Electron-microscopic and immunocytochemical analyses of Weibel-Palade bodies in the human umbilical vein during pregnancy

Summary The present study was done to elucidate the biological significance of the Weibel-Palade body of human umbilical vein endothelial cells. Quantitative determinations of these endothelial-specific granules throughout pregnancy revealed that their numbers and size per cell profile were maintained at low levels from 12 to 19 weeks of gestation; then both rapidly increased from 33 weeks to full term. This increase coincided with the development of the rough endoplasmic reticulum and an increase in the number of endothelial cell pinocytotic vesicles. Light-microscopic peroxidase anti-peroxidase and electron-microscopic protein A-gold techniques provided evidence that factor VIII-related antigen was localized in the Weibel-Palade bodies. Furthermore, in vitro treatment of incubated umbilical vein tissue with compound 48/80, a histamine releaser, induced degranulation of Weibel-Palade bodies from the endothelium. The present study indicates that Weibel-Palade bodies are storage sites of both histamine and factor VIII-related antigen and have an important role in the obliteration of this vessel.     

Rabbit autoantibodies to actin induced by immunization with modified homologous actins

Actin is a major antigen involved in the reaction of smooth muscle antibody positive sera from patients with chronic active hepatitis. In the present study, actin extracted from rabbit skeletal muscle was denatured by sodium dodecyl sulfate and was immunized into the rabbit, a homologous animal for actin. The rabbits, thus immunized, produced antibodies reactive with actins of homologous and heterologous animals. In addition, the antibodies showed reactivity with autologous actin. It indicates that the denatured homologous actin is capable of terminating immunological tolerance to actin and induces formation of autoantibody to rabbit actin. This phenomenon may be implicated in the occurrence of anti-actin antibody in sera from patients with chronic liver disease and several other diseases.     

An extremely acidic amino-terminal presequence of the precursor for the human mitochondrial hinge protein

Mitochondrial hinge protein is a subunit of ubiquinol-cytochrome-c reductase in the respiratory chain and 'hinges' cytochrome c with cytochrome c1. The protein is encoded in the nuclear genome, synthesized in the cytosol and then imported into the mitochondria. The cDNA of the human hinge protein has been cloned and its nucleotide sequence was determined. The deduced primary structure of the amino-terminal presequence consists of 13 amino acid residues, of which 4 amino acids are acidic and only one is basic. Since the presequences of most other precursors are rich in basic amino acids, this sequence is unique for targeting mitochondria. Expression of the gene was repressed in the presence of a phorbol ester in human promyelocyticleukemia cells (HL-60), and this repression was greater than that of the ADP/ATP translocator. These findings suggest that the hinge protein, the expression of which is well regulated, is imported into mitochondria via a specific pathway.     

In vitro mutated beta subunits from the F1-ATPase of the thermophilic bacterium, PS3, containing glutamine in place of glutamic acid in positions 190 or 201 assembles with the alpha and gamma subunits to produce inactive complexes

Using site-directed mutagenesis, Glu-190 or Glu-201 of the beta subunit of the F1-ATPase from the thermophilic bacterium PS3 were replaced with glutamine. It was possible to reconstitute complexes of the mutated beta subunits with alpha and gamma subunits, but the complexes did not have ATPase activity. It is concluded that carboxylic acid side chains of Glu-190 and Glu-201 of the beta subunit are essential for catalytic activity of F1-ATPase.     

Quantitative analysis of DNA secondary structure from solvent-accessible surfaces: the B- to Z-DNA transition as a model

Solvent structure and its interactions have been suggested to play a critical role in defining the conformation of polynucleotides and other macromolecules. In this work, we attempt to quantitate solvent effects on the well-studied conformational transition between right-handed B- and left-handed Z-DNA. The solvent-accessible surfaces of the hexamer sequences d(m5CG)3, d(CG)3, d(CA)3, and d(TA)3 were calculated in their B- and Z-DNA conformations. The difference in hydration free energies between the Z and the B conformations (delta delta GH(Z-B] was determined from these surfaces to be -0.494 kcal/mol for C-5 methylated d(CG), 0.228 kcal/mol for unmethylated d(CG), 0.756 kcal/mol for d(CA)-d(TG), and 0.896 kcal/mol for d(TA) dinucleotides. These delta delta GH(Z-B) values were compared to the experimental B- to Z-DNA transition energies of -0.56 kcal/mol that we measured for C-5 methylated d(CG), 0.69-1.30 kcal/mol reported for unmethylated d(CG), 1.32-1.48 kcal/mol reported for d(CA)-d(TG), and 2.3-2.4 kcal/mol for d(TA) dinucleotides. From this comparison, we found that the calculated delta delta GH(Z-B) of these dinucleotides could account for the previous observation that the dinucleotides were ordered as d(m5CG) greater than d(CG) greater than d(CA)-d(TG) greater than d(TA) in stability as Z-DNA. Furthermore, we predicted that one of the primary reasons for the inability of d(TA) sequences to form Z-DNA results from a decrease in exposed hydrophilic surfaces of adjacent base pairs due to the C-5 methyl group of thymine; thus, d(UA) dinucleotides should be more stable as Z-DNA than the analogous d(TA) dinucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)     

A potential approach for gene therapy targeting hepatoma using a liver-specific promoter on a retroviral vector.

Recent technological advances made in molecular biology and in vitro culture of human and other mammalian cells have led to broad medical and scientific acceptance of the feasibility of gene therapy for genetic diseases. Cancer might practically be one of the attractive targets for such therapy. For the treatment of cancer, it is important to manipulate the gene of interest such that it is expressed solely in cancer cells. We have developed a tissue-specific gene expression system, based on a tissue-specific promoter on a retroviral vector. A murine ecotropic retroviral vector was constructed in which the Escherichia coli beta-galactosidase gene served as a reporter; it was expressed under control of the albumin enhancer element and promoter. The tissue specificity of this vector was first assessed in vitro, and beta-galactosidase activity was detected exclusively in hepatoma cell lines. This recombinant retrovirus was injected directly into a subcutaneous tumor composed of transplantable murine MH-134 hepatoma cells, and expression of the gene was observed in vivo. Then this recombinant retrovirus was injected via the spleen or directly into the liver, resulting in the gene expression in dividing hepatocytes in partially hepatectomized mice, but not in nondividing hepatocytes in normal mice. Gene transfer specific to dividing hepatocytes and expression by means of retroviral vectors should possess high potential for selective elimination of hepatoma cells surrounded by nondividing normal hepatocytes.     

Distribution pattern of the dinoflagellateCeratium hirundinella (O. F. Müller) Bergh in a reservoir

Horizontal distribution of the dinoflagellateCeratium hirundinella (O. F. Müller) Bergh in the Ishitegawa Reservoir, Ehime Prefecture, Japan, was investigated. Water quality was also surveyed. It was observed that the population ofC. hirundinella exponentially decreased in number from the head of the reservoir to the dam site. Further investigation proved thatC. hirundinella initiated growth at the head of the reservoir, and later gradually expanded downstream. It was found during the period of increase in water temperature that the cell density ofC. hirundinella at the uppermost station exponentially increased.     

Sequence and over-expression of subunits of adenosine triphosphate synthase in thermophilic bacterium PS3

The primary structures of all the subunits of thermophilic ATP synthase were determined, and its alpha, beta and gamma subunits could be over-expressed in Escherichia coli, because these subunits were stable and reconstitutable. DNA of 7500 base pairs in length was found to contain a cluster of nine genes for subunits of ATP synthase. The order of their reading frames (size in base pairs) was: I(381): a(630): c(216): b(489): delta(537): alpha(1507): gamma(858): beta(1419): epsilon(396), I being a gene for a small hydrophobic, basic protein expressed in vitro. All the termini of TF0F1 subunits were confirmed by peptide sequencing. Large quantities of the overexpressed thermophilic alpha, beta and gamma subunits were prepared from the extract of E. coli, by a few purification steps.     

Solubilization and reconstitution of voltage-dependent calcium channel from bovine cardiac muscle. Ca2+ influx assay using the fluorescent dye Quin2

Highly purified sarcolemmal membranes, prepared from fresh bovine heart left ventricle, were solubilized by n-octyl beta-D-glucopyranoside and reconstituted into proteoliposomes with soybean phospholipids by the detergent-dialysis method. Ca2+ flux into the proteoliposomes was determined using the fluorescent probe Quin2. A membrane potential (negative in the proteoliposome interior) that was created by K+ diffusion mediated by valinomycin accelerated the Ca2+ influx. The voltage-dependent Ca2+ influx was dependent on pretreatment of the sarcolemmal membranes with Bay K 8644 and was inhibited by various calcium antagonists including nicardipine (K0.5 = 4.5.10(-7) M), verapamil (K0.5 = 9.2.10(-9) M), diltiazem (K0.5 = 26.10(-8) M) and omega-conotoxin (K0.5 = 9.5.10(-9) M).     

Succession of phytoplankton species in indoor reservoir models varying in sediment composition and water inlet site

Using four continuous-flow indoor reservoir models, the combined effects of varying water inlet depth and sediment composition on phytoplankton species composition and its succession pattern were studied. Species which are known to bloom in eutrophicated reservoirs were dominant in the species succession in each tank. At any one time, a step-like pattern was observed in the composition of the dominant species of each tank, wherein all the species which appeared in a tank with a lower phosphorus concentration were included in the species composition of the tank with a higher phosphorus concentration in an additive relationship.