Isolation of enriched carp spermatogonial stem cells from Labeo rohita testis for in vitro propagation

The in vitro culture system for spermatogonial stem cells (SSCs) is a powerful tool for exploring molecular mechanisms of male gametogenesis and gene manipulation. Very little information is available for fish SSC biology. Our aim was to isolate highly pure SSCs from the testis of commercially important farmed carp, Labeo rohita. The minced testis of L. rohita was dissociated with collagenase. Dissociated cells purified by two-step Ficoll gradient centrifugation followed by magnetic activated cell sorting (MACS) using Thy1.2 (CD90.2) antibody dramatically heightened recovery rate for spermatogonial cells. The purified cells were cultured in vitro conditions for more than two months in L-15 media containing 10% fetal bovine serum (FBS), 1% carp serum, and other nutrients. The proliferative cells were dividing as validated by 5-bromo-2′-deoxyuridine (BrdU) incorporation assay and formed colonies/clumps with the typical characteristics of SSCs A majority of enriched cell population represented a Vasa⁺, Pou5f1/pou5f1⁺, Ssea-1⁺, Tra-1-81⁺, plzf⁺, Gfrα1/gfrα1⁻, and c-Kit/c-kit⁻ as detected by immunocytochemical and/or quantitative real-time polymerase chain reaction (RT-PCR) analyses. Thus, Thy1⁺ SSCs were enriched with greater efficiency from the mixed population of testicular cells of L. rohita. A population of enriched spermatogonial cells could be cultured in an undifferentiated state. The isolated SSCs could provide avenue for undertaking research on basic and applied reproductive biology.     

Responses of neurons in the rostral ventrolateral medulla to whole body rotations: comparisons in decerebrate and conscious cats

The responses to vestibular stimulation of brain stem neurons that regulate sympathetic outflow and blood flow have been studied extensively in decerebrate preparations, but not in conscious animals. In the present study, we compared the responses of neurons in the rostral ventrolateral medulla (RVLM), a principal region of the brain stem involved in the regulation of blood pressure, to whole body rotations of conscious and decerebrate cats. In both preparations, RVLM neurons exhibited similar levels of spontaneous activity (median of ～17 spikes/s). The firing of about half of the RVLM neurons recorded in decerebrate cats was modulated by rotations; these cells were activated by vertical tilts in a variety of directions, with response characteristics suggesting that their labyrinthine inputs originated in otolith organs. The activity of over one-third of RVLM neurons in decerebrate animals was altered by stimulation of baroreceptors; RVLM units with and without baroreceptor signals had similar responses to rotations. In contrast, only 6% of RVLM neurons studied in conscious cats exhibited cardiac-related activity, and the firing of just 1% of the cells was modulated by rotations. These data suggest that the brain stem circuitry mediating vestibulosympathetic reflexes is highly sensitive to changes in body position in space but that the responses to vestibular stimuli of neurons in the pathway are suppressed by higher brain centers in conscious animals. The findings also raise the possibility that autonomic responses to a variety of inputs, including those from the inner ear, could be gated according to behavioral context and attenuated when they are not necessary.     

Polymorphism and nucleotide sequencing of BMPR1B gene in prolific Assam hill goat

Assam hill goat (Capra hircus) is a prolific local goat in India. bone morphogenetic protein receptor (BMPR1B) gene was studied as a candidate gene for the prolificacy of goats. The objective of the present study was to detect the incidence of mutation in the exonic region of BMPR1B gene of Assam hill goat. Total 90 blood samples were collected randomly from different parts of Assam and genomic DNA were extracted using phenol–chloroform method. The quantity and quality of extracted DNA was examined by spectrophotometry and gel electrophoresis, respectively. PCR amplicon showed a product of 140 bp fragment of BMPR1B gene. The purified product upon digestion with AvaII showed monomorphic banding pattern and revealed wild type alleles with AA genotype. Nucleotide sequencing showed one new mutation 773 (G→C) which is found to be unique in Assam hill goat. Construction of tree at nucleotide level generates from the present experiment lies in common cluster which differs from the other breeds of goat. The analysis of polymorphism for BMPR1B in Assam hill goat indicates that the genetic factor responsible for prolificacy or multiple kidding rates is not related to the reported mutated alleles of BMPR1B gene. Therefore, attempts to be made to detect other SNPs for BMPR1B gene or otherwise effort should be made towards other fecundity gene which might be responsible for the prolificacy of Assam hill goat.     

Passive immunity with multi‐serotype heat‐killed Shigellae in neonatal mice

The short‐ and long‐term passive protective efficacy of a mixture of heat‐killed cells of six serogroups/serotypes of Shigella strains (Shigella dysenteriae 1, S. flexneri 2a, S. flexneri 3a, S. flexneri 6, S. boydii 4, and S. sonnei) were studied in neonatal mice. Neonatal mice from immunized dams exhibited significant short‐ and long‐term passive protection against individual challenge by each of the six Shigella strains. High IgG and IgA titers against the lipopolysaccharide from each of the six Shigella strains were observed in sera from immunized dams.     

The β-Prism Lectin Domain of Vibrio cholerae Hemolysin Promotes Self-assembly of the β-Pore-forming Toxin by a Carbohydrate-independent Mechanism

Vibrio cholerae cytolysin/hemolysin (VCC) is an amphipathic 65-kDa β-pore-forming toxin with a C-terminal β-prism lectin domain. Because deletion or point mutation of the lectin domain seriously compromises hemolytic activity, it is thought that carbohydrate-dependent interactions play a critical role in membrane targeting of VCC. To delineate the contributions of the cytolysin and lectin domains in pore formation, we used wild-type VCC, 50-kDa VCC (VCC⁵⁰) without the lectin domain, and mutant VCC^D617A with no carbohydrate-binding activity. VCC and its two variants with no carbohydrate-binding activity moved to the erythrocyte stroma with apparent association constants on the order of 10⁷ m⁻¹. However, loss of the lectin domain severely reduced the efficiency of self-association of the VCC monomer with the β-barrel heptamer in the synthetic lipid bilayer from ∼83 to 27%. Notably, inactivation of the carbohydrate-binding activity by the D617A mutation marginally reduced oligomerization to ∼77%. Oligomerization of VCC⁵⁰ was temperature-insensitive; by contrast, VCC self-assembly increased with increasing temperature, suggesting that the process is driven by entropy and opposed by enthalpy. Asialofetuin, the β₁-galactosyl-terminated glycoprotein inhibitor of VCC-induced hemolysis, promoted oligomerization of 65-kDa VCC to a species that resembled the membrane-inserted heptamer in stoichiometry and morphology but had reduced global amphipathicity. In conclusion, we propose (i) that the β-prism lectin domain facilitated toxin assembly by producing entropy during relocation in the heptamer and (ii) that glycoconjugates inhibited VCC by promoting its assembly to a water-soluble, less amphipathic oligomer variant with reduced ability to penetrate the bilayer.     

Multidrug resistant staphylococci isolated from pigs with exudative epidermitis in North eastern Region of India

Exudative epidermatitis or greasy pig disease (GPD) is a contagious disease of pig and endemic worldwide caused by toxigenic strains under genus Staphylococcus. The present study reported an outbreak of GPD in Champhai district of Mizoram adjoining to the southern border of Myanmar. A total of 60 samples were collected from 22 clinically affected animals and processed for isolation and identification of Staphylococcus spp. All the isolates were subjected to antimicrobial sensitivity assay, biofilm production assay and detection of virulence genes, biofilm genes and mec genes followed by cloning and sequencing for phylogenetic analysis. A total of 44 staphylococci belonged to four species (S. sciuri, S. aureus,S. lentus, and S. hyicus) were isolated. Majority of the isolates were multidrug resistant with maximum resistance against ampicillin, penicillin including vancomycin. None of the S. hyicus isolates was methicillin resistant (MRSH) but 66·67% isolates were MRSA. By PCR, mecA gene was detected in S. aureus (n = 2), S. sciuri (n = 4) and S. lentus (n = 3). Biofilm associated gene icaD was detected in S. aureus (n = 3), S. sciuri (n = 5), S. hyicus (n = 4) and S. lentus (n = 6). The exfoliative toxin genes (ehxB, shetA and tsst1) were detected in S. hyicus (n = 3) and S. aureus (n = 1) isolates. All the isolates were closely related with the isolates from pigs of China, Germany, Japan and USA. The pathogens might be transmitted through illegal migration of pigs from Myanmar to India.