptake of [(14)C]alpha-methylglucopyranoside into the cells was three times greater when label was added to the apical side than when label was added to the basolateral side. Apical uptake of glucose was sodium dependent and inhibited by more than 90% with phloridzin. Thus, kidney proximal tubule cells in the filter insert culture system display functional polarity which appears to mimic function in vivo and may be useful for examining mechanisms of nephrotoxicity.

Developmental regulation of insulin-like growth factor-I-stimulated glucose transporter in rat brain astrocytes.

Astrocytic glial cells from 1- and 21-day-old rat brains were established in primary culture to study the expression of insulin-like growth factor-I (IGF-I) receptors and IGF-I-stimulated glucose transporter (Glut-1). Astrocytes from both age groups expressed specific high affinity IGF-I receptors, whose relative affinities for IGF-I, IGF-II, and insulin were comparable. However, the total number of binding sites and IGF-I receptor mRNA levels were 148% and 240% higher in astrocytes from 21-day-old compared with 1-day-old brains. IGF-I caused a dose-dependent stimulation of [3H]2-deoxy-D-glucose [( 3H]dGlc) uptake in astrocytes from 1-day-old brains. This was associated with increases in Glut-1 protein and mRNA levels. In contrast, astrocytes from 21-day-old brains exhibited a 58% decrease in the binding capacity and a 77% decrease in the steady state levels of Glut-1 protein and its mRNA. In addition, IGF-I failed to stimulate the Glut-1 system in these cells. This lack of IGF-I effect is not due to an alteration inherent to the Glut-1 system, since 12-O-tetradecanoyl-phorbol-13-acetate stimulated [3H]dGlc uptake and Glut-1 protein and its mRNA levels. These observations suggest that changes in basal and IGF-I-stimulated Glut-1 system in brain astrocytes may be developmentally regulated.

Insulin-like growth factor I (IGF-I) receptors and IGF-I action in oligodendrocytes from rat brains.

Oligodendrocyte progenitor cells were prepared by mechanical dissociation of 1-day-old rat brain cultures. These cells undergo proliferation and differentiation into oligodendrocytes as demonstrated by the expression of proliferation and differentiation-related specific antigens. We have used this unique culture system to characterize insulin-like growth factor I (IGF-I) receptors and their action in the central nervous system (CNS). 125I-IGF-I specifically binds to these cultures with high affinity. Competition-inhibition data suggest that IGF-I is most potent in competing for 125I-IGF-I binding, followed by IGF-II and insulin. Scatchard analyses of the binding data indicate a curvilinear plot with a Kd for high affinity of 0.2 nM, and a Bmax of 247 fmol/mg, and a Kd for low affinity of 3.2 nM and Bmax of 1213 fmol/mg protein. Covalent cross-linking followed by SDS-PAGE analysis demonstrated a radioactive band of Mr 135,000 which corresponds to the alpha subunit of the IGF-I receptor. Solution hybridization/RNase protection assay produced a single protected band corresponding to IGF-I receptor messenger RNA, further confirming the presence of these receptors. Incubation of progenitor cells with IGF-I resulted in a time- and concentration-dependent increase in [3H]thymidine incorporation and cell numbers. This effect appears to be mediated by IGF-I receptors since IGF-II and insulin were proportionately less potent. In addition to its effect on proliferation, IGF-I also increased the number of 4E7- and GC-antigen positive cells. These observations indicate that oligodendrocytes in primary culture express specific IGF-I receptors and that the interaction of IGF-I with these receptors results in the proliferation as well as differentiation of oligodendrocytes.

Effects of cis-diamminedichloroplatinum(II) on rabbit kidney in vivo and on rabbit renal proximal tubule cells in culture.

The nephrotoxic potential of cis-diamminedichloroplatinum(II) (CDDP) in rabbits, as well as its effect on cell viability, cellular synthetic activity, and specific enzyme activities in rabbit renal proximal tubule cells, was investigated. Male New Zealand White rabbits were given a single i.v. dose of either 2.5 or 5.0 mg/kg CDDP via the ear vein and sacrificed 5 days later. No drug-induced changes were observed in the kidneys of rabbits given 2.5 mg/kg CDDP. However, histopathological examination of kidneys from rabbits administered 5.0 mg/kg CDDP revealed marked tubular degeneration and necrosis, with the majority of lesions being situated in the outer zone of the cortex. This is in contrast to the effect of CDDP in the kidney of the rat where the necrosis is reported to be predominantly localized to the pars recta of the proximal tubule in the outer stripe of the medulla. The results from the in vitro experiments indicated that the viability of cells after a 6-h exposure to CDDP at concentrations up to 100 microM was greater than 95%. However, a dose-dependent decrease in cell viability was obtained after 24 h exposure with a TD50 (50% viability) of approximately 90 microM. In addition, the results after 24 h exposure to CDDP also indicated that Na+, K+-ATPase, a basolateral membrane marker enzyme, and alkaline phosphatase, a brush-border marker enzyme, were inhibited by 35-40% and 20%, respectively. No effect on succinic dehydrogenase, a mitochondrial marker enzyme, was obtained. Inhibition of all three marker enzymes was minimal at 6 h posttreatment. On the other hand, inhibition of DNA, RNA, and protein syntheses was evident as early as 6 h posttreatment with DNA (48-77%) and RNA (36-77%) syntheses being inhibited to a greater extent than protein synthesis (14-33%). These results demonstrate that inhibition of renal synthetic activity by CDDP, rather than its effect on enzyme activity, precedes the onset of cell lethality and may therefore be an important event in the initiation of CDDP-induced nephrotoxicity.

Insulin receptors in the brain: structural and physiological characterization.

The present study was conducted to characterize insulin receptors and to determine the effects of insulin in synaptosomes prepared from adult rat brains. Binding of 125I-insulin to synaptosome insulin receptors was highly specific and time dependent: equilibrium binding was obtained within 60 minutes, and a t1/2 of dissociation of 26 minutes. Cross-linking of 125I-insulin to its receptor followed by SDS-PAGE demonstrated that the apparent molecular weight of the alpha subunit of the receptor was 122,000 compared with 134,000 for the liver insulin receptor. In addition, insulin stimulated the dose-dependent phosphorylation of exogenous tyrosine containing substrate and a 95,000 MW plasma membrane associated protein, in a lectin-purified insulin receptor preparation. The membrane associated protein was determined to be the beta subunit of the insulin receptor. Incubation of synaptosomes with insulin caused a dose-dependent inhibition of specific sodium-sensitive [3H]norepinephrine uptake. Insulin inhibition of [3H]norepinephrine uptake was mediated by a decrease in active uptake sites without any effects in the Km, and was specific for insulin since related and unrelated peptides influenced the uptake in proportion to their structural similarity with insulin. These observations indicate that synaptosomes prepared from the adult rat brain possess specific insulin receptors and insulin has inhibitory effects on norepinephrine uptake in the preparation.

Insulin receptors and insulin action in dissociated brain cells.

The present study was conducted to characterize insulin receptors and insulin action in rat brain cells. Binding of [125I]insulin to cells obtained by mechanically dissociating rat brains was 86% specific, time-dependent and reached equilibrium within 90 min. The t1/2 of association was 14 min and t1/2 of dissociation was 8 min. Scatchard analysis demonstrated the typical curvilinear plot providing high affinity (0.03 nM) and low affinity (6.6 nM) binding sites. The total number of binding sites were 0.15 pmol/mg protein. Crosslinking of [125I]insulin to its receptors on dissociated brain cells followed by SDS-PAGE and autoradiography showed that the alpha-subunit of the receptor had a molecular weight of 122,000. This was in contrast with a molecular weight of 134,000 for the liver alpha-subunit. Incubation of dissociated brain cells with insulin resulted in a concentration-dependent inhibition of total [3H]norepinephrine (NE) uptake. This inhibitory effect of insulin on [3H]NE uptake was sodium ion-dependent suggesting that 80-90% of the sodium ion-dependent uptake was insulin-sensitive. Incubation of lectin-purified insulin receptors with insulin resulted in a time- and concentration-dependent stimulation of phosphorylation of the tyrosine residue of an exogenous substrate poly (Glu, Tyr) (4:1). In addition, insulin also stimulated the autophosphorylation of the beta-subunit of the insulin receptors. These observations corroborate our contention that insulin exerts neuromodulatory effects mediated by the specific insulin receptors in the brain.

Insulin-like growth factor I receptors in neuronal and glial cells. Characterization and biological effects in primary culture.

Primary cultures of neuronal and glial cells from 1-day-old neonatal rats contain high affinity receptors for insulin-like growth factor I (IGF-I). The IC50 for displacement of 125I-IGF-I binding by unlabeled IGF-I was 3 nM for neuronal cells and 4 nM for glial cells. Unlabeled insulin was 20-50 times less potent. Apparent molecular mass of the alpha subunits of the IGF-I receptor was 125 kDa in neuronal and 135 kDa in glial cells. IGF-I induced autophosphorylation of the IGF-I receptor beta subunit in lectin-purified membrane preparations in a dose-dependent manner. The major phosphoamino acid of the beta subunit in both cell types was tyrosine in the IGF-I-stimulated state and serine in the basal state. Apparent molecular mass of the beta subunits of the IGF-I receptors was 91 kDa for neuronal and 95 kDa for glial cells. Tyrosine kinase activity of the IGF-I receptors was demonstrated by IGF-I-induced phosphorylation of the exogenous substrate poly(Glu, Tyr) 4:1 in both cell types. IGF-I had no effect on 2-deoxyglucose uptake in neuronal cells. In contrast, in glial cells, IGF-I stimulated 2-deoxyglucose uptake at very high doses, presumably acting via the insulin receptor. The effect of IGF-I as a neurotrophic growth factor in both neuronal and glial cells was demonstrated by its stimulation of [3H]thymidine incorporation. These findings suggest the IGF-I is an important growth factor in nervous tissue-derived cells.