Phenotypic modulation of cultured bladder smooth muscle cells and the expression of inducible nitric oxide synthase

Phenotypic modulation of smooth muscle is associated with various pathological conditions, including bladder dysfunction. Cytoskeletal dynamics modulate the cell phenotype and were recently shown to be involved in regulation of inducible nitric oxide synthase (iNOS). We tested the hypothesis that the cell differentiation status affects iNOS expression, and that iNOS is preferentially expressed in immature dedifferentiated bladder smooth muscle cells (BSMC). Isolated at BSMC were put into different stages of differentiation by serum deprivation on laminin-coated plates in the presence of IGF-I and by interaction with Rho signaling and actin polymerization. iNOS and smooth muscle-myosin heavy chain (SM-MHC) protein expression were investigated with Western blot analysis. Our results showed iNOS protein in BSMC exposed to interleukin-1 beta (2 ng/ml) + TNF-alpha (50 ng/ml). Growth of BSMC in serum-free medium on laminin in the presence of IGF-I increased SM-MHC expression, whereas cytokine-induced iNOS was inhibited. Disruption of F-actin with latrunculin B (0.5 microM) potentiated iNOS expression and decreased SM-MHC expression. Rho inhibition with C3 (2.5 microg/ml) increased iNOS expression, whereas SM-MHC expression was slightly decreased. Rho-kinase inhibition with Y-27632 (10 microM) mediated a decrease in iNOS and a slight increase in SM-MHC expression. In conclusion, the capacity of BSMC to express iNOS was negatively correlated to differentiation status measured as SM-MHC expression. Actin cytoskeletal dynamics and Rho signaling are involved in regulation of cytokine-induced iNOS expression in BSMC. Phenotypic changes and impairment in actin cytoskeleton formation may potentiate cytokine activation and in turn increase nitric oxide production in the bladder during disease.     

A drop of intracellular pH stimulates citric acid accumulation by some strains of Aspergillus niger

By comparing kinetic parameters of plasma membrane proton pumps from two Aspergillus niger strains, significant differences in specific activities were observed. In low citric acid producing A158 strain the H+ -ATPase activity was about four-fold higher than in a high yielding A60 strain. Previously pH homeostasis was reported in A158 strain while in A60 strain spontaneous drop of intracellular pH was observed. During the growth in the medium with ammonium ions more rapid drop of extracellular pH was recorded with A158 strain and not so fast proton accumulation in the medium with A60 strain, indicating that proton pumps from later strain perhaps can not extrude all the protons that are released in the cytosol after the assimilation of ammonium ions. Vanadium ions were found to be potent inhibitors of both H+ -ATPases. By adding sodium vanadate in millimolar concentrations to the chemically defined medium that induces citric acid accumulation by A. niger, reduced pHi and increased rate of acid production was observed in A158 strain while in A60 strain intracellular pH decreased below 6.5 and concomitantly citric acid overflow was suppressed. The presented results suggest that one of the mechanisms stimulating citric acid accumulation by A. niger could be also a slight cytoplasmic acidification.     

Sex chromosome evolution and speciation in Ficedula flycatchers

Speciation is the combination of evolutionary processes that leads to the reproductive isolation of different populations. We investigate the significance of sex-chromosome evolution on the development of post- and prezygotic isolation in two naturally hybridizing Ficedula flycatcher species. Applying a tag-array-based mini-sequencing assay to genotype single nucleotide polymorphisms (SNPs) and interspecific substitutions, we demonstrate rather extensive hybridization and backcrossing in sympatry. However, gene flow across the partial postzygotic barrier (introgression) is almost exclusively restricted to autosomal loci, suggesting strong selection against introgression of sex-linked genes. In addition to this partial postzygotic barrier, character displacement of male plumage characteristics has previously been shown to reinforce prezygotic isolation in these birds. We show that male plumage traits involved in reinforcing prezygotic isolation are sex linked. These results suggest a major role of sex-chromosome evolution in mediating post- and prezygotic barriers to gene flow and point to a causal link in the development of the two forms of reproductive isolation.     

Physical Mapping of the Bacillus thuringiensis subsp. kurstaki and alesti Chromosomes

Two strains of the well-known insect pathogen and biopesticide, Bacillus thuringiensis (Bt), belonging to subspecies alesti (strain Bt5) and kurstaki (strain Bt213), were chosen for genetic characterization. The two strains belong to different serotypes and are currently classified into different subspecies, although their insecticidal activity is similar. Physical maps were constructed of Bt alesti and Bt kurstaki using Pulsed Field Gel Electrophoreses (PFGE), and the map positions of several genes were determined. The 5.5 Mb combined genetic and physical chromosome maps of the two strains were found to be indistinguishable, and the only differences detected between the strains were of extrachromosomal origin. A cryIA toxin gene probe hybridised to a chromosome fragment and to two extrachromosomal elements in both strains, migrating as 100 kb and 350 kb, respectively. In addition a cry hybridizing extrachromosomal element migrating as 80 kb was present only in Bt alesti. Both strains were also found to contain sequences hybridizing to an enterotoxin (hbla) gene probe. Such sequences were positioned on the 350 kb extrachromosomal element, as well as on the chromosome. Received: 20 April 2001 / Accepted: 29 May 2001     

Apoptosis within bovine follicular cells and its effect on oocyte development during in vitro maturation

Developmental competence of oocytes is compromised if they originate from atretic follicles. Apoptosis is the underlying process of atresia. Apoptotic changes in follicular cells are thought to influence the outcome of IVF. The aim of this study was to investigate apoptosis in different compartments of single bovine follicles (follicular wall, granulosa and cumulus cells (CC)) in relation to COC morphology, and to determine whether the addition, in vitro, of exogenous follicular cells from atretic follicles to maturing cumulus oocyte complexes (COCs) influenced the development of oocytes.Antral follicles were dissected from bovine ovaries and opened to obtain COCs and free floating granulosa cells (GC). The COCs were classified according to morphology. Apoptosis was determined in cumulus and granulosa cells and in homogenates of the remaining follicular wall.For every morphological class of COCs, a large variability of apoptotic expression was found in all follicle compartments. Follicular wall apoptosis was not correlated to COC morphology or to the percentage of apoptotic granulosa or cumulus cells. In grade 1 (best morphology) COCs, the degree of apoptosis in granulosa cells was comparable to cumulus cell apoptosis (P<0.01). The overall expression of apoptosis in granulosa cells of follicles containing grade 3 COCs (median+/-median absolute deviation: 37.8+/-13.8%) was significantly higher (P<0.05) than in follicles with grade 1 (22.7+/-10.4%) or grade 2 COCs (20.0+/-17.0%). About 48.3% of grade 3 COCs possessed strongly apoptotic cumulus cells compared to 27.8 and 28.2% of grade 1 or grade 2 COCs, respectively. Nonapoptotic cumulus complexes were observed in grades 1 and 2 COCs only.Adding exogenous follicular cells from atretic follicles to bovine COCs (grades 1 and 2) during in vitro maturation (IVM) had no impact on fertilization, blastocyst formation or hatching after IVF. This is of particular practical relevance to embryo production after ovum pick up (OPU), as during this process, good quality COCs are cultured together with simultaneously collected slightly atretic COCs.     

Nuclear ribosomal DNA sequence polymorphism and hybridization in checker mallows (Sidalcea, Malvaceae)

Checker mallows (Sidalcea, Malvaceae) constitute a western North American genus of annuals and perennials that have been regarded as taxonomically difficult because of complex patterns of morphological variation putatively stemming from hybridization and polyploidy. In recent molecular phylogenetic investigations extensive polymorphism was observed in the internal and external transcribed spacers (ITS and ETS) of 18S-26S nuclear ribosomal DNA for some Sidalcea samples. To resolve the evolutionary basis for this polymorphism and to readdress the evolutionary impact of hybridization in Sidalcea we cloned and sequenced the polymorphic DNAs and included the clones in phylogenetic analyses together with direct sequences of non-polymorphic samples. The positions of cloned spacer sequences in the phylogenetic trees suggest that S. reptans and two subspecies of S. malviflora may have been influenced by past hybridization with lineages of the "glaucescens" clade. Polymorphic sequence patterns in other taxa may be a result of extensive interbreeding within young clades, in keeping with the minimal sequence divergence, largely overlapping geographic distributions and morphology, and ploidy variation in these groups. Other possible explanations for polymorphic sequences in members of Sidalcea include slow concerted evolution relative to mutation rates, incomplete lineage sorting, and recent pseudogene formation.     

Secoiridoids and xanthones in the shoots and roots of <Emphasis Type="Italic">Centaurium pulchellum</Emphasis> cultured <Emphasis Type="Italic">in vitro</Emphasis>

Summary The qualitative and quantitative composition of secondary metabolites was studied in the shoots and roots of Centaurium pulchellum cultured in vitro. Secoiridoids (gentiopierin, swertiamarin, and sweroside) and xanthones (methylbellidifolin, demethyleustomin, and deccussatin) were isolated. Sweroside was found to be the major secoiridoid compound in the aerial parts of plants growing in nature. while swertiamarin dominated in plants cultured in vitro. In roots of all plants, genciopicrin was the major compound. Xanthone demethyleustomin was the major compound both in the shoots and roots of plants growing in nature and cultured in vitro. Different sugars (glucose, fructose, and sucrose) added in different concentrations in the medium affected the production of secondary compounds.     

Rapid genotyping of the osteoporosis-associated polymorphic transcription factor Sp1 binding site in the COL1A1 gene by pyrosequencing

We describe a novel polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) sequencingbased assay for rapid genotyping of the polymorphic Sp1 binding site in the COL1A1 gene (1). A single nucleotide G-->T substitution polymorphism at this GC-rich site has recently been reported to be a predictive genetic marker for low bone mineral density (BMD). To simplify screening for this marker, we optimized PCR conditions and subjected the amplicons to pyrosequencing, which is a convenient high-throughput sequence analysis technique, readily amenable to automation. The analysis of 200 deidentified convenience DNA samples extracted from blood revealed genotype frequences in Hardy-Weinberg equilibrium (SS 68.0%, Ss 28.5%, and ss 3.5%) in agreement with other studies of European populations. This study demonstrates for the first time that pyrosequencing can be used for rapid identification of the osteoporosis-associated single nucleotide polymorphism (SNP) in the COL1A1 gene.