Preservation of ejaculated and epididymal stallion spermatozoa by cooling and freezing

The suitability of ejaculated and epididymal stallion spermatozoa for cooled storage (5 degrees C) and cryopreservation was examined in 5 ejaculates from each of 6 stallions and in spermatozoa recovered from the cauda epididymidis after castration of these stallions. The percentage of progressively motile spermatozoa, examined by subjective estimation (cooled samples) or by computerized analysis (frozen-thawed samples), was used as parameter. In ejaculated semen samples containing 5 and 25% seminal plasma in a skim milk glucose extender, the lower amount of seminal plasma supported spermatozoal motility significantly better throughout storage at 5 degrees C. Addition of 5 or 25% seminal plasma to perfused epididymal spermatozoa (0% seminal plasma) resulted in a significant stimulation of spermatozoal motility by 25% seminal plasma at 0 h (P<0.05) and to a lesser extent at 24 and 48 h. Post-thaw motility of ejaculated as well as epididymal spermatozoa was not influenced by slow cooling to 15 degrees or 5 degrees C with or without glycerol prior to rapid freezing in liquid nitrogen vapor. During cooled storage, seminal plasma had a stimulatory effect on epididymal spermatozoa and depressed motility in ejaculated spermatozoa. Results on cryopreservation indicate that freezability of equine spermatozoa is already determined when spermatozoa leave the tail of the epididymis.     

In vitro maturation of equine oocytes collected by follicle aspiration and by the slicing of ovaries

The aim of this study was to examine 2 techniques for oocyte recovery from equine ovaries at slaughter: by aspiration of follicles and by additional slicing of ovaries. The morphology and nuclear configuration of oocytes recovered with either technique, and the time course of nuclear maturation during in vitro maturation were evaluated. Recovery rates were 1.75 and 4.14 oocytes per ovary for aspiration and slicing (total 145 and 344 oocytes from 83 ovaries), respectively. The oocytes were classified according to their cumulus/ooplasm morphology into 4 groups: compact/circular(A), compact/semicircular(B), expanded(C) and others(D). The percentages of oocytes in Groups A, B, C and D were 34, 38, 25 and 3% (aspiration) and 55, 26, 17 and 3% (slicing), respectively. The proportions of oocytes with a germinal vesicle in Groups A, B, C and D were 28 29 (97%), 23 35 (66%), 11 23 (48%) and 2 4 (50%) in oocytes from aspiration and 91 100 (91%), 52 65 (80%), 15 29 (52%) and 1 2 (50%) in oocytes from slicing, respectively. Group A and B oocytes recovered by aspiration (n=212) and slicing (n=312) were cultured in TCM199 supplemented with 10% fetal bovine serum, 1 mug/ml estradiol-17beta, and 0.02 AU/ml FSH at 38.5 degrees C in 5% CO(2) in air (5 to 10 oocytes per 50- mu l microdrop). At 8, 16, 24, 32 and 40 h of culture, the oocytes were fixed and stained. There were no significant differences in the percentages of Metaphase II stage (MII)-oocytes between recovery techniques at any time points examined. The proportions of MII-oocytes were 1 42 (2%), 4 43 (9%), 21 42 (50%), 28 45 (62%), and 28 40 (70%) at the respective time point in oocytes from aspiration and 0 51 (0%), 3 54 (6%), 22 59 (37%), 43 72 (60%), and 51 76 (67%) in oocytes from slicing, respectively. In most of the oocytes, resumption of meiosis occurred between 8 and 16 h of culture. The proportions of MII-oocytes increased significantly between 16 and 24 h and between 24 and 32 h of culture.     

Reactions of the bis(p-tolylsulfonylhydrazone) of periodate-oxidized methyl 4,6-O-benzylidene-α-D-glucopyranoside with borohydride and cyanoborohydride: novel syntheses of unsaturated and deoxy sugars

Periodate-oxidized methyl 4,6-O-benzylidene-α-D-glucopyranoside (1) reacted with p-toluenesulfonylhydrazine to give the substituted bis(hydrazone) 2, which was converted into an N-substituted epimino derivative (3) by treatment with sodium borohydride in ethanol. Compound 3 was further converted into the glyc-2-enoside 4 by heating it with sodium borohydride in 1,4-dioxane. Sodium cyanoborohydride in ethanol reduced 2 to an epimeric mixture of 2-deoxy-D-arabino (5) and D-ribo (6)-hexoside derivatives. In the presence of an acidic resin in the same solvent, however, compound 2 underwent hydrogenation to the bis(hydrazino) derivative (7). The mechanisms of these reactions are discussed.     

Synthesis and preliminary biological evaluation of truncated zoanthenol analogues

Zoanthamines are a family of marine alkaloids that have complex heptacyclic structures and are reported to be interleukin-6 modulators. While the structure of zoanthamines, especially the ABC-ring portion, is similar to that of steroids, the CDEFG-ring portion, composed of aminoacetal and lactone core, is a unique structural element. In this report, we designed and synthesized ABC-ring 6 and CDEFG-ring 7, which are truncated analogues of the northern and southern hemispheres of zoanthenol 5, respectively, and which incorporate all of the functionality of each hemisphere. A preliminary SAR study suggested that the hydrochloride of the CEFG-ring portion is an active pharmacophore for suppressing the growth of interleukin-6-dependent MH60 cells.     

Participation of syndecan 2 in the induction of stress fiber formation in cooperation with integrin alpha5beta1: structural characteristics of heparan sulfate chains with avidity to COOH-terminal heparin-binding domain of fibronectin

The present study provides direct evidence that syndecan 2 participates selectively in the induction of stress fiber formation in cooperation with integrin alpha5beta1 through specific binding of its heparan sulfate side chains to the fibronectin substrate. Our previous study with Lewis lung carcinoma-derived P29 cells demonstrated that the cell surface heparan sulfate proteoglycan, which binds to fibronectin, is syndecan 2 (N. Itano et al., 1996, Biochem. J. 315, 925-930). We here report that in vitro treatment of the cells by antisense oligonucleotide for syndecan 2 resulted in a failure to form stress fibers on fibronectin substrate in association with specific suppression of its cell surface expression. Instead, localization of actin filaments in the cytoplasmic cortex occurred. A similar response of the cells was observed when the cells were treated to eliminate functions of cell surface heparan sulfates, including exogenous addition of heparin and pretreatment with anti-heparan sulfate antibody, F58-10E4, and with proteinase-free heparitinase I. Size- and structure-defined oligosaccharides prepared from heparin and chemically modified heparins were utilized as competitive inhibitors to examine the structural characteristics of the cell surface heparan sulfates involved in organization of the actin cytoskeleton. Their affinity chromatography on a column linked with a recombinant H-271 peptide containing a C-terminal heparin-binding domain of fibronectin demonstrated that 2-O-sulfated iduronates were essential for the binding. Inhibition studies revealed that a heparin-derived dodecasaccharide sample enriched with an IdoA(2OS)-GlcNS(6OS) disaccharide completely blocked binding of the syndecan 2 ectodomain to immobilized H-271 peptide. Finally, the dodecasaccharide sample was shown to inhibit stress fiber formation, triggered by adhesion of P29 cells to a CH-271 polypeptide consisting of both the RGD cell-binding and the C-terminal heparin-binding domains of fibronectin in a fused form. All these results consistently suggest that syndecan 2 proteoglycan interacts with the C-terminal heparin-binding domain of fibronectin at the highly sulfated cluster(s), such as [IdoA(2OS)-GlcNS(6OS)](6) present in its heparan sulfate chains, to result in the induction of stress fiber formation in cooperation with integrin alpha5beta1.     

Detection of Norovirus and Sapovirus from diarrheic dogs and cats in Japan

Norovirus (NoV) and sapovirus (SaV) are important causes of human diarrhea. In this study, between 2007 and 2014 fecal samples were collected from 97 dogs and 83 cats with diarrhea and examined to determine the prevalence of NoV and SaV infections in Japan. To detect caliciviruses, approximately 300 bases targeting the polymerase gene were amplified using RT‐PCR and subjected to phylogenetic and homology analyses. Specific PCR products were obtained from four canine and nine feline samples: two canine and one feline isolate were classified as NoV, two canine isolates as SaV and the remaining eight feline isolates as vesivirus (VeV). The three NoV isolates were classified into the same clade as that of known canine and feline NoVs; their homologies (75.9–92.3%) were higher than those with human genogroup IV (GIV) NoVs (59.1–65.9%). The homology of the feline NoV isolate with previously reported feline NoV isolates was particularly high (91.7–92.3%). Regarding SaV, the two canine isolates were classified into the same clade as known canine SaVs and their homologies (72.5–86.5%) were higher than those with other mammal SaVs (20.7–58.0%). The eight feline VeV isolates were assumed to be feline calicivirus. The present study is the first report of the presence of NoV‐ and SaV‐infected dogs and cats in Japan. The findings suggest there are species‐specific circulations of NoV and SaV among dogs and cats, in Japan.     

Intrinsically fluorescent PAMAM dendrimer as gene carrier and nanoprobe for nucleic acids delivery: bioimaging and transfection study

This study successfully evaluated gene delivery and transfection toward rat C6 glioma cell lines mediated by intrinsic blue fluorescent poly(amido amine) (PAMAM) dendrimer. We used three antisense oligonucleotides, (AS-ODN) p75, NGF1, and NGF2 for knocking down specific protein expressions. The three oligonucleotides were electrostatically associated with the photoluminescent amino-terminated PAMAM dendrimer to yield fluorescent complexes at various nitrogen-to-phosphorus (N/P) ratios. Compared with pristine PAMAM dendrimer and hyperbranched polyethylenimine (PEI), the fluorescent PAMAM dendrimer revealed lower in vitro cytotoxicity toward C6 cells, allowing us to transfect the cells with the AS-ODN complexes under a higher N/P ratio. Due to the intrinsic fluorescence, cellular uptake behavior could be directly analyzed by fluorescence microscopy and flow cytometry, without additional fluorescence labeling. As expected, the result clearly suggested that the uptake efficiency increased as the N/P value increased. Furthermore, the quantified data obtained from flow cytometry indicated relatively higher uptake efficiency for the p75 complex, which is mainly due to different association patterns between the fluorescent dendrimer and AS-ODNs. At N/P = 20, atomic force microscopic analysis confirmed that the p75 complex formed well-condensed, spherical particles with dimensions less than 200 nm, but that NGF2 AS-ODN associated poorly with the dendrimer. Finally, Western blot analysis indicated that these complexes were capable of knocking down the specific protein expression to a certain level, being comparable to the hyperbranched PEI-mediated gene transfection. Our preliminary results clearly indicated that intrinsic fluorescent PAMAM dendrimers show promise as gene vehicles that can achieve delivery, transfection, and bioimaging at the same time.