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Biomedical subjects

R W Jansen

Publications and source records attributed to R W Jansen.

At least 55 records · Page 3Linked to original sources
PubMed1993

Novel, negatively charged, human serum albumins display potent and selective in vitro anti-human immunodeficiency virus type 1 activity.

We prepared a series of modified proteins and peptides by derivatizing the positively charged epsilon-amino groups of the lysine amino acids through reaction with anhydrides of succinic acid (Suc) and aconitic acid (Aco). Human serum albumin (HSA) was modified by introduction of a single carboxylic group (Suc-HSA) or two carboxylic groups (Aco-HSA) per amine function, yielding strongly negatively charged compounds. The in vitro anti-human immunodeficiency virus (HIV)-1 IC50 of Suc-HSA was about 1 microgram/ml, and the most polyanionic modified albumin of the series (Aco-HSA) exhibited an IC50 as low as 0.02 microgram/ml. Similar derivatization of the plasma protein orosomucoid or the synthetic polypeptide polylysine did not produce compounds with significant anti-HIV-1 activity, indicating an HSA-specific effect. The mechanism of action of Suc-HSA was reported to be the inhibition of a post-binding virus-cell fusion event, probably due to interference with the gp41-mediated fusion process. In the present study we demonstrate that the more potent Aco-HSA also interferes with this fusion process but, additionally, this compound inhibits (i) the binding of soluble CD4 to HIV-infected cells, (ii) the binding of HIV particles to MT-4 cells, and (iii) the binding of anti-gp120 monoclonal antibody to the gp120 molecule. This indicates that Aco-HSA, apart from post-binding fusion, also inhibits virus-cell binding by shielding viral gp120. The simultaneous inhibition of binding and fusion may lead to a synergistic effect, explaining the extreme potency of Aco-HSA. The polyanionic HSAs are significantly less active against HIV-2 and do not interfere with the replication of feline immunodeficiency virus or 12 other DNA or RNA viruses, indicating a HIV-1-specific effect. In contrast, another polyanionic compound, the sulfated polysaccharide dextran sulfate, inhibits the replication of various viruses in a more nonspecific way, as a general polyanion. Dextran sulfate also exhibits strong anticoagulant activity, whereas Suc-HSA and Aco-HSA do not show this unwanted side effect.

Anions↗
PubMed1992

Simultaneous analysis of azidothymidine and its mono-, di- and triphosphate derivatives in biological fluids, tissue and cultured cells by a rapid high-performance liquid chromatographic method.

A rapid high-performance liquid chromatographic (HPLC) method for the simultaneous analysis of the antiviral drug azidothymidine (AZT), AZT monophosphate, AZT diphosphate and AZT triphosphate, with ultraviolet detection in the nanomolar range, is described. Determination of these compounds in vitro in the human MT-4 lymphocyte cell line did not require a prior extraction, and AZT and its phosphorylated derivatives could be accurately analysed in one HPLC run. However, plasma, bile, liver homogenate and urine samples could not be injected directly into the chromatograph. Therefore, a solid-phase extraction procedure was developed, using azidodideoxyinosine as internal standard. The extractions of the compounds of interest from all but urine samples were reproducible, with recoveries between 65% (AZT triphosphate from plasma) and 100% (AZT from plasma).

Animals↗
PubMed1992

Genetic, antigenic and biological differences between strains of hepatitis A virus.

Recent studies have documented a considerable degree of genetic divergence among wild-type hepatitis A virus (HAV) strains recovered from different geographical locations. Human HAV strains can be grouped into four genotypes (I, II, III and VII) and unique simian strains belong to three additional genotypes (IV, V and VI). Between each of these genotypes, the nucleotide sequence varies at 15-25% of base positions in the P1 region. Despite this, there is good evidence that most, if not all, human strains of HAV are closely related antigenically. In contrast, although simian strains recovered from Old World monkeys are cross-reactive in immunoassays employing polyclonal antibodies, these strains have significant antigenic differences from human HAV strains. Nonetheless, because biological differences in the host range of these strains apparently preclude significant human infection, this is unlikely to pose a problem in controlling HAV infections with active immunization. Inactivated and attenuated vaccines produced from genotype I human strains (HM175 or CR326) are likely to provide protection against all relevant human HAV strains.

Animals↗
PubMed1992

Genetic relatedness of hepatitis A virus strains recovered from different geographical regions.

A pairwise comparison of the nucleic acid sequence of 168 bases from 152 wild-type or unique cell culture-adapted strains of hepatitis A virus (HAV) revealed that HAV strains can be differentiated genetically into seven unique genotypes (I to VII). In general, the nucleotide sequence of viruses in different genotypes differs at 15 to 25% of positions within this segment of the genome. Viruses from four of the genotypes (I, II, III and VII) were recovered from cases of hepatitis A in humans, whereas viruses from the other three genotypes (IV, V and VI) were isolated only from simian species developing a hepatitis A-like illness during captivity. Among non-epidemiologically related human HAV strains, 81 were characterized as genotype I, and 19 as genotype III. Within each of these major genotypes, there were two distinct groups (subgenotypes), which differed in sequence at approximately 7.5% of base positions. Each genotype and subgenotype has a characteristic amino acid sequence in this region of the polyprotein, with the most divergent genotypes differing at 10 of 56 residues. Strains recovered from some geographical regions belonged to a common (endemic) genotype, whereas strains from other regions belonged to several, probably imported, genotypes. Thus, HAV strains recovered in North America were for the most part closely related at the nucleotide sequence level, whereas in other regions, such as Japan and Western Europe, HAV strains were derived from multiple genotypes or sub-genotypes. These data indicate that patterns of endemic transmission can be differentiated from situations in which infections are imported due to travel.

Amino Acid Sequence↗
PubMed1991

Formaldehyde treated albumin contains monomeric and polymeric forms that are differently cleared by endothelial and Kupffer cells of the liver: evidence for scavenger receptor heterogeneity.

Formaldehyde treated albumin (F-HSA) was found to consist of a monomeric and a polymeric fraction. Both fractions were primarily endocytosed by rat liver sinusoidal cells. However, immunohistochemical staining of endocytosed material showed that the relative contribution of the endothelial and Kupffer cells in uptake of the monomer and the polymer differed significantly, with the monomer mainly having an endothelial cell- and the polymer predominantly having a Kupffer cell pattern of distribution. To directly confirm these heterogeneous patterns, we injected in vivo the 125I-labeled F-HSA fractions and isolated the endothelial and Kupffer cells by centrifugal elutriation. 73.7% of the monomeric F-HSA was found in endothelial cells and only 14.9% was found in Kupffer cells. In contrast, the polymeric F-HSA (1500 kD) was mainly endocytosed by Kupffer cells (71%), whereas the endothelial cells contributed only for 24% in hepatic uptake. In vivo studies and isolated perfused rat liver experiments showed that endocytosis of both monomer and polymer was inhibited by co-administration of polyinosinic acid, a well known inhibitor for scavenger receptors, indicating that these receptors on endothelial and Kupffer cells are mainly involved in this uptake process.

Animals↗
PubMed1991

Hepatic endocytosis of various types of mannose-terminated albumins. What is important, sugar recognition, net charge, or the combination of these features.

We synthesized several para-aminophenyl (pap-) mannose-terminated albumins with varying sugar density (Man7-HSA, Man22-HSA, and Man40-HSA) and compared hepatic uptake with (thio-)mannose-terminated bovine serum albumin (Man-43-AI-BSA) The rate of uptake in isolated perfused rat livers was found to be positively correlated with the sugar density (Man40-HSA = Man22-HSA greater than Man7-HSA greater than HSA). Immunohistochemical staining of liver sections showed for both types of neoglycoproteins that uptake occurred in nonparenchymal cells only. Competition experiments with a 500-fold excess of mannan, a known ligand for the mannose/N-acetylglucosamine receptor, that is predominantly localized in endothelial cells, showed complete inhibition of the (thio-)Man43-AI-BSA uptake. In the case of (pap-)mannose-terminated albumins, however, the extent of inhibition by mannan was moderate and decreased markedly with increasing sugar density, being only 20% for (pap-)Man40-HSA. Therefore, we hypothesized that one or more additional removal systems contributed to the clearance of these (pap-)mannose glycoproteins. We found that net negative charge of the (pap-)mannose albumins clearly increased with increasing sugar density, as shown on fast protein liquid chromatography anion-exchange chromatograms. To determine whether the scavenger receptor system that is also mainly present on endothelial cells is involved, we performed competition studies with strongly negatively charged substrates, such as dextran sulfate and formaldehyde-treated human serum albumin (fHSA). An excess of dextran sulfate (500 kDa), indeed blocked the (pap-)mannose-albumin uptake for more than 95%. Dextran sulfate completely inhibited the hepatic uptake of mannan as well, indicating that the polyanion does not discriminate between the scavenger system and the mannose receptor system and should be regarded as an aspecific inhibitor of receptor-mediated endocytotic pathways. Surprisingly, a 500-fold excess of fHSA only moderately (20%) inhibited the clearance of (pap-)Man40-HSA in spite of its high affinity for the scavenger receptor. However, a combination of mannan and fHSA strongly inhibited the uptake of (pap-)Man22-HSA (90%) and to a lesser extent (pap-)Man40-HSA (80%), indicating that a third uptake mechanism may exist that recognizes both mannose groups (or other sugars) and net negative charge. This so far unnoticed receptor system apparently is strongly affected by dextran sulfate and, as shown by immunohistochemistry, is mainly localized on Kupffer cells rather than on the endothelial cells of the liver.

Albumins↗
PubMed1991

Genetic variability within the 5' nontranslated region of hepatitis A virus RNA. Implications for secondary structure and function.

The RNA genome of hepatitis A virus (HAV) contains a lengthy and relatively well conserved 5' nontranslated region (5'NTR). In other picornaviruses, the 5'NTR has been shown to have important functions related to the initiation of viral translation and replication of viral RNA, functions which are critically dependent on both primary and secondary RNA structure. We have utilized a phylogenetic approach to construct a model of the secondary structure of the HAV 5'NTR. By comparing the nucleotide sequences of genetically divergent simian and human HAV strains, we identified a series of covariant nucleotide substitutions which are predictive of conserved, double-stranded helical structures within the 5'NTR, and which thus permitted improved thermodynamic modeling of the secondary structure. The model was further refined based on the observed sites of cleavage of synthetic RNA by single- and double-strand specific RNAses. The results of these studies suggest that the 5'NTR of HAV has a general organization similar to that of other picornaviruses, and shares certain structural features and perhaps specific functions with the 5'NTRs of the cardioviruses and aphthoviruses.

Base Composition↗
PubMed1991

Neoglycoproteins as carriers for antiviral drugs: synthesis and analysis of protein-drug conjugates.

In order to investigate whether neoglycoproteins can potentially act as carriers for targeting of antiviral drugs to certain cell types in the body, various neoglycoproteins were synthesized using thiophosgene-activated p-aminophenyl sugar derivatives. These neoglycoproteins were conjugated with the 5'-monophosphate form of the antiviral drug AZT. For a proper characterization of these preparations, both protein and drug content have to be determined. Comparison of the Lowry and the Bio-Rad protein assays revealed that for both the neoglycoprotein carriers themselves and the AZTMP conjugates, the Lowry assay yielded the most reliable and reproducible results. It was demonstrated that both the reagent used for drug conjugation (ECDI) as well as the introduction of phenyl-sugar groups in the protein interfered with the analysis of bound nucleotide as based on spectral differences between protein and protein-drug conjugate. Therefore, we developed a rapid HPLC system for determination of the drug-protein coupling ratio through acid hydrolysis of the covalently bound nucleotide. With the ECDI-mediated conjugation of 5'-monophosphate drug derivatives to neoglycoproteins, products with molar ratios of drug to protein ranging from 1.2 to 5.6 were obtained. The drug-neoglycoprotein conjugates appeared to be fairly stable during storage, in lyophilized form, at -20 degrees C. The anti-HIV-1 activity of the neoglycoprotein-drug conjugates, as determined in vitro in MT-4 cells, was shown to be dependent on glycosylation of the albumin and also on the kind of sugar present in the neoglycoprotein. The anti-HIV-1 activity of the AZTMP-mannose-albumin conjugate exceeded that of the parent drug by more than 4 times.

Antiviral Agents↗
PubMed1991

Mild dehydration and atrial natriuretic peptide in young and elderly subjects.

The influence of mild dehydration on plasma levels of atrial natriuretic peptide (ANP) was studied in both young (aged 18 to 25 years) and elderly (aged 72 to 86 years) subjects. We expected that dehydration would lower ANP concentrations due to the ensuing volume contraction. A different response of the ANP hormonal system in the elderly might help to explain the observation that elderly subjects are more predisposed to dehydration as compared to young subjects. Dehydration was induced by restriction of fluid intake to 25% of normal for one day. During the study, urinary osmolality increased while osmolar clearance and body weight decreased. Basal ANP concentrations were higher in the elderly subjects. However, these levels did not change during the dehydration study neither in the young nor in the elderly subjects. This may be explained by the activation of counter-regulatory systems preventing a decrease in central blood volume and hence a decrease in ANP concentration.

Adolescent↗
PubMed1991

The role of secretory immunity in hepatitis A virus infection.

Because the role of intestinal immunity remains uncertain in hepatitis A, samples of feces and saliva from infected primates and humans were tested for virus neutralizing activity. Only two of eight owl monkeys infected by the intragastric route developed neutralizing antibody detectable in extracts of feces collected up to 88 days after viral challenge, although serum neutralizing antibody was present in all monkeys by day 33. Similarly, neutralizing antibody was detected in fecal extracts from none of three experimentally infected human volunteers and only 1 of 15 naturally infected humans. The single positive human specimen contained occult blood. Only 2 of 19 saliva samples from naturally infected humans had significant viral neutralizing activity. In contrast, neutralizing antibody to type 2 poliovirus was present in most human fecal or saliva specimens tested. These data suggest that intestinal immunity does not play a significant role in protection against hepatitis A.

Animals↗
PubMed1991

The influence of food temperature on postprandial blood pressure reduction and its relation to substance-P in healthy elderly subjects.

Blood pressure (BP) in the elderly may decrease after a meal or oral glucose loading. The mechanism of this phenomenon is still unclear. In addition, the effect of the temperature of a meal on postprandial BP is unknown. However, it has been suggested that vasoactive gastrointestinal peptides are involved in the etiology of postprandial BP reduction. Therefore, we studied the effects of a cold and a warm glucose solution on BP, heart rate, plasma glucose, insulin, and substance-P levels in 15 healthy elderly subjects with a mean age of 74 +/- 3 (SD) years. With an interval of at least 2 days, a warm (50 degrees C) and a cold (5 degrees C) solution (75 g glucose/300 mL water) were given in random order. After the cold glucose loading mean arterial pressure increased by a maximum of 3.9 +/- 1.3 mmHg (P less than 0.01). In contrast, BP decreased after the warm solution by a maximum of 8.0 +/- 1.1 mmHg (P less than 0.001). Neither test had an influence on plasma substance-P levels. Our data suggest that postprandial blood pressure reduction in the elderly is dependent on food temperature. Substance-P does not seem to play a role in this phenomenon.

Administration, Oral↗
PubMed1991

The 5' nontranslated region of hepatitis A virus RNA: secondary structure and elements required for translation in vitro.

Although the lengthy 5' nontranslated regions (5'NTRs) of other picornaviral RNAs form highly ordered structures with important functions in viral translation, little is known about the 5'NTR of hepatitis A virus (HAV). We determined the nearly complete 5'NTR nucleotide sequences of two genetically divergent HAV strains (PA21 and CF53) and included these data in a comparative phylogenetic analysis of the HAV 5'NTR. We identified covariant nucleotide substitutions predictive of conserved secondary structures and used this information to develop a model of the 5'NTR secondary structure, which was further refined by thermodynamic predictions and nuclease digestion experiments. According to this model, the 5'NTR comprises six major structural domains. Domains I and II (bases 1 to 95) contain a 5'-terminal hairpin and two stem-loops followed by a single-stranded and highly variable pyrimidine-rich tract (bases 96 to 154). The remainder of the 5'NTR (domains III to VI, bases 155 to 734) contains several complex stem-loops, one of which may form a pseudoknot, and terminates in a highly conserved region containing an oligopyrimidine tract preceding the putative start codon by 13 bases. To determine which structural elements might function as an internal ribosome entry site, RNA transcripts representing the HAV 5'NTR with progressive 5' deletions were translated in rabbit reticulocyte lysates. The translation product was truncated, unprocessed P1 polyprotein. Removal of the 5'-terminal 354 bases of the 5'NTR had little effect on translation. However, deletion to base 447 slightly decreased translation, while deletion to base 533 almost completely abolished it. These data indicate that sequences 3' of base 355 play an important role in the translation mechanism utilized by genomic-length HAV RNA. Significantly, this region shares several conserved structural features with the internal ribosome entry site element of murine encephalomyocarditis virus.

Animals↗
PubMed1991

Antigenic and genetic variation in cytopathic hepatitis A virus variants arising during persistent infection: evidence for genetic recombination.

Variants of hepatitis A virus (pHM175 virus) recovered from persistently infected green monkey kidney (BS-C-1) cells induced a cytopathic effect during serial passage in BS-C-1 or fetal rhesus kidney (FRhK-4) cells. Epitope-specific radioimmunofocus assays showed that this virus comprised two virion populations, one with altered antigenicity including neutralization resistance to monoclonal antibody K24F2, and the other with normal antigenic characteristics. Replication of the antigenic variant was favored over that of virus with the normal antigenic phenotype during persistent infection, while virus with the normal antigenic phenotype was selected during serial passage. Viruses of each type were clonally isolated; both were cytopathic in cell cultures and displayed a rapid replication phenotype when compared with the noncytopathic passage 16 (p16) HM175 virus which was used to establish the original persistent infection. The two cytopathic virus clones contained 31 and 34 nucleotide changes from the sequence of p16 HM175. Both shared a common 5' sequence (bases 30 to 1677), as well as sequence identity in the P2-P3 region (bases 3249 to 5303 and 6462 to 6781) and 3' terminus (bases 7272 to 7478). VP3, VP1, and 3Cpro contained different mutations in the two virus clones, with amino acid substitutions at residues 70 of VP3 and 197 and 276 of VP1 of the antigenic variant. These capsid mutations did not affect virion thermal stability. A comparison of the nearly complete genomic sequences of three clonally isolated cytopathic variants was suggestive of genetic recombination between these viruses during persistent infection and indicated that mutations in both 5' and 3' nontranslated regions and in the nonstructural proteins 2A, 2B, 2C, 3A, and 3Dpol may be related to the cytopathic phenotype.

Animals↗
PubMed1991

Potent in vitro anti-human immunodeficiency virus-1 activity of modified human serum albumins.

A series of neoglycoproteins was synthesized by coupling of thiophosgene-activated p-aminophenyl derivatives [Biol. Cell. 47:95-110 (1983); J. Histochem. Cytochem. 32:1091-1094 (1984)] of various sugars to human serum albumin. The compounds were evaluated for their in vitro activity against human immunodeficiency virus (HIV). Neoglycoproteins with the highest sugar content were found to be the most potent inhibitors of HIV-1-induced cytopathogenicity. However, this was not due to the nature of the sugar used but, rather, was related to the extra negative charge of the neoglycoproteins. To investigate whether the antiviral activity of the neoglycoproteins exhibited sugar specificity, increased with increasing negative charge, or depended on both sugar specificity and negative charge, we synthesized albumins and neoglycoproteins with an enhanced negative charge, by treatment with formaldehyde or succinic anhydride. Succinylated human serum albumin had the most pronounced net negative charge and had an IC50 of about 1 microgram/ml. No cytotoxicity was observed at concentrations up to 1 mg/ml, implicating a selectivity index (CC50/IC50) of at least 10(3). To elucidate the mechanism of action of these anionic albumins, we investigated whether they interfered with HIV-1 adsorption to the cells, binding of anti-OKT4A monoclonal antibody (mAb) to the CD4 receptor, binding of anti-gp120 mAb to gp120, or inhibition of syncytium formation in co-cultures of HIV-1-infected HUT-78 cells with MOLT-4 cells. From these experiments, we conclude that albumins with an increased negative charge (a) are potent and nontoxic anti-HIV-1 agents, (b) cause a 50% reduction of syncytium formation in the same concentration range as their IC50 in the antiviral assay, and (c) do not bind to the OKT4A epitope of the CD4 receptor and only partly inhibit anti-gp120 mAb-gp120 interaction and virus-cell binding at concentrations that are 100 times higher than their IC50 in the antiviral assay. Therefore, we conclude that the modified albumins interfere with a post-binding event, of which one of the potential mechanisms is an interaction with the gp41 fusion protein, which is necessary for syncytium formation but is not involved in initial virus binding.

Adsorption↗
PubMed1990

Targeting of antiviral drugs to T4-lymphocytes. Anti-HIV activity of neoglycoprotein-AZTMP conjugates in vitro.

The delivery of the anti-HIV agent 3'-azido-3'-deoxythymidine (AZT), in its 5'-monophosphate form, (in) to human T-lymphocyte MT-4 cells in vitro through covalent coupling to neoglycoproteins was investigated. In vivo application of this drug targeting concept may lead to increased efficacy and/or diminished side effects caused by AZT during the treatment of AIDS and ARC patients. The rationale for the design of the neoglycoprotein carriers is based on the existence of sugar recognizing lectins on T-lymphocytes. Using a phenyl-linkage between sugar and Human Serum Albumin (HSA), various mannose-, fucose-, galactose-and glucose-containing neoglycoproteins were synthesized. The intrinsic anti-HIV activity of these neoglycoproteins was tested in vitro in HIV-1 infected MT-4 cells. Only the derivative having 40 moles mannose per mole protein (Man40HSA) shows pronounced anti-HIV-1 activity itself. This effect may be caused by interference of the Man40HSA with the gp120-CD4 mediated virus/MT-4 cell interaction. After conjugation with AZTMP, the mannose- as well as the fucose- and galactose-containing conjugates exhibited a pronounced activity. Conjugates of glucose-HSA and HSA displayed much less activity in spite of the fact that drug loading was considerably higher, compared with the galactose, mannose and fucose derivatives. In the series of mannose-neoglycoproteins, the Man22HSA-AZTMP conjugate was shown to be more than 30 times as active against HIV-1 compared to HSA-AZTMP. Selectivity indices of Man7 and Man22HSA-AZTMP were exceeding the AZT and AZTMP indices, indicating that these conjugates possess a more selective action. Stability experiments indicate that the potent action of the galactose-, mannose- and fucose-HSA-AZTMP conjugates is not due to a complete extracellular hydrolysis of the covalent drug-protein bond. Since Man22HSA has no intrinsic activity in the concentration range used, the antiviral effect is unlikely to be explained by synergism of the neoglycoprotein by a component of the cell membrane and subsequent internalization and release of the drug from the conjugate may play a role.

Antiviral Agents↗