Intracellular expression of the proliferative marker Ki-67 and viral proteins (NS3, NS5A and C) in chronic, long lasting hepatitis C virus (HCV) infection

Hepatitis C virus (HCV) continues to represent the main causative agent of the hepatitis, which leads to chronic transformation of the process in 60-80% individuals. It remains unclear how far cellular expression of HCV proteins in vivo may represent an index of progression of the disease and of proliferative activity in the liver in chronic hepatitis C. Aim of the studies included detection and subcellular localization of three HCV proteins (NS3, NS5A and C) in liver biopsies from adults (n=19) with chronic, long lasting hepatitis C as related to hepatocyte proliferative activity. The immunocytochemical ABC (avidin biotin-peroxidase complex) technique was applied, alone or associated with the ImmunoMax technique. Results of the immunocytochemical tests were compared to histological alterations in liver biopsies, proliferation index and with selected clinical data. A significantly higher expression of NS3 protein was noted, as compared to expressions of NS5A and C proteins. In all the patients, cytoplasmic localization of all proteins dominated over nuclear localization (p0.05). At the level of electron microscopy, protein localization in endoplasmic reticulum (ER) membranes, mitochondria, perinuclear region and/or in hepatocyte cell nucleus was observed. No direct relationships could be demonstrated between expressions of HCV proteins and of Ki-67 antigen. No correlations could also be demonstrated between cellular expression of any HCV protein on one hand and grading or staging, alanine transaminase (ALT), serum level of HCV RNA or alpha-fetoprotein (AFP) on the other. However, positive correlations were disclosed between proliferative activity of hepatocytes on one hand and patient's age, grading and staging on the other. Advanced hepatic fibrosis correlated also with serum levels of AFP. The studies were supplemented with data on subcellular localization of HCV proteins. Moreover, they indicated that in HCV infection grading and staging, proliferative activity of hepatocytes and serum AFP level represent more valuable indices of the disease progress than those provided by cellular expression of three potentially oncogenic HCV proteins in vivo.     

ULTRASTRUCTURE OF UNICELLULAR N2 FIXING CYANOBACTERIA FROM THE TROPICAL NORTH ATLANTIC AND SUBTROPICAL NORTH PACIFIC OCEANS1

Nitrogen fixing unicellular marine cyanobacteria may have a major role in the global biogeochemistry of N; nevertheless, little is known about their phylogeny and morphology. We isolated N₂ fixing unicellular cyanobacteria from the tropical North Atlantic and subtropical North Pacific Oceans and examined ultrastructural dynamics during dark:light cycles when grown in incubators. The isolate from the subtropical North Pacific was larger and showed a size variation from 3 to 7 μm but had similar morphology and cell division‐plane characteristics as the isolate from the North Atlantic (2.5 μm). Nitrogen fixation only occurred during the dark phase, and ultrastructural analysis demonstrated changes in the appearance and quantity of large carbohydrate‐like granules present in the cells. To verify the composition of these carbohydrate‐like granules, staining with periodic acid, thioacetic acid, and silver was carried out, and a positive reaction was obvious in all cells. The cells from the Atlantic seemed to empty their polysaccharide granules during the night, whereas those from the Pacific showed a decrease in the number of their granules. Our work suggests that phylogenetically related strains of unicellular N₂ fixing cyanobacteria from different oceans showed similar carbohydrate‐like granules that could be used to fuel N₂ fixation during darkness.     

Expression, purification and catalytic activity of Lupinus luteus asparagine beta-amidohydrolase and its Escherichia coli homolog.

We describe the expression, purification, and biochemical characterization of two homologous enzymes, with amidohydrolase activities, of plant (Lupinus luteus potassium-independent asparaginase, LlA) and bacterial (Escherichia coli, ybiK/spt/iaaA gene product, EcAIII) origin. Both enzymes were expressed in E. coli cells, with (LlA) or without (EcAIII) a His-tag sequence. The proteins were purified, yielding 6 or 30 mg.L(-1) of culture, respectively. The enzymes are heat-stable up to 60 degrees C and show both isoaspartyl dipeptidase and l-asparaginase activities. Kinetic parameters for both enzymatic reactions have been determined, showing that the isoaspartyl peptidase activity is the dominating one. Despite sequence similarity to aspartylglucosaminidases, no aspartylglucosaminidase activity could be detected. Phylogenetic analysis demonstrated the relationship of these proteins to other asparaginases and aspartylglucosaminidases and suggested their classification as N-terminal nucleophile hydrolases. This is consistent with the observed autocatalytic breakdown of the immature proteins into two subunits, with liberation of an N-terminal threonine as a potential catalytic residue.     

Polymorphisms of stress-related genes and the risk of nonsyndromic cleft lip with or without cleft palate

BACKGROUND: Nonsyndromic cleft lip with or without cleft palate (NCL/P) is a common structural malformation with a complex and multifactorial etiology. It has been shown that maternal psychological stress in the periconceptional period can contribute to an increase in the risk of NCL/P affecting pregnancy. METHODS: Twenty‐four single nucleotide polymorphisms of 11 stress‐related genes (COMT, CRHR1, FKBP5, GABRA6, HSD11β2, MAOA, NPY, NR3C1, SERPINA6, SLC6A4, and TPH2) were investigated in 220 healthy mothers of children with facial clefts and 210 matched controls using restriction fragment‐length polymorphism and high‐resolution melting analysis. RESULTS: We found that polymorphisms in SLC6A4, TPH2, and SERPINA6 appear to be maternal factors increasing the risk of having a child with facial clefts. The closest correlations with NCL/P were found for the SLC6A4 rs2020942 and TPH2 rs10879357 gene variants (odds ratio [OR], 1.720; 95% confidence interval [CI], 1.158–2.553; p = 0.0069; p_trend = 0.0036; and OR, 1.837; 95% CI, 1.226–2.753, p = 0.0030, p_trend = 0.0057; respectively). Moreover, haplotype analysis revealed that several combinations of markers in SLC6A4, TPH2, and SERPINA6 might be significantly associated with the risk of NCL/P affected pregnancies. However, these associations were not statistically significant after correction for multiple testing. CONCLUSION: This study suggests that nucleotide variants of genes encoding components of the hypothalamus‐pituitary‐adrenal axis and serotoninergic system have a role in the etiology of NCL/P in the Polish population. SLC6A4, TPH2, and SERPINA6 might be novel candidate genes for this common congenital anomaly. Birth Defects Research (Part A), 2011. © 2011 Wiley‐Liss, Inc.     

Heart mechanical and hemodynamic parameters of a beetle,Tenebrio molitor,at selected ages

The physiological processes that occur during the aging of insects are poorly understood. The aim of this study was to describe the changes in contractile activity and hemodynamic parameters of the heart that take place as the coleopteran beetle, Tenebrio molitor, ages. The frequency of heart contractions in beetles that had just undergone metamorphosis (median 24.7 beats/min) was significantly lower than the frequency of heart contractions in older beetles. In 56% of beetles that were < 1 week of age, a pattern of contractile activity with alternating periods of higher and lower contraction frequency was detected, suggesting that some posteclosion developmental processes occur during the first week of adulthood. All beetles that were 1 week of age showed a regular rhythm of heart contractions (median 72 beats/min). In older beetles, abnormalities such as heart arrhythmias or heart arrest were observed. The incidence of arrhythmia as well as the arrhythmicity index was highest in beetles that were 8–18 weeks old. The calculated stroke volume (SV) was also found to increase from eclosion to 12 weeks of age, and then decreased as adults aged further. Interestingly, cardiac output increased gradually, but the ejection fraction did not change significantly with age.     

Steroidal saponins from the genus <Emphasis Type="Italic">Allium</Emphasis>

Steroidal saponins are widely distributed among monocots, including the Amaryllidaceae family to which the Allium genus is currently classified. Apart from sulfur compounds, these are important biologically active molecules that are considered to be responsible for the observed activity of Allium species, including antifungal, cytotoxic, enzyme-inhibitory, and other. In this paper, literature data concerning chemistry and biological activity of steroidal saponins from the Allium genus has been reviewed.     

Diversity of Fusarium Species Isolated from Weeds and Plant Debris in Croatia

Weeds are alternative hosts of plant pathogens and when colonized may not exhibit disease symptoms. In 2008 and 2009, samples of weeds and plant debris were collected from 12 locations in eastern Croatia, and 300 Fusarium isolates colonizing them were identified. Strains were grouped and identified based on morphology and amplified fragment length polymorphism (AFLP) patterns. Portions of the β‐tubulin and translocation elongation factor 1‐α genes were sequenced from representative strains of each group to confirm the identifications. Fourteen Fusarium species were identified with F. graminearum (20%), F. verticillioides (18%), F. oxysporum (16%), F. subglutinans (13%) and F. proliferatum (11%) all present as more than 10% of the population. Fusarium acuminatum, F. avenaceum, F. concolor, F. crookwellense (F. cerealis), F. equiseti, F. semitectum, F. solani, F. sporotrichioides and F. venenatum, were all present at frequencies < 8%. Our results indicate that economically important Fusarium spp. may be isolated from numerous alternative hosts during the off season and that weeds and plant debris can serve as a reservoir of genetically diverse inoculum.     

Probing the Monophyly of the Sphaeropleales (Chlorophyceae) Using Data From Five Genes

Molecular phylogenetic analyses have had a major impact on the classification of the green algal class Chlorophyceae, corroborating some previous evolutionary hypotheses, but primarily promoting new interpretations of morphological evolution. One set of morphological traits that feature prominently in green algal systematics is the absolute orientation of the flagellar apparatus in motile cells, which correlates strongly with taxonomic classes and orders. The order Sphaeropleales includes diverse green algae sharing the directly opposite (DO) flagellar apparatus orientation of their biflagellate motile cells. However, algae across sphaeroplealean families differ in specific components of the DO flagellar apparatus, and molecular phylogenetic studies often have failed to provide strong support for the monophyly of the order. To test the monophyly of Sphaeropleales and of taxa with the DO flagellar apparatus, we conducted a molecular phylogenetic study of 16 accessions representing all known families and diverse affiliated lineages within the order, with data from four plastid genes (psaA, psaB, psbC, rbcL) and one nuclear ribosomal gene (18S). Although single‐gene analyses varied in topology and support values, analysis of combined data strongly supported a monophyletic Sphaeropleales. Our results also corroborated previous phylogenetic hypotheses that were based on chloroplast genome data from relatively few taxa. Specifically, our data resolved Volvocales, algae possessing predominantly biflagellate motile cells with clockwise (CW) flagellar orientation, as the monophyletic sister lineage to Sphaeropleales, and an alliance of Chaetopeltidales, Chaetophorales, and Oedogoniales, orders having multiflagellate motile cells with distinct flagellar orientations involving the DO and CW forms.