Utility of peptide–protein affinity complexes in proteomics: identification of interaction partners of a tumor suppressor peptide*

Abstract: We used a N‐biotinylated peptide analog of the C‐terminal domain of the tumor suppressor protein, p21^cip1/waf1 to elucidate peptide/protein interacting partners. The C‐terminal domain of p21^cip1/waf1 protein spanning 141–160 amino acid residues is known to bind PCNA and this interaction is important in many biological processes including cell‐cycle control. This C‐terminal 20‐mer efficiently extracts PCNA in the presence of a variety of N‐ or C‐terminally attached affinity tags. Using difference silver stained 2D gels combined with in‐gel tryptic digests, we identified the difference spots using MALDI‐TOF mass spectrometry‐based peptide mass fingerprinting followed by a database search using profound against NCBIs human nonredundant protein sequence data bank. Identified spots include the p48 subunit of chromatin assembly factor‐1, the heat shock 70 protein analog BiP, calmodulin, nucleolin and a spot similar in size to dimeric PCNA. In contrast, microcapillary ion‐trap LC‐MS/MS analysis of a tryptic digest of entire affinity extracts derived from both control and experimental runs followed by database searches using sequest confirmed the presence of most of the above proteins. This strategy also identified hnRNPA1, HPSP90α, HSP40 and T‐complex protein 1, a protein similar to prothymosin, and a possible allelic variant of the p21^cip1/waf1 protein. The use of N‐biotinylated peptide derived from the C‐terminal domain of p21^cip1/waf1 protein in proteomic analysis exemplified here suggests that peptides obtained from intracellular functional screens could also potentially serve as efficient baits to discover new drug targets.     

胃粘膜瓣成型预防食管胃吻合术后反流的观察

目的:介绍胃粘膜瓣成型预防食管胃吻合术后反流的临床应用.方法:食管、贲门癌50例,行部分食管部分胃切除,用吻合器作食管胃吻合后行胃粘膜瓣成型.结果:成型组术后无反流症状,上消化道造影及放射性核素显像表明该手术方法抗反流作用明显.食管镜检查示胃粘膜瓣成型组吻合口粘膜充血、水肿、糜烂、溃疡较常规手术组明显减轻.结论:胃粘膜瓣成型术可有效预防食管胃吻合术后胃食管反流.     

Lanzoprazole promotes gastric carcinogenesis in rats with duodenogastric reflux

Background Duodenogastric reflux is known to cause an increased frequency of cancer in the glandular portion of the stomach in rats. Furthermore, it is debated whether inhibition of gastric acid secretion may promote gastric carcinogenesis. In the present study we examined the combined effect of gastroduodenal reflux and acid inhibition with respect to the development of gastric carcinoma in the rat.Methods Following the construction of a gastrojejunostomy in male Wistar rats, half of them were given the proton pump inhibitor lanzoprazole for 1 year. The rats were then killed and the pH in the stomach and gastrin in blood were measured. The stomach was examined macroscopically as well as histologically.Results Gastrin levels at autopsy were significantly increased in treated rats compared to the control group, confirming an effect of lanzoprazole on gastric acid secretion. Body weight was significantly reduced in the treated rats. Thirty of 79 rats developed gastric cancer, and they were all adenocarcinomas of the Lauren intestinal type. Gastric cancers occurred significantly more often in lanzoprazole-treated rats (50%) compared with controls (27%).Conclusion Lanzoprazole given orally enhances the carcinogenic effect of duodenogastric reflux in rats.     

Assessment of adolescent varicocele

Varicocele is the most important male factor responsible for decreased fertility potential in married couples. From March through June 1994, 2,470 school boys aged 10–20 years were examined to establish the incidence of consecutive grades of varicocele and to develop a protocol for diagnosis and treatment of adolescents with varicocele. Grade 1 varicocele was found in 18%, grade 2 in 12%, and grade 3 in 5% of the population examined. An original protocol of ultrasonographic (US) examination (previously verified by angioscintigraphy) was introduced to assess boys with clinically diagnosed varicocele. The volume of each testis, testicular volume decrease (TVD), pampiniform vein diameter (PVD), and basal (BBF) and maximum blood flow (MBF) velocities were measured in 625 boys. In 74 cases a semen analysis was performed. The statistical analysis revealed that the presence of venous reflux and PVD correlated with the grade of varicocele. Decreases in testicular volume were highly dependent on the grade of varicocele, PVD, and BBF and MBF velocities. Analysis of the relationship between spermatic (boys over 17 years) and US findings revealed that the quality of spermatogenesis can be predicted by US examination in adolescents with varicocele. The authors recommend multiparametric US examination as a reliable, objective, and repeatable technique for establishing criteria for operative treatment in boys under 18 years of age with varicocele as well as for postoperative evaluation.     

Intracytoplasmic injection of fresh and cryopreserved testicular spermatozoa in patients with nonobstructive azoospermia—a comparative study

Objective: To compare the outcome of intracytoplasmic sperm injection (ICSI) with fresh and frozen-thawed testicular spermatozoa in patients with nonobstructive azoospermia.Design: Retrospective analysis of consecutive ICSI cycles.Setting: In Vitro Fertilization Unit, Assaf Harofeh Medical Center.Patient(s): Eighteen with nonobstructive azoospermia in whom testicular sperm was found after testicular sperm extraction.Intervention(s): Testicular sperm retrieval, cryopreservation, and ICSI with fresh or frozenthawed testicular spermatozoa.Main Outcome Measure(s): Two-pronuclear fertilization; embryo cleavage rates, mean number of embryos transferred per cycle, and their relative quality, embryo implantation, clinical pregnancy, and ongoing pregnancy rates (PRs) per ET.Result(s): No statistically significant differences were noted in all parameters examined between ICSI cycles with fresh or cryopreserved testicular spermatozoa from the same nine patients and comparing all ICSI cycles performed; with fresh (25 cycles) and thawed (14 cycles) testicular spermatozoa, respectively: two-pronuclear fertilization, 47% versus 44%; embryo cleavage rates, 94% versus 89%; implantation rates, 9% versus 11%; and clinical PR, 26% versus 27%. The delivery or ongoing PR using fresh sperm was better (21% versus 9%), but the difference did not reach statistical significance. The cumulative clinical PRs and ongoing PRs per testicular sperm extraction procedure were 36% and 24%, respectively.Conclusion(s): Testicular sperm cryopreservation using a simple freezing protocol is promising in patients with nonobstructive azoospermia augmenting the overall success achieved after surgical sperm retrieval. (Fertility Sterility 1997;68:892-7. C 1997 by American Society for Reproductive Medicine.)     

An infant case of bilateral small kidneys with both proximal and distal tubular dysfunction

Abstract A male infant with bilateral small kidneys associated with both proximal and distal tubular dysfunction, who showed chronic renal failure soon after birth, is reported. He was also noted to have both proximal and distal type of renal tubular acidosis. The small kidneys were thought to be due to renal hypodysplasia associated with bilateral severe vesicoureteral reflux, by radiological findings. An alkalization therapy with chemo-prophylaxis seemed to be of benefit in slowing the progression of renal failure in this case.     

Performing Testicular or Epididymal Sperm Retrieval Prior to the Injection of hCG

Purpose: Our purpose was to determine the feasibility and efficacy of performing testicular or epididymal sperm retrieval prior to the injection of human chorionic gonadotropin (hCG). Methods: This report deals with 87 sperm-positive percutaneous epididymal sperm aspiration (PESA), percutaneous testicular sperm aspiration (PTSA), or testicular sperm extraction (TESE) cycles. All sperm retrieval procedures were performed prior to administration of hCG to the women. Retrieved spermatozoa were cultured in vitro in simple medium for approximately 40 hr prior to intracyto-plasmic sperm injection. Results: In all but one cycle in which TESE was performed for nonobstructive azoospermia, motile sperm were available for ICSI. The overall fertilization rate was 53%. Pregnancy rate per transfer and implantation rate per embryo were 41.2 and 15.7%, respectively. Conclusions: Satisfactory fertilization and pregnancy rates can be achieved when PESA, PTSA, or TESE is performed prior to the injection of hCG followed by in vitro culture of spermatozoa approximately 40 hr before ICSI. Scheduling of testicular or epididymal sperm retrieval cases in this way appears to ease the workload on laboratory and operating room personnel. Furthermore, withholding hCG when sperm is absent may obviate the unnecessary risk of ovarian hyperstimulation when spermatozoa cannot be retrieved.     

RP-HPLC法测定兔眼各组织中多柔比星的浓度

目的建立测定兔眼各组织中多柔比星浓度的RP-HPLC法。方法采用RP-HPLC法,色谱柱为DiamonsilC18柱(250mm×4.6mm,5μm),流动相为乙腈∶0.2mol/L磷酸二氢钾缓冲液∶三乙胺(30∶70∶0.2),磷酸调pH到4.0;检测波长234nm;内标为柔红霉素;甲醇沉淀蛋白,固相萃取富集。结果在兔眼各组织中,多柔比星和内标保留时间合适,分离效果良好;在0.1~10μg/ml的浓度范围内线性关系良好,r>0.999;各组织中日内与日间精密度分别小于3.70%和3.39%,低、中、高浓度,各组织回收率接近100%,提取率、定量限和检测限均符合要求。结论所建立的RP-HPLC法测定兔眼各组织中多柔比星的含量准确可靠。     

超声循环法提取扁蓄总黄酮

目的在超声条件下寻找提取扁蓄总黄酮的优化条件。方法采用正交设计法,以超声功率、超声时间、提取温度、溶剂体积分数为因素,每个因素3个水平,用紫外分光光度法测定总黄酮作为评价指标。结果最佳提取条件为超声功率800W,超声时间105(35×3)min,提取温度40℃,溶剂体积分数65%。最佳提取条件下总黄酮的质量分数为1.908%。结论采用超声循环提取技术提取扁蓄中总黄酮,提取温度低、时间短,提取效率高,具有广阔前景。     

Automated solid-phase extraction and two-step derivatisation for simultaneous analysis of basic illicit drugs in serum by GC/MS

A combination of automated solid-phase extraction (SPE) and subsequent two-step derivatisation has been developed for the simultaneous analysis of basic drugs of abuse and cocaine metabolites in serum samples. Substances included in this procedure are morphine, codeine, methadone, cocaine, benzoylecgonine, methylecgonine, amphetamine, methamphetamine, MDMA, MDEA and MDA. SPE with mixed-mode cartridges (RP-C8 and cation-exchange) was fully automated with a Zymark RapidTrace SPE robot. GC/MS analysis was performed after derivatisation with a new two-step reaction by trifluoroacetic anhydride and 2,2,3,3,3-pentafluoropropanol. High recoveries (> 85%) with high reproducibility (CV 1.1-3.8%) were found for all drugs. High correlation coefficients (r > 0.998) were obtained due to the addition of deuterated standards prior to extraction. Experience obtained over 2 years of applying this method to drug analysis in serum is discussed.