HETEROGENEITY OF HISTAMINE Hi-RECEPTORS: SPECIES VARIATIONS IN [3H]MEPYRAMINE BINDING OF BRAIN MEMBRANES

[³H]Mepyramine binds with high affinity to membranes from brain of human, rat, guinea-pig, rabbit and mouse with drug specificity indicating an association with histamine H₁receptors. Considerable species differences occur in the affinity of [³H]mepyramine, with guinea-pig and human having 34 times greater affinity than rat, mouse or rabbit. The greater affinity of [³H]mepyramine in guinea-pig than in rat is attributable both to faster association and slower dissociation rates in guinea-pig. Species differences in affinity for H₁ receptor sites occur for some antihistamines but not for others. Some tricyclic antidepressant and neuroleptic drugs are extremely potent inhibitors of [³H]mepyramine binding, exceeding in potency any H₁ antihistamines examined. The tricyclic antidepressant doxepin and the neuroleptic clozapine are the most potent of all drugs examined in competing for [³H]mepyramine binding. The regional distribution of specific [³H]mepyramine binding differs considerably in the various species examined.     

Baked-Bean Waste: a Potential Substrate for Producing Fungal Amylases

Baked-bean waste was found to be a favorable substrate for amylase production by Aspergillus foetidus NRRL 337. Under optimum conditions, the yields of α-amylase (EC 3.2.1.1) and glucoamylase (EC 3.2.1.3) were 47 and 226 U, respectively, per ml of the waste fermented.     

Binding of 4-methylumbelliferyl-galactosides to peanut agglutinin

The binding of 4-methylumbelliferyl-α-D-galactopyranoside, -β-D-galactopyranoside and -D-Galβ(1→3)DGalNac to peanut agglutinin was studied by fluorescence. Peanut agglutinin quenched the fluorescence intensity of 4-methylumbelliferyl-α-D-galactopyranoside but enhanced that of the two 4-methylumbelliferyl-β-galactosides. For α-D-galactopyranoside, the association constants measured at 4 and 25°C were 3.4 × 10³ and 1.7 × 10³ M^?1 respectively, and for D-Galβ(1→3)DGalNac, 1.5 × 10⁵ and 3.3 × 10⁴ M^?1. The binding enthalpies estimated from these values are consistent with the existence of extended sugar binding sites in the peanut agglutinin molecule.     

Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases.

We describe the development of an expression-secretion system in Bacillus subtilis to improve the quality and quantity of the secreted foreign proteins. This system consists of a strain (WB600) deficient in six extracellular proteases and a set of sacB-based expression vectors. With the inactivation of all six chromosomal genes encoding neutral protease A, subtilisin, extracellular protease, metalloprotease, bacillopeptidase F, and neutral protease B, WB600 showed only 0.32% of the wild-type extracellular protease activity. No residual protease activity could be detected when WB600 was cultured in the presence of 2 mM phenylmethylsulfonyl fluoride. By using TEM beta-lactamase as a model, we showed that WB600 can significantly improve the stability of the secreted enzyme. To further increase the production level we constructed an expression cassette carrying sacY, a sacB-specific regulatory gene. This gene was placed under the control of a strong, constitutively expressed promoter, P43. With this cassette in the expression vector, an 18-fold enhancement in beta-lactamase production was observed. An artificial operon, P43-sacY-degQ, was also constructed. However, only a partial additive enhancement effect (24-fold enhancement) was observed. Although degQ can stimulate the production of beta-lactamase in the system, its ability to increase the residual extracellular protease activity from WB600 limits its application. The use of the P43-sacY cassette and WB600 would be a better combination for producing intact foreign proteins in high yield.     

DNA binding and dimerization determinants for thyroid hormone receptor alpha and its interaction with a nuclear protein.

The gel retardation assay was used to analyze the role of the thyroid hormone receptor alpha (TR alpha) ligand-binding domain (LBD) in controlling receptor interaction with a thyroid hormone responsive element (TRE). While wild type receptor TR alpha binds to the TRE mainly as monomer, deletion of 85 amino acids from its C-terminus results in a mutant receptor with enhanced DNA binding that forms several slow mobility complexes as revealed by gel retardation assay. Receptor deletion mutants that lack most of the LBD show significantly elevated DNA binding and are still able to bind to DNA as two complexes. Thus, the C-terminal end of TR alpha appears to interfere with the dimerization/oligomerization function and DNA binding of TR alpha. All C-terminal deletion mutants have lost their T3-responsive activator function, but some show constitutive activity. Nuclear factor from several cell lines, including CV-1, F9, and GC cells, interacts with TR alpha receptor to form a larger molecular weight complex as determined by gel retardation assay. This factor could not be detected in HeLatk- cells, where TR alpha does not activate a TRE-containing reporter gene. The nuclear factor is heat sensitive and does not bind to TRE itself but can interact with TR alpha in the absence of DNA. Deletion analysis demonstrates that the leucine zipper-like sequence located in the LBD of TR alpha is involved in this interaction. Together, our data suggest that TR alpha contains a dimerization function outside the LBD which is inhibited by the carboxy-terminal region, while the leucine zipper-like sequence in the LBD is required for interaction with a nuclear factor.     

Les Radiolaires d'âge jurassique supérieurà crétacé supérieur dans les Radiolarites du Pinde-Olonos (Presqu'île de Koroni; Péloponnèse méridional,Grèce)

Radiolarians of several radiolarites sections of the Pindos-Olonos zone, southern Peloponnesus, lead to propose direct datations of mesozoic sediments. The acuity of such datations allow to distinguish two periods for depositionof radiolarites s.s. in the tethyan region. The first period would be Upper Jurassic and general in tethyan realm, radiolarites depositing under various latitudes (0–35°N). This would be the result of a strong ocean surface current. The second period would be Upper Cretaceous (Vraconian—Coniacian) and be geographically much more restricted (0–15°N). This could result from a weaker current. The absence of radiolaritic sedimentation is possibly the result of the destruction or insulation of the accurate basins during the obduction of ophiolites on the apulo-african realm. The inventory of Late Cretaceous radiolarian fauna, rarely done on alpine series so far, shows similar result to those obtained in Central American and California.     

Properties and biosynthetic connection of the nucleotide pyrophosphatases of rat liver plasma membrane and endoplasmic reticulum

The detergent-solubilized nucleotide pyrophosphatases of the rat liver plasma membrane and endoplasmic reticulum fractions were purified by lectin affinity chromatography. They have the same molecular mass of 148 000 dalton; their catalytic properties are also very similar and correspond to those of the trypsin-solubilized activities from the same membrane preparations. Pulse-chase experiments on isolated perfused livers using [3H]leucine indicated different labelling kinetics of the proteins isolated from plasma membrane and endoplasmic reticulum. The plasma membrane enzyme became only slightly labelled in the presence of 100 microM vinblastine. The data support a precursor-product relationship of the nucleotide pyrophosphatases from endoplasmic reticulum and plasma membrane.     

Down-regulation of miR-17 family expression in response to retinoic acid induced neuronal differentiation

Whole-genome microRNA and gene expression analyses were used to monitor changes during retinoic acid induced differentiation of neuroblasts in vitro. Interestingly, the entire miR-17 family was over-represented among the down-regulated miRNA. The implications of these changes are considerable, as target gene prediction suggests that the miR-17 family is involved in the regulation of the mitogen-activated protein kinase (MAPK) signaling pathway, synaptic plasticity and other markers of neuronal differentiation. Significantly, many of the target responses predicted by changes in miRNA expression were supported by the observed changes in gene expression. As expected, markers of neuronal differentiation such as anti-apoptotic protein B-cell lymphoma 2 (BCL2), myocyte enhancer factor-2D (MEF2D) and zipper protein kinase (MAP3K12; aka ZPK/MUK/DLK) were each up-regulated in response to differentiation. The expression of these genes was also reduced in response to miR-17 and miR-20a transfection, and more specifically they were also shown to contain functional miRNA recognition elements for members of the miR-17 family by reporter gene assay. This suggests that the miR-17 family have an integral role in fine-tuning the pathways involved in the regulation of neuronal differentiation.