The role of Asn14 in the stability and conformation of the reactive-site loop of winged bean chymotrypsin inhibitor: crystal structures of two point mutants Asn14-->Lys and Asn14-->Asp.

A double-headed chymotrypsin inhibitor, WCI, from winged bean seeds was cloned for structural and biochemical studies. The inhibitor was subjected to two point mutations at a conserved position, Asn14. This residue, known to have a pivotal role in stabilizing the first reactive-site loop (Gln63-Phe68) of the inhibitor, is highly conserved in the sequences of the other members of Kunitz (STI) family as well as in the sequences of Kazal family of serine protease inhibitors. The mutants, N14K and N14D, were subjected to biochemical assay and their characteristics were compared with those of the recombinant inhibitor (rWCI). Crystallographic studies of the recombinant and the mutant proteins are discussed. These studies were primarily aimed at understanding the importance of the protein scaffolding towards the conformational rigidity of the reactive-site loop. Our analysis reveals that, as the Lys14 side chain takes an unusual fold in N14K and the Asp14 side chain in N14D interacts with the loop residues by water-mediated hydrogen bonds, the canonical conformation of the loop has remained effectively intact in both the mutant structures. However, minor alterations such as a 2-fold increase in the inhibitory affinity towards the cognate enzyme were observed.     

Conserved water-mediated H-bonding dynamics of catalytic Asn 175 in plant thiol protease

The role of invariant water molecules in the activity of plant cysteine protease is ubiquitous in nature. On analysing the 11 different Protein DataBank (PDB) structures of plant thiol proteases, the two invariant water molecules W1 and W2 (W220 and W222 in the template 1PPN structure) were observed to form H-bonds with the O_b atom of Asn 175. Extensive energy minimization and molecular dynamics simulation studies up to 2 ns on all the PDB and solvated structures clearly revealed the involvement of the H-bonding association of the two water molecules in fixing the orientation of the asparagine residue of the catalytic triad. From this study, it is suggested that H-bonding of the water molecule at the W1 invariant site better stabilizes the Asn residue at the active site of the catalytic triad.     

A yeast's eye view of mammalian reproduction: cross-species gene co-expression in meiotic prophase

Background  

Meiotic prophase is a critical stage in sexual reproduction. Aberrant chromosome recombination during this stage is a leading cause of human miscarriages and birth defects. However, due to the experimental intractability of mammalian gonads, only a very limited number of meiotic genes have been characterized. Here we aim to identify novel meiotic genes important in human reproduction through computational mining of cross-species and cross-sex time-series expression data from budding yeast, mouse postnatal testis, mouse embryonic ovary, and human fetal ovary.     

IMMUNOCYTOCHEMICAL LOCALIZATION OF δ-ZEIN IN THE PROTEIN BODIES OF MAIZE ENDOSPERM CELLS

The δ-zein, a minor component of the maize prolamin, shows extensive immunological cross-reactivity with α- and β-zeins. The adsorption of an anti-δ-zein serum sequentially with cross-reacting antigens revealed that only about 18% of the reactivity of the antiserum was directed to epitopes unique to δ-zein. The localization of the various zein classes within the protein bodies of endosperm cells is important to understanding the synthesis, sequestering, and utilization of these storage proteins. Sections of 28 days after pollination (DAP) isolated protein bodies and 18 and 40 DAP whole endosperms were reacted sequentially with whole anti-δ-zein serum and gold-conjugated protein A. The results showed intense gold labeling in the core (inside the peripheral zone) and weak labeling in the periphery of the sections. This localization was not definitive in view of the above-mentioned cross-reactivities. To obtain an unequivocal localization, the whole antiserum was adsorbed with α-, β-, and γ-zeins and rendered monospecific for δ-zein. Immunostaining of protein body sections with monospecific antiserum showed that gold label was exclusively in the core region of the protein body and appeared to be in discrete lines and zones especially in 18 DAP protein bodies. The data from localizations using the monospecific antiserum indicated that δ-zein occurs throughout the core region of the protein body, probably interspersed with α- and β-zeins. The location of δ-zein is consistent with that predicted from its order of degradation during seed germination.     

Primer-dependent synthesis of covalently linked dimeric RNA molecules by poliovirus replicase.

Poliovirus-specific RNA-dependent RNA polymerase (replicase, 3Dpol) was purified from HeLa cells infected with poliovirus. The purified enzyme preparation contained two proteins of apparent molecular weights 63,000 and 35,000. The 63,000-Mr polypeptide was virus-specific RNA-dependent RNA polymerase, and the 35,000-Mr polypeptide was of host origin. Both polypeptides copurified through five column chromatographic steps. The purified enzyme preparation catalyzed synthesis of covalently linked dimeric RNA products from a poliovirion RNA template. This reaction was absolutely dependent on added oligo(U) primer, and the dimeric product appeared to be made of both plus- and minus-strand RNA molecules. Experiments with 5' [32P]oligo(U) primer and all four unlabeled nucleotides suggest that the viral replicase elongates the primer, copying the poliovirion RNA template (plus strand), and the newly synthesized minus strand snaps back on itself to generate a template-primer structure which is elongated by the replicase to form covalently linked dimeric RNA molecules. Kinetic studies showed that a partially purified preparation of poliovirus replicase contains a nuclease which can cleave the covalently linked dimeric RNA molecules, generating template-length RNA products.     

An immunodominant site of γ-zein1 is in the region of tandem hexapeptide repeats

The immunochemical data from studies with polyclonal antisera to -zein₁, the 27 kD component of the maize prolamin, indicated that the region containing 8 tandem repeats of the sequence PPPVHL is an immunodominant site. In one case, the entire antibody repertoire of an antiserum recognized epitope(s) within this region. Three 17-mer oligopeptides corresponding to the predicted antigenic epitopes of -zein₁ were synthesized and reacted with three different anti--zein₁ sera in order to map antigenic sites in the intact protein. These antisera yielded positive reactions with a 17-mer peptide (peptide 37), which was not in a hydrophilic maximum but derived from the repeat region. The same antisera gave little or no reaction with other peptides (peptides 38 and 39), both of which were in a hydrophilic maximum. In addition, an antiserum to peptide 37 reacted strongly with both the homologous antigen and the intact -zein₁. Peptide 37 also blocked the binding of antisera to -zein₁ in competition assays. Subsequently, the shorter 6-mer (peptide 82) and 12-mer (peptide 80) versions of peptide 37 were synthesized, and both reacted with anti-peptide 37 serum and also with each of the three anti--zein₁ sera. In these reactions and in competition assays, the reactivity and the blocking ability increased in proportion to the length of the peptide. Based on these data, it was concluded that the repeat region of -zein₁ is the site of one or more continuous immunodominant epitopes. The data also suggest that the repeat region is exposed on the surface of the folded protein and probably occur as a mobile, random coil.     

Anti-GM3-lactam monoclonal antibodies of the IgG type recognize natural GM3-ganglioside lactone but not GM3-ganglioside

Immunization of mice with a synthetic GM₃-lactam-BSA (bovine serum albumin) conjugate (designed to emulate the corresponding natural GM₃-lactone conjugate), followed by fusion of splenocytes with myeloma cells, gave rise to more than 300 monoclonal hybridomas producing antibodies to GM₃-lactam-BSA, which did not react with Glc-BSA and BSA. Eight antibody clones were randomly chosen from the positive 300 hybridomas. The eight clones, all belonging to the IgG class, were unreactive against GM₃-ganglioside, whereas two antibodies (P5-1 and P5-3, both IgG₁, ) reacted with GM₃-ganglioside lactone. Binding of these two antibodies to the GM₃-lactam-BSA conjugate was inhibited by soluble glycosides of GM₂-, GM₃-, and GM₄-lactam and by GM₃- and GM₄-lactam, respectively, but not by Gb₃ or asialo-GM₁ and GM₂-saccharides. A third antibody (P3; IgG_2b, ) was inhibited by GM₂-, GM₃-, and GM₄-lactam, but did not recognize GM₃-ganglioside lactone.     

Herpes simplex virus type 1-specific cytotoxic T lymphocytes recognize immediate-early protein ICP27.

The identity of herpes simplex virus type 1 (HSV-1) antigens that serve as targets for cytotoxic T lymphocytes (CTL) and their ability to induce protective immunity remain uncertain. In this article, we report the identification of the immediate-early protein ICP27 as a CTL antigen in H-2d mice but not in H-2k or H-2b mice. Calculation of the frequencies of H-2d-restricted virus-specific CTL demonstrated that approximately one-fourth of the total HSV-1-specific response was directed against ICP27. To define the location of this CTL epitope, four truncated derivatives of the ICP27 gene which place the epitope in a 217-amino-acid region (amino acids 189 to 406) near the central portion of the protein were constructed. Mice immunized with ICP27 were able both to induce HSV-1-specific CTL and to survive a lethal intraperitoneal challenge with virulent HSV-1. However, neither appreciable antibody nor delayed-type hypersensitivity responses were induced in immunized mice, and they were also unable to clear a local epithelial virus challenge. It appears that ICP27, although capable of inducing several aspects of the immune response, is by itself unable to provide complete immunity.     

Response of microbial populations to environmental disturbance

Taxonomic and genetic diversities of microbial communities disturbed by chemical pollutants were lower than in undisturbed reference communities. The dominant populations within the disturbed communities had enhanced physiological tolerances and substrate utilization capabilities, indicating that generalized physiological versatility is an adaptive characteristic of populations that successfully compete within disturbed communities.