Factors affecting cryopreservation-associated damages in sperm motility of cockerels (Gallus gallus domesticus).

1. Sperm are exposed to severe osmotic stress during cryopreservation, which results in impairment of fertilisation ability, including motility and viability, in poultry. Sperm osmotolerance is regulated by many extracellular factors and varies widely in birds, leading to uncertainty in the nature of the osmotic injury.2. Tail bending is a primary response resulting from cell swelling from excessive osmotic stress. However, the underlying mechanism responsible for tail bending is largely unknown. This study examined the relationship between osmotic stress and post-thaw motility, with a particular focus on the role of Na+/K+ ATPase (NKA) in the tail bending response.3. Cryopreserved sperm exhibited rapidly reduced motility when maintained at 37°C. The combination of temperature change and osmotic stress was a primary factor responsible for tail bending. This work tested a hypothesis known to be associated with post-thaw tail abnormality in other species and found that cold shock, that is not accompanied by an apoptotic response, may occur. Ouabain inhibition of Na+/K+ ATPase activity alleviated the tail bending response in fresh and post-thaw sperm.4. These results demonstrated that the combination of temperature change and osmotic stress has a primary impact on the reduction of post-thaw motility, with a particular role in NKA activity, in the tail bending response of chicken sperm. These results provide a foundation for establishing cryopreservation methodology to ensure the optimal fertilisation potential of cryopreserved chicken sperm.

Developmental potential of cryopreserved gonadal germ cells from 7-day-old chick embryos recovered using the PBS(-) method.

1. A series of experiments were conducted to examine the developmental potential of cryopreserved gonadal germ cells (GGCs) recovered from both males and females on embryo day 7 (7 d-GGCs) using the PBS(-) method. Germline chimeras were produced by transferring 200 frozen/unfrozen 7 d-GGCs recovered from female/male Rhode Island Red (RIR) embryos into the dorsal aorta of 2-day-old female and male white leghorn (WL) embryos.2. Germ-cell recipient embryos were hatched and raised to sexual maturity and progeny testing was conducted by mating with RIR of the opposite sex. Brown-feathered progeny chicks were hatched in all eight possible progeny testing combinations, except for male GGC recipients produced by transferring female GGCs. Furthermore, brown-feathered progeny chicks were hatched when frozen-thawed sperm from male germline chimeras, produced by transferring unfrozen 7d-GGCs, were inseminated in normal female RIR and female WL germline chimeras.3. The results indicated that cryopreserved female/male GGCs from 7-day-old chick embryos, recovered using the PBS(-) method, were fully capable of developing into normal spermatozoa and ova in the gonad of recipient embryos under appropriate GGC donor/recipient combinations.

Sugar expression in the mucosae of the canine uterus and vagina during the oestrous cycle and with pyometra.

The pathogenesis of canine pyometra is still unclear, but bacterial infection of the endometrium, mediated by bacterial lectins, is suspected to induce pyometra. The aim of this study was to investigate sugar expression in the mucosae of the uterus and vagina of healthy dogs with normal oestrous cycles and in dogs with pyometra, using a panel of lectins to investigate the pathogenesis of pyometra. In dogs with pyometra, the uterine and vaginal mucosae were positive for lectins that selectively bind to glucose or mannose, especially during days 7-10 and 30-40 of dioestrus. These results suggest that temporal changes in sugar expression in the uterus and vagina present an opportunity for pathogens to infect the endometrium, causing pyometra.

Multigenic control of resistance to Sendai virus infection in mice.

Experimental infection of mice with Sendai virus (SeV) is frequently used as a model of viral pathogenesis of human respiratory disease. To understand the differences in host response to SeV among mice strains, we carried out genetic mapping studies in DBA/2 (D2) (susceptible) and C57BL/6 (B6) (resistant) mice. F(1), F(2), and N(2) backcrossed mice were generated and examined for their disease resistance and susceptibility. For the determination of virulence, percentage body weight loss and survival time were used as phenotypes. We, then, carried out a genome wide scan on 108 backcrossed mice for linkage with percentage body weight loss as phenotype. A major quantitative trait locus (QTL) showing significant linkage was mapped to the distal portion of Chr 4 (SeV1). In addition, two other QTLs showing suggestive statistical linkage were also detected on Chr 8 and 14. We, further, performed genome scan for interactions with least squares analysis of variance of all pairs of informative makers in backcrossed progenies. We identified a highly significant epistatic interaction between D3Mit182 and D14Mit10, then denoted as SeV2 and SeV3, respectively, and the latter was the same locus showing a suggestive level on Chr 14 in QTL analysis. Considered genotypes of these three loci, we could account for more than 90% of genetic effect on the differential response to SeV infection between B6 and D2 mice. These findings revealed a novel gene interactions controlling SeV resistance in mice and will enable the identification of resistance genes encoded within these loci.

Demographics, health-related behaviors, eating habits and knowledge associated with vegetable intake in Japanese adults.

OBJECTIVES: To analyze demographic, health-related behaviors, eating habit and knowledge associated with vegetable intake. METHODS: Secondary analyses using the dataset from the National Health and Nutrition Survey 2003. Food intake data measured by the food-weighing method in one-day and a questionnaire assessed the dietary intake and health-related behaviors, eating habit and knowledge. This study was made in Japan. The data of 1742 men and 2519 nonpregnant/nonlactating women, aged 20-69 years, energy intake between percentiles 1 and 99 were included. Vegetable intake was analyzed according to the Japanese vegetable recommendation (>or=350 g/day) after age adjustment. RESULTS: Average of VI was 307 g/day in men and 297 g/day in women. Only 35% of men and 31% of women met the recommended amount of vegetable intake. Japanese from city areas, aged 60-69 years, had the highest vegetable intake and subjects from metropolitan areas had the lowest vegetable intake. Depending on the age groups, risks for low vegetable intake in Japanese were found in subjects with skipping meals, alcohol intake and history of smoking. CONCLUSIONS: To increase vegetable intake, it is necessary to provide more nutritional education and lifestyle-related diseases education.

A computer-aided diagnosis system to screen for osteoporosis using dental panoramic radiographs.

OBJECTIVES: An eroded inferior cortex of the mandible detected on dental panoramic radiographs is useful for identifying post-menopausal women with low skeletal bone mineral density (BMD) or osteoporosis. The purposes of this study were to develop a computer-aided diagnosis (CAD) system that automatically determines cortical erosion of the mandible on dental panoramic radiographs and to assess the validation of this CAD system. METHODS: A CAD system was developed based on mathematical morphology for identifying post-menopausal women with low skeletal BMD or osteoporosis, based on World Health Organization criteria, by identifying whether the endosteal margin of mandibular cortical bone was eroded. The sensitivity, specificity, predictive value, accuracy and likelihood for a positive risk result were calculated using dichotomous 2 x 2 tables using 100 panoramic radiographs. RESULTS: For identifying women with low skeletal BMD, the sensitivity and specificity were 76.8% and 61.1%, respectively, the positive and negative predictive values were 90.0% and 36.7%, accuracy was 74.0% and the likelihood for positive risk was 1.96. The respective values for identifying women with osteoporosis were 94.4% and 43.8%, the positive and negative predictive values were 48.6% and 93.3%, respectively, the accuracy was 62.0% and the likelihood for positive risk was 1.68. CONCLUSION: Our results suggest that a CAD system applied to dental panoramic radiographs may be useful for identifying post-menopausal women with low skeletal BMD or osteoporosis.

Observer performance in diagnosing osteoporosis by dental panoramic radiographs: results from the osteoporosis screening project in dentistry (OSPD).

Mandibular cortical erosion detected on dental panoramic radiographs (DPRs) may be useful for identifying women with osteoporosis, but little is known about the variation in diagnostic efficacy of observers worldwide. The purpose of this study was to measure the accuracy in identifying women at risk for osteoporosis in a worldwide group of observers using DPRs. We constructed a website that included background information about osteoporosis screening and instructions regarding the interpretation of mandibular cortical erosion. DPRs of 100 Japanese postmenopausal women aged 50 years or older who had completed skeletal bone mineral measurements by dual energy X-ray absorptiometry were digitized at 300 dpi. These were displayed on the website and used for the evaluation of diagnostic efficacy. Sixty observers aged 25 to 66 years recruited from 16 countries participated in this study. These observers classified cortical erosion into one of three groups (none, mild to moderate, and severe) on the website via the Internet, twice with an approximately 2-week interval. The diagnostic efficacy of the Osteoporosis Self-Assessment Tool (OST), a simple clinical decision rule based on age and weight, was also calculated and compared with that of cortical erosion. The overall mean sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the 60 observers in identifying women with osteoporosis by cortical erosion on DPRs were 82.5, 46.2, 46.7, and 84.0%, respectively. Those same values by the OST index were 82.9, 43.1, 43.9, and 82.4%, respectively. The intra-observer agreement in classifying cortical erosion on DPRs was sufficient (weighted kappa values>0.6) in 36 (60%) observers. This was significantly increased in observers who specialized in oral radiology (P<0.05). In the 36 observers with sufficient intra-observer agreement, the overall mean sensitivity, specificity, PPV, and NPV in identifying women with osteoporosis by any cortical erosion were 83.5, 48.7, 48.3, and 85.7%, respectively. The mean PPV and NPV were significantly higher in the 36 observers with sufficient intra-observer agreement than in the 24 observers with insufficient intra-observer agreement. Our results reconfirm the efficacy of cortical erosion findings in identifying postmenopausal women at risk for osteoporosis, among observers with sufficient intra-observer agreement. Information gathered from radiographic examination is at least as useful as that gathered from the OST index.

Computer-aided system for measuring the mandibular cortical width on dental panoramic radiographs in identifying postmenopausal women with low bone mineral density.

INTRODUCTION: Mandibular inferior cortical width manually measured on dental panoramic radiographs may be useful for identifying postmenopausal women with low skeletal bone mineral density (BMD). Automatic measurement of cortical width may enable us to identify a large number of postmenopausal women with suspected low skeletal BMD. The purposes of this study were to develop a computer-aided system for measuring mandibular cortical width on dental panoramic radiographs and clarify the diagnostic efficacy of this system. METHODS: Panoramic radiographs of 100 postmenopausal women who had had BMD assessments of the lumbar spine and the femoral neck were used in this study. Experienced oral radiologist determined the position of the mental foramen on 100 digitized dental panoramic radiographs. After determination of the mental foramen, mandibular cortical width below the mental foramen was measured automatically with a computer-aided system by identifying the area of interest, enhancing the original image, determining inner and outer margins of the cortex, and selecting an appropriate point. Cortical width measured by this system was compared with BMD of the lumbar spine and the femoral neck. RESULTS: There were statistically significant correlation between cortical width measured by the computer-aided system and spinal BMD (r=0.50) and femoral neck BMD (r=0.54). These correlations were similar with those between cortical width by manual measurement and skeletal BMD. Sensitivity and specificity for identifying postmenopausal women with low spinal BMD by the computer-aided system were about 88.0% and about 58.7%, respectively. Those for identifying postmenopausal women with low femoral neck BMD by this system were about 87.5% and about 56.3%, respectively. CONCLUSION: Our results suggest that our computer-aided system may be useful for identifying postmenopausal women with low skeletal BMD.

Relationship of lipoprotein lipase and hepatic triacylglycerol lipase activity to serum adiponectin levels in Japanese hyperlipidemic men.

OBJECTIVE: The aim of this study was to determine how lipoprotein lipase (LPL) and hepatic triacylglycerol lipase (HTGL) activity relate to serum adiponectin levels. RESEARCH DESIGN AND METHODS: Fifty-five hyperlipidemic Japanese men were recruited for this study. LPL and HTGL activity in post-heparin plasma (PHP) was measured using Triton X-100 emulsified-[14C] triolein. The remaining activity in the presence of 1M NaCl was defined as HTGL activity. Serum adiponectin levels were determined by an enzyme-linked immunosorbent assay system. RESULT: LPL activity had a positive relationship with HDL2, but had no relation with HDL3, while HTGL had positive relationship with HDL3, but had no relationship with HDL2. LPL activity showed a positive relationship [r = 0.345, p = 0.010] to serum adiponectin levels, while and HTGL activity showed an inverse relationship [r = - 0.365 p = 0.006]. Multiple regression analysis with LPL and HTGL as dependent variables and age, BMI, serum adiponectin and the homeostasis model assessment of insulin resistance (HOMA-IR) as independent variables showed LPL and HTGL's association to adiponectin did not persist after adjustments for these covariants. However, the association of LPL activity to HOMA-IR was found to persist after adjustments of age, BMI, and serum adiponectin. CONCLUSIONS: There was a co-linearity between insulin sensitivity and adiponectin as well as insulin sensitivity and LPL/HTGL activity.

Structure and property of self-assemble valinyl bolaform amides having different chirality.

Bolaform amides were designed from N,N'-bis(carboethoxy-L-valinyl)-diaminoethane (1) by linking t-butyloxycarbonyl-valine through ethylenediamine (EDA) to enable spectroscopic and X-ray diffraction analyses. N,N'-Bis(Boc-L-valinyl)-diaminoethane (2) and N,N'-bis(Boc-D-valinyl)-diaminoethane (3) were composed of L-Val and D-Val, respectively. N-(Boc-L-valinyl)-N'-(Boc-D-valinyl)-diaminoethane (4) was composed of both L-Val and D-Val, and was achiral (meso-peptide). Peptide 5 was a 1:1 mixture of 2 and 3, and was also achiral (racemate). These peptides mediated gelation of corn oil at a concentration of approximately 1%. Within crystals, the peptides formed beta-sheet ribbons, but differences were observed in hydrogen-bonding patterns and side-chain arrangements. These differences were also deduced from temperature dependence of amide protons. Force-field calculations based on the crystal structures indicated that association of beta-sheet ribbons had energy benefits, and it was assumed that molecular aggregation progressed spontaneously. These structural studies indicated the chirality of amino acids affected for the properties of bolaform amides.

Epitope analysis of monoclonal antibody E5/G6, which binds to a linear epitope in the nucleocapsid protein of hantaviruses.

Monoclonal antibody E5/G6 recognized a linear epitope common to hantavirus nucleocapsid proteins. Using synthetic peptides, we identified epitope E5/G6 as the 9 mer YEDVNGIRK (NP 165-173), in which D167, G170, I171, and R172 are indispensable. Furthermore, all the peptides synthesized using various hantavirus sequences bound MAb E5/G6 consistently, despite the existence of several amino acid variations in this region. These results indicate that MAb E5/G6 is a useful tool for detecting hantavirus antigen in rodent or patient tissues using Western blotting or other immunohistochemical assays.

Central motor conduction in patients with anti-ganglioside antibody associated neuropathy syndromes and hyperreflexia.

OBJECTIVES: Several serum antibodies against gangliosides are diagnostically important, particularly in Guillain-Barré syndrome (GBS), Miller Fisher syndrome (MFS), and multifocal motor neuropathy (MMN). Although hyperreflexia is an atypical symptom in these disorders, it has been found in some patients with GBS, MFS, and MMN. The aim of the study was to determine whether hyperreflexia corresponds to corticospinal tract dysfunction in these patients. METHODS: The study examined central and peripheral motor conduction in patients with hyperreflexia who exhibited acute paralysis (group 1, n=5), acute ataxia and ophthalmoplegia (group 2, n=7), or chronic paralysis with conduction block (group 3, n=2). The clinical symptoms are similar to those in patients with GBS, MFS, and MMN, respectively, and serum anti-ganglioside antibodies were found to be positive in all patients. Using magnetic and electrical stimulation techniques, central and peripheral motor conduction were compared in patients in groups 1, 2, and 3 and patients with GBS (n=7), MFS (n=8), and MMN (n=6). RESULTS: Central motor conduction times (CMCTs) in patients in groups 1, 2, and 3 were significantly delayed compared with those in patients with GBS, MFS, and MMN (p<0.01, p<0.05, p<0.05, respectively), and the delayed CMCTs significantly improved in the recovery periods (p<0.01, p<0.01, p<0.05, respectively). However, motor conduction velocity, compound muscle action potential, and F wave conduction velocity were not significantly different between the patients. CONCLUSION: These findings indicate that corticospinal tract is functionally involved in patients with anti-ganglioside antibody associated neuropathy syndromes and hyperreflexia

Effect of asymmetric modification on the conformation of ascidiacyclamide analogs.

Ascidiacyclamide (ASC), cyclo(-Ile1-Oxz2-d-Val3-Thz4-)2 (Oxz=oxazoline and Thz=thiazole) has a C2-symmetric sequence, and the relationships between its conformation and symmetry have been studied. In a previous study, we performed asymmetric modifications in which an Ile residue was replaced by Gly, Leu or Phe to disturb the symmetry [Doi et al. (1999) Biopolymers49, 459-469]. In this study, the modifications were extended. The Ile1 residue was replaced by Gly, Ala, aminoisobutyric acid (Aib), Val, Leu, Phe or d-Ile, and the d-Val3 residue was replaced by Val. The structures of these analogs were analyzed by X-ray diffraction, 1H NMR and CD techniques. X-Ray diffraction analyses revealed that the [Ala1], [Aib1] and [Phe1]ASC analogs are folded, whereas [Val1]ASC has a square form. These structures are the first examples of folded structures for ASC analogs in the crystal state and are similar to the previously reported structures of [Gly1] and [Phe1]ASC in solution. The resonances of amide NH and Thz CH protons linearly shift with temperature changes; in particular, those of [Aib1], [d-Ile1] and [Val3]ASCs exhibited a large temperature dependence. DMSO titration caused nonlinear shifts of proton resonances for all analogs and largely affected [d-Ile1] and [Val3]ASCs. A similar tendency was observed upon the addition of acetone to peptide solutions. Regarding peptide concentration changes, amide NH and Thz CH protons of [Gly1]ASC showed a relatively large dependence. CD spectra of these analogs indicated approximately two patterns in MeCN solution, which were related to the crystal structures. However, all spectra showed a similar positive Cotton effect in TFE solution, except that of [Val3]ASC. In the cytotoxicity test using P388 cells, [Val1]ASC exhibited the strongest activity, whereas the epimers of ASC ([d-Ile1] and [Val3]ASCs), showed fairly moderate activities.

Capacitation status and in vitro fertility of boar spermatozoa: effects of seminal plasma, cumulus-oocyte-complexes-conditioned medium and hyaluronan.

In the present study, the effects of seminal plasma (SP), cumulus-oocyte-complexes (COCs) conditioned medium (CCM) and hyaluronan (HA) on functional changes and in vitro fertilizing ability of porcine spermatozoa were examined. In in vitro fertilization (IVF) experiments, 10% (v/v) of exogenous SP in the fertilization medium prevented sperm penetration (using fresh-extended and frozen-thawed ejaculated spermatozoa). Analysis of frozen-thawed CCM revealed a HA content to levels of 30 ng/mL per incubated COC. Presence of frozen-thawed CCM did not, however, prove effective to increase (furthermore decreasing) oocyte penetration in vitro, and neither did supplementation with exogenous HA at the same concentration as that present in the CCM (secreted by COCs). Analysis of sperm capacitation using the chlortetracycline (CTC) assay showed that frozen-thawed CCM had no elevating effect on 'B-pattern' spermatozoa (implying capacitation-like changes) and that addition of 10% (v/v) SP held spermatozoa in the 'F-pattern' (intact) status. Dose of 500 microg/mL HA and freshly prepared CCM increased, however, the frequency of capacitated spermatozoa (B-pattern) without resulting in increased rates of 'AR-pattern' (acrosome-reacted) spermatozoa, compared with controls. The present results confirm the decapacitating effect of SP and suggest capacitating actions of HA (dose-related) and CCM (freshly prepared) on boar spermatozoa in vitro. The unclear effects of frozen-thawed CCM and a low dose of HA on penetration rates of boar spermatozoa call for further researches of their function in vivo.

Outcomes of radiotherapy for inoperable locally advanced (T4) esophageal cancer-retrospective analysis-.

To assess the treatment outcome after radiotherapy with or without chemotherapy for inoperable locally advanced (T4) esophageal cancer (EC), a retrospective analysis was performed. We enrolled 37 patients with T4 EC and analyzed 35 patients. A total of 28 patients were treated with radiotherapy and chemotherapy (median dose, 60 Gy) and seven patients were treated with radiation alone (median dose, 60 Gy). Many mainly received high-dose cisplatin (CDDP) and 5-fluorouracil (FU) or continuous infusion of low-dose CDDP and 5-FU. Among the 35 patients, there were eight survivors and 27 deaths. The median follow-up period was 15.5 months (3.5 to 74 months). The one-year, 3-year, and 5-year survival rates were 37.5%, 10.0%, and 10.0%, respectively, with a median survival time (MST) of 7.3 months. When the patients were divided into two groups, a complete response (CR) group and a non-CR group, MST was 16.5 and 6.2 months, respectively, showing significant differences between the two groups (p=0.0317). CR patients showed more satisfactory long-term outcomes than non-CR patients.

Three types of polymorphisms in exon 14 in porcine Mx1 gene.

Much is known about the antiviral activity of Mx proteins in species such as mouse and human. In the mouse, loss of resistability to influenza virus has been shown to be due to specific polymorphisms in the Mx gene. This gene is therefore an interesting candidate gene for disease resistance in farm animals. The porcine Mx1 gene has already been identified and characterized based on its homology with mouse Mx1; however, until now no evidence of polymorphisms in the porcine gene has been reported. In this study, we have found two new polymorphisms in exon 14 of porcine Mx1 by DNA sequencing and confirmed their presence in different breeds, using polymerase chain reaction (PCR)-restriction fragment length polymorphisms (RFLP) with NarI and NaeI restriction enzymes. On the basis of the deduced amino acid sequence, one allele contains a deletion that may result in a frameshift to yield several amino acid substitutions and extension of the carboxyl terminal region of Mx1 protein. The deletion allele, Mx1c, was found to be segregating in Landrace, Berkshire, Duroc, Hampshire, and Yucatan miniature pig. A second point mutation, Mx1b, was detected in Meishan and two Vietnamese native pig breeds. All other breeds tested were fixed for the Mx1a allele that is identical to the sequence reported previously. It will be interesting to determine if the Mx1c deletion is associated with variation in resistance to the myxovirus family in the pig.

Developmental study of the different effects on the hybrid sterility of Kit and KitW-v alleles paired with Kit from Mus spretus.

The combination of the KitW or KitW-n mutant alleles and KitS from Mus spretus results in male hybrid sterility with small testes. In the present study, reproduction of the combination between KitW-v and KitS alleles was examined. The KitW-v/KitS male was fertile and the histologic structure was normal; the seminiferous tubules showed all of the normal stages of spermatogenesis. The postnatal development of the testis at 8, 12, 16 and 20 days was also studied in the fertile +Kit/+Kit and KitW-v/KitS males and the sterile KitW/KitS. The results showed that at 8 days there was no noticeable difference among the three genotype combinations, while from 12 to 20 days spermatogenesis in the KitW/KitS male nearly stopped before the meiosis stage. The expression of Kit receptor protein from the KitS allele in the sterile testis of the KitW/KitS male was confirmed using western blot analysis. The Kit ligand derived from M. spretus showed two amino acid changes in the extracellular domain compared with that from C57BL and it appears that the ligand-receptor interaction between C57BL and SPR may influence the male hybrid sterility of KitW/KitS.