Addition of Cryoprotectant Significantly Alters the Epididymal Sperm Proteome

Although cryopreservation has been developed and optimized over the past decades, it causes various stresses, including cold shock, osmotic stress, and ice crystal formation, thereby reducing fertility. During cryopreservation, addition of cryoprotective agent (CPA) is crucial for protecting spermatozoa from freezing damage. However, the intrinsic toxicity and osmotic stress induced by CPA cause damage to spermatozoa. To identify the effects of CPA addition during cryopreservation, we assessed the motility (%), motion kinematics, capacitation status, and viability of epididymal spermatozoa using computer-assisted sperm analysis and Hoechst 33258/chlortetracycline fluorescence staining. Moreover, the effects of CPA addition were also demonstrated at the proteome level using two-dimensional electrophoresis. Our results demonstrated that CPA addition significantly reduced sperm motility (%), curvilinear velocity, viability (%), and non-capacitated spermatozoa, whereas straightness and acrosome-reacted spermatozoa increased significantly (p < 0.05). Ten proteins were differentially expressed (two decreased and eight increased) (>3 fold, p < 0.05) after CPA, whereas NADH dehydrogenase flavoprotein 2, f-actin-capping protein subunit beta, superoxide dismutase 2, and outer dense fiber protein 2 were associated with several important signaling pathways (p < 0.05). The present study provides a mechanistic basis for specific cryostresses and potential markers of CPA-induced stress. Therefore, these might provide information about the development of safe biomaterials for cryopreservation and basic ground for sperm cryopreservation.     

Design,synthesis, and evaluation of curcumin analogues as potential inhibitors of bacterial sialidase

Sialidases are key virulence factors that remove sialic acid from the host cell surface glycan, unmasking receptors that facilitate bacterial adherence and colonisation. In this study, we developed potential agents for treating bacterial infections caused by Streptococcus pneumoniae Nan A that inhibit bacterial sialidase using Turmeric and curcumin analogues. Design, synthesis, and structure analysis relationship (SAR) studies have been also described. Evaluation of the synthesised derivatives demonstrated that compound 5e was the most potent inhibitor of S. pneumoniae sialidase (IC₅₀?=?0.2?±?0.1?µM). This compound exhibited a 3.0-fold improvement in inhibitory activity over that of curcumin and displayed competitive inhibition. These results warrant further studies confirming the antipneumococcal activity 5e and indicated that curcumin derivatives could be potentially used to treat sepsis by bacterial infections.     

Catalysis by dienelactone hydrolase: A variation on the protease mechanism

Dienelactone hydrolase (DLH), an enzyme from the β-ketoadipate pathway, catalyzes the hydrolysis of dienelactone to maleylacetate. Our inhibitor binding studies suggest that its substrate, dienelactone, is held in the active site by hydrophobic interactions around the lactone ring and by the ion pairs between its carboxylate and Arg-81 and Arg-206. Like the cysteine/serine proteases, DLH has a catalytic triad (Cys-123, His-202, Asp-171) and its mechanism probably involves the formation of covalently bound acyl intermediate via a tetrahedral intermediate. Unlike the proteases, DLH seems to protonate the incipient leaving group only after the collapse of the first tetrahedral intermediate, rendering DLH incapable of hydrolyzing amide analogues of its ester substrate. In addition, the triad His probably does not protonate the leaving group (enolate) or deprotonate the water for deacylation; rather, the enolate anion abstracts a proton from water and, in doing so, supplies the hydroxyl for deacylation. © 1993 Wiley-Liss, Inc.     

Systematic Screening of Drosophila Deficiency Mutations for Embryonic Phenotypes and Orphan Receptor Ligands

This paper defines a collection of Drosophila deletion mutations (deficiencies) that can be systematically screened for embryonic phenotypes, orphan receptor ligands, and genes affecting protein localization. It reports the results of deficiency screens we have conducted that have revealed new axon guidance phenotypes in the central nervous system and neuromuscular system and permitted a quantitative assessment of the number of potential genes involved in regulating guidance of specific motor axon branches. Deficiency “kits” that cover the genome with a minimum number of lines have been established to facilitate gene mapping. These kits cannot be systematically analyzed for phenotypes, however, since embryos homozygous for many deficiencies in these kits fail to develop due to the loss of key gene products encoded within the deficiency. To create new kits that can be screened for phenotype, we have examined the development of the nervous system in embryos homozygous for more than 700 distinct deficiency mutations. A kit of ∼400 deficiency lines for which homozygotes have a recognizable nervous system and intact body walls encompasses >80% of the genome. Here we show examples of screens of this kit for orphan receptor ligands and neuronal antigen expression. It can also be used to find genes involved in expression, patterning, and subcellular localization of any protein that can be visualized by antibody staining. A subset kit of 233 deficiency lines, for which homozygotes develop relatively normally to late stage 16, covers ∼50% of the genome. We have screened it for axon guidance phenotypes, and we present examples of new phenotypes we have identified. The subset kit can be used to screen for phenotypes affecting all embryonic organs. In the future, these deficiency kits will allow Drosophila researchers to rapidly and efficiently execute genome-wide anatomical screens that require examination of individual embryos at high magnification.     

Soil carbon sequestration and land‐use change: processes and potential

When agricultural land is no longer used for cultivation and allowed to revert to natural vegetation or replanted to perennial vegetation, soil organic carbon can accumulate. This accumulation process essentially reverses some of the effects responsible for soil organic carbon losses from when the land was converted from perennial vegetation. We discuss the essential elements of what is known about soil organic matter dynamics that may result in enhanced soil carbon sequestration with changes in land‐use and soil management. We review literature that reports changes in soil organic carbon after changes in land‐use that favour carbon accumulation. This data summary provides a guide to approximate rates of SOC sequestration that are possible with management, and indicates the relative importance of some factors that influence the rates of organic carbon sequestration in soil. There is a large variation in the length of time for and the rate at which carbon may accumulate in soil, related to the productivity of the recovering vegetation, physical and biological conditions in the soil, and the past history of soil organic carbon inputs and physical disturbance. Maximum rates of C accumulation during the early aggrading stage of perennial vegetation growth, while substantial, are usually much less than 100 g C m^?2 y^?1. Average rates of accumulation are similar for forest or grassland establishment: 33.8 g C m^?2 y^?1 and 33.2 g C m^?2 y^?1, respectively. These observed rates of soil organic C accumulation, when combined with the small amount of land area involved, are insufficient to account for a significant fraction of the missing C in the global carbon cycle as accumulating in the soils of formerly agricultural land.     

Detection of asymptomatic herpes simplex virus infections after vaccination.

Twenty-two volunteers seronegative for antibodies to herpes simplex virus (HSV) were enrolled in a trial to determine tolerance and immunogenicity of an HSV-2 glycoprotein subunit vaccine. Vaccine was administered at days 0, 28, and 140, and sera were obtained on days 0, 7, 14, 21, 28, 35, 49, 56, 140, 147, and 365 for determination of HSV neutralizing antibody activity and antibody-dependent cell cytotoxicity (ADCC). Sera were also tested by immunoprecipitation of radiolabeled HSV-2-infected cell proteins and polyacrylamide gel electrophoresis to identify the viral proteins which elicited antibody responses in vaccine recipients. After vaccination two male volunteers presented with atypical first-episode genital herpes: patient 1 with a culture-negative genital lesion at day 53 and patient 3 with urethritis at day 68. Seroconversion to wild-type viral proteins not present in the vaccine was detectable by radioimmunoprecipitation-polyacrylamide gel electrophoresis within 10 days in both patients. Two additional volunteers, one a sex contact of patient 1, seroconverted asymptomatically to nonvaccine proteins during the trial. All four vaccine breakthrough patients were indistinguishable from the other volunteers in the time required to develop neutralizing and ADCC antibodies, in the titer of these antibodies, and the time to seroconversion to gB and gD vaccine proteins. However, only one of the four breakthrough patients had antibodies to g80 (a complex of gC-2 and gE) after vaccination as compared with 15 of the other 18 volunteers (P = 0.05). Neither neutralizing antibody nor ADCC titers consistently identified acquisition of wild-type viral infection; therefore, protein-specific serologies were required to detect wild-type antibodies in these four patients. These data underscore the importance of using serologic assays which will distinguish naturally acquired infection from the immune response to vaccination.     

Cloning of rat brain succinyl-CoA:3-oxoacid CoA-transferase cDNA. Regulation of the mRNA in different rat tissues and during brain development.

3-Oxoacid CoA-transferase, which catalyses the first committed step in the oxidation of ketone bodies, is uniquely regulated in developing rat brain. Changes in 3-oxoacid CoA-transferase activity in rat brain during the postnatal period are due to changes in the relative rate of synthesis of the enzyme. To study the regulation of this enzyme, we identified, with a specific polyclonal rabbit anti-(rat 3-oxoacid CoA-transferase), two positive cDNA clones (approx. 800 bp) in a lambda gtll expression library, constructed from poly(A)+ RNA from brains of 12-day-old rats. One of these clones (lambda CoA3) was subcloned into M13mp18 and subjected to further characterization. Labelled single-stranded probes prepared by primer extension of the M13mp18 recombinant hybridized to a 3.6 kb mRNA. Rat brain mRNA enriched by polysome immunoadsorption for a single protein of size 60 kDa which corresponds to the precursor form of 3-oxoacid CoA-transferase was also found to be similarly enriched for the hybridizable 3.6 kb mRNA complementary to lambda CoA3. Affinity-selected antibody to the lambda CoA3 fusion protein inhibited 3-oxoacid CoA-transferase activity present in rat brain mitochondrial extracts. The 3.6 kb mRNA for 3-oxoacid CoA-transferase was present in relative abundance in rat kidney and heart, to a lesser extent in suckling brain and mammary gland and negligible in the liver. The specific mRNA was also found to be 3-fold more abundant in the brain from 12-day-old rats as compared with 18-day-old foetuses and adult rats, corresponding to the enzyme activity and relative rate of synthesis profile during development. These data suggest that 3-oxoacid CoA-transferase enzyme activity is regulated at a pretranslational level.     

Nonhomologous synapsis of the XY during early pachynema in In(X)1H male mice

It has been previously supposed that meiotic synapsis is restricted to homology during early, but not late pachynema. The synaptic begavior of an inverted X chromosome, In(X)1H as reflected in the synaptonemal complexes of the sex chromosomes has been examined in microspread spermatocytes by electron microscopy and evidence of extensive nonhomologus synapsis between the X and Y during early pachynema has been obtained.