The purpose of this study was to establish a rapid, reliable diagnostic method detecting Encephalomyocarditis virus(EMCV) RNA in formalin-fixed, paraffin-embedded tissues of EMCV naturally infected pigs by cDNA probe of EMC K3, the EMCV strain isolated from Korea. Using a biotin-labelled nick translated probe for the cDNA marker. We made up for some defects of radiolabeled method. In situ hybridization(ISH) technique, differently from theother nucleic acid hybridization methods, is able to detect the virus genome specifically in the state of the intact shapes of cells and/or tissues. We succeeded in performing the experiment to detect the EMCV within 1~2 hours usign the MicroProbeTM capaillary action system. In this study, we ovserved highly specific positive sighals of red color by staining the paraffinembedded tissue sections of naturally EMCV-infected pig organs or tissues, including brain, heart, kidney and lacrimal gland with the Fast Red TR salt/Naphtol phosphate chromogen. The results suggested that this ISH method is considered as a highly sensitive and reliable tool for molecular biologic diagnosis of the EMC viral disease.

While the C-terminal cytoplasmic tail of anion exchanger 1 (AE1, band 3) has been reported to possess important physiological roles, including one for proper membrane trafficking, its precise characteristics remain unclear. To clarify the overall structural consequences of the conserved sequence EL(K/Q)(L/C)LD(A/G)DD, containing the core binding sequence LDADD for carbonic anhydrase II, in the C-terminal region, we analyzed the membrane expression and turnover of bovine AE1 with a series of truncation and substitution mutations in HEK293 cells. Immunofluorescence microscopy and cell-surface biotinylation demonstrated that truncation mutants missing 18 C-terminal residues targeted the plasma membrane, but the one lacking the conserved region, by truncation of 28 amino acid residues, was retained inside the cells. Substitutions of Ala for Glusup(901), Leusup(902), Leusup(905), and Aspsup(906) in the sequence E901L(K/Q)(L/C)LDADD909 of bovine AE1 or those in the corresponding murine sequence also caused intracellular retention, though these mutants had half-lives comparable to that for wild-type AE1. These data demonstrate that the conserved amino acid residues Glusup(1), Leusup(2), Leusup(5), and Aspsup(6) in the EL(K/Q)(L/C)LD(A/G)DD region have essential structural consequences in stable expression of AE1 at the plasma membrane regardless of the ability in binding to carbonic anhydrase II of this region.

The hooded phenotype is one of the coat color phenotype seen peculiarly in the rat. The hooded locus showing autosomal recessive inheritance is mapped to chromosome (Chr) 14 and that the hooded phenotype receives modification by hooded-modifier gene showing the linkage to the hooded locus. However, a gene responsible for either the hooded or hooded-modifier gene is not yet identified. To clarify genetic control of hooded phenotype, we carried out genetic linkage studies using BN and LEA rats. For determination of phenotypic variation, we measured ratio of pigmented coat area in parental and their Fsub(1) and Fsub(2) rats. We, then, conducted a genome-wide scan on 152 Fsub(2) rats for linkage with ratio of pigmented coat area for the dorsal, ventral and total regions. A major quantitative trait locus (QTL), D14Got40, showing highly significant linkage contributing 70-90% of the variance for hooded phenotype was detected on Chr 14, which may be correspondent to the hooded locus. In addition, another QTL, D17Rat2, showing highly significant linkage was also detected on Chr 17 in dorsal region phenotype as well as a QTL showing suggestive linkage on Chr15 in ventral region phenotype. We, further, investigated a genome-wide scan for epistatic interactions and detected significant interactions between D14Got40 and D20Mit1, and between D14Got40 and D17Rat2 in the dorsal region phenotype. These results suggest that a major QTL in Chr 14, which is possibly correspondent to the hooded locus, mainly regulates the hooded phenotype with some modifier loci, two of which show epistatic interactions with the hooded locus.

To characterize the roles of C-peptide in vascular homeostatic processes, we examined the genes regulated by C-peptide in LEII mouse lung microvascular endothelial cells. Treatment of the cells with C-peptide increased the expression of c-Jun N-terminal kinase 1 (JNK1) mRNA dose-dependently, accompanied by an increase in JNK1 protein content. Prior treatment of the cells with PD98059, an ERK kinase inhibitor or SB203580, a p38MAPK inhibitor, abrogated the C-peptide-elicited JNK1 mRNA expression. These results indicate that C-peptide increases JNK1 protein levels, possibly through ERK- and p38MAPK-dependent activation of JNK gene transcription.

An R664X nonsense mutant AE1 is responsible for dominant hereditary spherocytosis in cattle and is degraded by the proteasomal endoplasmic reticulum-associated degradation. The present study demonstrated that R664X AE1 translated in vitro had the trypsin-sensitve site identical to that of the wild-type AE1. The P661S/R664X mutant containing a possible N-glycosylation site at Asnsup(660) showed an increase in size by 3 kDa both in the cell-free translation system and in transfected HEK293 cells. Moreover, steady state levels of R664X and P661S/R664X in HEK293 cells were markedly increased in the presence of a proteasome inhibitior. These findings indicate that the truncated C-terminal region of R664X AE1 has lumenal localization in the endoplasmic reticulum and is not accessible to proteasomal machineries in the cytosol.

The aim of this study was to clarify the role of the renal renin-angiotensin system (RAS) in diabetic nephropathy (DN) , which was induced by injection of streptozotocin (STZ). Male CBA/N and CBA/J mice were compared in this study. The former possesses a single renin gene, Ren1. whereas the latter carries two renin genes, Ren1 and Ren2. To examine the molecular dynamics of renal RAS, including renin, angiotensinogen (Agt), angiotensin-converting enzyme (Ace), angiotensin type 1 (Agtr1) and type 2 (Agtr2) receptors in experimental DN, we performed laser-microdissection (LMD) followed by reverse transcriptase nested polymerase chain reaction using each specific primer pairs and immunohistochemistry for renin and angiotensin 2. CBA/N mice had a higher response after injection of STZ than CBA/J mice, showing a significant increase of the kidney/body weight ratio, although there was no significant difference between the two strains for the blood glucose level or pancreatic beta-cell response. The onset of renal pathological changes associated with DN was earlier and more severe in CBA/N mice than in CBA/J mice. Distinct immunoreactivities for renin and angiotensin 2 were newly distributed on the flattered epithelial cells in the dilated distal tubules in the cortex as well as the collecting ducts in the cortex and medulla, and were demonstrated more intensity in CBA/N mice than in CBA/J mice. Micro dissectional analysis in both models revealed a higher incidence of RAS-related gene expression in CBA/J, Ren 2 mice than in CBA-N, Ren 1 mice.

A total of 253 samples from cattle (52), buffalo (51), sheep (50), goat (50) and chicken (50) were screened for the presence of Pasteurella multocida. Out of these, 17 samples were found positive by species specific PCR (PM-PCR) with ~460 bp amplified product. Overall prevalence of P. multocida was found to be 6.7%. Out of 17 PM-PCR positive samples, 12 (70%) were found positive by capsular type-B specific (HSB-PCR) and multiplex PCR assays with amplification of ~620 bp product in both the cases. A total of 5 isolates of P. multocida were obtained in 17 PM-PCR positive samples of which 4 were of serotype B:2 and 1 was of serotype A:3. The PM-PCR and HSB-PCR assays can be performed directly on clinical samples from animals with reproducible results without any non-specific amplification. Multiplex PCR further reduces the time for the species and type specific detection of P. multocida.

Thirty five human sputum collected from TB hospital Bareilly were investigated for Mycobacteria based on direct microscopy, culture and by multiplex peR targeting 12.7 kb fragment and IS 611O. DNA was isolated directly forms putums amples. Outof35 samples,25 were smear positive and 18 yielded culture and 16 were positive by the multiplex PeR. 10 samples were negative on smear mircoscopy, culture and PCR.

The causative agent of bovine tuberculosis (BTB) is <em>Mycobacterium bovis</em>, a bacteria belonging to <em>M. tuberculosis</em> complex (MTC). The definitive diagnosis is realized by isolation and identification of the <em>M. bovis</em> from clinical samples, using a combination of traditional culture and biochemical methods, which is considered the “gold standard”. This procedure is cumbersome and time-consuming. We evaluated a PCR assay for the direct detection of MTC DNA in naturally contaminated milk, using primers that were previously tested and proven reliable to target the <em>IS6110 </em>element. Milk previously seeded with <em>M. bovis</em> was used as the starting material, for padronization of the technique. The procedure involved extracting the DNA by enzymatic lysis (proteinase K and lysozyme), phenol, chloroform, isoamyl alcohol, followed by ethanol precipitation and PCR. The PCR assay allowed us to detect BTB in artificially contaminated milk, with a detection limit of 100 CFU/mL, and was also able to detect the bacillus in 50% (75/150) of samples from naturally infected animals. This procedure could be used to assist the <em>in vivo</em> diagnosis for BTB, complementing the sorological or microbiological tests and becomes an alternative option for implementation in epidemiological studies of BTB transmission and to prevent contaminated milk from entering the food supply. | O agente causador da tuberculose bovina (BTB) é o <em>Mycobacterium bovis</em>, uma bactéria pertencente ao complexo <em>M. tuberculosis</em> (CMT). O diagnóstico definitivo é realizado através do isolamento e identificação de <em>M. bovis</em> em amostras clínicas, utilizando uma combinação de cultura bacteriológica e métodos bioquímicos, que são considerados como "padrão de ouro". Entretanto esses procedimentos são trabalhosos e demorados. No seguinte estudo avaliamos um ensaio de PCR para a detecção direta de DNA de CMT em leite de vacas positivas ao teste cutâneo, utilizando <em>primers</em> previamente testados e comprovadamente confiáveis, para amplificação da região de interesse <em>IS6110</em>. Leite previamente contaminado com <em>M. bovis</em> foi utilizado como material de partida, para a padronização da técnica. O procedimento envolveu a extração do DNA por lise enzimática (proteinase K e lisozima), fenol, clorofórmio, álcool isoamílico, seguido de precipitação com etanol e PCR. O ensaio de PCR permitiu detectar BTB em leite artificialmente contaminado, com um limite de detecção de 100 UFC/mL, e também foi capaz de detectar o bacilo em 50% (75/150) de amostras de leite testadas. A técnica de PCR pode ser utilizada para auxiliar o diagnóstico <em>in vivo</em> da BTB, além de complementar os testes sorológicos ou microbiológicos, tornando-se uma alternativa para estudos epidemiológicos da transmissão de BTB, prevenindo que o leite contaminado entre na cadeia de alimentos.