Disease-associated Mutations in the Prion Protein Impair Laminin-induced Process Outgrowth and Survival

Prions, the agents of transmissible spongiform encephalopathies, require the expression of prion protein (PrP^C) to propagate disease. PrP^C is converted into an abnormal insoluble form, PrP^Sc, that gains neurotoxic activity. Conversely, clinical manifestations of prion disease may occur either before or in the absence of PrP^Sc deposits, but the loss of normal PrP^C function contribution for the etiology of these diseases is still debatable. Prion disease-associated mutations in PrP^C represent one of the best models to understand the impact of PrP^C loss-of-function. PrP^C associates with various molecules and, in particular, the interaction of PrP^C with laminin (Ln) modulates neuronal plasticity and memory formation. To assess the functional alterations associated with PrP^C mutations, wild-type and mutated PrP^C proteins were expressed in a neural cell line derived from a PrP^C-null mouse. Treatment with the laminin γ1 chain peptide (Ln γ1), which mimics the Ln binding site for PrP^C, increased intracellular calcium in cells expressing wild-type PrP^C, whereas a significantly lower response was observed in cells expressing mutated PrP^C molecules. The Ln γ1 did not promote process outgrowth or protect against staurosporine-induced cell death in cells expressing mutated PrP^C molecules in contrast to cells expressing wild-type PrP^C. The co-expression of wild-type PrP^C with mutated PrP^C molecules was able to rescue the Ln protective effects, indicating the lack of negative dominance of PrP^C mutated molecules. These results indicate that PrP^C mutations impair process outgrowth and survival mediated by Ln γ1 peptide in neural cells, which may contribute to the pathogenesis of genetic prion diseases.     

Blocking αvβ3 integrin by a recombinant RGD disintegrin impairs VEGF signaling in endothelial cells

Vascular endothelial growth factor (VEGF) and αvβ3 integrin are key molecules that actively participate in tumor angiogenesis and metastasis. Some integrin-blocking molecules are currently under clinical trials for cancer and metastasis treatment. However, the mechanism of action of such inhibitors is not completely understood. We have previously demonstrated the anti-angiogenic and anti-metastatic properties of DisBa-01, a recombinant His-tag RGD-disintegrin from Bothrops alternatus snake venom in some experimental models. DisBa-01 blocks αvβ3 integrin binding to vitronectin and inhibits integrin-mediated downstream signaling cascades and cell migration. Here we add some new information on the mechanism of action of DisBa-01 in the tumor microenvironment. DisBa-01 supports the adhesion of fibroblasts and MDA-MB-231 breast cancer cells but it inhibits the adhesion of these cells to type I collagen under flow in high shear conditions, as a simulation of the blood stream. DisBa-01 does not affect the release of VEGF by fibroblasts or breast cancer cells but it strongly decreases the expression of VEGF mRNA and of its receptors, vascular endothelial growth factor receptors 1 and 2 (VEGFR1 and VEGFR2) in endothelial cells. DisBa-01 at nanomolar concentrations also modulates metalloprotease 2 (MMP-2) and 9 (MMP-9) activity, the latter being decreased in fibroblasts and increased in MDA-MB-231 cells. In conclusion, these results demonstrate that αvβ3 integrin inhibitors may induce distinct effects in the cells of the tumor microenvironment, resulting in blockade of angiogenesis by impairing of VEGF signaling and in inhibition of tumor cell motility.     

Adipokine levels are altered by shiftwork: a preliminary study

Shiftwork is often associated with metabolic diseases, and in the past few years, several cytokines have been postulated to contribute to various diseases, including insulin resistance. The aim of this study was to compare the concentrations of adiponectin, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in samples of young adult men exposed to a fixed (i) night shift (n = 9), working from 22:00 to 06:00 h; (ii) early morning shift (n = 6), working from 06:00 to 14:00 h; and (iii) day shift (n = 7), working from 08:00 to 17:00 h. The fixed night-shift and early-morning-shift samples were considered collectively as a shiftworker group given their work times. Blood samples were collected during the regular working day at 4-h intervals over the course of 24 h, thus totaling six samples. Morphological and physical activity parameters did not differ between the three groups. Total energy intake was lowest on the early morning shifts (p 相似文献   

A Mutant of Metarhizium anisopliae var. acridum with Enhanced Submerged Conidiation

Summary An insertional mutant of Metarhizium anisopliae is described with enhanced submerged conidiation. In a 500 ml submerged culture, this mutant produces a mean of 4.05 × 10⁸ propagules ml⁻¹ from an inoculum of 1 × 10⁶ conidia, where the parental strain accumulates only 3.75 × 10⁴ propagules ml⁻¹.     

2-Benzoylpyridine-N(4)-tolyl thiosemicarbazones and their palladium(II) complexes: Cytotoxicity against leukemia cells

The palladium(II) complexes [Pd(2Bz4oT)Cl], [Pd(2Bz4mT)Cl], and [Pd(2Bz4pT)Cl] were prepared with N(4)-ortho- (H2Bz4oT) N(4)-meta- (H2Bz4mT) and N(4)-para- (H2Bz4pT) tolyl-thiosemicarbazones derived from 2-benzoylpyridine. The free thiosemicarbazones proved to be highly cytotoxic against Jurkat, HL60 and the resistant HL60.Bcl-X_L leukemia cell lines at nanomolar concentrations, but were much less cytotoxic to HepG2 human hepatoma cells. Upon coordination to palladium(II) the cytotoxic activity against all studied cell lines decreases. However, the high cytotoxicity of the free thiosemicarbazones against leukemia, together with their hepatotoxic profile similar to that of cisplatin suggest that N(4)-tolyl thiosemicarbazones have potential as chemotherapeutic drug candidates.     

Structural studies on zinc(II) complexes with 2-benzoylpyridine-derived hydrazones

2-Benzoylpyridine-phenylhydrazone (H2BzPh), 2-benzoylpyridine-para-chloro-phenylhydrazone (H2BzpClPh), and 2-benzoylpyridine-para-nitro-phenyl (H2BzpNO₂Ph) hydrazone were obtained and fully characterized, as well as their zinc(II) complexes [Zn(H2BzPh)Cl₂] (1), [Zn(H2BzClPh)Cl₂] (2) and [Zn(H2BzpNO₂Ph)Cl₂] (3). During the syntheses of complex 1 a second product crystallized, which was characterized as [Zn(2BzPh)₂] (1a). Upon re-crystallization in 1:9 DMSO:acetone conversion of 2 into [Zn(H2BzpClPh)Cl₂] · H₂O (2a) and of 3 into [Zn(2BzpNO₂Ph)Cl(DMSO)] (3a) occurred. The crystal structures of 1a, 2a and 3a were determined. In 1a the two nearly perpendicular H2BzPh ligands give rise to a distorted octahedral environment around the metal. The 5-fold coordination around the metal is completed with two chloride ions in 2a and with one chloride and one oxygen atom from DMSO in 3a.     

Relationship between Spontaneous Aminoglycoside Resistance in Escherichia coli and a Decrease in Oligopeptide Binding Protein

Changes in the amount of oligopeptide binding protein (OppA) in spontaneous kanamycin-resistant mutants of Escherichia coli were investigated. Among 20 colonies obtained from 10⁸ cells cultured in the presence of 20 μg of kanamycin/ml, 1 colony had no detectable OppA and 7 colonies were mutants with reduced amounts of OppA. Sensitivity of wild-type cells to kanamycin increased slightly by transformation of the oppA gene, but the sensitivity of the mutants increased greatly by the transformation. A mutant with no OppA was found to be a nonsense mutant of the oppA gene at amino acid position 166. In a mutant having a reduced level of OppA, the reduction was due to the decrease in OppA synthesis at the translational level. These mutants were also resistant to other aminoglycoside antibiotics, including streptomycin, neomycin, and isepamicin. Isepamicin uptake activities decreased greatly in these two kinds of mutants. The results support the proposition that aminoglycoside antibiotics are transported into cells by the oligopeptide transport system, and that transport is an important factor for spontaneous resistance to aminoglycoside antibiotics.     

Overexpression, purification, biochemical characterization, and molecular modeling of recombinant GDP-mannosyltransferase (GumH) from Xylella fastidiosa

The GumH enzyme from Xylella fastidiosa catalyzes the transfer reaction of a mannose from GDP-mannose to the carrier lipid cellobiose-pyrophosphate-polyprenol (Glc(2)-PP-Lip), an intermediary in the reaction for the synthesis of the exopolysaccharide (EPS) fastidian gum. The gumH gene was subcloned in the pMal-c2x vector, allowing the expression of the GumH-MBP fusion protein. Various attempts were made to obtain protein with the necessary degree of purity for crystallographic studies but the yield was very low. The gumH gene was then subcloned in the pET28a vector allowing the expression of the GumH enzyme in fusion with a histidine-rich peptide. The protein was purified and characterized. The three-dimensional structure of the X. fastidiosa GumH enzyme was modeled by threading studies. The model consists of N- and C-terminal domains similar in size and topology and separated by a deep cleft, which includes the EX(7)E motif that can be involved in the catalysis of GumH.     

Recombinant human insulin IX. Investigation of factors,influencing the folding of fusion protein-S-sulfonates,biotechnological precursors of human insulin

The peculiarities of molecular structures and the influence of reaction conditions on the folding efficiency of fusion proteins-biotechnological precursors of human insulin, expressed in Escherichia coli as inclusion bodies have been investigated. The fusion proteins contained proinsulin sequence with various leader peptides connected by an Arg residue to the insulin B-chain. The kind and the size of leader peptide do not have essential influence on folding efficiency. However, the efficiency of protein folding depends on the location of the (His)6 site, which is used for metal-chelating affinity chromatography. In our study the protein folding depends on the reaction medium composition (including additives), the presence of accompanied cell components, pH, temperature, concentrations of protein, and redox agents. A negative influence of nucleic acid and heavy metal ions on folding has been found. S-sulfonated fusion protein has proinsulin-like secondary structure (by CD-spectroscopy data) that is the key point for 95% efficient folding proceeding. Folded fusion proteins are transformed into insulin by enzymatic cleavage.