Identification of the DDAH2 protein in pig reproductive tract mucus: a putative oestrus detection marker.

Precisely detecting oestrus is important for artificial insemination. The aims of this study were to identify oestrus-specific sow mucus proteins to determine the optimal time for artificial insemination. The proestrous- and oestrous-stage mucus proteins were purified and analysed with proteomic tools such as two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight analyses. Among the differentially expressed proteins, the dimethylarginine dimethylaminohydrolase 2 (DDAH2) protein showed a 3.6-fold increase during the proestrous stage compared to that during the oestrous stage. A western immunoblot study revealed that two of three sow mucus samples clearly showed negative anti-DDAH2 antibody activity during the oestrous stage. This study demonstrated that the pig DDAH2 mucus protein exists during the proestrous stage, but not during the oestrous stage, suggesting that mucus DDAH2 could be useful as an oestrus detection marker.

Effects of Dietary Fat Types on Growth Performance, Pork Quality, and Gene Expression in Growing-finishing Pigs.

This study was performed to determine the effects of dietary fat sources, i.e., beef tallow, soybean oil, olive oil and coconut oil (each 3% in feed), on the growth performance, meat quality and gene expression in growing-finishing pigs. A total of 72 crossbred pigs (Landrace×Large White×Duroc) were used at 71±1 kg body weight (about 130 d of age) in 24 pens (320×150 cm) in a confined pig house (three pigs per pen) with six replicate pens per treatment. The growing diet was given for periods of 14±3 d and the finishing diet was given for periods of 28±3 d. The fat type had no significant effect either on growth performance or on chemical composition or on meat quality in growing-finishing pigs. Dietary fat type affected fatty acid composition, with higher levels of unsaturated fatty acids (UFAs) and monounsaturated fatty acids (MUFAs) in the olive oil group. Microarray analysis in the Longissimus dorsi identified 6 genes, related to insulin signaling pathway, that were differentially expressed among the different feed groups. Real time-PCR was conducted on the six genes in the longissimus dorsi muscle (LM). In particular, the genes encoding the protein kinase, cAMP-dependent, regulatory, type II, alpha (PRKAR2A) and the catalytic subunit of protein phosphatase 1, beta isoform (PPP1CB) showed the highest expression level in the olive oil group (respectively, p<0.05, p<0.001). The results of this study indicate that the type of dietary fat affects fatty acid composition and insulin signaling-related gene expression in the LM of pigs.

Use of fouling resistant nanofiltration and reverse osmosis membranes for dyeing wastewater effluent treatment.

Dyeing wastewater was post-treated by using nanofiltration (NF) and reverse osmosis (RO) membranes. To reduce membrane fouling, poly (vinyl alcohol) (PVA) with a neutral charge was coated on NF and RO membranes. The effect of surface charge and surface roughness on membrane fouling was investigated. Dyeing wastewater was pre-treated by using coagulation, activated sludge process, and MF process to investigate the effect of the pre-treatment on the membrane fouling. It is demonstrated that the extent of fouling is significantly influenced by the surface roughness and the surface charge on the NF and RO membranes. A membrane with a smooth and neutral surface was fouled less. The pre-treatment was essential for avoiding NF and RO membranes fouling. The quality of the final permeate was acceptable for water reuse.

The production of transforming growth factor-beta activity by rat granulosa cell cultures.

We have examined whether granulosa cells (GC) secrete transforming growth factor-beta (TGF beta)-like activity using cell cultures prepared from diethylstilbestrol-primed female rats. Our results indicate that a significant level of active as well as latent TGF beta activity is found in defined GC culture medium as assessed by 1) potentiation of FSH-induced differentiation of rat GC, 2) neutralization of its activity by anti-TGF beta immunoglobulin, 3) inhibition of DNA synthesis in mink lung epithelial cells (CCl 64), and 4) activation of latent TGF beta activity by either acid or heat treatment. TGF beta production was more pronounced when the cells were seeded on fibronectin-coated plates. There was no difference in the level of TGF beta secretion by GC preparations derived from either diethylstilbestrol-primed immature or normal immature rats or adult rats. Furthermore, rat GC-conditioned medium contained much more TGF beta activity than medium from normal rat kidney cells (NRK 49-F), human prostatic adenocarcinoma cells (PC-3), or porcine GC. Rat thecal/interstitial cell culture medium contained activity comparable to that of GC medium. We conclude that rat GC preparations secrete a high level of TGF beta activity in vitro. Taken together with previous results, this indicates the possibility that TGF beta may be an autocrine regulator as well as a paracrine one within the ovarian follicle. Moreover, because of the high level of TGF beta activity produced, the rat GC culture system appears to be a useful experimental model for further exploring relationships between TGF beta production and its action.

Human platelet-derived growth factor preparations contain a separate activity which potentiates follicle-stimulating hormone-mediated induction of luteinizing hormone receptor in cultured rat granulosa cells: evidence for transforming growth factor-beta.

The ability of platelet-derived growth factor (PDGF) preparations to potentiate FSH-mediated LH receptor induction in rat granulosa cell cultures was shown to be due to a component distinct from PDGF. Purification of heat-treated platelet lysate by carboxymethyl-Sephadex C-50 and Cibacron blue-Sepharose chromatography, followed by Bio-Gel P-60 chromatography, resulted in the separation of two activities: 1) a growth-promoting activity, P60-PDGF, defined on the basis of increased DNA synthesis in BALB/c-3T3 cells, and 2) a differentiation-promoting activity which enhanced FSH-dependent LH receptor induction in granulosa cells. On the basis of electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels, inhibition of tritiated thymidine uptake by epithelial cells, and attenuation of LH/hCG receptor expression in the presence of antitransforming growth factor-beta (anti-TGF beta) immunoglobulin G, the differentiation-promoting component of the preparations appears to be TGF beta. The Bio-Gel fractions that contained TGF beta did not stimulate LH receptor induction of cAMP production in the absence of FSH. PDGF prepared free of TGF beta did not potentiate receptor induction. We conclude, therefore, that the differentiative effects of PDGF previously described in this system are due to TGF beta.

RcsC-mediated induction of colanic acid by secretion of streptokinase in Escherichia coli K-12.

The introduction of a plasmid containing skc (streptokinase-coding gene) fused with ompA signal sequence into Escherichia coli K-12 strains, rendered the bacteria mucoid. Measurement of the synthesis of beta-galactosidase from a cps-lacZ fusion (lacZ fusion to a gene necessary for capsule synthesis) showed that the mucoid phenotype was due to induction of the capsular polysaccharide colanic acid synthesis. The introduction of a plasmid carrying skc fused with malE (gene encoding maltose-binding protein) also induced cps-lacZ expression, but intracellular expression of streptokinase in E. coli did not. The cps expression by secretion of streptokinase was diminished to the basal level in a cps-lacZ strain carrying a rcsC mutation. These results show that the secretion of streptokinase in E. coli induces colanic acid synthesis through the RcsC-dependent pathway.

Asp41-His48 region of streptokinase is important in binding to a substrate plasminogen.

Streptokinase is a plasminogen activator protein produced by several strains of beta-hemolytic streptococci. Random mutagenesis of streptokinase was carried out for the determination of critical amino acid residues in plasminogen activation. We selected and sequenced 14 streptokinase mutants with no plasminogen activation activity on skim milk-plasminogen overlay plate. Specific activities of the selected streptokinase mutants were determined with chromogenic assay. Eight mutants (V19F, V35E, E85D, L292R, D325P, D341E, I345N, and M369L) resulted in greatly decreased amidolytic activities. However, unexpectedly, six mutants (D41C, S44K, S44P, R45P, H48T, and D220G) showed substantial amidolytic activities comparable to that of wild type. Moreover, five-point mutations were concentrated on the Asp41-His48 region. These data indicate that the Asp41-His48 region in a streptokinase-plasminogen binary complex plays an important role in binding to a substrate plasminogen.

Inhibition of LDH-X by gossypol optical isomers.

Racemic gossypol is an effective male antifertility agent in several mammalian species. However, (+)-gossypol is not an effective male antifertility agent in the rat or the hamster. Previous studies have demonstrated the ability of racemic gossypol to inhibit the testis-specific LDH-X enzyme derived from various mammalian species and have suggested LDH-X as the potential site of gossypol antifertility action. In the present study, the effects of racemic gossypol and the (+) and (-) optical isomers of gossypol on LDH-X derived from rat and hamster testicular cytosol are compared to determine if there is any correlation between the in vitro inhibition of the LDH-X enzyme and in vivo antifertility effects. Both optical isomers of gossypol as well as racemic gossypol inhibit rat and hamster testicular cytosolic LDH-X activity. Inhibition of hamster testicular cytosolic LDH-X activity by (-)-gossypol was less than by either racemic gossypol or (+)-gossypol. Based on the previous reports of racemic gossypol inhibition of LDH-X, therefore, it cannot be simply concluded that LDH-X is the specific site of antifertility action of gossypol since, in the present study, (+)-gossypol, which is not an effective male antifertility agent, also inhibited rat and hamster testicular cytosolic LDH-X.

Effects on fertility of rabbits after active immunization with rat testicular cytochrome ct.

A Male rabbit was immunized with rat testicular cytochrome (Cyt) ct and mated with normal, unimmunized females. The matings resulted in abnormal pregnancies: no offspring or stillborn or undersized liveborn offspring weighing 25-30 gm each. Another unusual observation was that fur-pulling behavior, normally exhibited by pregnant female rabbits at the end of the gestational period, was absent in all of these pregnancies. Therefore, immunization of a normal rabbit with testicular cyt ct appeared to interfere with physiological and behavioral aspects of pregnancy in normal female rabbits. The immunological basis of these findings remains to be determined.

Antigenic analysis of human trophoblast membrane: detection of a lymphocyte cross-reactive antigen.

The antigenicity of human syncytiotrophoblast membrane (TM) was analyzed using rabbit antiserum raised against TM. From immunoblot analysis, about ten protein bands in TM were recognized by the anti-TM. These included placental alkaline phosphatase and TM-bound albumin. From limited antigenic specificity studies, these antigens, with the exception of albumin, were not detectable in membranes of liver, kidney, heart, and erythrocyte, or in human normal serum. Therefore, these antigens appear to be placental membrane specific. Analysis of lymphocyte membrane by immunoelectrophoresis, crossed immunoelectrophoresis, and immunoblot techniques revealed that a single protein band, designated "40 kDa," was cross-reactive with the anti-TM antiserum, indicating that this antigen is shared commonly between placenta and lymphocyte membranes. Experimental evidence suggesting that the 40-kDa membrane antigen is probably a unique trophoblast-lymphocyte cross-reactive antigen is based on the following observations: the 40-kDa antigen was not detectable in liver, heart, and kidney membrane; beta 2-microglobulin (12,000 daltons) was not detectable in our TM preparation; and the lymphocyte membrane showed only a single protein band at 40,000 daltons, not at 12,000 for beta 2-microglobulin.

Comparative in vitro spermicidal effects of (+/-)-gossypol, (+)-gossypol, (-)-gossypol and gossypolone.

The comparative in vitro spermicidal effects of (+)-gossypol, (-)-gossypol and (+/-)-gossypol were evaluated on the spermatozoa of human, monkey, rabbit, mouse, rat and hamster. The spermicidal effects of gossypol isomers were also compared with those of gossypolone, which is a proposed major metabolite of gossypol. Gossypol isomers and gossypolone were all spermicidal. (+)- and (-)-Gossypol demonstrated spermicidal activities at the same concentration at which (+/-)-gossypol shows spermicidal effects on the spermatozoa of all species tested. However, gossypolone was less potent than the gossypol isomers. The spermicidal action of gossypol may be a nonspecific effect unrelated to the antifertility mechanism of orally administered gossypol, since (+)-gossypol which is not an effective male antifertility agent also showed the equivalent spermicidal effect to that of (+/-)-gossypol.

Hybrid estimation of navigation parameters from aerial image sequence.

This work presents a hybrid method for navigation parameter estimation using sequential aerial images, where navigation parameters represent the position and velocity information of an aircraft for autonomous navigation. The proposed hybrid system is composed of two parts: relative position estimation and absolute position estimation. Computer simulation with two different sets of real aerial image sequences shows the effectiveness of the proposed hybrid parameter estimation algorithm.

Endothelial F-actin changes in the alkali burned rabbit cornea.

The healing mechanism of corneal endothelium after alkali burn was not completely understood. Rabbit cornea was burned with 1N sodium hydxoside for 1 minute. Endothelial F-actin was stained with NBD-phallacidin in regular sequence to find out the details of endothelial healing after alkali burn. F-actin was completely destroyed leaving a sharp margin against the unaffected area 1 hour after the burn. In the 3, 5 and 7 day specimens, highly active F-actin reactions were noted at the wound margin. New multiple F-actin layers, arising from the intact endothelium near the wound margin, were noted in the 9 day specimen. In the 8 1/2 month specimen, the endothelial defected area was covered by large primitive cells, each of which showed F-actin fiber bundles in the cytoplasm with a large nuclear shadow. Nearly all of the large primitive cells showed F-actin fibers arranged in shapes of cell junctions. Twelve months after the burn, endothelial defects were not found. Nearly all of the endothelial cells were normal in size and shape except for some mushroom-like projections toward the anterior chamber in some areas. Nineteen months after the burn, the endothelial cells were normal. Endothelial wound healing process can be continued even 1 year after the alkali burn in rabbit cornea.

Synergistic inhibitory effect of angiotensin-converting enzyme inhibitor and heparin on intimal hyperplasia after rat aorta injury.

The respective efficacies of angiotensin-converting enzyme (ACE) inhibitor and standard heparin were investigated with respect to their inhibitory effects on intimal hyperplasia after balloon denudation of rat aorta. Local angiotensin II effects in the artery wall may participate in regulation of the vascular response to arterial injury, apparently independent of the plasma renin and angiotensin system. ACE inhibitors have been shown to block intimal hyperplasia after arterial injury in rats. Increasing evidence points toward an inhibitory effect of heparin on intimal hyperplasia independent of anticoagulation. Balloon catheter aortic denudation was performed in 25 rats pharmacologically treated from six days or one day before to fourteen days after surgery and split into four groups: group A (control group), normal feeding; group B (ramipril group), ramipril 10 mg/kg/day orally; group C (heparin group), heparin 1200 IU/kg/day subcutaneously; group D (combined group), both ramipril and heparin. Animals were killed and aortas were perfused and fixed at physiologic pressure fourteen days after denudation. Cross-sectional intima-to-media area ratios (I-M ratio) were calculated by an image analyze system.

Trigger digits in children.

Seven thousand, seven hundred newborn children were examined prospectively to determine the congenital incidence of trigger thumb and finger. No cases were found. The case histories of 43 trigger digit cases (35 trigger thumbs and eight trigger fingers) noted in 40 children diagnosed at our center between 1995 and 1998 were reviewed with special reference to the spontaneous recovery rate, treatment outcome, and age at presentation. Of the 35 thumb cases, 23 underwent surgical release and all responded satisfactorily to surgical treatment. Spontaneous recovery was noted in 12 trigger thumb cases and in all eight trigger finger cases. Trigger finger developed earlier in life than trigger thumb and the spontaneous recovery rate was higher in trigger finger than trigger thumb.

Cryopreservation of porcine spermatogonial stem cells by slow-freezing testis tissue in trehalose.

Spermatogonial stem cells provide the foundation for continued adult spermatogenesis and their manipulation can facilitate assisted reproductive technologies or the development of transgenic animals. Because the pig is an important agricultural and biomedical research animal, the development of practical application techniques to manipulate the pig Spermatogonial stem cell is needed. The ability to preserve porcine Spermatogonial stem cell or testis tissue long term is one of these fundamental techniques. The objective of this study was to optimize methods to cryopreserve porcine Spermatogonial stem cell when freezing testis cells or testis tissue. To identify the most efficient cryopreservation technique, porcine testis cells (cell freezing) or testis tissue (tissue freezing) were frozen in medium containing dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS) or DMSO, FBS, and various concentrations of trehalose (50, 100, or 200 mM). After thawing, undifferentiated germ cells were enriched and treatments were evaluated for cryopreservation efficiency. The tissue freezing method resulted in significantly greater germ cell recovery (P = 0.041) and proliferation capacity (P < 0.001) compared to the cell freezing treatment. Regardless of freezing method (cell vs. tissue), addition of 200 mM trehalose to freezing medium increased germ cell recovery and proliferation capacity compared to cells frozen using the same freezing method without trehalose. Interestingly, addition of trehalose to the tissue freezing medium significantly increased germ cell recovery (P = 0.012) and proliferation capacity (P = 0.004) compared to the cell freezing treatment supplemented with trehalose. To confirm that cryopreservation in trehalose improves the survival of Spermatogonial stem cell, testis cells enriched for undifferentiated germ cells were xenotransplanted into recipient mouse testes. Germ cells recovered from tissue frozen with 200 mM trehalose generated significantly more (P < 0.001) donor derived colonies than tissue frozen without trehalose. Regardless of cryopreservation medium or freezing method, testis cell recovery, viability, and proliferation capacity of germ cells after thawing were significantly lower compared to those of untreated fresh control. Nevertheless, these data demonstrate that undifferentiated porcine germ cells can be efficiently cryopreserved in the presence of 200 mM trehalose.