Preliminary studies on the zinc-induced metallothionein protein with antibacterial activity in housefly larvae, Musca domestica.

Three isoforms of metallothionein protein induced with Zinc were isolated and purified from housefly larvae, Musca domestica, by gel filtration on Sephadex G-75, G-25 and anion exchange on DEAE-52 chromatography. Among them, one was found to possess antibacterial activity, and was further characterized by SDS-polyacrylamide gel electrophoresis, sulphydryl group determination, enzyme hydrolysis, and spectra property. Our results showed that the novel protein has the characteristics of heat-stable, low-molecular weight (6 kDa), rich-cysteine (approximately 12 cysteine residues in one molecule), metal affinity, and antibacterial activity. This paper was the first to report that metallothionein had antibacterial activity. We expect that this characteristic would give some help to investigate definite physiological functions of metallothionein.

ATP-sensitive K+ current and its modulation by substance P in gastric myocytes isolated from guinea pig.

To investigate whether ATP-sensitive K+ channels exist in gastric smooth muscle of the guinea pig and whether they are modulated by substance P, we recorded lemakalim-activated K+ currents from freshly isolated cells using the standard whole-cell configuration. With 0.1 mM ATP and 140 mM K+ in the pipette and 90 mM K+ in the bath solution and a holding potential of -80 mV, lemakalim (10 microM) activated a glibenclamide-sensitive inward current with a mean amplitude of -224+/-34 pA. These currents were voltage-independent from -90 to 0 mV and K+-selective. Increasing the intracellular ATP concentrations from 0.1 to 3 mM reduced the lemakalim-activated currents by about five-fold. External barium and cesium inhibited the lemakalim-activated currents in a dose-dependent manner. External tetraethylammonium (10 mM) inhibited the lemakalim-activated currents by 66+/-15%. Bath application of substance P (5 microM) inhibited the lemakalim-activated currents by 53+/-13% and this inhibition was absent when 0.5 mM guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS) was in the pipette. Phorbol 12,13-dibutyrate (PDB) inhibited the lemakalim-activated currents by 71+/-3%. Chelerythrine (1 microM) reduced the substance P-induced inhibition of lemakalim-activated currents by 22.2+/-11.3%. These results suggest the presence of ATP-sensitive K+ channels in gastric smooth muscle and that substance P inhibits ATP-sensitive K+ channels via G-protein through protein kinase C activation.

Capsaicin inhibits the voltage-operated calcium channels intracellularly in the antral circular myocytes of guinea-pig stomach.

Studies of the effect of capsaicin (CAP) on the smooth muscle contractions have shown both contraction and relaxation in various preparations. The direct effect of CAP on gastric smooth muscle itself has not yet been reported, though CAP was reported to relax the isolated guinea-pig stomach by releasing nitric oxide from the CAP-sensitive sensory neurons. Here we showed an evidence that CAP evokes a prolonged relaxation of gastric antral circular smooth muscle (CAP-induced relaxation) by blocking the voltage-operated Ca2+ channels (VOCC) from inside of the cell. CAP suppressed dose-dependently the spontaneous contractions of guinea-pig gastric circular muscle strip under the condition without neural influence (IC50 = 5.8 microM). The inhibitory effects of CAP both on the high K+ contracture induced by 50 mM K+ Tyrode solution and on the slow waves recorded using a conventional intracellular microelectrode technique were similar to those of Ca2+ channel antagonists, indicating that Ca2+ influx through the VOCC is decreased by CAP. Ca2+ channel current (I(Ba)) decreased in a concentration-dependent manner on superfusing the physiological salt solution containing various concentrations of CAP. The steady-state activation and inactivation curves of I(Ba) were not affected by the treatment with CAP. The experiment using a synthetic water-soluble analog of CAP, DA-5018 x HCl, suggested that the acting site of CAP is present in the intracellular side. Spontaneous transient outward K+ currents (STOCs) recorded at a holding potential of 0 mV were also inhibited by CAP and verapamil, Ca channel blocker. Taken together, these results indicate that CAP-induced relaxation is associated with the direct inhibitory action on the VOCC from inside of the cell.

The p53-inducible gene 3 (PIG3) contributes to early cellular response to DNA damage.

The p53-inducible gene 3 (PIG3) is originally isolated as a p53 downstream target gene, but its function remains unknown. Here, we report a role of PIG3 in the activation of DNA damage checkpoints, after UV irradiation or radiomimetic drug neocarzinostatin (NCS). We show that depletion of endogenous PIG3 sensitizes cells to DNA damage agents, and impaired DNA repair. PIG3 depletion also allows for UV- and NCS-resistant DNA synthesis and permits cells to progress into mitosis, indicating that PIG3 knockdown can suppress intra-S phase and G2/M checkpoints. PIG3-depleted cells show reduced Chk1 and Chk2 phosphorylation after DNA damage, which may directly contribute to checkpoint bypass. PIG3 exhibited diffuse nuclear staining in the majority of untreated cells and forms discrete nuclear foci in response to DNA damage. PIG3 colocalizes with gamma-H2AX and 53BP1 to sites of DNA damage after DNA damage, and binds to a gamma-H2AX. Notably, PIG3 depletion decreases the efficient induction and maintenance of H2AX phosphorylation after DNA damage. Moreover, PIG3 contributes to the recruitment of 53BP1, Mre11, Rad50 and Nbs1 to the sites of DNA break lesions in response to DNA damage. Our combined results suggest that PIG3 is a critical component of the DNA damage response pathway and has a direct role in the transmission of the DNA damage signal from damaged DNA to the intra-S and G2/M checkpoint machinery in human cells.

[The effects of programmed jogging on metabolism and cardio-pulmonary function of type II diabetic patients].

This study was done for the purpose of testing effects of programmed jogging as one of the self care activities on glucose and lipid metabolism and cardio-pulmonary function in type II diabetic patients. Research design was a repeated measure as non-equivalent control group pre-post test quasi-experimental design. Thirty type II diabetic patients who received follow-up care regularly at the out patient department of internal medicine in two general hospitals which had diabetic clinic were studied. Fourteen were assigned to the experimental group and sixteen to the control group. The experimental group participated in the programmed jogging 3 times per week for 4 weeks and the control group didn't have the programmed jogging. The programmed was developed from a review of the literature done by the researcher. Data were analyzed by X2-test, t-test, paired t-test and MANOVA using SPSS/PC The results are summarized as follows; 1. There were no significant difference between the experimental group and the control group for general characteristics and the pre-test of metabolism and cardiopulmonary function. 2. Hypothesis 1) that type II diabetic patients who participate in the programmed jogging will have a higher level of glucose metabolism than type II diabetic patients who don't do programmed jogging was partly supported(FBS; between groups F = 9.57, p < .05, time within the experimental group F = 24.28, p < .001, .05, HbA1C; between groups t = 1.09, p > .05, time within the experimental group t = 2.32, p < .05). 3. Hypothesis 2) that type II diabetic patients who participate in the programmed jogging will have a higher level of lipid metabolism than type II diabetic patients who don't do programmed jogging was rejected(triglyceride; between groups F = .85, p > .05, time within the experimental group F = 7.87, p < .05, total cholesterol; between groups F = 4.07, p > .05, time within the experimental group F = .02, p > .05, HDL cholesterol; between groups F = 2.72, p. > 05, time within the experimental group F = 9.81, p < .05, body weight; between groups F = 2.72, p > .05, time within the experimental group F = 15.38, p < .001).(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of pacemaker currents in interstitial cells of Cajal from murine small intestine by cyclic nucleotides.

1. Electrical rhythmicity (slow waves) in gastrointestinal muscles (GI) is generated by interstitial cells of Cajal (ICC). Cultured ICC from the murine small intestine were studied with the patch-clamp technique to characterize regulation of pacemaker currents by cyclic nucleotides. Cyclic nucleotide agonists were also tested on intact strips of murine small intestine. 2. Nitric oxide donors slowed the frequency of pacemaker currents in a concentration-dependent manner. These effects depended on cGMP formation and were reduced by 1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). The effects of nitric oxide donors were mimicked by membrane-permeable analogues of cGMP. The specific cGMP phosphodiesterase inhibitor zaprinast reduced the frequency of spontaneous pacemaker currents. 3. The cGMP-dependent effects on pacemaker currents were not affected by okadaic acid or KT-5823, an inhibitor of protein kinase G. 4. Forskolin, but not dideoxy forskolin, reduced the frequency of spontaneous pacemaker activity and activated a sustained outward current. The latter was likely to be due to ATP-dependent K+ channels because it was blocked by glibenclamide. 5. The effects of forskolin were not mimicked by membrane-permeable cAMP analogues. A membrane-permeable inhibitor of protein kinase A, myristoylated PKA inhibitor, and the adenylyl cyclase inhibitor SQ-22536, had no effect on responses to forskolin. 6. Responses of intact muscles to cGMP and cAMP agonists were similar to the responses of pacemaker cells. Changes in resting membrane potential and slow wave amplitude, however, were noted in intact jejunal muscles that were not observed in ICC. Differences in responses may have been due to the effects of cyclic nucleotide agonists on smooth muscle cells that would sum with responses of ICC in intact jejunal muscle strips. 7. A cGMP-dependent mechanism regulates slow wave frequency, but this occurs through direct action of cGMP not via protein phosphorylation. Regulation of pacemaker currents by cAMP-dependent mechanisms was not observed.

Bakuchiol: a hepatoprotective compound of Psoralea corylifolia on tacrine-induced cytotoxicity in Hep G2 cells.

Bioassay-guided fractionation of the H(2)O extract of the seeds of Psoralea corylifolia furnished one hepatoprotective compound, bakuchiol (1), together with two moderately active compounds, bakuchicin (2) and psoralen (3), on tacrine-induced cytotoxicity in human liver-derived Hep G2 cells. The EC(50) values of compounds 1 - 3 are 1.0, 47.0, 50.0 microg/ml, respectively. Silymarin as a positive control showed the EC(50) value with 5.0 microg/ml.

Regulation of ATP-sensitive K(+) channels by protein kinase C in murine colonic myocytes.

We investigated the regulation of ATP-sensitive K(+) (K(ATP)) currents in murine colonic myocytes with patch-clamp techniques. Pinacidil (10(-5) M) activated inward currents in the presence of high external K(+) (90 mM) at a holding potential of -80 mV in dialyzed cells. Glibenclamide (10(-5) M) suppressed pinacidil-activated current. Phorbol 12,13-dibutyrate (PDBu; 2 x 10(-7) M) inhibited pinacidil-activated current. 4-alpha-Phorbol ester (5 x 10(-7) M), an inactive form of PDBu, had no effect on pinacidil-activated current. In cell-attached patches, the open probability of K(ATP) channels was increased by pinacidil, and PDBu suppressed openings of K(ATP) channels. When cells were pretreated with chelerythrine (10(-6) M) or calphostin C (10(-7) M), inhibition of the pinacidil-activated whole cell currents by PDBu was significantly reduced. In cells studied with the perforated patch technique, PDBu also inhibited pinacidil-activated current, and this inhibition was reduced by chelerythrine (10(-6) M). Acetylcholine (ACh; 10(-5) M) inhibited pinacidil-activated currents, and preincubation of cells with calphostin C (10(-7) M) decreased the effect of ACh. Cells dialyzed with protein kinase C epsilon-isoform (PKCepsilon) antibody had normal responses to pinacidil, but the effects of PDBu and ACh on K(ATP) were blocked in these cells. Immunofluorescence and Western blots showed expression of PKCepsilon in intact muscles and isolated smooth muscle cells of the murine proximal colon. These data suggest that PKC regulates K(ATP) in colonic muscle cells and that the effects of ACh on K(ATP) are largely mediated by PKC. PKCepsilon appears to be the major isozyme that regulates K(ATP) in murine colonic myocytes.

Pacemaking in interstitial cells of Cajal depends upon calcium handling by endoplasmic reticulum and mitochondria.

Pacemaker cells, known as interstitial cells of Cajal (ICC), generate electrical rhythmicity in the gastrointestinal tract. Pacemaker currents in ICC result from the activation of a voltage-independent, non-selective cation conductance, but the timing mechanism responsible for periodic activation of the pacemaker current is unknown. Previous studies suggest that pacemaking in ICC is dependent upon metabolic activity 1y1yand1 Ca2+ release from intracellular stores. We tested the hypothesis that mitochondrial Ca2+ handling may underlie the dependence of gastrointestinal pacemaking on oxidative metabolism. Pacemaker currents occurred spontaneously in cultured ICC and were associated with mitochondrial Ca2+ transients. Inhibition of the electrochemical gradient across the inner mitochondrial membrane blocked Ca2+ uptake and pacemaker currents in cultured ICC and blocked slow wave activity in intact gastrointestinal muscles from mouse, dog and guinea-pig. Pacemaker currents and rhythmic mitochondrial Ca2+ uptake in ICC were also blocked by inhibitors of IP3-dependent release of Ca2+ from the endoplasmic reticulum and by inhibitors of endoplasmic reticulum Ca2+ reuptake. Our data suggest that integrated Ca2+ handling by endoplasmic reticulum and mitochondria is a prerequisite of electrical pacemaking in the gastrointestinal tract.

Expression of vimentin and cytokeratin in eutopic and ectopic endometrium of women with adenomyosis and ovarian endometrioma.

PROBLEM: To determine the expression of vimentin and cytokeratin in eutopic and ectopic endometrium of women with both adenomyosis and ovarian endometrioma and to evaluate their cyclic changes during the menstrual cycle. METHOD OF STUDY: Twenty patients requiring hysterectomy with salpingo-oophorectomy were studied by immunohistochemistry according to their menstrual cycles. RESULTS: Cyclic expression of vimentin was noted in eutopic endometrium and adenomyosis, but not in endometrioma. Cytokeratin expression did not change during the menstrual cycles. The mean intensities of epithelial vimentin were significantly different from each other, being the lowest in endometrioma, intermediate in adenomyosis, and the highest in eutopic endometrium. There was no significant difference in intensities of cytokeratin between adenomyosis and endometrioma, but these intensities were significantly lower than that of eutopic endometrium. CONCLUSIONS: Lower intensities of cytokeratin in adenomyosis and endometrioma than in eutopic endometrium suggest that the ectopic endometria may have a lower degree of differentiation regardless of the site. The lower intensity of epithelial vimentin in endometrioma than in adenomyosis during the proliferative phase may reflect decreased functional activity, probably because of a pressure effect on the lining epithelium within the endometrioma.

Protein kinase C mediates the desensitization of CCh-activated nonselective cationic current in guinea-pig gastric myocytes.

The possibility of the protein kinase C (PKC) pathway being a mechanism underlying the desensitization of carbachol- (CCh-)activated nonselective cationic current (ICCh) was investigated in a study of guinea-pig gastric myocytes. Using the conventional whole-cell patch-clamp technique with symmetrical CsCl-rich solution in pipette and bath, ICCh was induced by bath application of 50 microM CCh. With 0.5 mM EGTA [ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] in the pipette solution (0.5 mM [EGTA]i), ICCh decayed spontaneously (desensitization of ICCh) to around 20% within 10 min. Desensitization of ICCh was significantly attenuated with 2 mM [EGTA]i. At a concentration of 20 microM OAG (1-oleoyl-2-acetyl-sn-glycerol), a PKC activator, inhibited ICCh at 0.5 mM [EGTA]i but far less at 2 mM [EGTA]i (18% and 81% of control, respectively). The same cationic current induced by intracellular guanosine-5'-O-(3-thiotriphosphate) (GTP[gamma-S]) was not inhibited by OAG with 0.5 mM [EGTA]i. The pretreatment of gastric myocytes with PKC inhibitors, either 1 microM chelerythrine or 1 microM peptide inhibitor, attenuated the desensitization of ICCh. [Ca2+]i was also measured by single cell microfluorometry using fura-2. Under CCh stimulation with 2 mM [EGTA]i, [Ca2+]i did not increase above 100 nM while it increased to around 260 nM with 0.5 mM [EGTA]i. These results suggest that the desensitization of ICCh is partly due to the Ca2+-dependent PKC pathway in guinea-pig gastric myocytes.

Efficacy of microsurgical epididymal sperm aspiration (MESA) and intracytoplasmic sperm injection (ICSI) in obstructive azoospermia.

PURPOSE: Our purpose was to test whether micromanipulation using subzonal insemination and intracytoplasmic sperm injection could improve the poor fertilization and pregnancy rates obtained when attempting in vitro fertilization in patients with congenital absence of the vas deferens and unreconstructable obstructive azoospermia with microsurgically retrieved epididymal spermatozoa. RESULTS: Conventional in vitro fertilization (group A; 14 cycles), subzonal insemination (group B; 13 cycles), and intracytoplasmic sperm injection (group C; 28 cycles) were carried out in 55 treatment cycles. Fertilization rates for groups A, B, and C were 16.1, 31.4, and 48.6%, respectively (P < 0.05). Clinical pregnancy rates for groups A, B, and C were 7.1, 7.7, and 32.1% (P < 0.05), respectively. In five cycles, intracytoplasmic sperm injection using epididymal sperm from alloplastic spermatoceles was performed and two clinical pregnancies (40%) were obtained. CONCLUSIONS: The combined microsurgical epididymal sperm aspiration and intracytoplasmic sperm injection procedure is highly effective in improving the fertilization and pregnancy rate in congenital absence of the vas deferens and unreconstructable obstructive azoospermia. Furthermore, alloplastic spermatoceles may be useful for repeat sperm aspirations.