Structural basis of calcification inhibition by alpha 2-HS glycoprotein/fetuin-A. Formation of colloidal calciprotein particles

Genetic evidence from mutant mice suggests that alpha(2)-HS glycoprotein/fetuin-A (Ahsg) is a systemic inhibitor of precipitation of basic calcium phosphate preventing unwanted calcification. Using electron microscopy and dynamic light scattering, we demonstrate that precipitation inhibition by Ahsg is caused by the transient formation of soluble, colloidal spheres, containing Ahsg, calcium, and phosphate. These "calciprotein particles" of 30-150 nm in diameter are initially amorphous and soluble but turn progressively more crystalline and insoluble in a time- and temperature-dependent fashion. Solubilization in Ahsg-containing calciprotein particles provides a novel conceptual framework to explain how insoluble calcium precipitates may be transported and removed in the bodies of mammals. Mutational analysis showed that the basic calcium phosphate precipitation inhibition activity resides in the amino-terminal cystatin-like domain D1 of Ahsg. A structure-function analysis of wild type and mutant forms of cystatin-like domains from Ahsg, full-length fetuin-B, histidine-rich glycoprotein, and kininogen demonstrated that Ahsg domain D1 is most efficient in inhibiting basic calcium phosphate precipitation. The computer-modeled domain structures suggest that a dense array of acidic residues on an extended beta-sheet of the cystatin-like domain Ahsg-D1 mediates efficient inhibition.     

Requirement of domain-domain interaction for conformational change and functional ATP hydrolysis in myosin

Coordination between the nucleotide-binding site and the converter domain of myosin is essential for its ATP-dependent motor activities. To unveil the communication pathway between these two sites, we investigated contact between side chains of Phe-482 in the relay helix and Gly-680 in the SH1-SH2 helix. F482A myosin, in which Phe-482 was changed to alanine with a smaller side chain, was not functional in vivo. In vitro, F482A myosin did not move actin filaments and the Mg2+-ATPase activity of F482A myosin was hardly activated by actin. Phosphate burst and tryptophan fluorescence analyses, as well as fluorescence resonance energy transfer measurements to estimate the movements of the lever arm domain, indicated that the transition from the open state to the closed state, which precedes ATP hydrolysis, is very slow. In contrast, F482A/G680F doubly mutated myosin was functional in vivo and in vitro. The fact that a larger side chain at the 680th position suppresses the defects of F482A myosin suggests that the defects are caused by insufficient contact between side chains of Ala-482 and Gly-680. Thus, the contact between these two side chains appears to play an important role in the coordinated conformational changes and subsequent ATP hydrolysis.     

Orally active cephalosporins. Part 3: synthesis, structure-activity relationships and oral absorption of novel C-3 heteroarylmethylthio cephalosporins

A series of 7beta-[(Z)-2-(2-aminothiazol-4-yl)-2-hydroxyiminoacetamido]-3-(heteroarytmethylthio)cephalosporins was designed, synthesized and evaluated for antibacterial activity and oral absorption in rats. Antibacterial activity was markedly influenced by the structure of the heteroaromatic ring moiety. Oral absorption was influenced by the heteroaromatic ring moiety as well as by the arrangement of heteroatoms. Among these compounds, FK041 (2o), having a 4-pyrazolylmethylthio moiety, showed potent antibacterial activity against both gram-positive and gram-negative bacteria including Haemophilus influenzae. Further, it showed higher oral absorption than CFDN.     

The antioxidant effect of DL-alpha-lipoic acid on copper-induced acute hepatitis in Long-Evans Cinnamon (LEC) rats

The Long-Evans Cinnamon (LEC) rats, due to a genetic defect, accumulate excess copper (Cu) in the liver in a manner similar to patients with Wilson's disease and spontaneously develop acute hepatitis with severe jaundice. In this study we examined the protective effect of DL-alpha-Lipoic acid (LA) against acute hepatitis in LEC rats. LA was administered to LEC rats by gavage in doses of 10, 30 and 100 mg/kg five times per week, starting at 8-weeks-old and continuing till 12-weeks-old. Although LA had little effect against the increases in serum transaminase activities, it suppressed the loss of body weight and prevented severe jaundice in a dose-dependent manner. Antioxidant system analyses in liver showed that LA treatment significantly suppressed the inactivations of catalase and glutathione peroxidase, and the induction of heme oxygenase-1, an enzyme which is inducible under oxidative stress. Furthermore, LA showed dose-dependent suppressive effect against increase in nonheme iron contents of both cytosolic and crude mitochondrial fractions in a dose-dependent manner. Although at the highest dose, LA slightly suppressed the accumulation of Cu in crude mitochondrial fraction, it had no effect on the accumulation of Cu in cytosolic fraction. While LA completely suppressed the increase in lipid peroxidation (LPO) in the microsomal fraction at the highest dose, the suppressive effect against LPO in crude mitochondrial fractions was slight. From these results, it is concluded that LA has antioxidant effects at the molecular level against the development of Cu-induced hepatitis in LEC rats. Moreover, mitochondrial oxidative damage might be involved in the development of acute hepatitis in LEC rats.     

Effects of ionic valency of interacting metal elements in ion uptake by carrot (Daucas carota cv. U.S. harumakigosun)

Interaction of elements in the course of element uptake by carrot (Daucas carota cv. U.S. harumakigosun) exerted by the addition of elements, such as Rb, Zn, and Al, was investigated. For the purpose of precise evaluation of uptake behavior, the simultaneous determination of absorption of Na, Be, Sr, Mn, Co, Zn, Ce, Pm, and Gd was conducted by the multitracer technique. For root uptakes, Al exhibited its influence on the uptake of essential elements and on the uptake of toxic or unbeneficial ones, presumably as a result of the large electric valency that caused cell membrane disintegrity. On the other hand, Zn as a divalent cation only affected the uptake of essential and beneficial elements. Rubidium, which is a monovalent cation, did not exhibit any effect on the uptake of other ions. Concerning shoot uptakes, inhibition by Zn and Al, but not by Rb, was observed for the uptake of Sr, Mn, Co, and Zn. From the present investigation, it is suggested that there exists an interaction between added ions and the elements taken into plants and that the degree of interaction increases in the increasing order of ionic valency: M+ (Rb), M2+ (Zn), and M3+ (Al).     

Rat tripeptidyl peptidase I: molecular cloning, functional expression, tissue localization and enzymatic characterization

We purified tripeptidyl peptidase I (TPP I) to homogeneity from a rat kidney lysosomal fraction and determined its physicochemical properties, including its molecular weight, substrate specificity and partial amino acid sequence. The molecular weight of the enzyme was calculated to be 280,000 and 290,000 by non-denaturing PAGE and gel filtration, respectively, and to be 43 000 and 46 000 on SDS-PAGE in the absence and presence of beta-ME, respectively. These findings suggest that the enzyme is composed of six identical subunits. The Km, Vmax, kcat and kcat/Km values of TPP I at optimal pH (pH 4.0) were 680 microM, 3.7 micromol x mg(-1) x min(-1), 33.1 s(-1) and 4.87 x 10(4) s(-1) x M(-1) for Ala-Ala-Phe-MCA, respectively. TPP I was significantly inhibited by PCMBS and HgCl2, and moderately by DFP. These findings also suggest that TPP I is an exotype serine peptidase that is regulated by SH reagent. TPP I released the tripeptide Arg-Val-Tyr from angiotensin III more rapidly than from Ala-Ala-Phe-MCA, and also released Gly-Asn-Leu from neuromedin B with the same velocity as from Ala-Ala-Phe-MCA. Angiotensin III and neuromedin B have recently been found to be good natural substrates for lysosomal TPP I. Furthermore, we determined the rat liver cDNA structure and deduced the amino acid sequence. The cDNA, designated as lambdaRTI-1, is composed of 2485 bp and encodes 563 amino acids in the coding region. By Northern blot analysis, the order for TPP I mRNA expression was kidney > or = liver > heart > brain > lung > spleen > skeletal muscle and testis. In parallel experiments, the TPP I antigen was detected in various rat tissues by immunohistochemical staining.     

A sensitive non-radioactive in situ hybridization method for the detection of chicken IgG γ-chain mRNA: a technique suitable for detecting of variety of mRNAs in tissue sections

We established a sensitive non-radioactive in situ hybridization (ISH) method for the detection of chicken IgG γ-chain mRNA in paraffin sections. RNA probes were transcribed in vitro fromcloned chicken IgG CH1 nucleotide sequences with SP6/T7 RNA polymerases in the presence of DIG-UTP. These probes were used for hybridization and were immunodetected using anti-DIG antibodies conjugated to horseradish peroxidase. The immunoreactive products were visualized with DAB-H₂O₂. IgG γ-chain mRNA-expressing cells were localized in both the spleen and oviductal tissues. This method demonstrated an excellent sensitivity since the ISH signal was clear and the background was negligible. We found that in the spleen IgG γ-chain mRNA-expressing cells were present mainly in the red pulp, whereas in the oviduct they appeared mainly in the mucosal stroma and not in the mucosal epithelium. Published: May 14, 2001.