Modelo animal de ectasia corneal en conejo mediante inyección intraestromal de colagenasa tipo ii / Animal model of corneal ectasia in rabbits by intrastromal injection of type II collagenase

Introducción La colagenasa ii ha sido utilizada para inducir queratocono experimental en modelos animales. Sin embargo, no ha sido estudiado su efecto cuando se administra por inyección intraestromal, por lo que el propósito de este estudio fue estudiar los efectos de la inyección intraestromal de colagenasa ii sobre la superficie corneal y la morfología de la córnea. Método Se trabajó con 6 conejos Nueva Zelanda, se administró colagenasa ii por inyección intraestromal (5μL de 2,5mg/mL) en los ojos derechos y solución salina balanceada en los ojos izquierdos. Se realizaron queratometrías para evaluar la alteración de la curvatura, también al séptimo día se obtuvieron las córneas y se realizó tinción hematoxilina-eosina para examinar los cambios morfológicos. Asimismo, se investigaron los cambios en la expresión de colágeno tipo i por tinción rojo sirio y PCR semicuantitativa. Resultados K1, K2 y Km presentaron diferencias en los promedios con cambios estadísticamente significativos. Los cambios morfológicos que se demostraron fueron degradación y disposición irregular del estroma corneal, incremento en la densidad celular de queratocitos y ligera infiltración celular. Finalmente se demostró que hay mayor expresión de fibras de colágeno tipo i en el grupo experimental a diferencia de los controles y el grosor de las fibras también aumentó por acción de la colagenasa ii; sin embargo, en cuestión génica no hubo cambios en la expresión de colágeno tipo i a nivel molecular entre el grupo control y experimental. Conclusiones La colagenasa ii administrada por inyección intraestromal es capaz de inducir cambios en la superficie corneal y el estroma, pudiendo simular un modelo de queratocono (AU)

Introduction Collagenase II has been used to induce experimental keratoconus in animal models. However, its effect when administered by intrastromal injection has not been studied, so the purpose of this study was to study the effects of intrastromal injection of collagenase II on corneal surface and corneal morphology. Method Six New Zealand rabbits were used, collagenase II was administered by intrastromal injection (5μL of 2.5mg/mL) in the right eyes and balanced salt solution in the left eyes. Keratometry was performed to evaluate curvature alteration, also at day 7 corneas were obtained and hematoxylin–eosin staining was performed to examine morphologic changes. Likewise, changes in type I collagen expression were investigated by Sirius Red staining and semi-quantitative PCR. Results K1, K2, and Km presented differences in the means with statistically significant changes. The morphological changes that were demonstrated were degradation and irregular arrangement of the corneal stroma, increase in the cellular density of keratocytes and slight cellular infiltration. Finally, it was demonstrated that there is greater expression of type I collagen fibers in the experimental group as opposed to the controls and the thickness of the fibers also increased due to the action of collagenase II, however, in terms of genetics there were no changes in the expression of type I collagen at molecular level between the control and experimental groups. Conclusions Collagenase II administered by intrastromal injection is able to induce changes in the corneal surface and stroma, being able to simulate a model of keratoconus (AU)

Animal model of corneal ectasia in rabbits by intrastromal injection of type II collagenase.

INTRODUCTION: Collagenase II has been used to induce experimental keratoconus in animal models. However, its effect when administered by intrastromal injection has not been studied, so the purpose of this study was to study the effects of intrastromal injection of collagenase II on corneal surface and corneal morphology. METHODS: Six New Zealand rabbits were used, collagenase II was administered by intrastromal injection (5µL of 2.5mg/mL) in the right eyes and balanced salt solution in the left eyes. Keratometry was performed to evaluate curvature alteration, also at day 7 corneas were obtained and Hematoxylin-Eosin staining was performed to examine morphologic changes. Likewise, changes in type I collagen expression were investigated by Sirius Red staining and semiquantitative PCR. RESULTS: K1, K2 and Km presented differences in the means with statistically significant changes. The morphological changes that were demonstrated were degradation and irregular arrangement of the corneal stroma, increase in the cellular density of keratocytes and slight cellular infiltration. Finally, it was demonstrated that there is greater expression of type I collagen fibers in the experimental group as opposed to the controls and the thickness of the fibers also increased due to the action of collagenase II, however, in terms of genetics there were no changes in the expression of type I collagen at molecular level between the control and experimental groups. CONCLUSIONS: Collagenase II administered by intrastromal injection is able to induce changes in the corneal surface and stroma, being able to simulate a model of keratoconus.

Lifetime prevalence of mood and anxiety disorders in fragile X premutation carriers.

OBJECTIVE: The authors studied the lifetime prevalence of DSM-IV-TR psychiatric disorders in a population of adults with the fragile X premutation. METHOD: The Structured Clinical Interview for DSM-IV was conducted, from 2007-2008, in 85 individuals with the fragile X premutation, 47 with the fragile X-associated tremor/ataxia syndrome (FXTAS; 33 male, 14 female; mean age = 66 years) and 38 without FXTAS (16 male, 22 female; mean age = 52 years). Lifetime prevalence for mood and anxiety disorders among carriers with and without FXTAS was compared to available age-specific population estimates from the National Comorbidity Survey Replication (NCS-R). RESULTS: Among participants with FXTAS, 30 (65%) met lifetime DSM-IV-TR criteria for a mood disorder; 24 (52%) met lifetime DSM-IV-TR criteria for an anxiety disorder. Among the non-FXTAS participants, there were 15 instances of lifetime mood disorder (42%) and 18 of lifetime anxiety disorder (47%). When compared to age-specific NCS-R data, the lifetime prevalences of any mood disorder (P < .0001), major depressive disorder (P < .0001), any anxiety disorder (P < .0001), panic disorder (P = .006), specific phobia (P = .0003), and posttraumatic stress disorder (P = .004) were significantly higher in participants with FXTAS. The lifetime rates of social phobia in individuals with the premutation without FXTAS were significantly higher than NCS-R data (P = .001). CONCLUSIONS: This sample of carriers of the fragile X premutation had a notably high lifetime risk of mood and anxiety disorders. Mood and anxiety disorders may be part of the clinical phenotype of the fragile X premutation conditions, especially in carriers with FXTAS. Clinicians encountering these patients are advised to consider FXTAS as a neuropsychiatric syndrome as well as a neurologic disorder.

High-risk fragile x screening in Guatemala: use of a new blood spot polymerase chain reaction technique.

BACKGROUND: Because fragile X syndrome (FXS) is prevalent, it has become the subject of newborn and high-risk screening efforts. International screening, however, can be financially and logistically prohibitive, particularly in countries where resources may be scarce. Recently, we have developed a screening test on blood spot that can detect expanded alleles from the normal through the full mutation range in both males and females. It is accurate, rapid, inexpensive, and applicable on blood spots and therefore ideal for international screening. The use of this blood spot screening technique was piloted in "a high-risk screening" study of individuals in Guatemala. METHODS: One hundred and five blood spots from subjects from Guatemala were screened for the Fragile X Mental Retardation 1 mutation. They were classified as "high-risk" through placement into one of the following five categories: (a) relatives of someone with a previous FXS diagnosis, (b) individuals with confirmed autism, (c) individuals with confirmed intellectual disability, (d) individuals with Parkinson's-like presentation, and (e) individuals with a family history of intellectual disability but no confirmed cases of FXS. RESULTS: Fifteen of the individuals tested yielded an expanded allele, 10 premutations and 5 full mutations. All 15 expansions were found in individuals with a relative with a confirmed FXS diagnosis. No expansions were found in the other clinical groups. CONCLUSIONS: Blood spot polymerase chain reaction screening is an effective, cost-efficient method to conduct cascade testing in families with a known history of FXS, even in small screening cohorts.

Assessment of seasonal variability of cytochemical responses to contaminant exposure in the blue mussel Mytilus edulis (complex).

A selected suite of cytochemical parameters in Mytilus edulis are altered in response to field and laboratory exposure to chemical contaminants. These biomarkers include lysosomal stability, nicotinamide adenine dinucleotide phosphate (NADPH)-ferrihemoprotein reductase activity, liposfuscin deposition, and accumulation of lysosomal and cytoplasmic unsaturated neutral lipid. Normal variations in physiological processes (influenced by exogenous seasonal changes in temperature, salinity, food availability, etc.) may alter the sensitivity of these biomarkers to contaminant exposure. To address this issue, M. edulis (complex) were sampled monthly from a reference nonurban site (Coupeville, Penn Cove) and a polluted urban site (Seacrest, Elliott Bay) in Puget Sound, WA, for a period of 15 months. Physiological measurements including total length, total weight, somatic and mantle weights (an indication of gonadal development and reproductive status), condition index, and the presence or absence of hemic neoplasia (HN, or leukemia) were recorded. Significant differences in lysosomal stability, lysosomal and cytoplasmic unsaturated neutral lipids, lipofuscin deposition, and NADPH-ferrihemoprotein reductase activity in cells of the digestive gland or digestive tubules were generally found in mussels taken throughout the year from Seacrest compared to mussels sampled from Coupeville, consistent with exposure to chemical contaminants. No seasonally influenced suppression of the entire suite of parameters as measures of contaminant exposure was evident. Therefore these biomarkers can be used to evaluate contaminant exposure in mussels throughout the entire year.

Intervención psicológica en catástrofes: ¿una necesidad? / Psychological counselling in catastrophes: a necessity?

En el presente artículo se expone una revisión de la investigación sobre Intervención Psicológica en Catástrofes. En primer lugar se ha realizado un screening sobre las experiencias publicadas de la intervención en desastres a nivel mundial y además en el ámbito de la intervención psicológica en España. Posteriormente en base al estudio epidemiológico de la incidencia de casos clínicos que se asocian a la eventualidad de un suceso de estas características, se plantea un modelo de intervención, dividiendo la línea de acción entre las víctimas (primeros auxilios psicológicos, intervención en crisis, tratamiento del estrés postraumático) e intervinientes (el debriefing, bunout) siguiendo el modelo preventivo dividido en fases (AU)

From sediment bioassay to fish biomarker--connecting the dots using simple trophic relationships.

Two common problems in applying and interpreting invertebrate bioassays and fish biomarkers in sediment toxicology are the wide gap between significant effects concentrations determined by these two approaches, and a general lack of ecological context. We have devised an exposure system that is able to reconcile much of the disparity between invertebrate bioassay and fish biomarker results by incorporating realistic ecological processes based on deposit feeding and predator-prey interactions. This system relates the disturbance of interest (sediment contamination) to biologically meaningful effects in a resource of interest (marine flatfish) via a realistic contaminant vector (a deposit-feeding polychaete worm). In this pilot study, polychaetes (Armandia brevis) were exposed for 28 days to clean sediments supplemented with benzo(a)pyrene (BaP), para-para dichlorodiphenyldichloroethylene (pp'DDE), Aroclor 1254, or field sediments collected from two sites in Puget Sound, Washington, contaminated predominantly with polcyclic aromatic hydrocarbons (PAHs) or chlorinated compounds. Exposed worms were then fed live to juvenile English sole (Pleuronectes vetulus) for 10 or 12 days. At the end of the exposure period, fish were measured for length and weight, sacrificed, and preserved for either routine histopathology and immunohistochemical analysis of cytochrome P450 1A induction, or 32P post-labeling determination of hepatic PAH-DNA adducts. Growth of predatory flatfish was lower than reference in all but one of eight groups fed contaminant-exposed polychaetes; however, statistically significant reductions in growth were only observed in three of these eight groups, at least in part due to low statistical power. Juvenile sole from all contaminant-exposed groups showed increased expression of CYP1A, and fish exposed to BaP-exposed worms showed clear evidence of hepatic PAH-DNA adducts. This method allows the concurrent evaluation of sediment contamination at multiple biological and ecological levels. These results indicate that sediments determined to be nontoxic by common invertebrate bioassays may have the potential to cause adverse effects at higher trophic levels.

Environmental contaminants and the prevalence of hemic neoplasia (leukemia) in the common mussel (Mytilus edulis complex) from Puget Sound, Washington, U.S.A.

The relationship between hemic neoplasia, a blood cell disorder in bivalve molluscs, and chemical contaminants was evaluated in the common mussel (Mytilus edulis complex). Hemic neoplasia (HN) is endemic to mussel populations in Puget Sound. The prevalence of hemic neoplasia ranged from 0 to 30% in mussels from nine sites in Puget Sound, Washington. Organic chemical contamination in sediment from these sites range from 0.1 to 64.0 ppm of polycyclic aromatic hydrocarbons (PAHs) and 0.07 to 0.50 ppm chlorinated hydrocarbons. No relationship between the body burden of environmental contaminants and the prevalence of HN in mussels was identified. To evaluate the short-term ability of chemical contaminants to induce HN in mussels, mussels, from a site where mussels were previously determined to be HN free, were fed microencapsulated PAHs (composed of a mixture of phenanthrene, flouranthene, and benzo[a]pyrene) or PCBs (Aroclor 1254) and the prevalence of HN was assessed after 30 days of exposure. Although an apparent increase in HN prevalence (20 to 30%) was observed in all treatments groups except the untreated controls, no significant difference in the prevalence of HN was observed between the control group of mussels fed corn oil (vehicle) and mussels fed either PAHs or PCBs in corn oil. A long-term (180-day) exposure study was conducted to evaluate the influence of PAHs or PCBs in modulating the prevalence of HN in a mussel population already exhibiting a moderate HN prevalence. Mussels, from a site where mussels were previously determined to exhibit a background prevalence of HN, fed microencapsulated PAHs, PCBs, and corn oil (vehicle) over a long time period (180 days), revealed an apparent increased prevalence of HN (30 to 40%) above the low levels (20%) initially present. However, no significant difference in the prevalence of HN was observed between the control group of mussels fed corn oil (vehicle) and mussels fed either PAHs or PCBs in corn oil. Although chemical contaminants have been proposed as a modulating factor in the development and promotion of HN in bivalve molluscs from environmentally stressed and degraded habitats, we find no evidence that chemical contaminants induce or promote the development of HN in the mussel M. edulis complex.

Comparative bioaccumulation of chlorinated hydrocarbons from sediment by two infaunal invertebrates.

Bioaccumulation of chlorinated hydrocarbons (CHs) from field-contaminated sediments by two infaunal invertebrates, Rhepoxynius abronius (a non-deposit feeding amphipod) and Armandia brevis (a nonselective, deposit-feeding polychaete), was examined and species responses were compared. Sediments were selected over a large geographical area of the Hudson-Raritan estuary to assess the potential for bioaccumulation from a typical urban estuary. Unlike polycyclic aromatic hydrocarbons (PAHs) from these sediments, concentrations of CHs in interstitial water (IW) indicated that partition coefficients (Koc) were generally as expected, especially when based on predicted, nonsorbed, interstitial water CH concentrations (IWfree). Correlations between amphipod and polychaete tissue residues revealed that these species were responding similarly to a gradient of CH concentrations in sediment. While tissue residues and BAFloc (lipid/organic carbon normalized bioaccumulation factor) values for the trichlorobiphenyls were similar for both species, accumulation in the polychaete was three to 10 times higher for the more hydrophobic PCBs, which was attributed to differences in the route of exposure. A negative correlation between the bioaccumulation factor (BAF) and total organic carbon (TOC) was found for both species, which was expected according to equilibrium partitioning theory. Because it was assumed that the amphipod was not feeding in these tests and the polychaete was ingesting sediment, comparison of their tissue residues and bioaccumulation factors was useful for highlighting the importance of sediment ingestion, especially for short-term, nonequilibrium exposures. These results may also help elucidate the limitations associated with assessing bioaccumulation and the resultant toxic response in standard 10-day toxicity tests with similar invertebrates.

Purification and characterization of versicolorin B synthase from Aspergillus parasiticus. Catalysis of the stereodifferentiating cyclization in aflatoxin biosynthesis essential to DNA interaction.

The absolute configuration of the dihydrobisfuran ring system characteristic of aflatoxin B1 is essential to the covalent reaction of its metabolically activated form with double-stranded DNA. The biosynthesis of this potent mycotoxin proceeds through three configurationally labile intermediates to racemic versiconal hemiacetal. Subsequent enzymatic cyclization establishes the stereochemistry of this, critical ring fusion in (-)-versicolorin B and is catalyzed by versicolorin B synthase (VBS). The isolation and purification of VBS from Aspergillus parasiticus (SU-1, ATCC 56775) and its kinetic characterization and attempted inactivation are described. Initial purification trials were plagued both by a chromophoric impurity which was difficult to remove and by low recoveries of active protein. The discovery of a remarkably broad pH range of enzyme stability and catalytic activity led to an efficient procedure involving preparative isoelectric focusing and ion exchange FPLC chromatography. The enzyme behaved as a dimer upon gel filtration and migrated with M(r) 78000 Da during denaturing gel electrophoresis. The UV spectrum of pure VBS gave no evidence of a bound chromophore. Detailed kinetic analysis of VBS revealed that this protein selects from two equilibrating enantiomers of versiconal hemiacetal to cyclize the appropriate antipode to optically pure versicolorin B. By varying the amount of enzyme to a fixed concentration of substrate, the rate of enzymic cyclization could be limited by the intrinsic rate of enantiomerization of the substrate under the conditions of reaction. It was possible to quantitate the dynamics of this substrate enantiomerization/cyclization process, to establish the role played by VBS, and to evaluate the significance of each to the overall biosynthesis of aflatoxin. The potential role of an acidic residue of the enzyme in catalysis was supported by analysis of the pH-rate profile of VBS and chemical labeling studies. Successful demonstration of competitive inhibition of VBS by a simple substrate analogue led to the design and synthesis of a potential mechanism-based inactivator of the protein.

Suppression of B-cell mediated immunity in juvenile chinook salmon (Oncorhynchus tshawytscha) after exposure to either a polycyclic aromatic hydrocarbon or to polychlorinated biphenyls.

Juvenile chinook salmon (Oncorhynchus tshawytscha) were injected intraperitoneally with either the polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[a]anthracene (DMBA)1 or with the commercial polychlorinated biphenyl (PCB) mixture, Aroclor 1254, to assess effects on the B-cell mediated immune response. B-cell mediated immunity was assessed by examination of the primary and secondary plaque-forming cell (PFC) responses of anterior kidney and splenic leukocytes to a T-independent antigen, TNP-keyhole limpet hemocyanin (TNP-KLH). Salmon exposed to DMBA at dosages of 20% or 1% of the 96 hr LD50 (12.7 mg and 0.6 mg/kg of salmon, respectively) or to PCBs at a dosage of 20% of the 96 hr LD50 (54.0 mg/kg of salmon) exhibited a suppressed PFC response. The secondary PFC response of anterior kidney and splenic leukocytes to both antigens and the primary splenic PFC response to TNP-LPS were suppressed in salmon exposed to either DMBA or PCBs. However, only the primary PFC response of anterior kidney leukocytes to TNP-LPS was suppressed in salmon exposed to PCBs and no suppression of this response was observed in salmon exposed to DMBA. Neither anterior kidney or splenic leukocytes from salmon exposed to DMBA or PCBs showed an altered primary PFC response to the T-dependent antigen, TNP-KLH. These results suggest that B-cell mediated immunity in salmon is suppressed by known mammalian immunosuppressants and that suppression of the PFC response observed previously in salmon from an urban estuary may be due to contaminant exposure.

Polyamines in ejaculated ram spermatozoa and their relationship with sperm motility.

Intracellular polyamine levels in ejaculated spermatozoa and seminal fluid from rams were determined by fluorescent spectroscopy of their dansyl derivatives. Relationships between the sperm polyamine content and sperm motility of six mature and eight pubescent rams were studied. Samples were collected from both groups once a month from August through October. Mature rams had a greater percentage of motile sperm cells than lambs (94% versus 73% in September and 92% versus 78% in October); higher spermidine content (36 versus 9 pmol/10(8) cells in September and 162 versus 55 pmol/10(8) cells in October); higher spermine content (984 versus 205 pmol/10(8) cells in September and 1,229 versus 414 pmol/10(8) cells in October); and higher total sperm polyamine content (1,021 versus 216 pmol/10(8) cells in September and 2,258 versus 973 pmol/10(8) cells in October). In the lambs, spermidine content increased (55 versus 9 pmol/10(8) cells); spermine content increased (414 versus 205 pmol/10(8) cells); and total sperm polyamine content increased (973 versus 215 pmol/10(8) cells) in October compared to September. Ejaculates with sperm motility higher than 85% had greater spermine (848 versus 234 pmol/10(8) cells in September and 1064 versus 449 pmol/10(8) cells in October), and total sperm polyamine content (882 versus 244 pmol/10(8) cells in September and 2,015 versus 1,008 pmol/10(8) cells in October) than ejaculates with less than 450 pmol total sperm polyamines/10(8) cells was 68% +/- 6% compared to 90% +/- 4% in cells with greater than 450 pmol (average for all ejaculates) total sperm polyamines/10(8) cells. These data suggest a positive relationship between sperm polyamine constant and sperm motility.(ABSTRACT TRUNCATED AT 250 WORDS)

Isotypes of protein kinase C in bovine sperm.

Protein kinase C is present in bovine epididymal sperm. The enzyme was partially purified by gel filtration on Sephacryl S-300. The Ca2+/phosphatidylserine-dependent histone phosphotransferase activity elutes from the gel filtration column in a manner corresponding to a Mr approximately 80 kDa. The activity peak also corresponds with [3H]phorbol 12,13-dibutyrate binding activity. Immunoblot analysis of the partially purified enzyme with isozyme-specific monoclonal antibodies revealed the presence of alpha-, beta-, and gamma-subspecies of protein kinase C. Indirect immunofluorescence showed that the antibodies against alpha-, beta-, and gamma-subspecies produced prominent staining of the postacrosomal region of the sperm head. In addition, beta-subspecies antibodies produced minor staining of the midpiece and gamma-subspecies antibodies produced a minor staining of the acrosomal region.

Casein kinase I in bovine sperm: purification and characterization.

A highly purified preparation of sperm casein kinase I was obtained by sequential chromatography with phosphocellulose, gel filtration on sephacryl S-300, Affi-gel blue and DEAE-Cellulose. The chromatographic behavior and properties of the enzyme suggest that the sperm enzyme is similar to casein kinase I from other tissues. Antibodies against calf thymus casein kinase I cross-react with the sperm enzyme. A special feature of the sperm enzyme is that the activity is stimulated by spermine.

Purification and characterization of polyamine-stimulated protein kinase (casein kinase II) from bovine spermatozoa.

Casein kinase II from bovine epididymal spermatozoa was purified to apparent homogeneity by repeated chromatography with phosphocellulose and gel filtration with sephacryl S-200. The purified enzyme exhibited a molecular mass of 130 kDa by gel filtration and displayed three polypeptide bands with molecular masses of 26, 33, and 36 kDa by SDS-polyacrylamide gel electrophoresis. Antibodies raised against calf thymus casein kinase II cross reacted with the three sperm polypeptides. Incubation of the holoenzyme with either [gamma-32P]ATP or [gamma-32P]GTP resulted in the phosphorylation of the 26-kDa subunit. The enzymatic activity with casein as substrate was strongly inhibited by nanomolar heparin and greatly stimulated by micromolar spermine. With casein as substrate, the specific activity of the pure enzyme (0.5 mumol/min/mg protein) was comparable to that of casein kinase II from other sources. Endogenous substrates of the kinase were demonstrated by incubating sperm cytosolic extracts with [gamma-32P]GTP, under conditions that limit the expression of other protein kinases, and analyzing the products by SDS-PAGE and autoradiography. Similar results were obtained when sperm extracts, suitably diluted to minimize endogenous casein kinase II, were incubated with [gamma-32P]GTP and aliquots of pure sperm casein kinase II. Low concentrations (50 microM) spermine strongly enhanced the phosphorylation of 92- and 106-kDa cytosolic proteins. Our results clearly show that casein kinase II is present in spermatozoa and that it shares many of the properties of the enzyme from other sources. Further, they indicate that the enzyme plays a role in mediating the phosphorylation state of sperm proteins.

Partial purification and characterization of an acetylcarnitine hydrolase from bovine epididymal spermatozoa.

We previously reported that intact epididymal spermatozoa from bulls and hamsters oxidize [1-14C]acetyl-L-carnitine to 14CO2 at about the same rate as they oxidize [1-14C]acetate. In addition, we showed that acetylcarnitine is hydrolyzed by a hydrolase present in the plasma membrane and that the carnitine moiety does not enter the cell. Here we report the partial purification of the acetylcarnitine hydrolase from bovine spermatozoa and describe some of its properties. The detergent-extracted enzyme was purified by FPLC using an anion-exchange Mono-Q column. The hydrolase activity eluted from the column with the application of 0.22 to 0.30 M NaCl and was separated from acetylcholinesterase activity, which eluted with 0.35 to 0.40 M NaCl. Specific inhibitors of acetylcholinesterase had little effect on acetylcarnitine hydrolase but p-hydroxymercuriphenylsulfonate was a potent inhibitor of the hydrolase. Kinetic studies of the hydrolase yielded a K'm of 6-10 mM for acetylcarnitine and a V'max of 0.16 nmol min-1 mg protein-1. Similar studies with the acetylcholinesterase yielded a K'm for acetylcholine of about 300 microM and a V'max of 165 nmol min-1 mg protein-1. Acetylcarnitine was a poor substrate for the acetylcholinesterase. Several acyl-L-carnitines were tested as substrates for the hydrolase and the preferred substrate was acetylcarnitine. The role of acetylcarnitine hydrolase in the metabolism of acetylcarnitine by epididymal spermatozoa is discussed.

The metabolism of acetylcarnitine and acetate by bovine and hamster epididymal spermatozoa.

Since acetylcarnitine has been identified in the epididymal plasma of many mammalian species, we investigated whether acetylcarnitine could serve as an energy substrate for epididymal bull and hamster spermatozoa. Intact caudal cells from both species oxidized [I-14C]acetyl-l-carnitine to 14CO2, in vitro, and the amount oxidized was dependent on time, substrate concentration, and cell number. Within each species, the rate of oxidation was the same as the rate at which free [1-14C]acetate was oxidized. Spermatozoa incubated with [3H]acetyl-L-carnitine hydrolyzed the compound and [3H]acetate accumulated in the medium. Unlabeled acetate added to the incubation medium competed with cellular uptake of [3H]acetate and resulted in further increase in [3H]acetate accumulation in the medium. Furthermore, the acetyl group of acetylcarnitine was oxidized by spermatozoa without concomitant uptake of the carnitine group. Purified plasma membrane vesicles contained an acetylcarnitine hydrolase activity that was solubilized from whole cells by detergents and that could be distinguished from acetylcholinesterase also present in the cells. The solubilized acetylcarnitine hydrolase activity was inhibited by p-hydroxymercuriphenylsulfonate, but not by the specific acetylcholinesterase inhibitors, eserine or BW63C47. The sulfhydryl blocker also inhibited the production of 14CO2 from [1-14C]acetylcarnitine by intact cells; acetylcholinesterase inhibitors did not. From estimates of sperm energy requirements, our results indicate that extracellular acetylcarnitine serves as a physiologically important energy substrate for maturing sperm cells.

Casein kinase II activity and polyamine-stimulated protein phosphorylation of cytosolic and plasma membrane proteins in bovine sperm.

A highly purified preparation of sperm cytosolic protein kinase was obtained by repeated chromatography with phosphocellulose. The preferred substrate of the enzyme was casein and the activity was not stimulated by added Ca2+, calmodulin, or cAMP. With casein as substrate, both ATP and GTP served as phosphate donors and the activity was inhibited by low micromolar heparin and stimulated by low millimolar spermine and spermidine. These properties are characteristic of casein kinase II from other cells. Endogenous protein substrates of the enzyme in sperm cytosolic fractions and in plasma membranes were demonstrated by incubating the preparations with [gamma-32P]GTP, under conditions unfavorable to other protein kinases, and analyzing the products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Spermine greatly enhanced the phosphorylation of three (55, 92, and 106 kDa) proteins in both cytosolic and plasma membrane preparations. Our results indicate that polyamines play a role in modulating the phosphorylation state of proteins in sperm and may further regulate sperm function through this mechanism.

Effect of bromobenzene and O-bromophenol on kidney and liver of English sole (Parophrys vetulus).

1. English sole (Parophrys vetulus) were injected intraperitoneally with a single dose of 9.8 mmol bromobenzene/kg of fish or 1.9 mmol O-bromophenol/kg of fish, both known renal toxicants in mammals. 2. Kidney, liver, gill spleen, intestines, heart and blood samples were subsequently obtained up to 48 hr post-injection for determination of microscopic lesions, concentrations of selected tissue antioxidants (glutathione and ascorbic acid), and selected serum parameters. 3. Bromobenzene and O-bromophenol were both found to be hepatotoxic in English sole, as indicated by the presence of hepatocellular coagulation necrosis and fatty change in the liver, altered glutathione and ascorbic acid levels in liver tissue, elevated serum aspartate aminotransferase and alkaline phosphatase activity and increased serum glucose and triglyceride levels. 4. No evidence of nephrotoxicity was found in English sole exposed to either toxicant. 5. It is concluded that bromobenzene and O-bromophenol cannot be used as model nephrotoxicants but can be used as hepatotoxicants in English sole.

Calmodulin-stimulated cyclic nucleotide phosphodiesterases in plasma membranes of bovine epididymal spermatozoa.

Cyclic nucleotide phosphodiesterase in the plasma membranes of bovine epididymal spermatozoa was stimulated by added Ca2+ and calmodulin. The rate of hydrolysis and responsiveness toward calmodulin was greater for cAMP than for cGMP. The kinetic analysis of the activity revealed two forms of phosphodiesterase with apparent Km values of 7.5 and 95 microM for cAMP. Calmodulin stimulated both of the activities by increasing the Vmax without affecting the Km's. The activity response with respect to Ca2+ concentration appears to be biphasic in both the absence and presence of added calmodulin. Trifluoperazine inhibited the Ca2+- and calmodulin-sensitive enzyme activity in a dose-dependent manner. The calmodulin-stimulated phosphodiesterase activity in the sperm plasma membranes can be solubilized and absorbed to a Calmodulin-Sepharose affinity column in the presence of Ca2+.