B-Raf acts via the ROCKII/LIMK/cofilin pathway to maintain actin stress fibers in fibroblasts

Recent data have shown that the BRAF gene is mutated at a high frequency in human malignancies. We have analyzed the migratory characteristics of B-raf(-/-) mouse embryonic fibroblasts (MEFs) and compared these with the organization of the actin cytoskeleton and the activity of signaling pathways that are known to influence this organization. Disruption of B-raf significantly reduced the levels of phospho-ERK1/2 and, surprisingly, induced an approximately 1.5-fold increase in cell migration. Consistent with these findings, the high level of actin stress fibers normally present in MEFs was considerably reduced following disruption of B-raf, and the F-actin content of B-raf(-/-) cells was less than half that of B-raf(+/+) cells. Phosphorylation of the myosin light chain on Thr18/Ser19 residues was not reduced in B-raf(-/-) cells. Rather, reduced ROCKII expression and attenuated phosphorylation of ADF/cofilin on serine 3 occurred. Normal stress fiber and phosphocofilin levels were restored by the expression of human B-Raf and catalytically active MEK and by the overexpression of LIM kinase (LIMK). These results have important implications for the role of the B-Raf/ERK signaling pathway in regulating cell motility in normal and malignant cells. They suggest that B-Raf is involved in invasiveness by regulating the proper assembly of actin stress fibers and contractility through a ROCKII/LIMK/cofilin signaling pathway.     

Selenium supplementation on plasma glutathione peroxidase activity in patients with end-stage chronic renal failure

Patients with chronic renal failure (CRF) usually have a lower than healthy level of selenium (Se) in whole blood and plasma. Plasma glutathione peroxidase (GSH-Px) is synthesized mostly in the kidney. In CRF patients, activity of this enzyme is significantly reduced and its reduction increases with the progress of the disease. The aim of the study was to evaluate the effect of Se supplementation to CRF patients at various stages of the disease on Se concentration in blood components and on plasma GSH-Px activity. The study group comprised 53 CRF patients at various stages of the disease supplemented with Se (200 μg/d for 3 mo as Se-enriched yeast, containing about 70% l-selenomethionine [SeMet]). The control group consisted of 20 healthy subjects. The Se concentration in blood components was measured spectrofluorometrically with 2,3-diaminonaphthalene as a complexing reagent. GSH-Px activity in red cell hemolysates and plasma was assayed by the coupled method with tert-butyl hydroperoxide as a substrate. The Se concentration in whole blood and plasma of CRF patients is significantly lower as compared with healthy subjects, but similar at all stages of the disease. In the patients’ plasma, total protein and albumin levels are also significantly lower than in healthy subjects. Plasma GSH-Px activity in patients is extremely low, and contrary to Se concentration, it decreases linearly with the increasing stage of the illness. Se-supplied patients show an increased Se concentration in all blood components and at all disease stages, whereas plasma GSH-Px activity is enhanced only at the incipient stage of the disease. Se supply has no effect on plasma GSH-Px activity in uremic patients at the end stage of the disease. Total plasma protein and albumin levels did not change after Se supplementation. Our data seem to show that in patients with CRF lower total protein and albumin levels in plasma may be the chief cause of the low blood and plasma Se concentrations. GSH-Px activity decreases along with the kidney impairment. At the end stage of the disease, Se supplementation in the form of Se-enriched yeast has no effect on the increase in plasma GSH-Px activity.     

Immunoexpression of androgen receptors and aromatase in testes of patient with Klinefelter's syndrome

Klinefelter's syndrome (47, XXY) is the most common chromosome aneuploidy in men and is usually characterized by underdeveloped testes and sterility. The aim of the present study was to detect cellular distribution of androgen receptors (AR) and aromatase in testes of patient with KS. The tissue sections were processed for morphological and immunohistochemical staining. Additionally, levels of FSH, LH, PRL, estradiol, and testosterone were measured in the plasma. Morphological analysis revealed a complete absence of spermatogenesis. No germ cells were present in seminiferous tubules. In some tubules, nests of apparently degenerating Sertoli cells were found. In the interstitium, Leydig cell hyperplasia was observed. Using immunohistochemistry, nuclear AR staining was detected in Sertoli cells and peritubular cells, whereas in Leydig cells the staining was exclusively cytoplasmic. The immunostaining of aromatase was detected in the cytoplasm of Sertoli cells and Leydig cells. Increased levels of gonadotropins and decreased level of testosterone concomitantly with the cytoplasmic localization of AR in Leydig cells might contribute to the impaired testicular function in patient with KS.     

Prevalence of agglutinating antibodies against<Emphasis Type="Italic"> Listeria monocytogenes</Emphasis> in chicks of the white stork (<Emphasis Type="Italic">Ciconia ciconia</Emphasis> Linnaeus, 1758) in Poland

In 2002 and 2003, blood samples from white stork (Ciconia ciconia) chicks were examined for the presence of antibodies against Listeria monocytogenes. Listeria antibodies were detected in 121 (59%) of 205 chick samples. The probability of Listeria antibodies being present increased with chick age; chicks detected with Listeria antibodies were in better condition than those without the bacterium.     

Local and long-range structural effects caused by the removal of the N-terminal polypeptide fragment from immunoglobulin L chain lambda

The role of the N-terminal polypeptide fragment of the immunoglobulin l-chain in V domain packing stability, and the flexibility of the whole chain was approached by molecular dynamics simulation. The observations were supported by experimental analysis. The N-terminal polypeptide fragment appeared to be the low-stability packing element in the V domain. At moderately elevated temperature it may be replaced at its packing locus by Congo red and then removed by proteolysis. After removal of Congo red by adsorption to (diethylamino)ethyl (DEAE) cellulose, the stability of complete L chain and of L chain devoid of the N-terminal polypeptide fragment were compared. The results indicated that the N-terminal polypeptide fragment plays an essential role in the stability of the V domain. Its removal makes the domain accessible for ANS and Congo red dye binding without heating. The decreased domain stability was registered in particular as increased root mean square (RMS) fluctuation and higher susceptibility to proteolytic attack. The long-range effect was most clearly manifested at 340 K as independent V and C domain fluctuation in the l-chain devoid of the N-terminal polypeptide fragment. This is likely due to the lack of direct connections between the N- and C-termini of the V domain polypeptide. In a complete V domain the connection involves residues 8-12 and 106-110 in particular. Partial or complete disruption of this connection increases the freedom of V domain rotation, while its increased cohesion strengthens the coupling of the V and C domains, making the whole L chain less flexible.     

13C-NMR study of 4-azasteroids in solution and solid state

A group of biologically active 4-azasteroids was studied by 13C-NMR spectroscopy in solution and in the solid phase. A full assignment of signals in the spectra of samples in chloroform was performed for thirteen 4-azasteroids using two-dimensional techniques. Substituent and steric effects of a nitrogen atom, and their influence on chemical shifts of the neighboring carbon atoms are discussed. CP MAS spectra were obtained for five 4-azasteroids including finasteride. The spectra confirmed polymorphism of the latter compound. In addition to the polymorphic forms that are already known, a new molecular complex of finasteride with dioxane is reported.     

Synthesis of orthogonally protected vicinal diamines with amino acid-based skeleton

A convenient route to amino acid-based orthogonally protected 1,2-diamines starting from materials readily available for a peptide chemist is presented. The key step of the procedure is the Mitsunobu reaction of N-protected aminoalcohol, obtained by the reduction of commercially available Z- or Boc-protected amino acid, with imidodicarbonate or sulfonylcarbamate related to standard amino-protecting groups used in peptide chemistry yielding triprotected vicinal diamines.     

RNA:(guanine-N2) methyltransferases RsmC/RsmD and their homologs revisited – bioinformatic analysis and prediction of the active site based on the uncharacterized Mj0882 protein structure

Background  

Escherichia coli guanine-N2 (m²G) methyltransferases (MTases) RsmC and RsmD modify nucleosides G1207 and G966 of 16S rRNA. They possess a common MTase domain in the C-terminus and a variable region in the N-terminus. Their C-terminal domain is related to the YbiN family of hypothetical MTases, but nothing is known about the structure or function of the N-terminal domain.     

Bioinformatic analyses of the tRNA: (guanine 26, N2,N2)-dimethyltransferase (Trm1) family

Functional analyses of the tRNA:(guanine 26, N2,N2)-dimethyltransferase (Trm1) have been hampered by a lack of structural information about the enzyme and by low sequence similarity to better studied methyltransferases. Here we used computational methods to detect novel homologs of Trm1, infer the evolutionary relationships of the family, and predict the structure of the Trm1 methyltransferase. The N-terminal region of the protein is predicted to form an S-adenosylmethionine-binding domain, which harbors the active site. The C-terminal region is rich in predicted alpha-helices and, in analogy to other nucleic acid methyltransferases, may constitute the target recognition domain of the enzyme. Interposing these two domains, most Trm1 homologs possess a highly variable inserted sequence that is delimited by a Cys4 cluster, likely forming a Zn-finger structure. The residues of Trm1 predicted to participate in cofactor binding, target recognition, and catalysis, were mapped onto a preliminary structural model, providing a platform for designing new experiments to better understand the molecular functions of this protein family. In addition, identification of novel, atypical Trm1 homologs suggests candidates for cloning and biochemical characterization.