Cultured human granulosa cells as a model for corpus luteum function: relative roles of gonadotrophin and low density lipoprotein studied under defined culture conditions.

In primates, corpus luteum development involves both gonadotrophin stimulation and exposure to low density lipoprotein (LDL) delivered through vascularization of the granulosa cell-derived layer. These regulatory influences were modelled in vitro using granulosa cells obtained during in-vitro fertilization (IVF) cycles controlled with gonadotrophin releasing hormone (GnRH) analogue, human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin (HCG). Granulosa cells were cultured in defined medium on extracellular matrix. Without gonadotrophin or LDL in the medium, progesterone production declined progressively. With LDL alone, there was a short-lived elevation of progesterone output which subsequently declined. Culture with HCG alone resulted in a relatively unchanged rate of steroid production over 5 days despite morphological development. This contrasted with a marked and sustained increase in progesterone output over the same time when granulosa cells were cultured with combined HCG/LDL. Cultures were challenged with combined HCG/LDL on day 5. Where initial incubation included HCG, the challenge resulted in a recovery of progesterone output to values comparable to those of granulosa cells exposed to continuous HCG/LDL. Initial incubation without gonadotrophin led to a reduced response. Results suggest that LDL delivery to granulosa cells of the early corpus luteum causes a short-lived period of progesterone production. Sustained luteinization of granulosa cells and maintenance of gonadotrophin responsiveness requires continued exposure to gonadotrophin in the luteal phase.     

Osteoclastogenesis in the nonadherent cell population of human bone marrow is inhibited by rhBMP-2 alone or together with rhVEGF.

During bone development and repair, angiogenesis, osteogenesis, and bone remodeling are closely associated processes that share some common mediators. In the present study nonadherent human bone marrow mononuclear cells under the induction of sRANKL and M-CSF, differentiated into osteoclasts with TRAP-positive staining, VNR expression, and Ca-P resorptive activity. The effects of various combinations of rhBMP-2 (0, 3, 30, and 300 ng/mL) and rhVEGF (0 and 25 ng/mL) on osteoclastogenesis potentials were examined in this experimental system. The percentages of TRAP-positive multiple nucleated cells represent osteoclast differentiation potential, and the percentages of resorptive areas in the Ca-P coated plates resemble osteoclast resorption capability. The presence of rhBMP-2 at 30 and 300 ng/mL showed inhibitory effects on osteoclast differentiation and their resorptive capability in the human osteoclast culture system. rhVEGF (25 ng/mL) enhanced the resorptive function of osteoclast whenever it was used alone or combined with 3 ng/mL rhBMP-2. However, rhVEGF-induced resorptive function was inhibited by 30 ng/mL and 300 ng/mL rhBMP-2 in a dose-dependent manner. Statistical analysis demonstrated that an interactive effect exists between rhBMP-2 and rhVEGF on human osteoclastogenesis. These findings suggested that an interactive regulation may exist between BMPs and VEGF signaling pathways during osteoclastogenesis; exact mechanisms are yet to be elucidated.     

Pharmacokinetics of peptide T in patients with Acquired Immunodeficiency Syndrome (AIDS)

1. The pharmacokinetics of Dalal-peptide T-NH2 (peptide T) was determined during phase I clinical trials in patients with acquired immunodeficiecy disease (AIDS) and AIDS related complex (ARC). Drug levels were determined by specific RIA, and in some cases with HPLC analysis, after intraveneous (i.v.) or intranasal (i.n.), via metered sprayer, administration.

2. The plasma kinetics appeared to be bi-phasic with a first compartment half-life of 30 to 60 minutes and a second plasma clearence rate of 4 to 6 hours, observed for both routes of administration. Peptide T, in one individual was confirmed to be present at 6 hrs in plasma, determined after HPLC isolation followed by specific RIA.

3. Bioavailabilty, determined for a 2 mg test dose in six individuals was 9.3 ± 6.9 nmol/L. Peak plasma levels of 41 ± 30 nmol/L after 10 mg i.n., 2.8 ± 5.9 nmol/L after 2mg i.n., and 0.13 ± 0.07 nmol/L after 0.4 mg i.n. were observed. In two individuals tested, peptide T was detected in CSF at levels 20% of the corresponding plasma level 90 and 145 minutes post i.v. administration. Peptide T was not detected in urine. I.N. administration was well tolerated for times up to 21 months.     

Prevalence of hepatitis C virus antibody in an area endemic for hepatitis B virus and human T cell leukaemia virus

To clarify the prevalence of concurrent infection with hepatitis C virus (HCV), hepatitis B virus (HBV) and human T cell leukaemia virus (HTLV), we measured HCV antibody in the population of a district endemic for HBV and HTLV infection. Blood samples were collected in June 1990 from 579 inhabitants of four islands of Uwa Bay in the southwest of Ehime Prefecture in Japan. Anti-HCV antibody against C100-3 protein was detected using an enzyme-linked immunosorbent assay kit (Ortho Diagnostics). Thirteen of the 579 inhabitants (2.2%) were positive for anti-HCV, and this prevalence rate was not significantly different from the frequency of anti-HCV in Tokyo blood donors. A total of 11% (64 of 579) of the subjects were positive for HBsAg and 3.3% (19 of 579) were positive for anti-HTLV. These frequencies of HBsAg and anti-HTLV positivity were distinctly higher than the respective means of Japanese. All anti-HCV positive individuals were negative for HBsAg and anti-HTLV, while 54% (7 of 13) had increased alanine aminotransferase levels. These data suggest that the prevalence of HCV infection is not high even in an area endemic for HBV and HTLV infection.     

Two-step hard acid deprotection/cleavage procedure for solid phase peptide synthesis

A new two-step deprotection/cleavage procedure for t-butoxycarbonyl (Boc) based solid phase peptide synthesis is reported. First the protective groups are removed from 4-(oxymethyl)-phenylacetamidomethyl (PAM) resin attached peptide with the weak hard acid, trimethylsilyl bromide-thioanisole/trifluoroacetic acid (TFA). In the second step, the peptide is cleaved from the resin with a stronger hard acid such as trimethylsilvl trifluoromethanesulfonate in TFA or with HF. The method is also shown to deformylate Nⁱⁿ-formyltryptophan moiety efficiently. The usefulness of this procedure for practical solid phase peptide synthesis is demonstrated by comparison with other deprotection methods in the synthesis of urotensin II and human endothelin.     

Interactions Between Cytomegalovirus, Human Herpesvirus-6, and the Recurrence of Hepatitis C After Liver Transplantation

Recurrence of hepatitis C (HCV) following liver transplantation is common. Herpesvirus reactivation following transplant may have an immunomodulatory effect resulting in increased HCV replication. We studied whether cytomegalovirus (CMV) and human herpesvirus-6 (HHV-6) may be associated with HCV recurrence and viral load after transplant. We prospectively followed 66 HCV liver-transplant recipients with serial viral load testing for CMV and HHV-6. Infection and viral load were correlated with the development of biopsy-proven HCV recurrence and HCV viral loads. Histologic recurrence of HCV occurred in 41/66 (62.1%) patients. In the primary analysis, CMV infection and disease, and HHV-6 infection were not associated with HCV recurrence. Peak CMV and HHV-6 viral loads were not significantly different in patients with and without recurrence. No correlation was observed between HCV viral loads at 1 and 3 months post-transplant and peak HHV-6 or CMV viral loads. In a subgroup analysis, HHV-6 infection was associated with the development of more severe recurrence (hepatitis and/or fibrosis score > or = 2) (p = 0.01). Also, fibrosis scores at last follow up were higher in patients with CMV disease (1.67 vs. 0.56; p = 0.016) and in patients with HHV-6 infection (1.18 vs. 0.55; p = 0.031). In conclusion, HHV-6 and CMV infection and viral load were not associated with increased overall rates of HCV recurrence or HCV viral load after liver transplantation but may be associated with more severe forms of recurrence.     

Inward rectifying potassium channels in human malignant glioma cells

Human glioma cells obtained from established cell lines (Tp-276MG, Tp-301MG, Tp-378MG, Tp-483MG and U-251MG) were analyzed for the presence of ion channels with the tight-seal voltage clamp technique. The current-voltage relation revealed a marked inward rectification at hyperpolarizing voltages, due to the presence of inward rectifying K-channels in cells from all studied cell lines. These channels were conducting when the membrane potential was more negative than the K-equilibrium potential. The slope conductance for the inward K-currents (g_Ki) was affected both by [K⁺]_i and [K⁺]₀. g_Ki was proportional to [K⁺]₀ raised to 0.35 or 0.50, of which the larger value was measured in the presence of low [K⁺]_i (25mM). The rectification was not significantly different in cells perfused with Mg-free EDTA-buffered internal solution. Tl⁺ was 3.5 times more permaant than K⁺. g_ki was blocked by Cs⁺ (1 mM) in a voltage-dependent way (more effective in the hyperpolarized membrane), and by Na⁺ (154 mM) depending on voltage and time. From measurements of unitary current events in membrane patches (outside out or cell attached) the conductance of the single inward rectifying channel was estimated to be 27 ± 7 pS. This type of ion channel may be important for K-uptake by glial cells and hence for the K-homeostasis in the brain.     

有性生殖物种的单亲繁殖

生物科技的进步，如转基因技术与细胞核转移技术，不但改变了生物学与医药学的研究模式，更加深了和革新着我们对人类自身生物学特性的认识。通过定义线粒体DNA为转基因，我们曾提出核化线粒体组学与转基因人的新概念。鉴于有关克隆人的技术安全性与伦理学的争论。探讨了是否存在天然克隆人的可能性及检验这一可能性的相关研究途径；同时还探讨了单亲繁殖在遗传诊断中可能具有的指导作用。     

Anaesthesia monitoring: the human factors component of technology transfer

Resistance to change in monitoring practices from within the anaesthesiology community is a formidable obstacle, and coercive and exhortatory solutions are likely to be unsuccessful in some situations. An analysis of publications about technology transfer and professional obsolescence, and application of this data to the practice of anaesthesia, reveals various stresses that technology transfer from research areas to the workplace may induce in vulnerable anaesthesiologists and account for their attitudes. It is suggested that the invaluable pronouncements of high profile anaesthesiologist groups must be supplemented by supportive behaviour by physicians and administrators at an institutional level. The human factors issues to be addressed include: (i) Monitored data acquisition skills. (ii) Possibility of acting on monitored data. (iii) Assistance for personal insight into attitudinal difficulties that may be encountered. (iv) Data supporting the value of the device. (v) Ergonomically effective integration of the monitor into the work station.Alternatively the perceptions of potential users may accurately reflect changes in their status in the new work situation created by monitors, and decision making aids that may or may not be derived from them. Thus, plans to present job satisfaction in related clinical areas or to associate the proposed new system with evaluation of its effect on patient outcome will be necessary. In this way the clinician becomes involved in clinical research, a quality of personal and quality care development.     

Effect of sperm preparation with TEST yolk buffer on sperm-binding capacity under hemizona assay conditions

Summary. The study was conducted to evaluate the diverse effect and clinical significance of TEST yolk buffer treatment on sperm samples of 128 infertile men. Sperm samples were incubated with TEST yolk buffer and control medium (Ham's F-10) at room temperature for 2 h. The hemizona indices (mean ± SE) of the TEST yolk buffer and medium-treated sperm samples were 29 ± 2.3% and 22 ± 1.6%, respectively. Inspection of the individual response of each sperm sample to TEST yolk buffer revealed that 63 samples (49%) improved (double the interassay variation = 28%) their binding to zona pellucida, 36 (28%) remained unchanged, whereas the binding capacity of 29 samples (23%) decreased. Furthermore, TEST yolk buffer treatment of 24 samples (19%) resulted in an increased binding beyond the hemizona index threshold set up at 23%. This level was previously shown to be the cut-off point between fertile and infertile sperm samples. It was concluded that when applied to an unselected group of infertile men, TEST yolk buffer significantly increased sperm binding capacity to the zona pellucida. However, only 19% of the sperm samples showed improvement with clinical significance. The other sperm samples may have improved, remained unchanged or even deteriorated independently on basic sperm variables. Thus, the effect of TEST yolk buffer treatment on sperm binding should be tested prior to its clinical use to avoid possible damage to certain sperm samples.