Differential distribution of alpha subunits and beta gamma subunits of heterotrimeric G proteins on Golgi membranes of the exocrine pancreas

Heterotrimeric G proteins are well known to be involved in signaling via plasma membrane (PM) receptors. Recent data indicate that heterotrimeric G proteins are also present on intracellular membranes and may regulate vesicular transport along the exocytic pathway. We have used subcellular fractionation and immunocytochemical localization to investigate the distribution of G alpha and G beta gamma subunits in the rat exocrine pancreas which is highly specialized for protein secretion. We show that G alpha s, G alpha i3 and G alpha q/11 are present in Golgi fractions which are > 95% devoid of PM. Removal of residual PM by absorption on wheat germ agglutinin (WGA) did not deplete G alpha subunits. G alpha s was largely restricted to TGN- enriched fractions by immunoblotting, whereas G alpha i3 and G alpha q/11 were broadly distributed across Golgi fractions. G alpha s did not colocalize with TGN38 or caveolin, suggesting that G alpha s is associated with a distinct population of membranes. G beta subunits were barely detectable in purified Golgi fractions. By immunofluorescence and immunogold labeling, G beta subunits were detected on PM but not on Golgi membranes, whereas G alpha s and G alpha i3 were readily detected on both Golgi and PM. G alpha and G beta subunits were not found on membranes of zymogen granules. These data indicate that G alpha s, G alpha q/11, and G alpha i3 associate with Golgi membranes independent of G beta subunits and have distinctive distributions within the Golgi stack. G beta subunits are thought to lock G alpha in the GDP-bound form, prevent it from activating its effector, and assist in anchoring it to the PM. Therefore the presence of free G alpha subunits on Golgi membranes has several important functional implications: it suggests that G alpha subunits associated with Golgi membranes are in the active, GTP-bound form or are bound to some other unidentified protein(s) which can substitute for G beta gamma subunits. It further implies that G alpha subunits are tethered to Golgi membranes by posttranslational modifications (e.g., palmitoylation) or by binding to another protein(s).     

兰科盆距兰属（Gastrochilus）植物的修订

本文对盆距兰属（Ｇａｓｔｒｏｃｈｉｌｕｓ）植物作了修订,共分３个组,含４６种和１变种,其中１个组（Ｓｅｃｔ．Ｃａｅｓｐｉｔｏｓｉ）和８个种（Ｇ．ｃａｒｎｓｕｓ,Ｇ．ｇａｒｈｗａｌｅｎｓｉｓ,Ｇ．ｌｉｎｅａｒｉｆｏｌｉｕｓ,Ｇ．ｇｕａｎｇｔｕｎｇｅｎｓｉｓ,Ｇ．ｓｕｂｐａｐｉｌｌｏｓｕｓ,Ｇ．ｎａｎｃｈｕａｎｅｎｓｉｓ,Ｇ．ｓａｃｃａｔｕｓａｎｄＧ．ｇｏｎｇｓｈａｎｅｎｓｉｓ）为新的,首次在本文作了描述报导。本属属的形态特征,研究历史和订正后属下的分类群检索表,种的文献引证、简短的特征记要和地理分布以及在属中被排除的分类单位索引均提供在本文。     

Missense mutations in ABCG5 and ABCG8 disrupt heterodimerization and trafficking

Mutations in ABCG5 (G5) or ABCG8 (G8) cause sitosterolemia, an autosomal recessive disease characterized by sterol accumulation and premature atherosclerosis. G5 and G8 are ATP-binding cassette (ABC) half-transporters that must heterodimerize to move to the apical surface of cells. We examined the role of N-linked glycans in the formation of the G5/G8 heterodimer to gain insight into the determinants of folding and trafficking of these proteins. Site-directed mutagenesis revealed that two asparagine residues (Asn(585) and Asn(592)) are glycosylated in G5 and that G8 has a single N-linked glycan attached to Asn(619). N-Linked glycosylation of G8 was required for efficient trafficking of the G5/G8 heterodimer, but mutations that abolished glycosylation of G5 did not prevent trafficking of the heterodimer. Both G5 and G8 are bound by the lectin chaperone, calnexin, suggesting that the calnexin cycle may facilitate folding of the G5/G8 heterodimer. To determine the effects of 13 disease-causing missense mutations in G5 and G8 on formation and trafficking of the G5/G8 heterodimer, mutant forms of the half-transporters were expressed in CHO-K1 cells. All 13 mutations reduced trafficking of the G5/G8 heterodimer from the endoplasmic reticulum to the Golgi complex, and most prevented the formation of stable heterodimers between G5 and G8. We conclude that the majority of the molecular defects in G5 and G8 that cause sitosterolemia impair transport of the sterol transporter to the cell surface.     

NMR structures of r(GCAGGCGUGC)2 and determinants of stability for single guanosine-guanosine base pairs

Nucleotides in RNA that are not Watson-Crick-paired form unique structures for recognition or catalysis, but determinants of these structures and their stabilities are poorly understood. A single noncanonical pair of two guanosines (G) is more stable than other noncanonical pairs and can potentially form pairing structures with two hydrogen bonds in four different ways. Here, the energetics and structure of single GG pairs are investigated in several sequence contexts by optical melting and NMR. The data for r(5'GCAGGCGUGC3')(2), in which G4 and G7 are paired, are consistent with a model in which G4 and G7 alternate syn glycosidic conformations in a two-hydrogen-bond pair. The two distinct structures are derived from nuclear Overhauser effect spectroscopic distance restraints coupled with simulated annealing using the AMBER 95 force field. In each structure, the imino and amino protons of the anti G are hydrogen bonded to the O6 and N7 acceptors of the syn G, respectively. An additional hydrogen-bond connects the syn G amino group to the 5' nonbridging pro-R(p) phosphate oxygen. The GG pair fits well into a Watson-Crick helix. In r(5'GCAGGCGUGC3')(2), the G4(anti), G7(syn) structure is preferred over G4(syn), G7(anti). For single GG pairs in other contexts, exchange processes make interpretation of spectra more difficult but the pairs are also G(syn), G(anti). Thermodynamic data for a variety of duplexes containing pairs of G, inosine, and 7-deazaguanosine flanked by GC pairs are consistent with the structural and energetic interpretations for r(5'GCAGGCGUGC3')(2), suggesting similar GG conformations.     

Type I glycogen storage diseases: disorders of the glucose-6-phosphatase complex

Glycogen storage disease type I (GSD-I) is a group of autosomal recessive disorders with an incidence of 1 in 100,000. The two major subtypes are GSD-Ia (MIM232200), caused by a deficiency of glucose-6-phosphatase (G6Pase), and GSD-Ib (MIM232220), caused by a deficiency in the glucose-6-phosphate transporter (G6PT). Both G6Pase and G6PT are associated with the endoplasmic reticulum (ER) membrane. G6PT translocates glucose-6-phosphate (G6P) from the cytoplasm into the lumen of the ER, where G6Pase hydrolyses the G6P into glucose and phosphate. Together G6Pase and G6PT maintain glucose homeostasis. G6Pase is expressed in gluconeogenic tissues, the liver, kidney, and intestine. However G6PT, which transports G6P efficiently only in the presence of G6Pase, is expressed ubiquitously. This suggests that G6PT may play other roles in tissues lacking G6Pase. Both GSD-Ia and GSD-Ib patients manifest phenotypic G6Pase deficiency, characterized by growth retardation, hypoglycemia, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, and lactic academia and the current treatment is a dietary therapy. GSD-Ib patients also suffer from chronic neutropenia and functional deficiencies of neutrophils and monocytes, which is treated with granulocyte colony stimulating factor to restore myeloid function. The GSD-Ia and GSD-Ib genes have been cloned. To date, 76 G6Pase and 69 G6PT mutations have been identified in GSD-I patients. A database of the residual enzymatic activity retained by the G6Pase missense mutants is facilitating the correlation of the disease phenotype with the patients' genotype. While the molecular basis for the GSD-I disorders are now known and symptomatic therapies are available, many aspects of the diseases are still poorly understood, and there are no cures. Recently developed animal models of the disorders are now being exploited to delineate the disease more precisely and develop new, more causative therapies.     

植物激素作用中的G蛋白调节

Guanine nucleotide-binding proteins known as G proteins or GTPases are universal molecular switches that play a pivotal role in signal transduction. Signal transducing GTPases include heterotrimeric G proteins composed of Gα, Gβ and Gγ and monomeric small GTPases. Small GTPases are related to the α subunit of heterotrimeric G proteins but differ from heterotrimeric G proteins in the mechanisms by which they are regulated by upstream factors as well as those by which they activate downstream targets (Yang,2002).     

云南扁担杆属植物评注

云南扁担杆属植物计有14种3变种,本文对其中9种1变种作了分类学评注,并确认中国新记录2种,云南新记录1种,新组合1变种及新异名6个。     

G(o) controls the hyperpolarization-activated current in embryonic stem cell-derived cardiocytes

Hyperpolarization current (I(f)) is an important player in controlling heart rate and is stimulated by cAMP and inhibited by members of the pertussis toxin-sensitive G-protein G(i)/G(o) family. We have successfully derived cardiocytes from embryonic stem cells lacking G(o) or G(i2) and G(i3). We have established that both basal and isoproterenol-stimulated activities of I(f) in these cardiocytes have typical nodal-atrial characteristics and are unaffected by targeted gene inactivation of the G proteins G(o) or G(i2) and G(i3). Under basal conditions, both G(o) and G(i) are required for muscarinic inhibition of I(f) activity via a mechanism that involves the generation of nitric oxide, whereas, with prior stimulation by beta-agonists, only G(o) is required and G(i) and nitric oxide production are not. Our findings establish an essential role for G(o) in the antiadrenergic effect of muscarinic agent on I(f).     

野生植物根围的丛枝菌根真菌Ⅱ

本文主要报道了野生植物根围Glomus属的17个种,聚球囊霉G.aggregatumSchenck＆Smith,苏格兰球囊霉G.caledonium（Nicol.＆Gerd.）Trappe＆Gerd,近明球囊霉G．claroideumSchenck＆Smith,明球囊霉G．clarumNicolson＆Schenck,缩球囊霉G．constrictumTrappe,透光球囊霉G．diaphanumMorton,幼套球囊霉G．etunicatumBecker＆Gerdemann,集球囊霉G．fasciculatum（Thaxter）Gerd．＆Trappe,何氏球囊霉G．hoiBerch＆Trappe,地球囊霉G.geosporum（Nicol．＆Derd.）Warker,根内球囊霉G．intraradicesSchenck＆Smith,摩西球囊霉G．mosseae（Nicol．＆Gerd.）Gerd.＆Trappe,隐球囊霉G.occultumWalker,网状球囊霉G.reticulatumBhattcharjee＆Mukerji,地表球囊霉G.versiforme（Karsten）Berch,台湾球囊霉G．formosanumWu＆Chen,悬钩子球囊霉G．rubiformeGerdemann＆Trappe）Almeida＆Schenck;内养囊霉属1个种,稀有内养囊霉Entrophosporainfrequens（Hall）Ames＆Schenider。其中,网状球囊霉为我国新记录种。     

Multigene analysis suggests ecological speciation in the fungal pathogen Claviceps purpurea

Claviceps purpurea is an important pathogen of grasses and source of novel chemical compounds. Three groups within this species (G1, G2 and G3) have been recognized based on habitat association, sclerotia and conidia morphology, as well as alkaloid production. These groups have further been supported by Random Amplification of Polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers, suggesting this species may be more accurately described as a species complex. However, all divergent ecotypes can coexist in sympatric populations with no obvious physical barriers to prevent gene flow. In this study, we used both phylogenetic and population genetic analyses to test for speciation within C. purpurea using DNA sequences from ITS, a RAS-like locus, and a portion of beta-tubulin. The G1 types are significantly divergent from the G2/G3 types based on each of the three loci and the combined dataset, whereas the G2/G3 types are more integrated with one another. Although the G2 and G3 lineages have not diverged as much as the G1 lineage based on DNA sequence data, the use of three DNA loci does reliably separate the G2 and G3 lineages. However, the population genetic analyses strongly suggest little to no gene flow occurring between the different ecotypes, and we argue that this process is driven by adaptations to ecological habitats; G1 isolates are associated with terrestrial grasses, G2 isolates are found in wet and shady environments, and G3 isolates are found in salt marsh habitats.     

云南扁担杆属植物评注

云南扁担杆属植物计有14种3变种,本文对其中9种1变种作了分类学评注,并确认中国新记录2种．云南新记录1种,新组合1变种及新异名6个。     

中国太宇谷蛾属分类研究(鳞翅目,谷蛾科)(英文)

记述中国太宇谷蛾属Gerontha Walker10种,其中有4新种:拟华太宇谷蛾G.similihoenei sp.nov.,喙太宇谷蛾G.rostriformis sp.nov.,梯缘太宇谷蛾G.trapezia sp.nov.,褶太宇谷蛾G.rugulosa sp.nov.和3新纪录种:暹罗太宇谷蛾G.siamensis Moriuti,1989(图7,17～18)、清迈太宇谷蛾G.nawapuriensis Moriuti,1989(图8,19)、钻太宇谷蛾G.borea Moriuti,1977(图9,20).首次发现和报道了弯茎太宇谷蛾Gerontha flexura Huang et al .(图6,15-16)和暹罗太宇谷蛾G.siamensis Moriuti的雌性个体.文中给出了所有中国种的检索表.研究标本保存于南开大学生命科学学院昆虫标本室.     

Pollen and seed morphology of Gueldenstaedtia and Tibetia (Leguminosae) - with a special reference to the taxonomic significance

The pollen morphology of Gueldenstaedtia gansuensis. G. gracilis, G. henryi, G. monophylla. G. mutijlora, G. stenophylla. and G. verna and Tibetia liangshanensis, T. tongolensis, T. yadongensis. T. coelestis, and T yunnanensis are reported for the first time. The seed morphology of G. gracilis, G. maritima. G. monophylla, G. mutiflora, G. taihangensis, and G. verna and L coelestis, T. himalaica, T. yunnanensis, and T. yadongensis are firstly described here. In pollen morphology, the differences of pollen grains of Gueldenstaedtia and Tibetia are as follows: Gueldenstaedtia with pollen grains 3–colporate, psilate, and shapes spheroidal, sometimes subprolate, prolate or oblong; and Tibetia with pollen grains 3– and 4–colporate, perforate, shapes spheroidal, sometimes subprolate or prolate. These results, combined with the data of the basic chromosome number x=7 of Gueldenstaedtia and x=8 of Tibetia, support that the two genera should be recognized as two distinct genera, which are consistent with their morphological characters: Gueldenstaedtia with 2 upper lobes of calyx free, stipules free, adnate to petiole, and Tibetia with 2 upper lobes of calyx connate, stipules connate and opposite to leaves. In Tibetia, two types of pollen grains, 3– and 4–colporate pollen grains, are found. Regarding seed morphology: Gueldenstaedtia has circular depression, irregular circular depression or irregular circular reticulation on the surface; Tibetia has smooth surface. The differences in seed morphology of the two genera also support that they should be kept separate. The pollen morphology supports that G. gansuensis, G. gracilis, G. multiflora, G. stenophylla, and G. verna should be reduced into one species consistent with their morphological characteristics. The pollen grains of G. henryi are different from those of the other species in having wide colpi.     

Alteration of cell cycle kinetics by reducing agents in human peripheral blood lymphocytes from adult and senescent donors

For improving cell proliferation reducing agents are routinely used as medium supplements in murine cell cultures, however, they are rarely used for human peripheral blood lymphocytes (PBLs). Data on changes in cell kinetics induced by reducing agents are not available. Here cell kinetic alterations induced by reducing agents in human lymphocytes are revealed by applying flow cytometric BrdUrd/Hoechst cell cycle analysis and by using the exit kinetic model of Smith and Martin. Applying alpha-thioglycerol (a-TG) as a model compound it was shown that the major cell kinetic effect is a shortening of the mean duration of the G0/G1 phase. The minimum G0/G1 phase duration and the percentage of the non-cycling G0/G1 cell fraction decrease only slightly. Moreover, a lower number of PBL's are arrested in the G2/M phase of the 1st cell cycle. The durations of the S and G2/M phase in the 1st and G1 phase in the 2nd cycle are not affected. These cell kinetic effects are identical for lymphocytes from both adult and senescent donors. The supplementation of the cell cultures with recombinant IL-2 did not induce similar cell kinetic alterations compared with a-TG. This indicates that the variation of the cell cycle progression factor IL-2 is not solely responsible for improvement of the cell activation process in the G0/G1 phase.     

An analysis of the expression of the mutant angora-Y gene in the mouse

We studied the rate and duration of the growth of G1 and G3 hairs in mice homozygous for angora-Y mutant gene (goY). The follicular diameter of G3 hairs and the growth rate of G1 and G3 hairs in goY/goY mice do not differ from normal. However, the duration of growth period of all four studied types of hairs in goY/goY mice is longer than in the normal phenotype. Growth of the guard hairs G1 and G3 in mutants continues longer than in the normal phenotype by 7 and 3 days, respectively. For other hair types G1 and G3 (awl, auchene, zigzag) the duration of the growth period is approximately 3 days longer than in the control. As a result in goY/goY mice guard hairs G1 and G3 which have completed growth are 2 and 1.5 times longer than in +/+ mice. Other types of G1 hairs in mutants are longer by 50% and G3 hairs by 30% than in the wild type.     

Studies in Garcinia, dioecious tropical forest trees: agamospermy

RICHARDS, A. J., 1990. Studies in Garcinia , dioecious tropical forest trees: agamospermy . In Garcinia , agamospermy is known or suspected for ten species. Most are facultative agamosperms, with males occurring, but males are probably lacking in G. mangostana . In G. mangostana and G. hombroniana , adventitious embryony occurs autonomously, and haploid parthenogenesis may also occur rarely. In G. parvifolia , it is reported previously that gametophytic agamospermy occurs, and adventitious embryos are suppressed. Autonomous endospermy is found in G. mangostana and G. parvifolia . In G. hombroniana , endosperm probably only develops after PEN fertilization. In G. hombroniana , some proembryos are formed precociously, and further proembryo formation is inhibited by sexual embryos. Asexual proembryos tend to occur in large ovules in small ovaries, and sexual embryos tend to occur in small ovules in large ovaries. It is considered that facultative agamospermy renders the genus Garcinia particularly suitable for the development of new types of fruit. Agamospermy in a dioecious genus, adventitious embryony in the absence of fertilization and/or pseudogamy, and the co-occurrence of gametophytic and sporophytic agamospermies in the same genus are unusual phenomena, which are discussed in the context of the lifestyle of a tropical forest tree. For G. hombroniana, n = 24, and for G. cowa, n = 26 are reported.     

The importance of N-terminal polycysteine and polybasic sequences for G14alpha and G16alpha palmitoylation, plasma membrane localization, and signaling function

Plasma membrane targeting of G protein alpha (Galpha) subunits is essential for competent receptor-to-G protein signaling. Many Galpha are tethered to the plasma membrane by covalent lipid modifications at their N terminus. Additionally, it is hypothesized that Gq family members (Gqalpha,G11alpha,G14alpha, and G16alpha) in particular utilize a polybasic sequence of amino acids in their N terminus to promote membrane attachment and protein palmitoylation. However, this hypothesis has not been tested, and nothing is known about other mechanisms that control subcellular localization and signaling properties of G14alpha and G16alpha. Here we report critical biochemical factors that mediate membrane attachment and signaling function of G14alpha and G16alpha. We find that G14alpha and G16alpha are palmitoylated at distinct polycysteine sequences in their N termini and that the polycysteine sequence along with the adjacent polybasic region are both important for G16alpha-mediated signaling at the plasma membrane. Surprisingly, the isolated N termini of G14alpha and G16alpha expressed as peptides fused to enhanced green fluorescent protein each exhibit differential requirements for palmitoylation and membrane targeting; individual cysteine residues, but not the polybasic regions, determine lipid modification and subcellular localization. However, full-length G16alpha, more so than G14alpha, displays a functional dependence on single cysteines for membrane localization and activity, and its full signaling potential depends on the integrity of the polybasic sequence. Together, these findings indicate that G14alpha and G16alpha are palmitoylated at distinct polycysteine sequences, and that the adjacent polybasic domain is not required for Galpha palmitoylation but is important for localization and functional activity of heterotrimeric G proteins.