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The closed-state crystal structure of prokaryotic inward rectifier, KirBac1.1, has implicated four inner helical phenylalanines near the cytoplasmic side as a possible locus of the channel gate. In the present study, we investigate whether this structural feature corresponds to the physiological pH gate of the renal inward rectifier, Kir1.1 (ROMK, KCNJ1). Kir1.1 is endogenous to the mammalian renal collecting duct and the thick ascending limb of Henle and is strongly gated by internal pH in the physiological range. It has four leucines (L160-Kir1.1b), homologous to the phenylalanines of KirBac1.1, which could function as steric gates near the convergence of the inner (M2) helices. Replacing these Leu-160 residues of Kir1.1b by smaller glycines abolished pH gating; however, replacement with alanines, whose side chains are intermediate in size between leucine and glycine, did not eliminate normal pH gating. Furthermore, a double mutant, constructed by adding the I163M-Kir1.1b mutation to the L160G mutation, also lacked normal pH gating, although the I163M mutation by itself enhanced the pH sensitivity of the channel. In addition to size, side-chain hydrophobicity at 160-Kir1.1b was also important for normal pH gating. Mutants with polar side chains (L160S, L160T) did not gate normally and were as insensitive to internal pH as the L160G mutant. Hence, either small or highly polar side chains at 160-Kir1.1b stabilize the open state of the channel. A homology model of the Kir1.1 closed state, based on the crystal structure of KirBac1.1, was consistent with our electrophysiological data and implies that closure of the Kir1.1 pH gate results from steric occlusion of the permeation path by the convergence of four leucines at the cytoplasmic apex of the inner transmembrane helices. In the open state, K crosses the pH gate together with its hydration shell.

The effect of external potassium (K) and cesium (Cs) on the inwardly rectifying K channel ROMK2 (Kir1.1b) was studied in Xenopus oocytes. Elevating external K from 1 to 10mM increased whole-cell outward conductance by a factor of 3.4±0.4 in 15min and by a factor of 5.7±0.9 in 30min (n=22). Replacing external Na by Cs blocked inward conductance but increased whole-cell conductance by a factor of 4.5±0.5 over a period of 40min (n=15). In addition to this slow increase in conductance, there was also a small, rapid increase in conductance that occurred as soon as ROMK was exposed to external cesium or 10mM K. This rapid increase could be explained by the observed increase in ROMK single-channel conductance from 6.4±0.8 pS to 11.1±0.8 pS (10mM K, n=8) or 11.7±1.2 pS (Cs, n=8). There was no effect of either 10mM K or cesium on the high open probability (Po=0.97±0.01; n=12) of ROMK outward currents. In patch-clamp recordings, the number of active channels increased when the K concentration at the outside surface was raised from 1 to 50mM K. In cell-attached patches, exposure to 50mM external K produced one or more additional channels in 9/16 patches. No change in channel number was observed in patches continuously exposed to 50mM external K. Hence, the slow increase in whole-cell conductance is interpreted as activation of pre-existing ROMK channels that had been inactivated by low external K. This type of time-dependent channel activation was not seen with IRK1 (Kir2.1) or in ROMK2 mutants in which any one of 6 residues, F129, Q133, E132, V121, L117, or K61, were replaced by their respective IRK1 homologs. These results are consistent with a model in which ROMK can exist in either an activated mode or an inactivated mode. Within the activated mode, individual channels undergo rapid transitions between open and closed states. High (10mM) external K or Cs stabilizes the activated mode, and low external K stabilizes the inactivated mode. Mutation of a pH-sensing site (ROMK2-K61) prevents transitions from activated to inactivated modes. This is consistent with a direct effect of external K or Cs on the gating of ROMK by internal pH.

A very simple application of Bayes's theorem provides a flexible, adaptable and reliable method of deriving confidence limits for nominated functions of several binomial random variables. Tables of confidence coefficients are presented to illustrate the reliability of the method when the functions represent log-odds ratios

The parameters of the growth of human embryos in vitro between fertilization and the biastocyst are described. The data on the cleavage of embryos were subjected to statistical analysis. All the pronucleate eggs examined had two pronuclei. There was no evidence of any oocyte with three or more pronuclei, except in one examined 3 days after insemination, which may have been degenerating. Most eggs which were unfertilized remained in metaphase of the second meiotic division, with an extruded polar body. Spermatozoa were found embedded deeply in the zona pellucida of unfertilized and pronucleate eggs. A total of 417 embryos cleaved in vitro. Mary were in the two-cell stage by 28 hours, four-cell stage at 43 hours, and eight-cell stage at 54 hours. Cleavage patterns were regular in the great majority. Compaction began at the 16 cell stage; the blastocyst began to form between 4 and 5 days after fertilization. Forty-two embryos were examined sufficiently often to make a statistical analysis of their growth in vitro. The mean doubling time of the cells was 15.82 hours approximately. Estimates of the mean midstage times for each stage of division were calculated. The results are discussed in relation to the cure of infertility by replanting human embryos, to the possibility of chromosomal and other disorders arising in the embryos, and to potential scientific studies on early human development.

A direct, rapid radioimmunoassay was developed to measure progesterone in a small sample of milk taken from full milk collected at afternoon milking. Samples were preserved with potassium dichromate and mercuric chloride and transported to the laboratory for assay by a semi-automated procedure. Milk progesterone concentrations were significantly higher in pregnant cows compared with those in non-pregnant cows at 21, 24, 28, 42 and 60 days after insemination. The concentrations were greater in full milk taken at afternoon milking than in first milk taken at morning milking. The concentration of progesterone in milk was found to be correlated with fat content in samples with low progesterone values but not in those with high progesterone values. The success rate of the pregnancy test from a single milk sample 21, 24, 28 or 42 days after insemination ranged from 77·5 to 85·8% for pregnant cows and 85·7 to 100% for non-pregnant cows. The highest success rate was found at 24 days when 142 of 176 pregnant cows (80%) and 25 of 25 non-pregnant (100%) were correctly diagnosed. The use of paired instead of single milk samples gave negligible improvement in success rate. The accuracy of the test in three herds was compared and the incidence of false positive results was analyzed.

Ovulation induction alone, and ovulation induction in conjunction with one of four assisted procreation methods [intra-uterine insemination (IUI), intra-peritoneal insemination (IPI), gamete intra-Fallopian transfer (GIFT) or in-vitro fertilization (TVF)] were tested as a treatment for male infertility in a prospective randomized trial. The trial when completed had generated data on 499 cycles from 346 patients. There was overwhelming evidence that some form of assisted procreation was beneficial, when compared to the spontaneous natural conception rate, which is generally assumed to be between 0.01 and 0.02 for male infertility. There was also firm evidence that IUI, GIFT or FVF (mean per cycle pregnancy rate 0.212) were superior to the remaining two treatments of IPI and ovulation induction alone (mean per cycle pregnancy rate 0.068). Duration of infertility and a diagnosis of endometriosis were the most important (adverse) pre-treatment factors detected.