Gene therapy is expected to lead to new and useful methods to treat diseases. The development of assays to quantitate gene-therapy-derived proteins circulating in blood will be essential to investigate the effects and side effects of the introduced proteins. The purpose of this study is to evaluate whether a protein circulating at trace concentrations in blood can be measured by tagging a peptide corresponding to glucagon residues 19-29 onto its C-terminal end. We constructed plasmids encoding chimeric proteins and transferred them into rats by hydrodynamics-based delivery. When plasmids encoding human IL8-glucagon 19-29 chimeric protein were injected into rats to evaluate the accuracy of this method, there was a high correlation between chimeric proteins measured by an enzyme-linked immunosorbent assay for human IL8 and one by a radioimmunoassay for glucagon. Furthermore, when plasmids coding rat IFN gamma receptor IgG-Fc glucagon 19-29 chimeric protein were injected to evaluate the time course of chimeric proteins in blood plasma, we could calculate the concentrations in blood from 10 μl plasma samples using glucagon 19-29 tag as follows: 2815±2318 ng/ml after 4 hours (mean±S.D.), 6061±2789 ng/ml after 8 hours, 5752±2270 ng/ml after 12 hours, 2870±1062 ng/ml after one day, 1440±334 ng/ml after three days, 1120±433 ng/ml after seven days, and 281±162 ng/ml after 16 days. Blood sugar levels which might have been increased by glucagon did not increase even at peak chimeric protein concentrations. These results demonstrate a useful and convenient method to assay gene therapy products circulating in blood using a glucagon 19-29 tagging vector.
We measured lipid peroxidation and antioxidant enzymes in erythrocytes of types IIb and IV hyperlipoproteinemic (HLP) human subjects in comparison with age-matched controls. Thiobarbituric acid-reactive substances (TBARS), a measure of lipid peroxidation, glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), glutathione reductase (GR), and catalase (CAT) were determined in erythrocytes. We also measured lipid parameters including triglycerides (TG), total cholesterol (TC), HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C), apolipoprotein AI, and apolipoprotein B, and antioxidant related substances such as serum albumin, free iron, ferritin, ceruloplasmin. Thirty-two subjects (females 15, males 17) with type IIb (the mean age 45.6±8 [S.E.]), 34 with type IV (females 16, males 18) (the mean age 47±10 [S.E.]), and 36 normolipidemic voluntary subjects (females 18, males 18) (the mean age 46±8 [S.E.]) were included in the study. Erythrocytes were prepared by classical washing method (0.9%NaCl) from venous blood samples. The mean TBARS levels in plasma and erythrocyte suspensions were found to be significantly higher in both types IIb and IV hyperlipoproteinemics. Erythrocyte SOD and GSH-Px activities were decreased but erythrocyte GR activity did not change in both types IIb and IV hyperlipoproteinemics. Erythrocyte CAT activity was decreased in type IIb, but it was increased in type IV hyperlipoproteinemics. Erythrocyte SOD activity was negatively correlated with plasma TG level, whereas plasma free iron was positively correlated with plasma TBARS level in type IV hyperlipoproteinemics. These results suggest the presence of oxidative injury in patients with type IIb or IV hyperlipoproteinemia, and that the responses of erythrocyte antioxidant enzymes to oxidant stress are different in these conditions.
The aim of this study was to evaluate decrease in waist circumference in obese patients receiving different anti-obesity treatments. The study was designed as a short-term (12 weeks), open-label, and randomized trial. Eighty six patients (70 females, 81.4%; mean age 41.09±8.73 years, mean BMI 36.1±4.3 kg/m2) were randomized to four different therapy groups. The primary outcome parameters were waist circumference and body mass index (BMI). The therapy groups were a) diet+sibutramine 1×10 mg/d (n=22), b) diet+orlistat 3×120 mg/d (n=25), c) combination of diet+sibutramine+orlistat (n=20) and d) diet (n=19). Combination therapy was more effective than diet and orlistat mono-therapy (p<0.0001 for all), but not significantly superior to sibutramine mono-therapy (p=0.072) in decreasing BMI. Sibutramine mono-therapy was significantly more effective in inducing BMI decrease compared with orlistat mono-therapy (p=0.039). The association between change in BMI and change in waist circumference was strongest in the orlistat mono-therapy group (P interaction=0.003). This means that patients taking orlistat experienced more decrease in waist circumference (3.4 cm, R2=0.29) per unit decrease in BMI compared to patients under combination therapy (2.6 cm, R2=0.25, P interaction = 0.015) and patients taking sibutramine (1.8 cm, R2=0.19, P interaction=0.026). In the diet therapy group decline in waist circumference was independent of BMI (1.9 cm, R2=0.02, P interaction=0.076). Although combination therapy and sibutramine mono-therapy were more effective in decreasing BMI, reduction in waist circumference and BMI was most significantly associated with the orlistat mono-therapy group. This may hint at the possibility of orlistat inducing weight loss mainly in the abdominal area targeted to reduce cardiovascular risk.
The purpose of this study was to assess the validity of an adaptive filter, the scaled Fourier linear combiner (SFLC), in the impedance cardiography (ICG). Eight healthy males underwent constant-load bicycle exercise at different intensities from unloaded to near maximal intensity. The stroke volume (SV) and cardiac output (Q) measured by ICG at each condition were compared with those by the CO2 rebreathing method. We found that the noises were greatly reduced in the impedance waveform and that the inflection points, so-called the B- and X-points, were clearly detected even during strenuous exercise using the SFLC. Although a high correlation was observed between Qs measured by the two methods, the mean values of Qs in each method differed significantly and the regression line also differed significantly from the identity line. Likewise, a significant correlation was observed between SVs obtained by the two methods, but a significant difference in the group mean values and a trend of the regression line were observed. These findings suggest that the use of SFLC in ICG improves the performance in eliminating the noises and in detecting the inflection points in the waveform, thereby contributing to the accurate and beat-to-beat measurements of SV and Q especially during exercise.
To clarify the effect of androgen on the microcirculation in seminal vesicles of adult Wistar rats, we investigated the organ blood flow and morphological features in the capillaries after castration and subsequent testosterone supplementation. Testosterone (T) was subcutaneously injected every 12 hours after castration and its doses were set to 10−2, 10−1, 100 and 101 mg/kg-body weight (T10−2, T10−1, T100 and T101 groups, respectively). Organ blood flow was measured using laser Doppler flowmetry, and the subepithelial capillaries were analyzed using transmission electron microscopy. The capillaries were morphologically classified into 3 types; oval opened (type 1), intermediate (type 2) and collapsed (type 3), and their luminal areas were measured using a computed image analyzer. The organ blood flow was significantly reduced from 36.3±5.1 to 21.9±2.7 ml·min−1/100 g tissue, and the luminal area of the capillaries was significantly reduced from 9.02±1.28 to 4.85±0.82 μm2 with the shift of the type 1 and type 2 to type 3 after castration. The reduction of the luminal area and the blood flow reduction, and shift of the capillary type were significantly protected by gradated testosterone supplementation. These results indicate that the maintenance of the blood flow and morphological profiles in capillaries depend on androgen-supplementation levels in seminal vesicles.
We studied the changes in urinary bicarbonate, urinary pH and some physical parameters such as minute ventilation (VE), oxygen consumption (VO2), respiratory carbon dioxide (VCO2), heart rate, blood pressure, and blood lactate, before and after the submaximal exercise. Six male subjects aged 28-33 years were involved in the study. They performed the incremental exercise test using a bicycle ergometer until exhaustion. Levels of VE, VO2, VCO2, heart rate, and blood pressure increased continuously with an increase in cycling intensity. These parameters markedly decreased and reached the baseline levels within 5-10 minutes after the termination of exercise. According to an increase in cycling intensity, blood lactate increased continuously during exercise, but after termination of exercise the return of lactate to the baseline level was markedly retarded. Urinary bicarbonate and pH were within the range of those at 0 time (baseline levels) from the beginning until 30 minutes after the exercise. However, they began to increase abruptly about 30 minutes after the exercise, and continued to increase extensively for 2 hours thereafter. Such marked increase in urinary bicarbonate and pH seemed to be correlated with the aerobic metabolism of lactate in the muscles, liver, and kidney, finally producing CO2. It was also suggested that the measurement of urinary bicarbonate and pH may be useful for the estimation of physiological changes in the body after submaximal incremental cycling exercise loading.
Cancer-associated retinopathy (CAR) is an ocular manifestation of a paraneoplastic syndrome whereby immunological reactions toward recoverin, a retina-specific calcium binding protein, and other retinal antigens aberrantly expressed in tumor cells lead to the degeneration of retinal photoreceptor cells. Recently we reported that aberrant expression of recoverin was identified in more than 50% of tumor cells and their cell lines from several kinds of cancers, including gastric cancer, lung carcinoma, and other cancers. To elucidate the clinicopathological significance of aberrantly expressed recoverin in cancer cells, we performed immunocytochemical analysis using a monoclonal antibody against human recoverin. Within 18 patients with different clinical stages (I-IV) of gastric cancer, the aberrant expression of recoverin in tumor cells was recognized in 6 out of 18 patients (2 out of 2 stage IA, 1 out of 1 stage IB, 2 out of 3 stage II, 0 out of 7 stage IIIA, 0 out of 1 stage IIIB, and 1 out 4 stage IV). The present data are consistent in part with the previous observations that recoverin-expressing cancer cells induced tumor immunity and provide a favorable prognosis for primary cancer in CAR patients.
This research was performed in an effort to understand the decrease in fertility that has occurred over the past few decades. The objective of the study was to analyze female fertility according to maternal age; data were based on the number of children born per mother. The records of 18-year-old college students were obtained, and the mothers of the students were categorized into age groups according to the year of their birth (1915 to 1949). The number of children born to each mother was then analyzed. The total sample size was 4078. The results showed that an increase in two-children families led to a reduction in the mean number of children per mother. While the decrease in the maternal age at the time of the birth of the last child in the family was observed, the maternal age at the time of the first birth did not increase. Thus, the reduction in fertility may not be the result of delayed motherhood. The group of mothers, who gave birth to the largest number of children, had their highest fertility rate in the twenties. In addition, their fertility rate in the thirties was almost equal to other groups, who had the same fertility level in their twenties.
Several birth cohort studies have shown adverse effects of perinatal exposures to methylmercury (MeHg) and environmentally persistent organic pollutants (POPs). These chemicals are ingested mainly through fish consumption, but little is known about the hazardous effects in Japanese, whose fish consumption is high. The present study, the Tohoku Study of Child Development, was designed to examine the effects of perinatal exposures to MeHg, polychlorinated biphenyls (PCB), dioxins, pesticides, and other chemicals in Japanese children. Six hundred eighty-seven pregnant women were participated in this study with their written informed consent. Maternal peripheral blood, cord blood, cord tissue, placenta, and breast milk samples were collected for chemical analysis. Maternal hair was also taken for MeHg analysis. Infants born at full term were assessed by neurobehavioral tests: the Brazelton Neonatal Behavioral Assessment Scale at three days old, the Kyoto Scale of Psychological Development and the Bayley Scales of Infant Development at 7 and 18 months old, and the Fagan Test of Infant Intelligence at 7 months old. The children will be continuously followed up to ages 6-7 years. Maternal food intake frequency, maternal IQ, socioeconomic status, and home environment were assessed as covariates. The results of this cohort study will allow us to evaluate associations between the neurobehavioral development of children and perinatal exposures to MeHg and environmentally POPs in Japan.
Isolated torsion of the fallopian tube in premenarcheal girls is very rare. However, correct diagnosis and treatment are needed in order to optimize salvage of fallopian tube. Here, we report a case of fallopian tube torsion in a premenarcheal girl. A 12-year-old premenarcheal girl was admitted with a rapid history of colicky lower abdominal pain. MR image demonstrated normal ovaries and a large spherical cystic tumor with hemorrhage-like contents. The differential diagnosis included torsion of a para-ovarian cyst or hydrosalpinx, hemorrhage within a non-communicating uterine horn or a pelvic hemorrhage of unknown origin. Laparoscopic inspection/operation was performed. The distal portion of the left fallopian tube was swollen with two twists evident in the middle portion. The distal portion of the left fallopian tube was laparoscopically removed with the aid of a YAG laser. The mechanisms underlying disorders of the left fallopian tube are not well understood. However, some intrinsic/extrinsic causative factors are discussed. Although torsion of the fallopian tube occurs rarely and exhibits variable clinical features, the diagnosis should be considered in all young girls presenting with sudden colicky abdominal pain. Laparoscopic inspection/surgery appears to be the most suitable management.