贵州苗族新生儿HbF中Gγ/Aγ比值测定与AγT基因频率分析

用含Triton X-100的酸性尿素聚丙烯酰胺凝胶电泳(PAGE)法测定了贵州省108例苗族新生儿脐带血胎儿血红蛋白(HbF)中Gγ/Aγ值.显示107例(占99.07%)新生儿的Gγ值在55-78%之间,均值与标准差为70.42±3.45;高Gγ值者1例(占0.925%);低Gγ值者未发现。用Large-pore Vydac C4柱反相高效液相层析法对108例样本进行了AγT基因频率（fAγT）分析,fAγT为0.0648。 The ratio of Gγ to Aγ of fetal hemoglobin inn 108 cord blood samples of Miao nationality newborns in Guizhou province was determined by the polyacrylamide gel electrophoresis(PAGE).The result showed that one case had high Gγ value(0.925%) and 107 babies the normal Gγ values of 55 to 78%(70.42+3.45)。The AγT gene frequency(fAγT) analysis in 108 samples was done by high pressure liquid chromatography using a large pore Vydac C4 column,the fAγT was 0.0648.     

满族新生儿血红蛋白F中Gγ／Aγ比值测定及其异常者γ—珠蛋白基…

以聚丙烯酰胺凝胶电泳方法测定了３３１例辽宁满族新生儿脐带血血红蛋白Ｆ中Ｇγ／Ａγ比值。结果显示：高Ｇγ（＞８０％）者４例,占１．２１％,其基因频率（ｆ）为０．００６０４,低Ｇγ（３０－４８％）者６例,占１．８１％,其基因频率为０．００９０６,其余正常,Ｇγ平均值为６５．２３±６．３８％。未发现ＡγＴ链。对其中八例异常者（４例高Ｇγ值,４例低Ｇγ值）的染色体ＤＮＡ进行了基因图谱分析,确定４例高Ｇγ值     

Mycobacterium tuberculosis-Specific IL-21+IFN-γ+CD4+ T Cells Are Regulated by IL-12

In the current study of Mycobacterium tuberculosis (MTB)-specific T and B cells, we found that MTB-specific peptides from early secreted antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) induced the expression of IL-21 predominantly in CD4⁺ T cells. A fraction of IL-21-expressing CD4⁺ T cells simultaneously expressed Th1 cytokines but did not secrete Th2 or Th17 cytokines, suggesting that MTB-specific IL-21-expressing CD4⁺ T cells were different from Th1, Th2 and Th17 subpopulations. The majority of MTB-specific IL-21-expressing CD4⁺ T cells co-expressed IFN-γ and IL-21⁺IFN-γ⁺CD4⁺ T cells exhibited obviously polyfunctionality. In addition, MTB-specific IL-21-expressing CD4⁺ T cells displayed a CD45RO⁺CD62L^lowCCR7^lowCD40L^highICOS^high phenotype. Bcl-6-expression was significantly higher in IL-21-expressing CD4⁺ T cells than IL-21^-CD4⁺ T cells. Moreover, IL-12 could up-regulate MTB-specific IL-21 expression, especially the frequency of IL-21⁺IFN-γ⁺CD4⁺ T cells. Taken together, our results demonstrated that MTB-specific IL-21⁺IFN-γ⁺CD4⁺ T cells from local sites of tuberculosis (TB) infection could be enhanced by IL-12, which have the features of both Tfh and Th1 cells and may have an important role in local immune responses against TB infection.     

Gαq,Gγ1 and Plc21C Control Drosophila Body Fat Storage

mobilization of body fat is essential for energy homeostasis in animals. In insects, the adipokinetic hormone （Akh） systemically controls body fat mobilization. Biochemical evidence supports that Akh signals via a G protein-coupled receptor （GPCR） called Akh receptor （AkhR） using cyclic-AMP （cAMP） and Ca2＋ second messengers to induce storage lipid release from fat body cells. Recently, we provided genetic evidence that the intracellular calcium （iCa2＋） level in fat storage cells controls adiposity in the fruit fly Drosophila melanogaster. However, little is known about the genes, which mediate Akh signalling downstream of the AkhR to regulate changes in iCa2＋. Here, we used thermogenetics to provide in vivo evidence that the GPCR signal transducers G protein α q subunit （Gαq）, G protein γ1 （Gγ1） and Phospholipase C at 21C （Plc21C） control cellular and organismal fat storage in Drosophila. Transgenic modulation of Gαq, Gγ1 and Plc21C affected the iCa2＋ of fat body cells and the expression profile of the lipid metabolism effector genes midway and brummer, which results in severely obese or lean flies. Moreover, functional impairment of Gαq, Gγ1 and Plc21C antagonised Akh-induced fat depletion. This study characterizes Gαq, Gγ1 and Plc21C as anti-obesity genes and supports the model that Akh employs the Gαq/Gγ1/Plc21C module of iCa2＋ control to regulate lipid mobilization in adult Drosophila.     

新疆蒙古族、锡伯族新生儿HbF中Gγ/Aγ、AγI/AγT比值测定及基因图谱分析

用HPLC法测定新疆蒙古族78例、锡伯族60例新生儿胎儿血红蛋白(HbF)中Gγ/Aγ以及AγI/AγT比值。%Gγ均值为:蒙古族73.99% ,锡伯族74.59%。%AγI均值分别为蒙古族56.04%和锡伯族64.33%。在蒙古族中发现AγT纯合体4例、杂合体12例;锡伯族中发现AγT杂合体6例。蒙古族中AγT基因频率(fAγT)为0.128,锡伯族为0.05。 Abstract: Gγ/Aγ、AγI/Aγ Ratios of fetal hemoglobin(HbF) in 138 cases of newborns of Megnol (n=78)and Sibo(n=60)ethnic groups in Urumuqi,Xinjiang were determined by HPLC.The means of % Gγ of Mongol and sibo ethnic groups were 73.99% and 7459% respectively.12 Aγ/T cases of heterozygotes in Mongol,6 in Sibo and 4 cases of AγT homozygotes in Mongol were found.The frequencies of AγT gene were 0.128 in Mongol and 0.05 in Sibo respectively.The means of AγT of Mongol and Sibo ethnic groups were 56.04% and 64.33% respectively.     

反义磷脂酶Dγ(Anti-PLDγ)基因转化美洲黑杨G2的研究

通过农杆菌介导法将反义磷脂酶DT(Anti-PLDγ)基因转入美洲黑杨G2中以提高其耐盐性。本研究建立了美洲黑杨G2组培再生体系,确立了美洲黑杨G2叶片分化最适宜培养基和生根培养基,进行了卡那霉素(选择性抗生素)敏感性试验,改良了传统的农杆菌介导转化法。经诱导不定芽及生根阶段卡那霉素(选择性抗生素)连续筛选,获得了21株卡那霉素抗性植株,抗性植株经PCR及PCR-Southern杂交检测,有13株均呈阳性,证明Anti-PLDγ基因成功整合到美洲黑杨G2基因组中。耐盐性实验表明,4株转基因植株抗NaCl能力比对照有不同程度提高。     

Enzymatic relationship between human Tγ+ lymphocytes and thymocytes

The lactate dehydrogenase isoenzyme pattern has been determined in human thymocytes and in peripheral blood T lymphocytes, Tγ⁺, and Tμ⁺ lymphocytes. Thymocytes have a highly significant lower activity in LDH-1 and LDH-2 together with a highly significant higher activity in LDH-3 and LDH-4 than peripheral T lymphocytes. The same differences are seen when Tγ⁺ and Tμ⁺ cells are compared. On the contrary there are almost no differences in pattern between the peripheral T lymphocytes and Tμ⁺ cells on one hand and between thymocytes and Tγ⁺ cells on the other hand. These findings suggest that both thymocytes and Tγ⁺ cells exhibit a very immature LDH pattern with regard to the Tμ⁺ cell population.     

Abnormally High Levels of Virus-Infected IFN-γ+CCR4+CD4+CD25+ T Cells in a Retrovirus-Associated Neuroinflammatory Disorder

Background

Human T-lymphotropic virus type 1 (HTLV-1) is a human retrovirus associated with both HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), which is a chronic neuroinflammatory disease, and adult T-cell leukemia (ATL). The pathogenesis of HAM/TSP is known to be as follows: HTLV-1-infected T cells trigger a hyperimmune response leading to neuroinflammation. However, the HTLV-1-infected T cell subset that plays a major role in the accelerated immune response has not yet been identified.

Principal Findings

Here, we demonstrate that CD4⁺CD25⁺CCR4⁺ T cells are the predominant viral reservoir, and their levels are increased in HAM/TSP patients. While CCR4 is known to be selectively expressed on T helper type 2 (Th2), Th17, and regulatory T (Treg) cells in healthy individuals, we demonstrate that IFN-γ production is extraordinarily increased and IL-4, IL-10, IL-17, and Foxp3 expression is decreased in the CD4⁺CD25⁺CCR4⁺ T cells of HAM/TSP patients as compared to those in healthy individuals, and the alteration in function is specific to this cell subtype. Notably, the frequency of IFN-γ-producing CD4⁺CD25⁺CCR4⁺Foxp3⁻ T cells is dramatically increased in HAM/TSP patients, and this was found to be correlated with disease activity and severity.

Conclusions

We have defined a unique T cell subset—IFN-γ⁺CCR4⁺CD4⁺CD25⁺ T cells—that is abnormally increased and functionally altered in this retrovirus-associated inflammatory disorder of the central nervous system.     

普通外科常见G+病原菌对抗生素耐药的分析

目的：研究普通外科感染的G^ 病原菌及其对常用抗生素的耐药性，以指导临床用药。方法：采用API—ATB System和Kirby—Bauer法，对外科临床标本进行细菌培养并检测其对多种抗生素的耐药性。结果：普通外科病房24个月共分离得到265株G^ 球菌，位列前3位的分别是金黄色葡萄球菌、表皮葡萄球菌、肠球菌。金葡菌对常用抗生素的耐药性较高，万古霉素仍是治疗G^ 球菌的有效药物。结论：监测G^ 球菌的耐药性，对指导外科临床抗生素的合理应用，提高外科抗感染疗效有重要意义。     

用高效液相色谱法进行G+C的测定

1　概述细菌分类是以形态学特征和生理生化特性等表型特征为基础的。虽然这些方法也成功地应用至今 ,但从不同细菌类群中区别表型相似性来确定它们的系统发育关系 ,还是不够精确的。近 2 0多年来 ,随着分子生物学的迅速发展和各项新技术的广泛应用 ,有关细菌种属间亲缘关系的分类鉴定工作 ,已从一般有表型特征的鉴定 ,深化为遗传型特性的鉴定。细菌 DNA中 (G+C) m ol%含量的测定是细菌分类鉴定中的一个反映属、种间亲缘关系的遗传指征。目前已成为属、种鉴定的常用方法。鸟嘌呤 (G)、胞嘧啶 (C)含量是 DNA的特征值中最简单的一项 ,因为…     

抗生素对临床常见G+致病菌的效价分析

目的:调查我院常用抗G~ 致病菌的抗生素的种类效价情况,为抗G~ 致病菌的合理用药提供依据。方法:从2978个样本中分离到953株G~ 致病菌,用全自动微生物分析仪-VITEK32进行菌种鉴定及16种抗生素的药敏实验。结果:953株G~ 致病菌经鉴定属于11大类,其中金黄色葡萄球菌、表皮葡萄球菌、溶血葡萄球菌、草绿色链球菌和肠球菌属是五类主要的G~ 致病菌,依次分别占总分离量的30.3%、17.3%、15.1%、9.8%和6.8%;在检测的16种抗生素对排在前五位的G~ 致病菌的平均效价最低的五类抗生素是:青霉素(24.22%)、红霉素(26.22%)、头孢唑林(28.06%)、与链霉素(30.52%)和庆大霉素(31.06%)。而抗G~ 致病菌整体效果最好的是:螯合新泰林(93.72%)、万古霉素(82.62%)和四环素(78.68%)。结论:研究结果表明越来越多抗生素抗临床常见G~ 致病菌的效价呈现逐年递减的趋势。     

G��i and G�¦� Jointly Regulate the Conformations of a G�¦� Effector, the Neuronal G Protein-activated K+ Channel (GIRK)

Stable complexes among G proteins and effectors are an emerging concept in cell signaling. The prototypical Gβγ effector G protein-activated K⁺ channel (GIRK; Kir3) physically interacts with Gβγ but also with Gα_i/o. Whether and how Gα_i/o subunits regulate GIRK in vivo is unclear. We studied triple interactions among GIRK subunits 1 and 2, Gα_i3 and Gβγ. We used in vitro protein interaction assays and in vivo intramolecular Förster resonance energy transfer (i-FRET) between fluorophores attached to N and C termini of either GIRK1 or GIRK2 subunit. We demonstrate, for the first time, that Gβγ and Gα_i3 distinctly and interdependently alter the conformational states of the heterotetrameric GIRK1/2 channel. Biochemical experiments show that Gβγ greatly enhances the binding of GIRK1 subunit to Gα_i3^GDP and, unexpectedly, to Gα_i3^GTP. i-FRET showed that both Gα_i3 and Gβγ induced distinct conformational changes in GIRK1 and GIRK2. Moreover, GIRK1 and GIRK2 subunits assumed unique, distinct conformations when coexpressed with a “constitutively active” Gα_i3 mutant and Gβγ together. These conformations differ from those assumed by GIRK1 or GIRK2 after separate coexpression of either Gα_i3 or Gβγ. Both biochemical and i-FRET data suggest that GIRK acts as the nucleator of the GIRK-Gα-Gβγ signaling complex and mediates allosteric interactions between Gα_i^GTP and Gβγ. Our findings imply that Gα_i/o and the Gα_iβγ heterotrimer can regulate a Gβγ effector both before and after activation by neurotransmitters.     

Kinetically governed polymorphism of d(G₄T₄G₃) quadruplexes in K+ solutions

It has been generally recognized that understanding the molecular basis of some important cellular processes is hampered by the lack of knowledge of forces that drive spontaneous formation/disruption of G-quadruplex structures in guanine-rich DNA sequences. According to numerous biophysical and structural studies G-quadruplexes may occur in the presence of K(+) and Na(+) ions as polymorphic structures formed in kinetically governed processes. The reported kinetic models suggested to describe this polymorphism should be considered inappropriate since, as a rule, they include bimolecular single-step associations characterized by negative activation energies. In contrast, our approach in studying polymorphic behavior of G-quadruplexes is based on model mechanisms that involve only elementary folding/unfolding transitions and structural conversion steps that are characterized by positive activation energies. Here, we are investigating a complex polymorphism of d(G(4)T(4)G(3)) quadruplexes in K(+) solutions. On the basis of DSC, circular dichroism and UV spectroscopy and polyacrylamide gel electrophoresis experiments we propose a kinetic model that successfully describes the observed thermally induced conformational transitions of d(G(4)T(4)G(3)) quadruplexes in terms of single-step reactions that involve besides single strands also one tetramolecular and three bimolecular quadruplex structures.     

γ-Synuclein Interacts with Phospholipase Cβ2 to Modulate G Protein Activation

Phospholipase Cβ2 (PLC β2) is activated by G proteins and generates calcium signals in cells. PLCβ2 is absent in normal breast tissue, but is highly expressed in breast tumors where its expression is correlated with the progression and migration of the tumor. This pattern of expression parallels the expression of the breast cancer specific gene protein 1 which is also known as γ-synuclein. The cellular function of γ-synuclein and the role it plays in proliferation are unknown. Here, we determined whether γ-synuclein can interact with PLCβ2 and affect its activity. Using co-immunprecitation and co-immunofluorescence, we find that in both benign and aggressive breast cancer cell lines γ-synuclein and PLCβ2 are associated. In solution, purified γ-synuclein binds to PLCβ2 with high affinity as measured by fluorescence methods. Protease digestion and mass spectrometry studies show that γ-synuclein binds to a site on the C-terminus of PLCβ2 that overlaps with the Gαq binding site. Additionally, γ-synuclein competes for Gαq association, but not for activators that bind to the N-terminus (i.e. Rac1 and Gβγ). Binding of γ-synuclein reduces the catalytic activity of PLCβ2 by mechanism that involves inhibition of product release without affecting membrane interactions. Since activated Gαq binds more strongly to PLCβ2 than γ-synuclein, addition of Gαq(GTPγS) to the γ-synuclein -PLCβ2 complex allows for relief of enzyme inhibition along with concomitant activation. We also find that Gβγ can reverse γ-synuclein inhibition without dissociating the γ-synuclein- PLCβ2- complex. These studies point to a role of γ-synuclein in promoting a more robust G protein activation of PLCβ2.     

NAD+-dependent formation ofγ-aminobutyrate (GABA) from glutamate

Mitochondria and nuclei of various tissues, including brain and liver, are capable of producing-aminobutyrate (GABA) fromL-glutamate, but poorly, if at all, fromD-glutamate. The amino nitrogen of glutamate is found in the reaction product. The enzymes responsible for GABA formation were solubilized from crude liver cell nuclei by Triton X-100. The reaction is NAD⁺ dependent Oxygen, FMN, Mg²⁺, and pyridoxalphosphate enhanced GABA formation. NADP⁺, coenzyme A, ornithine, 2-oxoglutarate, and aminooxyacetic acid, among others, inhibited the formation of GABA. On the basis of the available information the reaction sequence, is formulated tentatively as follows:     

临床常见G+致病菌的种类及耐药性的研究

目的:调查我院常见G 致病菌的种类及其对目前常用抗生素的耐药现状,为临床医生合理选用抗生素提供数据资料.方法:从3286个样本中分离到206株G 致病菌,用全自动微生物分析仪-VITEK 32进行菌种鉴定及药敏实验.结果:206株G 致病菌经鉴定属于6大类,其中金黄色葡萄球菌,74株,占35.9%;溶血葡萄球茵,47株,占22.8%;表皮葡萄球茵,47株,占22.8%;肠球菌属,33株,占16.0%;肺炎链球茵,3株,占1.5%;β-溶血链球菌,2株,占1.0%.肺炎链球茵和β-溶血链球菌仅对检测的14种抗生素中的少数敏感.在对14种抗生素的耐药性实验中,药效普遍较差的抗生素为:青霉素、庆大霉素、苯唑西林.它们三者的平均药敏感率依次为:16.52%、18.42%和21.07%.结论:临床G 致病菌的耐药性问题已非常严峻,合理的选择和使用抗生素已十分重要.     

弗兰克氏菌的G+C含量和DNA杂交

对一些弗兰克氏菌的遗传和生理特性进行了研究。结果表明,7株菌可以分为3个基因群。从木麻黄(Caswarina equisetifolia)分离的菌株可以划为一个基因群(与菌株S-103的DNA同源性在74％以上),这是国外尚未报道的新基因群。其他两株弗兰克氏菌菌株Ar14(从赤杨根瘤分离)和Ptll(从Purshia根瘤分离)分别形成另外两个基因群,这与An的报道一致。7株弗兰克氏菌DNA的G+C mol％在69-73。S-103基因群菌株不利用糖类,仅利用简单的有机酸盐。而菌株ArI4和Ptll则利用一些糖和有机酸盐作为碳源。     

G protein β1γ2 subunits purification and their interaction with adenylyl cyclase

A preliminary study on the interaction of G protein (guanine triphosphate binding pro- tein) β1γ2 subunits and their coupled components in cell signal transduction was conducted in vitro. The insect cell lines, Sf9 (Spodoptera frugiperda) and H5 (Trichoplusia ni) were used to express the recombinant protein Gβ1γ2. The cell membrane containing Gβ1γ2 was isolated through affinity chromatography column with Ni-NTA agarose by FPLC method, and the highly purified protein was obtained. The adenylyl cyclase 2 (AC2) activity assay showed that the purified Gβ1γ2 could significantly stimulate AC2 activity. The interaction of β1γ2 subunits of G protein with the cytoplasmic tail of various mammalian adenylyl cyclases was monitored by BIAcore technology using NTA sensor chip, which relies on the phenomenon of surface plasmon resonance (SPR). The experiments showed the direct binding of Gβ1γ2 to the cytoplasmic tail C2 domain of AC2. The specific binding domain of AC2 with Gβ1γ2 was the same as AC2 activity domain which was stimulated by β1γ2.