A Comparative Evaluation and Utilization of Entrapped Bacillus sp. (Gen Bank MN 243657) and Pseudomonas aeruginosa SU-1 in Poultry Waste Degradation

Bacillus sp. (Genbank MN243657) was obtained from the Center for Bioscience and Nanoscience Research, and P. aeruginosa SU-1 were used in this study. Initially, biomass was optimized for the incubation period, pH, and temperature, following biomass optimization with RSM. The obtained organisms were able to produce keratinase, which was found to be a 64 KDa protein and 56KDa. Now the biomass produced using the optimized condition was immobilized and checked for its ability to degrade keratinase, and efficiency was compared. Immobilized P. aeruginosa SU-1 was degrading keratin and feather better than Bacillus sp. (Genbank MN243657). It was found to reduce featherweight by 57.3% and 41%. Hence it was found that both the immobilized bacterium can be used for poultry waste treatment, and P. aeruginosa SU-1 was more efficient than Bacillus sp. (Genbank MN243657).

Optimization of Biomass of Keratinase Producing Bacillus sp CBNRBT2 to Utilize in Whole-Cell Immobilization for Feather Degradation

Bacillus sp CBNRBT2, obtained from Centre for Bioscience and Nanoscience Research, was used in this study. Initially, biomass was optimized for the incubation period, pH, and temperature, following biomass optimization with RSM. The obtained organism was able to produce keratinase, which was found to be a 64 KDa protein. Now the biomass produced using the optimized condition was immobilized and checked for its ability to degrade keratinase. Efficiency was compared with the whole organism. Immobilized organisms were degrading keratin and feather better. It was found to reduce featherweight by 41 %. Thus, this immobilized bacterium can be used for poultry waste treatment.

Alternative splicing of the Menkes copper Atpase (Atp7a) transcript in the rat intestinal epithelium

The intestinal Menkes copper Atpase ( Atp7a) gene is strongly induced by iron deficiency in the rat intestine. We sought to develop an in vitro model to understand the mechanism of this induction by performing molecular studies in native rat intestine and in intestinal epithelial (IEC-6) cells. IEC-6 cells express Atp7a, and induction was noted with iron deprivation. 5′ Rapid amplification of cDNA ends and PCR experiments revealed three splice variants in rat intestine and IEC-6 cells; all variants were strongly induced during iron deprivation (five- to sevenfold). The splice variants presumably encode proteins that would either contain the extreme NH2 terminus of the protein (containing copper binding domain 1) or not. We thus hypothesized that more than one version of Atp7a protein exists. Antibodies against this NH2-terminal region of the protein were developed (named N-term) and used along with previously reported antibodies (against more COOH-terminal regions, termed 54–10) to perform immunoblotting and immunolocalization studies. Results with the 54–10 antiserum revealed an Atp7a protein variant of ∼190 kDa that localized to the trans-Golgi network of IEC-6 cells and trafficked to the plasma membrane with copper loading. Using the N-term antiserum, however, we noted protein of ∼97 and 64 kDa. The 97-kDa protein was cytosolic and nuclear, whereas the 64-kDa protein was nuclear specific. Immunolocalization analyses with the N-term antiserum showed strong staining of nuclei in IEC-6 and Caco-2 cells and in rat intestine. We conclude that novel Atp7a protein variants may exist in rat and human intestinal epithelial cells, with different intracellular locations and potentially distinct physiological functions.

Genes involved in the methyl tert-butyl ether (MTBE) metabolic pathway of Mycobacterium austroafricanum IFP 2012

Methyl tert-butyl ether (MTBE) is a persistent pollutant of surface and groundwater, and the reasons for its low biodegradability are poorly documented. Using one of the rare bacterial strains able to grow in the presence of MTBE, Mycobacterium austroafricanum IFP 2012, the protein profiles of crude extracts after growth in the presence of MTBE and glucose were compared by SDS-PAGE. Ten proteins with molecular masses of 67, 64, 63, 55, 50, 27, 24, 17, 14 and 11 kDa were induced after growth in the presence of MTBE. Partial amino acid sequences of N-terminal and internal peptide fragments of the 64 kDa protein were used to design degenerate oligonucleotide primers to amplify total DNA by PCR, yielding a DNA fragment that was used as a probe for cloning. A two-step cloning procedure was performed to obtain a 10 327 bp genomic DNA fragment containing seven ORFs, including a putative regulator, mpdR, and four genes, mpdC, orf1, mpdB and orf2, in the same cluster. The MpdB protein (64 kDa) was related to a flavoprotein of the glucose–methanol–choline oxidoreductase family, and the MpdC protein (55 kDa) showed a high similarity with NAD(P) aldehyde dehydrogenases. Heterologous expression of these gene products was performed in Mycobacterium smegmatis mc2 155. The recombinant strain was able to degrade an intermediate of MTBE biodegradation, 2-methyl 1,2-propanediol, to hydroxyisobutyric acid. This is believed to be the first report of the cloning and characterization of a cluster of genes specifically involved in the MTBE biodegradation pathway of M. austroafricanum IFP 2012.

Identification, Subviral Localization, and Functional Characterization of the Pseudorabies Virus UL17 Protein

ABSTRACT Homologs of the UL17 gene of the alphaherpesvirus herpes simplex virus 1 (HSV-1) are conserved in all three subfamilies of herpesviruses. However, only the HSV-1 protein has so far been characterized in any detail. To analyze UL17 of pseudorabies virus (PrV) the complete 597-amino-acid protein was expressed in Escherichia coli and used for rabbit immunization. The antiserum recognized a 64-kDa protein in PrV-infected cell lysates and purified virions, identifying PrV UL17 as a structural virion component. In indirect immunofluorescence analyses of PrV-infected cells the protein was predominantly found in the nucleus. In electron microscopic studies after immunogold labeling of negatively stained purified virion preparations, UL17-specific label was detected on single, mostly damaged capsids, whereas complete virions and the majority of capsids were free of label. In ultrathin sections of infected cells, label was primarily found dispersed around scaffold-containing B-capsids, whereas on DNA-filled C-capsids it was located in the center. Empty intranuclear A-capsids were free of label, as were extracellular capsid-less L-particles. Functional characterization of PrV-ΔUL17F, a deletion mutant lacking codons 23 to 444, demonstrated that cleavage of viral DNA into unit-length genomes was inhibited in the absence of UL17. In electron microscopic analyses of PrV-ΔUL17F-infected RK13 cells, DNA-containing capsids were not detected, while numerous capsidless L-particles were observed. In summary, our data indicate that the PrV UL17 protein is an internal nucleocapsid protein necessary for DNA cleavage and packaging but suggest that the protein is not a prominent part of the tegument.

The 64-Kilodalton Capsid Protein Homolog of Beet Yellows Virus Is Required for Assembly of Virion Tails

ABSTRACT The filamentous virion of the closterovirus Beet yellows virus (BYV) consists of a long body formed by the major capsid protein (CP) and a short tail composed of the minor capsid protein (CPm) and the virus-encoded Hsp70 homolog. By using nano-liquid chromatography-tandem mass spectrometry and biochemical analyses, we show here that the BYV 64-kDa protein (p64) is the fourth integral component of BYV virions. The N-terminal domain of p64 is exposed at the virion surface and is accessible to antibodies and mild trypsin digestion. In contrast, the C-terminal domain is embedded in the virion and is inaccessible to antibodies or trypsin. The C-terminal domain of p64 is shown to be homologous to CP and CPm. Mutation of the signature motifs of capsid proteins of filamentous RNA viruses in p64 results in the formation of tailless virions, which are unable to move from cell to cell. These results reveal the dual function of p64 in tail assembly and BYV motility and support the concept of the virion tail as a specialized device for BYV cell-to-cell movement.

Proteolytic processing of a human astrovirus nonstructural protein

To analyse the activity of the putative 3C-like serine protease encoded in open reading frame (ORF)-1a of human astrovirus serotype 1 (HAstV-1), ORF-1a was transcribed and translated in vitro. Translation products, identified by immunoprecipitation with specific antibodies against recombinant C-terminal ORF-1a fragments, include the full-length 101 kDa (p101) protein and a 38 kDa band (p38). In addition, a 64 kDa protein (p64) was immunoprecipitated by an anti-FLAG antibody when a FLAG epitope was inserted at the N terminus of the ORF-1a product. Mutation of the amino acids predicted to form the catalytic triad of the HAstV-1 3C-like serine protease (Ser-551, Asp-489, His-461) resulted in undetectable levels of p38, supporting the involvement of the HAstV-1 3C-like serine protease and the importance of these amino acids in the processing of p101 into p38 and p64. N-terminal deletions of up to 420 aa of p101 that did not involve the predicted 3C-like serine protease motif did not alter levels of p38 compared to wild-type. C-terminal deletion analysis localized p38 to the C terminus of ORF-1a. Mutation of the P1 residue of the predicted cleavage site, which is conserved among known human and sheep astrovirus sequences, resulted in no detectable p38, supporting cleavage at the Gln-567↓Thr-568 dipeptide. These results suggest that p101 is cleaved into an N-terminal p64 fragment and a C-terminal p38 product at Gln-567↓Thr-568 in a process that is dependent on the viral 3C-like serine protease.

Upregulated expression of human membrane type-5 matrix metalloproteinase in kidneys from diabetic patients

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade the extracellular matrix (ECM). The membrane-type matrix metalloproteinases (MT-MMPs) are a new family of MMPs that differ from other MMPs in that they have a transmembrane domain that anchors them to the cell surface. MT-MMPs have been shown to function as receptors and activators for other MMPs and to localize extracellular matrix proteolysis at the pericellular region. Here we report on mRNA and protein expression of the fifth human MT-MMP (MT5-MMP), a 64-kDa protein that is capable of converting pro-MMP-2 to its active form, in human kidney as well as its upregulation in diabetes. We also demonstrate upregulation of the active form of MMP-2 in kidney samples from patients with diabetes. Through immunohistochemistry, MT5-MMP expression was localized to the epithelial cells of the proximal and distal tubules, the collecting duct, and the loop of Henle. Furthermore, the tubular epithelial cells that expressed MT5-MMP were associated with tubular atrophy. Because renal tubular atrophy is a significant factor in the pathogenesis of diabetic nephropathy and renal failure and the molecular mechanisms regulating this process remain unknown, it is hypothesized that the elevated expression of MT5-MMP contributes to the activation of pro-MMP-2, which participates in the remodeling of the proximal and distal tubules as well as in the collecting duct. These results provide the first evidence of the expression of a MT-MMP in diabetes and suggest a novel role for MT5-MMP in the pathogenesis of renal tubular atrophy and end-stage renal disease.

Posttranslational Phosphorylation and Ubiquitination of the Saccharomyces cerevisiae Poly(A) Polymerase at the S/G2 Stage of the Cell Cycle

ABSTRACT The poly(A) polymerase of the budding yeast Saccharomyces cerevisiae (Pap1) is a 64-kDa protein essential for the maturation of mRNA. We have found that a modified Pap1 of 90 kDa transiently appears in cells after release from α-factor-induced G1 arrest or from a hydroxyurea-induced S-phase arrest. While a small amount of modification occurs in hydroxyurea-arrested cells, fluorescence-activated cell sorting analysis and microscopic examination of bud formation indicate that the majority of modified enzyme is found at late S/G2 and disappears by the time cells have reached M phase. The reduction of the 90-kDa product upon phosphatase treatment indicates that the altered mobility is due to phosphorylation. A preparation containing primarily the phosphorylated Pap1 has no poly(A) addition activity, but this activity is restored by phosphatase treatment. A portion of Pap1 is also polyubiquitinated concurrent with phosphorylation. However, the bulk of the 64-kDa Pap1 is a stable protein with a half-life of 14 h. The timing, nature, and extent of Pap1 modification in comparison to the mitotic phosphorylation of mammalian poly(A) polymerase suggest an intriguing difference in the cell cycle regulation of this enzyme in yeast and mammalian systems.

Regulation of Closterovirus Gene Expression Examined by Insertion of a Self-Processing Reporter and by Northern Hybridization

ABSTRACT A reporter open reading frame (ORF) coding for a fusion of bacterial β-glucuronidase (GUS) with a proteinase domain (Pro) derived from tobacco etch potyvirus was utilized for tagging individual genes of beet yellows closterovirus (BYV). Insertion of this reporter ORF between the first and second codons of the BYV ORFs encoding the HSP70 homolog (HSP70h), a major capsid protein (CP), and a 20-kDa protein (p20) resulted in the expression of the processed GUS-Pro reporter from corresponding subgenomic RNAs. The high sensitivity of GUS assays permitted temporal analysis of reporter accumulation, revealing early expression from the HSP70h promoter, followed by the CP promoter and later the p20 promoter. The kinetics of transcription of the remaining BYV genes encoding a 64-kDa protein (p64), a minor capsid protein (CPm), and a 21-kDa protein (p21) were examined via Northern blot analysis. Taken together, the data indicated that the temporal regulation of BYV gene expression includes early (HSP70h, CPm, CP, and p21 promoters) and late (p64 and p20 promoters) phases. It was also demonstrated that the deletion of six viral genes that are nonessential for RNA amplification resulted in a dramatic increase in the level of transcription from one of the two remaining subgenomic promoters. Comparison with other positive-strand RNA viruses producing multiple subgenomic RNAs showed the uniqueness of the pattern of closterovirus transcriptional regulation.