Localization of ROMK channels in the rat kidney

Renal potassium secretion occurs in the distal segments of the nephron through apically located secretory potassium (SK) channels. SK may correspond to the ROMK channels cloned from rat kidney. In this study, the localization of ROMK at the cellular level in the rat kidney was examined using an affinity-purified polyclonal antibody raised against a C-terminal peptide of ROMK. The specificity of the antibody was demonstrated by immunoblots of membranes of Xenopus oocytes expressing ROMK2. Immunoblots of homogenates from rat renal outer medulla and cortex revealed predominant bands of 70 to 75 kD, which were ablated by preadsorption with an excess of peptide. These bands were specific for the rat kidney. Immunolocalization studies revealed that ROMK is expressed in specific nephron segments in both the cortex and medulla. In the cortex, ROMK was found in the apical domain of the thick ascending limb of Henle's loop, the connecting tubule, and in some, but not all, cells of cortical collecting tubules. In the medulla, expression in the apical membrane of the thick ascending limbs of Henle's loop was strong, whereas outer medullary collecting ducts were weakly stained. Expression in the thick ascending limb was also heterogeneous; some cells that expressed the Na-K-Cl cotransporter were weakly stained with the anti-ROMK antibody. No staining of glomeruli, proximal tubules, or inner medullary collecting ducts was found. The localization of ROMK agrees well with the findings of SK in patch-clamp studies and supports the view that ROMK is the SK channel of the distal segments of the nephron.     

A neurophysiological study in children and adolescents with Crigler-Najjar syndrome type I

BACKGROUND: Paclitaxel, a natural product from Taxus brevifolia, is a microtubule stabilizing agent, which has been shown to block different cells in the G2/M phase of the cell cycle and consequently, to modulate their radioresponsiveness. Our aim was to test the cytotoxic and radiosensitizing potential of paclitaxel, with respect to different gynecological tumors with varying radiosensitivities. MATERIAL AND METHOD: We performed clonogenic assays and flow cytometry on 2 cell lines, MCF-7 (breast) and CaSki (cervix) cells, and on 2 primary ovarian tumor samples (OC-I and OC-II). The cells were irradiated with 200 kV X-rays, radiation doses of up to 8 Gy were applied either as single doses or in 2 Gy fractions. Paclitaxel concentrations varied from 0.07 to 700 nM, incubation times varied from 3 to 120 h. RESULTS: Paclitaxel alone changed the cell cycle distribution of the cells tested and was cytotoxic in a time and concentration dependent manner. When combined with radiation, most schedules resulted in additive effects of the combined treatments. However, for MCF-7 cells, when 7 nM paclitaxel, applied 24 h before irradiation, were combined with fractionated irradiation a supra-additive effect with a SER of 1.2 was found. For CaSki cells, under comparable conditions the SER was 1.13 but the effects were not statistically significant. CONCLUSION: Under specific conditions, paclitaxel exerted a weak radiosensitizing effect on breast and cervical carcinoma cells. A therapeutic gain may be possible on the basis of an optimal paclitaxel/radiation scheduling.     

Molecular dynamics of the anti-fluorescein 4-4-20 antigen-binding fragment. 2. Time-resolved fluorescence spectroscopy

Time-resolved fluorescence experiments were performed to investigate the dynamic aspects of the antigen-binding fragment (Fab) of a high-affinity monoclonal antibody (4-4-20) which binds the fluorescent hapten fluorescein. Both the unliganded Fab and a complex of the Fab with a nonfluorescent analog of fluorescein (fluoresceinamine, FLM) were examined. A fluorescence polarization probe [5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid, AEDANS] was covalently attached to the C-terminus of the Fab. Experiments were performed at three different temperatures (10, 25, and 35 degrees C), and phase-modulation data sets were collected for five different molar ratios of FLM to Fab at each temperature. Global analyses were then used to extract values for fluorescence lifetime and rotational correlation time from these data. In the lifetime analysis the best fit was obtained when the emission of AEDANS was described by a Lorentzian distribution of lifetimes (tau = 15.6 ns, distribution width = 3.4 ns, both at 25 degrees C), which suggested that the probe experienced a heterogeneous environment. Anisotropy analyses suggested that two different rotational components were present. The first was attributed to the global motion of the Fab and exhibited a rotational correlation time (theta 1) of ca. 33 ns at 25 degrees C. This component was relatively unaffected by antigen binding. The second rotational component was attributed to the local or segmental motion within the Fab and exhibited a rotational correlation time (theta 2) of 1.1 ns at 25 degrees C. This value increased by more than 50% upon antigen binding, a result which was consistent with molecular dynamics simulations of the same Fab--fluorescein system [Lim & Herron (1995) Biochemistry 34, 6962-6974]. Furthermore, statistical analysis showed that this increase was significant at the 95% confidence level.     

An exploration of sexual knowledge, attitudes and behavior in aboriginal elementary school students in the Ping-Tung area

The purpose of this study was to investigate sexual knowledge, attitudes and behavior of fifth and sixth grade students in aboriginal elementary schools in the Ping-Tung area. A structured questionnaire was administered to 1091 students who were selected by cluster sampling. The results showed: (1) The sexual knowledge score was low but sexual attitudes showed a positive trend. (2) 64.7% and 67.4% of students had at some time seen pictures of male or female sexual organs. (3) About 61% of students had seen sexual magazines or videotapes. (4) 66.2% of male and 88.1% of female students had heard about wet dreams or menstruation before their first experience; more than half of the students thought that wet dreams need treatment. (5) 17.8% of students had masturbation experience, and after that 59.3% of students had fear or guilt feeling. (6) Female students had significantly higher knowledge and attitude scores than male students, Demographic variables produced no significant difference in the above scores. (7) 42.4% of students most desired to know what phenomena indicate sexual maturity. (8) Sex knowledge had significantly positive correlation with sex attitude.     

Differences in anaerobic performance between boys and men

The purpose of this study was to compare the anaerobic performance of 11-12 year old prepubescent boys (stage 1; n = 18) using a maximal effort 90 s cycling test to that of adult men (n = 19). Performance was separated into short-term anaerobic power (SAP; work in 10 s), intermediate-term anaerobic capacity (IAC; work in 30 s) and long-term anaerobic capacity (LAC; work in 90 s). Resistance on the Monark cycle ergometer was chosen as the highest tolerable load for the 90 s test and set at 2.6 and 3.8 J. rev-1.kg body mass-1 for boys and men, respectively. Power drop-off (%PD) was determined as the change in 5 s power outputs from 0-90 s. Blood lactates were measured at rest (pretest; BL) and at 2 (BL2) and 5 (BL5) min post test. Absolute and relative (to weight or thigh volume) scores for SAC, IAC and LAC were 33-40% lower in the boys compared to the men (p < 0.001). The men demonstrated significantly greater %PD over the 90 s (32.2%) compared to the 25.3% PD of the boys (p < 0.001), suggesting that the development of fatigue was greater in the men even though power and work outputs were high. Resting BL values were similar for both groups. Mean BL2 and BL5 were higher (p < 0.01) in the men (13.5, SD = 3.4; 12.5, SD = 2.8 mmol.l-1) than in the boys (9.1, SD = 1.7; 8.3, SD = 1.6 mmol.l-1). The anaerobic performance of the boys compared to the men cannot be completely explained by the lower resistance setting used. Although the boys were 50% lighter than the men, their resistance was 70% of that used for the men. The results do not support the contention that children and adults have similar abilities in short term, exhaustive work when corrected for size. Prepubescent boys appear to be limited in their ability to perform short-term as well as intermediate and long term anaerobic exercise compared to adult males.     

Selective aortic arch perfusion using serial infusions of perflubron emulsion

With the aim of developing foetal gene therapy for cystic fibrosis, we have investigated the possibility of gene targeting to the mouse foetus with two different viral vector systems and at different times of gestation. We report here that recombinant retrovirus producing cells administered into the intra-amniotic cavity of mid- to late-gestation mouse MF1 foetuses survive in the amniotic fluid and are able to engraft to a certain extent in foetal tissues. By production of infectious virus they mediate transduction and beta-galactosidase transgene expression in neighbouring foetal tissues 24 to 72 h following injection. Retrovirus producer cells could, therefore, become a means to overcome the limitations of low retroviral titre, for in vivo foetal gene transfer. To investigate the developmental stage at which transduction of the airways and enteral systems can be obtained we also administered a highly infective first generation adenoviral vector (AdRSV beta gal) into the amniotic cavity of foetal mice between 13 to 16 days post coitus, beta-galactosidase activity was detected between 24 to 120 h after injection. The highest levels of transgene expression were generally observed between 48 to 72 h following injection of the adenoviral vector. We demonstrate that infection of the pulmonary airways is dependent on the developmental stage of the foetus and can be achieved on the 15th day of gestation.