An arginine-like effect of the "fumarate + glutamate + pyruvate" mixture on glucagon release.

The “fumarate + glutamate + pyruvate” mixture (FGP), currently used to supply energy in isolated tissues, was found to stimulate glucagon release in the $\text{in vitro}$ perfused rat pancreas. The stimulation of glucagon release by FGP resembles that induced by arginine, in that it is biphasic and its amplitude inversely related to the environmental glucose concentration. Both glutamate and pyruvate — but not fumarate — seemed to be involved in the stimulation induced by the FGP mixture. The glucagonotropic effect of the FGP mixture suggests that glucagon release is not invariably suppressed by energy-yielding nutrients.     

Mapping the BKCa channel's "Ca2+ bowl": side-chains essential for Ca2+ sensing

There is controversy over whether Ca(2+) binds to the BK(Ca) channel's intracellular domain or its integral-membrane domain and over whether or not mutations that reduce the channel's Ca(2+) sensitivity act at the point of Ca(2+) coordination. One region in the intracellular domain that has been implicated in Ca(2+) sensing is the "Ca(2+) bowl". This region contains many acidic residues, and large Ca(2+)-bowl mutations eliminate Ca(2+) sensing through what appears to be one type of high-affinity Ca(2+)-binding site. Here, through site-directed mutagenesis we have mapped the residues in the Ca(2+) bowl that are most important for Ca(2+) sensing. We find acidic residues, D898 and D900, to be essential, and we find them essential as well for Ca(2+) binding to a fusion protein that contains a portion of the BK(Ca) channel's intracellular domain. Thus, much of our data supports the conclusion that Ca(2+) binds to the BK(Ca) channel's intracellular domain, and they define the Ca(2+) bowl's essential Ca(2+)-sensing motif. Overall, however, we have found that the relationship between mutations that disrupt Ca(2+) sensing and those that disrupt Ca(2+) binding is not as strong as we had expected, a result that raises the possibility that, when examined by gel-overlay, the Ca(2+) bowl may be in a nonnative conformation.     

Ca2+ signals and T lymphocytes; "New mechanisms and functions in Ca2+ signalling"

Abstract Interaction between a T cell and an antigen‐presenting cell leads to the rapid formation of an immunological synapse allowing antigen detection by the T cell and the development of an immune response. Antigen detection triggers various cellular responses including a modest but sustained T cell Ca²⁺ increase. In this review are discussed a series of related questions. What are the various molecular events by which a T cell Ca²⁺ response can be triggered in the immunological synapse by a very small amount of antigen ? How is Ca²⁺ released from intracellular stores and how can these stores remain empty for hours ? Through which channels does Ca²⁺ influx takes place, and how is Ca²⁺ influx coupled to Ca²⁺ release from intracellular stores ? What are the main immediate and indirect cellular targets of the Ca²⁺ increase ?     

Synthesis and assembly of H+-ATPase complex by isolated "rough" thylakoids

The synthesis and assembly of chloroplast H+-ATPase complex were studied by analyzing the incorporation of [35S]methionine into the constituent subunits with isolated intact chloroplasts and with thylakoid membranes that had been prepared from the chloroplasts so that they would retain ribosomes. The complex was isolated from thylakoids after labeling and identified by immunoprecipitation with an antiserum specific to CF1. The mechanism for the assembly of the complex was demonstrated to be active in the isolated chloroplasts by the following observations: the plastid genome-regulated subunits (alpha, beta, epsilon, I, and III) were labeled by in organello translation and recovered with the complex, and three other subunits (gamma, delta, and II) were labeled when intact chloroplasts were incubated with translation products from polyadenylated RNA. The two largest subunits, alpha and beta, were translated on thylakoid-bound ribosomes when the thylakoid membranes were incubated with soluble factors from Escherichia coli. They were recovered with the H+-ATPase complex, suggesting that they are translated on the bound ribosomes in the chloroplast, and that the isolated membranes retain the ability to assemble a complete complex. Provided that these observations are the result of de novo assembly of the complex, the imported and processed nuclear-coded subunits are presumed to be pooled not in stroma but on the membrane.     

pH alterations "reset" Ca2+ sensitivity of brain Na+ channel 2, a degenerin/epithelial Na+ ion channel, in planar lipid bilayers

Members of the degenerin/epithelial Na(+) channel superfamily of ion channels subserve many functions, ranging from whole body sodium handling to mechanoelectrical transduction. We studied brain Na(+) channel 2 (BNaC-2) in planar lipid bilayers to examine its single channel properties and regulation by Ca(2+). Upon incorporation of vesicles made from membranes of oocytes expressing either wild-type (WT) BNaC-2 or BNaC-2 with a gain-of-function (GF) point mutation (G433F), functional channels with different properties were obtained. WT BNaC-2 resided in a closed state with short openings, whereas GF BNaC-2 was constitutively activated; a decrease in the pH in the trans compartment of the bilayer activated WT BNaC-2 and decreased its permeability for Na(+) over K(+). Moreover, these maneuvers made the WT channel more resistant to amiloride. In contrast, GF BNaC-2 did not respond to a decrease in pH, and its amiloride sensitivity and selectivity for Na(+) over K(+) were unaffected by this pH change. Buffering the bathing solutions with EGTA to reduce the free [Ca(2+)] to <10 nm increased WT single channel open probability 10-fold, but not that of GF BNaC-2. Ca(2+) blocked both WT and GF BNaC-2 in a dose- and voltage-dependent fashion; single channel conductances were unchanged. A drop in pH reduced the ability of Ca(2+) to inhibit these channels. These results show that BNaC-2 is an amiloride-sensitive sodium channel and suggest that pH activation of these channels could be, in part, a consequence of H(+) "interference" with channel regulation by Ca(2+).     

The role of voltage gated T-type Ca2+ channel isoforms in mediating "capacitative" Ca2+ entry in cancer cells

The mechanism by which Ca2+ enters electrically non-excitable cells is unclear. The sensitivity of the Ca2+ entry pathway in electrically non-excitable cells to inhibition by extracellular Ni2+ was used to direct the synthesis of a library of simple, novel compounds. These novel compounds inhibit Ca2+ entry into and, consequently, proliferation of several cancer cell lines. They showed stereoselective inhibition of proliferation and Ca2+ influx with identical stereoselective inhibition of heterologously expressed Cav3.2 isoform of T-type Ca2+ channels. Proliferation of human embryonic kidney (HEK)293 cells transfected with the Cav3.2 Ca2+ channel was also blocked. Cancer cell lines sensitive to our compounds express message for the Cav3.2 T-type Ca2+ channel isoform, its delta25B splice variant, or both, while a cell line resistant to our compounds does not. These observations raise the possibility that clinically useful drugs can be designed based upon the ability to block these Ca2+ channels.     

Predominance of NK1.1+TCR alpha beta+ or DX5+TCR alpha beta+ T cells in mice conditioned with fractionated lymphoid irradiation protects against graft-versus-host disease: "natural suppressor" cells

We developed a nonmyeloablative host conditioning regimen in a mouse model of MHC-mismatched bone marrow transplantation that not only reduces radiation toxicity, but also protects against graft-vs-host disease. The regimen of fractionated irradiation directed to the lymphoid tissues and depletive anti-T cell Abs results in a marked change in the residual host T cells, such that NK1.1+ or DX5+asialo-GM1+ T cells become the predominant T cell subset in the lymphoid tissues of C57BL/6 and BALB/c mice, respectively. The latter "natural suppressor" T cells protect hosts from graft-vs-host disease after the infusion of allogeneic bone marrow and peripheral blood cells that ordinarily kill hosts conditioned with sublethal or lethal total body irradiation. Protected hosts become stable mixed chimeras, but fail to show the early expansion and infiltration of donor T cells in the gut, liver, and blood associated with host tissue injury. Cytokine secretion and adoptive transfer studies using wild-type and IL-4(-/-) mice showed that protection afforded by NK1.1+ and DX5+asialo-GM1+ T cells derived from either donors or hosts conditioned with lymphoid irradiation is dependent on their secretion of high levels of IL-4.     

Topology and function of "stalk" proteins in the bovine mitochondrial H+-ATPase

Proton translocating ATPases comprise a hydrophilic sector F1, a membrane sector F0, and, in the case of bovine mitochondria, a connecting "stalk" which is believed to contain the oligomycin sensitivity-conferring protein (OSCP) and coupling factor 6 (F6). The present study was undertaken to verify the accessibility of F6 and OSCP to trypsin and to examine the functional consequences of such treatment. Our data show that F1 binds equally to trypsin-treated F0 and untreated F0, but the former complexes exhibit cold lability and only partial sensitivity to oligomycin. Furthermore, these complexes fail to exhibit ATP-driven proton translocation or ATP-32Pi exchange activity. Trypsinization of F0 does not, however, inhibit passive proton conductance through the membrane sector but actually enhances it. Immunological data indicate extensive degradation of OSCP under conditions where F6 proteolysis is insignificant. Intact H+-ATPase complexes are relatively resistant to both the structural and functional effects of trypsin. We conclude that OSCP is predominantly an extrinsic protein which is shielded by F1 in the native membrane. F6 may also be an extrinsic protein but is shielded from trypsinization by OSCP and/or other F0 polypeptides. The exposed, trypsin-sensitive segments of OSCP are not required for passive proton conductance through F0 but may be required for ATP-driven reactions. We propose that bovine mitochondrial OSCP is a functional analogue of subunit b in the Escherichia coli H+-ATPase.     

基于临床路径的“3+3”信息系统初探

医院信息化管理已成为当代医院管理的重要手段,信息化平台也成为临床运作的重要工具。临床路径信息化管理是医院发展的主要趋势。上海交通大学附属第六人民医院作为卫生部试行临床路径管理的试点医院,在临床路径实施过程中,创新管理运作模式,提出了基于临床路径的“3+3”信息系统。介绍了该系统的架构、设计理念及其应用成效。该系统是临床路径实施信息化管理的实践应用。实践证明,使用信息化手段推动临床路径的试点实施,达到了强化医院精细化管理的目的。     

Binding of water to "types I and II" Cu2+ in proteins

Water proton spin-lattice relaxation times have been measured at 30MHz between 280 – 333 K in aqueous solutions of proteins containing Type I Cu²⁺ ions (azurin and umecyanin) and Type II Cu²⁺ ions (benzylamine oxidase and superoxide dismutase). These measurements show that Type II Cu²⁺ is accessible to exchangeable water molecules but Type I is not. This behaviour is consistent with the EPR and optical properties of these ions and their likely biochemical functions.     

Australian Lasioglossum + Homalictus form a monophyletic group: resolving the "Australian enigma"

The bee genus Lasioglossum includes > 1,000 species of bees distributed on all continents except Antarctica. Lasioglossum is a major component of the bee fauna in the Holarctic, Ethiopian, and Asian regions and is an important group for investigating the evolution of social behavior in bees. Given its cosmopolitan distribution, the historical biogeography of the genus is of considerable interest. We reconstructed phylogenetic relationships among the subgenera and species within Lasioglossum s.s., using DNA sequence data from a slowly evolving nuclear gene, elongation factor-1 alpha. The entire data set includes > 1,604 aligned nucleotide sites (including three exons plus two introns) for 89 species (17 outgroups plus 72 ingroups). Parsimony and maximum likelihood analyses provide strong evidence that the primarily Indoaustralian subgenera (Homalictus, Chilalictus, Parasphecodes) form a monophyletic group. Bootstrap support for the Australian clade ranged from 73% to 77%, depending on the method of analysis. Monophyly of the Australian Lasioglossum suggests that a single colonization event (by way of Southeast Asia and New Guinea) gave rise to a lineage of > 350 native Indoaustralian bees. We discuss the implications of Australian monophyly for resolving the "Australian enigma"--the similarity in social behavior among the Australian halictine bees relative to that of Holarctic groups.     

Frequency-dependent excitability of "membrane" slow responses of Rabbit left atrial trabeculae in the presence of Ba2+ and high K+

Small trabeculae of rabbit left atrium immersed in TKBa solution (Tyrode with 10 mM K+ and 1 mM Ba2+) were used to study frequency dependence of "membrane" slow response excitability at long cycle lengths (greater than 1 s). In TKBa, stimuli generate graded, low- amplitude (2-15 mV) subliminal responses of variable long duration (up to 450 ms). A full all-or-none slow response is generated when a subliminal response depolarizes the membrane to about--35 mV. Subliminal response amplitude and rate of rise augment with stimulus intensity-duration product. For a fixed stimulus, the subliminal response is larger and faster at higher frequencies. Sudden changes in stimulus frequency or time course induce changes in subliminal response tha take four to eight cycles to attain steady state. For a fixed stimulus, slow response latency shortens progressively during the first few cycles after a sudden increase in frequency or when a rested preparation is excited (latency adaptation phenomenon, LAP). Slow response threshold stimulus requirements decrease during LAP (excitability hysteresis). The degree of excitability hysteresis is dependent on stimulation frequency and is more pronounced at higher frequencies. Frequency sensitivity of subliminal response (which causes frequency sensitivity of slow response excitability) is explained in terms of a transient state of enhancement set up by each stimulus. The enhanced state decays between stimuli with a half-time of approximately 4 s, thus allowing cumulative effects to become evident at rates above 0.1 Hz.     

Mechanism of action of "ruthenium red" compounds on Ca2+ ionophore from sarcoplasmic reticulum (Ca2+ + Mg2+)- adenosine triphosphatase and lipid bilayer.

Sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase was previously shown to have Ca2+-dependent and -selective ionophoric activity when tested in oxidized cholesterol lipid bilayer membranes (Shamoo, A. E., and MacLennan, D. H. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 3522). ruthenium red, a known inhibitor of (Ca2+ + Mg2+)-ATPase, is found to inhibit the Ca2+-ionophoric activity associated with (Ca2+ + Mg2+)-ATPase. Furthermore, ruthenium red alone acts as an anion-selective ionophore in lipid bilayers with the the following selectivity sequence for anions: l- greater than Cl-, Br- greater than F- greater than NO3-. The PCl-/PNa+ ratio was approximately 4/l. The presence of ruthenium red in excess of Ca2+ ionophore in lipid bilayer experiments converts the cation selectivity of the bilayer due to Ca2+ ionophore into anion selectivity.     

Identification and characterization of "basal" Ca2+-independent Mg2+-dependent ATP-hydrolase enzymatic activity in smooth muscle cell plasma membrane fractions

It is shown, that for correct definition of "basal" Ca(2+)-independent Mg(2+)-dependent ATPase ac-activity (10-13 mmol Pi/hour on 1 mg of protein) in a fraction of uterus smooth muscle cell plasma membranes is necessary to use in medium without calcium of an incubation not only EGTA and digitonin--of the factor of infringement in activity by this subcellular structure, but inhibitors of others Mg(2+)-dependent ATP-hydrolyse enzymatic systems localized as in plasma membrane (Na+, K(+)-ATPase) and in others subcellular frames, first of all, in mitochondria (Mg(2+)-ATPase) and endoplasmic reticulum (transport Ca2+, Mg(2+)-ATPase). In the case of a sacolemal fraction of a smooth muscle the contribution of others Mg(2+)-dependent ATP-hydrolyse systems in a common enzymatic hydrolysis ATP, which unconnected to functioning "basal" Ca(2+)-independent Mg(2+)-dependent ATPase, is very appreciable and achieves 35%. The researches, carried out in the frameworks of definition of initial velocity of enzymatic reaction, have enabled to define its some properties--cationic and anionic specificity, and also sensitivity to action of some inhibitors. It has appeared, that the "basal" Ca(2+)-independent Mg(2+)-dependent ATP-hydrolyse reaction is nonspecific rather both in relation to cations of divalent metals Me2+, and cations of monovalent metals and anions, which were utilized for support of ionic strength. The cations La--antagonist of cations Ca--practically did not influence enzymatic activity. The non-specific inhibitors transport of ATPases--p-chloromercuribenzoate, o-vanadate and eosine Y with a various degree of efficiency inhibited "basal" Ca(2+)-independent Mg(2+)-dependent ATP-hydrolyse reaction. On the basis of the analysis of the own and literary data the conclusion is made that "basal" Ca(2+)-independent Mg(2+)-dependent ATPase of a smooth muscle cell plasma membrane is considerably less sensitive to action of nonspecific inhibitors of the Ca(2+)-transporting systems, than these systems.     

Mn2+ does not uncouple adenosine "Ra" receptors from the liver adenylate cyclase

Four analogs of oxymorphone, oxymorphaminoethylthiol, oxymorphamino-ethyldisulfide, oxymorphaminoethyl-nitrobenzoic acid disulfide and oxymorphone thiazolidine, as well as the enkephalin analogs, enkephalin-thiol, Tyr-D-Ala-Gly-Phe-Leu-Lys(?-NH)COCH₂CH₂SH and the enkephalin-dimer, [Tyr-D-Ala-Gly-Phe-Leu-Lys(?-NH)COCH₂CH₂S-]₂, were examined for binding to enkephalin and morphine receptors. The analogs gained substantial affinity for enkephalin and lost affinity for morphine receptors. The affinity of the dimers of both opiates and enkephalins was slightly greater than that achieved by the corresponding thiol monomers. However, in the guinea pig ileum the dimeric analogs were much more active than the monomers. Receptor dimerization or cross-linking may be involved in the biological activity of opiates and opioid peptides.