Biomedical subjects

V Bosch

Publications and source records attributed to V Bosch.

At least 37 records · Page 2Linked to original sources

PubMed1997

Transfer of endoplasmic reticulum and Golgi retention signals to human immunodeficiency virus type 1 gp160 inhibits intracellular transport and proteolytic processing of viral glycoprotein but does not influence the cellular site of virus particle budding.

In this study, specific signals known to mediate endoplasmic reticulum or Golgi localization of transmembrane proteins have been transferred to the human immunodeficiency virus type 1 (HIV-1) env gene product. The intracellularly retained recombinant glycoproteins were not proteolytically processed to gp120 and gp41, which is further evidence that this process occurs at a later stage in the transport pathway, presumably within or near the trans-Golgi network. Since the subcellular localization of the viral glycoproteins of enveloped viruses can be one of the factors determining the cellular site of particle assembly and release, experiments were performed to determine if this property was altered by coexpression of the recombinant HIV-1 glycoproteins. When wild-type virus was compared to mutant virus encoding the intracellularly retained glycoproteins, the extent of HIV-1 particle release into the extracellular medium remained unaffected, and electron-microscopic analysis did not reveal any significant alteration in the cellular sites of particle assembly and budding. Thus, in COS-7 cells, altered subcellular localization of the viral glycoprotein does not exert a dominant influence on the assembly site of the HIV-1 particle.

Amino Acid Sequence↗

PubMed1996

Generation of lymphocyte cell lines coexpressing CD4 and wild-type or mutant HIV type 1 glycoproteins: implications for HIV type 1 Env-induced cell lysis.

To gain more insight into the processes leading to HIV-1 Env-induced cell death, we aim to coexpress stably wild-type and relevant mutant variants of both HIV-1 Env and human CD4 in lymphocyte cell lines. Here we report on the generation and characterization of several cell lines inducibly or constitutively expressing wild-type or cleavage-defective HIV-1 glycoproteins and human CD4 either singly or in combination. Coexpression of CD4 and wild-type Env led to the formation of multinucleated syncytia, to growth arrest and cell death, effects that all could be prevented by cultivation in the presence of monoclonal antibodies that inhibit cell surface membrane fusion. Cell lines coexpressing CD4 and mutated, noncleavable Env, detectable at the cell surface and still retaining CD4-binding capacity, were not retarded in their growth and cytolysis did not occur. These results indicate that cell lysis requires cell surface interaction of CD4 and gp120/41 and cleavage of gp160 to gp120 and gp41.

B-Lymphocytes↗

PubMed1996

Glycoprotein incorporation and HIV-1 infectivity despite exchange of the gp160 membrane-spanning domain.

We have examined the role of the membrane-anchoring domain of the HIV-1 glycoproteins in viral glycoprotein function, glycoprotein incorporation, and viral infectivity. For this purpose, we initially exchanged the entire membrane-spanning region with that from a cellular glycoprotein (CD22). Subsequently, the strictly conserved arginine in the central position of the transmembranal alpha-helix was replaced by a neutral residue (R696 --> I696). We have further examined the requirements within the cytoplasmic C-terminus for glycoprotein incorporation and replaced this region of gp160 with the long cytoplasmic C-terminus (118 amino acids) from CD22. Our results show that the specific amino acid sequence of the membrane-spanning region of gp160 is not necessary for viral infectivity, thus making it unlikely that this region is specifically involved in membrane fusion, in glycoprotein incorporation, or in infectivity of the cell lines tested. In contrast, recombinant gp160 with the CD22 C-terminal region, although present at the cell surface and membrane fusion-competent, was excluded from incorporation into particles. This could indicate that steric exclusion, and no pseudotyping, occurs when the heterologous, cytoplasmic C-terminal region is too long and not fitting.

Amino Acid Sequence↗

PubMed1996

Early response to the effect of iron fortification in the Venezuelan population.

In Venezuela a severe economic crisis beginning in 1983 provoked a progressive reduction of the quality and quantity of food consumed by the low socioeconomic strata of the population. In these strata, which represent > or = 80% of the Venezuelan population, we had seen a continuous increase in the prevalence of iron deficiency during that recent decade. As a result, in 1993 the Venezuela Government created the Special Commission for Enrichment of Foods. That same year a fortification program began in which precooked yellow and white maize and wheat flours were enriched with 20 and 50 mg Fe (as ferrous fumarate)/kg flour, respectively. The corn flour was also enriched with vitamin A, thiamine, riboflavin, and niacin, whereas the wheat flour was enriched with these same vitamins, except vitamin A. These two cereals represent 45% of the total energy consumed daily by the low socioeconomic strata of the population. A preliminary survey carried out in Caracas in 1994 in a population of 307 children aged 7, 11, and 15 y showed that the prevalence of iron deficiency determined by measuring the serum ferritin concentration and the prevalence of anemia were reduced from 37% and 19%, respectively, in 1992 to 15% and 10%, respectively in 1994.

Adolescent↗

PubMed1996

Unimpaired function of a naturally occurring C terminally truncated vif gene product of human immunodeficiency virus type 1.

In approximate 10 percent of natural human immunodeficiency virus 1 (HIV-1) vif gene populations, sequences of shortened vif open reading frames with premature stop codons have been found. Here we report the functional analysis of two patient-derived vif genes. Vif45-2 encodes a C terminally truncated Vif protein of only 173 instead of 192 amino acids and additionally contains several rare amino acid substitutions which are in part shared by vifA65-5. HIV-1 pNL4-3-derived recombinant A45-2 and A65-5 virions were fully infectious in H9 cells and human PBMC, both known to be non-permissive for vif-defective HIV-1. Furthermore, A45-2 virions produced in primary human monocyte-derived macrophages were infectious for MT-4 cells. This study unequivocally demonstrates that the C-terminal region (19 amino acids) of the Vif protein is dispensable for Vif function in the in vitro cell culture systems employed. Additionally, we investigated whether the Vif protein might be phosphorylated in vivo and obtained no evidence for this.

Amino Acid Sequence↗

PubMed1996

Dietary fatty acids in athero-thrombogenesis: influence of palm oil ingestion.

Dietary experiments, performed in metabolic wards, gave rise to predictive regression equations relating changes of plasma cholesterol concentration to the intake of fatty acids of the diet. It has been established that polyunsaturated fatty acids diminish and most saturated fatty acids increase plasma cholesterol concentration. This information led to expect that dietary use of palm oil may induce an unfavorable plasma lipoprotein profile. This has not been the case as shown in various dietary experiments. The reasons for this discrepancy is discussed. The influence of palm oil enriched diets on prothrombotic variables show that platelets are not affected in their function during prolonged dietary intervention. It is important to continue research on the effects of palm oil based diet on plasma fibrinogen, factor VII. There is still discordant information in this field.

Adult↗

PubMed1994

Dietary fish oil affects food intake, growth and hematologic values of weanling rats.

The object of this study was to evaluate the effect of increasing amounts of dietary fish oil on growth and hematological variables of the weanling male Sprague-Dawley rat. Animals were fed diets containing either fish oil (FO) or sesame oil (SO) at 5, 10 or 15% (w/w) for 31 d. Growth retardation and reduced food intake was noted in groups fed FO. Hemoglobin (Hb) concentration diminished when the dietary FO was above 5% (w/w). FO is a poor source of (n-6) fatty acids. We postulate that a partial deficiency in (n-6) polyenic family, is a consequence of the increasing amounts of FO in the diets, that may affect growth and erytropoiesis. In this report we show evidence supporting the hypothesis that diets enriched with fish oil can alter normal growth and induced hematological changes in the male weanling rat.

Animals↗

PubMed1993

Analysis of protein expression and virus-like particle formation in mammalian cell lines stably expressing HIV-1 gag and env gene products with or without active HIV proteinase.

Cell lines stably releasing noninfectious virus-like particles containing wild type or mutant gene products represent useful tools for a biochemical, immunological, and structural analysis of virus assembly. Human immunodeficiency virus (HIV) type 1 gag and env gene products were transiently and stably expressed in mammalian cells and the formation of virus-like particles incorporating viral glycoproteins was analyzed. Transient cotransfection of plasmids directing the synthesis of gag and env gene products yielded efficient release of particles but specific incorporation of HIV glycoproteins was not detected. A stable cell line expressing wild type HIV-1 glycoproteins was generated and transient transfection of this cell line with gag-encoding constructs led to the release of virus-like particles incorporating HIV surface and transmembrane glycoproteins. Attempts to establish stable cell lines expressing wild type HIV gag and pol genes were unsuccessful and only highly unstable lines primarily expressing uncleaved precursor polyproteins were obtained. This result appears to be caused by the cytotoxic effects of the viral proteinase since stable lines were readily selected after transfection of constructs either encoding an inactive mutant of the proteinase or a mutated frameshift signal which prevented expression of the pol reading frame. Stable coexpression of uncleaved Gag polyprotein and wild type env gene products yielded efficient release of immature virus-like particles incorporating HIV glycoproteins. Electron micrographs revealed lentiviral budding structures with the typical surface projections of viral glycoprotein oligomers.

Animals↗

PubMed1993

The relationship between iron deficiency and anemia in Venezuelan children.

The iron status of 3228 subjects from the nutrition survey Proyecto Venezuela was studied. The sample included children from 1 to 16 y of age grouped by age and sex. Values for three indicators of iron status were compared: hemoglobin concentration, serum ferritin concentration, and percentage saturation of serum transferrin. In all groups there was a strong overlap in the hemoglobin concentration distribution curves for non-iron-deficient and iron-deficient subjects classified as such according to the other two indexes. The prevalence of iron deficiency ranged from 35% in 1-3 y olds to 10% in adolescent males, the values being almost identical in the nonanemic group compared with the total population. In the different groups, 80-97% of the subjects with abnormal values of at least one of these two indexes were not anemic. The difficulties involved in establishing a state of iron deficiency according to these indexes are discussed.

Adolescent↗

PubMed1992

Inhibition of furin-mediated cleavage activation of HIV-1 glycoprotein gp160.

The envelope glycoprotein of human immunodeficiency virus (HIV) initiates infection by mediating fusion of the viral envelope with the cell membrane. Fusion activity requires proteolytic cleavage of the gp160 protein into gp120 and gp41 at a site containing several arginine and lysine residues. Activation at basic cleavage sites is observed with many membrane proteins of cellular and viral origin. We have recently found that the enzyme activating the haemagglutinin of fowl plague virus (FPV), an avian influenza virus, is furin. Furin, a subtilisin-like eukaryotic endoprotease, has a substrate specificity for the consensus amino-acid sequence Arg-X-Lys/Arg-Arg at the cleavage site. We show here that the glycoprotein of HIV-1, which has the same protease recognition motif as the FPV haemagglutinin, is also activated by furin.

Amino Acid Chloromethyl Ketones↗

PubMed1992

Expression of biologically active HIV glycoproteins using a T7 RNA polymerase-based eucaryotic vector system.

Bacteriophage T7 RNA polymerase and a derivative containing a nuclear localization signal were transiently expressed in CV-1 cells and were shown to localize to the cytoplasm and nucleus, respectively. A vector was constructed containing T7 promoter and transcription terminator sequences flanking a picornaviral 5' untranslated sequence for cap-independent translation and a polyA signal. Expression of the HIV-1 envelope glycoproteins in this vector system gave high levels of specific transcripts and translation products, independent of the subcellular localization of T7 RNA polymerase. The synthesis of HIV glycoproteins was also completely independent of the coexpression of the HIV rev protein, which is normally required for the expression of HIV structural proteins. In addition, a polyA signal was not required, whereas the presence of the picornaviral 5' untranslated region was necessary for efficient expression. Different possibilities to account for these findings are discussed. The HIV glycoproteins synthesized in this system were normally processed and assembled; they could induce syncytium formation and complement an env-deletion mutant of HIV-1.

Animals↗

PubMed1992

Retained in vitro infectivity and cytopathogenicity of HIV-1 despite truncation of the C-terminal tail of the env gene product.

Five in-frame stop mutations in the HIV-1 env gene, which lead to the production of env gene products truncated within the cytoplasmic C-terminal tail, have been generated and their effects on membrane fusion capacity, glycoprotein incorporation into virus particles, infectivity, and cytopathogenicity were analyzed. The resulting truncated glycoproteins were processed normally, were transported to the cell surface, and were able to induce CD4-dependent membrane fusion. The membrane fusion capacity of one of the mutant glycoproteins with a truncation of 144 amino acids was increased to about double of that induced by wild-type glycoprotein. With a single exception, the truncated viral glycoproteins were incorporated into virus particles which were infectious and cytopathic for permissive MT-4 cells. The infection kinetics with the mutated viruses were, however, delayed to varying degrees in comparison to infection with wild-type virus. Nevertheless, in each case, PCR amplification and direct sequencing of viral DNA in the infected cultures confirmed the presence of the mutant and the absence of revertant DNA. The mutant virus encoding a viral glycoprotein with the longest truncation (144 amino acids), in which only 7 cytoplasmic C-terminal amino acids in gp41 remain, resulted in infection kinetics in MT-4 cells which were only marginally delayed in comparison to those induced by wild-type virus. This means that these C-terminal 144 amino acids of gp41 are not necessary for glycoprotein incorporation into virus particles nor do they significantly contribute to the infectivity nor the cytopathogenicity of HIV-1 in MT-4 cells.

Biological Transport, Active↗

PubMed1992

HIV-1-induced cytopathogenicity in cell culture despite very decreased amounts of fusion-competent viral glycoprotein.

In order to examine the potential role of env-induced membrane fusion in the cytopathogenic properties of HIV-1 in cell culture, the effects of mutations within the proteolytic cleavage site of gp160, which result in a reduction but not a complete absence of proteolytic processing have been further studied. Cells expressing the mutant glycoproteins were shown to be severely reduced in their capacity to form syncytia. However, viruses encoding these glycoproteins could infect cell culture cells, albeit with delayed kinetics, and, at late infection time points, resulted in complete cytolysis of the infected culture. Since amplification by polymerase chain reaction and direct sequencing of the DNA in the infected cultures confirmed the presence of the mutant and the absence of revertant DNA, this shows that the amount of fusion competent viral glycoprotein does not influence HIV-1 cytopathogenicity, but rather that other parameters must be involved in inducing cell death.

Cell Line↗

PubMed1990

Mutational analysis of the human immunodeficiency virus type 1 env gene product proteolytic cleavage site.

The structural requirements for proteolytic cleavage of the human immunodeficiency virus type 1 env gene product, gp160, to gp120 and gp41 have been assessed by specific mutagenesis of the sequence Lys Ala Lys Arg Arg Val Val Glu Arg Glu Lys Arg located between amino acids 500 and 511, i.e., at the putative C terminus of gp120. The basic amino acids underlined have been mutated, individually and in combination, to neutral amino acids, and the cleavability of the mutated env gene products was examined after expression in CV-1 cells. The results show that the replacement of Arg-511 (cleavage presumably occurs C terminal to this amino acid) with Ser completely abolishes recognition and cleavage by the cellular protease(s), i.e., the remaining basic amino acids in the vicinity do not serve as alternative substrates. However, Arg-508 and Lys-510 are important features of the recognition site since, when they are individually changed to neutral amino acids, cleavage is severely impaired. The basic amino acids 500, 502, and 504 are, individually, not important for cleavage, since their individual replacement by neutral amino acids does not impair cleavage. However, when all four basic amino acids 500, 502, 503, and 504 are changed to neutral amino acids, cleavage is almost completely abolished. This shows that the sequence Arg Glu Lys Arg at the cleavage site is alone not sufficient for cleavage but that a contribution of other amino acids is required, whether the other amino acids provide a basic character or a certain structure in the vicinity of the cleavage site. When noncleavable or poorly cleavable mutant env genes are expressed from the infectious plasmid pNL4-3 in CD4+ human lymphoblastoid cells, noninfectious virus, incapable of spread throughout the culture, is produced.

Amino Acid Sequence↗

PubMed1988

The duck hepatitis B virus DNA polymerase is tightly associated with the viral core structure and unable to switch to an exogenous template.

The duck hepatitis B virus (DHBV) has a DNA polymerase associated with it which uses the incomplete viral genome as endogenous template. A prerequisite for studying this polymerase is the availability of conditions to open viral cores without destroying their enzymatic activity. In this study, this was achieved by a brief treatment with low pH. DHBV DNA in low-pH-treated cores was susceptible to digestion with deoxyribonuclease I and restriction enzymes, and large restriction fragments diffused out of the viral cores. However, the DHBV polymerase remained tightly associated with its DNA template in the viral core structure and could still incorporate nucleotides into those DNA fragments which carried the DNA-bound protein and remained in the core. The DHBV polymerase could not switch to any of several exogenously supplied templates although these were most likely accessible to it. The manner in which this tight association of the DHBV polymerase with the core may occur, and the possible implications of this interaction during the DHBV replication cycle, is discussed.

Animals↗

PubMed1988

The duck hepatitis B virus P-gene codes for protein strongly associated with the 5'-end of the viral DNA minus strand.

A number of antisera, elicited against different segments of the duck hepatitis B virus (DHBV) P-gene translation product, were used to immunoprecipitate the protein that is covalently bound to the 5'-end of the DHBV DNA minus strand. For monitoring purposes, a small DNA minus-strand fragment, carrying this protein, was radioactively labeled. All of the P-specific antisera specifically immunoprecipitated this DNA fragment demonstrating that the protein species attached to the immunoprecipitated DNA fragment were products of the DHBV P-gene. The electrophoretic behavior, in SDS gels, of the DNA minus-strand fragment-protein complex indicated that it was present mostly in the form of aggregates. However, a small fraction consisted of DNA minus-strand fragments carrying P-gene proteins, encoded solely within the 5'-region of the P-gene. This indicated that different P-gene proteins, presumably covalently bound at a common region and subsequently processed, were bound to the 5'-end of the DHBV DNA minus strand. The DHBV P-gene presumably codes for the virus-associated reverse transcriptase and DNA polymerase activities. Using the P-gene-specific antisera, it was not possible to detect putative P-gene-coded polymerase proteins in a free form, i.e., not bound to viral DNA. This may be due to insufficient sensitivity or to the polymerase protein(s) being heterogeneous and/or aggregated. In addition, it is possible that the genome-bound protein itself may have polymerase activity.

Animals↗

PubMed1988

[Fats in the diet].

Fats are important constituents of the human diet since on the one hand, they contribute to the caloric density of the diet, and on the other, they serve as vehicles of essential nutrients such as linoleic and alpha-linolenic acids, as well as fat-soluble vitamins. The existence of human populations subsisting on diets with values as low as 10% or more than 50% of the calories represented by fats, has been documented, demonstrating the great adaptability of man to a wide availability of this type of food. Nevertheless, extensive epidemiological and experimental research in relation to a frequent degenerative diseases of man, arteriosclerosis, have consistently demonstrated that the proportion of saturated fats in the diet has a positive correlation with the frequency of these alterations. Mortality and fat availability in Latin America is consistent with these results. In consequence, and taking into consideration the present level of fat availability in the Region, we propose that no more than 25% of the caloric requirement should be covered by fats. Additionally, this amount of fat should have equal proportions of saturated, monounsaturated and polyunsaturated fatty acids. Cholesterol, which is contained in animal fats, is not a necessary nutrient for humans, so that no minimum consumption needs to be established. Daily ingestion of cholesterol should be restrained to no more than 100 mg/1,000 calories. Introduction of new fatty foods for human consumption should be preceded by a thorough investigation of the metabolic consequences.

Cardiovascular Diseases↗

PubMed1983

Differential expression of endogenous virus glycoprotein in fibroblasts and sera of some adult chickens.

A rapid and convenient assay for the expression of endogenous retrovirus glycoprotein in adult chickens has been developed based on the enzyme-linked immunosorbent assay (ELISA) principle. This method has been standardized using the conventional chick helper factor test. In the course of establishing this method with a large number of specific pathogen-free (VALO) chickens, an interesting diversity became apparent; about 20% of the birds which, according to chick helper factor tests performed with feather follicle fibroblast cultures were negative for endogenous virus glycoprotein expression, exhibited relatively high titres of reactive glycoprotein in serum. However, in no case was a chick helper factor-positive animal negative in serological tests. The possibility of endogenous virus antigen expression which either cannot be detected in fibroblasts, or is incapable of functioning in the chick helper factor complementation, is discussed.

Animals↗