siRNAs and miRNAs: small RNA molecules for big tasks

Small RNAs have been recently discovered as important regulators of gene expression in Eukaryota. This review compares two categories of small RNAs existing in plants: short interfering RNAs (siRNAs) and microRNAs (miRNAs) and reveals similarities and differences between two intriguing processes: RNA degradation and translational repression directed by small RNAs. The disruption of miRNA-mediated regulation causes developmental abnormalities in plants, proving a fundamental role of miRNAs.     

Neuropeptide Y, ubiquitous and elusive

This paper reviews aspects of NPY research that were emerging in 1985, shortly after the isolation and characterization of the peptide. NPY had become known for its widespread distribution especially in the central and peripheral nervous systems, but also in the gastro-intestinal and respiratory tracts and in fibers innervating smooth muscle around blood vessels. Consistent with its distribution, it was determined that NPY is a potent vasoconstrictor, affects neuroendocrine systems and is involved in appetite regulation--areas of research still relevant today. Through advances in technology knowledge about NPY's role in these and newly discovered physiological functions has deepened considerably. Successful cloning of a series of NPY receptors has opened up new and complex research vistas. Lately, the creation of mice genetically modified for NPY as well as for several receptor subtypes has brought many puzzling observations--followed by questions yet to be answered.     

Correlation of HLA-Cw*06 allele frequency with some clinical features of psoriasis vulgaris in the population of northern Poland

Psoriasis is a common skin disease with a genetic background and significant human leukocyte antigen (HLA) associations. HLA-Cw6 is the most frequently described association, particularly with psoriasis of the early onset type. Few studies of its correlation with various psoriasis clinical phenotypes and severity of the disease have been published so far; none in the Polish population. In this study 78 patients with psoriasis vulgaris were evaluated clinically and subdivided according to the age of onset and the type of psoriasis. A system of disease severity evaluation was applied to each patient. All patients and the control group (70 unrelated persons) were typed for HLA-Cw*06. The results show that Cw*06 allele frequency was higher in psoriatics than in the control group. Our investigation confirms that the Cw*06 allele is positively associated with psoriasis vulgaris of the early onset type, with a positive family history and its more severe form.     

Heavy metal ions inhibition of jack bean urease: potential for rapid contaminant probing

The kinetics of heavy metal ions inhibition of jack bean urease was studied by progress curve analysis in a reaction system without enzyme-inhibitor preincubation. The inhibition was found to be biphasic with an initial, small inhibitory phase changing over the time course of 5-10 min into a final linear steady state with a lower velocity. This time-dependent pattern was best described by mechanism B of slow-binding inhibition, involving the rapid formation of an EI complex that subsequently undergoes slow conversion to a more stable EI* complex. The kinetic parameters of the process, the inhibition constants Ki and Ki* and the forward k5 and reverse k6 rate constants for the conversion, were evaluated from the reaction progress curves by nonlinear regression treatment. Based on the values of the overall inhibition constant Ki*, the heavy metal ions were found to inhibit urease in the following decreasing order: Hg2+ > Cu2+ > Zn2+ > Cd2+ > Ni2+ > Pb2+ > Co2+ > Fe3+ > As3+. With the Ki* values as low as 1.9 nM for Hg2+ and 7.1 nM for Cu2+, 100-1000 times lower than those of the other ions, urease may be utilized as a bioindicator of the trace levels of these ions in environmental monitoring, bioprocess control or pharmaceutical analysis.     

Toxicity of microcystin from cyanobacteria growing in a source of drinking water

Microcystin-LR (MC-LR) is a cyanobacterial heptapeptide that presents acute and chronic hazards to animal and human health. The morphological changes in mitochondria are the primary effect induced by MC-LR leading to cell death. We investigated the toxicity of cyanobacterial microcystin-containing extract (CEM) on the respiratory complex of mammalian mitochondria from Bos taurus. Cyanobacterial blooms of Microcystis aeruginosa were harvested from Sulejow Reservoir, a source of drinking water in central Poland. The concentration of microcystin-LR (MC-LR(CEM)) in CEM extract was determined by high-performance liquid chromatography (HPLC). Commercially available microcystin-LR (Sigma) was used as a standard (MC-LR(S)); both standard and CEM extract were incubated with mitochondria in different doses and time of exposure. MC-RL(CEM) at 1 nM, maximal acceptable dose of microcystin (WHO) in drinking water, provoked activation of cytochrome c oxidase complex in mitochondria. We suggest that it might be considered as a defensive signal of mitochondria against low concentration of a toxic compound. In contrast 1 iM MC-RL(CME) inhibited the activity of mitochondrial oxidase complex much stronger than the same concentration of standard MC-RL(S) (58% vs. 87% of control activity, P<0.05), and this may cause a similar effect to long-term consumption of water. In conclusion, we affirm that CEM extract is highly toxic, and mitochondria could be used as an indicator of this toxicity in vivo, especially during long-term consumption of water from reservoirs where microcystin is produced.     

Persistence of Mhc heterozygosity in homozygous clonal killifish,Rivulus marmoratus: implications for the origin of hermaphroditism

The mangrove killifish Rivulus marmoratus, a neotropical fish in the order Cyprinodontiformes, is the only known obligatorily selfing, synchronous hermaphroditic vertebrate. To shed light on its population structure and the origin of hermaphroditism, major histocompatibility complex (Mhc) class I genes of the killifish from seven different localities in Florida, Belize, and the Bahamas were cloned and sequenced. Thirteen loci and their alleles were identified and classified into eight groups. The loci apparently arose approximately 20 million years ago (MYA) by gene duplications from a single common progenitor in the ancestors of R. marmoratus and its closest relatives. Distinct loci were found to be restricted to different populations and different individuals in the same population. Up to 44% of the fish were heterozygotes at Mhc loci, as compared to near homozygosity at non-Mhc loci. Large genetic distances between some of the Mhc alleles revealed the presence of ancestral allelic lineages. Computer simulation designed to explain these findings indicated that selfing is incomplete in R. marmoratus populations, that Mhc allelic lineages must have diverged before the onset of selfing, and that the hermaphroditism arose in a population containing multiple ancestral Mhc lineages. A model is proposed in which hermaphroditism arose stage-wise by mutations, each of which spread through the entire population and was fixed independently in the emerging clones.     

Changes in the cellular behaviour of human colonic cell line Caco-2 in response to butyrate treatment

Gut-derived adenocarcinoma Caco-2 cells were treated with sodium butyrate (NaB) at physiologically relevant concentrations. We characterized its effects on proliferation, differentiation, apoptosis, adhesion to the solid support and interleukin-8 secretion. Differentiation was determined by brush border alkaline phosphatase activity. Apoptosis was assessed by acridine orange and Hoechst stains. Differentiation and apoptosis were analyzed in both adherent and floating cell populations. The transformed Caco-2 cells did not retain their malignant phenotype in the presence of NaB. They appeared to undergo a change in the phenotype induced by NaB, as indicated by reduced proliferation, enhanced differentiation, stimulation of apoptosis leading to decreased viability of cells, and stimulation of interleukin-8 secretion. Considering all the above facts and data, we postulate that Caco-2 cells cultured in NaB supplemented medium could regain the phenotypic characteristics of the phenotype of the parent cell from which originated the Caco-2 line.     

Functional characterization of Gne (UDP-N-acetylglucosamine-4-epimerase), Wzz (chain length determinant), and Wzy (O-antigen polymerase) of Yersinia enterocolitica serotype O:8

The lipopolysaccharide (LPS) O-antigen of Yersinia enterocolitica serotype O:8 is formed by branched pentasaccharide repeat units that contain N-acetylgalactosamine (GalNAc), L-fucose (Fuc), D-galactose (Gal), D-mannose (Man), and 6-deoxy-D-gulose (6d-Gul). Its biosynthesis requires at least enzymes for the synthesis of each nucleoside diphosphate-activated sugar precursor; five glycosyltransferases, one for each sugar residue; a flippase (Wzx); and an O-antigen polymerase (Wzy). As this LPS shows a characteristic preferred O-antigen chain length, the presence of a chain length determinant protein (Wzz) is also expected. By targeted mutagenesis, we identify within the O-antigen gene cluster the genes encoding Wzy and Wzz. We also present genetic and biochemical evidence showing that the gene previously called galE encodes a UDP-N-acetylglucosamine-4-epimerase (EC 5.1.3.7) required for the biosynthesis of the first sugar of the O-unit. Accordingly, the gene was renamed gne. Gne also has some UDP-glucose-4-epimerase (EC 5.1.3.2) activity, as it restores the core production of an Escherichia coli K-12 galE mutant. The three-dimensional structure of Gne was modeled based on the crystal structure of E. coli GalE. Detailed structural comparison of the active sites of Gne and GalE revealed that additional space is required to accommodate the N-acetyl group in Gne and that this space is occupied by two Tyr residues in GalE whereas the corresponding residues present in Gne are Leu136 and Cys297. The Gne Leu136Tyr and Cys297Tyr variants completely lost the UDP-N-acetylglucosamine-4-epimerase activity while retaining the ability to complement the LPS phenotype of the E. coli galE mutant. Finally, we report that Yersinia Wzx has relaxed specificity for the translocated oligosaccharide, contrary to Wzy, which is strictly specific for the O-unit to be polymerized.