Enhanced leaching of product at elevated temperatures: alpha-amylase produced by Bacillus licheniformis M27 in solid state fermentation system

Leaching of alpha-amylase from bacterial bran, produced by Bacillus licheniformis M27 in solid state fermentation was about 2.2 times higher at 50°C as compared to that at 30°C. Further increase by about 19% in leaching efficiency was observed when contact time was extended from 60 to 120 min. The overall increase of 2.54 times under these strategies is of economic importance and no information was available earlier on enhanced leaching of enzyme from fermented bran at elevated temperatures.     

Mapping of a region of dengue virus type-2 glycoprotein required for binding by a neutralizing monoclonal antibody.

Envelope glycoprotein E of flaviviruses is exposed at the surface of the virion, and is responsible for eliciting a neutralizing antibody (Ab) response, as well as protective immunity in the host. In this report, we describe a method for the fine mapping of a linear sequence of the E protein of dengue virus type-2 (DEN-2), recognized by a type-specific and neutralizing monoclonal Ab (mAb), 3H5. First, an Escherichia coli expression vector containing a heat-inducible lambda pL promoter was used to synthesize several truncated, and near-full length E polypeptides. Reactivities of these polypeptides with polyclonal mouse hyperimmune sera, as well as the 3H5 mAb revealed the location of the 3H5-binding site to be within a region of 166 amino acids (aa) between aa 255 and 422. For fine mapping, a series of targeted deletions were made inframe within this region using the polymerase chain reaction (PCR). The hydrophilicity pattern of this region was used as a guide to systematically delete the regions encoding the various groups of surface aa residues within the context of a near-full-length E polypeptide by using PCR. The 3H5-binding site was thus precisely mapped to a region encoding 12 aa (between aa 386 and 397). A synthetic peptide containing this sequence was able to bind to the 3H5 mAb specifically, as shown by enzyme-linked immunosorbent assay. In addition, we show that rabbit Abs raised against the synthetic peptide of 12 aa were able to bind to the authentic E protein, and to neutralize DEN-2 virus in a plaque reduction assay.     

Exencephaly and axial skeletal dysmorphogenesis induced by maternal exposure to cadmium in the mouse

To investigate the axial skeletal dysmorphogenesis associated with exencephaly and facial abnormalities, two doses of cadmium chloride (4 mg/kg and 6 mg/kg) were administered subcutaneously to MF1 mice on day 7 of gestation (sperm-positive day = day 0). Fetuses were collected on day 18. Gross examination revealed a very high incidence of cranioschisis aperta with exencephaly, maxillary and mandibular hypoplasia, low-set microtia, edema, and growth retardation of fetuses in both treatment groups. Alizarin red S-stained and cleared skeletal preparations of these embryos revealed hypoplasia of the premaxilla, maxilla, nasal bone, zygoma, and mandible of the facial skeleton. The orbital plate represented the frontal bone. The vault of the skull was conspicuously absent. In cranioschisis, the exoccipitals had splayed and fused with the atlas. The basicranial bones were hypoplastic and crowded, thus reducing the cranial cavity. The vertebral bodies were more affected than the arches. Hemivertebrae and longitudinal fusion of centra and arches were also noted. The ribs were usually rudimentary. Agenesis, fusion, and forking of ribs were frequently observed. The sternebrae were rudimentary, bipartite, or longitudinally fused. These data clearly establish the association between neural tube and axial mesodermal abnormalities and emphasize the interdependence of the neurectoderm and mesoderm in normal morphogenesis.     

Non-standard amino acids in peptide design and protein engineering

The introduction of non-coded amino acids with well defined stereochemical and functional properties will greatly enhance the scope of protein design and engineering. The present state of methodologies for incorporation of non-coded residues into proteins is examined. The prospects for conformationally constrained amino acid residues are evaluated in the light of peptide structural studies. Templates for secondary-structure nucleation and recent experiences in the incorporation of novel residues into proteins are considered.