Production of multimeric forms of CD4 through a sugar-based cross-linking strategy

We have developed a three-step cross-linking procedure that is specifically targeted at the carbohydrate on a protein and applied it to CD4 as a model system for studying the role of multivalent interactions in function. In the first step CD4 was oxidized with periodate, creating aldehydes that served as targets for the subsequent chemistry. Next the aldehydes were modified with cystamine, converting the reactive group into a thiol. Finally cross-linking through the thiol moiety was generated with the homobifunctional cross-linker bismaleimidohexane. With this procedure, approximately 60% of the CD4 was converted into higher molecular weight complexes that were soluble and retained function as assessed by glycoprotein gp120 binding activity. CD4 dimers and tetramers by mass were 4 and 15 times as active as CD4 monomer in blocking virus infection with HTLV-IIIB in an in vitro cellular assay. The cross-linking chemistry provides an efficient method for producing homomultimers of a glycoprotein.     

Synthesis and in vitro anti Mycobacterium tuberculosis activity of a series of phthalimide derivatives

New phthalimide derivatives were easily prepared through condensation of phthalic anhydride and selected amines with variable yields (70–90%). All compounds (3a–l) were evaluated against Mycobacterium tuberculosis H₃₇Rv using Alamar Blue susceptibility. The compounds 3c, 3i, and 3l have the minimum inhibitory concentrations (MICs) of 3.9, 7.8, and 5.0 μg/mL, respectively, and could be considered new lead compounds in the treatment of tuberculosis and multi-drug resistant tuberculosis.     

Mutational analysis of slyD, an Escherichia coli gene encoding a protein of the FKBP immunophilin family

slyD encodes a 196 amino acid polypeptide that is a member of the FKBP family of cis–trans peptidyl–prolyl isomerases (PPIases). slyD mutations affect plaque formation by the phage φX174 by blocking the action of the phage lysis protein E. Here we describe the selection of a set of spontaneous slyD mutations conferring resistance to the expression of gene E from a plasmid. These mutations occur disproportionately in residues of SlyD that, based on the structure of the prototype mammalian FKBP12, make ligand contacts with immunosuppressing drug molecules or are conserved in other FKBP proteins. A wide variation in the plating efficiency of φX174 on these E  ^R strains is observed, relative to the parental, indicating that these alleles differ widely in residual SlyD activity. Moreover, it is found that slyD mutations cause significant growth rate defects in Escherichia coli B and C backgrounds. Finally, overexpression of slyD causes filamentation of the host. Thus, among the FKBP genes found in organisms across the evolutionary spectrum, slyD is unique in having three distinct drug-independent phenotypes.     

A strategy for high cell density culture of heterotrophic microalgae with inhibitory substrates

Substrate inhibition is one of the major problems preventing high cell densities of microalgae in heterotrophic culture, so the possibility of overcoming the problem by various culture techniques was examined. It was found that perfusion culture may be the most appropriate technique for high cell densities in heterotrophic culture using inhibitory substrates. An experimental example in which a hollow fibre cell recycle system (HFCRS) was employed to achieve high cell densities of Chlamydomonas reinhardtii on acetate under heterotrophic conditions of growth was demonstrated. The cell density in the HFCRS was much higher than that reported in the literature for this species.     

Assembly of transcriptionally active chromatin in vitro: a possible role for topoisomerase II

Some models of in vitro chromatin assembly suggest a biphasic molecular mechanism. The first phase, nucleosome formation, is comprised of the formation of histone-DNA complexes which mature into a canonical nucleosome structure. The second phase represents the process by which these nucleosomes become properly spaced with a regular periodicity on the DNA. In this report, we examine the role of DNA topoisomerases in the latter phase of chromatin assembly. To study this process, we use a Xenopus laevis cell-free extract, which assembles quantitative amounts of chromatin on circular DNA templates, and the type II topoisomerase-specific antitumor drugs VM-26 and endrofloxicin. Our results suggest that nucleosome formation is unaffected by the presence of VM-26 or endrofloxicin. However, periodic spacing of nucleosomes is inhibited significantly by these drugs. In the absence of proper chromatin assembly, circular DNA molecules are processed into nucleoprotein complexes which are transcribed poorly. Taken together, these results indicate that the antitumor drugs VM-26 and endrofloxicin influence gene expression indirectly by blocking the periodic spacing of nucleosomes.     

Prediction of the three-dimensional structure of the rap-1A protein from its homology to theras-gene-encoded p21 protein

rap-1A, an anti-oncogene-encoded protein, is aras-p21-like protein whose sequence is over 80% homologous to p21 and which interacts with the same intracellular target proteins and is activated by the same mechanisms as p21, e.g., by binding GTP in place of GDP. Both interact with effector proteins in the same region, involving residues 32–47. However, activated rap-1A blocks the mitogenic signal transducing effects of p21. Optimal sequence alignment of p21 and rap-1A shows two insertions of rap-1A atras positions 120 and 138. We have constructed the three-dimensional structure of rap-1A bound to GTP by using the energy-minimized three-dimensional structure ofras-p21 as the basis for the modeling using a stepwise procedure in which identical and homologous amino acid residues in rap-1A are assumed to adopt the same conformation as the corresponding residues in p21. Side-chain conformations for homologous and nonhomologous residues are generated in conformations that are as close as possible to those of the corresponding side chains in p21. The entire structure has been subjected to a nested series of energy minimizations. The final predicted structure has an overall backbone deviation of 0.7 å from that ofras-p21. The effector binding domains from residues 32–47 are identical in both proteins (except for different side chains of different residues at position 45). A major difference occurs in the insertion region at residue 120. This region is in the middle of another effector loop of the p21 protein involving residues 115–126. Differences in sequence and structure in this region may contribute to the differences in cellular functions of these two proteins.     

Oat leaf base: tissue with an efficient regeneration capacity

Summary An efficient short term regeneration system using seedling derived oat (Avena sativa) leaf tissue has been developed. Callus derived from the leaf base showed a higher response of plant regeneration than callus initiated from mesocotyls and more mature parts of the leaves. A correlation between the nuclear DNA content of the donor material, as analysed with flow cytometry, and its ability to form callus was observed. Somatic embryogenesis was histologically recognised from callus derived from tissue close to the apical meristem. Plant regeneration media with various concentrations of auxin were tested. Callus from three different cultivars had a similar regeneration potential with an optimal regeneration frequency of 60%. About 2 months after inoculation regenerated plantlets could be moved to a greenhouse for cultivation.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 6-diamidino-2-phenylindole - IAA indole-3-acetic acid - KT kinetin - MS Murashige and Skoog's medium - NAA naphthalene acetic acid     

Interactions of chloride and amiloride with the renal Na+/H/ antiporter

Amiloride is a reversible inhibitor of the Na+/H+ antiporter which acts at the external aspect of the transport system. The kinetics of inhibition of the Na+/H+ antiporter with amiloride have been controversial, with the usual finding of simple competitive inhibition, but with other reports of mixed and noncompetitive inhibition of the transporter by amiloride. The present experiments demonstrate that the chloride content of the external transport buffer affects the kinetics of amiloride inhibition. Either simple competitive or mixed inhibition by amiloride was observed in the same vesicle preparations depending on the presence of chloride or gluconate in the buffer. The effect of chloride on the inhibitory effect of amiloride was dependent on the concentration of chloride and amiloride. Similar effects were observed with more potent analogues of amiloride. These findings suggest that the external aspect of the antiporter has a site or sites at which the inhibitory effects of amiloride on the Na+/H+ antiporter can be modified by chloride, even though chloride has only slight effects on the kinetics of the Na+/H+ antiporter in the absence of amiloride.