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.     

B组轮状病毒WH-2株vp7基因的原核表达与抗体的制备

目的：在大肠杆菌中表达人B组轮状病毒WH-2株VP7蛋白并制备其兔抗血清。方法：根据B组轮状病毒（GBRV）WH-2株vp7基因的全序列设计引物,用PCR的方法扩增得到vp7基因的编码区。将其克隆到原核GST融合表达载体pGEX-KG内,转化大肠杆菌E.coliDH5α,IPTG诱导表达人B组轮状病毒WH-2株VP7蛋白,经SDS-PAGE分离纯化表达的蛋白免疫新西兰兔,制备人B组轮状病毒WH-2株VP7蛋白抗血清。结果：经鉴定确认,vp7基因以正确的方式插入到载体中,此重组表达载体经IPTG诱导后,可表达相对分子量为53.4 kDa的GST-VP7融合蛋白。制备的抗血清经同样诱导表达的表达载体pGEX-KG表达产物吸收后1：500倍稀释后用Western Blot分析,与53.4 kDa的GST-VP7融合蛋白获得特异性显色信号。结论：人B组轮状病毒WH-2株VP7蛋白成功在大肠杆菌中GST融合表达,所表达的蛋白和制备的抗体不但为研究结构与功能提供了物质基础,也为GBRV所引起的疾病预防、诊断和治疗等流行病学研究和临床诊断奠定了基础,具有重要实际应用价值。     

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.     

The spermatozoal volume as indicative of the plasma membrane integrity (modification of the hypoosmotic swelling test). I. Methods

Summary. The aim of the present study was to establish a more differentiating indicator of plasma membrane integrity of spermatozoa than the classic version of the hypoosmotic swelling test according to Jeyendraan. Spermatozoa were prepared by density gradient centrifugation (90% Percoll) to select 'fertilization competent' spermatozoa only. After a second washing procedure sufficiently pure sperm cell suspensions were obtained. The volume distributions of these sperm cells were measured with a Coulter Counter at 25 °C after adaptation in 300 mosmolar NaCl solution resp. 150 mosmolar NaCl solution for 5 min. These volume distributions showed significantly different patterns for the isotonic and hypotonic stress situation in the simple salt solution. Moreover, the comparison of the response to hypoosmotic stress showed more than four reproducible characteristic patterns, promising well differentiated results for different sperm populations. The new method for the detection of hypoosmotic swelling effect might be a real and valuable functional parameter.     

Testicular amplificaion and impaired transmission of human butyrylcholinesterase cDNA in transgenic mice

Gene amplification occurs frequently in tumour tissues yet is,in general, non-inheritable. To study the molecular mechanismsconferring this restraint, we created transgenic mice carryinga human butyrylcholinesterase (BCHE) coding sequence, previouslyfound to be amplified in a father and son. Blot hybridizationof tail DNA samples revealed somatic transgene amplificationswith variable restriction patterns and intensities, suggestingthe occurrence of independent amplification events, in 31% (11/35)of mice from the FII generation but in only 3.5% (2/58) of theFII and FIV generations. In contrast, >10-fold amplificationsof the BCHE transgene and the endogenous acetylcholinesteraseand c-raf genes appeared in both testis and epididymis DNA from>80% of FIII mice. Drastic, selective reductions in testisBCHEmRNA but not in actin mRNA were detected by the PCR amplificationof testis cDNA from the transgenic mice, and apparently resultedin the limited transmission of amplified genes. The testicularamplification of the BCHE transgene may potentially representa general phenomenon with clinical implications in human infertility.     

Low-dose erythropoietin is effective and safe in children on continuous ambulatory peritoneal dialysis

Hypertension is one of the most important complications of erythropoietin (rHuEPO) therapy in dialysis patients. In this study, the effect of two different dosage regiments of subcutaneous rHuEPO on blood pressure [BP] was evaluated in 20 anemic children on continuous ambulatory peritoneal dialysis (CAPD). Patients were randomized to receive rHuEPO 50 U/kg, either once a week (group 1, 50 U/kg per week) or three times a week (group 2, 150 U/kg per week). At the beginning of the study, 8 patients in group 1 and 8 patients in group 2 were on antihypertensive therapy. In group 1, the hematocrit increased gradually and significantly from 18.98%±1.79% to 30.1%±1.62% after 6 months, while in group 2 it rapidly increased from 19.53%±1.86% to 32.4%±1.11% after 3 months. A significant increase in the mean arterial BP was observed in group 2. Antihypertensive therapy had to be increased in all of the 8 previously hypertensive patients and had to be initiated in 1 of the 2 originally normotensive patients in the same group. None of the patients in group 1 required a change in antihypertensive medication. We conclude that during treatment with rHuEPO pre-existing hypertension and the dose of rHuEPO are the most important risk factors for the development or worsening of hypertension in children on CAPD, and gradual elevation of hematocrit by low-dose rHuEPO avoids the development of severe hypertension. Received December 11, 1995; received in revised form September 16, 1996; accepted September 19, 1996     

人皮质骨矿化基质中骨盐框架结构

目的：研究人皮质骨矿化基质中骨盐的框架结构及框架中骨微间隙。方法：应用透射电镜、场发射扫描电镜观察、电脑图像分析及能谱分析，分析无骨病成人长骨、扁骨２００例骨盐分布特征。结果：骨盐框架结构由微柱、微梁、微小梁、弓状梁、致密点、隔板和骨微间隙构成。骨微间隙由洞、内衬和壁组成，洞平均直径为８４．４±７５．６ｎｍ，与骨小管相比有显著差异（Ｐ值＜０．００１），平均密度为１１～１７个／μｍ２，与骨小管之比超过１０：１。骨盐分针形结晶和微颗粒结晶。结论：骨盐框架结构及骨微间隙是骨盐在人皮质骨矿化基质中的存在形式，可能与骨盐吸收、沉着有关。