Heterogeneity of intercellular adhesion in rat liver cells in culture

The intercellular homotypic adhesive properties of 14 clones derived from a nontumorigenic rat liver epithelial cell line (LEC), derived from neonatal Fischer rats, were examined and compared to those of the hepatoma H4-II-E cell line. Each clone was assayed also for the degree of chromosomal aneuploidy and the ability to grow in soft agar. Over 100-fold differences in adhesive properties were observed among the clones, but no correlation was observed between the degree of aneuploidy in the clones and intercellular adhesive properties. The parent LEC cell line and the clones derived from it were unable to grow in soft agar. The H4-II-E cells showed negligible capacity to reaggregate after dissociation into single cells and these cells readily formed colonies in soft agar. Many of the LEC clones were similar to the H4-II-E cells in their adhesive properties, which suggests that reduced cell-to-cell adhesiveness per se is not a necessary prerequisite of epithelial cells to be able to grow independent of anchorage. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of concanavalin A (Con A)-binding glycoproteins in the "most adhesive" clone 67 and the "least adhesive" clone 201 showed markedly elevated amounts of acidic 105 and 67-kDa glycoproteins in clone 67. Proteins with similar migration patterns in 2D-PAGE have previously been reported to participate in specific homotypic intercellular adhesion of liver cells. The Con A-binding glycoprotein pattern in H4-II-E cells was markedly different from that of LEC cells with a set of six proteins missing and nine proteins appearing new in the H4-II-E cells. It is suggested that, in addition to identifying known epithelial cell polypeptides, systematic screening of cell surface-associated glycoproteins in normal and transformed epithelial cells in vitro and in vivo may lead to identification of novel polypeptides intimately associated with the transformed phenotype.     

Combined effect of light and temperature on triacylglycerol accumulation in Dicranum elongatum

In the subarctic moss Dicranum elongatum Schleich & Schwaegr., the level of total lipids and triacylglycerols (TAG) was high in late winter and spring and low in autumn and winter. Four-week exposure of field material to continuous light (135μmol m⁻²s⁻¹) at 1°C resulted in a considerable increase in the amount of TAG in the autumn material acclimated to low temperatures and rhythmic light in the field. In contrast, the same treatment did not cause any increase in TAG in the spring material, acclimated to low temperatures and continuous light in the field. Results from experiments, in which moss cultivated for 4 months at 9°C on 12-h photoperiods (135μmol m⁻²s⁻¹) was kept for 3 weeks at low temperatures (9°C and −3°C) either in continuous light (135 or 70 μmol m⁻²s⁻¹) or with 12-h photoperiods (135 μmol m⁻²s⁻¹), indicated that the TAG level was higher at higher light intensity. At 9°C it was also higher in continuous light of both intensities than in rhythmic light. These results strongly suggest that decreasing irradiance and decreasing daylength limits the accumulation of TAG in D. elongatum during autumn in the subarctic.     

Effects of methylbenzenes on microsomal enzymes in rat liver,kidney and lung

The effects of pretreatment with toluene, o-, m-, p-xylene and mesitylene were investigated on the microsomal enzymes of liver, kidney and lung in rats. The activities of aminopyrine N-demethylase, aryl hydrocarbon hydroxylase, aniline hydroxylase, NADPH-cytochrome c reductase, as well as the concentrations of cytochrome P-450 and cytochrome b₅ were determined. The effects were most marked in the liver, where toluene caused increase in aniline hydroxylase and cytochrome P-450; o-xylene in aminopyrine N-demethylase and cytochrome b₅; m-xylene and mesitylene in all the enzymes investigated. In kidneys, all the compounds increased the activity of aniline hydroxylase; m-xylene induced cytochrome P-450 and b₅ as well as NADPH-cytochrome c reductase; p-xylene induced cytochrome P-450, and mesitylene cytochrome P-450 and b₅. Aminopyrine N-demethylase activity was decreased by toluene. In lungs, only mesitylene caused any significant differences from the controls: increase in aminopyrine N-demethylase and aryl hydrocarbon hydroxylase, decrease in aniline hydroxylase. The methylbenzenes tested induced the microsomal enzymes in a rough correlation to the number of their methyl groups and their hydrophobic properties.     

Chromosomes and reproductive behaviour of Finnish Taraxacum agamospecies

Material consisting of 157 accessions of 69 Finnish Taraxacum agamospecies, representing 7 sections, was grown in an experimental field for comparison of certain botanical and nutritional characteristics. This paper gives the collecting data as well as cytological and reproductive characteristics of the material. The largest section Taraxacum (Vulgariu ) was represented by 60 agamospecies, 59 of them triploid (2n = 24) and one tetraploid (T. penicilliforme , 2n = 32). The tetraploid and 37 triploids were chromosome counted for the first time. The diploid sexual species T. obtusilobum from Sweden was included for comparison. Other sections consisted of 3 triploids and 6 tetraploids, 3 tetraploid species of the section Erythrosperma being chromosome counted for the first time. Seedlings with deviating numbers were occasionally found. A banding technique of the chromosomes was developed but it did not allow the exact identification of other than the satellite chromosomes. Species-specific features were found in the flowering phenology and in several pollen characteristics (stainability, mean and variability of the size). There were indications of genetic variation within certain agamospecies in pollen characteristics. Isolated and emasculated (mostly only isolated) flower heads gave mostly a full seed set, which indicates obligate, autonomous agamospermy. Lower seed sets of T. adami, T. dahlstedtii and T. hamatiforme in isolation may be a sign of facultative apomixis in these species, requiring further study.     

Naturally Acquired Picornavirus Infections in Primates at the Dhaka Zoo

The conditions in densely populated Bangladesh favor picornavirus transmission, resulting in a high rate of infection in the human population. Data suggest that nonhuman primates (NHP) may play a role in the maintenance and transmission of diverse picornaviruses in Bangladesh. At the Dhaka Zoo, multiple NHP species are caged in close proximity. Their proximity to other species and to humans, both zoo workers and visitors, provides the potential for cross-species transmission. To investigate possible interspecies and intraspecies transmission of picornaviruses among NHP, we collected fecal specimens from nine NHP taxa at the Dhaka Zoo at three time points, August 2007, January 2008, and June 2008. Specimens were screened using real-time PCR for the genera Enterovirus, Parechovirus, and Sapelovirus, and positive samples were typed by VP1 sequencing. Fifty-two picornaviruses comprising 10 distinct serotypes were detected in 83 fecal samples. Four of these serotypes, simian virus 19 (SV19), baboon enterovirus (BaEV), enterovirus 112 (EV112), and EV115, have been solely associated with infection in NHP. EV112, EV115, and SV19 accounted for 88% of all picornaviruses detected. Over 80% of samples from cages housing rhesus macaques, olive baboons, or hamadryas baboons were positive for a picornavirus, while no picornaviruses were detected in samples from capped langurs or vervet monkeys. In contrast to our findings among synanthropic NHP in Bangladesh where 100% of the picornaviruses detected were of human serotypes, in the zoo population, only 15% of picornaviruses detected in NHP were of human origin. Specific serotypes tended to persist over time, suggesting either persistent infection of individuals or cycles of reinfection.     

Molecular Evolution of the Human Enteroviruses: Correlation of Serotype with VP1 Sequence and Application to Picornavirus Classification

Sixty-six human enterovirus serotypes have been identified by serum neutralization, but the molecular determinants of the serotypes are unknown. Since the picornavirus VP1 protein contains a number of neutralization domains, we hypothesized that the VP1 sequence should correspond with neutralization (serotype) and, hence, with phylogenetic lineage. To test this hypothesis and to analyze the phylogenetic relationships among the human enteroviruses, we determined the complete VP1 sequences of the prototype strains of 47 human enterovirus serotypes and 10 antigenic variants. Our sequences, together with those available from GenBank, comprise a database of complete VP1 sequences for all 66 human enterovirus serotypes plus additional strains of seven serotypes. Phylogenetic trees constructed from complete VP1 sequences produced the same four major clusters as published trees based on partial VP2 sequences; in contrast to the VP2 trees, however, in the VP1 trees strains of the same serotype were always monophyletic. In pairwise comparisons of complete VP1 sequences, enteroviruses of the same serotype were clearly distinguished from those of heterologous serotypes, and the limits of intraserotypic divergence appeared to be about 25% nucleotide sequence difference or 12% amino acid sequence difference. Pairwise comparisons suggested that coxsackie A11 and A15 viruses should be classified as strains of the same serotype, as should coxsackie A13 and A18 viruses. Pairwise identity scores also distinguished between enteroviruses of different clusters and enteroviruses from picornaviruses of different genera. The data suggest that VP1 sequence comparisons may be valuable in enterovirus typing and in picornavirus taxonomy by assisting in the genus assignment of unclassified picornaviruses.Human enteroviruses (family Picornaviridae) infect millions of people worldwide each year, resulting in a wide range of clinical outcomes ranging from inapparent infection to mild respiratory illness (common cold), hand-foot-and-mouth disease, acute hemorrhagic conjunctivitis, aseptic meningitis, myocarditis, severe neonatal sepsis-like disease, and acute flaccid paralysis (reviewed in references 43 and 45). In the United States, enteroviruses are responsible for 30,000 to 50,000 meningitis hospitalizations per year as a result of 30 million to 50 million infections. Serologic studies have distinguished 66 human enterovirus serotypes on the basis of an antibody neutralization test (43), and additional antigenic variants have been defined within several of the serotypes on the basis of reduced or nonreciprocal cross-neutralization between prototype and variant strains (6, 8, 68, 71, 72). On the basis of their pathogenesis in humans and experimental animals, the enteroviruses were originally classified into four groups, polioviruses, coxsackie A viruses (CA), coxsackie B viruses (CB), and echoviruses, but it was quickly realized that there were significant overlaps in the biological properties of viruses in the different groups (8). The more recently isolated enteroviruses have been named with a system of consecutive numbers: EV68, EV69, EV70, and EV71 (42).A comparison of nucleotide and deduced amino acid sequences at the 5′ end of VP2 has identified four major phylogenetic groups within the Enterovirus genus: CA16-like viruses (cluster A), a CB-like group containing all CB and echoviruses as well as CA9 and EV69 (cluster B), poliovirus-like viruses (cluster C), and EV68 and EV70 (cluster D) (23, 24, 49, 53, 54, 73). However, pairwise alignments and phylogenetic analyses within these groups demonstrated that the VP2 sequence does not fully correlate with serotype, as viruses known to belong to the same serotype often failed to cluster together (2, 49). (E22 and E23 are genetically distinct from enteroviruses [24], and their reclassification into a separate genus has been proposed [45]).VP1 is the most external and immunodominant of the picornavirus capsid proteins (58). A number of major neutralization sites reside in the VP1 proteins of many picornaviruses (reviewed in references 40 and 44), but the specific epitopes responsible for serotype specificity and intratypic variation have not been identified. Similarly, the genetic correlates of serotype identity remain unknown. If the important serotype-specific neutralization sites reside in VP1, then the VP1 sequence or some portion thereof would be predicted to correlate with serotype. Studies on the three serotypes of poliovirus have shown that a partial VP1 sequence correlates well with serotype (32). In addition, genetic lineages based on the VP1 sequence can be used to define poliovirus reservoirs and chains of transmission (reviewed in reference 30). To test whether the VP1 sequence might be applied to the classification of nonpolio enteroviruses and to the analysis of the phylogenetic relationships among the human enteroviruses, we determined the complete VP1 nucleotide sequences for 47 human enterovirus prototypes and 10 well-characterized antigenic variants. These data, together with previously available sequences, comprise a database of complete VP1 sequences for all known human enterovirus serotypes and 12 natural antigenic variants. This database will be useful for molecular epidemiologic studies of enteroviral disease outbreaks, to obtain a better understanding of the genetic correlates of serotype, and for the development of enteroviral molecular diagnostic reagents.     

Htd2p/Yhr067p is a yeast 3-hydroxyacyl-ACP dehydratase essential for mitochondrial function and morphology

Among the recently recognized aspects of mitochondrial functions, in yeast as well as humans, is their ability to synthesize fatty acids in a malonyl-CoA dependent manner. We describe here the identification of the 3-hydroxyacyl-ACP dehydratase involved in mitochondrial fatty acid synthesis. A colony-colour-sectoring screen was applied in Saccharomyces cerevisiae in a search for mutants that, when grown on a non-fermentable carbon source, were unable to lose a plasmid that carried a chimeric construct coding for mitochondrially localized bacterial analogue. Our mutants, which are respiratory deficient, lack cytochromes and display abnormal mitochondrial morphology, were found to have a lesion in the yeast YHR067w/RMD12 gene. The Yhr067p is predicted to be a member of the thioesterase/thioester dehydratase-isomerase superfamily enzymes. Hydratase 2 activity in mitochondrial extracts from cells overexpressing YHR067w was increased. These overexpressing cells also display a striking mitochondrial enlargement phenotype. We conclude that YHR067w encodes a novel mitochondrial 3-hydroxyacyl-thioester dehydratase 2 and suggest renaming it HTD2. The mitochondrial phenotypes of the null and overexpression mutants suggest a crucial role of YHR067w in maintenance of mitochondrial respiratory competence and morphology in yeast.     

Sex steroid hormone metabolism and prostate cancer

The growth and function of the prostate is dependent on androgens. The two predominant androgens are testosterone, which is formed in the testis from androstenedione and 5alpha-dihydrotestosterone, which is formed from testosterone by 5alpha-reductases and is the most active androgen in the prostate. Prostate cancer is one of the most common cancers among men and androgens are involved in controlling the growth of androgen-sensitive malignant prostatic cells. The endocrine therapy used to treat prostate cancer aims to eliminate androgenic activity from the prostatic tissue. Most prostate cancers are initially responsive to androgen withdrawal but become later refractory to the therapy and begin to grow androgen-independently. Using LNCaP prostate cancer cell line we have developed a cell model to study the progression of prostate cancer. In the model androgen-sensitive LNCaP cells are transformed in culture conditions into more aggressive, androgen-independent cells. The model was used to study androgen and estrogen metabolism during the transformation process. Our results indicate that substantial changes in androgen and estrogen metabolism occur in the cells during the process. A remarkable decrease in the oxidative 17beta-hydroxysteroid dehydrogenase activity was seen whereas the reductive activity seemed to increase. The changes suggest that during transformation estrogen influence is increasing in the cells. This is supported by the cDNA microarray screening results which showed over-expression of several genes up-regulated by estrogens in the LNCaP cells line representing progressive prostate cancer. Since local steroid metabolism controls the bioavailability of active steroid hormones in the prostate, the variations in steroid-metabolizing enzymes during cancer progression may be crucial in the regulation of the growth and function of the organ.