Some peptide-like colocalizations in endocrine cells of the pyloric caeca and the intestine of Oncorhynchus mykiss (Teleostei)

Summary The coexistence of immunoreactivities to cholecystokinin, glucagon, glucagon-like peptide 1, salmon pancreatic polypeptide, neuropeptide tyrosine, and peptide tyrosine tyrosine was studied immunocytochemicaly, revealing for the first time in fish intestine the existence in the same cell of immunoreactivities to cholecystokinin-glucagon/glucagon-like peptide 1, cholecystokinin-salmon pancreatic polypeptide, glucagon/glucagon-like peptide 1-salmon pancreatic polypeptide, glucagon/glucagon-like peptide 1-neuropeptide tyrosine, salmon pancreatic polypeptide tyrosine tyrosine, and glucagon/glucagon-like peptide 1-peptide tyrosine tyrosine. Colocalization of cholecystokinin-salmon pancreatic polypeptide was observed only in the pyloric caeca of the rainbow trout Oncorhynchus mykiss, while the other colocalizations also occurred in proximal and middle intestinal segments. In all cases, endocrine cells immunoreactive to only one of the paired antisera were detected except for anti-glucagon and anti-glucagon-like peptide 1, which always immunostained the same cells.     

alpha-Tocopherol modifies tyrosine phosphorylation and capacitation-like state of cryopreserved porcine sperm

Sperm cryopreservation is associated with the production of reactive oxygen species (ROS) leading to membrane destabilization, which induces capacitation-like changes, increases protein tyrosine phosphorylation, and decreases their fertilizing ability. alpha-Tocopherol, a lipid peroxidation inhibitor, preserves the functionality of cryopreserved porcine sperm. Our aim was to evaluate the effect of alpha-tocopherol on sperm quality parameters as well as capacitation-like changes and modifications in protein tyrosine phosphorylation. Boar sperm frozen with or without 200 microg/mL of alpha-tocopherol were thawed and maintained at 37 degrees C for 10 min in BTS. Routine parameters of semen quality were evaluated by optical microscopy and membrane changes were determined by the epifluorescence chlortetracycline technique. Changes in protein tyrosine phosphorylation were examined using a specific anti-phosphotyrosine monoclonal antibody. Motility was higher (18%, P<0.05) in semen with alpha-tocopherol. Viability did not differ (P>0.05) between treatments. However, there was less (P<0.05) capacitation-like changes in semen with alpha-tocopherol compared to control samples. A MW 32 kDa tyrosine-phosphorylated protein was detected in extracts of cryopreserved sperm; the intensity of immunostaining was lower in semen containing alpha-tocopherol compared to the control (0.211+/-0.030 versus 0.441+/-0.034 arbitrary units). Additionally, this band was not detected in fresh sperm. The addition of alpha-tocopherol to the extender prior to cryopreservation of boar semen protected sperm membranes against oxidative damage and reduced both tyrosine phosphorylation and the capacitation-like state.     

Replication slippage may cause parallel evolution in the secondary structures of mitochondrial transfer RNAs

Presence of the dihydrouridine (D) stem in the mitochondrial cysteine tRNA is unusually variable among lepidosaurian reptiles. Phylogenetic and comparative analyses of cysteine tRNA gene sequences identify eight parallel losses of the D-stem, resulting in D-arm replacement loops. Sampling within the monophyletic Acrodonta provides no evidence for reversal. Slipped-strand mispairing of noncontiguous repeated sequences during replication or direct replication slippage can explain repeats observed within cysteine tRNAs that contain a D-arm replacement loop. These two mechanisms involving replication slippage can account for the loss of the cysteine tRNA D-stem in several lepidosaurian lineages, and may represent general mechanisms by which the secondary structures of mitochondrial tRNAs are altered.      

Phosphorylant capacity study and lactate mitochondrial oxidation in frozen bovine sperm

Frozen-stored bovine sperm-pellets of proven fertility were used, and the response to respiratory chain effectors was studied, thus demonstrating the energy conservation capacity. It was further observed that the assayed suspensions used lactate oxidatively, which proves the LDH-X mitochondrial activity (the presence of oxidative substrates is fundamental in capacitation and acrosome reaction processes). The suspensions were treated with 10mM phosphate buffer hypotonic medium to eliminate plasmalema and cytoplasmic content. Lactate respiration was sensitive to respiratory chain effectors, such as oligomycin and antimycin. To evaluate the LDH-X contribution to mitochondrial respiration, lipoate dehydrogenase was inhibited through 5-methoxyindole-2-carboxylic acid (MICA) in the presence of pyruvate-malate and citrate-malate, obtaining with the addition of lactate, oxygen uptakes of 18% and 51% with respect to respiration with the mentioned substrates. In the MICA dose-effect curve, a major sensitivity to inhibitor in active state mitochondrial respiration is obtained when pyruvate-malate is used. Lactate competence with pyruvate by mitochondrial LDH-X was observed. The results obtained would allow the thorough study of the necessity of oxidative energy in the capacitation and fertilization processes, and of the LDH-X role in frozen-stored bovine sperm.     

cDNA cloning of a developmentally regulated hemocyanin subunit in the crustacean Cancer magister and phylogenetic analysis of the hemocyanin gene family

The complete cDNA sequence and protein reading frame of a developmentally regulated hemocyanin subunit in the Dungeness crab (Cancer magister) is presented. The protein sequence is aligned with 18 potentially homologous hemocyanin-type proteins displaying apparent sequence similarities. Functional domains are identified, and a comparison of predicted hydrophilicities, surface probabilities, and regional backbone flexibilities provides evidence for a remarkable degree of structural conservation among the proteins surveyed. Parsimony analysis of the protein sequence alignment identifies four monophyletic groups on the arthropodan branch of the hemocyanin gene tree: crustacean hemocyanins, insect hexamerins, chelicerate hemocyanins, and arthropodan prophenoloxidases. They form a monophyletic group relative to molluscan hemocyanins and nonarthropodan tyrosinases. Arthropodan prophenoloxidases, although functionally similar to tyrosinases, appear to belong to the arthropodan hexamer- type hemolymph proteins as opposed to molluscan hemocyanins and tyrosinases.      

Microsatellite allele frequencies in humans and chimpanzees, with implications for constraints on allele size

The distributions of allele sizes at eight simple-sequence repeat (SSR) or microsatellite loci in chimpanzees are found and compared with the distributions previously obtained from several human populations. At several loci, the differences in average allele size between chimpanzees and humans are sufficiently small that there might be a constraint on the evolution of average allele size. Furthermore, a model that allows for a bias in the mutation process shows that for some loci a weak bias can account for the observations. Several alleles at one of the loci (Mfd 59) were sequenced. Differences between alleles of different lengths were found to be more complex than previously assumed. An 8-base-pair deletion was present in the nonvariable region of the chimpanzee locus. This locus contains a previously unrecognized repeated region, which is imperfect in humans and perfect in chimpanzees. The apparently greater opportunity for mutation conferred by the two perfect repeat regions in chimpanzees is reflected in the higher variance in repeat number at Mfd 59 in chimpanzees than in humans. These data indicate that interspecific differences in allele length are not always attributable to simple changes in the number of repeats.      

Monoclonal antibodies against chicken type V collagen: production, specificity, and use for immunocytochemical localization in embryonic cornea and other organs

Two monoclonal antibodies have been produced against chick type V collagen and shown to be highly specific for separate, conformational dependent determinants within this molecule. When used for immunocytochemical tissue localization, these antibodies show that a major site for the in situ deposition of type V is within the extracellular matrices of many dense connective tissues. In these, however, it is largely in a form unavailable to the antibodies, thus requiring a specific “unmasking” treatment to obtain successful immunocytochemical staining. The specificity of these two IgG antibodies was determined by inhibition ELISA, in which only type V and no other known collagen shows inhibition. In ELISA, mixtures of the two antibodies give an additive binding reaction to the collagen, suggesting that each is against a different antigenic determinant. That both antigenic determinants are conformational dependent, being either in, or closely associated with, the collagen helix is demonstrated by the loss of antibody binding to molecules that have been thermally denatured. The temperature at which this occurs, as assayed by inhibition ELISA, is very similar to that at which the collagen helix melts, as determined by optical rotation. This gives strong additional evidence that the antibodies are directed against the collagen. The antibodies were used for indirect immunofluorescence analyses of cryostat sections of corneas and other organs from 17 to 18-day-old chick embryos. Of all tissues examined only Bowman’s membrane gave a strong staining reaction with cryostat sections of unfixed material. Staining in other areas of the cornea and in other tissues was very light or nonexistent. When, however, sections were pretreated with pepsin dissolved in dilute HAc or, surprisingly, with the dilute HAc itself dramatic new staining by the antibodies was observed in most tissues examined. The staining, which was specific for the anti-type V collagen antibodies, was largely confined to extracellular matrices of dense connective tissues. Experiments using protease inhibitors suggested that the “unmasking” did not involve proteolysis. We do not yet know the mechanism of this unmasking; however, one possibility is that the dilute acid causes swelling or conformational changes in a type-V collagen-containing supramolecular structure. Further studies should allow us to determine whether this is the case.     

Influence of antioxidants on SOD activity in bovine sperm

SOD activity and susceptibility to peroxidation on spermatozoa from frozen and fresh bovine semen were determined either in presence or not of synthetic (BHA) or natural (vitamin E) antioxidants. In sperm suspensions incubated with vitamin E, SOD activity was higher than in the control samples and the ones treated with BHA. It was found a highly significative correlation between malondialdehyde production and SOD activity. SOD activity could be used in bovine spermatozoa as a metabolic indicator of membrane integrity.     

A novel deconvolution method for modeling UDP-N-acetyl-D-glucosamine biosynthetic pathways based on 13C mass isotopologue profiles under non-steady-state conditions

This article is a response to Wang and Luo. See correspondence article http://www.biomedcentral.com/1741-7007/10/30/ [WEBCITE] and the original research article http://www.biomedcentral.com/1741-7007/9/24 [WEBCITE].     

Colocalization of immunoreactivities to serotonin, calcitonin and CGRP in endocrine pulmonary cells of the iberian lizard Podarcis hispanica (Reptilia)

The coexistence of immunoreactivities to serotonin (5HT), calcitonin (CT) and calcitonin gene-related peptide (CGRP) was studied in pulmonary endocrine cells of the Iberian lizard by immunocytochemistry and in semithin/thin sections under light and electron microscope. Immunostaining of serial sections revealed coexistence of 5HT/CT/CGRP immunoreactivities in some cells, while in others only 5HT/CT or CGRP immunoreactivities were found. Appropriate absorption controls excluded crossreactivity between the antisera used. Ultrastructurally, cells immunoreactive to 5HT/CT and CGRP share similar features, with round or slightly ovoid secretory granules of mean diameter from 165 to 180 nm. The possible functional significance of the copresence of 5HT, CT and CGRP is discussed.