Primary structure of heat-stable enterotoxin produced by Yersinia enterocolitica

A heat-stable enterotoxin was isolated and purified from the culture supernatant of $\text{Yersinia enterocolitica}$ by reversed-phase high-performance liquid chromatography. The amino acid sequence of the purified toxin was determined to be as follows: Gln-Ala-Cys(X)-Asp-Pro-Pro-Ser-Pro-Pro-Ala-Glu-Val-Ser-Ser-Asp-Trp-Asp-Cys-Cys-Asp-Val-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys (X: not determined). The C-terminal sequence containing 6 half-cystine residues was highly homologous to that of heat-stable enterotoxin of enterotoxigenic $\text{Escherichia coli.}$     

Induction of endoreduplication in Chinese hamsters V79 cells by cytosine arabinoside

Endoreduplication (ER) could be induced very effectively in Chinese hamster V79 cells exposed to cytosine arabinoside (1-β-D-arabinofuranosylcytosine; Ara-C). Cells were cultured for 48 hours in Ara-C containing medium. ER frequency increases rapidly after Ara-C release. About 60% of metaphase cells were endoreduplicated at 8–10 hours after release from Ara-C (5 μg/ml). Induction of ER also depends on Ara-C concentrations.     

Cytological evidence for holocentric chromosomes of the silkworms,Bombyx mori and B. mandarina, (Bombycidae,Lepidoptera)

The nature of the centromere and the orientation in meiosis of silkworm chromosomes were investigated using the trivalent of the F₁ hybrid between the wild and domestic silkworm and X-ray-induced aberrant chromosomes as well as normal silkworm chromosomes. The results of the experiments were as follows: (1) Pro-metaphase chromosomes showed no distinct primary constriction even after treatment with hypotonic solution, (2) sister chromatids separated in parallel along the entire length of the chromosome at mitotic anaphase, (3) chiasmata underwent complete terminalization during diakinesis and thus chromosome dyads were always connected end-to-end by a terminal chiasma at metaphase I, (4) radiation-induced aberrant chromosomes were stably transmitted throughout a number of cell generations, and (5) although the homomorphic bivalents generally orientated axially at metaphase I and equatorially at metaphase II, this normal sequence tended to be inverted or modified in the X-ray-induced aberrant chromosomes and in the trivalent of the F₁ hybrid silkworms. These observations may be best interpreted by assuming the holocentric nature of silkworm chromosomes.     

Solution properties of synthetic polypeptides. V. Helix–coil transition in poly(β-benzyl L-aspartate)

Simple approximate expressions have been derived from the theory of Zimm and Bragg for use in the analysis of experimental data on the helix-coil transition in polypeptide. On the basis of the resulting expressions practical procedures are proposed to determine two basic parameters characterizing a thermally induced transition, i.e., helix initiation parameter σ and enthalpy change for helix formation, ΔH. They have been applied to the data for poly(β-benzyl L -aspartate) (PBLA) with the result: σ = 1.6 × 10^?4 and ΔH = ?450 cal/mole for PBLA in m-cresol; σ = 0.6 × 10^?4 and ΔH = 260 cal/mole for PBLA in chloroform containing 5.7 vol-% of dichloroacetic acid. This result gives evidence that σ may change not only from one polypeptide to another but also for a given polypeptide in different solvents. The change in limiting viscosity number [η] accompanying the transition was measured in the same solvents. The curve of [η] versus helical content had a relatively monotonic shape for the chloroformdichloroacetic acid solutions as compared with that for the m-cresol solutions, indicating that [η] depended largely on σ. Provided that [η] is a direct measure of the mean-square radius of gyration, 〈S²〉, the results are consistent with the theoretical predictions of Nagai and of Miller and Flory for 〈S²〉.     

Chemiluminescent assay of various enzyme activites and its application to enzyme immunoassays

A highly sensitive chemiluminescent assay for NAD(P)H have been developed. The principle of the method is as follows; NAD(P)H reduces molecular oxygen to superoxide anion (O) and hydrogen peroxide (H₂O₂) in the presence of 1-methoxy-5-methylphenazinium methyl sulphate (1-MPMS) as electron mediator. The produced O and H₂O₂ can be measured by chemiluminescent reaction using isoluminol (IL) and microperoxidase (m-POD). A linear relationship between chemiluminescence intensity and NAD(P)H concentration (log/log) was obtained ranged from 10^?9 mol/I to 10^?5 mol/I. This chemiluminescent reaction has been coupled to the assay of glucose-6-phosphate dehydrogenase (G6PDH), β-D -galactosidase (β-Gal) and alkaline phosphatase (ALP). The detection limits of G6PDH, β-Gal and ALP were 10^?18 mol, 10^?20 mol and 10^?18 mol per assay, respectively. The chemiluminescent assay of these enzymes applied to chemiluminescent enzyme immunoassay for 17α-hydroxy-progesterone and DNA hybridization assay using these enzymes as label.     

A single amino acid substitution in the A subunit of Escherichia coli enterotoxin results in a loss of its toxic activity

A plasmid encoding a mutant gene of heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, was induced by treatment of plasmid EWD 299 with hydroxylamine. A mutant strain of E. coli HB 101 carrying the mutant plasmid pTUH 6A produced a low toxic LT analogue (mutant LT), which was cross-reactive with anti-LT antibody. The mutant LT activity was less than 0.15 and 0.006% of the normal LT in the rabbit ileal loop test and in the rabbit skin permeability test, respectively. The amino acid composition of the mutant LT-B subunit was the same as that of the normal B subunit. Though the A2 fragment of the mutant LT was identical to normal LT by DNA analysis, the A1 fragment of the mutant LT differed from the normal A1 fragment in one amino acid at position 112; namely it had lysine instead of glutamic acid from the N terminus. These data suggest that glutamic acid at position 112 from the N terminus of the A1 fragment is important for the A subunit to express its biological activity.     

Tumor cytotoxicity of human monocyte membrane-bound interleukin-1α induced by synergistic actions of interferon-γ and synthetic acyltripeptide,FK-565

Summary Human blood monocytes were isolated by counter-flow centrifugal elutriation from healthy donors and these noncytotoxic monocytes were rendered tumoricidal to allogeneic melanoma (A375) cells by activation with a synthetic acyltripeptide (FK-565), as assessed by measuring release of [¹²⁵I]iododeoxyuridine in 72 h. When monocytes were treated with FK-565 for 16 h, and then fixed with paraformaldehyde, they showed cytotoxicity to A375 melanoma cells. The fixed-monocyte-mediated cytotoxicity to A375 cells was induced by the synergistic actions of FK-565 and recombinant interferon- (rIFN-), but not other cytokines [rIFN-A, rIFN-, tumor necrosis factor (TNF), interleukin (IL)-2, -3 and -6]. For synergistic activation of monocytes with induction of a membrane-associated antitumor monokine, the monocytes had to be incubated first with rIFN- and then with FK-565. FK-565 also acted synergistically with rIFN- to stimulate monocytes to produce membrane-associated IL-1 activity, which induced C3H/HeJ thymocyte blastogenesis in response to phytohemagglutinin P. The tumoricidal and thymocytestimulating activities of the fixed monocytes were almost completely inhibited by a specific anti-(IL-1) antiserum, but not by a specific anti-(IL-1) antiserum or monoclonal anti-TNF antibody. These results suggest that membrane-associated IL-1 of human blood monocytes can be induced by two activation signals (rIFN- then FK-565) at their suboptimal concentrations.Abbreviations IL interleukin - IFN interferon - TNF tumor necrosis factor     

Carbachol-Induced Cosecretion of Immunoreactive Atrial Natriuretic Peptides with Catecholamines from Cultured Bovine Adrenal Medullary Cells

The distribution and secretion of atrial natriuretic peptides (ANPs) were investigated in bovine adrenal medulla. (1) Cultured bovine adrenal medullary cells (2 x 10(6)/dish) contained 100.4 +/- 6.0 fmol of immunoreactive ANP (IR-ANP) and 207.3 +/- 6.6 nmol of catecholamines as epinephrine plus norepinephrine. (2) Stimulation of nicotinic but not muscarinic acetylcholine receptors caused a cosecretion of IR-ANP and catecholamines corresponding to the ratio of IR-ANP to catecholamines in cultured bovine adrenal medullary cells. (3) Carbachol-stimulated secretion of IR-ANP was dependent on the presence of extracellular Ca2+. (4) Chromaffin granules isolated from bovine adrenal medulla contained large amounts of IR-ANP and catecholamines, in the same ratio as did cultured adrenal medullary cells. (5) Reverse-phase HPLC analysis showed that both stored and secreted IR-ANP consisted of two components, which eluted at the position of ANP(99-126) or ANP(1-126). These results indicate that ANPs are stored as ANP(99-126) and ANP(1-126) in chromaffin granules, and are cosecreted in parallel with catecholamines in a Ca2+-dependent manner by the stimulation of nicotinic acetylcholine receptors.