Assessment of synchronization measures for effective ventricular support by using the shape memory alloy fibred artificial myocardium in goats.

Thromboembolic and haemorrhagic complications are the primary causes of mortality and morbidity in patients with artificial hearts, which are known to be induced by the interactions between blood flow and artificial material surfaces. The authors have been developing a new mechanical artificial myocardial assist device by using a sophisticated shape memory alloy fibre in order to achieve the mechanical cardiac support from outside of the heart without a direct blood contacting surface. The original material employed as the actuator of artificial myocardial assist devices was 100um fibred-shaped, which was composed of covalent and metallic bonding structure and designed to generate 4-7 % shortening by Joule heating induced by the electric current input. In this study, we focused on the synchronization of the actuator with native cardiac function, and the phase delay parameter was examined in animal experiments using Saanen goats. Total weight of the device including the actuator was around 150g, and the electric power was supplied transcutaneously. The device could be successfully installed into thoracic cavity, which was able to be girdling the left ventricle. The contraction of the device could be controlled by the originally designed microcomputer. The mechanical contraction signal input had been transmitted with the phase delay of 50-200 msec after the R-wave of ECG, and hemodynamic changes were investigated. Cardiac output and systolic left ventricular pressure were elevated with 20% delay of cardiac cycle by 27% and 7%, respectively, although there was smaller difference under the condition of the delay of over 30%. Therefore, it was suggested that the synchronization measures should be examined in order to achieve sophisticated ventricular passive/active support on physiological demand.

Daily chewing gum exercise for stabilizing the vertical occlusion.

We investigated the effects of a chewing gum exercise program on occlusal conditions and evaluated compliance of subjects. Thirty-five healthy adult volunteers (26 males and nine females) were asked to chew gum for 10-15 min before or after three meals daily for four weeks. Occlusal conditions were recorded as occlusal parameters, such as occlusal contact area, occlusal contact force, and pressure using dental prescale films. These parameters were evaluated by an Occluzer before the exercise period commenced, after four weeks of exercise, and then one month after the end of the exercise period. These parameters were statistically compared using one-way ANOVA. We found that: (i) after four weeks of exercise, anterior and posterior occlusal contact areas and forces were significantly (P < 0.05) increased and the increments were significantly (P < 0.05) higher in the anterior occlusal contact area and force than in the posterior occlusal contact area and force, (ii) the anteroposterior ratio of occlusal contact area and force increased, but not markedly, (iii) increased parameters had significantly (P < 0.05) decreased within one month after the end of the four-week exercise period, (iv) most participants did not complain for discomfort or stress during the exercise. The chewing gum exercise program could increase occlusal contact area and force and also move the anteroposterior occlusal balance forward. Patient compliance with the exercise is likely high enough to keep them exercising.

Sensorless control for a sophisticated artificial myocardial contraction by using shape memory alloy fibre.

The authors have been developing an artificial myocardium, which is capable of supporting natural contractile function from the outside of the ventricle. The system was originally designed by using sophisticated covalent shape memory alloy fibres, and the surface did not implicate blood compatibility. The purpose of our study on the development of artificial myocardium was to achieve the assistance of myocardial functional reproduction by the integrative small mechanical elements without sensors, so that the effective circulatory support could be accomplished. In this study, the authors fabricated the prototype artificial myocardial assist unit composed of the sophisticated shape memory alloy fibre (Biometal), the diameter of which was 100 microns, and examined the mechanical response by using pulse width modulation (PWM) control method in each unit. Prior to the evaluation of dynamic characteristics, the relationship between strain and electric resistance and also the initial response of each unit were obtained. The component for the PWM control was designed in order to regulate the myocardial contractile function, which consisted of an originally-designed RISC microcomputer with the input of displacement, and its output signal was controlled by pulse wave modulation method. As a result, the optimal PWM parameters were confirmed and the fibrous displacement was successfully regulated under the different heat transfer conditions simulating internal body temperature as well as bias tensile loading. Then it was indicated that this control theory might be applied for more sophisticated ventricular passive or active restraint by the artificial myocardium on physiological demand.

Morphological approach for the functional improvement of an artificial myocardial assist device using shape memory alloy fibres.

The authors have been developing a mechano-electric artificial myocardial assist system (artificial myocardium) which is capable of supporting natural contractile functions from the outside of the ventricle without blood contacting surface. In this study, a nano-tech covalent type shape memory alloy fibre (Biometal, Toki Corp, Japan) was employed and the parallel-link structured myocardial assist device was developed. And basic characteristics of the system were examined in a mechanical circulatory system as well as in animal experiments using goats. The contractile functions were evaluated with the mock circulatory system that simulated systemic circulation with a silicone left ventricular model and an aortic afterload. Hemodynamic performance was also examined in goats. Prior to the measurement, the artificial myocardial assist device was installed into the goat's thoracic cavity and attached onto the ventricular wall. As a result, the system could be installed successfully without severe complications related to the heating, and the aortic flow rate was increased by 15% and the systolic left ventricular pressure was elevated by 7% under the cardiac output condition of 3L/min in a goat. And those values were elevated by the improvement of the design which was capable of the natural morphological myocardial tissue streamlines. Therefore it was indicated that the effective assistance might be achieved by the contraction by the newly-designed artificial myocardial assist system using Biometal. Moreover it was suggested that the assistance gain might be obtained by the optimised configuration design along with the natural anatomical myocardial stream line.

Development of an Artificial Myocardium using a Covalent Shape-memory Alloy Fiber and its Cardiovascular Diagnostic Response.

The authors have been developing a newly-designed totally-implantable artificial myocardium using a covalent shape-memory alloy fibre (Biometal®, Toki Corporation), which is attached onto the ventricular wall and is also capable of supporting the natural ventricular contraction. This mechanical system consists of a contraction assistive device, which is made of Ti-Ni alloy. And the phenomenon of the martensitic transformation of the alloy was employed to achieve the physiologic motion of the device. The diameter of the alloy wire could be selected from 45 to 250μm. In this study, the basic characteristics of the fiber of 150μm was examined to design the sophisticated mechano-electric myocardium. The stress generated by the fiber was 400gf under the pulsatile driving condition (0.4W, 1Hz). Therefore it was indicated that the effective assistance might be achieved by using the Biometal shape-memory alloy fiber.

Papanicolaou tests and molecular analyses using new fluid-based specimen collection technology in 3000 Japanese women.

A fluid-based Papanicolaou test has been established to improve sample collection and preparation. This study was the first large-scale investigation in Japan to examine the feasibility of using fluid-based Papanicolaou specimens to detect human papillomavirus (HPV) using Hybrid Capture II and polymerase chain reaction (PCR). Three thousand patients who visited Keio University Hospital between October 2000 and February 2001 were enrolled in the study. The results of the fluid-based Papanicolaou tests corresponded well with those of conventional Papanicolaou smears (96.8% concordance). The sensitivities of cervical neoplasia detection using the fluid-based Papanicolaou test (73.9%) and Hybrid Capture II (76.3%, P=0.55) were not significantly different. Among the cervical intraepithelial neoplasia 3 and squamous cell carcinoma specimens, HPV 16 and HPV 52 were predominantly detected using the PCR method. Although some DNA samples extracted from the fluid-based specimens were degradaded, PCR and direct sequencing could be performed without difficulty even after 1 year of specimen storage. We conclude that fluid-based Papanicolaou specimens can be applied to investigate HPV infection.

Additional organizational features of the murine gamma-glutamyl hydrolase gene. Two remotely situated exons within the complement C3 gene locus encode an alternate 5' end and proximal ORF under the control of a bidirectional promoter.

Analysis of independently isolated clones from a mouse liver cDNA library identified a splice variant of gamma-GH mRNA with novel nucleotide sequence at the 5' end. Genomic sequencing now shows that this variant (variant II) incorporates two new alternates (exons Bla and Blb) of exon 1 in the murine gamma-GH gene remotely situated with respect to the rest of the gene. Further analysis of this variant also showed that it incorporates a small segment at the 3' end of exon A1, revealing that the previously described exon 1 consists of two individual exons (Ala and Alb) joined at a cryptic splice site. The 5' UTR and a segment of the ORF of variant II results from splicing of exon Bla to exon Blb which in turn is spliced to exon Alb and through this splicing to the rest of the exons within this gene. Remarkably, this splicing occurs even though exons Bla and Blb are located >45 Kb upstream of exons Ala and Alb. Our results also show that transcription starting at exons Bla and Blb is under the control of a separate and bidirectional promoter (promoter B). Exons Bla and Blb are located on the sense DNA strand within the complement C3 gene locus which is encoded on the antisense strand. This promoter is less efficient than the downstream promoter (promoter A) in regulating transcription at least in the context of reporter gene and primer extension assays. However, in these same contexts, this region of DNA sequence in the reverse orientation is markedly more efficient in driving transcription of an unidentified gene. Deletion of specific regions of sequence within this promoter have different effects depending upon the orientation (forward or reverse) within the reporter gene construct.

Amiloride-sensitive pressure-induced myogenic contraction in rat cerebral artery.

The inhibitory action of amiloride on the pressure-induced contraction was assessed in isolated rat cerebral artery. The artery was mounted in an arteriograph, and the change in intracellular Ca2+ concentration ([Ca2+]i) and vessel diameter were simultaneously measured. The contractile response elicited by intraluminal pressurization was independent of endothelium, i.e. myogenic in nature, and abolished by nicardipine, a Ca2+ antagonist or by removal of extracellular Ca2+, and was potentiated by 25 mM KCl. Cyclopiazonic acid and thapsigargin, inhibitors of the Ca2+-ATPase pump of the sarcoplasmic reticulum, and a protein kinase C inhibitor calphostin C did not suppress the pressure-induced contraction. Amiloride, a putative stretch-activated cation channel blocker, attenuated with an IC50 (50% inhibitory concentration) of about 3 microM the increase in [Ca2+]i and contractile activity in response to pressure, whereas the drug showed no apparent effect on the contraction produced by high KCl or 9,11-dideoxy-11alpha,9alpha-epoxymethano prostaglandin F2alpha (U46619). Furthermore, amiloride (100 microM) did not significantly affect intracellular pH in the artery. In spite of its multiple pharmacological actions, it seems possible that amiloride is a useful alternative tool at the cellular or tissue level to study the mechanotransduction mechanisms involved in the pressure-induced contraction in rat cerebral artery.

[A case of metastatic colon carcinoma in which a continuous intrahepatic artery-infusion of 5-FU leucovorin and cisplatin, and systemic chemotherapy with CPT-11 was very effective].

We report a patient with metastatic colon carcinoma who was treated effectively with a continuous intrahepatic artery-infusion of 5-FU, Leucovorin and cisplatin, and systemic chemotherapy with CPT-11. A 50-year-old man was diagnosed as having well differentiated adenocarcinoma of the sigmoid colon with multiple liver metastases in March, 1997. Left hemicolectomy and subsequent catheterization into the common hepatic artery via the gastroduodenal artery were performed in April, 1997. He was treated with 3 courses of continuous intrahepatic artery-infusion of 5-FU, Leucovorin and cisplatin, and two courses of systemic chemotherapy with CPT-11 during hospitalization, followed by 6 courses of a similar intraarterial therapy in an outpatient setting. Reinstallation of the catheter into the hepatic artery via the femoral artery was performed because of occlusion of the reservoir. During the 6th course of intraarterial therapy, diarrhea, nausea, and vomiting appeared and angiography revealed a narrowing of the hepatic artery. Therefore, the intrahepatic artery-infusion therapy was reinitiated with doses of 5-FU, Leucovorin and cisplatin reduced to approximately 80%. After 5 courses of this therapy, the computed tomography scan showed a marked decrease in the size of the metastatic hepatic lesions by 90%, and the serum level of CEA decreased from 657.7 ng/ml to 4.5 ng/ml. No severe side effects were seen during the treatment. Though multiple lung metastases were indicated during the intrahepatic artery-infusion therapy, both the liver and lung metastases have been well controlled with continuous intrahepatic artery-infusion chemotherapy and systemic chemotherapy. The continuous intrahepatic arterial infusion of 5-FU, leucovorin and cisplatin appears to be very effective for the treatment of colon carcinoma with liver metastasis without reducing the quality of life.

Attenuation of pressure-induced myogenic contraction and tyrosine phosphorylation by fasudil, a cerebral vasodilator, in rat cerebral artery.

The mechanism by which fasudil inhibits pressure-induced myogenic contraction was studied with regard to tyrosine phosphorylation in rat cerebral artery. Intracellular Ca(2+) concentration ([Ca(2+)](i)) and vessel diameter were simultaneously measured. Total tyrosine phosphorylation level and phosphorylation of tyrosine 419 on pp60(src) required for its full catalytic activity were immunocytochemically detected in situ. Fasudil (1 - 100 microM) partially suppressed the increase in [Ca(2+)](i), and totally attenuated contraction elicited by pressurization from 10 to 60 mmHg. Furthermore, fasudil (100 microM) significantly attenuated tyrosine phosphorylation and the activity of pp60(src) augmented in situ by pressure. Herbimycin A (1 - 100 nM) and genistein (3 - 30 microM), tyrosine kinase inhibitors, effectively attenuated the pressure-induced increase in [Ca(2+)](i), contraction, tyrosine phosphorylation, and activation of pp60(src). Both fasudil and herbimycin A directly inhibited the pp60(src) activity in a cell free system. Orthovanadate (100 microM), a tyrosine phosphatase inhibitor, significantly potentiated the pressure-induced increase in [Ca(2+)](i) and contraction. Nicardipine (100 nM), a Ca(2+) antagonist, completely inhibited pressure-induced increase in [Ca(2+)](i) and contraction, but affected neither tyrosine phosphorylation nor activity of pp60(src) in the pressurized arteries. Arginine-glycine-aspartic acid-serine peptide (1 - 100 microM) concentration-dependently reduced the pressure-induced contraction. In addition to the hitherto reported vasodilatory actions of fasudil, the present results suggest the inhibition by fasudil of pressure-induced tyrosine phosphorylation and pp60(src) activation. The wide spectrum of inhibitory actions of fasudil may contribute to the effective attenuation of the pressure-induced contraction in the cerebral artery.

[A case of advanced ovarian clear cell adenocarcinoma responding to cisplatin-cyclophosphamide and paclitaxel-carboplatin].

We report a case of stage IV ovarian clear cell adenocarcinoma successfully controlled by a combination of cisplatin-cyclophosphamide (CP) and paclitaxel-carboplatin (TJ). A 64-year-old female with advanced ovarian cancer and pleural effusion underwent 4 courses of combination chemotherapy with CP. The giant ovarian tumor showed a relative reduction in size and the pleural effusion disappeared. Serum CA-125 levels and CA 19-9 levels markedly decreased. Thus, the patient underwent a total hysterectomy and bilateral salphingoophorectomy. During the operation, a giant ovarian clear cell adenocarcinoma was found, but there was no peritoneal dissemination and no cancer cells in her peritoneal fluid. Thereafter, she underwent 3 courses of combination chemotherapy with TJ. She has had 13 months of stable, tumor-free survival since the operation and normal serum CA-125 and CA 19-9 levels. Ovarian clear cell adenocarcinoma generally responds poorly to cisplatin and carboplatin, but in combination chemotherapy with paclitaxel-carboplatin these may be effective.

Organization and structure of the mouse gamma-glutamyl hydrolase gene and the functional identification of its promoter.

The organization and structure of the murine GH gene encoding gamma-glutamyl hydrolase were determined. The murine GH gene spans 24kb and was found to be distributed in nine exons. The intron/exon coding junctions delineated conformed to the GT-AG rule. The 5' UTR and 3' UTR along with some coding sequences were incorporated in exons 1 and 9, respectively, whereas exons 2-8 incorporated only coding sequence. A relatively GC-rich region of the genome 5' of exon 1 was distinctly promoter-like and encoded a number of putative cis-acting elements, including six Sp1 sites known to be involved in the regulation of transcription but no TATA sequence motif. Primer extension analysis of this region with mouse liver and S180 cell mRNA revealed several tsps within the region encompassing the Sp1 sites. Functional analysis of this 5' upstream region of sequence was carried out by inserting it into the pGL3 reporter gene vector for transfection into NIH3T3 cells. The transcription of the luciferase gene that resulted in these cells established the identity of this region as an active promoter for the mouse GH gene.

Tyrosine phosphorylation of STAT3 by leptin through leptin receptor in mouse metaphase 2 stage oocyte.

Leptin is the product of the obese gene (ob), and is secreted in plasma from mature adipocytes. It has been recently reported that leptin is synthesized in granulosa and cumulus cells within the follicle of the ovary, and is present in mature human oocytes, suggesting possible roles of leptin in several aspects of pre- and post-ovulatory follicular development. On the other hand, STAT (Signal Transducer and Activator of Transcription) transcription factors are involved in leptin-associated signal transduction. In this report, we studied the expression of leptin receptor and STAT3 activation by leptin in metaphase 2 stage (M2) oocytes. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting showed that mRNA and protein of leptin receptor were expressed in M2 stage oocyte. Leptin at 15 ng/ml, the concentration observed in follicular fluid, caused tyrosine phosphorylation of STAT3 in mouse M2 stage oocytes. These results suggest possible roles of leptin in several aspects during oocyte maturation by activating the STAT signal transduction pathway.

v-src induces cisplatin resistance by increasing the repair of cisplatin-DNA interstrand cross-links in human gallbladder adenocarcinoma cells.

Activation of Src, which has an intrinsic protein tyrosine kinase (PTK) activity, has been demonstrated in human solid tumors, such as colorectal and breast cancers. To investigate the role of activated Src in drug resistance, we evaluated the effect of v-src on the resistance to various anti-cancer drugs using v-src-transfected HAG-1 human gallbladder adenocarcinoma cells. Compared with parental or mock-transfected HAG-1 cells, v-src-transfected HAG/src3-1 cells showed a 3.5-fold resistance to cis-diamminedichloroplatinum (II) (CDDP) but not to doxorubicin, etoposide or 5-fluorouracil. By contrast, activated H-ras, which acts downstream of src, failed to induce resistance to either of these drugs. Furthermore, wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, and H7, a protein kinase C (PKC) inhibitor, did not alter CDDP resistance. Evaluation of the kinetics of the removal of DNA interstrand cross-links (ICLs), measured by alkaline elution, showed a significant increase in this removal in HAG/src3-1 cells as compared with mock-transfected cells, though no differences were found in the formation of DNA ICLs between these cell lines. CDDP resistance in v-src-transfected cells was reversed, if not completely, by either herbimycin A or radicicol, specific inhibitors of Src-family PTKs, suggesting that Src tyrosine kinase activity induces CDDP resistance. Moreover, significant reduction in the repair of CDDP-induced DNA ICLs was observed upon treatment with radicicol. The intracellular glutathione content and mRNA expression of topoisomerase II and metallothionein were virtually identical between these cell lines, except for topoisomerase I mRNA. Our data strongly suggest that the ability of activated src, but not ras, to induce CDDP resistance is mediated by augmentation of DNA repair through Src to downstream signal-transduction pathways distinct from either the Ras, PI 3-kinase or PKC pathway.

Rab3B is essential for GnRH-induced gonadotrophin release from anterior pituitary cells.

Gonadotrophin-releasing hormone (GnRH) induces the release of gonadotrophins via an increase in cytosolic Ca2+ concentration ([Ca2+]). Rab3B, a member of the small GTP-binding protein Rab family, is known to be involved in Ca(2+)-regulated exocytosis in pituitary cells. However, it is not known whether Rab3B functions in the physiological process regulated by GnRH in gonadotrophs. In this study using antisense oligonucleotide against Rab3B (AS-Rab3B) we determined that Rab3B is involved in GnRH-induced gonadotrophin release. Rab3B immunopositive cells were reduced in 24% of pituitary cells by AS-Rab3B. This treatment did not affect the population of gonadotrophs or the intracellular contents of gonadotrophins. However, AS-Rab3B significantly inhibited the total amount of basal and GnRH-induced gonadotrophin released from pituitary cells. These results show that Rab3B is involved in basal and GnRH-induced gonadotrophins release but not the storage of gonadotrophins. Next, the changes in [Ca2+] and exocytosis in gonadotrophs treated with AS-Rab3B were compared among Rab3B-positive and -negative cells. The change in [Ca2+] was not different in the two groups, but exocytosis was significantly inhibited in Rab3B-negative cells. These results suggest that Rab3B is essential for GnRH-regulated exocytosis downstream of cytosolic Ca2+ in gonadotrophs.

SNAP-25 is essential for cortical granule exocytosis in mouse eggs.

Synaptosome-associated protein of 25 kDa (SNAP-25) has been shown to play an important role in Ca2+-dependent exocytosis in neurons and endocrine cells. During fertilization, sperm-egg fusion induces cytosolic Ca2+ mobilization and subsequently Ca2+-dependent cortical granule (CG) exocytosis in eggs. However, it is not yet clear whether SNAP-25 is involved in this process. In this study, we determined the expression and function of SNAP-25 in mouse eggs. mRNA and SNAP-25 were detected in metaphase II (MII) mouse eggs by RT-PCR and immunoblot analysis, respectively. Next, to determine the function of SNAP-25, we evaluated the change in CG exocytosis with a membrane dye, tetramethylammonium-1,6-diphenyl-1,3,5-hexatriene, after microinjection of a botulinum neurotoxin A (BoNT/A), which selectively cleaves SNAP-25 in MII eggs. Sperm-induced CG exocytosis was significantly inhibited in the BoNT/A-treated eggs. The inhibition was attenuated by coinjection of SNAP-25. These results suggest that SNAP-25 may be involved in Ca2+-dependent CG exocytosis during fertilization in mouse eggs.

Interferon-alpha and -gamma inhibit the growth and neoplastic potential of v-src-transformed human epithelial cells by reducing Src tyrosine kinase activity.

To investigate whether interferons (IFNs) selectively suppress the growth of solid tumor cells with elevated protein tyrosine kinase (PTK) activity, we evaluated the effect of recombinant IFN-alpha2a and IFN-gamma on the proliferative and neoplastic potentials triggered by p60v-src using v-src-transformed HAG-1 human epithelial cells. When compared with control cells harboring the pSV2neo gene, the monolayer growth of v-src-transformed cell lines was inhibited by both recombinant IFNs, in a dose-dependent manner, whereas growth of ras-transfected cell lines was not affected. Moreover, IFNs markedly reduced the clonogenic growth of v-src-transformed cells in soft-agar rather than monolayer growth, suggesting the preferential activity of IFNs on anchorage-independent growth. Pretreatment of cells with Src or the Src-like PTK inhibitor herbimycin A or radicicol, alleviated dose-dependently the growth-inhibitory activity of IFN-alpha2a against v-src-transformed cells, suggesting that IFNs may share a common inhibitory pathway with Src PTK inhibitors. Accordingly, like herbimycin A, IFNs were found to reduce tyrosine phosphorylation of p60v-src and suppressed in vitro p60v-src kinase activity in v-src-transformed cells. Our data, together with the fact that IFNs inhibit the growth potential driven by Src but not by activated Ras, suggest that inhibition of signal transduction pathway through Src to downstream transduction events may be a primary mechanism of IFN-induced anti-prolifeative and anti-tumoral activity.

Expression of Rab3A in the cortical region in mouse metaphase II eggs.

Rab3A, a member of the small GTP-binding protein superfamily, has been implicated in regulated exocytosis at presynapses. At fertilization, sperm-egg fusion induces cytosolic calcium mobilization and cortical granule (CG) exocytosis in the egg. However, it is not yet clear whether Rab3A is involved in this process. We previously reported that Rabphilin-3A functions in calcium-dependent CG exocytosis. Rabphilin-3A is known to bind with Rab3A, Rab3B, and Rab3C. In this study, we clarified which member of the Rab3 was expressed in mouse metaphase II eggs. Messenger RNA encoding Rab3A but not Rab3B or Rab3C, was detected in unfertilized metaphase II eggs by RT-PCR. Rab3A protein was also detected in unfertilized metaphase II eggs by immunoblot analysis. Next the expression and the localization of Rab3A in eggs at various stages of development were determined by immunofluorescence analysis using confocal laser scanning microscopy. Rab3A protein was specifically distributed in the cortical region in eggs from before fertilization to the two-cell stage. However, it was not detected at the three- or four-cell stage 40 hr after fertilization. These results suggest that Rab3A may function with Rabphilin-3A in CG exocytosis.

Specific attenuation of the pressure-induced contraction of rat cerebral artery by herbimycin A.

In order to determine whether protein tyrosine kinase mechanisms are involved in pressure-induced contraction, we compared effects of three structurally unrelated tyrosine kinase inhibitors and orthovanadate, a tyrosine phosphatase inhibitor, on the pressure-induced contraction of the posterior cerebral artery isolated from rats. The change in vessel diameter was continuously measured with a width analyzer. Herbimycin A inhibited the pressure-induced contraction, while it only slightly inhibited contractions produced by potassium chloride or 9,11-dideoxy-11alpha,9alpha-epoxymethano prostaglandin F2alpha (U46619). Genistein inhibited not only the pressure-induced contraction but also the U46619-induced one. Tyrphostin 23 significantly attenuated contractions in response to three different stimuli, i.e., pressure, potassium chloride and U46619. Orthovanadate potentiated the pressure-induced contraction. These results suggest that herbimycin A is a specific and potent inhibitor of the pressure-induced contraction and that a protein tyrosine kinase mechanism may play an important role in the genesis of the pressure-induced contraction of the rat cerebral artery.

Involvement of a sperm protein sensitive to sulfhydryl-depleting reagents in mouse sperm-egg fusion.

Effects of various sulfhydryl (SH)-depleting reagents on sperm-egg fusion were demonstrated. When sperm were treated with three plasma membrane-permeable SH-depleting reagents, N-ethyl-maleimide, sodium tetrathionate and 5,5'-dithiobis (2-nitro-benzoic acid), the rates of cleavage of eggs were significantly lower than those with control sperm. Neither the motility, penetration of the zona pellucida, acrosomal status of the sperm, nor sperm-egg binding was affected by the SH-depleting reagents. Fusion of sperm and zona-free egg was estimated by the sperm nuclear incorporation into the eggs, intracellular calcium mobilization, and cortical granule exocytosis in the eggs. Sperm-egg fusion was blocked dose-dependently when sperm were exposed to membrane-permeable SH-depleting reagents, but was not blocked by a membrane-impermeable SH-depleting reagent, eosin-5-maleimide. Blockage of fusion by sodium tetrathionate was completely reversed by an SH-reductant, dithiothreitol. These results suggest that a protein which is sensitive to SH-depleting reagents may play an important role in mouse sperm-egg fusion and that the functional SH region of the protein may be located at an intracellular site.