Amiloride fluxes across erythrocyte membranes

Amiloride is known to inhibit both the influx of Na+ and the activation of mitogenesis in many cultured cell lines. This paper describes experiments in which the permeability coefficient of amiloride was determined from measurements of tracer fluxes across human erythrocytes and resealed ghosts. From an analysis of these fluxes, a permeability coefficient of 10(-7) cm/s for the uncharged form of amiloride was deduced. Based upon this measured permeability value, we present calculations of intracellular accumulation times of amiloride in cells of differing surface-to-volume ratio.     

Developmental changes in rat liver alcohol dehydrogenase

Hepatic alcohol dehydrogenase activity and mass content change coordinately during development in male rats. Enzyme activity and mass content increase continuously after birth to 100 and 80% of maximal values within 6 weeks (2.6 ± 0.4 μmole/min/g liver and 92 ± 20 μg/g liver), respectively. When expressed per milligram of soluble proteins, both parameters peak at 3 weeks (0.052 ± 0.002 μmole/min/mg protein and 2.0 ± 0.4 μg/mg protein) and then decrease gradually to plateau levels. These decreases probably arise from a “surge” in soluble liver protein levels that occurs after weaning. Similar developmental patterns also occur in female rats. These findings are the first quantitative measurements of this enzyme in developing animals.     

Proton conductance of cell membranes

Several workers have suggested that cell membranes have a high proton conductance. Our interest in this concept arose from the possibility that the nutrient (submucosal-facing) membrane of the gastric mucosa may have a high proton or hydroxyl ion conductance which would play a role in the regulation of the acid-base balance of the cell. We found that wide changes in the H⁺ concentration of the fluid bathing the nutrient side of the in vitro frog gastric mucosa did not result in significant changes in p.d. However, a maintained change of the H⁺ concentration of the bathing fluid would be expected to produce only a temporary change in p.d. Since a diffusion barrier is present on the nutrient side the temporary change in p.d. might be masked. An analysis of this possibility was made on the basis of a conceptual model and as a result of the analysis it is concluded that the proton (and/or OH^?) conductance of the nutrient membrane of the frog gastric mucosa is not a significant fraction of its total conductance. The present status of the proton conductance hypothesis with respect to striated muscle and to the secretory membrane of the gastric mucosa is reviewed.     

人体单臂间歇运动对发汗调定点的影响

本工作系在微小气候相对恒定条件下,对10名健康男青年每人进行四项实验。实验 Ⅰ 为测定双侧腿足浸入43℃水中,诱发左前臂屈侧显现定量汗点时的口腔温度(舌下)阈值,作为发汗调定点参考值(ToSSP);实验 Ⅱ 为 Ⅰ 附加右臂间歇轻负荷运动(77W)时测定 ToSSP,部分对象还记录了皮肤电反应;实验 Ⅲ、Ⅳ 为 Ⅰ、Ⅱ 均附加4.5m/s 气流(22—25℃)直吹头面部,再分别测定 ToSSP。实验 Ⅰ 与 Ⅱ 同体对照22人次,Ⅲ 与 Ⅳ 同体对照24人次。结果表明,实验 Ⅱ、Ⅳ 的 ToSSP 均值及其潜伏期均值分别较 Ⅰ、Ⅲ 者降低(P<0.01)或缩短(P<0.001);Ⅰ、Ⅱ间的 ToSSP 均值差、潜伏期均值差,分别与 Ⅲ、Ⅳ 之间者无显著差异(P>0.2);Ⅱ、Ⅳ 的ToSSP 均值各与其实验开始前的口温均值亦无明显差异(P>0.5)。此结果支持运动时体温调定点下降的论点,并提示在研究体温调定点活动时,以 ToSSP 为指标较用发汗速率为优越,因 ToSSP 不为许多干扰因素所影响。     

RFLP mapping using near-isogenic lines in the soybean [Glycine max (L.) Merr.]

Summary A molecular marker analysis of a near-isogenic line (NIL), its donor parent (DP), and its recurrent parent (RP) can provide information about linkages between molecular markers and a conventional marker introgressed into the NIL. If the DP and RP possess different alleles for a given molecular marker, and if the NIL possesses the same allele as the DP, then it is reasonable to presume a linkage between that molecular marker and the introgressed marker. In this study, we examined the utility of RFLPs as molecular markers for the NIL genemapping approach. The allelic status of fifteen RFLP loci was determined in 116 soybean RP/NIL/DP line sets; 66 of the Clark RP type and 50 of the Harosoy RP type. Of the 1740 possible allelic comparisons (116 NILs x 15 RFLP loci), 1638 were tested and 462 (33.9%) of those were informative (i.e., the RP and DP had different RFLP alleles). In 15 (3.2%) of these 462 cases the NIL possessed the DP-derived RFLP allele, leading to a presumption of linkage between the RFLP locus and the introgressed conventional marker locus. Two presumptive linkages, pK-3 — and pK-472 — Lf _i, were subsequently confirmed by cosegregation linkage analysis. Although not yet confirmed, two other associations, pk-7 ab and pK-229 — y ₉ seemed to be plausible linkages, primarily because the pk-7 — ab association was detected in two independently derived NILs and both markers of the pK-229 — y ₉ association were known to be linked to Pb. The data obtained in this investigation indicated that RFLP loci were useful molecular markers for the NIL gene-mapping technique.Published as Paper no. 9101, Journal Series, Nebraska Agric. Res. Div. Project no. 12-091. Research partially funded by a grant from the Nebraska Soybean Development, Utilization, and Marketing Board     

Identification of the guanine binding domain peptide of the GTP-binding site of glucagon.

Glucagon, a peptide hormone synthesized and secreted by alpha islet cells, regulates glucose homeostasis by several mechanisms. Using [gamma 32P]8N3GTP, a proven photoaffinity probe for GTP, a specific nucleotide binding site on human glucagon was detected that showed preference for GTP. Half-maximal saturation of photoinsertion into the polypeptide hormone was at 8-12 microM with either [alpha 32P]8N3GTP or [gamma 32P]8N3GTP. GTP protected photolabeling with an apparent kd of 15 microM, whereas ATP was less effective as a protector, exhibiting an apparent kd of about 30 microM. Maximal protection by GTP and ATP was over 90%. UTP, CTP, GDP, ADP, GMP, AMP, guanosine, adenosine, guanine, and adenine were much less effective protectors, indicating that binding is specific for purine nucleoside triphosphates, particularly GTP. Mg2+ at 150 microM enhanced photoinsertion (twofold), whereas at 2-10 mM, it inhibited photoinsertion. Both Ca2+ and Zn2+ at 0.2 mM decreased photoinsertion about 45%. Purification of chymotryptic and tryptic digests of photolabeled glucagon by reverse-phase high performance liquid chromatography (HPLC) revealed that the N-terminal peptide, HSQGTF, was the only peptide region covalently photomodified by [32P]8N3GTP. GTP, if present during photolysis, greatly reduced both photoinsertion into glucagon and the amount of radiolabeled peptide recovered on HPLC. The specificity of binding to the N-terminal region is suggestive of a physiological role for a glucagon-GTP complex in the mechanism of action of this hormone.     

Direct repeats flanking the Bacteroides transposon Tn4351 are insertion sequence elements.

The clindamycin-erythromycin resistance (Ccr Emr) region of the Bacteroides transposon Tn4351 is flanked by direct repeats. This study showed that the direct repeats are insertion sequence (IS) elements. Although both IS elements can mediate transfer of the chloramphenicol (Cmr) marker on pBR328 by cointegrate formation with the conjugal IncW plasmid R388, IS4351R-mediated transfer of Cmr occurred at a consistently lower frequency than did the transfer mediated by IS4351L. Analysis of plasmids from the resultant transconjugants revealed IS-mediated activities such as deletions, tandem duplication of IS4351L, and excision of IS4351R.     

Tn4351 transposes in Bacteroides spp. and mediates the integration of plasmid R751 into the Bacteroides chromosome.

The gene for resistance to erythromycin and clindamycin, which is carried on the conjugative Bacteroides plasmid, pBF4, has been shown previously to be part of an element (Tn4351) that transposes in Escherichia coli. We have now introduced Tn4351 into Bacteroides uniformis 0061 on the following two suicide vectors: (i) the broad-host-range IncP plasmid R751 (R751::Tn4351) and (ii) pSS-2, a chimeric plasmid which contains 33 kilobases of pBF4 (including Tn4351) cloned into the IncQ plasmid RSF1010 and which is mobilized by R751. When E. coli HB101, carrying either R751::Tn4351 or R751 and pSS-2, was mated with B. uniformis under aerobic conditions, Emr transconjugants were detected at a frequency of 10(-6) to 10(-5) (R751::Tn4351) or 10(-8) to 10(-6) (R751 and pSS-2). In matings involving pSS-2, all Emr transconjugants contained simple insertions of Tn4351 in the chromosome, whereas in matings involving R751::Tn4351, about half of the Emr transconjugants had R751 cointegrated with Tn4351 in the chromosome. Of the Emr transconjugants, 13% were auxotrophs. Bacteroides spp. which had R751 cointegrated with Tn4351 in the chromosome did not transfer R751 or Tn4351 to E. coli HB101 or to isogenic B. uniformis, nor did the intergrated R751 mobilize pE5-2, an E. coli-Bacteroides shuttle vector that contains a transfer origin that is recognized by R751.