Hemato-biochemical alterations and urinalysis in dogs suffering from benign prostatic hyperplasia.

AIM: The study was designed to evaluate the hemato-biochemical alterations, urinalysis along with histomorphological and histological changes of prostate glands in dogs affected with benign prostatic hyperplasia (BPH) in and around Bhubaneswar, Odisha, India. MATERIALS AND METHODS: In toto, 445 dogs presented to the Teaching Veterinary Clinical Complex of the College of Veterinary Sciences and Animal Husbandry, one Government Veterinary Hospital and two pet clinics in and around Bhubaneswar screened for the presence of BPH. Most of the 57 dogs were 6 years and above as reported by the owners. Only 57 dogs found positive for BPH basing on the presence of typical clinical signs subjected for a detailed hemato-biochemical study. Most of the 57 dogs were 6 years and above as reported by the owners. Routine and microscopic urinalyses were done as per the routine procedure. Histomorphological evaluations of prostate glands were done through manual rectal palpation. Histological examinations of prostate tissue sections of two dead dogs were conducted with routine hematoxylin and eosin stain. RESULTS: The study revealed about 12.8% (57/445) of dogs was suffering from BPH. Typical clinical signs - such as passing small thin tape-shaped feces, holding tail away from backward, tenesmus, and straining during urination and defecation - were seen in most of the cases. Urine samples of affected dogs were positive for glucose, occult blood, and protein. A significant decrease in lymphocytes and increase in eosinophil counts in dogs with BPH was recorded. Serum biochemical analysis showed a nonsignificant increase in creatinine and blood urea nitrogen with a significant decrease in total protein, albumin, globulin, A:G ratio. Histology of prostate glands collected during postmortem was characterized by fibrosis of prostate gland, and hyperplasia of the acinar epithelium. CONCLUSIONS: High rate of the prevalence of BPH in dogs poses an alarming condition which if diagnosed at an early stage can certainly prolong the longevity of the dogs.

Development of Crystalline Cellulosic Fibres for Sustained Release of Drug.

Natural quinoline alkaloid camptothecin (CPT) is used for the treatment of colon, lung, breast and ovarian cancers still facing challenges due to low solubility in aqueous and biological fluids. Its lactone form easily converts into a toxic carboxylic form at slightly basic pH, typical in blood and tissue fluid has rapid clearance from systemic administration. We report a new approach based on micro crystalline cellulose (MCC) and nano crystalline cellulose (NCC) isolated from natural sources such as Cymbopogan flexuosus to stabilize and regulate the release kinetics of CPT in physiological solution. Langmuir and Freundlich isotherm studies approve that degree of crystallinity i.e. ratio of amorphous and crystalline cellulose regulate the adsorption of CPT. The freeze dried celluloses of Cymbopogan flexuosus origin (MCC and NCC) further were optimized for drug delivery with a mimicked physiologically relevant solution. Both carriers can significantly extend the release of drug as compared to reported values, however, NCC showed better results. Not only the crystallinity but crystal size and hydrogen bonding play critical role in drug release. Free diffusion of drug into physiological solution follows the Ritger- Peppes kinetic model. The coefficient of the model signifies the Fickian diffusion mechanism of release. The investigation indicates that NCC cellulosic matrix can act as a better carrier of CPT for its sustained release formulation.

Effect of Moringa oleifera on hematological parameters of calves reared in industrial fluorotic area.

AIM: The present study was undertaken to evaluate the ameliorative potential of dried Moringa oleifera fruit powder in fluorosis affected calves reared around the vicinity of aluminium smelter plant. MATERIALS AND METHODS: Total 107 calves were screened on the basis of clinical signs and higher plasma fluoride (more than 0.2 ppm) level for evidence of fluorosis. Out of that, 90 samples found positive and from them 18 calves of 6-12 months age group were selected and divided equally into three groups named as Group II, III, and IV. Group II remained as disease control group whereas Group III calves were supplemented with dried M. oleifera fruit powder of 25 g/calve for 60 days. Group IV calves were supplemented with calcium carbonate at 100 mg/kg body weight and boric acid at 10 mg/kg for the same experimental period. Group I consisted of six numbers of healthy calves taken from the non-fluorotic zone, i.e. Bhubaneswar. Plasma fluoride level, hemoglobin (Hb), packed cell volume (PCV), total leukocyte count (TLC), differential count (DC), total erythrocyte count, mean corpuscular volume (MCV), mean corpuscular Hb (MCH), and MCH concentration (MCHC) were estimated on day 0, 30, and 60 of the experiment. RESULTS: Supplementation of dried M. oleifera fruit powder to fluorosis affected calves resulted in significant reduction in plasma fluoride level and increase in Hb%, PCV, TLC and altered DC. Similar results were also recorded in calcium+boron group, except PCV and Hb. No significant changes were observed in MCV, MCH, and MCHC values. CONCLUSION: The present study concluded that supplementation of dried M. oleifera fruit powder daily for 60 days has shown protection against chronic fluoride toxicity in calves.

Flower synchrony, growth and yield enhancement of small type bitter gourd (Momordica charantia L.) through plant growth regulators and NPK fertilization.

Assessment of growth regulator and NPK fertilization effects are important tools for flower stimulation and yield improvement in cucurbits. This investigation demonstrates the comparative male-female flower induction and fruit yield of small sized bitter gourd treated with NPK fertilizers and plant growth regulators. Namely, two experiments having three replicates were conducted in a Randomized Complete Block Design (RCBD) with NPK fertilization and plant growth regulators-GA3, NAA and Ethophon application on small sized bitter gourd-genotype BG5 at the research field of the Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU). In experiment 1, different doses of NPK fertilizers comprised of 10 treatments and in that of experiment 2, different levels of plant growth regulators indicated 10 treatments. The results indicated that application of different doses of NPK fertilizer and plant growth regulators significantly (< or = 0.05) influenced over the flower initiation and fruit setting. The application of N90-P45-K60 fertilizer along with Ethophon spraying resulted in the better yield of small sized bitter gourd.

In vitro organogenesis and genetic transformation in popular Cucumis sativus L. through Agrobacterium tumefaciens.

The effect of growth regulators and culture conditions on the morphogenetic response of cotyledonary leaf discs was studied in popular cucumber variety (Cucumis sativus cv. Sheetal). Organogenesis was induced directly without any intervening callus phase on Murashige and Skoog medium supplemented with different concentrations of benzyladenine and indole propionic acid. Best results (93%) were obtained in the presence of the 4 mg/L benzyladenine and 1 mg/L IPA. The elongated shoots were rooted in basal medium with 1 mg/L indole butyric acid, hardened and transferred to the field conditions. Genetic transformation system has been established for Cucumis sativus cv. Sheetal, plants by infecting cotyledonary explants with Agrobacterium tumefaciens strain LBA4404 carrying binary plasmid pBI121, which contains scorable marker, beta-glucuronidase and selectable marker nptII under the CaMV 35S promoter. Infection was most effective when explants were infected with Agrobacterium for 15 min and co-cultivated for 2 days in the co-cultivation medium. Shoots were regenerated directly from cotyledonary leaf explants in the presence of kanamycin (50 microg/ml) and analysed. Southern blot analysis confirmed that transformation had occurred. This method will allow genetic improvement of this crop by the introduction of agronomically important genes.

Enhanced expression of heterotrimeric GTP-binding protein subunits in Zajdela ascitic hepatoma.

G protein beta subunit (Gbeta2) is over-expressed in Zajdela ascitic hepatoma (ZAH), a rat ascitic tumour, at mRNA as well as protein levels. Nuclear run-off transcription analysis suggests that the expression of Gbeta2 in ZAH is regulated mainly at transcriptional as well as post-transcriptional levels. Galphai3 is also over-expressed in ZAH. No amplification or any change in the organization of the genes for GBbeta2 or Galphai3 was observed. It is possible that the over-expression of G protein subunits in ZAH could provide proliferative advantage to the cells by virtue of their effects on second messenger systems.

Predominance of antibodies to hepatitis C virus envelope proteins in various disease statuses of hepatitis C.

The antibody profile to various proteins of hepatitis C virus (HCV) was studied in 113 patients positive for HCV RNA in various disease statuses of hepatitis C (HC). A single peptide (E2/NS1, aa 413-436 of HCV polyprotein) chosen from a conserved region at the C-terminus of the hypervariable region (HVR) HVR1 of HCV was found to be sufficient for reliable diagnosis of the infection, even in the acute phase. Six hundred and one suspected HC cases and 200 voluntary blood donors were tested by this peptide. The sensitivity of detection of HCV antibodies by this peptide did not increase with addition of peptides from other HCV proteins. Our results clearly demonstrate that antibodies to HCV envelope proteins occur in a higher percentage of the infected population than those to other proteins. This emphasizes the necessity of using representative sequences from HCV envelope proteins in diagnostic immunoassays of this viral infection.

Cloning, expression, and function of BLAME, a novel member of the CD2 family.

The CD2 family is a growing family of Ig domain-containing cell surface proteins involved in lymphocyte activation. Here we describe the cloning and expression analysis of a novel member of this family, B lymphocyte activator macrophage expressed (BLAME). BLAME shares the structural features of the CD2 family containing an IgV and IgC2 domain and clusters with the other family members on chromosome 1q21. Quantitative PCR and Northern blot analysis show BLAME to be expressed in lymphoid tissue and, more specifically, in some populations of professional APCs, activated monocytes, and DCS: Retroviral forced expression of BLAME in hematopoietic cells of transplanted mice showed an increase in B1 cells in the peripheral blood, spleen, lymph nodes, and, most strikingly, in the peritoneal cavity. These cells do not express CD5 and are CD23(low)Mac1(low), characteristics of the B1b subset. BLAME may therefore play a role in B lineage commitment and/or modulation of signal through the B cell receptor.

Syntheses of immunodominant peptide regions of hepatitis C viral pathogens using PS-BDODMA resin: a single peptide derived from the conserved domain (E2/NS1) was highly effective in detecting anti-HCV antibodies.

Peptides were synthesized with very high purity and yield on a highly solvating copolymer of 1,4-butanediol dimethacrylate cross-linked polystyrene support (PS-BDODMA). The polymer was synthesized by radical aqueous suspension polymerization using toluene as the diluent and 1% polyvinyl alcohol as the suspension stabilizer. The supports were highly resistant to various chemical reagents and solvents that are frequently used in polypeptide synthesis. The peptides synthesized on this support were designed from the core (C), envelope (E1 and E2) and nonstructural protein (NS1-NS5) regions of the prototype hepatitis C viral (HCV) polyprotein, and were used to develop a peptide-based immunoassay (PBEIA) for the detection of HCV antibodies. The purity of these peptides was tested by HPLC. Peptide identity was confirmed by amino acid analysis and MALDI-TOF-MS. A single peptide chosen from a conserved area (E2/NS1) at the C-terminus of the hypervariable region (HVRI) was found to be sufficient for effective and reliable diagnosis of HCV infection in infected individuals, as well as also apparently healthy individuals. The CD spectrum of the peptide shows no preference for any ordered secondary structure. When used, peptide mixtures from various protein regions of HCV reduced the sensitivity and reliability of the diagnosis partly because of epitope masking.

Syntheses of four peptides from the immunodominant region of hepatitis C viral pathogens using PS-TTEGDA support for the investigation of HCV infection in human blood.

Four peptides were designed and synthesized on a highly solvating copolymer of tetraethyleneglycol diacrylate cross-linked polystyrene (PS-TTEGDA) support with very high purity and yield. The polymer was synthesized in various cross-linking densities (1, 2, 3, 4, 5 and 10%) using radical aqueous suspension polymerization. Four per cent PS-TTEGDA resin showed rigidity and mechanical characteristics comparable with those of divinylbenzene cross-linked polystyrene (PS-DVB) support. Swelling and solvation characteristics of PS-TTEGDA were much higher than PS-DVB support in all solvents used in solid-phase peptide synthesis. Forty-eight hour treatment of the support with neat trifluoroacetic acid did not show any change in its infrared spectra. PS-TTEGDA could be functionalized with chloromethyl, aminomethyl and hydroxymethyl functional groups under various controlled conditions. Synthetic utility of the support was demonstrated by the synthesis of four peptides selected from the envelope and nonstructural protein region of the prototype hepatitis C virus (HCV). These peptides were later used successfully to develop a peptide-based immunoassay (PBEIA) for the detection of HCV immunity. Peptides designed from the NS1 and NS4 protein regions were found to be very promising for the development of a new diagnostic kit to detect HCV infection in human blood. Peptide purity was tested by RP-FPLC and the peptide identity was confirmed by amino acid analysis.

Analysis of human immune response to potential hepatitis C viral epitopes.

A peptide-based enzyme immunoassay (PBEIA) has been developed using synthetic peptides whose sequences were selected from the core, envelope and non-structural regions of the prototype hepatitis C virus (HCV) genome. Results of PBEIA of sera obtained from several patients with various liver disorders were compared to those of commercial enzyme-linked immunosorbent assays (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). A large number of samples, which were repeatedly negative in commercial ELISA, were positive in PBEIA. There was a good correlation between the results of PBEIA and RT-PCR. The developed PBEIA proved to be a sensitive assay that had high specificity and was capable of detecting antibodies in various HCV-related liver disorders including the acute phase of the infection.

Prior exposure to superantigen can inhibit or exacerbate autoimmune encephalomyelitis: T-cell repertoire engaged by the autoantigen determines clinical outcome.

Experimental allergic encephalomyelitis (EAE) is inducible in experimental animals immunized with myelin basic protein (MBP), proteolipid protein (PLP) or their peptides. We compared T-cell responses to encephalitogenic epitopes of PLP(43-64) and MBP(Ac1-11) in a single mouse strain, (PL/J x SJL)F1. MBP(1-11)-specific T-cell hybridomas expressed predominantly TCR V beta 8 or V beta 4, while PLP(43-64)-specific hybridomas expressed a diverse TCR repertoire. To analyze the biologic significance of the TCR repertoire (limited vs. diverse) to disease susceptibility, we pretreated mice with a superantigen (SEB), and then induced disease with these autoantigens. Mice injected with SEB and immunized with MBP(Ac1-11) showed significant inhibition of EAE, whereas SEB-pretreated mice immunized with PLP(43-64) had an increased severity of EAE and developed a chronic disease. These data demonstrate that prior exposure to microbial superantigens can significantly alter the autoimmune disease course depending upon the TCR repertoire used by the autoantigen.

Cell free ascitic fluid prevents loss of cell surface sialic acid from Zajdela Ascitic Hepatoma cells in culture.

In the Zajdela Ascitic Hepatoma (ZAH), a rat tumor, high levels of cell surface sialic acid residues are present which masked the immunogenicity of the cells. We have shown here that cell surface sialic acid level goes down rapidly when ZAH cells are put in culture. The reduction in surface sialic acid levels is due to a decrease in sialic acid residues on the major sialylated glycoprotein, gp 120, as well as a decrease in gp 120 polypeptide. The loss of sialic acid from the cultured cells is reduced if the cells are cultured in the presence of cell free ascitic fluid from ZAH tumor.

Reciprocal expression of co-stimulatory molecules, B7-1 and B7-2, on murine T cells following activation.

The co-stimulatory B7 molecules (B7-1 and B7-2) are expressed on professional antigen-presenting cells in mice. In this study, we demonstrate that B7-1 (CD80) and B7-2 (CD86) are also expressed on murine T cells in the absence of major histocompatibility complex class II molecules. The temporal expression of these two molecules on T cells varies with the state of activation where resting T cells express B7-2 but show little or no expression of B7-1. Following activation, B7-2 expression is down-regulated and there is a concomitant increase in the expression of B7-1 on the cell surface which peaks at about 72 h. Thus these two co-stimulatory molecules are reciprocally expressed on the T cell surface. This pattern of expression of B7-1 and B7-2 on T cells suggests that these two molecules may have different roles in the generation and regulation of immune responses.

Lactoferrin contains structural motifs of ribonuclease.

A high molecular weight ribonuclease isolated from human milk (hmRNAase) shares identical immunological, physical, structural features and considerable sequence homology with human lactoferrin; and it has been demonstrated to be an isoform of lactoferrin. We have analyzed the sequence data of lactoferrin looking for the existence of specific features corresponding to the consensus sequence of pyrimidine-specific ribonucleases. The analysis was done by comparing sequence features with respect to elements which are, in principle, responsible for RNAase activity. This revealed the existence of a ribonuclease-signature pattern in lactoferrin. Further analysis of X-ray data together with molecular modeling studies have revealed close similarities between the spatial geometry of the constituent groups of the active site of pyrimidine-specific ribonucleases and the corresponding groups comprising the potential active site of lactoferrin. Our results provide the strong structural basis for the existence of ribonuclease activity in lactoferrin.

A novel way to generate nonspecifically radiolabeled proteins for use as molecular-weight markers.

A method for labeling proteins using radioactive nucleotides in the presence of divalent cations such as Cu2+, Zn2+ and Cd2+ is described. Small amounts of marker proteins can be rapidly labeled using the method and they can be used for molecular-weight determination of radioactive proteins of unknown molecular weights, as for example, any protein labeled with 35S-methionine in vivo or any protein radiolabeled by 32P in protein phosphorylation experiments. The gels in which the labeled markers and the proteins of unknown molecular weight are electrophoresed could be directly exposed to X-ray films and comparisons made from a single autoradiogram, avoiding a two step procedure of Coomassie blue staining followed by autoradiography and aligning of the two sets of bands. This metal-ion-mediated labeling of proteins described in the present communication is not by any site-specific interaction of the nucleotide with the proteins and the protein is denatured after the complex formation. The adducts, once formed, are stable under conditions of SDS-PAGE or EDTA treatment. It is suggested that proteins labeled under conditions described in this communication are ternary complexes involving aromatic residues of the proteins, nucleotides and the divalent cations.

Negative correlation with liver cell division of a 38 kilodalton protein whose phosphorylation is enhanced by ras and G-proteins.

We showed earlier that the phosphorylation of a 38 kDa protein (p38) from rat liver plasma membrane is stimulated by ras or endogenous G-proteins. We have now estimated the level of expression of p38 in liver tissues from embryos at different stages of development, regenerating liver and also in tumor cell lines of hepatic origin. Our results indicate that the expression of p38 is negatively correlated with cell division. It is suggested that the phosphorylation of p38, an event which is regulated by ras proteins and G-proteins, could be involved in signal transduction processes associated with the inhibitory regulation of cell division.

Purification and characterization of a high molecular weight ribonuclease from human milk.

We have purified a high molecular weight ribonuclease (hmRNase) from human milk by a two-step column chromatographic procedure and characterized the enzyme. The molecular mass of hmRNase is 80 kDa as determined from SDS-polyacrylamide gel electrophoresis. The pH optimum of the enzyme is in the range of 7.5-8.0, similar to other secretory RNases. hmRNase is pyrimidine-specific and cleaves the phosphodiester bond 3' to a pyrimidine residue. It selectively degrades the pyrimidine strand in poly(rA):poly(rU) and poly(dA):poly(rU) double stranded substrates. The extent of degradation for naturally occurring RNAs vary in the order tRNA < rRNA < mRNA at low enzyme concentrations. hmRNase shows allosteric behavior with positive cooperativity in its reaction on polynucleotide substrates. The activity of the enzyme is enhanced in the presence of monoribonucleotides. Antiserum obtained against purified hmRNase did not cross-react with low molecular weight RNase which is also present in milk. In addition, an immunologically cross-reacting species could not be detected in the serum, suggesting the origin of hmRNase in the mammary gland but not blood.

Regulation of expression of beta-galactoside alpha 2,6-sialyltransferase in a rat tumor, Zajdela ascitic hepatoma.

Tumor cell surface sialic acid levels determine a number of important properties governing cellular interactions and cell-cell communication. Towards understanding the mechanism of regulation of sialic acid levels upon cellular transformation, we have studied the regulation of expression of beta-galactoside alpha 2,6-sialyltransferase in a rat tumor, the Zajdela ascitic hepatoma. We demonstrate distinct differences in the regulation of expression of the enzyme in the tumor cells as compared to normal liver cells. The expression of sialyltransferase is regulated both at the transcriptional and post-transcriptional level in a tissue-specific manner.