YscO of Yersinia pestis Is a Mobile Core Component of the Yop Secretion System

The Yersinia pestis low-Ca²⁺ response stimulon is responsible for the temperature- and Ca²⁺-regulated expression and secretion of plasmid pCD1-encoded antihost proteins (V antigen and Yops). We have previously shown that lcrD, yscC, yscD, yscG, and yscR encode proteins that are essential for high-level expression and secretion of V antigen and Yops at 37°C in the absence of Ca²⁺. In this study, we characterized yscO of the Yop secretion (ysc) operon that contains yscN through yscU by determining the localization of its gene product and the phenotype of an in-frame deletion. The yscO mutant grew and expressed the same levels of Yops as the parent at 37°C in the presence of Ca²⁺. In the absence of Ca²⁺, the mutant grew independently of Ca²⁺, expressed only basal levels of V antigen and Yops, and failed to secrete these. These defects could be partially complemented by providing yscO in trans in the yscO mutant. Overexpression of YopM and V antigen in the mutant failed to restore the export of either protein, showing that the mutation had a direct effect on secretion. These results indicated that the yscO gene product is required for high-level expression and secretion of V antigen and Yops. YscO was found by immunoblot analysis in the soluble and membrane fractions of bacteria growing at 37°C irrespective of the presence of Ca²⁺ and in the culture medium in the absence of Ca²⁺. YscO is the only mobile protein identified so far in the Yersinia species that is required for secretion of V antigen and Yops.     

A new species of Sorghastrum (Poaceae) from Isla Socorro, Colima, Mexico

A new species ofSorghastrum,S. pohlianum, from Mexico is described and illustrated. A numerical analysis comparing the new species to the closest species (S. nutans andS. nudipes) was undertaken. The first three principal components explain 84% of the variation of the taxa involved. In addition there is morphological evidence to distinguish the new species from its closest relatives. It differs fromS. nutans by showing smaller inflorescences, having sterile lemmas and anthers, and having somewhat smaller but wider blades and different flowering periods. In addition, it is distinguished fromS. nudipes due to the presence of auricles > 1 mm long and inflorescence branches bearing spikelets along their entire length.     

DESICCATION AND STARVATION TOLERANCE OF ADULT DROSOPHILA: OPPOSITE LATITUDINAL CLINES IN NATURAL POPULATIONS OF THREE DIFFERENT SPECIES

Desiccation and starvation tolerance were measured along latitudinal transects in three Drosophilid species (Drosophila ananassae, D. melanogaster, and Zaprionus indianus) of the Indian subcontinent. In each case, significant latitudinal clines were observed; desiccation tolerance increased with latitude while starvation tolerance decreased. Such field observations suggest that desiccation and starvation tolerance are fitness related traits that are independently selected in nature and genetically independent. It was, however, difficult to relate these genetic changes with precise climatic variables, except winter temperature. The overall negative correlation between the two traits, which was evidenced in natural populations, contrasts with a positive correlation generally observed in various laboratory selection experiments and that also seems to exist between different species. These observations point to the difficulty of interpreting correlations among fitness-related traits when different evolutionary levels are compared, and also different sets of data, that is, field versus laboratory studies.     

The Fibronectin Domain ED-A Is Crucial for Myofibroblastic Phenotype Induction by Transforming Growth Factor-β1

Transforming growth factor-β1 (TGFβ1), a major promoter of myofibroblast differentiation, induces α-smooth muscle (sn) actin, modulates the expression of adhesive receptors, and enhances the synthesis of extracellular matrix (ECM) molecules including ED-A fibronectin (FN), an isoform de novo expressed during wound healing and fibrotic changes. We report here that ED-A FN deposition precedes α-SM actin expression by fibroblasts during granulation tissue evolution in vivo and after TGFβ1 stimulation in vitro. Moreover, there is a correlation between in vitro expression of α-SM actin and ED-A FN in different fibroblastic populations. Seeding fibroblasts on ED-A FN does not elicit per se α-SM actin expression; however, incubation of fibroblasts with the anti-ED-A monoclonal antibody IST-9 specifically blocks the TGFβ1-triggered enhancement of α-SM actin and collagen type I, but not that of plasminogen activator inhibitor-1 mRNA. Interestingly, the same inhibiting action is exerted by the soluble recombinant domain ED-A, but neither of these inhibitory agents alter FN matrix assembly. Our findings indicate that ED-A–containing polymerized FN is necessary for the induction of the myofibroblastic phenotype by TGFβ1 and identify a hitherto unknown mechanism of cytokine-determined gene stimulation based on the generation of an ECM-derived permissive outside in signaling, under the control of the cytokine itself.     

Differential Modulation of SERCA2 Isoforms by Calreticulin

In Xenopus laevis oocytes, overexpression of calreticulin suppresses inositol 1,4,5-trisphosphate-induced Ca²⁺ oscillations in a manner consistent with inhibition of Ca²⁺ uptake into the endoplasmic reticulum. Here we report that the alternatively spliced isoforms of the sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA)2 gene display differential Ca²⁺ wave properties and sensitivity to modulation by calreticulin. We demonstrate by glucosidase inhibition and site-directed mutagenesis that a putative glycosylated residue (N1036) in SERCA2b is critical in determining both the selective targeting of calreticulin to SERCA2b and isoform functional differences. Calreticulin belongs to a novel class of lectin ER chaperones that modulate immature protein folding. In addition to this role, we suggest that these chaperones dynamically modulate the conformation of mature glycoproteins, thereby affecting their function.     

Isolation of Broad-Host-Range Replicons from Marine Sediment Bacteria

Naturally occurring plasmids isolated from heterotrophic bacterial isolates originating from coastal California marine sediments were characterized by analyzing their incompatibility and replication properties. Previously, we reported on the lack of DNA homology between plasmids from the culturable bacterial population of marine sediments and the replicon probes specific for a number of well-characterized incompatibility and replication groups (P. A. Sobecky, T. J. Mincer, M. C. Chang, and D. R. Helinski, Appl. Environ. Microbiol. 63:888–895, 1997). In the present study we isolated 1.8- to 2.3-kb fragments that contain functional replication origins from one relatively large (30-kb) and three small (<10-kb) naturally occurring plasmids present in different marine isolates. 16S rRNA sequence analyses indicated that the four plasmid-bearing marine isolates belonged to the α and γ subclasses of the class Proteobacteria. Three of the marine sediment isolates are related to the γ-3 subclass organisms Vibrio splendidus and Vibrio fischeri, while the fourth isolate may be related to Roseobacter litoralis. Sequence analysis of the plasmid replication regions revealed the presence of features common to replication origins of well-characterized plasmids from clinical bacterial isolates, suggesting that there may be similar mechanisms for plasmid replication initiation in the indigenous plasmids of gram-negative marine sediment bacteria. In addition to replication in Escherichia coli DH5α and C2110, the host ranges of the plasmid replicons, designated repSD41, repSD121, repSD164, and repSD172, extended to marine species belonging to the genera Achromobacter, Pseudomonas, Serratia, and Vibrio. While sequence analysis of repSD41 and repSD121 revealed considerable stretches of homology between the two fragments, these regions do not display incompatibility properties against each other. The replication origin repSD41 was detected in 5% of the culturable plasmid-bearing marine sediment bacterial isolates, whereas the replication origins repSD164 and repSD172 were not detected in any plasmid-bearing bacteria other than the parental isolates. Microbial community DNA extracted from samples collected in November 1995 and June 1997 and amplified by PCR yielded positive signals when they were hybridized with probes specific for repSD41 and repSD172 replication sequences. In contrast, replication sequences specific for repSD164 were not detected in the DNA extracted from marine sediment microbial communities.  The maintenance and horizontal transfer of extrachromosomal elements provide one mechanism by which microbial communities can rapidly adapt to changes in environmental conditions. This adaptation can be in the form of plasmid rearrangements and duplications (18, 40), a change in the plasmid copy number (40, 54), or lateral or horizontal movement of plasmids within bacterial populations. An example demonstrating the importance of plasmid-mediated genetic adaptation in natural microbial communities, likely caused by lateral transfer, is the increased frequencies (2- to 10-fold) of catabolic plasmids reported in bacterial isolates obtained from polluted marine and freshwater environments compared to isolates from nonpolluted or less impacted ecosystems (8, 23, 43). Plasmids also play a major role in promoting the widespread distribution of antibiotic resistance genes attributed to the intense and increased use of antibiotics (42).The ability of plasmids to self-transfer or to be mobilized by transfer-proficient plasmids and the ability to replicate in different bacterial hosts are key factors in the spread of plasmid-encoded genes within microbial communities. Plasmids which are considered to have broad host ranges in nature have the potential to significantly affect the microbial community structure and function due to their ability to replicate and be maintained in members of distantly related genera. Thus, to better understand gene flux in natural systems and hence the potential role of plasmids in promoting horizontal transfer within microbial communities, knowledge of the distribution, diversity, and host ranges of naturally occurring plasmids is necessary.At present, most indigenous plasmids from marine and freshwater systems have been only partially characterized with respect to host range, replication mechanisms, incompatibility groups, and conjugal abilities. Plasmids containing similar or related replication systems are considered incompatible if they cannot coexist in a host cell (12, 41). This trait has facilitated the grouping of plasmids from gram-negative bacteria, mainly members of the family Enterobacteriaceae, into more than 30 different incompatibility groups (3). While molecularly based plasmid classification or replicon typing by using DNA sequences of replication origins and incompatibility loci of well-characterized plasmids has been useful in classifying plasmids from bacterial isolates of medical importance (9, 10, 14), plasmids from various marine microbial communities, including sediments, biofilms, bulk water, and the marine air-water interface, have been recently shown to contain incompatibility and replication regions unrelated to those currently defined (11, 53).The present study was undertaken to characterize, at the molecular level, the replication and incompatibility loci of naturally occurring plasmids isolated from gram-negative marine heterotrophs for use as replicon probes to classify and type, at the molecular level, plasmids present in bacterial populations of marine sediments. Replication origins were obtained from plasmids ranging in size from 6 to 30 kb isolated from culturable bacteria of coastal California marine sediments (53). Phylogenetic analysis indicated that the plasmids were initially isolated from bacteria belonging to the α and γ-3 subclasses of the class Proteobacteria. Although a sequence and hybridization analysis of the replication origins from the marine plasmids confirmed the lack of homology with previously described plasmids, the replication regions contained features commonly found in previously characterized plasmid replication origins. The replication origins of the naturally occurring plasmids appear to have a broad host range, as indicated by their ability to replicate in members of diverse gram-negative marine genera. In addition to molecular characterization of the indigenous plasmids, the persistence of the replicons in marine sediment bacterial populations was determined by PCR amplification of microbial community DNA extracted on different dates and examined for the presence of homologous plasmid replication sequences.     

Region-Specific Targets of p42/p44MAPK Signaling in Rat Brain

Abstract: In vitro studies indicate that p42/p44^MAPK phosphorylate both nuclear and cytoplasmic proteins. However, the functional targets of p42/p44^MAPK activation in vivo remain unclear. To address this question, we localized activated p42/p44^MAPK in hippocampus and cortex and determined their signaling effects after electroconvulsive shock treatment (ECT) in rats. Phosphorylated p42/p44^MAPK content increased in the cytoplasm of hippocampal neurons in response to ECT. Consistent with this cytoplasmic localization, inhibition of ECT-induced p42/p44^MAPK activation by the extracellular signal-regulated kinase kinase inhibitor PD098059 blocked phosphorylation of the cytoplasmic protein microtubule-associated protein 2c (MAP2c), but failed to inhibit the induction of the nuclear protein c-Fos in response to ECT. In contrast to hippocampal neurons, cortical neurons exhibited an increase in amount of phosphorylated p42/p44^MAPK in both the nucleus and cytoplasm after ECT. Accordingly, PD098059 blocked the induction of Fos-like immunoreactivity in the nuclei of cortical neurons as well as MAP2c phosphorylation in the cytoplasm. Our data indicate that both nuclear and cytoplasmic substrates can be activated by p42/p44^MAPK in vivo. However, the functional targets of p42/p44^MAPK signaling depend on the precise location of p42/p44^MAPK within different subcellular compartments of brain regions. These results indicate unique functional pathways of p42/p44^MAPK-mediated signal transduction within different brain regions in vivo.     

Repeated Cocaine Administration Alters Extracellular Glutamate in the Ventral Tegmental Area

Abstract: The present study determined if repeated cocaine injections alter the effect of cocaine on extracellular glutamate in the ventral tegmental area (VTA). All rats were treated with daily cocaine (15 mg/kg i.p. × 2 days, 30 mg/kg i.p. × 5 days) or saline for 7 days. At 21 days after discontinuing the daily injections, a dialysis probe was placed into the VTA and the extracellular levels of glutamate were estimated. A systemic injection of cocaine (15 mg/kg i.p.) elevated extracellular glutamate in the VTA of rats pretreated with daily cocaine but not in the daily saline-pretreated subjects. No significant change in glutamate was produced by a saline injection in either pretreatment group. In a group of rats pretreated with daily cocaine, the D₁ antagonist SCH-23390 (30 µ M ) was infused through the dialysis probe prior to the acute injections of saline and cocaine. SCH-23390 prevented the increase in extracellular glutamate associated with the acute administration of cocaine. Behavioral data were collected simultaneously with the measures of extracellular glutamate. The behavioral stimulant effect of cocaine was greater in cocaine-pretreated than saline-pretreated subjects, and the behavioral augmentation in cocaine-pretreated rats was partly blocked by SCH-23390. These data support the hypotheses that repeated cocaine administration produces an increase in the capacity of D₁ receptor stimulation to release glutamate in the VTA and that this mechanism partly mediates behavioral sensitization produced in rats treated with daily cocaine injections.