A conserved inhibitory and differential stimulatory action of nucleotides on K(IR)6.0/SUR complexes is essential for excitation-metabolism coupling by K(ATP) channels.

The mechanism by which ubiquitous adenine nucleotide-gated K(IR)6.0(4)/SUR(4) channels link membrane excitability with cellular metabolism is controversial. Is a decreased sensitivity to inhibitory ATP required, or is the Mg-ADP/ATP-dependent stimulatory action of the ATPase, sulfonylurea receptor (SUR), on K(IR) sufficient to elicit a physiologically significant open channel probability? To evaluate the roles of nucleotide inhibition versus stimulation, we compared K(IR)6.1-based K(NDP) channels with K(IR)6.2-based K(ATP) channels and all possible K(IR)6.1/6.2 hybrids. Although K(NDP) channels are thought to be poorly sensitive to inhibitory ATP and to require Mg-nucleotide diphosphates for activity, we demonstrate that, like K(ATP), and hybrid channels, they are inhibited with an IC(50(ATP)) 100-fold lower than [ATP](i). K(IR)6.1 is, however, more efficiently stimulated by SUR than K(IR)6.2, thus providing a mechanism for differential nucleotide regulation, in addition to the known differential interactions of Mg-nucleotides with SUR isoforms. The on-cell and spontaneous activities of K(NDP), K(ATP), and hybrid channels identified in native cells, are different; thus, their similar IC(50(ATP)) values argue the regulatory "beta" SUR subunits play a preeminent role in coupling excitation to metabolism and pose questions about the physiologic significance of models, which assume the ATP insensitivity of open K(IR)s.     

The N-terminus of KIR6.2 limits spontaneous bursting and modulates the ATP-inhibition of KATP channels

KATP channels are heteromultimers of a sulfonylurea receptor SUR and KIR6.2 with the inward rectifier forming the pore which is regulated by SUR. We have examined the contributions of the cytoplasmic domains of KIR6.2 to control of spontaneous bursting and ATP-inhibition in human SUR1/KIR6.2 KATP channels. Truncations of the N-terminus of KIR6.2 nearly eliminate transitions to interburst closed states without affecting the open or intraburst closed states, thus producing SUR1/DeltaNKIR6.2 channels with an extremely high open probability in the absence of nucleotides. These channels have a decrease apparent ATP-sensitivity which is consistent with the involvement of the N-terminus in a transition to an interburst closed state that preferentially binds inhibitory ATP. Mutations in both the N- and proximal C-termini of KIR6.2 can synergistically attenuate the ATP-inhibition. The results identify the N-terminus of KIR6.2 as a determinant of the interburst kinetics of KATP channels and suggest that the two cytoplasmic domains of KIR6.2 participate in ATP-inhibitory gating through distinct mechanisms.     

Reduction of sialic acid O-acetylation in human colonic mucins in the adenoma-carcinoma sequence

The oligo-O-acetylation of sialic acids found in normal colonic mucins is greatly reduced in colorectal cancer. Mucins prepared from cancer tissue in adenocarcinoma showed this reduction, while normal O-acetylation was detected in resection margin and control cases and total mucin sialic acid content was significantly decreased in cancer vs control samples. A reduction of the O-acetyl transferase activity catalysing the O-acetylation reaction was also found. A series of cultured human colorectal cell lines derived from the same premalignant adenomatous line, and representative of the adenoma-carcinoma sequence were examined and revealed a depletion of oligo-O-acetylation in the original diploid premalignant line, re-expression in a further premalignant line and reduction in malignant mucinous and adenocarcinoma cell lines. Reduction of sialic acid O-acetylation appears as an early event in the process of malignant transformation in human colorectal cancer.     

Isolation and identification of a 92-kDa stress induced protein from Candida albicans

Mycopathologia - It was previously shown that the presence of estrogen enhances survival of Candida albicans under heat and oxidative stresses. A 92-kDa protein is inducible by heat shock and...     

Genomic modulation of diabetes (<Emphasis Type="Italic">db/db)</Emphasis> and obese (<Emphasis Type="Italic">ob/ob</Emphasis>) mutation-induced hypercytolipidemia: cytochemical basis of female reproductive tract involution

Infertility and hypercytolipidemic utero-ovarian involution are recognized consequences of the diabetes-obesity syndrome (DOS) in C57BL mice with either obese (ob/ob) or diabetes (db/db) single gene mutations. We have evaluated the interdependent deleterious influences of both mutation types and differences in the genomic background on utero-ovarian dysfunction in C57BL mice. Control (+/?) C57BL mice were matched with littermate ob/ob and db/db mutants expressed on either the /KsJ or /6 background. Both ob/ob and db/db mutations increased body weights of /KsJ and /6 background strains relative to +/? groups. In contrast, uterine and ovarian weights were depressed by ob/ob and db/dbmutations relative to +/?, regardless of the background strain, but especially when expressed on the /KsJ background. Functionally, both ob/ob and db/db mutations induced hyperglycemic-hyperinsulinemic states coupled with depressed serum estradiol-17-β and progesterone concentrations when expressed on a /KsJ background. Microscopic analysis of utero-ovarian tissue samples revealed marked hypercytolipidemia in the follicular granulosa and endometrial epithelial tissue layers of both ob/oband db/db mutant groups relative to normal +/? cytoarchitecture. The db/db mutation consistently promoted more severe hypercytolipidemic profiles than the ob/obmutation, regardless of background strain. Thus, the severity of utero-ovarian hypercytolipidemia following the expression ofob/ob and db/db mutations in C57BL mice is influenced, or moderated, by the genomic background on which the mutation is expressed.     

Coordinated functions of WSS1, PSY2 and TOF1 in the DNA damage response

The stabilization and processing of stalled replication forks is required to maintain genome integrity in all organisms. In an effort to identify novel proteins that might be involved in stabilizing stalled replication forks, Saccharomyces cerevisiae mutant wss1Δ was isolated from a high-throughput screening of ~5000 deletion strains for genes involved in the response to continuous, low-intensity UV irradiation. Disruption of WSS1 resulted in synergistic increases in UV sensitivity with null mutants of genes involved in recombination (RAD52) and cell cycle control (RAD9 and RAD24). WSS1 was also found to interact genetically with SGS1, TOP3, SRS2 and CTF4, which are involved in recombination, repair of replication forks and the establishment of sister chromatid cohesion. A yeast two-hybrid screen identified a potential physical interaction between Wss1 and both Psy2 and Tof1. Genetic interactions were also detected between PSY2 and TOF1, as well as between each gene and RAD52 and SRS2, and between WSS1 and TOF1. Tof1 is known to be involved in stabilizing stalled replication forks and our data suggest that Wss1 and Psy2 similarly function to stabilize or process stalled or collapsed replication forks.     

Eventual entrainment of the human circadian pacemaker by melatonin is independent of the circadian phase of treatment initiation: clinical implications

About 15% of the legally blind completely lack light perception. Most of these individuals have abnormally phased circadian rhythms and many free-run. Light treatment is not an option for them. However, melatonin treatment can be highly effective. A daily dose of 0.5 mg of melatonin usually results in entrainment. It has been suggested that treatment in individuals with circadian periods > 24 h should be initiated on the advance zone of the melatonin phase response curve, which was based on findings in which melatonin initiated on the delay zone were less likely to result in entrainment, even though treatment continued across all circadian phases. In the present study, 7 totally blind people started low-dose melatonin treatment (0.5 mg; 1 person was given 0.05 mg) on the delay zone. All entrained as circadian phase free-ran and the advance zone of the melatonin phase response curve coincided with the time of melatonin administration. These results are consistent with studies in other mammals. It does not appear that low-dose melatonin treatment needs to be initiated on the advance zone to induce eventual entrainment in blind people with free-running rhythms > 24 h. Therefore, it is not essential that circadian phase be ascertained before starting low-dose melatonin treatment of blind people.     

The cis-Pro touch-turn: a rare motif preferred at functional sites

A new motif of three-dimensional (3D) protein structure is described, called the cis-Pro touch-turn. In this four-residue, three-peptide motif, the central peptide is cis. Residue 2, which precedes the proline, has phi, psi values either in the "prePro region" of the Ramachandran plot near -130 degrees, 75 degrees or in the Lalpha region near +60 degrees, +60 degrees. The Calpha(1)-Calpha(4) distance is 4-5 A and the two flanking peptides lie parallel to one another, making van der Waals contact rather than a hydrogen bond. Apparently, this arrangement is locally unfavorable and therefore rare, usually occurring only if needed for biological function. Of the 12 examples in a 500-protein database, cis-Pro touch-turns are found at the catalytic sites of pectate lyase, Ni-Fe hydrogenase, glucoamylase, xylanase, and opine dehydrogenase and at the primary binding sites of ribonuclease H, type I DNA polymerase, ribotoxin, and phage gene 3 protein. In each of these protein families, the touch-turns serve different roles; their functional importance is supported by conservation and mutagenesis data. In analyzing the conservation patterns of these 3D motifs, new methods for in-depth quality evaluation of the structural bioinformatic data are employed to distinguish between significant exceptions and errors     

15Alpha-hydroxyprogesterone in male sea lampreys, Petromyzon marinus L

There is growing evidence that sea lampreys, Petromyzon marinus L., produce gonadal steroids differing from those of other vertebrates by possessing an additional hydroxyl group at the C15 position. Here we demonstrate that sea lamprey testes produce 15alpha-hydroxyprogesterone (15alpha-P) in vitro when incubated with tritiated progesterone, that 15alpha-P is present in the plasma of sea lampreys, and that plasma concentrations of immunoreactive (ir) 15alpha-P rise dramatically in response to injections of gonadotropin-releasing hormone (GnRH). The identity of the tritiated 15alpha-P produced in vitro was confirmed by co-elution with standard 15alpha-P on high performance liquid chromatography, co-elution with standard and acetylated 15alpha-P on thin layer chromatography, and specific binding to antibodies raised against standard 15alpha-P. The in vitro conversion was used to produce tritiated 15alpha-P label for a radioimmunoassay (RIA), which is able to detect 15alpha-P in amounts as low as 2 pg per tube. The RIA has been used to measure the plasma concentrations of 15alpha-P in males given two serial injections, 24 h apart, of either lamprey GnRH I or GnRH III (50, 100, or 200 microg/kg) or saline control, with plasma being sampled 8 and 24 h after the second injection. Plasma concentrations of ir-15alpha-P rose from < 1 to 36 ng/ml (mean of all treatments) 8 h after injection and declined within 24 h. This is the first time that an RIA has detected such high steroid concentrations in lampreys. This finding is suggestive of a role for 15alpha-P in control of reproduction in the sea lamprey.     

A second human antiretroviral factor, APOBEC3F, is suppressed by the HIV-1 and HIV-2 Vif proteins

The HIV-1 Vif protein suppresses the inhibition of viral replication caused by the human antiretroviral factor APOBEC3G. As a result, HIV-1 mutants that do not express the Vif protein are replication incompetent in 'nonpermissive' cells, such as primary T cells and the T-cell line CEM, that express APOBEC3G. In contrast, Vif-defective HIV-1 replicates effectively in 'permissive' cell lines, such as a derivative of CEM termed CEM-SS, that do not express APOBEC3G. Here, we show that a second human protein, APOBEC3F, is also specifically packaged into HIV-1 virions and inhibits their infectivity. APOBEC3F binds the HIV-1 Vif protein specifically and Vif suppresses both the inhibition of virus infectivity caused by APOBEC3F and virion incorporation of APOBEC3F. Surprisingly, APOBEC3F and APOBEC3G are extensively coexpressed in nonpermissive human cells, including primary lymphocytes and the cell line CEM, where they form heterodimers. In contrast, both genes are quiescent in the permissive CEM derivative CEM-SS. Together, these data argue that HIV-1 Vif has evolved to suppress at least two distinct but related human antiretroviral DNA-editing enzymes.