Stimulation of B-cells via the membrane immunoglobulin receptor or with phorbol myristate 13-acetate induces tyrosine phosphorylation and activation of a 42-kDa microtubule-associated protein-2 kinase.

Engagement of membrane IgM on a number of human and murine B-cell lines induced activation of a Mn(2+)-preferring serine/threonine kinase that phosphorylated microtubule-associated protein-2 (MAP-2) in vitro. B-cell MAP-2 kinase (MAP-2K) activity could be fractionated into two peaks by sequential DEAE and hydrophobic chromatography. Although peak I included two tyrosine phosphoproteins of molecular mass 36 and 38 kDa, peak II showed a single 42-kDa tyrosine phosphoprotein (pp42). Since all kinase activity could be removed from peak II material over an antiphosphotyrosine immune affinity column, it suggests that pp42 is identical with lymphoid MAP-2K. Although peak I activity showed a similarity to peak II with regard to its preference for Mn2+, sensitivity to phosphatase exposure, and resistance to a range of common serine kinase inhibitors, it is not clear whether these activities are related. MAP-2 kinase activity could also be induced by treatment with the phorbol ester, phorbol myristate 13-acetate, suggesting that protein kinase C may also be involved with MAP-2K regulation. Although MAP-2K activity reached a peak response within minutes of receptor ligation, there were differences in the rates of dephosphorylation of pp42 and decline of MAP-2K activity in different B-cell lines. The tyrosine phosphatase inhibitor, vanadate, transformed a rapidly reversible MAP-2K response in BAL 17.2 cells into a sustained state of activation that resembled the kinetics of activation in WEHI-231 cells. The latter finding implies involvement of a tyrosine phosphatase, which opposes the effect of an inducing tyrosine kinase.     

Structure determination of the Antp (C39----S) homeodomain from nuclear magnetic resonance data in solution using a novel strategy for the structure calculation with the programs DIANA, CALIBA, HABAS and GLOMSA.

The structure of a mutant Antennapedia homeodomain, Antp(C39----S), from Drosophila melanogaster was determined using a set of new programs introduced in the accompanying paper. An input dataset of 957 distance constraints and 171 dihedral angle constraints was collected using two-dimensional n.m.r. experiments with the 15N-labeled protein. The resulting high quality structure for Antp(C39----S), with an average root-mean-square deviation of 0.53 A between the backbone atoms of residues 7 to 59 in 20 energy-refined distance geometry structures and the mean structure, is nearly identical to the previously reported structure of the wild-type Antp homeodomain. The only significant difference is in the connection between helices III and IV, which was found to be less kinked than was indicated by the structure determination for Antp. The main emphasis of the presentation in this paper is on a detailed account of the practical use of a novel strategy for the computation of nuclear magnetic resonance structures of proteins with the combined use of the programs DIANA, CALIBA, HABAS and GLOMSA.     

Investigation of means to improve rates of fertilization in in vitro matured/in vitro fertilized bovine oocytes

Experiments were conducted with 5,979 oocytes to determine whether detaching some of the cumulus cells from oocytes either before or after maturation would improve the fertilization rate and proportion of oocytes that developed to expanded blastocysts. Oocytes were aspirated from ovaries of slaughtered cows and matured, fertilized and cultured in vitro. Pipetting immature oocytes before maturation to detach some of the cumulus, with all cumulus cells left in the maturation wells, significantly increased fertilization rates, especially of oocytes that initially had a full cumulus investment. In further experiments, pipetting oocytes either before or after maturation to detach most of the cumulus, or treating with hyaluronidase after maturation to disperse the cumulus, significantly increased fertilization rates and proportions of oocytes developing to expanded blastocysts.     

Mhc-DRB diversity of the chimpanzee (Pan troglodytes)

Fifty-four chimpanzee Patr-DRB and five human HLA-DRB second exons were cloned and sequenced from thirty-five chimpanzees and four B-cell lines and compared with known Mhc-DRB sequences of these two species. Equivalents of the HLA-DRB1 ^* 02,-DRB1 ^* 03, -DRB1 ^* 07 allelic lineages and the HLA-DRB3,-DRB4, -DRB5, -DRB6, and -DRB7 loci were all found in the chimpanzee. In addition, two chimpanzee Patr-DRB lineages (Patr-DRBX and -DRBY) were found for which no human counterparts have been described. None of the Patr-DRB sequences is identical to known HLA-DRB sequences. The Patr-DRB1 ^* 0702 and HLA-DRB1 ^* 0701 alleles are the most similar sequences in a comparison between the two species and differ by only two nucleotides out of 246 sequences. Equivalents of the HLA-DRB1 ^* 01,-DRB1 ^* 04, and -DRB1 ^* 09 alleles were not found in our sample of chimpanzees. A per locus comparison of the number of Patr-DRB alleles with the HLA-DRB alleles shows that the Patr-DRB3, -DRB4, -DRB5, and -DRB6 locus are, thus far, more polymorphic than ther human homologs. The polymorphism of the Patr-DRB1 locus seems to be less extensive than that reported for the HLA-DRB1 locus. Nevertheless, the Patr-DRB1 locus seems to be the most polymorphic of the Patr-DRB loci. Phylogenetic analyses indicate that the HLA-DRB1 ^* 09 allele may have originated from a recombination between a Mhc-DRB5 allele and the DRB1 allele of a Mhc-DR7 haplotype. Although recombination seems to increase the diversity of the Patr-DRB alleles, its contribution to the generation of Patr-DRB variation is probably low. Hence, most Patr-DRB diversity presumably accumulated via recurrent point mutations. Finally, two distinct PAtr-DRB haplotypes are deduced, one of which (the chimpanzee equivalent of the HLA-Dr7 haplotype) is probably older than 6–8 million years.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide database and have been assigned the accession numbers Mg6074-Mg6132. Correspondence to: M. Kenter.     

Determination of the three-dimensional structure of the Antennapedia homeodomain from Drosophila in solution by 1H nuclear magnetic resonance spectroscopy

The determination of the three-dimensional structure of the Antennapedia homeodomain from Drosophila in solution is described. The techniques used are 1H nuclear magnetic resonance spectroscopy for the data collection, and calculation of the protein structure with the program DISMAN followed by restrained energy minimization with a modified version of the program AMBER. A group of 19 conformers characterizes a well-defined structure for residues 7 to 59, with an average root-mean-square distance from the backbone atoms of 0.6 A relative to the mean of the 19 structures. The structure contains a helix from residues 10 to 21, a helix-turn-helix motif from residues 28 to 52, which is similar to those reported for several prokaryotic repressor proteins, and a somewhat flexible fourth helix from residues 53 to 59, which essentially forms an extension of the presumed recognition helix, residues 42 to 52. The helices enclose a structurally well-defined molecular core of hydrophobic amino acid side-chains.     

Genetically encoded amino acids with <Emphasis Type="Italic">tert</Emphasis>-butyl and trimethylsilyl groups for site-selective studies of proteins by NMR spectroscopy

The amino acids 4-(tert-butyl)phenylalanine (Tbf) and 4-(trimethylsilyl)phenylalanine (TMSf), as well as a partially deuterated version of Tbf (dTbf), were chemically synthesized and site-specifically incorporated into different proteins, using an amber stop codon, suppressor tRNA and the broadband aminoacyl-tRNA synthetase originally evolved for the incorporation of p-cyano-phenylalanine. The ¹H-NMR signals of the tert-butyl and TMS groups were compared to the ¹H-NMR signal of tert-butyltyrosine (Tby) in protein systems with molecular weights ranging from 8 to 54 kDa. The ¹H-NMR resonance of the TMS group appeared near 0 ppm in a spectral region with few protein resonances, facilitating the observation of signal changes in response to ligand binding. In all proteins, the R ₂ relaxation rate of the tert-butyl group of Tbf was only little greater than that of Tby (less than two-fold). Deuteration of the phenyl ring of Tbf made only a relatively small difference. The effective T ₂ relaxation time of the TMS signal was longer than 140 ms even in the 54 kDa system.     

Industrial wastewaters harbor a unique diversity of bacterial communities revealed by high-throughput amplicon analysis

Industrial wastewater effluents present a major source of water pollution, and can potentially alter the microbial ecological landscape. While there are numerous reports on the microbial quality of domestic municipal effluents and their perceived environmental effects, there are limited reports devoted to the study of bacterial diversity of effluents from individual industries before they are mixed up with other sources. This study analyzed both the physicochemical parameters and bacterial community structures of different industrial wastewaters using Illumina high-throughput sequencing platform. Industrial wastewater with temperature ranging from 18.9 to 21.5 °C, and total dissolved solid (TDS) levels at up to 4611 mg/L, appeared to be predominated by Proteobacteria (44.44–75.86%) with the exception of the Capegate sample where Actinobacteria (39.66%) were the highest. Sulfur levels were significantly higher (p?<?0.05) in Dixon wastewater constituting higher populations of sulfur reducing bacteria (SRB) compared to the other sites. Diversity index (Shannon-H index) and richness estimator (Chao1 index) ranged from 974 (Capegate) to 4552 (Dixon) and 6.04 (Dixon) to 4.15 (CWI), respectively. Multivariate analysis results highlighted that the bacterial communities were strongly shaped by physicochemical variables. The top 10 operational taxonomic units (OTUs) of each industrial sample had the potential to play important roles in the bioremediation and biodegradation of pollutants. Dominant OTUs belonging to the phyla Planctomyces from the Chemreem sample could not be classified to any genera and are likely to represent novel species.     

The amino-acid sequence of bovine glutathione peroxidase

The amino-acid sequence of the seleno-enzyme glutathione peroxidase from bovine erythrocytes was completely determined. Fragmentation of the carboxymethylated protein comprised cleavages with trypsin, with endoproteinase Lys-C, and with cyanogen bromide in 70% formic acid. The resulting peptides were separated by reversed-phase high-performance chromatography or by gel filtration. For sequence determination automated solid or liquid phase techniques of Edman degradation were used. The proper alignment of fragments was experimentally proven in all but one instance. In this case, consistent indirect evidence was provided. The monomer of glutathione peroxidase was shown to consist of 198 amino acids representing a molecular mass ob about 21 900 Da. The active site selenocysteine was localized at position 45. In addition, four cysteine residues were found at positions 74, 91, 111, and 152. The N-terminal part of the sequence obtained revealed a pronounced homology with a partial sequence of the rat liver enzyme. Moreover, tentative sequence data predicted from X-ray crystallographic analysis of bovine glutathione peroxidase were found to agree in about 80% of the residues with the sequence presented. Differences between the predicted and the experimentally determined sequence are discussed.     

Measurement of dissociation constants of high-molecular weight protein–protein complexes by transferred <Superscript>15</Superscript>N-relaxation

The use of ¹⁵N-relaxation data for determination of the dissociation constant of a protein–protein complex is proposed for the situation where a ¹⁵N-labeled protein is bound to an unlabeled protein of high molecular weight, and the chemical exchange between bound and free protein is fast on the NMR time scale. The approach is shown to be suitable for estimating dissociation constants in the micromolar to millimolar range, using protein solutions at relatively low concentration. An example is shown for the interaction between two subunits from the Escherichia coli DNA polymerase III complex, involving a ¹⁵N-labeled fragment of the C-terminal domain of the τ subunit (15 kDa) and the unlabeled α subunit (130 kDa).