REP-1 gene mutations in Japanese patients with choroideremia

· Background: Choroideremia (CHM) is an X-linked progressive dystrophy of the choroid, retinal pigment epithelium, and retina. Recently, the REP-1 gene was isolated and the causative mutations in the gene were detected in patients with CHM. In a previous study, we described a Japanese family with CHM who had a mutation in the REP-1 gene. In the present study, we performed extensive analysis of the REP-1 gene in patients with CHM from several institutions in Japan. · Methods: Twenty-six patients with CHM and 5 unaffected females from 22 independently ascertained families were examined. Exons 1–15 of the REP-1 gene were screened by single-strand conformation polymorphism. The DNA fragments suspected of any variations were directly sequenced. · Results: Fifteen different mutations, including one previously reported mutation, were detected in 18 families. In addition, carrier status was proven in four unaffected females found to be heterozygous for the mutant allele. · Conclusions: Fifteen different mutations of the REP-1 gene were detected in 18 Japanese families. There were no hot spots for the mutations and no missense mutations. The results show that REP-1 gene defects cause CHM in Japanese patients, and the mutations in these Japanese patients differed from the mutations reported for CHM patients in Europe, Canada, and America except for R267X and 1313delTC. These findings suggest that the mutations occurred independently in the Japanese patients. Received: 13 August 1998 Revised version received: 16 November 1998 Accepted: 9 December 1998     

Intraoperative laparoscopic complications

PURPOSE: The aim of this study was to assess various intraoperative and postoperative complications associated with laparoscopic colorectal surgery. Specifically, the impact of surgical experience and procedure type on complications was analyzed. METHODS: All patients who underwent laparoscopic surgery were analyzed by age, sex, surgical indications, procedure performed, procedure length, intraoperative and postoperative complications, incidence and causes for conversion, duration of postoperative ileus, and length of hospital stay. Patients were classified for type of procedure and chronologically into four consecutive groups. Procedures were also categorized into four different groups: GI, total abdominal colectomies; GII, segmental resections; GIII, diverting procedures; GIV, others (abdominoperineal resection, Hartmann's creation or closure, anterior resection, and rectopexy). RESULTS: Between August 1991 and October 1995, 167 patients of a mean age of 49.6 (15–88) years underwent laparoscopic colorectal procedures. All procedures were electively performed. Common indications for surgery included inflammatory disease in 70 (42 percent), neoplasia in 56 (33 percent), functional bowel disorders in 30 (18 percent), and other forms of colorectal disorders in 11 (7 percent) patients. The most significant variable affecting intraoperative laparoscopic complication rate was surgical experience measured as the time interval during which surgery was performed (P=0.02). Total complication rate decreased from 29 percent during the first period to 11 percent by the second period (P<0.04) and 7 percent during the third period (P<0.005). Thus, the learning curve appeared to have required more than 50 cases to achieve. Moreover, even after performance of 94 (1991–1993) procedures in GI and GIV, these procedures were associated with higher complication rates than were those procedures in GII and GIII (P=0.04). CONCLUSION: Surgical experience and case selection are the most critical variables by which the surgeon can decrease the intraoperative laparoscopic complication rate.     

Prospective comparison of open<Emphasis Type="Italic">vs</Emphasis>. laparoscopic colon surgery for carcinoma

Laparoscopy for colonic diseases began in 1990 and has established a role in benign disease. Early observations and experiences demonstrated feasibility of laparoscopic surgery for a variety of colonic disease processes, but the applicability to colonic carcinoma was unclear. METHODS: In 1990, we began a comparative study of open (OCR)vs.laparoscopic (LCR) approach to colon cancer. The study progressed 65 months, with 224 patients in OCR group and 191 patients in LCR group. Parameters studied are stage, location, length of specimen, number of lymph nodes resected, margins, postoperative course, wound complications, recurrence rates, and immediate and long-term survival. OCR were standardized by one group, and LCR were standardized by a second group. All patients undergoing LCR were given freedom to choose either OCR or LCR, and informed consent was obtained. RESULTS: Equal or greater lymph node retrieval, resections, and distal margins were evident with LCR. Benefits with LCR were shown with shorter hospitalization (5.7vs.9.7 days), less blood loss, less wound problems (1vs.14), and quicker return of bowel function. Survival, recurrence, and death rates were essentially the same. There were no trocar implants in the LCR group. CONCLUSION: After five years, this study shows that laparoscopy does no harm to the patient, offers comparable oncologic resections, and seems to be patient-friendly, with less pain, quicker return of bowel functions, shortened hospitalization, and quicker return to full activity.     

Immunolocalization of prolyl 4-hydroxylase in fibroblasts cultured from Tenon's capsule of humans

Background: The excessive accumulation of extracellular matrix (ECM) with the repopulation of fibroblasts may lead to an unsuccessful outcome of glaucoma filtering surgery. We examined the immunolocalization of ECM components and prolyl 4-hydroxylase, an enzyme involved in collagen biosynthesis, in cultured Tenon's capsule fibroblasts (TCFs) of humans to evaluate the production of ECM in the cells. Methods: We used light microscopy to evaluate the immunolocalization of prolyl 4-hydroxylase and ECM components, collagen types I, III, and IV, cellular fibronectin, and laminin in TCFs. Ultrastructural localization of the enzyme was also evaluated by electron microscopy. Results: Immunoreactivity with monoclonal antibodies against the and subunits of the enzyme or with the polyclonal antibody against it was detected in the cytoplasm of the cells in a fine granular pattern, indicating its localization in the indoplasmic reticulum (ER). Immunoreactivity for the enzyme was detected in the cisternae of the ER on electron microscopy. Types I and III collagen reactivities were also observed in the cytoplasm in a fine granular pattern. T reactivity was present diffusely on the cell surface. The distribution of laminin reactivity in the cytoplasm resembled that of types I and III collagen. Cellular fibronectin reactivity was observed in the ECM in a reticular pattern. Conclusion: Prolyl 4-hydroxylase was located in the cisternae of the ER. TCFs produced a variety of ECM components in vitro. The results provide insight into the fibrotic process during scar formation at the site of a bleb following filtering surgery.     

Circadian Changes in Urine Volume and Frequency In Elderly Men

Purpose

Circadian changes in urinary frequency, diuresis and bladder capacity were evaluated in middle-aged and elderly patients.

Materials and Methods

A total of 2,703 urinations from 50 men (median age 66 years) was recorded with a 24-hour uroflowmetry system.

Results

During a 24-hour period the elderly group showed increased frequency and decreased bladder capacity. No difference was observed in diuresis between the 2 age groups. From midnight to 6 a.m. frequency in the elderly group was significantly greater (p less than 0.01) than that in the middle-aged group.

Conclusions

Increased frequency in the elderly group during these hours was primarily due to an increase in diuresis.     

Kinetics of d-tubocurarine disposition and pharmacologic response in rats

After bolus intravenous dosing of d-tubocurarine (d-TC) to rats, the twitch heights of the tibialis anterior muscle indirectly stimulated were followed, and its decrease was defined as pharmacologic response of d-TC. The relation between dose and response intensity was found to be well described with Hill's equation. According to a theory proposed by Smolen, Hill's equation was also applicable to the biophase d-TC concentration-response relation; the time courses of the relative biophase d-TC concentration indicated linear kinetics with dose levels 0.15 mg/kg and the occurrence of dose-dependent disposition with 0.30 mg/kg. After bolus i.v. dosing of³H-d-TC, plasma d-TC concentration obeyed a dose-independent two compartment model with doses 0.15mg/kg, but not with 0.30 mg/kg. This finding matched the above estimated with pharmacologic data. The active metabolite was not found in plasma and urine. The extent of d-TC plasma protein binding was independent of the wide range of plasma levels and its mean (±SD) value was 30.5 (±3.8). Plasma d-TC levels and pharmacologie response intensity were well correlated by Hill's equation and a three compartment model (the general two and the biophase compartments) in the dose range 0.15 mg/kg.This work was presented at the First Japanese-American Symposium on Pharmacokinetics and Biopharmaceutics, Tokyo, July 1981, which was held in memory of Dr. Sidney Riegelman.     

New method for quantitative measurement of N-nitrosodimethylamine formation in the whole mouse

A simple method for the quantitative estimation of the formation of N-nitrosodimethylamine (NDMA) in mice has been developed. Mice were frozen in liquid nitrogen and homogenized. NDMA was then extracted and analyzed by a gas chromatograph equipped with a thermal energy analyzer. In normal mice NDMA (100 nmole) administered orally was rapidly metabolized and recovery of NDMA was about 10% after 60 min. However, when pyrazole (300 mg/kg) was injected i.p. to mice 60 min before the administration of NDMA, more than 80% of the administered NDMA could be recovered within 60 min. This result suggested that in pyrazole pretreated mice the accurate amount of NDMA formed could be estimated. Therefore the NDMA formation was measured in the pyrazole pretreated mice. When 0.25 mole of aminopyrine and from 0.25 to 2.0 umole of sodium nitrite were simultaneously administered orally, the amount of the NDMA formation in 20 min was found to be from 8.2 to 60.3 nmole. These values are equal to about from 30 to 200 g/kg of body weight which are nearly daily doses expected to cause the carcinogenic effect on mice or rats. This method of measuring NDMA in pyrazole pretreated mice appears to be useful for investigating the in vivo formation of NDMA quantitatively.     

The application of population pharmacokinetic analysis to large scale clinical efficacy trials

Summary Results have been presented that demonstrate the ability to conduct population pharmacokinetic analysis as a component of clinical efficacy and safety trials. This method of analysis offers the potential to determine the pharmacokinetics of a drug in the actual patients receiving medication and to evaluate relationships between pharmacokinetics and drug action. However, active involvement in the protocol design, and data collection process are required to ensure the quality of the resultant data set.     

Effect of tetrakis-μ-3,5-diisopropylsalicylatodiaquodicopper(II) on the status of reduced glutathione in freshly isolated hepatocytes

Effects of different concentrations of tetrakis--3,5-diisopropylsalicylatodiaquodicopper(II) (Cu(II)₂(3,5-DIPS)₄(H₂O)₂) on the reduced status of glutathione (GSH), the major nonprotein thiol in tissues, were investigated using freshly isolated hepatocytes. Cu(II)₂(3,5-DIPS)₄ below 100 M did not have any significant effects on either the GSH content or viability of the hepatocytes, but at 150–250 M it decreased both parameters after 1 h of incubation. The decrease in cellular GSH was not followed by an increase in the oxidized form of GSH (GSSG) in the cell suspension. The addition of deferoxamine with Cu(II)₂(3,5-DIPS)₄ to the hepatocyte suspension prevented depletion in GSH content and loss of cell viability by Cu(II)₂(3,5-DIPS)₄. Both GSH depletion and loss of cell viability were found to be Cu(II)₂(3,5-DIPS)₄ dose dependent. From these results, it appears that Cu(II)₂(3,5-DIPS)₄ penetrated the cell membrane and acted by decreasing the GSH level by forming a copper-glutathione complex.