The mini-exon gene of Trypanosoma (Nannomonas) simiae: sequence variation between isolates and a distinguishing molecular marker.

The mini-exon gene repeats from two isolates of Trypanosoma (Nannomonas) simiae were amplified by polymerase chain reaction (PCR). The products from each isolate differed in size, however they cross-hybridised in Southern blots. The nature of the size variation was revealed by comparison of the DNA sequence of each repeat: relative to the BAN7 strain, the mini-exon gene of KETRI 2431 contained three apparent deletions that were flanked by short (> or = 6-bp) direct repeats. Furthermore, one of the cloned repeats was used as a hybridisation probe against DNA from other closely-related African trypanosomes. The lack of hybridisation of the T. (N.) simiae mini-exon gene to genomic DNA from the Forest, Kilifi and Savannah subgroups of T. (N.) congolense and T. (N.) godfreyi indicate that this PCR-hybridisation assay may be useful for distinguishing T. (N.) simiae from other members of the subgenus Nannomonas.

Excellent outcome using selective criteria for rectocele repair.

PURPOSE: The aim of this study was to review our experience with patients with rectoceles using very selective criteria for operative repair and to critically review our surgical results. METHODS: This is a review of patients selected for rectocele repair between 1989 and 1994. RESULTS: Two hundred seventy-nine patients were evaluated for pelvic outlet symptoms in our clinic. Defecography was performed in 180 patients; rectocele was seen in 143 patients (79 percent; 135 females and 8 males). On physical examination, 132 patients had a palpable rectocele (73 percent). Rectocele repair was recommended for 35 patients (13 percent); 33 (32 females and 1 male) underwent this procedure. Mean age was 55 (range, 16-78) years. Although many patients complained of constipation, incontinence and pelvic pain, in these 33 patients criteria for repair included the sensation of a vaginal mass or bulge that required digital support and/or rectal digitizing for evacuation (58 percent), retention of barium in the rectocele on defecography (55 percent), or a very large rectocele with internal anterior rectal wall prolapse (6 percent). A hysterectomy had been performed previously in 47 percent of women repaired. Rectocele repair was performed by a standard transanal approach in 31 patients and transabdominally in 2 patients. Hospital stay averaged 3.7 (range, 1-8) days. Few postoperative complications occurred; urinary retention was the most common (18 percent). All patients were followed postoperatively, and 26 patients (79 percent) answered a standardized questionnaire. Mean follow-up was 31 (range, 5-64) months. Eighty percent of patients questioned who initially complained of a vaginal mass or bulge reported complete resolution (significant improvement by the sign test, P < 0.5). Subjectively, 92 percent of patients questioned reported improvement in their preoperative symptoms and satisfaction with the operation. CONCLUSION: Rectoceles are frequently identified during defecography, which is performed for pelvic floor complaints, yet are often asymptomatic. In contrast to other recent reports of rectocele repair, our data indicate that careful selection of patients using specific criteria may result in very good clinical results.

Mini-exon gene variation in human pathogenic Leishmania species.

We have used polymerase chain reaction to amplify the mini-exon gene repeat from 18 Leishmania strains. DNA sequence analysis of the cloned products reveals high conservation of both the exon and intron (i.e. transcribed region). In contrast, variation is evident in both the length and primary sequence of the non-transcribed spacers. Dermotropic species of the New World subgenus Leishmania possess a 0.3-kb gene that differs from the 0.25-kb gene of New World dermotropic species of the subgenus Viannia. The Old/New World viscerotropic species and Old World dermotropic species possess a 0.4-kb mini-exon gene. However, the genes from the viscerotropic and dermotropic groups may be distinguished on the basis of sequence differences in the non-transcribed spacer. Comparative analysis of the -86 to -1 region from all species has been used to measure relatedness within the genus. In general, all the observed differences correlate with the four major groups of Leishmania (New World dermotropic Leishmania, New World dermotropic Viannia, Old World dermotropic Leishmania and viscerotropic Leishmania). Two of the three repeats cloned from L. donovani show short deletions. The missing sequence is flanked by direct, 7-bp repeats suggesting that the sequences may have been deleted by homologous recombination. Such rearrangements could account for the diversity detected in the non-transcribed spacers of the mini-exon genes.

Delayed onset and decreased incidence of diabetes in BB rats fed free radical scavengers.

We tested the hypothesis that free radicals play a role in the selective destruction of pancreatic beta-cells in BB/Wor rats. Diabetes-prone BB rats of both sexes and 40 days of age were divided into three groups. The control group was fed ad libitum Purina rat chow powder, while the experimental group was fed ad libitum the rat chow powder blended with a mixture of four known free radical scavengers: allopurinol, mercaptopropionylglycine, dimethylthiourea and Vitamin E. A third group was pair-fed 10 g chow powder/rat/day, since in earlier experiments we observed that rats on the experimental diet consumed only about 10 g/rat/day. All rats were studied up to age 120 days. Body weight and food intake were measured daily. Urine was tested for glucose beginning at age 60 days. When glucosuria appeared, blood glucose and urinary ketones were measured. Body weight gain in the experimental and pair-fed groups was similar, but lower than the control group. Life table analysis of the data showed a decreased and a delayed onset of diabetes in the rats fed free radical scavengers. Thus, the results of this study demonstrated that calorie restriction and the related impaired growth did not affect the incidence of diabetes in the BB rat. In addition, the results suggested a role for free radicals in the spontaneous destruction of pancreatic beta-cells in the BB rat.

PCR amplification of mini-exon genes differentiates Trypanosoma cruzi from Trypanosoma rangeli.

Synthetic oligonucleotides corresponding to a conserved 22 nucleotide sequence within the tandemly repeated mini-exon gene have been used to amplify a single gene-containing repeat from Trypanosoma cruzi and Trypanosoma rangeli, two morphologically similar organisms with overlapping hosts and geographical distribution but different pathogenicity in humans. The T. cruzi repeat is 582 nucleotides long and the T. rangeli repeat is 858 nucleotides. The two organisms may therefore be distinguished primarily by the electrophoretic mobilities of their respective amplification products. Confirmation of the diagnosis can be obtained by Southern blot analysis using species-specific DNA probes from the unique intergenic regions. We also present a diagnostic assay in which the unique intergenic regions are immobilized on nylon membranes and differentiation is based on hybridization with a digoxigenin-labelled PCR product.

Effects of chronic non-ketotic diabetes and aging on myocardial function and fatty acid oxidation.

The effects of aging and chronic non-ketotic diabetes on contractile properties, oxygen consumption, palmitate oxidation and morphology were studied in isolated, perfused working hearts of 2, 9, 12 and 22 month old rats. The heart rate, coronary flow, and oxygen consumption were no different among the 9, 12 and 22 month control and diabetic hearts. Cardiac work was not depressed in control hearts until 22 months of age. Depression of cardiac output due to aging in the control hearts progressed in stages. The superimposition of chronic diabetes in the 9, 12 and 22 month rats did not further depress the cardiac work or cardiac output. [1-14C] palmitate oxidation in the 2 and 9 month control hearts was higher than that of the 12 and 22 month controls. Chronic diabetes did not affect fatty acid oxidation in the 9 and 12 month rats compared to their controls, but was diminished in the 22 month diabetic rat heart. These results suggest that impairments in the contractile properties of the isolated hearts of the chronically diabetic, senescent rats were primarily due to aging.

Exact parallel matrix inversion using para-Hensel codes with systolic processors.

The application of para-Hensel codes for exact inversion or generalized inversion of a matrix using massively parallel processors is described.

Stimulation by acetoacetate of hepatic triacylglycerol synthesis.

The factors responsible for the huge accumulation of hepatic triacylglycerols in the ketotic diabetic state are not established. Our earlier work suggested a role for ketone bodies in the increased hepatic triacylglycerol synthesis observed in the ketotic diabetic state. Isolated hepatocytes obtained from normal fed rats were incubated with sodium acetoacetate or sodium chloride (control) and [1-14C]palmitate in Krebs-albumin buffer. Acetoacetate stimulated triacylglycerol synthesis in a concentration-dependent manner without increasing palmitate uptake or inhibiting palmitate oxidation. Beta hydroxybutyrate showed no effect on palmitate esterification to triacylglycerols. Isolated hepatocytes of normal fed rats were incubated with either sodium acetoacetate or sodium chloride and the nuclear-free homogenate was incubated with [U-14C]glycero-3-phosphate and cofactors. The synthesis of triacylglycerol and the activity of the cytosolic phosphatidate phosphohydrolase were increased in the cells pre-incubated with acetoacetate. The results of this study demonstrate that the increases in triacylglycerol synthesis and the cytosolic activity of phosphatidate phosphohydrolase previously observed by us in the ketotic diabetic liver, could be reproduced in normal fed rat liver cells by incubating them with acetoacetate. The results identify acetoacetate as a potential factor, in the regulation of hepatic triacylglycerol synthesis and for hepatic accumulation of triacylglycerols observed in the ketotic diabetic state.

Error-free parallel rational arithmetic for optical and VLSI computing.

An error-free carry-free (parallel) rational arithmetic system based on residue and p-adic representation is introduced. In this system, Farey rational numbers, whose numerators and denominators are bounded, are encoded into Para-Hensel codes (parallel rational Hensel code), and the parallel element-wise arithmetic is performed using these codes. The algorithms for encoding into and decoding from the Para-Hensel code and the arithmetic algorithms are described. This system will have extensive applications in massively parallel processors.

Increased fatty acid uptake, a factor in increased hepatic triacylglycerol synthesis in aging rats.

The mechanisms responsible for the increased hepatic triacylglycerol synthesis in aging are not established. We studied [1-14C] palmitate uptake and its esterification to triacylglycerols in the isolated hepatocytes of 2-month, 10-month and 20-month-old normal rats. In all hepatocytes, palmitate uptake and its esterification were linearly related to medium palmitate concentration, but palmitate uptake and triacylglycerol synthesis by the hepatocytes of 10-month and 20-month-old rats were nearly double that observed with the cells of 2-month-old rats. These results suggest that increased fatty acid uptake by the liver cells was a contributory factor in the increased triacylglycerol synthesis observed in the liver of senescent rats. The changes in the hepatocyte leading to increased fatty acid uptake and hence increased triacylglycerol synthesis are detected as early as middle age of the rat.

A diagnostic tool with important implications for treatment of addiction: identification of factors underlying relapse and remission time distributions.

The results of analysis of successive relapse and remission times of many opiate addicts were examined. It is discovered that motivation is the preeminent factor that governs the distribution of lengths of remission times (periods of abstinence), differences in types of motivation leading to an underlying mixture of three or fewer Weibull distributions. On the other hand, two distinct factors-namely wear-out (dysfunction resulting from exhaustion or tiring out brought on by the addict's enforced life-style) and precipitation of events that may not be beyond the control of the addict-govern the distribution of lengths of relapse times (periods of using opiates), leading, typically, to an underlying competing-risk distribution. Methods for using this information to aid in treatment and in research are described.

Inhibition by adenosine triphosphate of heart microsomal neutral lipase activity.

Triacylglycerol lipolysis was inhibited by palmitate in the isolated perfused normal rat heart. Acetate or acetylcarnitine could reproduce the inhibitory effects of palmitate. Since heart neutral lipase plays an important role in the lipolysis of heart triacylglycerols, the effects of acetylcarnitine, acetyl CoA and related metabolites on the microsomal neutral lipase activity were studied. ATP inhibited the enzyme activity in a concentration-dependent manner without a lag phase. AMP and adenylyl imidodiphosphate, two compounds structurally related to ATP but whose phosphate groups cannot be transferred, did not inhibit the microsomal lipase activity. These results suggested that ATP inhibited the lipase activity through the transfer of its phosphate group. It is proposed that cellular ATP concentration is a determinant of tricylglycerol lipolysis in the heart.

Effects of epinephrine and perfusion pressure on the peak aortic pressure development and glucose transport in the isolated perfused heart of normal and diabetic rats.

The effects of increased perfusion pressure and epinephrine stimulation on the contractile parameters and glucose transport in the isolated perfused hearts of control and ketotic diabetic rats were studied. An increase in perfusion pressure from 60 mm Hg to 100 mm Hg resulted in increases in coronary flow and peak aortic pressure development in control and diabetic hearts. The responses of the diabetic heart were similar to the control. Epinephrine produced lower increments in peak aortic pressure development in control and diabetic hearts under the higher perfusion pressure. Glucose uptake, although stimulated about 4-fold in both control and diabetic hearts on increasing the perfusion pressure, was still lower in the diabetic heart. Epinephrine stimulated glucose uptake in both control and diabetic hearts at 60 mm Hg, but the control heart showed a greater response. At 100 mm Hg perfusion pressure, the stimulatory effect of epinephrine on glucose uptake was abolished in both control and diabetic hearts. The results of this study show that the contractile and glucose stimulatory effects of epinephrine were influenced by the perfusion pressure. Epinephrine did not correct the impairment in glucose transport in the diabetic heart.

New technique for detection of changes in QRS morphology of ECG signals.

With the use of the electrocardiogram (ECG) as a prototype signal, a new technique was devised for detecting signals embedded in noise. Averaged "normal" digitized ECG signals formed a template to which subsequent ECG QRS complexes were compared. The difference between the averaged template signals and subsequent normal beats was white noise, whereas the difference between the template and ectopic beats consisted of nonrandom signal variation. The template to new signal comparison for the zero-, first-, second-, and third-order differences utilized an approximate F test. Accurate detection of abnormal signals associated with high- and low-frequency noise is accomplished with this method, and the practical clinical utility of the method is under study.

Skeletal muscle carbohydrate metabolism during short-term and prolonged in vivo electrical stimulations.

Electrical stimulations were applied to the gastrocnemius muscle of intact frog. Rana hexadactyla (Lesson) for short-term (SMS) and prolonged (PMS) periods. Short-term muscular stimulations resulted in decreased glycogenolysis and glycolysis with depleted glycogen and lactic acid contents in the muscles and increased lactic acid content in the blood. The activity levels of SDH and MDH were decreased in SMS muscles. Prolonged muscular stimulations, on the other hand, increased glycogenolysis and glycogenolysis was suggested to be due to stepped-up glycogenesis. Tissue oxidative metabolism was also correlated with regulation of activities of enzymes concerned with anaerobic pathways.

Regulation of hepatic triglyceride synthesis in diabetic rats.

The syntheses of triglyceride and its precursors were increased when liver homogenates of ketotic diabetic rats were incubated with [U-14C]-glycero 3-phosphate and cofactors. Triolein sonicates produced a concentration-dependent inhibition of the synthesis of both diglyceride and triglyceride, whereas monoolein sonicates had no effect. Rat serum very low density lipoproteins, like triolein sonicates, inhibited the synthesis of diglyceride and triglyceride. Furthermore, the intracellular form of very low density lipoproteins, namely nascent very low density lipoproteins, also inhibited the synthesis of diglyceride and triglyceride. A higher apparent I50 (concentration of inhibitor that produces 50% inhibition of activity) was observed in liver homogenates of ketotic diabetic rats for inhibition of triglyceride or diglyceride synthesis by triolein sonicates, serum very low density lipoproteins, high density lipoproteins, and nascent very low density lipoproteins. Insulin treatment of the diabetic rats reversed the I50 values to control. In studies on the site of inhibition of triglyceride synthesis in the overall biosynthetic pathway, serum very low density lipoproteins produced a concentration-dependent inhibition of liver cytosolic phosphatidate phosphohydrolase activity. A higher I50 value was obtained with the hepatic enzyme of the diabetic rats. This higher I50 value was reversed to control by insulin treatment of the diabetic rats. These results indicated that the activity of this enzyme was less sensitive to inhibition by very low density lipoproteins in the ketotic diabetic state. The reduced sensitivity of phosphatidate phosphohydrolase activity to triglyceride inhibition observed in the present studies could explain our previous observation of an increased rate of triglyceride synthesis in ketotic diabetic liver homogenates.