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Biomedical subjects

N Madani

Publications and source records attributed to N Madani.

At least 19 recordsLinked to original sources

[Neuroimaging findings in eclamptic patients still symptomatic after 24 hours: a descriptive study about 19 cases].

OBJECTIVE: To describe the neuroimaging findings in eclamptic patients still symptomatic after 24 hours. PATIENTS AND METHODS: All parturients consecutively admitted over a 12-month period for eclampsia and presenting with neurological disorders (coma, focal neurological abnormalities, and eye sight disturbance) underwent cerebral CT-scan and/or MRI. RESULTS: Nineteen women were studied, and all had abnormal neuroradiological findings. The CT-scan was normal in three cases. Cerebral oedema was the predominant lesion (14 cases). It was localized in the parietal or occipital area (12 cases), paraventricular area (1 case), or was diffuse (1 case). Diffusion weighted MRI was performed in four cases and showed cytotoxic cerebral oedema in one case. There were three cases of cerebral venous thrombosis and two cases of intracerebral haemorrhage. CONCLUSION: Various neuroradiological lesions can be observed in eclamptic patients. A localized cerebral oedema is the most frequent. Diffusion weighted MRI should be systematically done when neurological disorders persist.

Adolescent↗

[Incidence, causes and prognostic factors of hyponatremia in intensive care].

PURPOSE: The incidence of hyponatremia is unknown, their causes are multiple. The higher mortality, especially in intensive care units, is currently unexplained. The objective of this article is to evaluate the incidence of hyponatremia, to assess their causes and to identify predictors of prognosis in intensive care units. METHODS: We included retrospectively all patients admitted at department of medical intensive care unit between January 1996 and February 2001, who presented at the admission, an hyponatremia (< 130 mmol/l). We excluded all patients who presented a hospital acquired hyponatremia, or hyponatremia associated with hyperglycemia > 13 mmol/l or with mannitol administration. Data were analysed by univariate methods, then by multivariate analysis. RESULTS: During the study period, 300 patients were identified among 2188: the incidence was 13.7% with 95% confidence interval (95% CI) between 9.8 % and 16.7%. Hypovolemic hyponatremia was observed in 25.7%, hypervolemic in 23.7% and normovolemic in 50.6%. In-hospital mortality was 37.7% (95% CI: 31.8% - 42.3%). Nine data were significantly associated with higher mortality in univariate analysis, but only 5 were identified as independant predictors of hospital mortality in multivariate analysis: hyponatremia < 125 mmol/l with a significant relative risk (RR) (RR = 2.10; 95% CI: 1.43-3.08; p < 0.001), Glasgow score < 9 (RR = 2.66; 95% CI: 1.25-5.66; p = 0.01), Glasgow score between 9 and 14 (RR = 1.94; 95% CI: 1.31-2.88; p < 0.001), shock (RR = 1.80; 95% CI: 1.10-3.05; p = 0.02) and blood urea concentration > 10 mmol/l (RR = 1.59; 95% CI : 1.08-2.34; p = 0.02). CONCLUSION: The frequency of hyponatremia is high; the normovolemic type represented 50%. Mortality is linked, in greater part, to organs dysfunction, but the severity of hyponatremia remained a significant predictor of mortality.

Adult↗

[Left ventricular thrombus and myocarditis induced by paraphenylenediamine poisoning].

We showed that the paraphenylenediamine (PPD) poisoning caused myocarditis but there is no data on the echocardiographic features. We report a case of myocarditis induced by PPD poisoning with echographic data. After ingestion of 5 g of PPD, a 18-year-old woman was admitted to the hospital with asphyxia and rhabdomyolysis. An electrocardiogram showed ventricular extrasystoles and negative T waves. The serum concentration of CK was 28,020 UI l(-1) (iso-enzyme MB = 840 UI l(-1)). A transthoracic echocardiography showed significant left and right ventricular hypokinesis (shortening fraction = 20% and left ventricular ejection fraction = 35%) and a left ventricular apical thrombus. Anticoagulation treatment with heparin was initiated. A follow-up echocardiogram performed on the 15th day showed normalization of ventricular function and disappearance of the thrombus. No embolic event was noted. Echocardiography is indicated in the myocarditis induced by PPD poisoning to prove the ventricular function as well as the presence of a thrombus.

Adolescent↗

[Comparative efficiency of nebulized adrenaline and salbutamol in severe acute asthma. A randomized, controlled prospective study].

OBJECTIVE: To compare nebulized salbutamol and nebulized adrenaline in acute severe asthma (ASA). STUDY DESIGN: Prospective controlled study. PATIENTS AND METHODS: October 1998 at May 99, 44 patients (31 women and 13 men, 35 +/- 11 yrs) with ASA (defined as peak expiratory flow rate (PEF) < 150 l min-1 and normo- or hypercapnia) were randomized to receive either nebulized salbutamol (n = 22), 10 mg/h-1 during 2 h then 5 mg every 4 h or nebulized adrenaline (n = 22), 6 mg/h-1 during 2 h then 3 mg every 4 h. The efficacy was assessed by PEF, forced expiratory volume in one second (FEV1) and Fischl's score during eight hours and by arterial blood gases during the first hour. Side-effects were evaluated by heart rate, systolic blood pressure, serum potassium and blood glucose. Statistical tests: Wilcoxon, Fischer exact, ANOVA and Scheffe's test. RESULTS: Both groups were similar with respect to age, sex, severity, duration of asthma and length of crisis. With the two treatments, PEF increased significantly but no statistical difference were observed between the two groups during the eight hours: 117.7 +/- 41.6 l min-1 to 203.3 +/- 56.9 l min-1 in the salbutamol group; 116.4 +/- 36.8 l min-1 to 217.3 +/- 188.8 l min-1 in the adrenaline group; p = 0.77. FEV1, Fischl's score and arterial blood gases did not differ significantly between treatments at every time interval. There were no significant difference between the two groups in terms of side-effects. The intravenous way was necessary at 3 cases of the salbutamol group and 4 cases of adrenaline group (NS). CONCLUSION: The results suggest that nebulized adrenaline is as effective as nebulized salbutamol in the ASA without significant side-effects. The nebulization could reduce systemic effects of adrenaline.

Acute Disease↗

Frequent substitution polymorphisms in African green monkey CCR5 cluster at critical sites for infections by simian immunodeficiency virus SIVagm, implying ancient virus-host coevolution.

In contrast to humans, several primate species are believed to have harbored simian immunodeficiency viruses (SIVs) since ancient times. In particular, the geographically dispersed species of African green monkeys (AGMs) are all infected with highly diversified SIVagm viruses at high prevalences (greater than 50% of sexually mature individuals) without evident diseases, implying that the progenitor monkeys were infected prior to their dispersal. If this is correct, AGMs would be expected to have accumulated frequent resistance-conferring polymorphisms in host genes that are important for SIV replication. Accordingly, we analyzed the coding sequences of the CCR5 coreceptors from 26 AGMs (52 alleles) in distinct populations of the four species. These samples contained 29 nonsynonymous coding changes and only 15 synonymous nucleotide substitutions, implying intense functional selection. Moreover, 24 of the resulting amino acid substitutions were tightly clustered in the CCR5 amino terminus (D13N in the vervets and Y14N in the tantalus species) or in the first extracellular loop (Q93R and Q93K in all species). The Y14N substitution was extremely frequent in the 12 wild-born African tantalus, with 7 monkeys being homozygous for this substitution and 4 being heterozygous. Although two of these heterozygotes and the only wild-type homozygote were naturally infected with SIVagm, none of the Y14N homozygotes were naturally infected. A focal infectivity assay for SIVagm indicated that all five tested SIVagms efficiently use CCR5 as a coreceptor and that they also use CXCR6 (STRL33/Bonzo) and GPR15 (BOB) with lower efficiencies but not CXCR4. Interestingly, the D13N, Y14N, Q93R, and Q93K substitutions in AGM CCR5 all strongly inhibited infections by the SIVagm isolates in vitro. The Y14N substitution eliminates a tyrosine sulfation site that is important for infections and results in partial N-linked glycosylation (i.e., 60% efficiency) at this position. Nevertheless, the CCR5(Y14N) component that lacks an N-linked oligosaccharide binds the chemokine MIP-lbeta with a normal affinity and is fully active in signal transduction. Similarly, D13N and Q93R substitutions did not interfere with signal transduction. Thus, the common substitution polymorphisms in AGM CCR5 strongly inhibit SIVagm infections while substantially preserving chemokine signaling. In contrast, polymorphisms of human CCR5 are relatively infrequent, and the amino acid substitutions are randomly situated and generally without effects on coreceptor function. These results support an ancient coevolution of AGMs and SIVagm viruses and establish AGMs as a highly informative model for learning about host proteins that play critical roles in immunodeficiency virus infections.

Amino Acid Sequence↗

Arginine 447 plays a pivotal role in substrate interactions in a neuronal glutamate transporter.

Glutamate transporters from the central nervous system play a crucial role in the clearance of the transmitter from the synaptic cleft. Glutamate is cotransported with sodium ions, and the electrogenic translocation cycle is completed by countertransport of potassium. Mutants that cannot interact with potassium are only capable of catalyzing electroneutral exchange. Here we identify a residue involved in controlling substrate recognition in the neuronal transporter EAAC-1 that transports acidic amino acids as well as cysteine. When arginine 447, a residue conserved in all glutamate transporters, is replaced by cysteine, transport of glutamate or aspartate is abolished, but sodium-dependent cysteine transport is left intact. Analysis of other substitution mutants shows that the replacement of arginine rather than the introduced cysteine is responsible for the observed phenotype. In further contrast to wild type, acidic amino acids are unable to inhibit cysteine transport in R447C-EAAC-1, indicating that the selectivity change is manifested at the binding step. Electrophysiological analysis shows that in the mutant cysteine, transport has become electroneutral, and its interaction with the countertransported potassium is impaired. Thus arginine 447 plays a pivotal role in the sequential interaction of acidic amino acids and potassium with the transporter and, thereby, constitutes one of the molecular determinants of coupling their fluxes.

Amino Acid Sequence↗

Cellular and viral specificities of human immunodeficiency virus type 1 vif protein.

The vif gene of human immunodeficiency virus type 1 (HIV-1) greatly enhances the infectivity of HIV-1 virions that are released from cells classified as nonpermissive (e.g., lymphocytes, macrophages, and H9 leukemic T cells) but is irrelevant in permissive cells (e.g., HeLa or COS cells). Recently, it was reported that vif expression in nonpermissive cells dramatically increases infectivity not only of HIV-1 but also of other enveloped viruses, including murine leukemia viruses (MLVs). This was surprising in part because MLVs and other murine retroviruses lack vif genes yet replicate efficiently in T lymphocytes. To investigate these issues, we first developed improved methods for producing substantial quantities of HIV-1 virions with vif deletions from healthy H9 cells. These virions had approximately the same amounts of major core proteins and envelope glycoproteins as the control wild-type virions but were only approximately 1% as infectious. We then produced H9 cells that contained wild-type or vif deletion HIV-gpt proviruses, which lack a functional env gene. After superinfection with either xenotropic or amphotropic MLVs, these cells released HIV-gpt virions pseudotyped with an MLV envelope plus replication-competent MLV. Interestingly, the pseudotyped HIV-gpt (vif deletion) virions were noninfectious, whereas the MLV virions simultaneously released from the same H9 cells were fully infectious. These results strongly suggest that the Vif protein functions in a manner that is both cell specific and at least substantially specific for HIV-1 and related lentiviruses. In addition, these results confirm that vif deletion HIV-1 virions from nonpermissive cells are blocked at a postpenetration stage of the infection pathway.

Animals↗

A critical site in the core of the CCR5 chemokine receptor required for binding and infectivity of human immunodeficiency virus type 1.

Like the CCR5 chemokine receptors of humans and rhesus macaques, the very homologous (approximately 98-99% identical) CCR5 of African green monkeys (AGMs) avidly binds beta-chemokines and functions as a coreceptor for simian immunodeficiency viruses. However, AGM CCR5 is a weak coreceptor for tested macrophage-tropic (R5) isolates of human immunodeficiency virus type 1 (HIV-1). Correspondingly, gp120 envelope glycoproteins derived from R5 isolates of HIV-1 bind poorly to AGM CCR5. We focused on a unique extracellular amino acid substitution at the juncture of transmembrane helix 4 (TM4) and extracellular loop 2 (ECL2) (Arg for Gly at amino acid 163 (G163R)) as the likely source of the weak R5 gp120 binding and HIV-1 coreceptor properties of AGM CCR5. Accordingly, a G163R mutant of human CCR5 was severely attenuated in its ability to bind R5 gp120s and to mediate infection by R5 HIV-1 isolates. Conversely, the R163G mutant of AGM CCR5 was substantially strengthened as a coreceptor for HIV-1 and had improved R5 gp120 binding affinity relative to the wild-type AGM CCR5. These substitutions at amino acid position 163 had no effect on chemokine binding or signal transduction, suggesting the absence of structural alterations. The 2D7 monoclonal antibody has been reported to bind to ECL2 and to block HIV-1 binding and infection. Whereas 2D7 antibody binding to CCR5 was unaffected by the G163R mutation, it was prevented by a conservative ECL2 substitution (K171R), shared between rhesus and AGM CCR5s. Thus, it appears that the 2D7 antibody binds to an epitope that includes Lys-171 and may block HIV-1 infection mediated by CCR5 by occluding an HIV-1-binding site in the vicinity of Gly-163. In summary, our results identify a site for gp120 interaction that is critical for R5 isolates of HIV-1 in the central core of human CCR5, and we propose that this site collaborates with a previously identified region in the CCR5 amino terminus to enable gp120 binding and HIV-1 infections.

Amino Acid Sequence↗

Weaning from mechanical ventilation: a model for extubation.

OBJECTIVE: To develop a model able to determine the right time for extubation and to validate its performance. paragraph sign DESIGN: A prospective clinical study. SETTING: 14-bed medical intensive care unit in a university hospital. PATIENTS: 101 patients (37 women/64 men) ventilated over more than 48 h (mean 10.4 +/- 10.3 days) and considered ready to be weaned by the medical team (February 1996-February 1998). METHODS: This study included two series: a development series with 53 patients and a validation series with 48 patients. Before extubation, a weaning test was performed measuring tidal volume (V(T)), respiratory rate (f), f/V(T) ratio, minute ventilation, vital capacity (VC) and maximum inspiratory and expiratory pressures (MIP and MEP). The success of extubation was assessed after 48 h. Receiver operating characteristic (ROC) curves allowed the analysis of the discriminating power of each parameter. Threshold values were determined using the Youden's index. To create the best predictive model, we performed a multiple logistic regression analysis. To assess the calibration and the discrimination of the model, the Hosmer- Lemeshow goodness-of-fit test and area under ROC curves (AUC) were adopted. MEASUREMENTS AND RESULTS: In a development series, 60 tests were carried out with 38 successful extubations and 22 extubation failures. The multivariate analysis found three significant variables: VC (threshold value = 635 ml), f/V(T) ratio (threshold value = 88 breaths/min.l) and MEP (threshold value = 28 cmH(2)O). The validation cohort included 59 tests (38 successes and 21 failures). The validation series shows a good discrimination (AUC = 0.855 +/- 0.059) and calibration (goodness-of-fit test C: p = 0.224) of the model. CONCLUSION: VC together with the f/V(T) ratio and MEP offer accurate prediction of early extubation.

Adolescent↗

Protective and therapeutic effect of DNA-based immunization against hepadnavirus large envelope protein.

BACKGROUND & AIMS: Studies in the murine model suggest that injection of DNA encoding hepatitis B virus structural proteins is promising for the induction of a specific immune response. We used the duck hepatitis B virus (DHBV) model to study the protective and therapeutic effects of naked DNA immunization against hepadnaviral large envelope protein. METHODS: A pCI-preS/S plasmid expressing the DHBV large protein was used for intramuscular immunization of ducks. The humoral response was tested by enzyme-linked immunosorbent assay, immunoblotting, neutralization, and in vivo protection tests. For DNA therapy, DHBV-carrier ducks received four injections of this plasmid. Viremia was monitored for 10 months; thereafter, liver biopsies were performed. RESULTS: Immunization with pCI-preS/S plasmid induced a specific, long-lasting, neutralizing, and highly protective anti-preS humoral response in uninfected animals. After pCI-preS/S treatment, a significant and sustained decrease in serum and liver DHBV DNA was observed for carrier ducks compared with the controls. CONCLUSIONS: DNA immunization against DHBV large protein results in a potent and protective anti-preS response in the duck model. The results of long-term follow-up of DNA-treated chronically infected ducks are promising and show the usefulness of this model for the study of genetic immunization in chronic hepatitis B therapy.

Animals↗

gp120 envelope glycoproteins of human immunodeficiency viruses competitively antagonize signaling by coreceptors CXCR4 and CCR5.

Signal transductions by the dual-function CXCR4 and CCR5 chemokine receptors/HIV type 1 (HIV-1) coreceptors were electrophysiologically monitored in Xenopus laevis oocytes that also coexpressed the viral receptor CD4 and a G protein-coupled inward-rectifying K+ channel (Kir 3.1). Large Kir 3.1-dependent currents generated in response to the corresponding chemokines (SDF-1alpha for CXCR4 and MIP-1alpha; MIP-1beta and RANTES for CCR5) were blocked by pertussis toxin, suggesting involvement of inhibitory guanine nucleotide-binding proteins. Prolonged exposures to chemokines caused substantial but incomplete desensitization of responses with time constants of 5-7 min and recovery time constants of 12-19 min. CXCR4 and CCR5 exhibited heterologous desensitization in this oocyte system, suggesting possible inhibition of a common downstream step in their signaling pathways. In contrast to chemokines, perfusion with monomeric or oligomeric preparations of the glycoprotein of Mr 120, 000 (gp120) derived from several isolates of HIV-1 did not activate signaling by CXCR4 or CCR5 regardless of CD4 coexpression. However, adsorption of the gp120 from a T-cell-tropic virus resulted in CD4-dependent antagonism of CXCR4 response to SDF-1alpha, whereas gp120 from macrophage-tropic viruses caused CD4-dependent antagonism of CCR5 response to MIP-1alpha. These antagonisms could be partially overcome by high concentrations of chemokines and were specific for coreceptors of the corresponding HIV-1 isolates, suggesting that they resulted from direct interactions of gp120-CD4 complexes with coreceptors and that they did not involve the desensitization pathway. These results indicate that monomeric or oligomeric gp120s specifically antagonize CXCR4 and CCR5 signaling in response to chemokines, but they do not exclude the possibility that gp120s might also function as weak agonists in some cells. The gp120-mediated disruption of CXCR4 and CCR5 signaling may contribute to AIDS pathogenesis.

Animals↗

An endogenous inhibitor of human immunodeficiency virus in human lymphocytes is overcome by the viral Vif protein.

The vif gene of human immunodeficiency virus type 1 (HIV-1) encodes a basic Mr 23,000 protein that is necessary for production of infectious virions by nonpermissive cells (human lymphocytes and macrophages) but not by permissive cells such as HeLa-CD4. It had been proposed that permissive cells may contain an unidentified factor that functions like the viral Vif protein. To test this hypothesis, we produced pseudotyped wild-type and vif-deleted HIV gpt virions (which contain the HIV-1 genome with the bacterial mycophenolic acid resistance gene gpt in place of the viral env gene) in permissive cells, and we used them to generate nonpermissive H9 leukemic T cells that express these proviruses. We then fused these H9 cells with permissive HeLa cells that express the HIV-1 envelope glycoprotein gp120-gp41, and we asked whether the heterokaryons would release infectious HIV gpt virions. The results clearly showed that the vif-deleted virions released by the heterokaryons were noninfectious whereas the wild-type virions were highly infectious. This strongly suggests that nonpermissive cells, the natural targets of HIV-1, contain a potent endogenous inhibitor of HIV-1 replication that is overcome by Vif.

Animals↗

Distal enhancer regulation by promoter derepression in topologically constrained DNA in vitro.

Long-range promoter-enhancer interactions are a crucial regulatory feature of many eukaryotic genes yet little is known about the mechanisms involved. Using cloned chicken betaA-globin genes, either individually or within the natural chromosomal locus, enhancer-dependent transcription is achieved in vitro at a distance of 2 kb with developmentally staged erythroid extracts. This occurs by promoter derepression and is critically dependent upon DNA topology. In the presence of the enhancer, genes must exist in a supercoiled conformation to be actively transcribed, whereas relaxed or linear templates are inactive. Distal protein-protein interactions in vitro may be favored on supercoiled DNA because of topological constraints. In this system, enhancers act primarily to increase the probability of rapid and efficient transcription complex formation and initiation. Repressor and activator proteins binding within the promoter, including erythroid-specific GATA-1, mediate this process.

Animals↗

CD4, CXCR-4, and CCR-5 dependencies for infections by primary patient and laboratory-adapted isolates of human immunodeficiency virus type 1.

We have used a focal infectivity method to quantitatively analyze the CD4, CXCR-4, and CCR-5 dependencies for infections by diverse primary patient (PR) and laboratory-adapted (LA) isolates of human immunodeficiency virus type 1 (HIV-1). Infectivities of T-cell-tropic viruses were analyzed in a panel of HeLa-CD4 cell clones that have distinct quantities of CD4 and in human astroglioma U87MG-CD4 cells that express a large quantity of CD4 and become highly susceptible to infection after transfection with a CXCR-4 expression vector. The latter analysis indicated that PR as well as LA T-cell-tropic viruses efficiently employ CXCR-4 as a coreceptor in an optimal human cell line that contains abundant CD4. Previous uncertainties regarding coreceptor usage by PR T-cell-tropic HIV-1 isolates may therefore have derived from the assay conditions. As reported previously, unrelated LA and PR T-cell-tropic HIV-1 isolates differ in infectivities for the HeLa-CD4 clonal panel, with LA viruses infecting all clones equally and PR viruses infecting the clones in proportion to cellular CD4 quantities (D. Kabat, S. L. Kozak, K. Wherly, and B. Chesebro, J. Virol. 68:2570-2577, 1994). To analyze the basis for this difference, we used the HeLa-CD4 panel to compare a molecularly cloned T-cell-tropic PR virus (ELI1) with six of its variants that grow to different extents in CD4-positive leukemic cell lines and that differ only at specific positions in their gp120 and gp41 envelope glycoproteins. All mutations in gp120 or gp41 that contributed to laboratory adaptation preferentially enhanced infectivity for cells that had little CD4 and thereby decreased the CD4 dependencies of the infections. There was a close correlation between abilities of T-cell-tropic ELI viruses to grow in an expanded repertoire of leukemic cell lines, the reduced CD4 dependencies of their infections of the HeLa-CD4 panel, and their sensitivities to inactivation by soluble CD4 (sCD4). Since all of the ELI viruses can efficiently use CXCR-4 as a coreceptor, we conclude that an increase in viral affinity for CD4 rather than a switch in coreceptor specificity is principally responsible for laboratory adaption of T-cell-tropic HIV-1. Syncytium-inducing activities of the ELI viruses, especially when analyzed on cells with low amounts of CD4, were also highly correlated with their laboratory-adapted properties. Results with macrophage-tropic HIV-1 were strikingly different in both coreceptor and CD4 dependencies. When assayed in HeLa-CD4 cells transfected with an expression vector for CCR-5, macrophage-tropic HIV-1 isolates that had been molecularly cloned shortly after removal from patients were equally infectious for cells that had low or high CD4 quantities. Moreover, despite their substantial infectivities for cells that had only a trace of CD4, macrophage-tropic isolates were relatively resistant to inactivation by sCD4. We conclude that T-cell-tropic PR viruses bind weakly to CD4 and preferentially infect cells that coexpress CXCR-4 and large amounts of CD4. Their laboratory adaptation involves corresponding increases in affinities for CD4 and in abilities to infect cells that have relatively little CD4. In contrast, macrophage-tropic HIV-1 appears to interact weakly with CD4 although it can infect cells that coexpress CCR-5 and small quantities of CD4. We propose that cooperative binding of macrophage-tropic HIV-1 onto CCR-5 and CD4 may enhance virus adsorption and infectivity for cells that have only a trace of CD4.

Acquired Immunodeficiency Syndrome↗

Infectious properties of human immunodeficiency virus type 1 mutants with distinct affinities for the CD4 receptor.

Recent evidence suggests that primary patient isolates of T-cell-tropic human immunodeficiency virus type 1 (HIV-1 ) have lower affinities for CD4 than their laboratory-adapted derivatives, that this may partly result from tighter gp120-gp41 bonds that constrain the CD4 binding sites of the primary viruses, and that selection for increased CD4 affinity may be the principal factor in laboratory adaptation of HIV-1 (S. L. Kozak, E. J. Platt, N. Madani, F. E. Ferro, Jr., K. Peden, and D. Kabat, J. Virol. 71:873-882, 1997). These conclusions were based on studies with a panel of HeLa-CD4 cell clones that differ in CD4 levels over a broad range, with laboratory-adapted viruses infecting all clones with equal efficiencies and primary T-cell-tropic viruses infecting the clones in proportion to cellular CD4 levels. Additionally, all of the primary and laboratory-adapted T-cell-tropic viruses efficiently used CXCR-4 (fusin) as a coreceptor. To test these conclusions by an independent approach, we studied mutations in the laboratory-adapted virus LAV/IIIB that alter the CD)4 binding region of gp120 and specifically reduce CD4 affinities of free gp 120 by 85 to 98% (U. Olshevsky et al., J. Virol. 64:5701-5707, 1990). These mutations reduced virus titers to widely varying extents that ranged from severalfold to several orders of magnitude and converted infectivities on the HeLa-CD4 panel from CD4 independency to a high degree of CD4 dependency that resembled the behavior of primary patient viruses. The relative infectivities of the mutants correlated closely with their sensitivities to inactivation by soluble CD4 but did not correlate with the relative CD4 affinities of their free gp120s. Most of the mutations did not substantially alter envelope glycoprotein synthesis, processing, expression on cell surfaces, incorporation into virions, or rates of gp120 shedding from virions. However, one mutation (D457R) caused a decrease in gp160 processing by approximately 80%. The fact that several mutations increased rates of spontaneous viral inactivation (especially D368P) suggests that HIV-1 life spans may be determined by structural stabilities of viral envelope glycoproteins. All of the wild-type and mutant viruses were only slowly and inefficiently adsorbed onto cultured CD4-positive cells at 37 degrees C, and the gradual declines in viral titers in the media were caused almost exclusively by spontaneous inactivation rather than by adsorption. The extreme inefficiency with which infectious HIV-1 is able to infect cultured susceptible CD4-positive cells in standard assay conditions casts doubt on previous inferences that the vast majority of retrovirions produced in cultures are noninfectious. Apparent infectivity of T-cell-tropic HIV-1 in culture is limited by productive associations with CD4 and is influenced in an interdependent manner by CD4 affinities of viral gp120-gp41 complexes and quantities of cell surface CD4.

Acquired Immunodeficiency Syndrome↗

Protein metabolism during weight reduction with very-low-energy diets: evaluation of the independent effects of protein and carbohydrate on protein sparing.

The aim of this study was to assess the independent effects of carbohydrate and protein intakes in protein sparing during weight reduction. Forty-eight obese women were randomly assigned to consume isoenergetic (2500 kJ/d) liquid diets that provided the following amounts (g/d) of protein and carbohydrate, respectively, for 28 d: 50 and 10, 50 and 76, 70 and 10, and 70 and 86. The effects of carbohydrate and protein were analyzed by repeated-measures analysis of variance (ANOVA). Carbohydrate significantly affected daily urinary ammonia and total nitrogen excretion, stool nitrogen, and nitrogen balance. Protein, independently of carbohydrate, significantly affected daily urinary ammonia, urea, and total nitrogen excretion but had no effect on nitrogen balance. Cumulative nitrogen losses (mmol/28 d) were lower in the high-carbohydrate groups than in the low-carbohydrate groups (1869 +/- 392 and 3611 +/- 328, P = 0.003) but were similar in the groups receiving 50 and 70 g protein/d (3171 +/- 327 and 2326 +/- 430, respectively, P = NS). These results indicate that carbohydrate and protein have independent but additive protein-sparing effects during weight reduction.

3-Hydroxybutyric Acid↗

The erythroid protein cGATA-1 functions with a stage-specific factor to activate transcription of chromatin-assembled beta-globin genes.

The chick beta-globin gene is regulated developmentally within erythroid cells by the interaction of multiple proteins with the promoter and the 3' enhancer. These interactions are correlated with changes in chromatin structure, which are characteristic of the actively expressed gene. Using in vitro chromatin assembly and transcription with staged erythroid extracts, we have determined the critical proteins required to activate expression of nucleosome-reconstituted beta-globin genes. These genes contain a specialized TATA box at -30 (GATA) through which the erythroid-restricted protein cGATA-1 and TFIID both function to regulate different steps in beta-globin expression. We find that TBP (TATA-binding protein) alone can activate transcription of beta-globin chromatin templates from promoters mutated to a canonical TATA box but is ineffective on those containing the normal -30 GATA site. The occupancy of this site by cGATA-1 also fails to generate efficient expression of beta-globin chromatin unless combined with a stage-specific protein, NF-E4, that binds to an adjacent site. However, NF-E4 does not function with TBP to derepress nucleosome-assembled beta-globin genes. We propose that the developmental regulation of beta-globin expression is achieved, in part, by the requirement of an erythroid protein and a stage-specific factor, rather than TBP, to activate chromatin through a specialized TATA box.

Animals↗