Localization of the gene for a third G protein β-subunit to mouse chromosome 6 near Raf-1

The large family of signal transducing proteins known as G proteins are heterotrimers that dissociate into an independent α-subunit and βγ-subunit complex after ligand binding or other stimulation. For Gα, at least 30 distinct sequences representing 10 different classes have been identified. On the other hand, cDNAs for only three Gβ-subunit genes have been isolated so far. All three of the Gβ genes have been chromosomally mapped in the human, but only two in the mouse. Using a human retinal cDNA for the third G protein β-subunit, we have mapped the corresponding gene, termed Gnb-3, to mouse Chromosome 6 with somatic cell hybrids and have positioned it distal to but near the marker Raf-1 by analysis of the progeny of three genetic crosses.     

Molecular cloning and characterization of the mouse UDP-N-acetylglucosamine:α-3- -mannoside β-1,2-N-acetylglucosaminyltransferase I gene

The biosynthesis of protein-bound complex N-glycans in mammals requires a series of covalent modifications governed by a large number of specific glycosyltransferases and glycosidases. The addition of oligosaccharide to an asparagine residue on a nascent polypeptide chain begins in the endoplasmic reticulum. Oligosaccharide processing continues in the Golgi apparatus to produce a diversity of glycan structures. UDP-N-acetylglucosamine:α-3- -mannoside β-1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101; GlcNAc-TI) is a key enzyme in the process because it is essential for the conversion of high-mannose N-glycans to complex and hybrid N-glycans. We have isolated the mouse gene encoding GlcNAc-TI (Mgat-1) from a genomic DNA library. The mouse sequence is highly conserved with respect to the human and rabbit homologs and exists as a single protein-encoding exon. Mgat-1 was mapped to mouse Chromosome 11, closely linked to the gene encoding interleukin-3 by the analysis of multilocus interspecies backcrosses. RNA analyses of Mgat-1 expression levels revealed significant variation among normal tissues and cells.     

Partition coefficients of plant cuticles for monoterpenes

Summary Cuticle/water partition coefficients (K_c/w) for d-limonene, -pinene and -pinene were determined by an extrapolation and a desorption method. The sorption experiments were carried out with isolated angiosperm and gymnosperm cuticles and with [¹⁴C]-labelled monoterpenes, which were obtained biosynthetically. Both methods were suitable for the determination of the K_c/w of volatile hydrophobic compounds. For the angiosperm cuticles the partition coefficients are of the order of 10⁴, which indicates a high accumulation of monoterpenes in the cuticle. The values of the conifer cuticles of Picea abies (L.) Karst. and Abies alba Mill., however, are lower due to their high lignin content. This is proved by the increase of the partition coefficients after removal of polar and phenolic components. The K_c/w can be estimated with good accuracy from the octanol/water partition coefficient, which was determined experimentally.     

The use of toluidine blue for in situ detection of micro-organisms in foods

The use of toluidine blue to stain cryosections of food samples to detect sites of microbial growth was examined. The method successfully detected colonies and single cells of both yeasts and bacteria at magnifications of x 400 or below in the majority of foods studied. The method demonstrates great potential for studying the micro-environments in which micro-organisms grow.     

Characterization of recombinant human renin: Kinetics,pH-stability, and peptidomimetic inhibitor binding

The kinetic behavior andpH-stability of recombinant human renin was analyzed using a new fluorogenic substrate based on the normal P₆-P₃ renin cleavage sequence in human angiotensinogen. The design of this fluorogenic substrate makes possible, for the first time, direct monitoring of the kinetics of proteolytic conversion of prorenin to renin. ThepH-stability profile for renin, measured with the substrate at 25°C, indicated a broad plateau of stability betweenpH 6.0 and 10.0. Analysis of thepH-activity profile of renin for the substrate indicated a minimumK _m (1.8 µM) atpH 7.4 and a maximumV _m betweenpH 7.4 and 8.0. The thermodynamics of the binding of a novel, soluble, peptidomimetic inhibitor to renin indicated it is possible to retain the tight-binding characteristics and enthalpy contributions to binding of larger peptide-derived inhibitors, while reducing inhibitor size and entropic contributions to binding. A novel derivative of the fluorogenic substrate, containing a 3-methyl histidine substitution at the P₂ site, was used to test the recent hypothesis that renin functions by virtue of substrate-directed catalysis.     

The origin of ovarian neuropeptide Y (NPY)-immunoreactive nerve fibres from the inferior mesenteric ganglion in the pig

Summary Applying a double-immunofluorescence technique, the porcine ovary is demonstrated to receive two populations of NPY-immunoreactive nerve fibres originating from the inferior mesenteric ganglion: one with colocalized tyrosine hydroxylase and supplying predominantly the ovarian vasculature, and a second, solely NPY-immunoreactive and almost exclusively associated with growing follicles. A third group of tyrosine hydroxylase-and dopamine--hydroxylase-positive, but NPY-negative nerve fibres is associated with ovarian blood vessels and, to a minor extent, with ovarian follicles. As revealed by retrograde tracing, the vast majority of postganglionic neurons projecting to the ovary is located in a discrete area of the ganglion, suggesting a somatotopic organization of the porcine inferior mesenteric ganglion. Moreover, the finding indicate that three subpopulations of postganglionic sympathetic neurons with different chemical codes supply different target components of the porcine ovary. The physiological relevance of the described neurons in the nervous control of ovarian functions remains to be elucidated.A portion of these results has been presented in abstract form (Majewski et al. 1991)     

Inhibition of cell proliferation with antibody-targeted liposomes containing methotrexate-γ-dimyristoylphosphatidylethanolamine

We have prepared liposomes containing methotrexate-γ-dimyristoylphosphatidylethanolamine (MTX-DMPE liposomes), to which protein A was covalently coupled, permitting specific association of these liposomes in vitro with murine cells preincubated with relevant protein A-binding monoclonal antibodies. In the absence of antibody the presence of externally-oriented methotrexate (MTX) in MTX-DMPE liposomes did not result in greater binding to cells than liposomes made without MTX-γ-DMPE. Derivation of methotrexate with phospholipid permits enhanced drug-liposome association. These liposomes are more resistant than conventional liposomes to repeated cycles of freezing and thawing. MTX-DMPE liposomes are comparable to antibody-targeted liposomes made with encapsulated water-soluble methotrexate both with respect to specific binding to target cells and drug effect. The inhibitory effects off MTX-liposomes, as well as free MTX, were reversible by either thiamin pyrophosphate (Tpp) or N⁵-formyltetrahydrofolate (F-THF), while the effects of MTX-DMPE liposomes were reversed only by N⁵-formyltetrahydrofolate. This suggests that the toxicity of non-targeted MTX-liposomes may be due to leakage of the encapsulated MTX. The absence of an effect of thiamin pyrophosphate on non-targeted MTX-DMPE liposomes indicates that they do not enter into the cell via the normal folate transport system.