Cleft palate in mice with a targeted mutation in the γ-aminobutyric acid-producing enzyme glutamic acid decarboxylase 67

The functions of neurotransmitters in fetal development are poorly understood. Genetic observations have suggested a role for the inhibitory amino acid neurotransmitter γ-aminobutyric acid (GABA) in the normal development of the mouse palate. Mice homozygous for mutations in the β-3 GABAA receptor subunit develop a cleft secondary palate. GABA, the ligand for this receptor, is synthesized by the enzyme glutamic acid decarboxylase. We have disrupted one of the two mouse Gad genes by gene targeting and also find defects in the formation of the palate. The striking similarity in phenotype between the receptor and ligand mutations clearly demonstrates a role for GABA signaling in normal palate development.

The Putative “Switch 2” Domain of the Ras-related GTPase, Rab1B, Plays an Essential Role in the Interaction with Rab Escort Protein

Posttranslational modification of Rab proteins by geranylgeranyltransferase type II requires that they first bind to Rab escort protein (REP). Following prenylation, REP is postulated to accompany the modified GTPase to its specific target membrane. REP binds preferentially to Rab proteins that are in the GDP state, but the specific structural domains involved in this interaction have not been defined. In p21 Ras, the α2 helix of the Switch 2 domain undergoes a major conformational change upon GTP hydrolysis. Therefore, we hypothesized that the corresponding region in Rab1B might play a key role in the interaction with REP. Introduction of amino acid substitutions (I73N, Y78D, and A81D) into the putative α2 helix of Myc-tagged Rab1B prevented prenylation of the recombinant protein in cell-free assays, whereas mutations in the α3 and α4 helices did not. Additionally, upon transient expression in transfected HEK-293 cells, the Myc-Rab1B α2 helix mutants were not efficiently prenylated as determined by incorporation of [3H]mevalonate. Metabolic labeling studies using [32P]orthophosphate indicated that the poor prenylation of the Rab1B α2 helix mutants was not directly correlated with major disruptions in guanine nucleotide binding or intrinsic GTPase activity. Finally, gel filtration analysis of cytosolic fractions from 293 cells that were coexpressing T7 epitope-tagged REP with various Myc-Rab1B constructs revealed that mutations in the α2 helix of Rab1B prevented the association of nascent (i.e., nonprenylated) Rab1B with REP. These data indicate that the Switch 2 domain of Rab1B is a key structural determinant for REP interaction and that nucleotide-dependent conformational changes in this region are largely responsible for the selective interaction of REP with the GDP-bound form of the Rab substrate.

Mutations in the C-terminal fragment of DnaK affecting peptide binding.

Escherichia coli DnaK acts as a molecular chaperone through its ATP-regulated binding and release of polypeptide substrates. Overexpressing a C-terminal fragment (CTF) of DnaK (Gly-384 to Lys-638) containing the polypeptide substrate binding domain is lethal in wild-type E. coli. This dominant-negative phenotype may result from the nonproductive binding of CTF to cellular polypeptide targets of DnaK. Mutations affecting DnaK substrate binding were identified by selecting noncytotoxic CTF mutants followed by in vitro screening. The clustering of such mutations in the three-dimensional structure of CTF suggests the model that loops L1,2 and L4,5 form a rigid core structure critical for interactions with substrate.

Description of the fossil manganese nodules found in the Oligocene clay deposits of Moravia, Brno area, Czech Republic

In the green Oligocene clay of Krizanovice (former Krzyzanowicz) the author found numerous black nodules. In the 3-4 centimeter thick black crust of a particular specimen the concentration in MnO2 is evaluated at 46.6% MnO2. The determination was done using the Volhard's method (precipitation of Fe by ZnO and titration with KMnO4). Only the dissoleved part in HCL was analysed. The non soluble residue was essentially a silica precipitate in the form of many gray flakes. The specific gravity of the crust was evaluated at 3.8. In the internal yellow core the amount of manganese is about 2.39% MnO2. Due to the light color it is judged to probably be in the form of Mn2O3.

A polymorphism in the agouti signalling protein (ASIP) is associated with decreased levels of mRNA

To date, a role for agouti signalling protein (ASIP) in human pigmentation has not been well characterized. It is known that agouti plays a pivotal role in the pigment switch from the dark eumelanin to the light pheomelanin in the mouse. However, because humans do not have an agouti banded hair pattern, its role in human pigmentation has been questioned. We previously identified a single polymorphism in the 3'-untranslated region (UTR) of ASIP that was found at a higher frequency in African-Americans compared with other population groups. To compare allele frequencies between European-Australians and indigenous Australians, the g.8818A -> G polymorphism was genotyped. Significant differences were seen in allele frequencies between these groups (P < 0.0001) with carriage of the G allele highest in Australian Aborigines. In the Caucasian sample set a strong association was observed between the G allele and dark hair colour (P = 0.004) (odds ratio 4.6; 95% CI 1.4-15.27). The functional consequences of this polymorphism are not known but it was postulated that it might result in message instability and premature degradation of the transcript. To test this hypothesis, ASIP mRNA levels were quantified in melanocytes carrying the variant and non-variant alleles. Using quantitative real-time polymerase chain reaction the mean ASIP mRNA ratio of the AA genotype to the AG genotype was 12 (P < 0.05). This study suggests that the 3'-UTR polymorphism results in decreased levels of ASIP and therefore less pheomelanin production.

Adiponectin multimerization is dependent on conserved lysines in the collagenous domain: Evidence for regulation of multimerization by alterations in posttranslational modifications

Adiponectin is a secreted, multimeric protein with insulin-sensitizing, antiatherogenic, and antiinflammatory properties. Serum adiponectin consists of trimer, hexamer, and larger high-molecular-weight (HMW) multimers, and these HMW multimers appear to be the more bioactive forms. Multimer composition of adiponectin appears to be regulated; however, the molecular mechanisms involved are unknown. We hypothesize that regulation of adiponectin multimerization and secretion occurs via changes in posttranslational modifications (PTMs). Although a structural role for intertrimer disulfide bonds in the formation of hexamers and HMW multimers is established, the role of other PTMs is unknown. PTMs identified in murine and bovine adiponectin include hydroxylation of multiple conserved proline and lysine residues and glycosylation of hydroxylysines. By mass spectrometry, we confirmed the presence of these PTMs in human adiponectin and identified three additional hydroxylations on Pro71, Pro76, and Pro95. We also investigated the role of the five modified lysines in multimer formation and secretion of recombinant human adiponectin expressed in mammalian cell lines. Mutation of modified lysines in the collagenous domain prevented formation of HMW multimers, whereas a pharmacological inhibitor of prolyl- and lysyl-hydroxylases, 2,2'-dipyridyl, inhibited formation of hexamers and HMW multimers. Bacterially expressed human adiponectin displayed a complete lack of differentially modified isoforms and failed to form bona fide trimers and larger multimers. Finally, glucose-induced increases in HMW multimer production from human adipose explants correlated with changes in the two-dimensional electrophoresis profile of adiponectin isoforms. Collectively, these data suggest that adiponectin multimer composition is affected by changes in PTM in response to physiological factors.

"Lájk első látásra?" – Mi inspirálja a márkák kedvelését az online térben, kis vendéglátóhelyek esetében? ("Like at first sight?" – What inspires liking brands in online social media in the case of small catering establishments?)

A márkatulajdonosok számára elsődleges fontosságú, hogy feltérképezzék saját márkaközösségüket, ezáltal is elősegítve a közösségi fókuszú kommunikációs tevékenységüket. Különösen igaz ez a jellemzően kis- és középvállalati formában működő vendéglátóhelyek számára, ahol a közösségi média sokszor az egyetlen rendelkezésre álló marketingkommunikációs eszköz. Empirikus kutatásunkban fogyasztói narratívákat (n=151) elemzünk a tartalomelemzés módszertanával. A válaszadókat arra kértük, hogy írjanak kedvenc vendéglátóhelyükkel való kapcsolatukról az online térben. Elemzésünkben arra keressük a választ, hogy a márkaközösségek tagjai milyen belső indíttatásból kerültek kapcsolatba kedvenc vendéglátóhelyeikkel a közösségi platformokon. Az elsősorban fogyasztási élményekhez kötött kapcsolat online közösségi térbe való kiterjesztésére olyan belső motivációkat tártunk fel, mint a pragmatikusabb információközpontú indíttatások (pl. kizárólagos információk, naprakészség, vállalati ajánlatok, közösségimédia-specifikus tartalmakhoz való hozzáférés igénye), az érzelmi kötődés kifejezése (pl. nosztalgia, ismerősöknek való ajánlás, személyes érintettség), a társas befolyás (oldalak kedvelése mint kommunikációs tartalom, konformitás, önkifejezés), valamint a vállalati alapképességeken túlmutató egyéb tematikus kapcsolódások. ____ It is of primary importance for brand owners to map their own brand community, in order to stimulate their social-focused communication activity. This is especially true for typically small and medium-sized catering enterprises, where in most cases, social media is the only available marketing communications tool. In our empirical research, we analyze consumer narratives (n=151) with the methodology of content analysis. We asked the respondents to write about their relationship with their most preferred catering establishments in the online social sphere. In our research, we search for the inner intention by which members of brand communities got connected with their most preferred catering establishments on social platforms. We revealed such inner motivations for users extending a primarily experience (an consumption) based relationship to the online social sphere like rather pragmatic, information-focused intentions (e.g. exclusive information, timeliness, company offers, access claim for social media -specific contents), the expression of emotional binding (e.g. nostalgia, reference for acquaintances, personal involvement), social influence (the like as communication content, conformity, self-expression), and other thematic linkages beyond core company competences.

Abundance and exploitation of loggerhead turtles nesting in Boa Vista Island, Cape Verde: The only substantial rookery in the eastern Atlantic.

[EN] The main nesting area for loggerhead turtles in the eastern Atlantic is in the Cape Verde Islands, largely restricted to the island of Boa Vista. Extensive monitoring demonstrated a globally significant population for the species despite a sustained high level of anthropogenic take of nesting females for local consumption. Through an extensive stratified monitoring program across the island in the seasons 2007-2009, we estimated a total of 13955, 12028 and 19950 clutches in the 3 years, respectively. These values indicate that the mean number of nesting females averaged 3700. Considering that a female breed, on average, every 2.4 years, we estimate that the overall number of adult females in the population during these three seasons was 8900.

Numerical solution of a Cauchy problem for Laplace equation in 3-dimensional domains by integral equations

A numerical method based on integral equations is proposed and investigated for the Cauchy problem for the Laplace equation in 3-dimensional smooth bounded doubly connected domains. To numerically reconstruct a harmonic function from knowledge of the function and its normal derivative on the outer of two closed boundary surfaces, the harmonic function is represented as a single-layer potential. Matching this representation against the given data, a system of boundary integral equations is obtained to be solved for two unknown densities. This system is rewritten over the unit sphere under the assumption that each of the two boundary surfaces can be mapped smoothly and one-to-one to the unit sphere. For the discretization of this system, Weinert’s method (PhD, Göttingen, 1990) is employed, which generates a Galerkin type procedure for the numerical solution, and the densities in the system of integral equations are expressed in terms of spherical harmonics. Tikhonov regularization is incorporated, and numerical results are included showing the efficiency of the proposed procedure.

Numerical solution of an elliptic 3-dimensional Cauchy problem by the alternating method and boundary integral equations

We consider the Cauchy problem for the Laplace equation in 3-dimensional doubly-connected domains, that is the reconstruction of a harmonic function from knowledge of the function values and normal derivative on the outer of two closed boundary surfaces. We employ the alternating iterative method, which is a regularizing procedure for the stable determination of the solution. In each iteration step, mixed boundary value problems are solved. The solution to each mixed problem is represented as a sum of two single-layer potentials giving two unknown densities (one for each of the two boundary surfaces) to determine; matching the given boundary data gives a system of boundary integral equations to be solved for the densities. For the discretisation, Weinert's method [24] is employed, which generates a Galerkin-type procedure for the numerical solution via rewriting the boundary integrals over the unit sphere and expanding the densities in terms of spherical harmonics. Numerical results are included as well.

Three-dimensional hydrogen-bonded structures in the 1:1 proton-transfer compounds of L-tartaric acid with the associative-group monosubstituted pyridines 3-minopyridine, 3-carboxypyridine (nicotinic acid) and 2-carboxypyridine (picolinic acid).

The 1:1 proton-transfer compounds of L-tartaric acid with 3-aminopyridine [3-aminopyridinium hydrogen (2R,3R)-tartrate dihydrate, C5H7N2+·C4H5O6-·2H2O, (I)], pyridine-3-carboxylic acid (nicotinic acid) [anhydrous 3-carboxypyridinium hydrogen (2R,3R)-tartrate, C6H6NO2+·C4H5O6-, (II)] and pyridine-2-carboxylic acid [2-carboxypyridinium hydrogen (2R,3R)-tartrate monohydrate, C6H6NO2+·C4H5O6-·H2O, (III)] have been determined. In (I) and (II), there is a direct pyridinium-carboxyl N+-HO hydrogen-bonding interaction, four-centred in (II), giving conjoint cyclic R12(5) associations. In contrast, the N-HO association in (III) is with a water O-atom acceptor, which provides links to separate tartrate anions through Ohydroxy acceptors. All three compounds have the head-to-tail C(7) hydrogen-bonded chain substructures commonly associated with 1:1 proton-transfer hydrogen tartrate salts. These chains are extended into two-dimensional sheets which, in hydrates (I) and (III) additionally involve the solvent water molecules. Three-dimensional hydrogen-bonded structures are generated via crosslinking through the associative functional groups of the substituted pyridinium cations. In the sheet struture of (I), both water molecules act as donors and acceptors in interactions with separate carboxyl and hydroxy O-atom acceptors of the primary tartrate chains, closing conjoint cyclic R44(8), R34(11) and R33(12) associations. Also, in (II) and (III) there are strong cation carboxyl-carboxyl O-HO hydrogen bonds [OO = 2.5387 (17) Å in (II) and 2.441 (3) Å in (III)], which in (II) form part of a cyclic R22(6) inter-sheet association. This series of heteroaromatic Lewis base-hydrogen L-tartrate salts provides further examples of molecular assembly facilitated by the presence of the classical two-dimensional hydrogen-bonded hydrogen tartrate or hydrogen tartrate-water sheet substructures which are expanded into three-dimensional frameworks via peripheral cation bifunctional substituent-group crosslinking interactions.

Hydrogen-bonded two- and three-dimensional polymeric structures in the ammonium salts of 3,5-dinitrobenzoic acid, 4-nitrobenzoic acid and 2,4-dichlorobenzoic acid

The structures of the ammonium salts of 3,5-dinitrobenzoic acid, NH4+ C7H3N2O6- (I), 4-nitrobenzoic acid, NH4+ C7H4N2O4- . 2H2O (II) and 2,4-dichlorobenzoic acid, NH4+ C7H3Cl2O2- . 0.5H2O (III), have been determined and their hydrogen-bonded structures are described. All salts form hydrogen-bonded polymeric structures, three-dimensional in (I) and two-dimensional in (II) and (III). With (I), a primary cation-anion cyclic association is formed [graph set R3/4(10)] through N-H...O hydrogen bonds, involving a carboxyl O,O' group on one side and a single carboxyl O-atom on the other. Structure extension involves both N-H...O hydrogen bonds to both carboxyl and nitro O-atom acceptors. With structure (II), the primary inter-species interactions and structure extension into layers lying parallel to (0 0 1) are through conjoined cyclic hydrogen-bonding motifs: R3/4(10) [one cation, a carboxyl (O,O') group and two water molecules] and centrosymmetric R2/4(8) [two cations and two water molecules]. The structure of (III) also has conjoined R3/4(10) and centrosymmetric R2/4(8) motifs in the layered structure but these differ in that he first involves one cation, a carboxyl (O,O') as well as a carboxyl (O) group and one water molecule, the second, two cations and two carboxyl O-groups. The layers lie parallel to (1 0 0). The structures of the salt hydrates (II) and (III) reported in this work, giving two-dimensional layered arrays through conjoined hydrogen-bonded nets provide further illustrations of a previously indicated trend among ammonium salts of carboxylic acids, but the anhydrous three-dimensional structure of (I) is inconsistent.

Structural and mechanistic motifs in membrane proteins: the three-dimensional modelling of rhodopsin, band 3, and the nicotinic acetylcholine receptor

Because so little is known about the structure of membrane proteins, an attempt has been made in this work to develop techniques by which to model them in three dimensions. The procedures devised rely heavily upon the availability of several sequences of a given protein. The modelling procedure is composed of two parts. The first identifies transmembrane regions within the protein sequence on the basis of hydrophobicity, β-turn potential, and the presence of certain amino acid types, specifically, proline and basic residues. The second part of the procedure arranges these transmembrane helices within the bilayer based upon the evolutionary conservation of their residues. Conserved residues are oriented toward other helices and variable residues are positioned to face the surrounding lipids. Available structural information concerning the protein's helical arrangement, including the lengths of interhelical loops, is also taken into account. Rhodopsin, band 3, and the nicotinic acetylcholine receptor have all been modelled using this methodology, and mechanisms of action could be proposed based upon the resulting structures.

Specific residues in the rhodopsin and iodopsin sequences were identified, which may regulate the proteins' wavelength selectivities. A hinge-like motion of helices M3, M4, and M5 with respect to the rest of the protein was proposed to result in the activation of transducin, the G-protein associated with rhodopsin. A similar mechanism is also proposed for signal transduction by the muscarinic acetylcholine and β-adrenergic receptors.

The nicotinic acetylcholine receptor was modelled with four trans-membrane helices per subunit and with the five homologous M2 helices forming the cation channel. Putative channel-lining residues were identified and a mechanism of channel-opening based upon the concerted, tangential rotation of the M2 helices was proposed.

Band 3, the anion exchange protein found in the erythrocyte membrane, was modelled with 14 transmembrane helices. In general the pathway of anion transport can be viewed as a channel composed of six helices that contains a single hydrophobic restriction. This hydrophobic region will not allow the passage of charged species, unless they are part of an ion-pair. An arginine residue located near this restriction is proposed to be responsible for anion transport. When ion-paired with a transportable anion it rotates across the barrier and releases the anion on the other side of the membrane. A similar process returns it to its original position. This proposed mechanism, based on the three-dimensional model, can account for the passive, electroneutral, anion exchange observed for band 3. Dianions can be transported through a similar mechanism with the additional participation of a histidine residue. Both residues are located on M10.