Comparative analyses of human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vif mutants.

Virion infectivity factor (vif), a gene found in all lentiviruses, plays an essential role in virus replication in certain target cells. We examined the replication competence of the human immunodeficiency virus type 2 (HIV-2) vif mutant in different T-cell lines and primary cells in comparison with that of the HIV-1 vif mutant. Both mutant viruses were unable to replicate in peripheral blood-derived mononuclear cells but replicated with wild-type efficiency in certain T-cell lines, such as SupT1 and MOLT-4/8. These results confirm the importance of vif in the infection of relevant target cells and imply that some cellular factor(s) could compensate for vif function. However, HIV-1 and HIV-2 vif mutant viruses also show differential replications in other cell lines, suggesting either different threshold requirements for the same cellular factor(s) or the involvement of different factors to compensate for vif-1 and vif-2 functions. By cross complementation experiments, we showed that vif-1 and vif-2 have similar functions. Our studies further indicate the existence of two kinds of nonpermissive cells: H9 is unable to complement HIV-1 delta vif but is susceptible to a one-round infection with HIV-1 delta vif produced from permissive cells. In contrast, U937 is nonpermissive for HIV-2 delta vif produced from permissive cells but, once infected, is able to complement the delta vif function. In both types of nonpermissive cells, a step prior to proviral DNA synthesis is affected.     

Evaluation of the use of cyclodextrins in chiral separation of basic drug substances by capillary electrophoresis

The influence of the choice of type and/or concentration of cyclodextrin, other additives, the temperature surrounding the capillary, and buffer pH on the separation of some chiral basic drug substances in capillary zone electrophoresis has been evaluated. It was found that pH of the buffer and type and concentration of cyclodextrin had a major influence on the separation. © 1995 Wiley-Liss, Inc.     

Starved Tetrahymena thermophila cells that are unable to mount an effective heat shock response selectively degrade their rRNA.

Tetrahymena thermophila cells that had been shifted from log growth to a non-nutrient medium (60 mM Tris) were unable, during the first few hours of starvation, to mount a successful heat shock response and were killed by what should normally have been a nonlethal heat shock. An examination of the protein synthetic response of these short-starved cells during heat shock revealed that whereas they were able to initiate the synthesis of heat shock proteins, it was at a much reduced rate relative to controls and they quickly lost all capacity to synthesize any proteins. Certain pretreatments of cells, including a prior heat shock, abolished the heat shock inviability of these starved cells. Also, if cells were transferred to 10 mM Tris rather than 60 mM Tris, they were not killed by the same heat treatment. We found no abnormalities in either heat shock or non-heat shock mRNA metabolism in starved cells unable to survive a sublethal heat shock when compared with the response of those cells which can survive such a treatment. However, selective rRNA degradation occurred in the nonsurviving cells during the heat shock and this presumably accounted for their inviability. A prior heat shock administered to growing cells not only immunized them against the lethality of a heat shock while starved, but also prevented rRNA degradation from occurring.     

Polyol accumulation by two filamentous fungi grown at different concentrations of NaCl

The accumulation of polyols by Aspergillus niger (van Tiegh) strain S 1 and Penicillium chrysogenum (Thom) strain S 30 was followed during growth in media of different concentrations of NaCl. The major polyols found were glycerol, erythritol and mannitol. The total polyol pool increased in both organisms in response to raised salinity, and the proportion of glycerol and erythritol was markedly enhanced at high salinity.     

The effect of glucagon on the kinetics of hepatic mitochondrial calcium uptake

Summary Previous work by this and other laboratories has shown that glucagon administration stimulates calcium uptake by subsequently isolated hepatic mitochondria. This stimulation of hepatic mitochondrial Ca²⁺ uptake byin vivo administration of glucagon was further characterized in the present report. Maximal stimulation of mitochondrial Ca²⁺ accumulation was achieved between 6–10 min after the intravenous injection of glucagon into intact rats. Under control conditions, Ca²⁺ uptake was inhibited by the presence of Mg²⁺ in the incubation medium. Glucagon treatment, however, appeared to obliterate the observed inhibition by Mg²⁺ of mitochondrial Ca²⁺ uptake. Kinetic experiments revealed the usual sigmoidicity associated with initial velocity curves for mitochondrial calcium uptake. Glucagon treatment did not alter this sigmoidal relationship. Glucagon treatment significantly increased the V_max for Ca²⁺ uptake from 292±22 to 377±34 nmoles Ca²⁺ /min per mg protein (n=8) but did not affect the K_0.5, (6.5–8.6 μM). Since the major kinetic change in mitochondrial Ca²⁺ uptake evoked by glucagon is an increase in V_max, the enhancement mechanism is likely to be an increase either in the number of active transport sites available to Ca²⁺ or in the rate of Ca²⁺ carrier movement across the mitochondrial membranes.     

Immunofluorescence microscopic demonstration of vimentin filaments in asteroid bodies of sarcoidosis. A comparison with electron microscopic findings

Asteroid bodies in multinucleate giant cells from sarcoid granulomas were investigated by immunofluorescence and electron microscopy. The following points have been established: 1. Asteroid bodies are made up of individual components of the so-called cytoskeleton, predominantly vimentin filaments. Microtubules are involved in smaller amounts in the formation of the asteroid bodies. 2. They arise within the area of the cytosphere. The body of the asteroid includes the centrioles while the arms of the asteroid usually extend into the Golgi area and occasionally up to the cell nuclei. 3. Asteroid bodies result from aggregation of the flexible filamentous and microtubular systems of the centrosphere. The processes of aggregation probably result from local fluid shifts and sol-gel transformations. 4. The stellate form of the aggregations is determined by the preexistent radial arrangement of the elements of the cytosphere. 5. The prevailing specific environment of the underlying granulomatous disease, together with the internal characteristics of the structure and function of the giant cells, in particular in states of exhaustion may play a part in their development.     

Role of the fusogenic peptide sequence in syncytium induction and infectivity of human immunodeficiency virus type 2.

Syncytium induction is a characteristic feature of infection by human immunodeficiency virus (HIV) in vitro. The hydrophobic amino terminus of the transmembrane glycoprotein of HIV type 1 is an essential determinant of virus entry into the target cell population and the formation of syncytia in cell culture. To define the role of the HIV type 2 fusion peptide during infection and syncytium formation, we introduced 8 amino acid substitutions into the hydrophobic amino terminus of gp41, changing either the hydrophobicity, the charge, or the polarity of the amino acid. Viruses containing the envelope mutations were analyzed for their syncytium-inducing capacities, levels of infectivity, and envelope processing and expression. Mutations that increased the hydrophobic nature of the fusion peptide increased syncytium formation, whereas mutations which increased the charge and the polarity and/or decreased the hydrophobicity of the fusion domain severely reduced the capacity of the virus to induce syncytia. However, viruses severely compromised for syncytium formation exhibit only slightly lower levels of infectivity.     

Rat cystathionine beta-synthase. Gene organization and alternative splicing.

We elucidated the structure and alternative splicing patterns of the rat cystathionine beta-synthase gene. The gene is 20-25 kilobase pairs long, and its coding region is divided into 17 exons. These are alternatively spliced, forming four distinct mRNAs (types I through IV). The predicted open reading frames encode proteins of 61.5, 39, 60, and 52.5 kDa, respectively. Exons 13 and 16 are used alternatively and mutually exclusively. Exon 13 includes a stop codon and encodes the unique carboxyl-terminal sequence found in types II and IV. Exon 16 is present only in type I. Types I and III, which differ by 42 nucleotides (exon 16), are the predominant synthase mRNA forms in rat liver. Seventeen arginine peptides from pure liver synthase matched the deduced amino acid sequences of types I and III. These two polypeptides are detectable in liver extracts; each exhibits enzymatic activity when expressed in transfected Chinese hamster cells. Synthase shows substantial sequence similarity with pyridoxal 5'-phosphate dependent enzymes from lower organisms. Similarity of synthase to Escherichia coli O-acetylserine (thiol)-lyase (cysK) is 52%; E. coli tryptophan synthase beta chain (trpB), 36%; yeast serine deaminase, 33%. Lysine 116 in synthase aligns with the established pyridoxyllysine residue of these enzymes suggesting that it is the pyridoxal 5'-phosphate binding residue.     

Multi-system approach to study mutagenesis induced by chemical carcinogens.

To understand molecular mechanisms of the mutation fixation process induced by a mutagen and carcinogen, a multi-system approach is suggested to reduce the probability that the results are biased by the assay used. In this light we described our different approaches to answer basic questions on the mutagenesis induced by the chemical carcinogen 4-Nitroquinoline-1-oxide. We determined mutations at the molecular level in three experimental systems: a) in prokaryotes (ss M13mp19 lacZ'/E. coli F'lacZ delta M15); b) in eukaryotes (i) ss and ds pZ189 supF/CV1-P/E.coli lacZam and (ii) HPRT in CHO cells with different repair capacity. We think this type of approach can be used to study the genetic effects of new cancer drugs for which the molecular mechanisms of action at the molecular level are still not well understood. We think to apply the know-how to study mutational spectra in tumor derived tumor suppressor genes.