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.

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.

New p53 target, phosphatase of regenerating liver 1 (PRL-1) downregulates p53.

Most of the p53 target genes, all except MDM2, COP1 and PIRH2, perform functions in apoptosis, differentiation and cell cycle arrest. The aforementioned oncogenes downregulate p53 through a negative feedback mechanism, and thus contribute to tumor development. In this study, we report a new p53 target, PRL-1, which is believed to be a significant regulator in the development and metastasis of a variety of cancer types. Phosphatase of regenerating liver 1 (PRL-1) overexpression reduced the levels of endogenous and exogenous p53 proteins, and inhibited p53-mediated apoptosis. On the other hand, the ablation of PRL-1 by small interfering RNA (siRNA) increased p53 protein levels. The p53 downregulation was mediated by p53 ubiquitination and subsequent proteasomal degradation. Furthermore, p53 ubiquitination by PRL-1 was achieved through two independent pathways, by inducing PIRH2 transcription and by inducing MDM2 phosphorylation through Akt signaling. In addition, we showed that the PRL-1 gene harbors a p53 response element in the first intron, and its transcription is regulated by the p53 protein. These findings imply that the new oncogenic p53 target, PRL-1, may contribute to tumor development by the downregulation of p53 by a negative feedback mechanism.

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.

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.

Endogenous glycosylphosphatidylinositol-specific phospholipase C releases renal dipeptidase from kidney proximal tubules in vitro.

Spontaneous enzymic release of renal dipeptidase (RDPase; EC 3.4.13.19), a glycosylphosphatidylinositol (GPI)-linked ectoenzyme, was observed in vitro during incubation of porcine proximal tubules at 37 degrees C. Triton X-114 phase separation of the released RDPase showed that the majority of the enzyme activity partitioned into the aqueous phase, indicating its hydrophilic nature. Immunoblot analyses using an antibody against the cross-reacting determinant (CRD) inositol 1,2-cyclic monophosphate, the epitope formed by phospholipase C (PLC) cleavage of the GPI anchor on a protein, detected the released RDPase. Reprobing the immunoblot with an anti-RDPase serum showed the RDPase band co-migrating with the CRD band. The release of RDPase from the proximal tubules was a Ca(2+)-dependent process and had a pH optimum of 9.0. These results indicate that RDPase is released from the proximal tubules by the action of a distinct endogenous GPI-specific PLC.

Reoperation of Recurrent Gastric Cancer.

PURPOSE: The aim of this study was to evaluate the outcome of reoperation in recurrent gastric cancers. MATERIALS AND METHODS: We conducted a retrospective analysis of 86 patients who underwent reoperation for recurrent gastric cancer. We reviewed the time interval between first operation and reoperation, as well as the recurrence pattern, type of reoperation, and survival following reoperation. RESULTS: the average time to reoperation following curative resection was 27.8+/-25.9 months (median 18.4 months). Fifty-three cases (61.6%) of reoperation were performed within 2 years follwoing the first operation. The most common reason for reoperation was intestinal obstruction followed by gastric remnant recurrence and intra-abdominal mass. Complete resection was possible in 14 cases (16.3%) and a palliative procedure such as partial resection or bypass procedures was performed in 54 cases. In 18 cases (20.9%), simple lapalotomy was done without any aid. The most common site of recurrence was the peritoneum followed by the gastric remnant, distant lymph node and hematogenous liver metastasis. Operative mortality was 10.5%. Excluding the 9 cases of operative mortality, the mean survival time after reoperation was 15.4+/-2.5 months (mean 8.6 months). Survival following complete resection was much longer than palliative procedure and exploration only (37.9+/-8.7 vs 10.9+/-1.5 vs 4.7+/-0.8 months, p=0.000). CONCLUSION: The complete resection of recurrent gastric cancer can prolong survival. Early detection of localized recurrence is important in order to increase the chance of complete resection.

The antithrombotic effects of CI-1028, an orally bioavailable direct thrombin inhibitor, in a canine model of venous and arterial thrombosis.

Direct thrombin inhibitors represent a new class of drug that may offer a therapeutic alternative for the treatment and prevention of thrombembolic conditions, especially on the venous side of the systemic circulation. CI-1028 (PD 172524/LB30057) is a potent, highly selective inhibitor of thrombin that is orally bioavailable. The efficacy of this compound has been demonstrated in animal models in which intra-venous administration was used. The objective of this study was to evaluate the efficacy of CI-1028 after oral administration in a canine electrolytic injury model of venous and arterial thrombosis. CI-1028 was administered via oral gavage, and animals received either saline or 10, 15, 20, or 30 mg/kg of drug. Fifteen minutes later, the dogs were anesthetized and a femoral artery and vein were exposed and instrumented to induce electrolytic injury and thrombosis while continuously monitoring blood flow in the vessels. Maximum blood CI-1028 concentrations of 0.88+/-0.27, 1.8+/-0.3, 2.2+/-0.5, and 3.2+/-0.5 microg/mL were generally achieved 15 to 30 minutes after administering the compound in the 10-, 15-, 20-, and 30-mg/kg groups, respectively. Administration of CI-1028 increased the time to occlusion (TTO), the principal efficacy end point, in a dose-dependent manner in both arteries and veins. The TTO in the control group (n=8) averaged 66+/-11 minutes in the arteries and 69+/-6 minutes in the veins. In dogs treated with 10 mg/kg (n=8), the TTO was not significantly different from that of the control group. In the 15-mg/kg group (n=9) TTO averaged 140+/-27 minutes in the arteries (p=not significant) and 125+/-15 minutes (p<0.05) in the veins. In the 20-mg/kg group (n=8), TTO was significantly longer than controls in both types of vessels, averaging 168+/-30 minutes in the arteries (p=0.05) and 155+/-21 minutes (p<0.05) in the veins. Likewise, at 30 mg/kg (n=8) both the arterial (179+/-17 minutes) and venous (188+/-15 minutes) TTO was significantly prolonged compared with controls. Surgical blood loss and template bleeding times tended to increase in a dose-dependent manner but a statistically significant elevation was detected for template bleeding time only at the highest dose. Dramatic changes in thrombin time were detected, consistent with the CI-1028 mechanism of action. Virtually no changes were detected in prothrombin time. Maximum activated partial thromboplastin time (aPTT) and activated clotting time changes were detected approximately 30 minutes after dosing, and they were approximately twofold and fivefold baseline values, respectively, at the highest dose. In conclusion, these results demonstrate that CI-1028 provides dose-dependent antithrombotic efficacy after oral administration in a canine model of venous and arterial thrombosis.

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.

Pharmacokinetics and tolerability of gemifloxacin (SB-265805) after administration of single oral doses to healthy volunteers.

Gemifloxacin (known as SB-265805 or LB-20304) is a potent, novel fluoroquinolone compound with a broad spectrum of antibacterial activity. The pharmacokinetics and tolerability of oral gemifloxacin were characterized in healthy male volunteers after a single dose of 20, 40, 80, 160, 320, 600, or 800 mg. Multiple serum and urine samples were collected and analyzed for gemifloxacin using high-performance liquid chromatography with fluorescence detection. Safety assessments included vital signs, 12-lead electrocardiogram readings, hematology, clinical chemistry, urinalysis, and adverse-experience monitoring. Gemifloxacin was rapidly absorbed after all doses. Maximum concentrations of gemifloxacin in serum (C(max)) were achieved approximately 1 h after dosing, after which concentrations in serum declined in a biexponential manner. Values of C(max) and the area under the concentration-time curve in serum from 0 h to infinity (serum AUC(0-infinity)) increased linearly with dose. Serum AUC(0-infinity) values (mean +/- standard deviation) were 0.65+/-0.01, 1.28+/-0.22, 2.54+/-0.31, 5.48+/-1.24, 9.82+/-2.70, 24.4+/-7.1, and 31.4+/-7.6 microg. h/ml following 20-, 40-, 80-, 160-, 320-, 600-, and 800-mg doses, respectively. The terminal phase elimination half-life was independent of dose, with an overall mean of 7.4+/-2.0 h. The profiles indicated that the pharmacokinetic profile is suitable for a once-daily dosing regimen. Approximately 25 to 40% of the administered dose was excreted unchanged in the urine, and renal clearance (ca. 150 ml/min) was independent of dose. There were no significant changes in clinical chemistry, hematology, or urinalysis parameters, vital signs, or 12-lead electrocardiogram readings in subjects, irrespective of dose. The results of these studies support the further investigation of once-daily administration of gemifloxacin.

A humanized anti--4-1BB monoclonal antibody suppresses antigen-induced humoral immune response in nonhuman primates.

The interaction of 4-1BB and its ligand plays an important role in the regulation of T-cell-mediated immune responses. In this study, the authors examined the effect of a humanized anti--4-1BB monoclonal antibody (H4B4) on ovalbumin-induced immune responses in baboons. Previously, a mouse monoclonal antibody, 4B4 against the human 4-1BB molecule, was generated and characterized. Based on this antibody, a humanized version of 4B4 monoclonal antibody was constructed and the resultant antibody, H4B4, showed full recovery of the binding activity of the original antibody 4B4: a 1.5-fold increase in affinity for 4-1BB. In addition, H4B4 mediated antibody-dependent cellular cytotoxicity of activated human peripheral blood T cells and CEM cells in a dose-dependent manner. Weekly administration of H4B4 at doses of 1 or 4 mg/kg could suppress immunoglobulin G production against ovalbumin. This was not a result of the overall immune suppression, because the numbers of B and T cells and the total immunoglobulin G production were not altered during treatment with H4B4. These findings suggest that treatment with H4B4 may be a valid therapeutic approach to control unwanted immune responses in persons with autoimmune diseases.

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.

High-performance liquid chromatographic determination of a new oral thrombin inhibitor in the blood of rats and dogs.

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of a new oral thrombin inhibitor (compound I) in the blood of rats and dogs. The analyte was deproteinized with a 1.5 volume of methanol and a 0.5 volume of 10% zinc sulfate, and the supernatant was injected into a 5-microm Capcell Pak C18 column (150 x 4.6 mm I.D.). The mobile phase was a mixture of acetonitrile and 0.2% triethylamine of pH 2.3 (31:69, v/v) with a flow-rate of 1.0 ml/min at UV 231 nm. The retention time of compound I was approximately 9.3 min. The calibration curve was linear over the concentration range of 0.05-100 mg/l for rat blood (r2>0.9995, n=6) and dog blood (r2>0.9993, n=6). The limit of quantitation was 0.05 mg/l for both bloods using a 100-microl sample. For the 5 concentrations (0.05, 0.1, 1, 10, and 100 mg/l), the within-day recovery (n=4) and precision (n=4) were 98.1-104.1% and 1.5-6.8% for rat blood and 95.4-105.7% and 1.4-5.3% for dog blood, respectively. The between-day recovery (n=6) and precision (n=6) were 99.8-105.3% and 3.7-12.6% for rat blood and 87.5-107.1% and 2.9-15.3% for dog blood, respectively. The absolute recoveries were 82.4-93.3%. No interferences from endogenous substances were observed. In conclusion, the presented simple, sensitive, and reproducible HPLC method proved and was used successfully for the determination of compound I in the preclinical pharmacokinetics.

Streptokinase secretion by Serratia marcescens signaled by the C-terminal 41 amino acid segment of metalloprotease.

In order to investigate the secretion signal of Serratia marcescens metalloprotease (SMP) and examine the ability of the secretion signal to secrete foreign proteins, hybrid genes encoding the passenger-SMP C-terminal segments were constructed. As a passenger protein, streptokinase (SK) deprived of its signal peptide was used. Three kinds of SMP C-terminal segments containing 41, 80, or 220 amino acid residues were fused to the C-terminus of SK as secretion signals. The SK-SMP chimeric proteins containing 41 or 220 amino acid segments of the SMP C-terminus were secreted into the culture medium by the SMP transporter of S. marcescens. This result suggests that cytoplasmic SK is secreted into the external medium by the C-terminal segments of SMP and also shows that the smallest, 41 amino acid segment of the SMP C-terminus functions as a secretion signal of foreign proteins as well as SMP.

Experience of orthotopic liver transplantation in 11 patients with liver cirrhosis from Korea: medical factors affecting outcome.

Orthotopic liver transplantation (OLT) has evolved to become a standard treatment of choice for end-stage liver diseases. The present study was performed to evaluate the peri-operative medical factors affecting transplantation outcome and to determine if patients with type B viral cirrhosis were acceptable for OLT. A total of 11 patients with end-stage cirrhosis, who have received OLT in Kangnam St. Mary's Hospital since May 1993, included 8 HBV-related cases, 1 Hepatitis C Virus (HCV)-related case, and 2 non-B, non-C cases. One-year cumulative survival rate by Kaplan-Meier method was 43.7%. Factors significantly associated with 1-year survival of the recipients during pre-OLT period were performance status and modified Pugh-Child score (p=0.015 and p=0.015, respectively). Among those 4 patients who lived longer than 1 year, 3 of 4 patients with HBsAg-positive had no HBV re-infection with our protocol. These results suggest that, to improve the outcome of OLT in cirrhosis patients, transplantation should be performed in the stage when patients maintain better performance and hepatic functional reserve during the end-stage of liver cirrhosis. In addition, patients with cirrhosis caused by HBV infection may be indicated for OLT, because HBV re-infection is preventable effectively with a high-dose hepatitis B immunoglobulin protocol.

The Leu-3 residue of Serratia marcescens metalloprotease inhibitor is important in inhibitory activity and binding with Serratia marcescens metalloprotease.

Serratia marcescens metalloprotease inhibitor (SmaPI) is a proteinase inhibitor toward Serratia marcescens metalloprotease (SMP). In sequential deletion analysis of the N-terminal region of the SmaPI, SmaPIs starting at Ser-2 and Leu-3 residues, respectively, had nearly a full inhibitory activity toward SMP. However, SmaPI starting at Ala-4 residue showed severely decreased inhibitory activity. Furthermore, kinetic analysis demonstrated that SmaPI starting at the Ala-4 residue had an inhibition constant for SMP approximately fourfold higher than that of wild-type SmaPI. The interactions of Leu-3 with SMP contribute 0.73 kcal mol-1 to the overall stability of the SMP-SmaPI complex (8.44 kcal mol-1). To elucidate the detailed role of the Leu-3 residue in inhibitory activity of SmaPI, several site-directed mutations were introduced. The inhibitory activities of Leu-3 mutants in which the Leu-3 has been converted to Ala, Asp, Gly, Ile, Lys, Phe, or Pro were correlated with the hydrophobicities of substituted amino acids. About 0.3 kcal mol-1 is attributable to the side chain of the Leu-3 residue in the binding with SMP. From these results, it is suggested that (i) in contrast with the Erwinia chrysanthemi inhibitor, Gly-1 and Ser-2 of SmaPI are not critical and (ii) the hydrophobicity of Leu-3 may be important in its inhibitory activity and binding with SMP.

Benzylamine-based selective and orally bioavailable inhibitors of thrombin.

A series of p-aminomethylphenylalanine derivatives were investigated as novel thrombin inhibitors. This study led to potent inhibitors of thrombin (Ki up to 3.3 nM) that are trypsin-selective, highly orally bioavailable in rats, and highly permeable across Caco-2 cells. The P1 benzylamine binding mode in the thrombin active site was identified by X-ray crystallographic analysis.