Analysis of the effects of polyphenols on human spermatozoa reveals unexpected impacts on mitochondrial membrane potential, oxidative stress and DNA integrity; implications for assisted reproductive technology.

The need to protect human spermatozoa from oxidative stress during assisted reproductive technology, has prompted a detailed analysis of the impacts of phenolic compounds on the functional integrity of these cells. Investigation of 16 individual compounds revealed a surprising variety of negative effects including: (i) a loss of mitochondrial membrane potential (Δψm) via mechanisms that were not related to opening of the permeability transition pore but associated with a reduction in thiol expression, (ii) a decline in intracellular reduced glutathione, (iii) the stimulation of pro-oxidant activity including the induction of ROS generation from mitochondrial and non-mitochondrial sources, (iv) stimulation of lipid peroxidation, (v) the generation of oxidative DNA damage, and (vi) impaired sperm motility. For most of the polyphenolic compounds examined, the loss of motility was gradual and highly correlated with the induction of lipid peroxidation (r=0.889). The exception was gossypol, which induced a rapid loss of motility due to its inherent alkylating activity; one consequence of which was a marked reduction in carboxymethyl lysine expression on the sperm tail; a post-translational modification that is known to play a key role in the regulation of sperm movement. The only polyphenols that did not appear to have adverse effects on spermatozoa were resveratrol, genistein and THP at doses below 100µM. These compounds could, therefore, have some therapeutic potential in a clinical setting.

The role of learning by a predator, Rivulus hartii, in the rare-morph survival advantage in guppies.

Negative frequency-dependent selection (NFDS), where rare types are favoured by selection, can maintain diversity. However, the ecological processes that mediate NFDS are often not known. Male guppies (Poecilia reticulata) exhibit extreme diversity of colour patterning and, in a previous field experiment, rare morphs had a survival advantage. Here, we test the hypothesis that predators impose NFDS because they are efficient at capturing familiar prey morphs, but are less efficient at capturing unfamiliar morphs. Over a series of trials, we presented Rivulus hartii, a natural predator of guppies, with male guppies with the same colour patterning (A trials); then, for a second series of trials, we presented the rivulus with guppies with a new colour pattern (B trials). The success of rivulus at capturing guppies on the first attack increased over successive A trials. First attack success decreased significantly for the early B trials, and then increased during successive B trials, eventually reaching the same level as in the best A trials. This experiment demonstrates that learning, perhaps through long-term search image formation, plays a role in predation success on familiar vs. unfamiliar prey morphs. These results support the hypothesis that predator learning contributes to the maintenance of the extreme male guppy polymorphism seen in nature.

Selection at the MHC class IIB locus across guppy (Poecilia reticulata) populations.

The highly diverse genes of the major histocompatibility complex (MHC) are important in the adaptive immune system and are expected to be under selection from pathogens. Thus, the MHC genes provide an exceptional opportunity to investigate patterns of selection within and across populations. In this study, we analyzed genetic variation at the MHC class IIB gene and six microsatellite loci across 10 populations of guppies (Poecilia reticulata) in the northern range of Trinidad. We found a high level of diversity at the MHC, with a total of 43 alleles in 142 individuals. At the population level, we found that neutral evolution could not fully account for the variability found at the MHC. Instead, we found that MHC F(ST) statistics were lower than F(ST) derived from the microsatellite loci; 33 of 45 population pairwise estimates for the MHC were significantly lower than those for the microsatellite loci, and MHC F(ST) estimates were consistently lower than those predicted by a coalescent model of neutral evolution. These results suggest a similar selection acting across populations, and we discuss the potential roles of directional and balancing selection. At the sequence level, we found evidence for both positive and purifying selection. Furthermore, positive selection was detected within and adjacent to the putative peptide-binding region (PBR) of the MHC. Surprisingly, we also found a purifying selection at two sites within the putative PBR. Overall, our data provide evidence for selection for functional diversity at the MHC class IIB gene at both the population and nucleotide levels of guppy populations.

MHC class IIB additive and non-additive effects on fitness measures in the guppy Poecilia reticulata.

The genetic architecture of fitness at the class IIB gene of the major histocompatibility complex (MHC) in the guppy Poecilia reticulata was analysed. Diversity at the MHC is thought to be maintained by some form of balancing selection; heterozygote advantage, frequency-dependent selection or spatially and temporally fluctuating selection. Here these hypotheses are evaluated by using an algorithm that partitions the effect of specific MHC allele and genotypes on fitness measures. The effect of MHC genotype on surrogate measures of fitness was tested, including growth rate (at high and low bulk food diets), parasite load following a parasite challenge and survival. The number of copies of the Pore_a132 MHC allele was inversely related to infection by Gyrodactylus flukes and it appeared to be positively related to faster growth. Also, genotypes combining the Pore_a132 or other relatively common alleles paired with rare MHC alleles produced both advantageous and detrimental non-additive effects. Thus, the genetic architecture underlying fitness at the MHC is complex in the P. reticulata.

Anaphylaxis to cisatracurium following negative skin testing.

General anaesthesia for the patient with a history of anaesthesia-related anaphylaxis is challenging. Precautions against anaphylaxis and the use of skin test negative drugs can reduce but not eliminate the risk. In the majority of such cases, subsequent anaesthesia is uneventful. However, the absence of a clearly identified triggering agent increases the difficulties facing the anaesthetist. We present a case of anaphylaxis to cisatracurium following a negative skin test.

Development of a physical characteristics assessment (PCA): a checklist for determining appropriate computer access for individuals with cerebral palsy.

The physical characteristics assessment (PCA), a newly developed illustrated checklist, is designed for use by allied health and education professionals to identify a viable method for an individual with cerebral palsy to access a computer. The PCA was developed in response to a documented void of such assessments in the literature. The value of PCA was assessed with respect to content validity, clinical utility, reliability (inter-rater), and opinion of persons with cerebral palsy and health and education professionals. Professionals (n = 72) and persons with cerebral palsy (n = 78) from 20 sites in Canada, Chile, England, Israel, Spain, and the United States participated in the study. Statistically, the PCA produced a highly significant (p < 0.001) assessment time savings compared with non-PCA methods. A statistically significant difference in the number of user/computer interface changes during five post-assessment computer training sessions was not shown between the PCA and non-PCA methods. Inter-rater reliability ranged between 90% and 100% (n = 33 pairs). Both persons with cerebral palsy assessed via the PCA and professionals using the PCA gave positive opinions. Overall results support the value of the PCA.

HPLC purification and separation of 5,5'-substituted-2,4-imidazolidinedithiones.

Several 5,5'-substituted-2,4-imidazolidinedithiones, synthesized from aldehydes or ketones, have been purified by HPLC using poly(styrene-divinylbenzene) packings. Purified 5,5'-substituted-2,4-imidazolidinedithiones have been identified in column effluent by UV absorbance and corroborated by mass spectrometry. Several silica-based, polymeric, and poly(styrene-divinylbenzene)-based packings were evaluated as matrices for resolution of a mixture of purified 5,5'-substituted-2,4-imidazolidinedithiones.

Identification of alpha-carboxamidated and carboxy-terminal glycine forms of peptides in bovine hypothalamus, bovine pituitary and porcine heart extracts.

Two chemical assays have been developed for identifying and quantifying peptides which either could be biologically active by virtue of their alpha-carboxamidation or could be substrates for peptidylglycine alpha-amidating mono-oxygenase. The first assay is specific for the alpha-carboxamide of peptides. Using bis[trifluoroacetoxy]iodobenzene, the alpha-carboxamide was converted via a Hoffman reaction into a primary amine, which was then quantified by ninhydrin. The second assay is specific for glycine at the carboxy-terminus of a peptide. Glycine at the carboxy-terminus was derivatized to form 2-thiohydantoin, which was then separated and quantified by reverse phase HPLC. These assays were used to detect peptides in HPLC-separated extracts of bovine hypothalamus, bovine anterior lobe pituitary and porcine heart which may be of biological interest.

Isolation and identification of a novel peptide from the accessory sex gland of the female house fly, Musca domestica.

A peptide of novel structure was isolated from accessory glands of the female house fly, Musca domestica by molecular-weight limit centrifugal ultrafiltration and reversed-phase HPLC. After amino acid and protein sequence analyses, the structure of this peptide was confirmed by chemical synthesis and plasma desorption mass spectral analysis to be Leu-Leu-Asn-Ala-Leu-Pro-Leu-Asp-Ala-Leu-Ser-Ser-Leu-Thr-Gly-NH2. The accessory gland peptide stimulated contraction of the house fly oviduct at concentrations of 10(-7) M and above.

Single photon emission computerized tomography in obsessive compulsive disorder: a preliminary study.

Patterns of cerebral perfusion in patients with obsessive compulsive disorder were evaluated using single photon emission computerized tomography. Eleven patients, who satisfied the DSM-III-R criteria and Research Diagnostic Criteria for the disorder, were evaluated using the distribution of 99m-Tc-HMPAO as a radiotracer. The Yale-Brown Obsessive Compulsive Scale was administered to each patient to assess the severity of their symptoms. The images obtained were evaluated qualitatively and semi-quantitatively by a physician in nuclear medicine who was blind to the patients' diagnoses. Eight of the 11 patients demonstrated asymmetric perfusion of the basal ganglia; the left side showed impaired perfusion in six patients.

Primary structure of two neuropeptide hormones with adipokinetic and hypotrehalosemic activity isolated from the corpora cardiaca of horse flies (Diptera).

The primary structures of two neuropeptides, Tabanus atratus adipokinetic hormone (Taa-AKH) and Tabanus atratus hypotrehalosemic hormone (Taa-HoTH), from the corpora cardiaca of horse flies (Diptera: Tabanidae) have been determined. Amino acid sequences of Taa-AKH (less than Glu-Leu-Thr-Phe-Thr-Pro-Gly-Trp-NH2) and Taa-HoTH (less than Glu-Leu-Thr-Phe-Thr-Pro-Gly-Trp-Gly-Tyr-NH2) (where less than Glu = pyroglutamic acid) were determined by automated gas-phase Edman degradation of the peptides deblocked by pyroglutamate aminopeptidase and by fast atom bombardment mass spectrometry. The hormones were synthesized, and the natural and synthetic peptides exhibited identical chromatographic, spectroscopic, and biological properties. When assayed in adult face fly males, Taa-AKH and Taa-HoTH demonstrated hyperlipemic activity, in addition, Taa-HoTH also demonstrated a significant hypotrehalosemic activity.

Surgical management of the severely multiply handicapped individual.

Thirty-four surgical procedures were performed over a 12-year period on a select population of severely multiply handicapped individuals. Heel cord lengthening, hamstring and adductor releases, as well as selected osteotomies, tenotomies, and arthrodeses (both wrist and foot) were performed. The objectives were improvement in sitting posture, care, and comfort. Independent evaluations of active function, passive function, cosmesis, self-esteem, limb posture, and ease of dressing and hygiene were assessed. Significant improvement was generally noted. No patient was made worse. Selected procedures in the severely multiply handicapped individual have a definite place in patient management.

cDNA sequence of rat liver fructose-1,6-bisphosphatase and evidence for down-regulation of its mRNA by insulin.

A coding-length clone of rat liver fructose-1,6-bisphosphatase (EC 3.1.3.11) was isolated by immunological screening of a cDNA library in lambda gt11. Its identity was verified by comparing the deduced amino acid sequence with that obtained by direct sequencing of a complete set of CNBr and proteolytic peptides from the purified protein. The enzyme subunit is composed of 362 amino acids and has N-acetylvaline as the amino-terminal residue. The cDNA, 1255 base pairs (bp) long, consisted of 1086 bp of coding region, 15 bp of 5' untranslated sequence, and 154 bp at the 3' untranslated end. The 3' untranslated sequence contained a polyadenylylation signal (AATAAA) followed after 30 bp by a stretch of 7 adenines at the end of the clone. The deduced amino acid sequence was identical to the primary sequence of the protein and confirmed the alignment of five nonoverlapping peptides. It also confirmed the 27-residue extension, unique to the rat liver subunit, ending with a carboxyl-terminal phenylalanine. RNA blot analyses using the radiolabeled liver cDNA as a probe revealed a single band of fructose-1,6-bisphosphatase mRNA, 1.4 kilobases long, in liver and kidney but not in nongluconeogenic tissues. Fructose-1,6-bisphosphatase mRNA was increased 10-fold in livers from diabetic rats and was reduced to control levels after 24 hr of insulin treatment, suggesting that the changes in enzyme activity observed in diabetes and after insulin treatment are due to alterations in mRNA abundance.

Isolation and primary structure of a neuropeptide hormone from Heliothis zea with hypertrehalosemic and adipokinetic activities.

A neuropeptide was isolated from the corpora cardiaca of the corn earworm moth Heliothis zea, and purified by sequential gradient elution in three reversed phase-high performance liquid chromatographic steps. The primary structure, pGlu-Leu-Thr-Phe-Ser-Ser-Gly-Trp-Gly-Asn-NH2 was determined by automated gas-phase Edman degradation of the peptide deblocked with pyroglutamate aminopeptidase, and confirmed by fast atom bombardment mass spectrometry. The hormone was synthesized and the natural and synthetic peptides had identical chromatographic and spectroscopic properties. Both natural and synthetic hormones caused the elevation of trehalose and lipid levels in the hemolymph of adult H. zea males.

Isolation of two neuropeptides in the AKH/RPCH-family from horseflies (Diptera).

Two neuropeptides (DCCI and DCCII) in the adipokinetic/red pigment concentrating hormone-family have been isolated and purified from the corpora cardiaca of horseflies (Diptera : Tabanidae). Both peptides were purified by a sequence of three reversed phase-high performance liquid chromatographic steps. Amino acid analysis of the purified peptides indicated the following composition for DCCI: Glx(l), Gly(1), Leu(1), Phe(1), Pro(1), Thr(2) and for DCCII: Glx(1), Gly(2), Leu(1), Phe(1), Pro(1), Thr(2), and Tyr(1). Photodiode array ultraviolet spectroscopy indicated the presence of tryptophan in both DCCI and II. Both DCCI and II had red pigment concentrating hormone activity in the crab, Uca pugilator.

Complete amino acid sequence of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

The complete amino acid sequence of 6-phospho-fructo-2-kinase/fructose-2,6-bisphosphatase from rat liver was determined by direct analysis of the S-carboxamidomethyl protein. A complete set of nonoverlapping peptides was produced by cleavage with a combination of cyanogen bromide and specific proteolytic enzymes. The active enzyme is a dimer of two identical polypeptide chains composed of 470 amino acids each. The NH2-terminal amino acid residue of the polypeptide chain was shown to be N-acetylserine by fast atom bombardment mass spectrometry of the purified N-terminal tetradecapeptide isolated after cleavage of the intact S-carboxamidomethylated protein with lysyl endoproteinase (Achromobacter protease I). Alignment of the set of unique peptides was accomplished by the analysis of selected overlapping peptides generated by proteolytic cleavage of the intact protein and the larger purified cyanogen bromide peptides with trypsin, Staphylococcus aureus V8 protease, and lysyl endoproteinase. Four nonoverlapping peptides were aligned by comparison with the amino acid sequence predicted from a partial cDNA clone encoding amino acid positions 166-470 of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Colosia, A.D., Lively, M., El-Maghrabi, M. R., and Pilkis, S. J. (1987) Biochem. Biophys. Res. Commun. 143, 1092-1098). The nucleotide sequence of the cDNA corroborated the peptide sequence determined by direct methods. A search of the Protein Identification Resource protein sequence database revealed that the overall amino acid sequence appears to be unique since no obviously homologous sequences were identified. However, a 100-residue segment of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (residues 250-349), including the active site histidine residue of the bisphosphatase domain, was found to be homologous to the active site regions of yeast phosphoglycerate mutase and human bisphosphoglycerate mutase.

Effect of culture conditions on the structure of Streptococcus pneumoniae type 19A(57) capsular polysaccharide.

The structural modifications and immunochemical activities of several Streptococcus pneumoniae type 19A polysaccharide (PS) preparations have been studied by sugar compositional analysis and immunodiffusion. The 19A PS preparations Lab-A-1 and Lab-A-3 and one PS isolated from 19A strain OB contained fucose (Fuc) and galactose (Gal) in addition to rhamnose (Rha) and glucose (Glc). In contrast, 19A PSs Lab-A-2 and Lab-B contained only Rha and Glc. Despite their different sugar compositions, these 19A preparations appeared to be identical in serologic activity as measured by immunodiffusion with rabbit 19A and 19F antisera. The 19A PS Lab-A-1 was separated into three fractions by DEAE-Sepharose CL-6B column chromatography with a NaCl gradient. Fraction II was the major peak with a yield of 72.9%. Fraction Ia contained Fuc and Gal, while fraction II contained Fuc, Gal, Rha, and Glc. Fractions Ia and Ib did not react with rabbit 19A antiserum. In contrast, 19A PS Lab-A-2 displayed only one peak, which was eluted by a NaCl gradient (0 to 0.6 M NaCl), and contained only Rha and Glc. The 19A PSs prepared from Lab-A and Centers for Disease Control (CDC) strains and grown in pneumococcal inoculum medium (PIM) and modified Holt medium were chromatographed on a DEAE-Sepharose CL-6B column, and the separated fractions were examined for their sugar composition. The fractions obtained from the 19A PSs Lab-A-PIM and CDC-PIM exhibited four sugar components, as observed for the PS Lab-A-1, while the separated fractions from the 19A PSs Lab-A-Holt and CDC-Holt displayed two sugar components, a pattern similar to that of PS Lab-A-2. Thus, the sugar compositions of 19A PS appeared to vary according to the type of culture medium used to grow the 19A organisms.

Use of immobilized exopeptidases and volatile buffers for analysis of peptides by fast atom bombardment mass spectrometry.

beta-Lipotrophin (62-77) or Ac-gastrin releasing peptide was incubated with immobilized carboxypeptidase Y or aminopeptidase M. Subsequent aliquots of each incubation mixture were analysed by fast atom bombardment mass spectrometry using a dithiothreitol/dithioerythritol liquid matrix. The use of immobilized enzymes and volatile buffers for exopeptidase digestions enabled rapid and facile separation of enzyme from digestion products. This approach to mass spectral peptide analysis reduced spectral background arising from a glycerol matrix, buffer salts, or enzyme proteins and contaminants, enabling analysis of as little as 200 picomoles of a suitable peptide.

Analysis of peptides containing oxidized methionine and/or tryptophan by fast atom bombardment mass spectrometry.

Treatment of peptides containing methionine and/or tryptophan with dimethylsulfoxide/hydrochloric acid/acetic acid resulted in oxidation of these amino acids respectively to methionine sulfoxide and oxyindolalanine. This reaction was monitored by fast atom bombardment mass spectrometry using a dithiothreitol/dithioerythritol liquid matrix. Under these conditions, only methionine and tryptophan were oxidized. Comparison of mass spectra of a sample before and after oxidation should provide a rapid screening procedure for determination of these residues in peptides.