Detection and localization of regucalcin in spermatozoa of water buffalo (Bubalus bubalis): A calcium‐regulating multifunctional protein

Regucalcin (RGN) is a calcium‐regulating, anti‐apoptotic, antioxidative and antiproliferative multifunctional protein predominantly seen in liver and kidney. All these functions are very crucial during spermatogenesis and sperm maturation process until fertilization of the ovum. Although many studies have reported the wide distribution of regucalcin in the male reproductive tract of the rat, human and bovine, its presence in spermatozoa is yet to be demonstrated wherein calcium has a pivotal role in the transport, capacitation, acrosomal reaction and further fusion with ova. Here, we detected the expression of regucalcin mRNA and protein in buffalo spermatozoa using real‐time PCR and Western blot, respectively. The study detected two new regucalcin isoforms of 44 kDa and 48 kDa size along with the reported 34‐kDa, 28‐kDa and 24‐kDa isoforms, wherein the 34‐kDa isoform was found to be membrane associated in spermatozoa. Further, immunocytochemistry study localized the regucalcin protein in the acrosomal region of the caudal and ejaculated buffalo spermatozoa while it was detected in both cytoplasm and acrosomal region of testicular spermatozoa. This discovery of RGN in spermatozoa and localization in the acrosomal region will help to focus researchers to see its role in calcium‐related functions like capacitation, acrosomal reaction and membrane fusion. Overall, regucalcin may be a new fertility marker in buffalo and can be utilized for infertility treatments.     

Primary structure of the abundant seed albumin of Theobroma cacao by mass spectrometry

The most abundant albumin present in seeds of Theobroma cacao was purified to apparent homogeneity as judged by high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and NH(2)-terminal sequence analysis. Tryptic peptide mass fingerprinting of the purified protein by HPLC/ESI-MS showed the presence of 16 masses that matched the expected tryptic peptides corresponding to 95% of the translated amino acid sequence from the cDNA of the 21 kDa cocoa albumin. Collision-induced dissociation MS/MS analysis of the C-terminal peptide isolated from the CNBr cleavage products provided unequivocal evidence that the mature cocoa albumin protein is nine amino acid residues shorter than expected from the reported cDNA of its corresponding gene. The experimentally determined M(r) value of 20234 was in excellent agreement with the truncated version of the amino acid sequence. The purified cocoa albumin inhibited the catalytic activities of bovine trypsin and chymotrypsin. The inhibition was stoichiometric with 1 mol of trypsin or chymotrypsin being inhibited by 1 mol of inhibitor with apparent dissociation constants (K(i)) of 9.5 x 10(-8) and 2. 3 x 10(-6) M, respectively, for inhibitor binding at pH 8.5 and 37 degrees C. No inhibition of the catalytic activities of subtilisin, papain, pepsin, and cocoa endoproteases was detected under their optimal reaction conditions.     

Isolation and characterization of 2S cocoa seed albumin storage polypeptide and the corresponding cDNA

The amine pool of cocoa is known to be an essential component for the development of the typical cocoa flavor. To better understand and to produce an intense in vitro cocoa flavor, identification of the polypeptides that are the source of the amine flavor precursor pool is essential. Chromatographic analysis of the polypeptide profile of unfermented cocoa resulted in identification of a novel storage polypeptide of M(r) 8515. The N-terminal sequence of the first 34 residues of the purified polypeptide shows similarity to 2S storage albumins of cotton and Brazil nut and sweet protein, Mabinlin. To identify the corresponding cDNA of the putative cocoa 2S albumin, 18 randomly chosen clones from the cDNA library of immature Theobroma cacao seed mRNA were sequenced, and a full-length cDNA clone encoding a protein harboring the N-terminal sequence of the novel polypeptide was selected. The open reading frame of the clone encodes a polypeptide of M(r) 17125. Comparison of the translated amino acid sequence of the precursor protein or the mature polypeptide against the Swiss-Prot and TrEMBL databases shows high sequence similarity (>52%) and identity (>38%) to many plant 2S albumins. Tryptic peptide mass fingerprinting of the purified polypeptide by high-performance liquid chromatography-electrospray ionization mass spectrometry shows 10 masses that match the expected tryptic peptides of the deduced sequence. Together with the published work on plant 2S albumin processing, the results presented here suggest that post-translational processing yields a 73-residue polypeptide (residue positions 78-150) corresponding to the 9 kDa subunit of the mature cocoa 2S albumin protein.     

Characterization of peptides formed during fermentation of cocoa bean.

Analysis by SDS-PAGE and GPC-MS of fermented cocoa extracts shows changes in the amount and composition of the major proteins, accompanied by formation of complex distributions of peptides. MS/MS studies and application of SEQUEST sequencing software have allowed identification of two related peptides, a hexapeptide and a nonapeptide, formed from vicilin, one of the cocoa storage proteins. Time course studies of the two peptides show different abundance profiles and indicate, in part, production of the hexapeptide from the nonapeptide.     

Minimal External Masculinization in a SRY-negative XX Male Podenco Dog

Normal mammalian sex differentiation takes place in three genetically controlled steps: chromosomal sex determination (XX or XY), gonadal differentiation and development of the phenotypic sex. Animals are considered to be sex reversed if chromosomal sex determination and gonadal development are not in agreement. In this report, sex reversal is described in a 1.5-year-old Podenco dog that was referred because of suspected recurrent growth of a previously removed os clitoridis in the vulva. With that exception the dog was phenotypically female, but had never been in oestrus and exhibited male behaviour. Abdominal ultrasonography showed a small tubular structure dorsal to the bladder, consistent with a uterus. An ovoid structure resembling a gonad was visible between the right kidney and inguinal canal. Plasma testosterone concentrations before and after GnRH administration indicated the presence of functional testicular tissue. Two testes, each with its epididymis and ductus deferens, and a complete bicornuate uterus were removed surgically. Cytogenetic analysis of peripheral blood lymphocytes showed a normal female karyotype (78, XX). These findings are consistent with the diagnosis of an XX male. PCR analysis of genomic DNA revealed that the SRY gene was absent. In summary, this report describes the first SRY-negative XX male Podenco dog with an almost complete female phenotype despite high basal and stimulated plasma testosterone concentrations. It is hypothesized that the clinical observations in this dog may have been caused by reduced and delayed Müllerian-inhibiting substance secretion and the absence of conversion of testosterone to dihydrotestosterone due to 5α-reductase deficiency.