Molecular cloning in Arabidopsis thaliana of a new protein phosphatase 2C (PP2C) with homology to ABI1 and ABI2

Trapeziectomy, ligament reconstruction and tendon interposition arthroplasty is one of the most commonly performed procedures to address pain and instability due to osteoarthritis at the basal joint of the thumb. To determine the effect of stress on first metacarpal subsidence, 15 ligament reconstruction and tendon interposition basal joint arthroplasties were evaluated after a mean follow-up of 32 months. Radiographs were obtained of the arthroplasty at rest and then with maximal effort key pinch stress, which is known to subject the first carpometacarpal joint to considerable axial compression stress. Compared with the preoperative x-rays, the first metacarpal had subsided 21% of the arthroplasty space at rest. Under stress, the first metacarpal was found to subside another 10.5% in height. No subluxation of the metacarpal base could be detected. Key pinch strength improved 17% from the preoperative strength. Tip-to-tip pinch strength improved 17% from the preoperative measurement. Grip strength improved 17% from the preoperative measurement. Grip strength was 9% greater than the preoperative grip strength. There was no statistical association between the amount of first metacarpal subsidence and follow-up key pinch, tip pinch, or grip strength. With axial compressive loading of the arthroplasty, such as in lateral pinch, there is some further proximal migration of the first metacarpal, but this is minimal and does not correlate with functional outcome.     

Phase change and the regulation of trichome distribution in Arabidopsis thaliana

Higher plants pass through several phases of shoot growth during which they may produce morphologically distinct vegetative structures. In Arabidopsis thaliana this phenomenon is apparent in the distribution of trichomes on the leaf surface. Leaves produced early in rosette development lack trichomes on their abaxial (lower) surface, leaves produced later have trichomes on both surfaces, and leaves in the inflorescence (bracts) may have few or no trichomes on their adaxial (upper) surface. Here we describe some of the factors that regulate this distribution pattern. We found that the timing of abaxial trichome production and the extent to which bracts lack adaxial trichomes varies in different ecotypes. The production of abaxial trichomes appears to be regulated by the age, rather than the size of the plant. This conclusion is based on the observation that mutations that affect either the rate (altered meristem programming1) or onset (paused) of leaf initiation respectively increase or decrease the number of leaves that lack abaxial trichomes, but have only a minor effect on the time at which the first leaf with abaxial trichomes is produced. The production of abaxial trichomes is coordinated with the reproductive development of the shoot as this trait is delayed by photoperiodic conditions and some mutations that delay flowering. The loss of adaxial trichomes is likely to be a consequence of floral induction, and is accelerated by terminal flower1-10, a mutation that accelerates inflorescence development. We demonstrate that gibberellins promote trichome production in Arabidopsis and present evidence indicating that abaxial trichome production is regulated by both the level of a trichome inducer and the competence of the abaxial epidermis to respond to this inducer.     

Molecular changes in Entamoeba histolytica in response to bacteria

Entamoeba histolytica, the protozoan parasite, is the causative agent of amoebiasis. The degree of virulence, as inferred from invasiveness, of potentially pathogenic strains may be regulated by both host and parasite factors that determine the gut environment. One such factor that plays an important role is the bacterial flora in the gut. Previous studies have clearly shown that bacterial flora is an important determinant of virulence in E. histolytica. However, the exact nature of changes induced in E. histolytica in response to bacteria and their role in virulence is not clear. In this study the levels of a number of molecules potentially important in virulence mechanisms were determined in E. histolytica cells grown with and without normal human bacterial flora, using enzyme-linked immunosorbent assay. Significant changes were observed only after the E. histolytica cells had been adapted to grow with bacterial flora for a number of generations, and not in short term culture.     

Molecular cloning, expression, and enzymatic characterization of rabbit hydroxysteroid sulfotransferase AST-RB2

Cytosolic sulfotransferases, which consist of at least three gene families, play a major role in activation and detoxification of both endogenous and exogenous chemicals. We recently purified a rabbit sulfotransferase, AST-RB2, showing high activities to both hydroxysteroids and amines. To characterize this enzyme, a rabbit cDNA library was screened using anti-AST-RB2 antibodies. The isolated cDNA was judged to encode AST-RB2 (ST2A8) based on the amino acid sequences of peptide fragments obtained from purified AST-RB2. The cDNA showed high similarity to other mammalian hydroxysteroid sulfotransferases (ST2) at the amino acid level (58-68%), but low similarity to aryl sulfotransferases (ST1) (less than 37%). The protein expressed in Escherichia coli catalyzed sulfation of typical ST2 substrates. Therefore, ST2A8 was judged to belong to the ST2 family from both its primary structure and substrate specificity. The ST2A8 protein expressed in E. coli clearly differed from rat ST2A1 and ST2A2 on its localization (cytosol/insoluble fraction ratio). ST2A8 had no activity to lithocholate, but showed the highest catalysis on dehydroepiandrosterone and testosterone among the four forms (ST2A1, ST2A2, ST2A3, and ST2A8), indicating a clear difference between ST2A forms in substrate specificity to endogenous chemicals.     

Molecular cloning and expression of human and mouse tyrosylprotein sulfotransferase-2 and a tyrosylprotein sulfotransferase homologue in Caenorhabditis elegans

Tyrosine O-sulfation, a common post-translational modification in eukaryotes, is mediated by Golgi enzymes that catalyze the transfer of the sulfuryl group from 3'-phosphoadenosine 5'-phosphosulfate to tyrosine residues in polypeptides. We recently isolated cDNAs encoding human and mouse tyrosylprotein sulfotransferase-1 (Ouyang, Y. B., Lane, W. S., and Moore, K. L. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2896-2901). Here we report the isolation of cDNAs encoding a second tyrosylprotein sulfotransferase (TPST), designated TPST-2. The human and mouse TPST-2 cDNAs predict type II transmembrane proteins of 377 and 376 amino acid residues, respectively. The cDNAs encode functional N-glycosylated enzymes when expressed in mammalian cells. In addition, preliminary analysis indicates that TPST-1 and TPST-2 have distinct specificities toward peptide substrates. The human TPST-2 gene is on chromosome 22q12.1, and the mouse gene is in the central region of chromosome 5. We have also identified a cDNA that encodes a TPST in the nematode Caenorhabditis elegans that maps to the right arm of chromosome III. Thus, we have identified two new members of a class of membrane-bound sulfotransferases that catalyze tyrosine O-sulfation. These enzymes may catalyze tyrosine O-sulfation of a variety of protein substrates involved in diverse physiologic functions.     

Molecular cloning of an Arabidopsis cDNA encoding a dynamin-like protein that is localized to plastids

Palaeontology provides the only direct record for morphological and genetic change through time and uniquely contributes to systematics in two ways: by providing access to denser taxon sampling than is otherwise possible and by dating divergence times. Claims that ancient DNA has survived millions of years in certain fossils suggested the possibility that palaeontology could contribute directly to molecular systematic studies. Unfortunately, none of the supposed geologically ancient DNA records stands up to detailed scrutiny and fossils therefore contribute primarily through the morphological information they preserve. Denser taxon sampling can improve the accuracy of phylogenetic estimates primarily through allowing better discrimination of homoplasy from homology. This in turn leads to more accurate hypotheses of character transformation. Denser taxon sampling also offers the opportunity for more accurate rooting, since more characters can be polarized by reference to a stem-group taxon than to an extant sister-group taxon. Missing data can be a problem for fossils, but is not crippling. Finally the temporal order of clade appearances in the fossil record can provide ancillary evidence for selecting a working phylogeny from among a number of equally most parsimonious cladograms.     

cDNA cloning and expression of a new form of human aryl sulfotransferase

Twenty-eight patients (with 30 primary and 8 revision total hip arthroplasties) completed a standardized questionnaire containing the Western Ontario and McMaster Universities (WOMAC) osteoarthritis index and Harris hip score questions prior to an office visit a minimum of 1 year after surgery. The range of hip motion measured by an orthopaedic surgeon was compared with the responses to questions on stiffness and function as well as with global scores in the WOMAC osteoarthritis index. Patient responses to the questions asking if they could cut their toenails on the operated side and the Harris hip score question asking if they could put on socks and tie a shoe correlated significantly with postoperative hip motion (P < .005). The WOMAC global pain and stiffness scores did not correlate with range of motion. The WOMAC physical function score correlated significantly only with hip flexion (P < .05). Of the WOMAC physical function questions, difficulty bending to pick an object off the floor (P < .05) and getting on and off the toilet (P < .05) correlated with the sum of the range of motion in all planes and weighted Harris hip score range of motion calculation. These data suggest that the points allocated in the Harris hip score for range of motion can be estimated reasonably accurately from questionnaire or phone response to a series of questions on a standardized questionnaire. The question on ability to cut toenails or the Harris hip score question regarding ability to put on socks and tie a shoe correlated with the most individual planes of motion, but several WOMAC physical function questions also correlated with total and weighted range of motion calculations.     

Dissection of sexual organ ontogenesis: a genetic analysis of ovule development in Arabidopsis thaliana

Understanding organogenesis remains a major challenge in biology. Specification, initiation, pattern formation and cellular morphogenesis, have to be integrated to generate the final three-dimensional architecture of a multicellular organ. To tackle this problem we have chosen the ovules of the flowering plant Arabidopsis thaliana as a model system. In a first step towards a functional analysis of ovule development, we performed a large-scale genetic screen and isolated a number of sterile mutants with aberrant ovule development, We provide indirect genetic evidence for the existence of proximal-distal pattern formation in the Arabidopsis ovule primordium. The analysis of the mutants has identified genes that act at an intermediate regulatory level and control initiation of morphogenesis in response to proximal-distal patterning. A second group of genes functions at a subordinate control level and regulates general cellular processes of morphogenesis. A large group of male and female sterile mutants shows defects restricted to early or late gametogenesis. In addition, we propose that the mature ovule obtains its overall curved shape by at least three different processes that act in only one domain of the ovule.     

儿童下呼吸道感染常见病原菌分布及耐药性变迁

目的:研究我院下呼吸道感染患儿病原菌分布及其耐药性变迁,为临床合理使用药物治疗提供参考依据.方法:对2008年1月到2010年12月住院的所有下呼吸道感染患儿,共907例,进行痰培养并对其鉴定和药敏结果进行回顾性分析.结果:3年中分离的革兰阴性杆菌以肺炎克雷伯菌(KPN)和大肠埃希菌(ECO)为主,KPN和ECO产超广谱β-内酰胺酶(ESBLs)率,分别达31.8%～40.0%和41.7%～54.1%;革兰阳性球菌以肺炎链球菌和金黄色葡萄球菌为主,其中耐甲氧西林金黄色葡萄球菌(MRSA)分离率达28.5%～54.4%.病原菌对大部分抗生素有耐药性.结论:儿童下呼吸道感染常见病原菌主要为革兰阴性杆菌,对头孢类耐药率较高,且有逐年升高趋势,故临床上应根据药敏结果合理使用抗生素.     

Molecular cloning and characterization of viruses isolated from chimpanzees with pathogenic human immunodeficiency virus type 1 infections

We have previously described the development of AIDS in a chimpanzee (C499) infected with human immunodeficiency virus type 1 (HIV-1) and the subsequent pathogenic HIV-1 infection in another chimpanzee (C455) transfused with blood from C499 (F. J. Novembre et al., J. Virol. 71:4086-4091, 1997). In the present study, two virus isolates were derived from these animals: HIV-1JC from peripheral blood mononuclear cells (PBMC) of C499, and HIV-1NC from plasma of C455. These virus isolates were used to generate two infectious molecular clones, termed HIV-1JC16 and HIV-1NC7 (JC16 and NC7, respectively). Comparative analyses of the sequences of the two clones showed that they were highly interrelated but distinct. Based on heteroduplex mobility assays, JC16 and NC7 appear to represent dominant viruses in the uncloned stock population. Compared with amino acid sequences of the parental viruses HIV-1SF2, HIV-1LAV-1b, and HIV-1NDK, JC16 and NC7 showed a number of differences, including insertions, deletions, and point mutations spread throughout the genome. However, insertion/deletion footprints in several genes of both JC16 and NC7 suggested that recombination between SF2 and LAV-1b could have occurred, possibly contributing to the generation of a pathogenic virus. Comparative in vitro analyses of the molecular clones and the uncloned stocks of HIV-1JC and HIV-1NC revealed that these viruses had strikingly similar replicative abilities in mitogen-stimulated PBMC and in macrophages. Compared to the SF2 and LAV-1b isolates of HIV-1, HIV-1JC and HIV-1NC isolates were more similar to LAV-1b with respect to the ability to replicate in mitogen-stimulated PBMC and macrophages. These viruses should prove to be useful in mapping determinants of pathogenesis.     

Molecular cloning and characterization of 12-oxophytodienoate reductase, an enzyme of the octadecanoid signaling pathway from Arabidopsis thaliana. Structural and functional relationship to yeast old yellow enzyme

Using partial amino acid sequence information for 12-oxophytodienoate-10,11-reductase obtained from Corydalis sempervirens we have cloned the homologous enzyme from Arabidopsis thaliana. The open reading frame of the cDNA encodes a polypeptide of 372 amino acids (Mr = 41,165) with significant similarity to the sequence of Old Yellow Enzyme from Saccharomyces carlsbergensis (Saito, K., Thiele, D. J., Davio, M., Lockridge, O., and Massey, V. (1991) J. Biol. Chem. 266, 20720-20724), a flavin (FMN)-protein catalyzing the NADPH-dependent reduction of the olefinic bond of alpha,beta-unsaturated carbonyls. Specifically, all residues required for binding of FMN in Old Yellow Enzyme are conserved in the A. thaliana sequence, as are all residues associated with catalytic activity. The enzyme was functionally expressed from its cDNA in Escherichia coli and thus proven to encode OPDA reductase. Further similarities of OPDA reductase and yeast Old Yellow Enzyme include their binding to and elution by reductant from N-(4-hydroxybenzoyl)aminohexyl-Sepharose, the immunoreactivity of yeast Old Yellow Enzyme with an antiserum raised against plant OPDA reductase and the demonstration that Old Yellow Enzyme is an active OPDA reductase. It is thus conceivable that the physiological role of Old Yellow Enzymes now known from bacteria, yeasts, and higher plants, is in oxylipin metabolism.     

Cell marking in Arabidopsis thaliana and its application to patch-clamp studies

Ion transport processes at the plasma membrane of plant cells are frequently studied by applying membrane-patch voltage-clamp (patch-clamp) electrophysiological techniques to isolated protoplasts. As plants are composed of many tissues and cell types, and each tissue and cell type may be specialized to a particular function and possess a unique complement of transport proteins, it is important to certify the anatomical origin of the protoplasts used for patch-clamp studies. This paper describes a general molecular genetic approach to marking specific cell types for subsequent patch-clamp studies and presents a specific example: a comparison of the K+ currents in protoplasts from cortical and stelar cells of Arabidopsis roots. Transgenic Arabidopsis were generated in which the expression of green fluorescent protein (GFP) from Aequoria victoria was driven by the CaMV 35S promoter (line mGFP3). In roots of the transgenic mGFP3 line, visible fluorescence was restricted to the stele. Protoplasts were generated from roots of the mGFP3 line and K+ currents in non-fluorescent (cortical/epidermal) and fluorescent (stelar) protoplasts were assayed using patch-clamp techniques. It was found that both the frequency of observing inward rectifying K+ channel (IRC) activity and the relative occurrence of IRC compared to outward rectifying K+ channels were significantly lower in protoplasts from cortical/epidermal cells compared to cells of the stele. The presence of GFP did not affect the occurrence or biophysical properties of K+ channels. It is concluded that the generation of transgenic Arabidopsis expressing GFP in a cell-specific fashion is a convenient and reliable way to mark protoplasts derived from contrasting cell types for subsequent patch-clamp studies.     

Functional cloning of a cDNA encoding Mei2-like protein from Arabidopsis thaliana using a fission yeast pheromone receptor deficient mutant

Aromatherapy has been defined as 'the art--and science--of using essential plant oils in treatments ... a truly holistic therapy, taking into account mind, body and spirit ...' (Davis 1991). Aromatherapy is a valuable means of maintaining optimum health, particularly when the dis-ease of the body or mind is related to stress. The process of hospitalization is a potentially stressful experience that has been well researched (Broome et al 1990, Kachoyeanos & Friedhoff 1993, Strachan 1993, Taylor 1991). This paper examines the ways in which massage and aromatherapy could be of benefit to hospitalized children, particularly those infected with Human Immunodeficiency Virus (HIV). Wright (1995) states that nurses should encourage self-healing by 'putting the patient in the best condition for nature to act'. Aromatherapy massage has the potential to achieve this through inducing relaxation and reducing the stressful aspects of hospitalization. Thus, the author would like to propose the use of this valuable skill as an extension of the nursing role.     

Isoform-dependent differences in feedback regulation and subcellular localization of serine acetyltransferase involved in cysteine biosynthesis from Arabidopsis thaliana

Serine acetyltransferase (SATase; EC 2.3.1.30), which catalyzes the formation of O-acetyl-L-serine (OAS) from acetyl-CoA and L-serine, plays a regulatory role in the biosynthesis of cysteine by its property of feedback inhibition by cysteine in bacteria and certain plants. Three cDNA clones encoding SATase isoforms (SAT-c, SAT-p, and SAT-m) have been isolated from Arabidopsis thaliana. However, the significance of the feedback regulation has not yet been clear in these different isoforms of SATase from A. thaliana. We constructed the overexpression vectors for cDNAs encoding three SATase isoforms of A. thaliana and analyzed the inhibition of SATase activity by cysteine using the recombinant SATase proteins. In the case of SAT-c, the activity was feedback-inhibited by a low concentration of cysteine (the concentration that inhibits 50% activity; IC50 = 1.8 microM). By contrast, SAT-p and SAT-m were feedback inhibition-insensitive isozymes. We also determined the subcellular localization of three SATase isozymes by the transient expression of fusion proteins of each SATase N-terminal region with jellyfish green fluorescent protein (GFP) in 4-week-old Arabidopsis leaves. The SAT-c-GFP fusion protein was stayed in cytosol, whereas SAT-p-GFP and SAT-m-GFP fusion proteins were localized in chloroplasts and in mitochondria, respectively. These results suggest that these three SATase isoforms, which are localized in the different organelles, are subjected to different feedback regulation, presumably so as to play the particular roles for the production of OAS and cysteine in Arabidopsis cells. Regulatory circuit of cysteine biosynthesis in the plant cells is discussed.     

Identification and characterization of a novel cap-binding protein from Arabidopsis thaliana

Cap-binding proteins specifically bind to the 7-methyl guanosine (m7G) functional group at the 5' end of eukaryotic mRNAs. A novel Arabidopsis thaliana protein has been identified that has sequence similarity to cap-binding proteins but is clearly a different form of the protein. The most obvious primary sequence difference is the substitution of two of the eight conserved tryptophan residues with other aromatic amino acids in the novel protein. Analogous forms of this novel protein appear to be present in other higher eukaryotes but not in yeast. Analysis of the native and recombinant forms of the novel protein by retention on m7GTP-Sepharose indicate that it is a functional cap-binding protein. Measurements of the dissociation constant for this protein indicate that it binds m7GTP 5-20-fold tighter than eukaryotic initiation factor (eIF)(iso)4E. The novel protein also supports the initiation of translation of capped mRNA in vitro. Biochemical analysis and yeast two-hybrid data indicate that it interacts with eIF(iso)4G to form a complex. Based on these observations, this protein appears to be able to function as a cap-binding protein and is given the designation of novel cap-binding protein (nCBP).     

Molecular cloning and hormonal regulation of PiT-1, a sodium-dependent phosphate cotransporter from rat parathyroid glands

The extracellular concentration of inorganic phosphate (Pi) is an important determinant of parathyroid cell function. The effects of Pi may be mediated through specific molecules in the parathyroid cell membrane, one candidate molecule for which would be a Na+-dependent Pi cotransporter. A complementary DNA encoding a Na+-Pi cotransporter, termed rat PiT-1, has now been isolated from rat parathyroid. The 2890-bp complementary DNA encodes a protein of 681 amino acids that shows sequence identities of 97% and 93% with the type III Na+-Pi cotransporters mouse PiT-1 and human PiT-1, respectively. Expression of rat PiT-1 in Xenopus oocytes revealed that it possesses Na+-dependent Pi cotransport activity. PiT-1 messenger RNA (mRNA) is widely distributed in rat tissues and is most abundant in brain, bone, and small intestine. The amount of PiT-1 mRNA in the parathyroid of vitamin D-deficient rats was reduced compared with that in normal animals and increased markedly after administration of 1,25-dihydroxyvitamin D3. Furthermore, the abundance of PiT-1 mRNA in the parathyroid was much greater in rats fed a low-Pi diet than in those fed a high-Pi diet. Thus, rat PiT-1 may contribute to the effects of Pi and vitamin D on parathyroid function.     

Genetic analysis of a seedling stress response to ethylene in Arabidopsis

A genetic framework has been devised for the action of genes within the ethylene-response pathway. This working model is based on the epistatic interactions among a variety of ethylene response mutations. Most of the mutations that have been described act in a linear pathway. Genes controlling cell elongation in response to ethylene must, at some level, act to affect the architecture of the cytoskeleton. Genes that act late in the pathway, in mutant form, may lead to highly specific phenotypes such as the increased sensitivity to taxol in the ein6 mutant. Analysis of these downstream components may provide critical insights into the nature of ethylene's effect on the cell elongation machinery.