Effect of low density lipoproteins in extender on freezability and fertility of buffalo (Bubalus bubalis) bull semen

This study was designed to determine whether low-density lipoporoteins (LDLs) extracted from egg yolk in extender improve the freezability and fertility of buffalo bull semen. Semen from three Nili-Ravi buffalo bulls was diluted at 37 °C with tris-citric acid extender (50 × 10⁶ motile spermatozoa mL⁻¹) containing LDLs 2.5%, 5%, 10%, and 15% extracted from egg yolk and extender containing 20% egg yolk was kept as control. Diluted semen was cooled to 4 °C in 2 h, equilibrated at 4 °C for 4 h, filled in 0.5 mL French straws, and kept on liquid nitrogen vapors for 10 min. Straws were then plunged and stored in liquid nitrogen (-196 °C). Sperm motility (visually; %), plasma membrane integrity (%; with supravital hypo-osmotic swelling test), and viability (%; with dual staining test using Trypan-blue Giemsa) were assessed at post-dilution, pre-freezing and post-thawing. At post-dilution and pre-freezing, sperm progressive motility, plasma membrane integrity and viability was similar (P > 0.05) in extender containing 10% LDLs or the control. However, at post-thaw the aforementioned parameters were higher (P < 0.05) in extender containing 10% LDLs compared with the control and other experimental extenders. The fertility rate of inseminations performed were higher (P < 0.05) with extender containing 10% LDLs than the control. It was concluded that LDLs (10%) in extender improved the freezability and fertility of buffalo bull spermatozoa.     

Calbindin D9k is not required for 1,25-dihydroxyvitamin D3-mediated Ca2+ absorption in small intestine

The exact role of calbindin D9k in vitamin D-mediated calcium absorption has been debated but remains unsettled. In 129/OlaHsd mice, calbindin D9k was found highest in duodenum (36-50%) and kidney (24-34%) followed by stomach, lung and uterus. Age does not affect the relative distribution of calbindin D9k but it does decline with age in duodenum of both male and female 129/Ola mice. Recently, we produced a null calbindin D9k mutant 129/OlaHsd mouse; this mouse proved to be indistinguishable from the wild-type in phenotype and in a serum calcium level regardless of age or gender. We have now examined directly whether the mutant mouse can absorb calcium from the intestine in response to the active form of vitamin D. The calbindin D9k null mutant mouse is fully able to absorb calcium from the intestine in response to 1,25-dihydroxyvitamin D3. It is, therefore, clear that calbindin D9k is not required for vitamin D-induced intestinal calcium absorption.     

Design, synthesis and pharmacological evaluation of cyclic mimetics of the insulin-like peptide 3 (INSL3) B-chain.

Insulin-like peptide 3 (INSL3) is a peptide hormone belonging to the relaxin-insulin superfamily of peptides that plays important roles in testes descent, oocyte maturation and the control of male germ cell apoptosis. These actions are mediated via a specific G-protein coupled receptor, LGR8. Previous structure-activity studies have shown that the key binding site of INSL3 is situated within its B-chain. Recent studies in our laboratory have led to the identification of a cyclic peptide mimetic 2 of the INSL3 B-chain, which we have shown to compete with the binding of [33P]-relaxin to LGR8 expressed in HEK293T cells, and to inhibit cAMP-mediated signaling in these cells, i.e. it is an antagonist of INSL3. In order to further define the structure-activity relationships of cyclic analogues of the INSL3 B-chain, we used a structure-based approach to design a series of cyclic, disulfide-constrained INSL3 B-chain mimetics. To do this, we first created a model of the 3D structure of INSL3 using the crystal structure of human relaxin as a template. This model of INSL3 was then used as a template to design a series of disulfide-constrained mimetics of the INSL3 B-chain. The peptides were synthesized by solid-phase peptide synthesis using pseudoproline dipeptides to improve the synthesis outcome. Of the seven prepared INSL3 B-chain mimetics, three compounds were found to have partial displacement activity, while four were able to completely displace [33P]-relaxin from LGR8, including compounds that were markedly shorter than compound 2. The best of these, mimetic 6, showed significantly greater affinity for LGR8 than compound 2, but still displayed around 1000-fold less affinity for LGR8 than native INSL3. Analysis of selected mimetics for their alpha-helical content using circular dichroism (CD) spectroscopy revealed that, generally, the mimetics showed less than expected helicity. The inability of the compounds to display true native INSL3 structure is likely contributing to their reduced receptor binding affinity. We are currently examining alternative INSL3 B-chain mimetics that might better present key receptor binding residues in the native INSL3-like conformation.     

Genetic diversity of Indian garlic core germplasm using agro-biochemical traits and SRAP markers

The characterization of garlic germplasm improves its utility, despite the fact that garlic hasn't been used much in the past. Garlic has an untapped genetic pool of immense economic and medicinal value in India. Hence, using heuristic core collection approach, a core set of 46 accessions were selected from 625 Indian garlic accessions based on 13 quantitative and five qualitative traits. The statistical measures (CV per cent, CR per cent, VR per cent) were used to sort the core set using Shannon-Wiener diversity index and the Nei diversity index. In addition, the variation within the core set was tested for 18 agro-morphological and six biochemical characteristics (allicin, phenol content, pyruvic acid, protein, allyl methyl thiosulfinate (AMTHS), and methyl allyl thiosulfinate (MATHS)). Further study of the core set's molecular diversity was performed using sequence related amplified polymorphism (SRAP) markers, which revealed a wide range of diversity among the core set's accessions, with an average polymorphism efficiency (PE) of 80.59 percent, polymorphism information content (PIC) of 0.29, effective multiplex ratio (EMR) of 3.51, and marker index (MI) of 0.99. The findings of this study will be useful in identifying high-yielding, elite garlic germplasm lines with the trait of interest. Since this core set is indicative of total germplasm, these selected breeding lines will be used for genetic improvement of garlic in the future.     

Courtship Behavior of Zaprionus indianus (Gupta) (Diptera: Drosophilidae) from Populations Colonizing South America

We describe for the first time the sexual behavior and the courtship song of males of the African fly Zaprionus indianus (Gupta), a recent invader of South America. The male courtship song is formed by monocyclic pulses and the courtship behavior is simple when compared to that of species of Drosophila. Two interpulse interval (IPI) distributions were observed: pre-mounting and mounting. No significant difference was observed between the pre-mounting IPIs of males that descended from three geographical populations from South America. We also observed the songs produced by females and the homosexual behavior exhibited by males. A sequence of bursts is produced by females as a refusal signal against males, while males emit a characteristic song that identifies sex genus, which differs from the courtship song. The short courtship and mating latencies recorded reveal vigorous males and receptive females, respectively.     

Plasmodium falciparum Rosetting Epitopes Converge in the SD3-Loop of PfEMP1-DBL1α

The ability of Plasmodium falciparum parasitized RBC (pRBC) to form rosettes with normal RBC is linked to the virulence of the parasite and RBC polymorphisms that weaken rosetting confer protection against severe malaria. The adhesin PfEMP1 mediates the binding and specific antibodies prevent sequestration in the micro-vasculature, as seen in animal models. Here we demonstrate that epitopes targeted by rosette disrupting antibodies converge in the loop of subdomain 3 (SD3) which connects the h6 and h7 α-helices of PfEMP1-DBL1α. Both monoclonal antibodies and polyclonal IgG, that bound to epitopes in the SD3-loop, stained the surface of pRBC, disrupted rosettes and blocked direct binding of recombinant NTS-DBL1α to RBC. Depletion of polyclonal IgG raised to NTS-DBL1α on a SD3 loop-peptide removed the anti-rosetting activity. Immunizations with recombinant subdomain 1 (SD1), subdomain 2 (SD2) or SD3 all generated antibodies reacting with the pRBC-surface but only the sera of animals immunized with SD3 disrupted rosettes. SD3-sequences were found to segregate phylogenetically into two groups (A/B). Group A included rosetting sequences that were associated with two cysteine-residues present in the SD2-domain while group B included those with three or more cysteines. Our results suggest that the SD3 loop of PfEMP1-DBL1α is an important target of anti-rosetting activity, clarifying the molecular basis of the development of variant-specific rosette disrupting antibodies.