Genetic studies of human apolipoproteins. V. A novel rapid procedure to screen apolipoprotein E polymorphism

A simple and new method has been developed to detect apolipoprotein E polymorphism directly from plasma or serum without prior ultracentrifugation and delipidation. The method combines the use of dialyzed plasma or serum samples with or without neuraminidase treatment followed by monodimensional isoelectric focusing in simple or 3 M urea gels at a constant low power and progressively increasing voltage over a 3-hr period, and finally protein blotting to a nitrocellulose membrane. Apolipoprotein E phenotypes are identified immunologically using a double antibody reaction, the primary antibody being a monospecific, polyclonal goat anti-apolipoprotein E, and the secondary antibody being a rabbit anti-goat IgG conjugated with alkaline phosphatase. The method was employed to screen apolipoprotein E polymorphism in two white populations in the United States. The frequency values are comparable to those reported previously by other investigators using conventional detection methods. The procedure is simple, accurate, suitable for large scale epidemiologic, clinical, and genetic studies.     

Initiation of morphogenic cell-suspension and protoplast cultures of barley (Hordeum vulgare L.)

Cell-suspension cultures were initiated from embryogenic calli of various barley cultivars. Seven fast-growing suspension lines were obtained from four different cultivars (cvs. Dissa, Emir, Golden Promise and Igri). Two of these cell suspensions showed morphogenic capacity. From a cell suspension of cv. Dissa, albino plantlets were regenerated when aggregates were cultured on solid medium. Aggregates of cv. Igri usually stopped differentiation at the globular stage, but occasionally formed scutellum-like structures. Five suspension lines were used for protoplast isolation and culture. Dividing protoplasts were obtained from all lines, but with cv. Igri a few divisions only and no further development were observed. Protoplasts from the various lines differed in the time of first division (2–14 d), division frequency (0.09–70.9%) and efficiency of colony formation (0.09–7.3%). Protoplasts isolated from the morphogenic cell suspension of cv. Dissa developed compact calli which sporadically regenerated albino plantlets.Abbreviations CC, MS, N6, SH, Kao8p culture media; see Material and methods - cv chltivar - dicamba 3,6-dichloro-o-anisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Mitochondrial boundary membrane contact sites in brain: points of hexokinase and creatine kinase location, and control of Ca2+ transport

The location of hexokinase at the surface of brain mitochondria was investigated by electron microscopy using immuno-gold labelling techniques. The enzyme was located where the two mitochondrial limiting membranes were opposed and contact sites were possible. Disruption of the outer membrane by digitonin did not remove bound hexokinase and creatine kinase from brain mitochondria, although the activity of outer membrane markers and adenylate kinase decreased, suggesting a preferential location of both enzymes in the contact sites. In agreement with that, a membrane fraction was isolated from osmotically lysed rat brain mitochondria in which hexokinase and creatine kinase were concentrated. The density of this kinase-rich fraction was specifically increased by immuno-gold labelling of hexokinase, allowing a further purification by density gradient centrifugation. The fraction was composed of inner and outer limiting membrane components as shown by the specific marker enzymes, succinate dehydrogenase and NADH-cytochrome-c-oxidase (rotenone insensitive). As reported earlier for the enriched contact site fraction of liver mitochondria the fraction from brain mitochondria contained a high activity of glutathione transferase and a low cholesterol concentration. Moreover, the contacts showed a higher Ca2+ binding capacity in comparison to outer and inner membrane fractions. This finding may have regulatory implications because glucose phosphorylation via hexokinase activated the active Ca2+ uptake system and inhibited the passive efflux, resulting in an increase of intramitochondrial Ca2+.     

Mutational specificity of a proof-reading defective Escherichia coli dnaQ49 mutator

Summary The dnaQ (mutD) gene product which encodes the -subunit of the DNA polymerase III holoenzyme has a central role in controlling the fidelity of DNA replication because both mutD5 and dnaQ49 mutations severely decrease the 3–5 exonucleolytic editing capacity.It is shown in this paper that more than 95% of all anaQ49-induced base pair substitutions are transversions of the types G:C-T:A and A:T-T:A. Not only is this unusual mutational specificity precisely that observed recently for a number of potent carcinogens such as benzo(a) pyrene diolepoxide (BPDE) and aflatoxin B1 (AFB1), which are dependent on the SOS system to mutagenize bacteria, but it is also seen for the constitutively expressed SOS mutator activity in E. coli tif-1 strains as well as for the SOS mutator activity mediated gap filling of apurinic sites. Because the G:C-T:A and A:T-T:A transversions can either result from the insertion of an adenine across from apurinic sites or arise due to the incorporation of syn-adenine opposite a purine base, we postulate that the DNA polymerase III holoenzyme also has a reduced discrimination ability in a dnaQ49 background.The introduction of a lexA (Ind^-) allele, which prevents the expression of SOS functions, led to a significant reduction in the dnaQ49-caused mutator effect.Both, the mutational specificity observed and the partial lexA ⁺ dependence of the mutator effect provoke a reanalysis of the hypothesis that the DNA polymerase III holoenzyme can be converted into the postulated but until now unidentified SOS polymerase.     

Human breast milk stimulates prostaglandin synthesis in cultured human skin fibroblasts

Effect of human breast milk or its fractions on prostaglandin synthesis was investigated in cultured human skin fibroblasts. Prostaglandins released into the media were measured by radioimmunoassay. Incorporation of breast milk (2% level) into 10% fetal calf serum media (for 48 hours) stimulated the synthesis of 6-keto-PGF1 alpha (stable product of prostacyclin) by 800%. This stimulating effect of milk persisted after cold acetone extraction to remove phospholipids and potentiated further after dialysis. Stimulation by one of the commercial formulas (Similac) was less than 50% of the milk effect. Milk also stimulated PGE2 synthesis, although to a much lesser degree. These studies show for the first time that a) human breast milk contains potent factor(s) capable of influencing prostaglandin synthesis and suggest that b) these factors might have a role in the development of lipid synthetic pathways during early life.     

Mucopolysaccharidosis-like alterations in cardiac valves of rats treated with tilorone

The purpose of this study was to examine whether the aortic and mitral valves of rats are involved in the mucopolysaccharidosis-like disorder induced by tilorone. Rats were treated with large doses of the drug for periods of 1-21 weeks. After chronic drug treatment the leaflets of both heart valves were thickened and opaque. In all treated animals the spongiosa layer of the stroma was crowded with vacuolated cells; the fibrosa layer was altered only after prolonged treatment. Ultrastructurally, the vacuolated cells of the spongiosa could be identified as histiocytes and fibroblasts, the former being the most susceptible cell type. The fibroblasts of the fibrosa represented the least sensitive cell type. The histochemical results showed that the clear cytoplasmic vacuoles in the spongiosa cells were due to lysosomal storage of polyanionic material with staining characteristics similar to cartilage matrix. After discontinuation of drug treatment the alterations persisted for several weeks. The present study shows that heart valves are involved in the mucopolysaccharidosis-like disorder induced by tilorone. The molecular pathomechanism of the disorder and the exact identification of the storage material must await further analysis.     

DNA-polymorphic patterns linked to the β-globin genes in German families affected with hemoglobinopathies and thalassemias: a comparison to other ethnic groups

Summary DNA haplotype constellations of the β-globin gene cluster have been analyzed in German families with hemoglobinopathies (Hb Freiburg, Hb K?ln, Hb Presbyterian) and β-thalassemias. The polymorphis patterns obtained were compared to those found in families from Greece, Italy, and Turkey affected by β-thalassemia syndromes. With the combined analysis of seven restriction site polymorphisms a DNA-diagnostic prediction for additional offspring could be made with an overall frequency of 75% in the four ethnic groups.