Liver fatty acid-binding protein in two cases of human lipid storage

Summary FABPs in the various tissues play an important role in the intracellular fatty acid transport and metabolism. Reye's syndrome (RS) and multisystemic lipid storage (MLS) are human disorders characterized by a disturbance of lipid metabolism of unknown etiology. We investigated for the first time L-FABP in these two conditions. Affinity purified antibodies against chicken L-FABP were raised in rabbits, and found to cross-react specifically with partially purified human L-FABP. L-FABP content in liver samples of two patients with RS and MLS was investigated by immuno-histochemistry, SDS-PAGE and ELISA. L-FABP immuno-histochemistry showed increased reactivity in the liver of RS patient and normal reactivity in MLS liver. L-FABP increase in RS liver was confirmed by densitometry of SDS-PAGE and ELISA method. By these two methods the increase amounted to 180% and 199% (p < 0.02), respectively, as compared to controls. A possible role of L-FABP in the pathogenesis of RS is discussed.     

Body size increase in insular rodent populations: a role for predators?

Insular mammalian populations living in areas of small size are often characterized by a drastic change in body mass compared to related continental populations or species. Generally, small mammals (less than 100 g) evolve into giant forms while large mammals (up to 100 g) evolve into dwarf forms. These changes, coupled with changes in other life, behavioural, physiological or demographic traits are referred to generally as the insular syndrome. We tested in this study the relative contribution of three factors — area of island, numbers of competitor species and number of predator species — to changes in body size of the woodmouse (Apodemus sylvaticus) in the Western Mediterranean Sea. Our results, based on a comparative analysis using the phylogenetic independent contrasts method, indicate that the increase in body size is related both to the decrease of island size and to the lower number of predator species. A decrease of competitor species does not seem to have an important effect.     

Interaction of diphtheria toxin fragment A and of elongation factor 2 with Cibacron blue

Diphtheria toxin fragment A interacts with Cibacron blue in solution, although it is not retained by blue Sepharose columns. Difference spectral titration of fragment A with the dye gives a dissociation constant of the order of 10^–5 M and a 11 stoichiometry for the complex. In equilibrium dialysis experiments Cibacron blue behaves as a competitive inhibitor of the binding of NAD to diphtheria toxin fragment A. The dye inhibits in a non-competitive way the fragment A-catalysed transfer of ADP-ribose from NAD to elongation factor 2 (EF2). By affinity chromatography on blue Sepharose a binding of EF2 and of ADP-ribosyl-EF2 with the dye is also demonstrated. GDP, GTP and GDP(CH₂)P are able to displace EF2 from blue Sepharose.     

Expression of HOX homeogenes in human neuroblastoma cell culture lines

Mammalian genes containing a class-I homeobox (HOX genes) are highly expressed in the embryonic nervous system. As a first step towards the molecular analysis of the role these genes play in neural cells, we studied the expression of four human HOX genes in five neuroblastoma (NB) cell lines - SK-N-BE, CHP-134, IMR-32, SK-N-SH and LAN-1 - during the process of differentiation induced by treatment with retinoic acid (RA). The four genes, HOX1D, 2F, 3E and 4B, located at corresponding positions in the four HOX loci, share a high degree of sequence similarity with the Drosophila Deformed homeotic gene and constitute a homology group, group 10. One of these genes, HOX1D, is not expressed in the cells used, whereas the other three are highly expressed in untreated and RA-induced NB cells, even though the expression pattern in the various lines is slightly different for the three genes. Our analysis reveals a complex and specific expression pattern in these lines, paving the way to an identification of different NB-cell populations by means of specific HOX gene expression schemes. On the other hand, in every line studied, morphological maturation toward a neuronal differentiated phenotype appears to be associated with increased HOX gene expression.     

A hemagglutinin specific for sialic acids in a rat brain synaptic vesicle-enriched fraction

A hemagglutinating activity was detected in a synaptic vesicle-enriched fraction prepared from adult rat brain, using trypsinized glutaraldehyde-fixed rabbit erythrocytes. The specific activity of the fraction, in two series of experiments, was 7.5 and 16-fold higher than in the other subcellular fractions. The activity was absent from the synaptosome cytosol. In a study using twenty-five different carbohydrates and glycoproteins, best inhibitors were N-acetylneuraminic acid and N-glycolylneuraminic acid, together with bovine submaxillary mucin and a glycopeptide fraction prepared from rabbit erythrocyte membranes. The activity was thermolabile and very sensitive to proteolytic enzymes (but insensitive to neuraminidase) indicating that a protein (agglutinin) is responsible for the activity. Experiments using detergents and high ionic strength showed that the agglutinin is tightly bound to membranes, inactivated by the so-called non denaturing detergents, and stable in diluted sodium dodecyl sulphate. Hypotheses are discussed on the possible function of the agglutinin.     

Regulation of the Pool Size of Valine in Escherichia coli K-12

Three mutations (ilvH611, ilvH612, and ilvH613) are described which make Escherichia coli K-12 resistant to valine inhibition and are located near leu. The expression of the ilv genes appears to be normal in these mutants since the isoleucine-valine biosynthetic enzymes are not derepressed relative to the wild type. The intracellular concentration of valine is, however, higher in the mutants than in the isogenic ilvH(+) strain. These mutants also excrete valine, probably because of the high intracellular concentration of this amino acid. The pool size of valine is regulated independently from that of isoleucine and leucine. The increased intracellular concentration of valine is due to a decreased feedback inhibition that valine exerts on its own biosynthetic pathway. In fact, acetolactate synthase activity assayed in extracts of ilvH612 and ilvH613 mutants is more resistant to valine inhibition than the activity assayed in the ilvH(+) isogenic strain. Two forms of acetolactate synthase activity can be separated from these extracts by adsorption and elution on hydroxylapatite. One of them is as sensitive to valine inhibition as that of the wild type, the other is more resistant to valine inhibition.     

Structural Genes for a Newly Recognized Acetolactate Synthase in Escherichia coli K-12

Evidence is reported that shows the presence in Escherichia coli K-12 of a newly found acetolactate synthase. This enzyme is the product of two genes, ilvH and ilvI, both located very close to leu. Amber mutations have been found in both genes and therefore their products are polypeptides. Mutations in the ilvH gene cause the appearance of an acetolactate synthase activity which is relatively resistant to valine inhibition and can be separated by adsorption on hydroxylapatite from another activity present in the extract and more sensitive to valine inhibition than the former. A mutant altered in the ilvI gene was isolated among the revertants sensitive to valine inhibition of an ilvH mutant. Such a mutant lacks the resistant acetolactate synthase. A temperature-sensitive revertant of the ilvI mutant contained a temperature-sensitive acetolactate synthase. Thus ilvI is the structural gene for a specific acetolactate synthase. The activity of the ilvH gene product has been measured by adding an extract containing it to a purified ilvI acetolactate synthase, which, upon incubation, became more sensitive to valine inhibition. Conversely, a valine-sensitive acetolactate synthase (the product of the ilvH and the ilvI genes) became more resistant to valine inhibition upon incubation with an extract of a strain containing a missense ilvH gene product.     

Effect of 17β-estradiol on calcium response to phytohaemagglutinin in human lymphocytes

Pre-treatment of human lymphocytes with 17-estradiol diminishes the increase in concentration of cytosolic free calcium after stimulation with phytohaemagglutinin. The effect is dependent on 17-estradiol concentration and on the preincubation time. The effect is not due to an interaction between 17-estradiol and phytohaemagglutinin, but appears to be a consequence of the binding of the hormone to the cell surface. The effect is specific for 17-estradiol, since the isomer and other steroid hormones (progesterone, testosterone, diethylstilbestrol and 5-androstan), have no effect. Since the effect of the 17-estradiol can be suppressed by treatment of lymphocytes with ouabain, it appears that the effect of estradiol on the rise of cytosolic calcium induced by phytohaemagglutinin is mediated by the (Na, K)-ATPase.     

Increased reliability of selective PCR by using additionally mutated primers and a commercialTaq DNA polymerase enhancer

A reliable selective PCR procedure that combines the use of additionally mutated primers with the specificity-enhancing properties of a commercial preparation (Perfect Match, Stratagene) is described. The human immunodeficiency virus type 1pol gene point mutations known to confer in vitro resistance to azidothymidine were examined as a model for optimization of the assay. The usual strategy of deliberately introducing an additional mismatch 1 residue from the 3′ end in the wild-type and mutant primers did not allow reproducible discrimination between wild-type and mutant target sequences. Addition of minimal amounts of Perfect Match to the same PCR mixtures resulted in a significantly enlarged range of selective annealing temperatures, providing a valuable and cost-effective means for reliable detection of known mutations by selectivePCR.