Multilocus sequence typing for characterization of Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius is an opportunistic pathogen in dogs. Four housekeeping genes with allelic polymorphisms were identified and used to develop an expanded multilocus sequence typing (MLST) scheme. The new seven-locus technique shows S. pseudintermedius to have greater genetic diversity than previous methods and discriminates more isolates based upon host origin.

Middle and late eocene mammal communities: a major discrepancy.

The multituberculate Parectypodus lovei has been found in late Eocene rocks in Montana, together with 11 other mammal species similar to those found in the late Eocene Tepee Trail Formation in Wyoming.The multituberculate and six other species are unknown in rocks of equivalent age or of middle Eocene age elsewhere. It is suggested that the known middle Eocene faunas are all taken from a similar ecological situation and do not reflect the true diversity of middle Eocene life. Middle Eocene faunas of different ecological aspect may be recovered from sediments along, and in, the mountain fronts of northwestern Wyoming.

Initiation of sulfate activation: a variation in c4 photosynthesis plants.

In leaves of plants with C(4) photosynthesis, sulfur assimilation is initiated in bundle sheath cells whereas carbon and nitrogen assimilation are initiated in mesophyll cells. The activation of sulfate by adenosine triphosphate sulfurylase in leaves of C(4) plants occurs in chloroplasts of bundle sheath cells and is effected by two isozymes of approximately equal activities that accounted for 95 to 100 percent of the total leaf activity.

Frequent requirement of hedgehog signaling in non-small cell lung carcinoma.

Although it had previously been suggested that the hedgehog (HH) pathway might be activated in some lung tumors, the dependence of non-small cell lung carcinomas (NSCLC) for HH activity had not been comprehensively studied. During a screen of a panel of 60 human tumor cell lines with an HH antagonist, we observed that the proliferation of a subset of NSCLC cell lines was inhibited. These NSCLC cell lines express HH, as well as key HH target genes, consistent with them being activated through an autocrine mechanism. Interestingly, we also identified a number of NSCLC cell lines that express high levels of the downstream transcription factor GLI1 and harbor enhanced levels of HH activity, but appear insensitive to known HH antagonists. We hypothesized that the high levels of GLI1 in these cells would function downstream of the HH antagonist target, allowing them to bypass the antagonist-mediated block in proliferation. Consistent with this hypothesis, when the levels of GLI1 are knocked down in such cells, they become sensitive to these inhibitors. We go on to show that a large percentage of primary NSCLC samples express GLI1, consistent with constitutive activation of the HH pathway in these samples. Taken together, these results establish the involvement of the HH signaling pathway in a subset of NSCLCs.

Occurrence of C(3) and C(4) photosynthesis in cotyledons and leaves of Salsola species (Chenopodiaceae).

Most species of the genus Salsola (Chenopodiaceae) that have been examined exhibit C(4) photosynthesis in leaves. Four Salsola species from Central Asia were investigated in this study to determine the structural and functional relationships in photosynthesis of cotyledons compared to leaves, using anatomical (Kranz versus non-Kranz anatomy, chloroplast ultrastructure) and biochemical (activities of photosynthetic enzymes of the C(3) and C(4) pathways, (14)C labeling of primary photosynthesis products and (13)C/(12)C carbon isotope fractionation) criteria. The species included S. paulsenii from section Salsola, S. richteri from section Coccosalsola, S. laricina from section Caroxylon, and S. gemmascens from section Malpigipila. The results show that all four species have a C(4) type of photosynthesis in leaves with a Salsoloid type Kranz anatomy, whereas both C(3) and C(4) types of photosynthesis were found in cotyledons. S. paulsenii and S. richteri have NADP- (NADP-ME) C(4) type biochemistry with Salsoloid Kranz anatomy in both leaves and cotyledons. In S. laricina, both cotyledons and leaves have NAD-malic enzyme (NAD-ME) C(4) type photosynthesis; however, while the leaves have Salsoloid type Kranz anatomy, cotyledons have Atriplicoid type Kranz anatomy. In S. gemmascens, cotyledons exhibit C(3) type photosynthesis, while leaves perform NAD-ME type photosynthesis. Since the four species studied belong to different Salsola sections, this suggests that differences in photosynthetic types of leaves and cotyledons may be used as a basis or studies of the origin and evolution of C(4) photosynthesis in the family Chenopodiaceae.

C4 plants in the vegetation of Mongolia: their natural occurrence and geographical distribution in relation to climate.

The natural geographical occurrence, carbon assimilation, and structural and biochemical diversity of species with C4 photosynthesis in the vegetation of Mongolia was studied. The Mongolian flora was screened for C4 plants by using 13C/12C isotope fractionation, determining the early products of 14CO2 fixation, microscopy of leaf mesophyll cell anatomy, and from reported literature data. Eighty C4 species were found among eight families: Amaranthaceae, Chenopodiaceae, Euphorbiaceae, Molluginaceae, Poaceae, Polygonaceae, Portulacaceae and Zygophyllaceae. Most of the C4 species were in three families: Chenopodiceae (41 species), Poaceae (25 species) and Polygonaceae, genus Calligonum (6 species). Some new C4 species in Chenopodiaceae, Poaceae and Polygonaceae were detected. C4 Chenopodiaceae species make up 45% of the total chenopods and are very important ecologically in saline areas and in cold arid deserts. C4 grasses make up about 10% of the total Poaceae species and these species naturally concentrate in steppe zones. Naturalized grasses with Kranz anatomy,of genera such as Setaria, Echinochloa, Eragrostis, Panicum and Chloris, were found in almost all the botanical-geographical regions of Mongolia, where they commonly occur in annually disturbed areas and desert oases. We analyzed the relationships between the occurrence of C4 plants in 16 natural botanical-geographical regions of Mongolia and their major climatic influences. The proportion of C4 species increases with decreasing geographical latitude and along the north-to-south temperature gradient; however grasses and chenopods differ in their responses to climate. The abundance of Chenopodiaceae species was closely correlated with aridity, but the distribution of the C4 grasses was more dependent on temperature. Also, we found a unique distribution of different C4 Chenopodiaceae structural and biochemical subtypes along the aridity gradient. NADP-malic enzyme (NADP-ME) tree-like species with a salsoloid type of Kranz anatomy, such as Haloxylon ammodendron and Iljinia regelii, plus shrubby Salsola and Anabasis species, were the plants most resistant to ecological stress and conditions in highly arid Gobian deserts with less than 100 mm of annual precipitation. Most of the annual C4 chenopod species were halophytes, succulent, and occurred in saline and arid environments in steppe and desert regions. The relative abundance of C3 succulent chenopod species also increased along the aridity gradient. Native C4 grasses were mainly annual and perennial species from the Cynodonteae tribe with NAD-ME and PEP-carboxykinase (PEP-CK) photosynthetic types. They occurred across much of Mongolia, but were most common in steppe zones where they are often dominant in grazing ecosystems.

The relationship of CO2 assimilation pathways and photorespiration to the physiological quantum requirement of green plant photosynthesis.

The quantum requirement of green cells for CO2 fixation has been evaluated and discussed in view of the recent discovery of photorespiration and of multiple biochemical pathways for photosynthetic CO2 fixation. The reported quantum requirement of algae generally is near 9 quanta per CO2 fixed. It is suggested that the high CO2 concentrations and low O2 concentrations used for these algae experiments would have completely suppressed photorespiration and, therefore, the minimum number of quanta required to fix 1 CO2 molecule was correctly determined in these experiments. With higher plant leaves, when measurements are made under physiological environments, quantum requirements range from about 12 to 20 quanta per CO2 fixed. It is suggested that these physiological quantum requirements are higher because photorespiration is functional in these leaves and that photorespiration requires energy. The energy requirement of photorespiration was derived using biochemical models of leaf photosynthesis combining photorespiration with specific biochemical pathways for CO2 fixation. The calculated physiological quantum requirements for C3, C4 and CAM plant photosynthesis are 13, 15 and 17 respectively. The literature values on quantum requirements correspond well with these biochemical models of net photosynthesis. However, it was concluded that the biochemical models fail to give a complete description of photosynthesis in plants using the C4-dicarboxylic acid cycle.

The role of mecA and blaZ regulatory elements in mecA expression by regional clones of methicillin-resistant Staphylococcus pseudintermedius.

Two major regional clones of methicillin-resistant Staphylococcus pseudintermedius (MRSP) have been identified in Europe and North America. They are designated multilocus sequence types (ST) 71 and 68 and contain staphylococcal chromosome cassette (SCCmec) types II-III and V(T), respectively. One notable difference between the two clones is a deletion in the mecI/mecR1 regulatory apparatus of ST 68 SCCmec V(T). This deletion in analogous methicillin-resistant Staphylococcus aureus (MRSA) results in more responsive and greater expression of the mecA encoded penicillin-binding protein 2a, and is associated with SCCmec types occurring in community-acquired MRSA lineages. The aim of this study was to characterize mec and bla regulatory apparatuses in MRSP and determine their effects on expression of mecA. Seventeen S. pseudintermedius isolates representing nine methicillin-resistant ST lineages were screened for the presence of the repressors blaI and mecI and sensors blaR1 and mecR1. The bla and mec operons for each isolate were sequenced and compared for homology between the repressor open-reading frames (ORF), sensor ORFs, and mecA promoter regions. A real-time reverse transcriptase PCR expression assay was developed, validated and applied to nine isolates determining the effect of oxacillin induction on mecA transcription. Significant differences were found in mecA expression between isolates with a full regulatory complement (mecI/mecR1 and blaI/blaR1) and those with truncated and/or absent regulatory elements. Isolates representative of European and North American MRSP ST regional clones have dissimilar mecA responses to oxacillin.

Identification of a predominant multilocus sequence type, pulsed-field gel electrophoresis cluster, and novel staphylococcal chromosomal cassette in clinical isolates of mecA-containing, methicillin-resistant Staphylococcus pseudintermedius.

Methicillin resistance encoded by the mecA gene is increasingly observed in Staphylococcus pseudintermedius. Little is known about the population genetics of veterinary staphylococci bearing methicillin resistance. The aim of this study was to determine the relatedness of resistant bacteria and to compare them with methicillin-susceptible isolates. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) fragment profiling were performed on methicillin-resistant S. pseudintermedius (MRSP) and methicillin-susceptible S. pseudintermedius (MSSP) isolates obtained from canine samples submitted to the veterinary teaching hospital bacteriology service between 2006 and 2008. Multilocus sequence typing detected 20 different sequence types, 16 of which were not previously described. Methicillin-resistant isolates were predominantly ST 68, possessed the Staphylococcus aureus-associated staphylococcal chromosomal cassette mec (SCCmec) type V(T) and fell within the largest PFGE cluster; whereas methicillin-susceptible strains were more genetically diverse. This suggests that most methicillin resistance within the population of isolates tested originated from a single source which has persisted and expanded for several years.

Electrophoretic analysis of protoplast, vacuole, and tonoplast vesicle proteins in crassulacean Acid metabolism plants.

Protoplasts and vacuoles were isolated and purified in large numbers from the CAM plants Ananas comosus (pineapple) and Sedum telephium for protein characterization. Vacuoles were further fractionated to yield a tonoplast vesicle preparation. Polypeptides of protoplasts, vacuoles, and tonoplast vesicles were compared to whole leaf polypeptides from both plants by one-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis. Approximately 100 vacuole polypeptides could be resolved of which 25 to 30% were enriched in the tonoplast vesicles. The proteins of protoplasts, vacuoles, and tonoplast vesicles from A. comosus were analyzed further by two-dimensional gel electrophoresis. When one-dimensional electrophoretograms of A. comosus polypeptides were stained with a glycoprotein-specific periodic acid Schiff stain, very few polypeptides appeared to be glycosylated, whereas a large number of glycosylated polypeptides were detected with a silver-based glycoprotein stain particularly in tonoplast vesicles. Analysis of the enzymic content of vacuoles from both plants indicated the presence of a variety of hydrolases, including bromelain as a major constituent of A. comosus. No substrate-specific ATPase, however, could be detected in vacuoles or tonoplast vesicles from either plant.

Biochemical and immunological properties of alkaline invertase isolated from sprouting soybean hypocotyls.

Alkaline invertase from sprouting soybean (Glycine max) hypocotyls was purified to apparent electrophoretic homogeneity by consecutive use of DEAE-cellulose, green 19 dye, and Cibacron blue 3GA dye affinity chromatography. This protocol produced about a 100-fold purification with about a 11% yield. The purified protein had a specific activity of 48 mumol of glucose produced mg-1 protein min-1 (pH 7.0) and showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) (58 kDa) and in native PAGE, as indicated by both protein and activity staining. The native enzyme molecular mass was about 240 kDa, suggesting a homotetrameric structure. The purified enzyme exhibited hyperbolic saturation kinetics with a Km (sucrose) near 10 mM and the enzyme did not utilize raffinose, maltose, lactose, or cellibose as a substrate. Impure alkaline invertase preparations, which contained acid invertase activity, on contrast, showed biphasic curves versus sucrose concentration. Combining equal activities of purified alkaline invertase with acid invertase resulted in a biphasic response, but there was a transition to hyperbolic saturation kinetics when the activity ratio, alkaline: acid invertase, was increased above unity. Alkaline invertase activity was inhibited by HgCl2, pridoxal phosphate, and Tris with respective Ki values near 2 microM, 5 microM, and 4 mM. Glycoprotein staining (periodic acid-Schiff method) was negative and alkaline invertase did not bind to two immobilized lectins, concanavalin A and wheat germ agglutinin; hence, the enzyme apparently is not a glycoprotein. The purified alkaline invertase, and a purified soybean acid invertase, was used to raise rabbit polyclonal antibodies. The alkaline invertase antibody preparation was specific for alkaline invertase and cross-reacted with alkaline invertases from other plants. Neither purified soybean alkaline invertases nor the crude enzyme from several plants cross-reacted with the soybean acid invertase antibody.

Differential Protein Composition and Gene Expression in Leaf Mesophyll Cells and Bundle Sheath Cells of the C(4) Plant Digitaria sanguinalis (L.) Scop.

The distribution and molecular weights of cellular proteins in soluble and membrane-associated locations were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining of leaf (Digitaria sanguinalis L. Scop.) extracts and isolated cell extracts. Leaf polypeptides also were pulse-labeled, followed by isolation of the labeled leaf cell types and analysis of the newly synthesized polypeptides in each cell type by electrophoresis and fluorography.Comparison of the electrophoretic patterns of crabgrass whole leaf polypeptides with isolated cell-type polypeptides indicated a difference in protein distribution patterns for the two cell types. The mesophyll cells exhibited a greater allocation of total cellular protein into membrane-associated proteins relative to soluble proteins. In contrast, the bundle sheath cells exhibited a higher percentage of total cellular protein in soluble proteins. Phosphoenolpyruvate carboxylase was the major soluble protein in the mesophyll cell and ribulose bisphosphate carboxylase was the major soluble protein in the bundle sheath cell. The majority of in vivo(35)S-pulse-labeled proteins synthesized by the two crabgrass cell types corresponded in molecular weight to the proteins present in the cell types which were detected by conventional staining techniques. The bundle sheath cell and mesophyll cell fluorograph profiles each had 15 major (35)S-labeled proteins. The major incorporation of (35)S by bundle sheath cells was into products which co-electrophoresed with the large and small subunits of ribulose bisphosphate carboxylase. In contrast, a major (35)S-labeled product in mesophyll cell extracts co-electrophoresed with the subunit of phosphoenolpyruvate carboxylase. Both cell types exhibited equivalent in vivo labeling of a polypeptide with one- and two-dimensional electrophoretic behavior similar to the major apoprotein of the light-harvesting chlorophyll a/b protein. Results from the use of protein synthesis inhibitors during pulse-labeling experiments indicated intercellular differences in both organelle and cytoplasmic protein synthesis. A majority of the (35)S incorporation by crabgrass mesophyll cell 70S ribosomes was associated with a pair of membrane-associated polypeptides of molecular weight 32,000 and 34,500; a comparison of fluorograph and stained gel profiles suggests these products resemble the precursor and mature forms of the maize chloroplast 32,000 dalton protein reported by Grebanier et al. (1978 J. Cell Biol. 28:734-746). In contrast, crabgrass bundle sheath cell organelle translation was directed predominantly into a product which co-electrophoresed with the large subunit of ribulose bisphosphate carboxylase.

Two-Dimensional Electrophoretic Mapping of Proteins of Bundle Sheath and Mesophyll Cells of the C(4) Grass Digitaria sanguinalis (L.) Scop. (Crabgrass).

Two-dimensional electrophoresis was performed on proteins of bundle sheath and mesophyll cells isolated from the C(4) grass Digitaria sanguinalis (L.) Scop. Two-dimensional maps of these proteins were constructed and ribulose-1,5-biphosphate carboxylase and phosphoenolpyruvate carboxylase were identified. Of the total number of proteins found in both cell types, 36% were found only in bundle sheath cells, 17% only in mesophyll cells, and 47% in both cell types. By comparison, the distributions of 48 enzymes assayed in these cell types were 35%, 21%, and 44%, respectively.Protein patterns were also compared with C(4) plants exhibiting different decarboxylation pathways and, in both bundle sheath and mesophyll cells, proteins were found which were unique to each species. Bundle sheath proteins of one C(4) species were found to be more like bundle sheath proteins of another C(4) species than like mesophyll proteins of the same species.

Comparative characterization of phosphoenolpyruvate carboxylase in c(3), c(4), and c(3)-c(4) intermediate panicum species.

Various properties of phosphoenolpyruvate carboxylases were compared in leaf preparations from C(3)-C(4) intermediate, C(3), and C(4)Panicum species. Values of V(max) in micromoles per milligram chlorophyll per hour at pH 8.3 were 57 to 75 for the enzyme from Panicum milioides, Panicum schenckii, and Panicum decipiens (all C(3)-C(4)). The values for Panicum laxum (C(3)) and Panicum prionitis (C(4)) were 20 to 40 and 952 to 1374, respectively. The V(max) values did not change at pH 7.3 except for the C(4) value, which increased about 24%. At pH 8.3, the phosphoenolpyruvate carboxylases from C(3) and C(3)-C(4) species had slightly higher K(m) HCO(3) (-) and lower K(') phosphoenolpyruvate values than did the C(4) enzyme. With each species at pH 7.3, all K(') phosphoenolpyruvate values were 2- to 4-fold greater.The enzyme from all species was inhibited 85 to 90% by 1 millimolar malate at rate-limiting phosphoenolpyruvate and Mg(2+) levels. With low levels of malate, 0.2 millimolar, the rate curve with respect to phosphoenolpyruvate was distinctly sigmoidal, and the inhibition was not eliminated at 5 millimolar phosphoenolpyruvate.Malate at 10 millimolar protected all phosphoenolpyruvate carboxylases from inactivation at 55 C at pH 5.5, but not at pH 8.3. Aspartate did not protect well. When incubated at 37 C at pH 8.3 without phosphoenolpyruvate, but with HCO(3) (-), the enzyme from several C(4) grasses lost 92 to 98% of the initial activity after 4 minutes, whereas the enzymes from C(3) and C(3)-C(4)Panicum species retained 60 to 70% of their activities. In contrast, 5 millimolar phosphoenolpyruvate stabilized the enzyme at 37 C in all plant extracts.The phosphoenolpyruvate carboxylase from C(3)-C(4) intermediate Panicum species has properties most similar to the enzyme from C(3)Panicum species. The higher leaf activity of the enzyme from the intermediate plants than from C(3) species is not due to any unusual property assayed other than a higher V(max.).

Phosphofructokinase activities in photosynthetic organisms : the occurrence of pyrophosphate-dependent 6-phosphofructokinase in plants and algae.

A pyrophosphate-dependent phosphofructokinase (PPi-PFK) activity is detectable in extracts of a wide variety of primitive and advanced plants, the Charalean algae, and in the photosynthetic bacterium, Rhodospirillum rubrum. Angiosperms with extractable PPi-PFK activities 4- to 70-fold higher than the respective ATP-PFK activities tend to be succulent and to exhibit CAM. Even though PPi-PFK activity is not detected in crude extracts of some well known CAM plants, e.g. plants in the Crassulaceae, gel filtration of the extract and/or inclusion of the PPi-PFK activator, fructose 2,6-bisphosphate, in the assay reveals that a PPi-PFK activity is present in these species. Fructose 2,6-bisphosphate likewise activates PPi-PFK activities in extracts of C(3) and C(4) plants. C(3) and C(4) plant PPi-PFK activities are roughly equivalent to ATP-PFK activities in the same species. PPi-PFK activity is also detected in some bryophytes, lower vascular plants, ferns, and gymnosperms. The Charophytes, advanced algae presumed to be similar to species ancestral to vascular plants, exhibit at least 4-fold higher PPi-PFK than ATP-PFK activities. R. rubrum also exhibits a much higher PPi-PFK activity than ATP-PFK activity. These data indicate that PPi-PFK may serve as an alternate enzyme to ATP-PFK in glycolysis in a wide range of photosynthetic organisms.

Diurnal changes in volume and specific tissue weight of crassulacean Acid metabolism plants.

The diurnal variations in volume and in specific weight were determined for green stems and leaves of Crassulacen acid metabolism (CAM) plants. Volume changes were measured by a water displacement method. Diurnal variations occurred in the volume of green CAM tissues. Their volume increased early in the light period reaching a maximum about mid-day, then the volume decreased to a minimum near midnight. The maximum volume increase each day was about 2.7% of the total volume. Control leaves of C(3) and C(4) plants exhibited reverse diurnal volume changes of 0.2 to 0.4%. The hypothesis is presented and supported that green CAM tissues should exhibit a diurnal increase in volume due to the increase of internal gas pressure from CO(2) and O(2) when their stomata are closed. Conversely, the volume should decrease when the gas pressure is decreased.The second hypothesis presented and supported was that the specific weight (milligrams of dry weight per square centimeter of green surface area) of green CAM tissues should increase at night due to the net fixation of CO(2). Green CAM tissues increased their specific weight at night in contrast to control C(3) and C(4) leaves which decreased their specific weight at night. With Kalanchoë daigremontiana leaves, the calculated increase in specific leaf weight at night based on estimates of carbohydrate available for net CO(2) fixation was near 6% and the measured increase in specific leaf weight was 6%.Diurnal measurements of CAM tissue water content were neither coincident nor reciprocal with their diurnal patterns of either volume or specific weight changes.

Inhibition of coral and algal photosynthesis by ca-antagonist phenothiazine drugs.

The effects of various calcium ion antagonists and ion transport inhibitors on photosynthetic O(2) evolution of corals, isolated zooxanthellae, sea anemone tentacles, and Chlorococcum oleofaciens were measured. Only the phenothiazine drugs were effective at inhibiting photosynthesis. Trifluoperazine, a calcium ion antagonist drug, inhibited at low concentrations, with 10(-4) molar and 8 x 10(-6) molar completely abolishing photosynthesis in the intact corals and isolated zooxanthellae, respectively. Net photosynthetic O(2) evolution of C. oleofaciens was eliminated by concentrations of trifluoperazine as low as 2.8 x 10(-5) molar.

Inhibition of oxalacetate decarboxylation during C4 photosynthesis by 3-mercaptopicolin acid.

3-Mercaptopicolinic acid specifically inhibits phosphoenolpyruvate carboxykinase in leaves of the C4 plant Panicum maximum. Both the ATP- and ADP-dependent decarboxylation of oxalacetate and the carboxylation activity of phosphoenolpyruvate carboxykinase are inhibited by 3-mercaptopicolinic acid while phosphoenolpyruvate carboxylase and ribulose-1,5-bisphosphate carboxylase are not inhibited. 3-Mercaptopicolinic acid inhibits the fixation of 14CO2 by illuminated P. maximum bundle sheath strands which is dependent upon oxalacetate and ATP but does not affect C3 photosynthesis in bundle sheath strands nor C4 photosynthesis in mesophyll cells. 3-Mercaptopicolinic acid treatment reduced P. maximum leaf photosynthesis 25% while raising the photosynthetic CO2 compensation point from near zero to 18 to 45 mul of CO2/liter of air.