Porcine high molecular weight kininogen: its purification and properties as a thiol proteinase inhibitor as compared to human high molecular weight kininogen

1. High mol. wt kininogen (HMW kininogen) was purified to a homogeneous state from porcine plasma. 2. The protein exhibited a strong inhibitory activity for thiol proteinases such as ficin, papain and calpain I, whereas it did not inhibit serine proteinases, trypsin and chymotrypsin. 3. The mol. wt, isoelectric point, amino acid and carbohydrate compositions, stabilities to temperature and pH, kinetic constants, and immunological properties of the porcine HMW kininogen were determined and compared with those of human HMW kininogen.     

Expression level of enzymes related to in situ estrogen synthesis and clinicopathological parameters in breast cancer patients

In order to evaluate the importance of estrogen production in tumor and surrounding tissues, we measured mRNA expression levels of 5 enzymes participating to estrogen synthesis in situ and 4 breast cancer-related proteins in 27 pairs of tumor and non-malignant tissues. Steroid sulfatase (STS) mRNA was more frequently detected in tumor tissues rather than in their non-malignant counterparts. Estrogen sulfotransferase (EST) was constantly expressed with high level not only in tumor tissues but also in their surrounding non-malignant counterparts. In contrast, mRNA expression levels of aromatase, and 17β-hydroxysteroid dehydrogenase type I and II were relatively low and detected only in small proportion of the patients. We also measured the mRNA expression levels of the same nine genes in tumor tissues of 197 breast cancer patients, and analyzed relationship between the mRNA expression level and the clinicopathological parameters. The mRNA expression levels of STS, aromatase and erbB2 in tumor tissues increased as breast cancer progressed. The tumoral mRNA expression levels of STS, estrogen receptor β, and erbB2 in patients with recurrence were higher than those in patients without recurrence. Upregulation of STS expression plays an important role in tumor progression of human breast cancer and is considered to be responsible for estrogen production in tumor and surrounding tissues.     

Expression of bovine lung prostaglandin F synthase in Escherichia coli

The full-length bovine lung prostaglandin(PG) F synthase cDNA was constructed from partial cDNA clones and ligated into bacterial expression vector pUC8 to develop expression plasmid pUCPF1. This plasmid permitted the synthesis of bovine lung PGF synthase in Escherichia coli. The recombinant bacteria overproduced a 36-KDa protein that was recognized by anti-PGF synthase antibody, and the expressed protein was purified to apparent homogeneity. The expressed protein reduced not only carbonyl compounds including PGD2 and phenanthrenequinone but also PGH2; and the Km values for phenanthrenequinone, PGD2, and PGH2 of the expressed protein were 0.1, 100, and 8 microM, respectively, which are the same as those of the bovine lung PGF synthase. The protein produced PGF2 alpha from PGH2, and 9 alpha, 11 beta-PGF2 from PGD2 at different active sites. Moreover, the structure of the purified protein from Escherichia coli was essentially identical to that of the native enzyme in terms of C-terminal sequence, sulfhydryl groups, and CD spectra except that the nine amino acids provided by the lac Z' gene of the vector were fused to the N-terminus. These results indicate that the expressed protein is essentially identical to bovine lung PGF synthase. We confirmed that PGF synthase is a dual function enzyme catalyzing the reduction of PGH2 and PGD2 on a single enzyme and that it has one binding site for NADPH.     

Simultaneous measurement of atrial natriuretic polypeptide (ANP) messenger RNA and ANP in rat heart--evidence for a preferentially increased synthesis and secretion of ANP in left atrium of spontaneously hypertensive rats (SHR)

Tissue levels of atrial natriuretic polypeptide (ANP) messenger RNA (ANPmRNA) and ANP in the rat heart were measured simultaneously. In Wistar rats, ANPmRNA of the same size (approximately 0.95 kbp) was detected in all four chambers of the rat heart. The ANPmRNA level was the highest in the right atrium, and the left atrial level was slightly lower than the right atrial level. Ventricular levels were more than two orders of magnitude lower than atrial levels. Tissue ANP concentrations of four chambers were roughly parallel to ANPmRNA levels. In spontaneously hypertensive rats (SHR) with the elevated plasma ANP level, the ANPmRNA level in the left atrium was substantially increased. The left/right ratio of atrial ANPmRNA level in SHR (150%) was higher than that in control Wistar Kyoto rats (WKY) (90%). In contrast, the left/right ratio of atrial ANP concentration was decreased in SHR (44%) compared with that in WKY (84%). The ratio of ANP to ANPmRNA levels in the left atrium of SHR was about three times smaller than that in the right atrium of SHR, and those in bilateral atria of WKY. These results indicate that the biosynthesis and secretion of ANP from the left atrium is preferentially increased in SHR. Thus, simultaneous determination of ANPmRNA and ANP levels is a refined strategy of investigation for the biosynthesis, storage and secretion of ANP.     

Heavy chain of human high molecular weight and low molecular weight kininogens binds calcium ion

An antibody subpopulation, anti high molecular weight (anti-HMW) kininogen-Ca2+ antibody able to bind specifically to the HMW kininogen-Ca2+ complex, was isolated from anti-HMW kininogen antiserum. Partially purified anti-HMW kininogen antibody was applied to a HMW kininogen-Sepharose column equilibrated with 40 mM tris(hydroxymethyl)aminomethane hydrochloride buffer, pH 7.5, containing 1.0 M NaCl and 1 mM CaCl2, and anti-HMW kininogen-Ca2+ antibody was eluted with 5 mM ethylenediaminetetraacetic acid. As a result of characterization by enzyme-linked immunosorbent assay, this antibody specifically recognized the cyanogen bromide cleaved fragment 1 (CB-1) region (1-160 amino acid sequence) of the heavy chain of kininogen molecules in the presence of Ca2+ or Mg2+. Furthermore, circular dichroism (CD) experiments showed that the conformational changes of HMW kininogen and heavy chain were induced by metal ions such as Ca2+ and Mg2+ and that these changes were due to the conformational change of the CB-1 region of the heavy chain. The dissociation constant (Kd) for the heavy chain-Ca2+ measured by CD analysis at 214 nm was found to be 0.33 +/- 0.09 mM (mean +/- SD). The number of Ca2+-binding sites of heavy chain calculated from the Hill plot was 1.15 +/- 0.04 (mean +/- SD). Then, a possible Ca2+-binding site was found in the amino-terminal portion of the heavy chain of kininogen molecules.     

Existence of multiple forms of cytochrome P-450scc purified from bovine adrenocortical mitochondria

Three fractions of cytochrome P-450scc (denoted as fractions a, b, and c) were purified by a new procedure from bovine adrenocortical mitochondria. The amino-acid content analyses of these three fractions showed no difference. NH2-terminal amino-acid sequences of cytochrome P-450scc fractions, a and b agreed completely with the sequence deduced by nucleotide sequence of cDNA of cytochrome P-450scc mRNA (Morohashi, K., Fujii-Kuriyama, Y., Okada, Y., Sogawa, K., Hirose, T., Inayama, S. and Omura, T. (1984) Proc. Natl. Acad. Sci. USA 81, 4647-4651), whereas the sequence of fraction c showed a missing of isoleucine at the NH2-terminal. COOH-terminal ámino-acid sequences of fractions a, b and c were -Gln-Ala-COOH, identical with the deduced sequence from the cDNA. Measurements of the enzymatic activities of cholesterol side-chain cleavage reaction revealed no distinct difference among these three fractions. Although each of these fractions appeared as a single protein staining band upon SDS-polyacrylamide gel electrophoresis, these fractions showed heterogeneities upon two-dimensional electrophoresis and chromatofocusing. Fraction a contained the major form of cytochrome P-450scc, and its isoelectric point was estimated to be pH 7.8 by isoelectric focusing under both native and denatured conditions, and this value was confirmed by chromatofocusing. Neither of the carbohydrate-specific stainings (such as periodic acid-Schiff staining and lectin-peroxidase stainings using concanavalin A, wheat-germ agglutinin, and soybean agglutinin) of purified cytochrome P-450scc fractions after the electrophoretic resolution on SDS-polyacrylamide gel could show cytochrome P-450scc fractions as glycoproteins, suggesting that the heterogeneities were not due to the glycosylation state.     

Biochemical and physiological characteristics of Xenorhabdus species,symbiotically associated with entomopathogenic nematodes including Steinernema kushidai and their pathogenicity against Spodoptera litura (Lepidoptera: Noctuidae)

The symbiotic bacterium strain, SK-1 isolated from Steinernema kushidai, a new species of entomopathogenic nematode, was compared with other strains of Xenorhabdus species. Like other Xenorhabdus nematophilus strains, this new strain is gram-negative, facultatively anaerobic, peritrichously flagellated rod and negative for catalase and nitrate reduction. It can be distinguished from the other Xenorhabdus spp. by differences in reactions to phenylalanine deaminase, no acid production from myo-inositol and utilizations of inosine, dl-malate, formate and methanol. Intra-haemocoelic injection of actual cells or liquid culture supernatant into sixth instar larvae of Spodoptera litura for either Phase I or II variants were not pathogenic. Other strains of X. nematophilus showed pathogenicity for whole cell injections. The supernatants of strain D-1 and ATCC 19061, which are symbionts of Steinernema carpocapsae were pathogenic, however pathogenicity decreased and then terminated by increases in temperature.     

Solution conformation of endothelin determined by means of 1H-NMR spectroscopy and distance geometry calculations

The structure of endothelin-1 (ET-1), an endothelial cell-derived peptide with vasoconstricting activity, was determined in an aqueous solution by means of a combination of NMR and distance geometry calculations. The resulting structure is characterized by an alpha-helical conformation in the sequence region, Lys9-Cys15. Furthermore, an extended structure and a turn structure exist in the Cys1-Ser4 and Ser5-Asp8 regions respectively, and no preferred conformation was found for the C-terminal part of the peptide which was not uniquely constrained by the NMR data. These structural elements, the alpha-helical structure in the sequence portion, Cys-X-X-X-Cys, and the extended structure in Cys-X-Cys, are homologous to those found commonly in several neurotoxic peptides.     

Morphological studies of the neuromuscular mechanism shifting from sucking to biting of mice

In order to give a neuroanatomical evidence to the mechanism of shifting from sucking to biting, we investigated in prenatal, newborn and postnatal mice whether there is a time difference in the neurogenesis of the neurons relative to sucking and biting or in the histogenesis of their peripheral effector organs by the HRP labeling technique and electron microscopy. The results obtained are as follows. (1) At birth the facial motoneurons exceed the trigeminal motoneurons in cell area and development. (2) After birth, the trigeminal motoneurons grow rapidly and outstrip the growth of the facial motoneurons at the age of 6 days. (3) Thereafter, the cell area of both neuron types continues to increase gradually. (4) The initial sign of the alpha motor end plates is found in the orbicularis oris muscle innervated by the facial nerve in 17-day-old fetuses, while that of the trigeminal nerve is delayed in the masseter muscle of 18-day-old fetuses. (5) The initial sign of the muscle spindle appears with the sensory terminals in the masseter muscle of 17-day-old fetuses and the fundamental structure of the muscle spindle is formed in 4-day-old youngs. (6) Myelination of the facial nerve begins in 3-day-old youngs, while that of the trigeminal nerve becomes apparent in 4- or 5-day-old youngs. From these bases, it is obvious that the facial nerve elements related to sucking are firstly developed at birth and that the differentiation of the trigeminal nerve elements related to biting is rapidly accelerated after birth.     

The production of anorexigenic substances by intestinal flora

The role of intestinal flora in the production of anorexigenic substance was investigated. Proteus mirabilis (P. mirabilis) and Escherichia coli (E. coli) were found to produce an anorexigenic substance, while Enterococcus faecalis (E. faecalis, type 1 and 2) and Staphylococcus intermedius (S. intermedius) did not. The anorexigenic substance was purified and was detected as, a single though broad band by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity of the final form of the purified substance was 120 units/mg carbohydrate. The substance contained no protein residue and appeared to be a lipopolysaccharide. The evidence that intestinal flora produces an anorexigenic substance leads to an interesting assumption that the intestinal flora may be responsible for regulating food intake.