Novel flutamide regulated genes in the rat ventral prostate: differential modulation of their expression by castration and flutamide treatments

Aim: To identify flutamide regulated genes in the rat ventral prostate. Methods: Total RNA from ventral prostates control and flutamide treated rats were isolated. Differentially expressed transcripts were identified using display reverse transcriptase polymerase chain reaction. The effect of castration on the expression of regulated transcripts was studied. Results: We have identified beta 2-microglobulin, cytoplasmic FMR1 protein 2 and pumilio 1 as flutamide induced and spermine binding protein and ribophorin II as flutamide targets in the rat ventral prostate. Although flutamide treatment caused an induction of pumilio I mRNA, had no effect. Conclusion: Castration and flutamide treatments exert differential effects on gene expression. might also have direct AR independent effects, which might have implications in the emergence of androgen dent prostate cancer and the failure of flutamide therapy.

PVA-PSSA Membrane with Interpenetrating Networks and its Methanol Crossover Mitigating Effect in DMFCs

A membrane with interpenetrating networks between poly(vinyl alcohol) (PVA) and poly(styrene sulfonic acid) (PSSA) coupled with a high proton conductivity is realized and evaluated as a proton exchange membrane electrolyte for a direct methanol fuel cell (DMFC). Its reduced methanol permeability and improved performance in DMFCs suggest the new blend as an alternative membrane to Nafion membranes. The membrane has been characterized by powder X-ray diffraction, scanning electron microscopy, time-modulated differential scanning calorimetry, and thermogravimetric analysis in conjunction with its mechanical strength. The maximum proton conductivity of 3.3×10−2 S/cm for the PVA–PSSA blend membrane is observed at 373 K. From nuclear magnetic resonance imaging and volume localized spectroscopy experiments, the PVA–PSSA membrane has been found to exhibit a promising methanol impermeability, in DMFCs. On evaluating its utility in a DMFC, it has been found that a peak power density of 90 mW/cm2 at a load current density of 320 mA/cm2 is achieved with the PVA–PSSA membrane compared to a peak power density of 75 mW/cm2 at a load current density of 250 mA/cm2 achievable for a DMFC employing Nafion membrane electrolyte while operating under identical conditions; this is attributed primarily to the methanol crossover mitigating property of the PVA–PSSA membrane.

Effects of membrane channel-forming polypeptides on mitochondrial oxidative phosphorylation. A comparison of alamethicin, gramicidin A, melittin and tetraacetyl melittin

Transmembrane channel-forming polypeptides can function as uncouplers of mitochondrial oxidative phosphorylation. The observed effects are dependent on the phosphate ion (Pi) concentration in the medium. At low Pi (2.5 mM) the order of uncoupling efficiencies is gramicidin A much greater than alamethicin greater than tetraacetyl melittin greater than melittin. The remarkably high activity of gramicidin A suggests insertion of preformed channel dimers into the membrane. It is also suggested that lipid phase association of peptides is necessary in the other cases. At Pi = 100 mM inhibitory effects are observed for alamethicin and tetraacetyl melittin. Less pronounced inhibition is seen for melittin, while no such effect is noted for gramicidin A. The site of inhibition is shown to be complex IV, and the differences in the behavior of the peptides are rationalized in terms of channel structures.

Effects of membrane channel-forming polypeptides on mitochondrial oxidative phosphorylation. A comparison of alamethicin, gramicidin A, melittin and tetraacetyl melittin.

Transmembrane channel-forming polypeptides can function as uncouplers of mitochondrial oxidative phosphorylation. The observed effects are dependent on the phosphate ion (Pi) concentration in the medium. At low Pi (2.5 mM) the order of uncoupling efficiencies is gramicidin A much greater than alamethicin greater than tetraacetyl melittin greater than melittin. The remarkably high activity of gramicidin A suggests insertion of preformed channel dimers into the membrane. It is also suggested that lipid phase association of peptides is necessary in the other cases. At Pi = 100 mM inhibitory effects are observed for alamethicin and tetraacetyl melittin. Less pronounced inhibition is seen for melittin, while no such effect is noted for gramicidin A. The site of inhibition is shown to be complex IV, and the differences in the behavior of the peptides are rationalized in terms of channel structures.

Zervamicins, a structurally characterised peptide model for membrane ion channels

Voltage dependent membrane channels are formed by the zervamicins, a group of α-aminoisobutyric acid containing peptides. The role of polar residues like Thr, Gln and Hyp in promoting helical bundle formation is established by dramatically reduced channel lifetimes for a synthetic apolar analog. Crystal structures of Leu1-zervamicin reveal association of bent helices. Polar contacts between convex faces result in an ‘hour glass’ like arrangement of an aqueous channel with a central constriction. The structure suggests that gating mechanisms may involve movement of the Gln11 carboxamide group. Gln3 may play a role in modulating the size of the channel mouth.

Synthesis of neoglycopeptides and analyses of their biodistributionin vivo to identify tissue specific uptake and novel putative membrane lectins

Complex typeN-linked oligosaccharides derived from fetuin, fibrinogen and thyroglobulin were coupled to acetyltyrosine affording a series of neoglycopeptides with retention of terminal structures and the beta-anomeric configuration of their reducing endN-acetylglycosamine residue. The neoglycopeptides thus synthesized could be labelled to high specific activities with125I in the aromatic side chain of tyrosine. Analysis of the fate of these neoglycopeptides in conjunction with inhibition with asialofetuin and oligosaccharides of defined structure in micein vivo revealed the uptake of galactosylated biantennary compound by kidneys, in addition to the known itinerary of triantennary galactosylated complex oligosaccharide from fetuin to liver and the galactosylated biantennary chain with fucosylation in the core to bone marrows. On the other hand, the agalacto, aglucosamino biantennary chains with and without fucosylation in the core region are taken up by submaxillary glands while the conserved trimannosyl core with fucose is primarily concentrated in stomach tissue. These studies thus define new routes for the uptake of complexN-linked glycans and also subserve to identify lectins presumably involved in their recognition.

Morphogenesis of Mammalian Endoplasmic Reticulum and Golgi Apparatus throughout the Cell Cycle

The endoplasmic reticulum (ER) and the Golgi apparatus are organelles that produce, modify and transport proteins and lipids and regulate Ca2+ environment within cells. Structurally they are composed of sheets and tubules. Sheets may take various forms: intact, fenestrated, single or stacked. The ER, including the nuclear envelope, is a single continuous network, while the Golgi shows only some level of connectivity. It is often unclear, how different morphologies correspond to particular functions. Previous studies indicate that the structures of the ER and Golgi are dynamic and regulated by fusion and fission events, cytoskeleton, rate of protein synthesis and secretion, and specific structural proteins. For example, many structural proteins shaping tubular ER have been identified, but sheet formation is much more unclear. In this study, we used light and electron microscopy to study morphological changes of the ER and Golgi in mammalian cells. The proportion, type, location and dynamics of ER sheets and tubules were found to vary in a cell type or cell cycle stage dependent manner. During interphase, ER and Golgi structures were demonstrated to be regulated by p37, a cofactor of the fusion factor p97, and microtubules, which also affected the localization of the organelles. Like previously shown for the Golgi, the ER displayed a tendency for fenestration and tubulation during mitosis. However, this shape change did not result in ER fragmentation as happens to Golgi, but a continuous network was retained. The activity of p97/p37 was found to be important for the reassembly of both organelles after mitosis. In EM images, ER sheet membranes appear rough, since they contain attached ribosomes, whereas tubular membranes appear smooth. Our studies revealed that structural changes of the ER towards fenestrated and tubular direction correlate with loss of ER-bound ribosomes and vice versa. High and low curvature ER membranes have a low and high density of ribosomes, respectively. To conclude, both ER and Golgi architecture depend on fusion activity of p97/p37. ER morphogenesis, particularly of the sheet shape, is intimately linked to the density of membrane bound ribosomes.

POP-inhibiittorin KYP-2047 vaikutukset angiogeneesiin in vitro

Angiogeneesi on tärkeä ilmiö elimistön fysiologiassa, mutta myös lukuisissa patologisissa tiloissa. Angiogeneesi on monivaiheinen prosessi, joka sisältää angiogeneesiä indusoivia ja sitä inhiboivia tekijöitä tasapainossa keskenään. Useat tutkimukset puoltavat sitä, että tymosiini ȕ4 (Tȕ4) ja tetrapeptidi Ac-SDKP (N-asetyyliseryyli- aspartyyli-lysyyli-proliini) indusoivat angiogeneesiä in vitro ja in vivo. Tutkimukset viittaavat myös siihen, että prolyylioligopeptidaasi (POP) hydrolysoi peptidifragmentin Ac- SDKP Tȕ4:n (43 ah) proliinin jälkeen. POP on laajalti esiintyvä seriiniproteaasi, joka pystyy pilkkomaan vain alle 30 aminohapon oligopeptidejä. Tȕ4:n tulee siksi pilkkoutua ensin jonkin, vielä tuntemattoman peptidaasin johdosta. POP:ia on löydetty eniten aivoista, minkä vuoksi sitä on tutkittu varsinkin muistin ja oppimisen häiriötiloissa sekä neurodegeneratiivisten sairausten yhteydessä. POP:in todellinen fysiologinen merkitys on kuitenkin vielä selvittämättä. Tämän pro gradun kirjallisuusosiossa selvitetään angiogeneesiin liittyvien tekijöiden yhteyksiä sekä kuvataan angiogeenisten Tȕ4:n, Ac-SDKP:n ja POP:in ominaisuuksia, esiintymistä ja toimintaa. Kokeellisen osion tarkoituksena oli osoittaa, osallistuvatko POP ja Tȕ4 tetrapeptidin Ac-SDKP muodostumiseen ja kapillaarimuodostumiseen ja edelleen, voidaanko POPaktiivisuutta, tetrapeptidi- ja kapillaarimuodostumista estää spesifisellä POP-inhibiittorilla, KYP-2047:llä. Kokeellinen osa oli kaksiosainen. Ensimmäisessä osassa tutkittiin POPaktiivisuutta ja suoritettiin Ac-SDKP –pitoisuusmittauksia ajanjaksolla 0-180 min Wistarkannan rotista tehdyillä homogenaateilla. Tutkimusryhminä olivat 0,1 ja 0,5 μM KYP-2047 (+2 μM Tȕ4), 1:20 (0,625 μM) humaaniperäinen rekombinantti-POP (+ 2 μM Tȕ4), 2 μM Tȕ4 (pos. kontrolli) ja raakahomogenaatti (neg. kontrolli). Toisessa osassa tutkittiin kapillaarimuodostumista ajanjaksolla 0-180 min humaaniperäisillä napanuoralaskimon primaariendoteelisoluilla MatrigelTM Matrix -päällystetyllä 48- kuoppalevyllä, jolle oli siirrostettu 50 000 solua/kuoppa. Naudan seerumilla ja antibiooteilla käsitellyt tutkimusryhmät olivat 5 ja 10 μM KYP-2047 (+4 μM Tȕ4), 1:20 (0,625 μM) humaaniperäinen rekombinantti-POP (+4 μM Tȕ4), 4 μM Tȕ4 (pos. kontrolli) ja DMEM (neg. kontrolli). Kuoppia inkuboitiin ja kapillaarimuodostuminen kuvattiin valomikroskoopilla digitaalikameralla. Kutakin tutkimusryhmää pipetoitiin kolmeen rinnakkaiseen kuoppaan ja kokeet toistettiin neljästi. Sulkeutuneiden kapillaarien lukumäärä laskettiin manuaalisesti ja tuloksista tehtiin tilastollinen analyysi. 7ȕ4:n ja POP:in havaittiin molempien osallistuvan tetrapeptidin AC-SDKP muodostumiseen munuaishomogenaateissa. Primaariendoteelisolut muodostivat selkeitä kapillaareja Matrigelilla, erityisesti POP- ja Tȕ4–ryhmissä. KYP-2047 inhiboi tehokkaasti POP:ia kaikissa kokeissa osoittautuen hyväksi antiangiogeeniseksi yhdisteeksi. Angiogeneesin mekanismien ja POP:in, Tȕ4:n ja Ac-SDKP:n yhteyksien selvittäminen vaatii luonnollisesti vielä lisätutkimuksia.

Functional role of the Mso1p-Sec1p complex in membrane fusion regulation

Sec1/Munc18 (SM) protein family members are evolutionary conserved proteins. They perform an essential, albeit poorly understood function in SNARE complex formation in membrane fusion. In addition to the SNARE complex components, only a few SM protein binding proteins are known. Typically, their binding modes to SM proteins and their contribution to the membrane fusion regulation is poorly characterised. We identified Mso1p as a novel Sec1p interacting partner. It was shown that Mso1p and Sec1p interact at sites of polarised secretion and that this localisation is dependent on the Rab GTPase Sec4p and its GEF Sec2p. Using targeted mutagenesis and N- and C-terminal deletants, it was discovered that the interaction between an N-terminal peptide of Mso1p and the putative Syntaxin N-peptide binding area in Sec1p domain 1 is important for membrane fusion regulation. The yeast Syntaxin homologues Sso1p and Sso2p lack the N-terminal peptide. Our results show that in addition to binding to the putative N-peptide binding area in Sec1p, Mso1p can interact with Sso1p and Sso2p. This result suggests that Mso1p can mimic the N-peptide binding to facilitate membrane fusion. In addition to Mso1p, a novel role in membrane fusion regulation was revealed for the Sec1p C-terminal tail, which is missing in its mammalian homologues. Deletion of the Sec1p-tail results in temperature sensitive growth and reduced sporulation. Using in vivo and in vitro experiments, it was shown that the Sec1p-tail mediates SNARE complex binding and assembly. These results propose a regulatory role for the Sec1p-tail in SNARE complex formation. Furthermore, two novel interaction partners for Mso1p, the Rab GTPase Sec4p and plasma membrane phospholipids, were identified. The Sec4p link was identified using Bimolecular Fluorescence Complementation assays with Mso1p and the non-SNARE binding Sec1p(1-657). The assay revealed that Mso1p can target Sec1p(1-657) to sites of secretion. This effect is mediated via the Mso1p C-terminus, which previously has been genetically linked to Sec4p. These results and in vitro binding experiments suggest that Mso1p acts in cooperation with the GTP-bound form of Sec4p on vesicle-like structures prior to membrane fusion. Mso1p shares homology with the PIP2 binding domain of the mammalian Munc18 binding Mint proteins. It was shown both in vivo and in vitro that Mso1p is a phospholipid inserting protein and that this insertion is mediated by the conserved Mso1p amino terminus. In vivo, the Mso1p phospholipid binding is needed for sporulation and Mso1p-Sec1p localisation at the sites of secretion at the plasma membrane. The results reveal a novel layer of membrane fusion regulation in exocytosis and propose a coordinating role for Mso1p in connection with membrane lipids, Sec1p, Sec4p and SNARE complexes in this process.

Wave propagation in a micropolar fluid contained in a visco-elastic membrane

The aim of the paper is to investigate the propagation of a pulse in a micropolar fluid contained in a visco-elastic membrane. It was undertaken with a view to study how closely we can approximate the flow of blood in arteries by the above model. We find that for large Reynolds number, the effect of micropolarity is hardly perceptible, whereas for small Reynolds numbers it is of considerable importance.

Integrin trafficking regulated by Rab21 is necessary for cytokinesis

Adherent cells undergo remarkable changes in shape during cell division. However, the functional interplay between cell adhesion turnover and the mitotic machinery is poorly understood. The endo/exocytic trafficking of integrins is regulated by the small GTPase Rab21, which associates with several integrin alpha subunits. Here, we show that targeted trafficking of integrins to and from the cleavage furrow is required for successful cytokinesis, and that this is regulated by Rab21. Rab21 activity, integrin-Rab21 association, and integrin endocytosis are all necessary for normal cytokinesis, which becomes impaired when integrin-mediated adhesion at the cleavage furrow fails. We also describe a chromosomal deletion and loss of Rab21 gene expression in human cancer, which leads to the accumulation of multinucleate cells. Importantly, reintroduction of Rab21 rescued this phenotype. In conclusion, Rab21-regulated integrin trafficking is essential for normal cell division, and its defects may contribute to multinucleation and genomic instability, which are hallmarks of cancer.

cAMP-regulated Protein Lysine Acetylases in Mycobacteria

Cyclic AMP synthesized by Mycobacterium tuberculosis has been shown to play a role in pathogenesis. However, the high levels of intracellularcAMP found in both pathogenic and nonpathogenic mycobacteria suggest that additional and important biological processes are regulated by characterization of novel cAMP-binding proteins in M. smegmatis and M. tuberculosis (MSMEG_5458 and Rv0998, respectively) that contain a cyclic nucleotide binding domain fused to a domain that shows similarity to the GNAT family of acetyltransferases. We detect protein lysine acetylation in mycobacteria and identify a universal stress protein (USP) as a substrate of MSMEG_5458. Acetylation of a lysine residue in USP is regulated by cAMP, and using a strain deleted for MSMEG_5458, we show that USP is indeed an in vivo substrate for MSMEG_5458. The Rv0998 protein shows a strict cAMP-dependent acetylation of USP, despite a lower affinity for cAMP than MSMEG_5458. Thus, this report not only represents the first demonstration of protein lysine acetylation in mycobacteria but also describes a unique functional interplay between a cyclic nucleotide binding domain and a protein acetyltransferase.