Effect of dextran sulfate on the interaction between very low density lipoprotein and lipoprotein lipase

The effect of dextran sulfate on the interaction between very low density lipoprotein (VLDL) and purified bovine milk lipoprotein was studied. Dextran sulfate increased VLDL-triacylglycerol hydrolysis by lipoprotein lipase about 2-fold, but did not alter the Km value for triacylglycerol in VLDL. Strong association of dextran sulfate with the VLDL-lipoprotein lipase complex was demonstrated by gel filtration on BioGel A-5m, although dextran sulfate did not bind to VLDL and only very slightly to lipoprotein lipase. These findings suggest that dextran sulfate increases triacylglycerol hydrolysis in VLDL by binding to the VLDL-lipoprotein lipase complex.     

Immunological studies on bovine milk lipoprotein lipase. Effects of Fab fragments on enzyme activity

Rabbit antiserum was prepared against purified bovine mild lipoprotein lipase. Immunoelectrophoresis of lipoprotein lipase gave a single precipitin line against the antibody which was coincident with enzyme activity. The gamma-globulin fraction inhibited heparin-releasable lipoprotein lipase activity of bovine arterial intima, heart muscle and adipose tissue. The antibody also inhibited the lipoprotein lipase activity from adipose tissue of human and pig, but not that of rat and dog. Fab fragments were prepared by papain digestion of the gamma-globulin fraction. Fab fragments inhibited the lipoprotein lipase-catalyzed hydrolysis of dimyristoylphosphatidylcholine vesicles and trioleoylglycerol emulsions to the same extent. The Fab fragments also inhibited the lipolysis of human plasma very low density lipoproteins. The change of the kinetic parameters for the lipoprotein lipase-catalyzed hydrolysis of trioleoylglycerol by the Fab fragments was accompanied with a 3-fold increase in Km and a 10-fold decrease in Vmax. Preincubation of lipoprotein lipase with apolipoprotein C-II, the activator protein for lipoprotein lipase, did not prevent inhibition of enzyme activity by the Fab fragments. However, preincubation with dipalmitoylphosphatidylcholine-emulsified trioleoylglycerol or Triton X-100-emulsified trioleoylglycerol had a protective effect (remaining activity 7.0 or 25.8%, respectively, compared to 1.0 or 0.4% with no preincubation). The addition of both apolipoprotein C-II and substrate prior to the incubation with the Fab fragments was associated with an increased protective effect against inhibition of enzyme activity; remaining activity with dipalmitoylphosphatidylcholine-emulsified trioleoylglycerol was 40.6% and with Triton X-100-emulsified trioleoylglycerol, 45.4%. Human plasma very low density lipoproteins also protected against the inhibition of enzyme activity by the Fab fragments. These immunological studies suggest that the interaction of lipoprotein lipase with apolipoprotein C-II in the presence of lipids is associated with a conformational change in the structure of the enzyme such that the Fab fragments are less inhibitory. The consequence of a conformational change in lipoprotein lipase may be to facilitate the formation of an enzyme-triacylglycerol complex so as to enhance the rate of the lipoprotein lipase-catalyzed turnover of substrate to products.     

Possible MIS Production by Follicle Cells in Spontaneous Oocyte Maturation of the Ascidian, Halocynthia roretzi

Outer and inner follicle cell-enclosed oocytes (oocyte complexes) of Halocynthia roretzi underwent germinal vesicle breakdown (GVBD) within 2 hr when transferred from ovaries to normal seawater of pH 8 (NSW). Extrusion of test cells (TC) into the perivitelline space and elevation of the chorion also occurred. This phenomenon was designated as spontaneous oocyte maturation.
Seawater of low pH, protease inhibitors such as leupeptin or soybean trypsin inhibitor (SBTI), and calcium deficiency inhibited the spontaneous maturation only when introduced to the NSW during the first 10 minutes of incubation. GVBD-blocked complexes underwent GVBD after addition of trypsin regardless of pH or the absence of calcium ions. The oocytes from which follicle cells were removed with glycosidase did not undergo GVBD in NSW, but addition of trypsin triggered GVBD in these defolliculated oocytes (TC oocytes). Furthermore, incubation media in which spontaneous maturation had occurred, induced GVBD in the TC oocytes. This GVBD-inducing activity was heat-labile and was inhibited by leupeptin.
These results indicate that in the first step of the spontaneous oocyte maturation, outer and/or inner follicle cells give a signal to the oocyte itself or TC oocyte. This signal is likely to be trypsin-like.     

Origin of Germ Cells and Early Differentiation of Gonads in the Starfish, Asterina pectinifera

The origin of the germ cells and the development of the genital system in the annually spawning starfish, Asterina pectinifera , were studied by light and electron microscopy. Characteristic germ cells were first characterized in gonads after spawning: the gonia are larger than somatic cells, have large nuclei (with electron-lucent nucleoplasm), and show mitochondrial aggregation associated with nuage (electron-dense bodies). In young starfish without gonads similar cells were detected in the haemal sinus, where they were termed primordial germ cells (PGCs). Brachiolariae and metamorphosed juveniles had a cellular cluster in the coelomic epithelium, near the hydroporic canal. The cluster was comprised of cells endowed with the above-mentioned characteristics of the germ cells. The germ cell counts indicated that PGCs migrate from the aboral haemal sinus near the hydroporic canal, through the haemal sinus to the gonads, where they settle, proliferate, and differentiate into gonia.     

Insulin binds to type V collagen with retention of mitogenic activity

The abilities of eight extracellular matrix proteins, fibronectin, vitronectin, laminin, and collagen types I, II, III, IV, and V to bind insulin were examined by binding studies with insulin conjugated with peroxidase. At a physiological pH and ionic strength, type V collagen bound to insulin most strongly. The other types of collagen, laminin, and vitronectin also bound insulin with affinity lower than that of type V collagen. The insulin-binding site of type V collagen was in a 30-kDa CNBr fragment of the alpha 1 (V) chain. Analysis of the amino acid sequence showed that this 30-kDa fragment was identical to the heparin-binding fragment of type V collagen. The insulin-binding sites of laminin and vitronectin were located in the A chain and in the heparin-binding domain, respectively. Insulin bound to type V collagen stimulated the synthesis of DNA by mouse mammary tumor MTD cells, indicating that bound insulin retained mitogenic activity.     

Evidence for insertion sequence-mediated spread of the thermostable direct hemolysin gene among Vibrio species.

The tdh gene of Vibrio parahaemolyticus which encodes the thermostable direct hemolysin has been found in some strains of other Vibrio species. Analysis of seven tdh genes cloned from V. parahaemolyticus, Vibrio mimicus, and non-O1 Vibrio cholerae revealed that all tdh genes were flanked by insertion sequence-like elements (collectively named ISVs) or related sequences derived from genetic rearrangement of ISVs. The ISVs possessed 18-bp terminal inverted repeats highly homologous to those of IS903 (2- to 4-bp mismatch) and were 881 to 1,058 bp long with less than 33.6% sequence divergence. These features and nucleotide sequence similarities among ISVs and IS903 (overall homologies between ISVs and IS903, ca. 50%) strongly suggest that they were derived from a common ancestral sequence. A family of ISVs were widely distributed in Vibrio species, often regardless of the possession of the tdh genes, and one to several copies of the ISVs per organism were detected. A strain of V. mimicus possessed two copies of the ISVs flanking the tdh gene and three copies unrelated to the tdh gene. However, the transposition activity of the ISVs could not be demonstrated, probably because they had suffered from base changes and insertions and deletions within the transposase gene. The possible mode of ISV-mediated spread of the tdh gene is discussed from an evolutionary standpoint.     

Site-specific integration of the actinophage R4 genome into the chromosome of Streptomyces parvulus upon lysogenization.

The lysogenization of Streptomyces parvulus by actinophage R4 occurs by site-specific integration of the phage genome into the chromosome. The DNA fragments containing the attachment sites on the host chromosome, the phage genome, and the two junctions created by insertion of the phage genome were cloned and sequenced. The attachment sites were found to share a common core of 12 bp. This common core sequence was not detected in chromosomal DNAs of S. coelicolor and S. lividans.     

Induction of cytotoxic T cells to a cross-reactive epitope in the hepatitis C virus nonstructural RNA polymerase-like protein.

Cytotoxic T lymphocytes (CTL) have been found to mediate protection in vivo against certain virus infections. CTL also may play an important role in control of infection by hepatitis C virus (HCV), but no CTL epitopes have yet been defined in any HCV protein. The nonstructural protein with homology to RNA polymerase should be a relatively conserved target protein for CTL. To investigate the epitope specificity of CTL specific for this protein, we used 28 peptides from this sequence to study murine CTL. Mice were immunized with a recombinant vaccinia virus expressing the HCV nonstructural region corresponding to the flavivirus NS5 gene (RNA polymerase), and the primed spleen cells were restimulated in vitro with peptides. CTL from H-2d mice responded to a single 16-residue synthetic peptide (HCV 2422 to 2437). This relatively conserved epitope was presented by H-2d class I major histocompatibility complex (MHC) molecules to conventional CD4- CD8+ CTL but was not recognized by CTL restricted by H-2b. Moreover, exon shuffle experiments using several transfectants expressing recombinant Dd/Ld and Kd demonstrated that this peptide is seen in association with alpha 1 and alpha 2 domains of the Dd class I MHC molecule. This peptide differs from the homologous segments of this nonstructural region from three other HCV isolates by one residue each. Variant peptides with single amino acid substitutions were made to test the effect of each residue on the ability to sensitize targets. Neither substitution affected recognition. Therefore, these conservative mutations affected peptide interaction neither with the Dd class I MHC molecule nor with the T-cell receptor. Because these CTL cross-react with all four sequenced isolates of HCV in the United States and Japan, if human CTL display similar cross-reactivity, this peptide may be valuable for studies of HCV diagnosis and vaccine development. Our study provides the first evidence that CD8+ CTL can recognize an epitope from the HCV sequence in association with a class I MHC molecule.     

Neutralization of microcystin shock in mice by tumor necrosis factor alpha antiserum.

Microcystis aeruginosa is a common cyanobacterium in water blooms that appear widely in nutrient-rich, fresh, and brackish waters, and its toxic blooms cause the death of domestic animals. The administration of a crude toxic cell extract of M. aeruginosa K-139 to mice can produce tumor necrosis factor (TNF) and prompt severe physiological disturbances, especially liver damage, which can lead to death. The in vitro production of TNF-alpha by peritoneal macrophages was observed after stimulation with the cell extract or the purified toxin from K-139 cells. The expression of a TNF-alpha mRNA was also detected in spleen cells and peritoneal macrophages after stimulation with the cell extract. However, a previous injection of rabbit anti-murine TNF-alpha serum could prevent the liver damage to some extent and protect the mice from death. These findings indicate the involvement of TNF in microcystin shock.