Bimodal activation of SMC ATPase by intra- and inter-molecular interactions.

Structural maintenance of chromosomes (SMC) proteins play fundamental roles in higher-order chromosome dynamics from bacteria to humans. It has been proposed that the Bacillus subtilis SMC (BsSMC) homodimer is composed of two anti-parallel coiled-coil arms, each having an ATP-binding domain at its distal end. It remains totally unknown, however, how the two-armed structure supports ATP-dependent actions of BsSMC. By constructing a number of mutant derivatives including 'single-armed' BsSMC, we show here that the central hinge domain provides a structural flexibility that allows opening and closing of the two arms. This unique structure brings about bimodal regulation of the SMC ATPase cycle. Closing the arm can trigger ATP hydrolysis by allowing an end-end interaction within a dimer (intramolecular mode). When bound to DNA, ATP promotes a dimer-dimer interaction, which in turn activates their DNA-dependent ATPase activity (intermolecular mode). Our results reveal a novel mechanism of ATPase regulation and provide mechanistic insights into how eukaryotic SMC protein complexes could mediate diverse chromosomal functions, such as chromosome condensation and sister chromatid cohesion.     

Analyses of azopigments obtained from the delta fraction of bilirubin from mammalian plasma (mammalian biliprotein).

Azopigments were obtained from the delta fraction of bilirubin (mammalian biliprotein) in cholestatic sera of men, rats and guinea pigs by diazo reaction with diazotized p-iodoaniline and analysed by t.l.c. Delta bilirubin of men and rats generated both unconjugated and glucuronide-conjugated azodipyrroles, whereas that of guinea pigs, in which the predominant form of conjugated bilirubin in serum was bilirubin monoglucuronide, generated only unconjugated azodipyrrole. We further analysed the azopigments by reversed-phase h.p.l.c. to distinguish their endovinyl and exovinyl isomers. The results indicated (a) that covalent binding of bilirubin to protein occurs exclusively on the conjugated dipyrrolic (either endovinyl or exovinyl) half of the parent conjugated bilirubin, (b) that both bilirubin monoglucuronide and bilirubin diglucuronide generate delta bilirubin, the latter yielding a 'conjugated' form of delta bilirubin that preserves the glucuronic acid moiety on the dipyrrolic half not bound covalently to protein, and (c) that therefore at least four forms of delta bilirubin exist in jaundiced sera of men and rats.     

A fragile X female with Down syndrome

Summary Clinical and cytogenetic aspects of a female infant with trisomy 21 and the fragile X [fra (X)] chromosome are reported. Most of the facial characteristics of the patient are those observed in Down syndrome, but some features such as long face with prominent forehead and lower jaw, and large ears are related to the fra (X) syndrome. The origin of an additional chromosome 21 may be ascribed to maternal first meiotic nondisjunction in our case. It has been suspected that female carriers of the fra (X) chromosome may be predisposed to meiotic nondisjunctional events. However, there is probably no relationship between the two chromosomal abnormalities in our case because of the maternal age at the delivery.     

Interleukin-6 triggers the association of its receptor with a possible signal transducer, gp130

Interleukin-6 mediates pleiotropic functions in various types of cells through its specific receptor (IL-6-R), the cDNA of which has already been cloned. We report here that an 80 kd single polypeptide chain (IL-6-R) is involved in IL-6 binding and that IL-6 triggers the association of this receptor with a non-ligand-binding membrane glycoprotein, gp130. The association takes place at 37 degrees C within 5 min and is stable for at least 40 min in the presence of IL-6, but does not occur at 0 degree C. Human IL-6-R can associate with a murine gp130 homolog and is functional in murine cells. Mutant IL-6-R lacking the intracytoplasmic portion is functional, suggesting that the two polypeptide chains interact to involve their extracellular portion. In fact, a soluble IL-6-R lacking the transmembrane and intracytoplasmic domains can associate with gp130 in the presence of IL-6 and mediate its function. These findings indicate that the complex of IL-6 and IL-6-R can interact with a non-ligand-binding membrane glycoprotein, gp130, extracellularly and can provide the IL-6 signal.     

Differences in lectin binding patterns of normal human endometrium between proliferative and secretory phases

Lectin binding patterns in normal human endometrium were examined by light and electron microscopy using seven different lectins (ConA, WGA, RCA, PNA, UEA-1, DBA, and SBA). For light microscopic observations, criteria based on the incidence and intensity of cells positive for the lectin staining were adopted to evaluate the different staining patterns of the proliferative and secretory endometria obtained by the avidin-biotin-peroxidase complex (ABC) technique. At the light microscopic level, ConA, WGA, and RCA stained endometrial glandular cells in both phases. The number of PNA-positive cells with the binding sites entirely limited to the apical surface tended to be reduced slightly in the secretory phase. UEA-1 weakly stained the apical surface of glandular cells in the proliferative phase but not in the secretory phase. Among the lectins used in this study, DBA and SBA displayed remarkable changes between the phases. That is, in the proliferative phase they produced only a faint or slight positive stain at the apical surface, but the incidence and intensity of DBA- and the SBA-positive glandular cells increased in the secretory phase. By electron microscopy, the reaction product of ConA was observed in the plasma membrane, endoplasmic reticulum, nuclear envelope, and the Golgi apparatus, and the binding sites of RCA and DBA were observed in the plasma and Golgi membranes. Between both phases, the reactivity of ConA and RCA showed almost no change. However, the secretory endometrial cells containing the DBA-positive Golgi apparatus were markedly increased in number compared with the proliferative ones bearing the lectin-positive organelles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chitin gels

Chitin dissolved in N,N-dimethylacetamide, N-methyl-2-pyrrolidone and their mixed solvents in the presence of 5% LiCl was treated with acetic anhydride-pyridine, and the mixture solution was heated at 100 degrees C for 6 h to give a partially O-acetylated chitin gel. Chitin dissolved in these solvents in the presence of 5% LiCl was mixed with pyridine, and the mixture solution was heated at 60 degrees C for 5 h to give a chitin gel. Both the gels were rigid and transparent, and their properties and the rate of the hydrolysis of the chitin xerogel by hen-egg white lysozyme were essentially similar to those of N-acetylchitosan gel prepared by chemical N-acetylation of chitosan.     

Determination of guanine by high-performance liquid chromatography with electrochemical detection: application to DNA and RNA assays

A highly sensitive assay for guanine was developed using high-performance liquid chromatography with electrochemical detection (ECD). Guanine was susceptible to the electrochemical oxidation, and ECD response was proportional to the amount of guanine in the range 0.25-4 pmol of guanine. The ECD of guanine was applicable to the analysis of nucleic acids. DNA and RNA were hydrolyzed in 0.03 and 3 M HCl, respectively, and guanine liberated from the nucleic acids was separated on a reverse-phase column and determined by ECD. The method allowed detection of 0.2 ng of calf thymus DNA or tRNA. An application of the method is shown for DNA and RNA assays in trichloroacetic acid extracts of rat adrenal and liver.     

Dual loading of the fluorescent indicator fura-2 and 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) in isolated myocytes

Isolated rat heart myocytes were loaded with both the Ca2+ sensitive fluorescent probe fura-2/AM and the fluorescent pH indicator 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF/AM). Changes in [Ca2+]i and pHi were measured simultaneously using digitized video fluorescence microscopy. In measurement of [Ca2+]i and pHi, the ratios of dual-loaded cells were not different from single-loaded cells. Using this method, [Ca2+]i and pHi in myocytes were 48 +/- 7 nM and 7.17 +/- 0.05. It is concluded that [Ca2+]i and pHi could be measured simultaneously in isolated myocyte using dual-loading of fura-2 and BCECF.     

Different preparations of natural and recombinant human interleukin-6 (IFN-beta 2, BSF-2) similarly stimulate acute phase protein synthesis and uptake of alpha-aminoisobutyric acid by cultured rat hepatocytes

1. Rat hepatocytes were cultured for 2 days in Williams E medium containing 1 microM insulin and dexamethasone. 2. Production of five plasma proteins was determined by electroimmunoassay in the media, and amino acid uptake was measured by [alpha-14C]aminoisobutyric acid accumulation in hepatocytes. 3. Supernatants from rat peritoneal macrophages and IL-6/IFN-beta 2/BSF-2 obtained from four different laboratories similarly stimulated synthesis of fibrinogen, alpha 1-cysteine proteinase inhibitor and alpha 2-macroglobulin, as well as [14C]-accumulation in cultured hepatocytes. 4. It is concluded that IL-6 is the principal hepatocyte stimulating factor responsible for typical features of the acute phase response of liver cells.