Niche-specificity and the variable fraction of the Pectobacterium pan-genome.

We compare genome sequences of three closely related soft-rot pathogens that vary in host range and geographical distribution to identify genetic differences that could account for lifestyle differences. The isolates compared, Pectobacterium atrosepticum SCRI1043, P. carotovorum WPP14, and P. brasiliensis 1692, represent diverse lineages of the genus. P. carotovorum and P. brasiliensis genome contigs, generated by 454 pyrosequencing ordered by reference to the previously published complete circular chromosome of P. atrosepticum genome and each other, account for 96% of the predicted genome size. Orthologous proteins encoded by P. carotovorum and P. brasiliensis are approximately 95% identical to each other and 92% identical to P. atrosepticum. Multiple alignment using Mauve identified a core genome of 3.9 Mb conserved among these Pectobacterium spp. Each core genome is interrupted at many points by species-specific insertions or deletions (indels) that account for approximately 0.9 to 1.1 Mb. We demonstrate that the presence of a hrpK-like type III secretion system-dependent effector protein in P. carotovorum and P. brasiliensis and its absence from P. atrosepticum is insufficient to explain variability in their response to infection in a plant. Additional genes that vary among these species include those encoding peptide toxin production, enzyme production, secretion proteins, and antibiotic production, as well as differences in more general aspects of gene regulation and metabolism that may be relevant to pathogenicity.

Performance of a high-rate/high-shear activated sludge bioreactor treating biodegradable wastewater.

A high-rate activated sludge (HRAS) bioreactor with high shear venturi aeration was operated in the laboratory at various organic loading rates to evaluate chemical oxygen demand (COD) removal efficiency and sludge production. Organic loading rates two orders of magnitude higher than conventional activated sludge loading were investigated in the modified HRAS reactor for a period of 41 weeks. Filtered COD removal efficiency varied from 81 to 92 % for organic loading rates of 3 to 85 kg COD m(-3) d(-1). Observed sludge yield was determined to be 0.10-0.25 g TSS produced g(-1) COD removed, under hydraulic and solids retention times (SRTs) approaching less than two hours and two days, respectively. Observed sludge yield actually declined as loading increased and SRT decreased. It was concluded that high-shear forces created in the reactor due to intense aeration at high volumetric organic loading rates increased substrate utilization rate, improved filtered COD removal efficiency, and kept sludge yields relatively low (despite the very low operating SRTs).

CaM kinase signaling induces cardiac hypertrophy and activates the MEF2 transcription factor in vivo.

Hypertrophic growth is an adaptive response of the heart to diverse pathological stimuli and is characterized by cardiomyocyte enlargement, sarcomere assembly, and activation of a fetal program of cardiac gene expression. A variety of Ca(2+)-dependent signal transduction pathways have been implicated in cardiac hypertrophy, but whether these pathways are independent or interdependent and whether there is specificity among them are unclear. Previously, we showed that activation of the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin or its target transcription factor NFAT3 was sufficient to evoke myocardial hypertrophy in vivo. Here, we show that activated Ca(2+)/calmodulin-dependent protein kinases-I and -IV (CaMKI and CaMKIV) also induce hypertrophic responses in cardiomyocytes in vitro and that CaMKIV overexpressing mice develop cardiac hypertrophy with increased left ventricular end-diastolic diameter and decreased fractional shortening. Crossing this transgenic line with mice expressing a constitutively activated form of NFAT3 revealed synergy between these signaling pathways. We further show that CaMKIV activates the transcription factor MEF2 through a posttranslational mechanism in the hypertrophic heart in vivo. Activated calcineurin is a less efficient activator of MEF2-dependent transcription, suggesting that the calcineurin/NFAT and CaMK/MEF2 pathways act in parallel. These findings identify MEF2 as a downstream target for CaMK signaling in the hypertrophic heart and suggest that the CaMK and calcineurin pathways preferentially target different transcription factors to induce cardiac hypertrophy.

Isolation of Y chromosome-specific sequences from Silene latifolia and mapping of male sex-determining genes using representational difference analysis.

The genomic subtraction method representational difference analysis (RDA) was used to identify male-specific restriction fragments in the dioecious plant Silene latifolia. Male-specific restriction fragments are linked to the male sex chromosome (the Y chromosome). Four RDA-derived male-specific restriction fragments were used to identify polymorphisms in a collection of X-ray-generated mutant plants with either hermaphroditic or asexual flowers. Some of the mutants have cytologically detectable deletions in the Y chromosome that were correlated with loss of male-specific restriction fragments. One RDA-derived probe detected a restriction fragment present in all mutants, indicating that each has retained Y chromosomal DNA. The other three probes detected genomic fragments that were linked in a region deleted in some hermaphroditic and some asexual mutants. Based on the mutant phenotypes and the correlation of cytologically visible deletions with loss of male-specific restriction fragments, these markers were assigned to positions on the Y chromosome close to the carpel suppression locus. This RDA mapping also revealed a Y-linked locus, not previously described, which is responsible for early stamen development.

Modulation of oligosaccharide processing in an exocrine secretory glycoprotein of rat parotid cells by beta-adrenoreceptor activation.

Such stimulation of rat parotid acinar cells in vitro modulated the rate of processing of N-linked oligosaccharides in a high-molecular weight (220 kdalton) secretory glycoprotein. Conversion of polymannose-type oligosaccharides to complex-type oligosaccharides was evaluated by sensitivity to endoglucosaminidase H and alpha-mannosidase, and with a specific inhibitor of glucosidases I/II. Oligosaccharide maturation in the 220 kdalton glycoprotein required one-third to half less time in cells exposed to the beta-adrenergic agonist isoproterenol than in controls.

Factors associated with improved biochemical response to neoadjuvant androgen deprivation therapy before definitive radiation therapy in prostate cancer patients.

BACKGROUND: In prostate cancer patients treated with androgen deprivation therapy (ADT) and radiation therapy (RT), a pre-RT PSA level 0.5 ng ml(-1), determined after neoadjuvant ADT and before RT, predicts for worse survival measures. The present study sought to identify patient, tumor and treatment characteristics associated with the pre-RT PSA in prostate cancer patients. METHODS: We reviewed the charts of all patients diagnosed with intermediate- and high-risk prostate cancer and treated with a combination of neoadjuvant (median, 2.2 and 2.5 months, respectively), concurrent, and adjuvant ADT and RT between 1990 and 2011. RESULTS: A total of 170 intermediate- and 283 high-risk patients met inclusion criteria. On multivariate analysis, both intermediate- and high-risk patients with higher pre-treatment PSA (iPSA) were significantly less likely to achieve a pre-RT PSA <0.5 ng ml(-1) (iPSA 10.1-20 ng ml(-1): P=0.005 for intermediate risk; iPSA 10.1-20 ng ml(-1): P=0.005, iPSA >20 ng ml(-1): P<0.001 for high risk). High-risk patients undergoing total androgen blockade were more likely to achieve a pre-RT PSA <0.5 ng ml(-1) (P=0.031). We observed an interaction between race and type of neoadjuvant ADT (P=0.074); whereas African-American men on total androgen blockade reached pre-RT PSA <0.5 ng ml(-1) as frequently as other men on total androgen blockade (P=0.999), African-American men on luteinizing hormone-releasing hormone (LH-RH) agonist monotherapy/orchiectomy were significantly less likely to reach pre-RT PSA <0.5 ng ml(-1) compared with other men on LH-RH monotherapy/orchiectomy (P=0.001). CONCLUSIONS: Our findings suggest that total androgen blockade in the neoadjuvant period may be beneficial compared with LH-RH monotherapy for achieving a pre-RT PSA <0.5 ng ml(-1) in African-American men with high-risk prostate cancer. In addition, men with higher iPSA are more likely to have a pre-RT PSA greater than 0.5 ng ml(-1) in response to neoadjuvant ADT and are therefore candidates for clinical trials testing newer, more aggressive hormone-ablative therapies.

Relationship between oligosaccharide-lipid synthesis and protein synthesis in mouse LM cells.

Previous studies from several laboratories have reported that inhibition of protein synthesis results in a concomitant reduction in synthesis of oligosaccharide-diphosphoryldolichol. We have investigated this phenomenon in LM cells grown in defined culture medium. The results of this study indicate that incubation of LM cell with cycloheximide at a concentration sufficient to totally arrest polypeptide synthesis, results in a rapid reduction in the synthesis of [3H]mannose-labeled or [3H]glucosamine-labeled oligosaccharide-lipid within 2 min. Cycloheximide treatment had only a slight inhibitory effect on synthesis of GDP-Man. Furthermore, during the first 5 min after the addition of cycloheximide to LM cells, when [3H]mannose incorporation into oligosaccharide-lipid was maximally reduced, the specific activity of the GDP-Man pool was identical to that observed in control cells. Labeling in vivo of the lipid-linked saccharide precursors of oligosaccharide-lipid revealed that cycloheximide had virtually no effect on the synthesis of Man-P-Dol, GlcNAc-PP-Dol, GlcNAc2-PP-Dol, and beta-Man-(GlcNAc)2-PP-Dol (Dol = dolichol). In agreement with this finding, results of experiments in vitro using microsomes prepared from LM cells indicate that cycloheximide did not directly inhibit the enzymes responsible for the synthesis of these lipid-linked saccharide precursors. Supplementation of mouse LM cells by preincubation for 1 h with dolichyl phosphate (5 micrograms/ml) resulted in a 300% stimulation of oligosaccharide-lipid synthesis when compared to non-supplemented cells. However, dolichyl phosphate supplementation of cycloheximide-treated cells failed to restore oligosaccharide-lipid synthesis to the level observed in control cells. In control cells pre-labeled oligosaccharide-lipid turned over rapidly and, as expected, cycloheximide addition to the chase medium significantly retarded this turnover process. However, preincubation of cells with exogenous dolichyl phosphate had little or no effect on the turnover of oligosaccharide-lipid in control or cycloheximide-treated cells. These findings argue against dolichyl phosphate deficiency as the primary cause of reduced oligosaccharide-lipid synthesis when protein synthesis is blocked. The implications of these results, and an alternative hypothesis to explain the effect of inhibition of protein synthesis on oligosaccharide-lipid synthesis based on elevation of intracellular GTP levels, are discussed.

Functional cis-element sequence requirements for suppression of gene expression by the TNPA protein of the Zea mays transposon En/Spm.

TNPA, one of the two transposition proteins encoded by the En/Spm transposable elements of Zea mays, suppresses the expression of genes that contain an appropriate cis element. Suppression can be monitored in tobacco protoplasts in a transient expression assay as follows. The plant promoter-driven expression of the Escherichia coli-glucuronidase (GUS)-encoding gene, uidA, is repressed in the presence of TNPA if the GUS gene contains a functional cis element in the untranslated RNA leader sequence. Earlier, we found that the minimal cis element is composed of two 12 bp sequences in a tail-to-tail inverted orientation. Each 12 bp sequence is sufficient to bind TNPA in vitro and can be thought of as a half-site in the cis element. Here, we investigated the sequence requirements of the minimal cis element. Our observations support our expectations that a functional cis element must provide a template to which two TNPA molecules can bind in the correct orientation. Sequences within the half-sites can be altered as long as the eight bases that make up the consensus binding sites are not changed. However, we found the following unexpected sequence specificities. Firstly, some changes to the consensus binding sequence can be tolerated in one half-site, as long as the other site matches the consensus. Secondly, although the region between the half-sites can vary in sequence and in length between two and four bases, a thymidine residue is not tolerated directly 5' preceding the second half-site.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the cell surface receptors for human B cell growth factor of 12,000 molecular weight.

Quiescent normal human B cells have been shown to require an activation step before proliferating in response to B cell growth factor (BCGF) of 12,000 m.w. (12 kd). One effect of cell activation has been the putative acquisition of specific cell surface growth factor receptors. In this report, the existence of such receptors has been confirmed by using purified radioiodinated BCGF-12 kd. BCGF-12 kd receptors on activated B cells have been shown to be distinct form those interacting with IL 2. Scatchard analysis revealed both high affinity receptor sites with an apparent Kd of 28.6 pM and low affinity receptor sites with Kd of 1.2 nM on freshly prepared, anti-IgM activated peripheral blood B cells. Human B cells grown in culture for extended periods of time in the presence of BCGF-12 kd also displayed high affinity receptor sites (Kd, 41.4 pM) and low affinity receptor sites (Kd, 0.9 nM). The action of BCGF-12 kd therefore appears to be mediated by binding to its lineage-specific receptors on the cell surface.

beta-Adrenergic receptor regulation of N-linked protein glycosylation in rat parotid acinar cells.

We have investigated the relationship between beta-adrenergic receptor stimulation and protein glycosylation and secretion in rat parotid gland cells in vitro. The potent beta-adrenergic agonist (-)-isoproterenol increases [3H]mannose incorporation into newly synthesized glycoproteins. This effect is enhanced if cells are first preincubated with dolichyl phosphate and is not observed after muscarinic-cholinergic or alpha-adrenergic stimulation of cells. The increase in [3H]mannose incorporation is abolished by incubation of cells with tunicamycin, suggesting that the glycosylation events being studied involved asparagine-linked oligosaccharides. The extent of increase in glycosylation is dependent on the concentration of (-)-isoproterenol to which cells are exposed. (+/-)-Propanolol totally abolishes the (-)-isoproterenol-induced increase in [3H]mannose incorporation, in a manner similar to its effects on exocrine secretion. Our findings suggest that beta-adrenergic receptor activation has a profound influence on N-linked protein glycosylation in rat parotid cells in addition to eliciting exocrine protein release.

Dolichyl phosphate supplementation increases N-linked protein glycosylation in rat parotid acinar cells without increasing glycoprotein secretion.

N-linked protein glycosylation was increased three- to five-fold, in dispersed rat parotid acinar cells in vitro, by supplementation with exogenous dolichylphosphate. Despite this increase, glycoprotein secretion from both control and dolichylphosphate-supplemented cells was comparable.

En/Spm encoded tnpA protein requires a specific target sequence for suppression.

The En/Spm encoded suppressor function has been reconstituted in transgenic tobacco protoplasts. The suppressor affects genes which contain an En/Spm responsive transposable element in the transcribed sequences. The En/Spm encoded protein tnpA binds a defined cis element in the inserted transposon, repressing expression of the adjacent gene. This was shown by monitoring transient expression of a bacterial marker gene (GUS) expressed from a strong plant viral promoter. Suppressible variants of the marker gene were produced by inserting I element sequences into the untranslated sequences of the GUS transcript. Comparison of transient expression of these variants in wildtype tobacco protoplasts with their expression in protoplasts transgenic for tnpA protein demonstrates that tnpA is the suppressor. In addition, the minimal cis element required for suppression has been defined as a dimer consisting of two 12 bp tnpA binding sequences in a particular inverted orientation. One of these dimers occurs in each En/Spm end close to the characteristic 13 bp terminal inverted repeat. TnpA binding sites in different arrangements do not respond as well to tnpA. The implications of this observation are discussed. This system can be used to analyse tnpA-DNA interactions involved in gene regulation further.

Tetracycline-inducible CaM kinase II silences hypertrophy-sensitive gene expression in rat neonate cardiomyocytes.

Recent work from this laboratory both in rat primary cardiomyocytes and in ventricular tissue of transgenic mouse models of induced hypertrophy has identified two Ca(2+)/calmodulin-dependent nuclear signaling cascades. The first involves the phosphatase calcineurin (CaN). The second is the CaM kinase kinase cascade which involves CaM kinase I and CaM kinase IV. Each of these signaling cascades strongly up-regulate transcription of hypertrophy-sensitive genes in the rat ventricular cardiomyocyte. We have documented that over-expression of an active form of CaM kinase II silenced transcriptional induction of hypertrophy-sensitive genes. The purpose of this study was to generate an inducible CaM kinase II expression system and correlate its expression with the silencing of hypertrophic-sensitive reporters. A truncated form of CaM KII, CaM KII (1-290) was subcloned downstream and proximal to a promoter under transcriptional control (induction) of the tetracycline-regulated transcription factor, tet-TransActivator (tTA). Hypertrophy-sensitive reporter activity in primary cardiomyocytes was silenced when tet-inducible CaM KII was co-expressed with plasmids harboring active forms of CaN, CaM KI or CaM KIV. For instance, induced CaM KII expression silenced CaN, CaM kinase I, or CaM kinase IV driven ANF reporter activity 4.9-, 2.9-, and 6.9-fold below their maximal values, respectively. Myocyte exposure to doxycycline (DOX) blocked tTA-driven CaM KII expression and restored CaN/CaM KI or CaN/CaM KIV driven reporter activation. This study demonstrates, for the first time, that active CaM KII silences Ca(2+)-sensitive nuclear signaling cascades for transcriptional up-regulation of cardiomyocyte hypertrophy.

Multicolor FISH mapping of the dioecious model plant, Silene latifolia.

Silene latifolia is a key plant model in the study of sex determination and sex chromosome evolution. Current studies have been based on genetic mapping of the sequences linked to sex chromosomes with analysis of their characters and relative positions on the X and Y chromosomes. Until recently, very few DNA sequences have been physically mapped to the sex chromosomes of S. latifolia. We have carried out multicolor fluorescent in situ hybridization (FISH) analysis of S. latifolia chromosomes based on the presence and intensity of FISH signals on individual chromosomes. We have generated new markers by constructing and screening a sample bacterial artificial chromosome (BAC) library for appropriate FISH probes. Five newly isolated BAC clones yielded discrete signals on the chromosomes: two were specific for one autosome pair and three hybridized preferentially to the sex chromosomes. We present the FISH hybridization patterns of these five BAC inserts together with previously described repetitive sequences (X-43.1, 25S rDNA and 5S rDNA) and use them to analyze the S. latifolia karyotype. The autosomes of S. latifolia are difficult to distinguish based on their relative arm lengths. Using one BAC insert and the three repetitive sequences, we have constructed a standard FISH karyotype that can be used to distinguish all autosome pairs. We also analyze the hybridization patterns of these sequences on the sex chromosomes and discuss the utility of the karyotype mapping strategy presented to study sex chromosome evolution and Y chromosome degeneration.

Oligosaccharyltransferase activity is markedly increased during differentiation of a nonfusing myoblast cell line.

We have studied several aspects of glycoprotein synthesis in myoblast differentiation by using a nonfusing myoblast cell line, BC3H1. Previous studies showed that transfer of proliferating undifferentiated BC3H1 cells to mitogen-depleted medium results in the cells' withdrawal from the cell cycle and induction of a variety of muscle-specific gene products [E. N. Olson, L. Glaser, J. P. Merlie, R. Sebane, and J. Lindstrom (1983) J. Biol. Chem. 258, 13946-13953]. Because cell surface glycoproteins have been implicated in myoblast differentiation, in the present study we measured the amount of oligosaccharyltransferase in microsomes isolated from BC3H1 cells at various stages of differentiation. By using an acceptor peptide containing the sequence-Asn-Leu-Thr-, enzyme activity was measured by formation of [3H]glycopeptide. In addition, active enzyme protein was measured with a 125I-labeled photoreactive derivative of the acceptor tripeptide. Both of these independent assay methods revealed a marked increase in oligosaccharyltransferase when differentiation was induced by serum depletion. Moreover, mitogenic stimulation of differentiated cells resulted in a return of oligosaccharyltransferase to near basal levels. This reversible increase in this key enzyme in protein glycosylation occurred despite the fact that both total protein and glycoprotein synthesis were depressed during differentiation. These data indicate that during myogenesis the level of oligosaccharyltransferase is regulated in parallel with a number of muscle-specific gene products. These results are discussed in the context of regulation of the pathway of glycoprotein synthesis.

Production, purification, and characterization of human recombinant IL-8 from the eucaryotic vector expression system baculovirus.

The cDNA for the human chemotactic interleukin, IL-8, was subcloned from a bacterial source into the eucaryotic vector expression system baculovirus. Recombinant human IL-8 (rhIL-8) was synthesized and secreted from Sf9 cells derived from Spodoptera frugiperda following infection of a recombinant virus harboring the full-length IL-8 structural gene. Infected Sf9 cells produced rhIL-8 in a range from 0.5 to 2.0 mg of rhIL-8/liter of postinfection cell culture media. The recombinant interleukin was purified (greater than 600-fold) to homogeneity using preparative HPLC. rhIL-8 retained all of the physical, immunological, and biochemical properties observed for the natural product, monocyte-derived IL-8. rhIL-8 was assessed for biological efficacy by three criteria: (a) ability to induce chemotaxis in human neutrophils, (b) ability to induce oxygen burst metabolism, and (c) ability to be recognized by purified rabbit antibody generated against monocyte-derived IL-8. Antibody generated against monocyte-derived IL-8 recognized rhIL-8 isolated during all stages of the purification protocol. rhIL-8 was strongly chemotactic for human neutrophils and exhibited a chemotactic index comparable to that reported for other strong chemotactic peptides. rhIL-8 was identified following sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single silver-stained band having an estimated molecular weight of 9.2 kDa and displayed amino acid residue molar abundance homology predicted for the mature form of the interleukin. Baculovirus vector expression coupled to preparative HPLC proved to be a very efficient method for large-scale recombinant interleukin production.

Prenatal diagnosis and outcome in sacrococcygeal teratomas: a review of cases between 1992 and 1998.

A review of sacrococcygeal teratomas diagnosed in the antenatal period in the West Midlands region over a six year interval is reported. The aim of the study was to assess the contribution of ultrasound scanning to the management of cases and to determine the outcome of prenatally diagnosed sacrococcygeal teratomas. A retrospective review of 10 cases was performed to obtain pregnancy details, ultrasound scan data and outcome information. Two fetuses were electively aborted. Perinatal mortality was 62.5% in the remaining cases with all stillbirths and neonatal deaths occurring in babies delivered preterm (at or before 34 weeks' gestation). Marked increase in tumour size (mainly vascular/solid) was observed in five of the fetuses, which was often associated with local compression effects and the development of hydrops. Eight out of 10 cases were delivered vaginally, one following aspiration of the large cystic tumour. Three of the four neonates surviving to surgery underwent successful resection of their benign tumours. As well as guiding prognosis, serial ultrasound scans may also allow the mode of delivery to be planned more effectively. The importance of a multidisciplinary team approach to these difficult cases is emphasized.

Conserved expression of a TASSELSEED2 homolog in the tapetum of the dioecious Silene latifolia and Arabidopsis thaliana.

To investigate the genetics of male sex determination and stamen development in the dioecious plant Silene latifolia (white campion), male-specific transcripts were isolated from developing flowers by cDNA subtraction. One of the cDNAs identified, STA1, had high DNA and amino acid sequence homology to the male sex determining gene of Zea mays (maize), TASSELSEED2. Both genes are expressed in male and not in female flowers, However, they do not share the same expression pattern. The TASSELSEED2 gene product is expressed in the gynoecium primordia of male maize flowers where it is necessary for pistil abortion. STA1 is not expressed in the gynoecium primordia of male white campion and therefore its gene product cannot perform the same function in sex determination that TASSELSEED2 performs in maize. STA1 is expressed in tapetal cells of white campion male flowers and of white campion hermaphroditic mutants. A homologous gene is also expressed in the tapetum of hermaphroditic Silene species. Tapetal expression of a homologous gene (named ATA1) was also found in Arabidopsis thaliana. The similarity in primary sequence and expression pattern of STA1 and ATA1 indicate that these genes have a conserved role in tapetum development.

Technical advance: single pollen typing combined with laser-mediated manipulation.

We combined single pollen typing with laser-mediated manipulation. After drilling a hole in the wall of a pollen grain from a dioecious plant (Silene latifolia) with a UV-laser microbeam, the single pollen grain was recovered directly in the cap of a PCR tube, using a non-contact method called laser pressure catapulting. The entire genome of the single pollen grain was then amplified with improved primer-extension-preamplification PCR (I-PEP PCR). Nested PCR with sequence tagged site (STS)-specific primers was used to analyze several loci in the haploid genome. The single copy gene MROS1 was detected in most of the single pollen grains analyzed. Bgl10, which is localized on the Y chromosome, was detected in approximately half of the pollen grains. MROS3 is reported to be localized on the X chromosome. Using inverse PCR, we isolated two genomic clones of MROS3: MROS3A and MROS3B. The single pollen analysis using nested PCR showed that MROS3A and MROS3B are derived from different loci that are not located on the X chromosome. Single pollen typing not only reveals sex chromosome-linkage within the haploid genome, but can also discriminate between alleles and different loci. This method should also be useful for measuring recombination frequencies without genetic crossover analysis.