Interaction of the serum amyloid A proteins with phospholipid

The serum amyloid A proteins (SAA) are transported in plasma in association with the high density lipoproteins. We have studied the solution properties of two of the polymorphic forms of SAA, SAA1 and SAA4, and compared the lipid-binding properties of SAA4 to those of the well characterized apolipoproteins, apo-A-I, apo-A-II, and apo-C-III. SAA4 was monomeric at pH 2.9 but considerable self-association was demonstrated at pH 8.2, even in the presence of 1.0 M guanidine HCl. SAA4 differed from the apolipoproteins in its ability to disrupt multilamellar dimyristoylphosphatidylcholine (DMPC) liposomes and generate bilayer discs. Apo-A-I, apo-A-II, and apo-C-III reduced the turbidity of DMPC dispersions at protein:lipid molar ratios of 1:200. SAA4, however, increased turbidity at molar ratios of 1:250 and 1:100 even when preincubated in guanidine HCl before addition to liposomes. Optical density decreased only at ratios of 1:50 and 1:25. At an SAA4:DMPC ratio of 1:50, discoidal particles (long axis, 28.1 nm; short axis, 4.4 nm) were formed which were similar to those produced by apo-C-III. Lipid binding induced changes in SAA4 conformation similar to those observed in the apolipoproteins. The alpha-helical content and intrinsic tryptophanyl fluorescence were increased and quenching of tryptophanyl fluorescence by acrylamide was reduced in the presence of DMPC. In addition, SAA4 as well as the apolipoproteins broadened the range and increased the temperature of the gel-liquid crystal transition temperature of DMPC.     

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.     

Asymmetric labeling of amino lipids in liposomes.

Fluorescamine and trinitrobenzenesulfonate were used as chemical probes to differentially label amino phospholipids in liposomes. At low concentrations, fluorescamine reacts primarily with amino lipids on the external half of the bilayer. Further increase in fluorescamine concentration resulted in a linear increase of labeling indicating penetration and reaction with the internal half of the bilayer. Because of the pH requirements of the fluorescamine reaction, internal labeling was eliminated with a H+ gradient: inside acidic/outside alkaline. Differential labeling was also achieved with trinitrobenzenesulfonate, which is normally not permeable but which can be transported by valinomycin-K+ complex and react with internal amines. Thus, either half of the bilayer can be labeled with the same or different reagents. When liposomes were double-labeled, the fluorescence of fluorescamine was quenched by the trinitrobenzenesulfonate label. This quenching was reversed by solubilizing the liposomes with acidic ethanol. No quenching occurred when fluorescamine-labeled liposomes were mixed with trinitrobenzenesulfonate-reacted liposomes (or trinitrophenylated methylamine) suggesting close proximity of two labels is required for quenching. Conditions which promoted vesicular fusion promptly produced quenching. These differential labeling procedures can be usefully applied to quantitate aminolipids on internal and external vesicular surface, monitor vesicular fusion, and assess liposomal structure.     

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.     

Cooperativity in nucleosomes assembly on supercoiled pBR322 DNA.

Many studies have shown that in reconstituted chromatin model systems, containing only purified DNA and histone octamer, nucleosomes can adopt well defined locations with respect to DNA nucleotide sequence. Recently, nucleosome-nucleosome interactions were suggested as one of the factors underlying preferential nucleosomes positioning. In the present paper this aspect has been studied by topological analysis and electron microscopy visualization of minichromosomes reconstituted at different histone/DNA ratios. Both methods suggest that cooperativity plays a role in nucleosomes formation. A linear cooperative model in which nucleosomes are formed on discrete sites with cooperative interactions occurring only between nearest neighbours allows to calculate the cooperative constant. The reported results show that basic interactions, which are of relevance in the process of chromatin folding, are present also in very simple model system.     

Expression of acetylcholinesterase during visual system development in Drosophila

As in other insects acetylcholine (ACh) and acetylcholinesterase (AChE) function in synaptic transmission in the central nervous system of Drosophila. Studies on flies mutant for AChE indicate that in addition to its synaptic function of inactivating acetylcholine, this neural enzyme is required for normal development of the nervous system (J.C. Hall, S.N. Alahiotis, D.A. Strumpf, and K. White, 1980, Genetics 96, 939-965; R.J. Greenspan, J.A. Finn, and J.C. Hall, 1980, J. Comp. Neurol. 189, 741-774). In order to understand what role AChE may play in neural development, it is necessary to know, in detail, where and when the enzyme appears. The use of monoclonal antibodies to localize AChE in the developing visual system of wild type Drosophila has yielded the novel observation that AChE appears in photoreceptor (retinula) cells 4-6 hr after they differentiate and 3 to 4 days before they are functional. Three days later the staining in the cell body of these cells is reduced. Because retinula cells have no functional connections at the time when AChE is first detected, AChE can not be performing its standard synaptic function. Subsequent to the reduction of AChE in the retinula cells, midway through the pupal stage, the enzyme accumulates rapidly in the neuropils of the optic lobes of the brain. Thus, there is a biphasic accumulation of AChE in the developing visual system with the enzyme initially being expressed in the retinula cells and accumulating later in the optic lobes.     

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.