Chimeric porcine circoviruses (PCV) containing amino acid epitope tags in the C terminus of the capsid gene are infectious and elicit both anti-epitope tag antibodies and anti-PCV type 2 neutralizing antibodies in pigs

A chimeric porcine circovirus (PCV1-2) with the capsid gene of pathogenic PCV2 cloned into the genomic backbone of nonpathogenic PCV1 is attenuated in pigs but elicits protective immunity against PCV2. In this study, short epitope tags were inserted into the C terminus of the capsid protein of the chimeric PCV1-2 vaccine virus, resulting in a tractable marker virus that is infectious both in vitro and in vivo. Pigs experimentally infected with the epitope-tagged PCV1-2 vaccine viruses produced tag-specific antibodies, as well as anti-PCV2 neutralizing antibodies, indicating that the epitope-tagged viruses could potentially serve as a positive-marker modified live-attenuated vaccine.     

QSAR study on antibacterial activity of sulphonamides and derived Mannich bases

The paper describes synthesis and comparative study on antibacterial activities of sulphonamides and Mannich bases derived from them. The compounds were screened for their antibacterial activity against various gram-positive and gram-negative bacteria and were analyzed statistically. The results have shown that the compounds are quiet active against pathogens under study and were nontoxic.     

Anti-Candida activity of two-peptide bacteriocins,plantaricins (Pln E/F and J/K) and their mode of action

The fungicidal effect of plantaricin peptides PlnE, -F, -J, and -K was studied against pathogenic yeast, Candida albicans. Dose-dependent inhibitory effect was observed by drop in cell viability, further demonstrated by measuring the fluorescence intensity of cells by exposing them to 5, (6)-carboxyfluorescein diacetate (CFDA). Live/dead staining by CFDA and propidium iodide (PI) also suggested the viability loss response. Also, the PI uptake by treated cells suggested the membrane damage. PlnJ was identified as most inhibitory among different plantaricins tested. PlnJ not only induced membrane potential dissipation but also resulted in the release of K⁺. In addition, enhanced production of reactive oxygen species (ROS) was also observed by fluorometry using 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA). Dual staining with Hoechst stain and PI depicted both early apoptotic and necrotic cells in the treated population. Terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) positive staining further confirmed the ROS-mediated apoptosis. Scanning electron microscopy and transmission electron microscopy also revealed characteristic apoptotic features such as appearance of blebs, indentations, and wrinkling of the cell wall, discontinuity of cell membrane, undefined and damaged nuclei, and shrinkage of protoplasm. Taken together the results suggest that Pln-treatment initiate the apoptosis cell death which may lead to necrosis due to toxicity of the plantaricin peptides.     

Expression of PDE11A in normal and malignant human tissues.

Cyclic nucleotide phosphodiesterase 11A (PDE11A) is the newest member in the PDE family. Although the tissue distribution of PDE11A mRNA has been shown, its protein expression pattern has not been well studied. The goal of this report is to investigate the distribution of PDE11A proteins in a wide range of normal and malignant human tissues. We utilized a polyclonal antibody that recognized all four PDE11A isoforms. Its specificity was demonstrated by Western blot analysis on a recombinant human PDE11A protein and native PDE11A proteins in various human tissues. Immunohistochemistry showed that PDE11A is widely expressed. Various degrees of immunoreactivity were observed in the epithelial cells, endothelial cells, and smooth muscle cells of all tissues examined. The highest expression was in the epithelial, endothelial, and smooth muscle cells of the prostate, Leydig, and spermatogenic cells of the testis, the tubule epithelial cells in the kidney, the epithelial and endothelial cells in the adrenal, the epithelial cells and macrophages in the colon, and the epidermis in the skin. Furthermore, PDE11A expression was also detected in several human carcinomas. Our results suggest that PDE11A might be involved in multiple physiological processes in various organs via its ability to modulate intracellular cAMP and cGMP levels.     

Plasmid-Encoded Sequestration of Copper by Pseudomonas pickettii Strain US321

Pseudomonas pickettii strain US321 appeared to elicit a copper resistance mechanism upon exposure to copper. The bacterial colonies turned blue in the presence of this metal in a chemically defined medium, suggesting accumulation of copper. Prolonged exposure to copper resulted in the characteristic “copper sink” morphology of the colonies. Atomic absorption spectrophotometric analysis confirmed that this organism can accumulate copper. The strain US321 exhibited a high-molecular-weight plasmid, pUS321. The plasmid-cured strain, PC25, is highly sensitive to copper owing to a poor copper management. A plasmid-encoded sequestration mechanism operating in the strain US321 is suggested. Received: 3 September 1996 / Accepted: 12 December 1996     

Effects of acetylcholine, TSH and other stimulators on intracellular calcium concentration in dog thyroid cells

The intracellular free calcium concentration, [Ca2+]i, has been measured in dog thyroid cells using the fluorescent Ca2+-indicator, quin2. Acetylcholine or its non-hydrolyzable analog, carbamylcholine rapidly increased [Ca2+]i by 40 +/- 4% (mean +/- SE) over the basal level of 81 +/- 2 nM. This increase was totally abolished by atropine, a muscarinic cholinergic receptor blocker, but was not influenced by verapamil, a voltage dependent-calcium channel blocker. Depletion of extracellular Ca2+ by the addition of EGTA, diminished but did not abolish the response to carbamylcholine. These data suggest that cholinergic effectors increase [Ca2+]i by mobilization of Ca2+ from intracellular stores rather than from an influx of Ca2+. Addition of TSH, isoproterenol, phorbol ester, dibutyryl cyclic GMP or cyclic AMP did not elicit any change in [Ca2+]i suggesting that their action may not involve any mobilization of intracellular Ca2+. These data provide direct evidence that in the thyroid cell, cholinergic agents act via their receptors to cause a rapid increase in [Ca2+]i, which may mediate their metabolic effects.     

Hydrolysis of concentrated sucrose syrups by invertase immobilized on anion exchanger waste cotton thread.

Yeast invertase was immobilized on polyethyleneimine-coated cotton thread by adsorption followed by crosslinking with glutaraldehyde. The thread-bound invertase was used as an easily retrievable system for the hydrolysis of 80% w/v commercial sucrose syrups. The immobilized enzyme was stable for over 90 days to a temperature of 50 degrees C, only when stored in 80% sucrose solution. Above this temperature, inactivation of enzyme was observed. The cotton threads were used in a batch reactor for hydrolysis of sucrose in about 30 batches carried out over a period of 50 days without loss in activity. The threads could also be used in a packed bed reactor (1.51) for 97% hydrolysis of 80% sucrose syrups at 50 degrees C at a rate of about 360 kg per month for a period of 3 months.     

Physico chemical alterations in desugared buffalo milk after fermentation withSaccharomyces fragilis

Summary Buffalo milk was completely desugared by fermentation for 17 h withSaccharomyces fragilis. A thin-layer chromatographic profile indicated the total absence of lactose, galactose, glucose and any oligosaccharides. Desugaring by fermentation did not alter the lipid composition or the protein content of the milk. Gas chromatographic studies indicated the formation of volatile compounds such as acetaldehyde, ethyl acetate, ethyl alcohol, n-propanol, n-butanol and isoamyl alcohol. A pleasant fruity odour observed in the initial stage of fermentation changed to an overripe fruity odour on fermentation. The volatile compounds formed disappeared on spraydrying the milk, leaving only a residual yeasty odour in the desugared milk powder.     

Batten disease: biochemical and molecular characterization revealing novel <Emphasis Type="Italic">PPT1</Emphasis> and <Emphasis Type="Italic">TPP1</Emphasis> gene mutations in Indian patients

Background

Neuronal ceroid lipofuscinoses type I and type II (NCL1 and NCL2) also known as Batten disease are the commonly observed neurodegenerative lysosomal storage disorder caused by mutations in the PPT1 and TPP1 genes respectively. Till date, nearly 76 mutations in PPT1 and approximately 140 mutations, including large deletion/duplications, in TPP1 genes have been reported in the literature. The present study includes 34 unrelated Indian patients (12 females and 22 males) having epilepsy, visual impairment, cerebral atrophy, and cerebellar atrophy.

Methods

The biochemical investigation involved measuring the palmitoyl protein thioesterase 1 and tripeptidy peptidase l enzyme activity from the leukocytes. Based on the biochemical analysis all patients were screened for variations in either PPT1 gene or TPP1 gene using bidirectional Sanger sequencing. In cases where Sanger sequencing results was uninformative Multiplex Ligation-dependent Probe Amplification technique was employed. The online tools performed the protein homology modeling and orthologous conservation of the novel variants.

Results

Out of 34 patients analyzed, the biochemical assay confirmed 12 patients with NCL1 and 22 patients with NCL2. Molecular analysis of PPT1 gene in NCL1 patients revealed three known mutations (p.Val181Met, p.Asn110Ser, and p.Trp186Ter) and four novel variants (p.Glu178Asnfs*13, p.Pro238Leu, p.Cys45Arg, and p.Val236Gly). In the case of NCL2 patients, the TPP1 gene analysis identified seven known mutations and eight novel variants. Overall these 15 variants comprised seven missense variants (p.Met345Leu, p.Arg339Trp, p.Arg339Gln, p.Arg206Cys, p.Asn286Ser, p.Arg152Ser, p.Tyr459Ser), four frameshift variants (p.Ser62Argfs*19, p.Ser153Profs*19, p.Phe230Serfs*28, p.Ile484Aspfs*7), three nonsense variants (p.Phe516*, p.Arg208*, p.Tyr157*) and one intronic variant (g.2023_2024insT). No large deletion/duplication was identified in three NCL1 patients where Sanger sequencing study was normal.

Conclusion

The given study reports 34 patients with Batten disease. In addition, the study contributes four novel variants to the spectrum of PPT1 gene mutations and eight novel variants to the TPP1 gene mutation data. The novel pathogenic variant p.Pro238Leu occurred most commonly in the NCL1 cohort while the occurrence of a known pathogenic mutation p.Arg206Cys dominated in the NCL2 cohort. This study provides an insight into the molecular pathology of NCL1 and NCL2 disease for Indian origin patients.