N-methyl-D-aspartate receptor-induced proteolytic conversion of postsynaptic class C L-type calcium channels in hippocampal neurons.

Ca2+ influx controls multiple neuronal functions including neurotransmitter release, protein phosphorylation, gene expression, and synaptic plasticity. Brain L-type Ca2+ channels, which contain either alpha 1C or alpha 1D as their pore-forming subunits, are an important source of calcium entry into neurons. Alpha 1C exists in long and short forms, which are differentially phosphorylated, and C-terminal truncation of alpha 1C increases its activity approximately 4-fold in heterologous expression systems. Although most L-type calcium channels in brain are localized in the cell body and proximal dendrites, alpha 1C subunits in the hippocampus are also present in clusters along the dendrites of neurons. Examination by electron microscopy shows that these clusters of alpha 1C are localized in the postsynaptic membrane of excitatory synapses, which are known to contain glutamate receptors. Activation of N-methyl-D-aspartate (NMDA)-specific glutamate receptors induced the conversion of the long form of alpha 1C into the short form by proteolytic removal of the C terminus. Other classes of Ca2+ channel alpha1 subunits were unaffected. This proteolytic processing reaction required extracellular calcium and was blocked by inhibitors of the calcium-activated protease calpain, indicating that calcium entry through NMDA receptors activated proteolysis of alpha1C by calpain. Purified calpain catalyzed conversion of the long form of immunopurified alpha 1C to the short form in vitro, consistent with the hypothesis that calpain is responsible for processing of alpha 1C in hippocampal neurons. Our results suggest that NMDA receptor-induced processing of the postsynaptic class C L-type Ca2+ channel may persistently increase Ca2+ influx following intense synaptic activity and may influence Ca2+-dependent processes such as protein phosphorylation, synaptic plasticity, and gene expression.

Toiminimen muuttaminen osakeyhtiöksi

Tutkielman tarkoituksena on selvittää, mitkä ovat keskeisimmät syyt, jotka johtavat halukkuuteen muuttaa yhtiömuotoa ja miten yhtiömuodon muutos vaikuttaa yhtiön verotukselliseen asemaan ja hallinnointiin. Tutkimus syventyy toiminimen muuttamiseen osakeyhtiöksi näiden kahden yhtiömuodon myös ollessa yleisimmät muodot harjoittaa yritystoimintaa.

A medium for inducing conversion of Histoplasma capsulatum var. capsulatum into its yeast-like form

Histoplasma capsulatum var. capsulatum is a dimorphic fungus that, under special conditions, converts from its more common mycelial form to a yeast-like form. Achieving this conversion, however, has been problematical for researchers. The present study tested conversion rates in ten Histoplasma capsulatum var. capsulatum strains using seven culture media, four of wich were conventional and three novel. One of our novel media, MLGema, induced complete conversion, of two strains within five days of incubation at 35 degrees centigrades, and of all strains that eventually converted by the time of the second subculturing transfer, under defined experimental conditions. MLGema is also inexpensive and easy to produce.

Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity.

Human Fas ligand (L) (CD95L) and tumor necrosis factor (TNF)-alpha undergo metalloproteinase-mediated proteolytic processing in their extracellular domains resulting in the release of soluble trimeric ligands (soluble [s]FasL, sTNF-alpha) which, in the case of sFasL, is thought to be implicated in diseases such as hepatitis and AIDS. Here we show that the processing of sFasL occurs between Ser126 and Leu127. The apoptotic-inducing capacity of naturally processed sFasL was reduced by >1,000-fold compared with membrane-bound FasL, and injection of high doses of recombinant sFasL in mice did not induce liver failure. However, soluble FasL retained its capacity to interact with Fas, and restoration of its cytotoxic activity was achieved both in vitro and in vivo with the addition of cross-linking antibodies. Similarly, the marginal apoptotic activity of recombinant soluble TNF-related apoptosis-inducing ligand (sTRAIL), another member of the TNF ligand family, was greatly increased upon cross-linking. These results indicate that the mere trimerization of the Fas and TRAIL receptors may not be sufficient to trigger death signals. Thus, the observation that sFasL is less cytotoxic than membrane-bound FasL may explain why in certain types of cancer, systemic tissue damage is not detected, even though the levels of circulating sFasL are high.

Chaperone-supervised conversion of prion protein to its protease-resistant form

Transmissible spongiform encephalopathies (TSEs) are lethal, infectious disorders of the mammalian nervous system. A TSE hallmark is the conversion of the cellular protein PrPC to disease-associated PrPSc (named for scrapie, the first known TSE). PrPC is protease-sensitive, monomeric, detergent soluble, and primarily α-helical; PrPSc is protease-resistant, polymerized, detergent insoluble, and rich in β-sheet. The “protein-only” hypothesis posits that PrPSc is the infectious TSE agent that directly converts host-encoded PrPC to fresh PrPSc, harming neurons and creating new agents of infection. To gain insight on the conformational transitions of PrP, we tested the ability of several protein chaperones, which supervise the conformational transitions of proteins in diverse ways, to affect conversion of PrPC to its protease-resistant state. None affected conversion in the absence of pre-existing PrPSc. In its presence, only two, GroEL and Hsp104 (heat shock protein 104), significantly affected conversion. Both promoted it, but the reaction characteristics of conversions with the two chaperones were distinct. In contrast, chemical chaperones inhibited conversion. Our findings provide new mechanistic insights into nature of PrP conversions, and provide a new set of tools for studying the process underlying TSE pathogenesis.

Catalytic conversion of wastes from the bioethanol production into carbon nanomaterials

This work addressed the production of carbon nanomaterials (CNMs) by catalytic conversion of wastes from the bioethanol industry, in the form of either sugarcane bagasse or corn-derived distillers dried grains with solubles (DDGS). Both bagasse and DDGS were pyrolysed at temperatures in the range of 600-1000 degrees C. The pyrolyzate gases were then used as CNM growth agents by chemical vapor deposition on stainless steel meshes, serving as both catalysts and substrates. CNM synthesis temperatures of 750-1000 degrees C were explored, and it was determined that their growth was most pronounced at 1000 degrees C. The nanomaterials produced from pyrolysis of bagasse were in the form of long, straight, multi-wall nanotubes with smooth walls and axially uniform diameters. Typical lengths were circa 50 mu m and diameters were in the range of 20-80 nm. The nanomaterials produced from pyrolysis of DDGS were in the form of long, entangled, rope-like structures with rugged walls, and axially non-uniform diameters. Typical diameters were in the range of 100-300 nm and their lengths were in the tens of microns. This process also produces a bio-syngas byproduct that is enriched in hydrogen. (C) 2011 Elsevier B.V. All rights reserved.

Experimental conversion of colony social organization in fire ants (Solenopsis invicta): worker genotype manipulation in absence of queen effects

Colony social organization in the fire ant Solenopsis invicta appears to be under strong genetic control. In the invasive USA range, polygyny (multiple queens per colony) is marked by the presence of the Gp-9(b) allele in most of a colony's workers, whereas monogyny (single queen per colony) is associated with the exclusive occurrence of the Gp-9(B) allele. Ross and Keller, Behav Ecol Sociobiol 51:287-295 (2002) experimentally manipulated social organization by cross-fostering queens into colonies of the alternate form, thereby changing adult worker Gp-9 genotype frequencies over time. Although these authors showed that social behavior switched predictably when the frequency of b-bearing adult workers crossed a threshold of 5-10%, the possibility that queen effects caused the conversions could not be excluded entirely. We addressed this problem by fostering polygyne brood into queenright monogyne colonies. All such treatment colonies switched social organization to become polygyne, coincident with their proportions of b-bearing workers exceeding 12%. Our results support the conclusion that polygyny in S. invicta is induced by a minimum frequency of colony workers carrying the b allele, and further confirm that its expression is independent of queen genotype or history, worker genotypes at genes not linked to Gp-9, and colony genetic diversity.

Increased Wnt signaling triggers oncogenic conversion of human breast epithelial cells by a Notch-dependent mechanism.

Wnt and Notch signaling have long been established as strongly oncogenic in the mouse mammary gland. Aberrant expression of several Wnts and other components of this pathway in human breast carcinomas has been reported, but evidence for a causative role in the human disease has been missing. Here we report that increased Wnt signaling, as achieved by ectopic expression of Wnt-1, triggers the DNA damage response (DDR) and an ensuing cascade of events resulting in tumorigenic conversion of primary human mammary epithelial cells. Wnt-1-transformed cells have high telomerase activity and compromised p53 and Rb function, grow as spheres in suspension, and in mice form tumors that closely resemble medullary carcinomas of the breast. Notch signaling is up-regulated through a mechanism involving increased expression of the Notch ligands Dll1, Dll3, and Dll4 and is required for expression of the tumorigenic phenotype. Increased Notch signaling in primary human mammary epithelial cells is sufficient to reproduce some aspects of Wnt-induced transformation. The relevance of these findings for human breast cancer is supported by the fact that expression of Wnt-1 and Wnt-4 and of established Wnt target genes, such as Axin-2 and Lef-1, as well as the Notch ligands, such as Dll3 and Dll4, is up-regulated in human breast carcinomas.

Basic properties of potassium oxide supported on zeolite y studied by pyrrole-tpd and catalytic conversion of methylbutynol

ABSTRACT We report on the basic properties of zeolite NaY and potassium supported on NaY (K/NaY) assessed by pyrrole-TPD and MBOH transformation. Pyrrole-TPD revealed that impregnation of zeolite NaY with potassium promoted additional adsorption sites for pyrrole compared to parent zeolite. For zeolite with various potassium loadings, pyrrole adsorbed on K/NaY decreased with increased potassium loading. Reduction in pyrrole adsorption could be due to potassium hindering intrinsic basic sites (lattice oxygen), to oxide of potassium occluding in zeolite cavities restricting access for pyrrole, or to K2O reacting with pyrrole to form nondesorbed pyrrolate anions. On MBOH transformation, potassium almost completely suppressed NaY acid sites while K/NaY basicity increased with potassium loading.

A rare case of solution and solid state inter-conversion of two copper(II) dimers and a copper(II) chain

Three Cu(II)-azido complexes of formula [Cu2L2(N-3)(2)] (1), [Cu2L2(N-3)(2)]center dot H2O (2) and [CuL(N-3)](n) (3) have been synthesized using the same tridentate Schiff base ligand HL (2-[(3-methylaminopropylimino)-methyl]-phenol), the condensation product of N-methyl-1,3-propanediamine and salicyldehyde). Compounds 1 and 2 are basal-apical mu-1,1 double azido bridged dimers. The dimeric structure of 1 is centro-symmetric but that of 2 is non-centrommetric. Compound 3 is a mu-1,1 single azido bridged 1D chain. The three complexes interconvert in solution and can be obtained in pure form by carefully controlling the synthetic conditions. Compound 2 undergoes an irreversible transformation to 1 upon dehydration in the solid state. The magnetic properties of compounds 1 and 2 show the presence of weak antiferromagnetic exchange interactions mediated by the double 1,1-N-3 azido bridges (J = -2.59(4) and -0.10(1) cm-(1), respectively). The single 1,1-N-3 bridge in compound 3 mediates a negligible exchange interaction.

Conversion of myoblasts to physiologically active neuronal phenotype.

Repressor element 1 (RE1)-silencing transcription factor (REST)/neuron-restrictive silencer factor (NRSF) can repress several terminal neuronal differentiation genes by binding to a specific DNA sequence (RE1/neuron-restrictive silencer element [NRSE]) present in their regulatory regions. REST-VP16 binds to the same RE1/NRSE, but activates these REST/NRSF target genes. However, it is unclear whether REST-VP16 expression is sufficient to cause formation of functional neurons either from neural stem cells or from heterologous stem cells. Here we show that the expression of REST-VP16 in myoblasts grown under muscle differentiation conditions blocked entry into the muscle differentiation pathway, countered endogenous REST/NRSF-dependent repression, activated the REST/NRSF target genes, and, surprisingly, activated other neuronal differentiation genes and converted the myoblasts to a physiologically active neuronal phenotype. Furthermore, in vitro differentiated neurons produced by REST-VP16-expressing myoblasts, when injected into mouse brain, survived, incorporated into the normal brain, and did not form tumors. This is the first instance in which myoblasts were converted to a neuronal phenotype. Our results suggest that direct activation of REST/NRSF target genes with a single transgene, REST-VP16, is sufficient to activate other terminal neuronal differentiation genes and to override the muscle differentiation pathways, and they suggest that this approach provides an efficient way of triggering neuronal differentiation in myoblasts and possibly other stem cells.

Conversion of DNA gyrase into a conventional type II topoisomerase

DNA gyrase is unique among topoisomerases in its ability to introduce negative supercoils into closed-circular DNA. We have demonstrated that deletion of the C-terminal DNA-binding domain of the A subunit of gyrase gives rise to an enzyme that cannot supercoil DNA but relaxes DNA in an ATP-dependent manner. Novobiocin, a competitive inhibitor of ATP binding by gyrase, inhibits this reaction. The truncated enzyme, unlike gyrase, does not introduce a right-handed wrap when bound to DNA and stabilizes DNA crossovers; characteristics reminiscent of conventional type II topoisomerases. This new enzyme form can decatenate DNA circles with increased efficiency compared with intact gyrase and, as a result, can complement the temperature-sensitive phenotype of a parCts mutant. Thus these results suggest that the unique properties of DNA gyrase are attributable to the wrapping of DNA around the C-terminal DNA-binding domains of the A subunits and provide an insight into the mechanism of type II topoisomerases.

Interaction of two arginine residues in lactate oxidase with the enzyme flavin: Conversion of FMN to 8-formyl-FMN

Two arginine residues, Arg-181 and Arg-268, are conserved throughout the known family of FMN-containing enzymes that catalyze the oxidation of α-hydroxyacids. In the lactate oxidase from Aerococcus viridans, these residues have been changed to lysine in two single mutations and in a double mutant form. In addition, Arg-181 has been replaced by methionine to determine the effect of removing the positive charge on the residue. The effects of these replacements on the kinetic and thermodynamic properties are reported. With all mutant forms, there are only small effects on the reactivity of the reduced flavin with oxygen. On the other hand, the efficiency of reduction of the oxidized flavin by l-lactate is greatly reduced, particularly with the R268K mutant forms. The results demonstrate the importance of the two arginine residues in the binding of substrate and its interaction with the flavin, and are consistent with a previous hypothesis that they also play a role of charge neutralization in the transition state of substrate dehydrogenation. The replacement of Arg-268 by lysine also results in a slow conversion of the 8-CH3- substituent of FMN to yield 8-formyl-FMN, still tightly bound to the enzyme, and with significantly different physical and chemical properties from those of the FMN-enzyme.

Direct conversion of an oligopeptide from a β-sheet to an α-helix: A model for amyloid formation

A 16-amino acid oligopeptide forms a stable β-sheet structure in water. In physiological solutions it is able to self-assemble to form a macroscopic matrix that stains with Congo red. On raising the temperature of the aqueous solution above 70°C, an abrupt structural transition occurs in the CD spectra from a β-sheet to a stable α-helix without a detectable random-coil intermediate. With cooling, it retained the α-helical form and took several weeks at room temperature to partially return to the β-sheet form. Slow formation of the stable β-sheet structure thus shows kinetic irreversibility. Such a formation of very stable β-sheet structures is found in the amyloid of a number of neurological diseases. This oligopeptide could be a model system for studying the protein conformational changes that occurs in scrapie or Alzheimer disease. The abrupt and direct conversion from a β-sheet to an α-helix may also be found in other processes, such as protein folding and protein–protein interaction. Furthermore, such drastic structure changes may also be exploited in biomaterials designed as sensors to detect environmental changes.

Malignant conversion of chemically transformed normal human cells.

Two structurally unrelated chemicals, aflatoxin B1 and propane sultone, transformed human foreskin cells to a stage of anchorage-independent growth. Isolation from agar and repopulation in monolayer culture of these transformed cells was followed by transfection with a cDNA library, which resulted in cells that exhibited an altered epithelioid morphology. Chemically transformed/nontransfected cells and transfected normal cells did not undergo a significant morphological change. These epithelioid-appearing, transfected cells, when inoculated into nude mice, form progressively growing tumors. The tumors are histopathologically interpreted as carcinomas. All of the first generation tumors in the surrogate hosts exhibited characteristic rates of growth similar to those of transplants of spontaneous human tumors. In the second generation of tumor xenografts, the progressively growing tumors derived from the transfected cells exhibited a more rapid rate of growth. Southern analysis and reverse transcription PCR confirmed that a 1.3-kb genetic element was integrated into the genome and was actively being transcribed. Examination of the metaphase chromosomes in normal human cells revealed that the genetic element responsible for this conversion was located at site 31-32 of the q arm of chromosome 7. The DNA sequence of this 1.3-kb genetic element contains a coding region for 79 amino acids and a long 3'-untranslated region and appears to be identical to CATR1.3 isolated from tumors produced by methyl methanesulfonate-converted, nontransplantable human tumor cells.