Enhanced expression of the ß4-galactosyltransferase 2 gene impairs mammalian tumor growth.

Altered N-glycosylation of membrane proteins is associated with malignant transformation of cells. We found that the expression of the ß4-galactosyltransferase 2 (ß4GalT2) gene is decreased markedly during the transformation. Here, we examined whether the tumor growth activity of B16-F10 mouse melanoma cells can be reduced by the enhanced expression of the ß4GalT2 gene. We isolated a clone, B16-ß4GalT2, showing its ß4GalT2 transcript 2.5 times higher than a control clone, B16-mock, by transducing its cDNA, and transplanted them subcutaneously into C57BL/6 mice to examine their tumor growth activity. The results showed that the average size of tumors formed with B16-mock cells is 13.1±0.76 mm, whereas that of tumors formed with B16-ß4GalT2 cells is 5.1±1.13 mm (P<0.01) 2 weeks after transplantation. Immunohistochemical analyses showed that the apoptosis and the suppression of angiogenesis are induced in the tumors upon transduction of the ß4GalT2 gene. To pursue a clinical usefulness of the ß4GalT2 gene for suppressing human tumor growth, we injected adenoviruses carrying the human ß4GalT2 cDNA into HuH-7 human hepatocellular carcinomas developed in severe combined immunodeficient mice, and observed marked growth retardation of the tumors. The enhancement of the ß4GalT2 gene expression in tumors is one of the promising approaches to suppress human tumor growth.

Oral symptoms predict mortality: a prospective study in Japan.

Several studies have reported positive associations between oral infections and systemic diseases. The purpose of the present study was to evaluate the effects of oral symptoms on mortality from cardiovascular disease (CVD) and pneumonia. Using data from a cohort study in Japan, we analyzed 4,139 individuals aged 40-79 years. The baseline questionnaire included the following items related to oral symptoms: 'sensitive teeth', 'difficulty in chewing tough food substances', 'bleeding gums', and 'mouth feels sticky'. We used the Cox proportional hazard model to estimate hazard ratios (HRs) and 95% confidence intervals (95%CIs) for mortality, after adjustments for lifestyle, socio-economic factors, and history of diseases. Persons complaining that their 'mouth feels sticky' had a two-fold higher risk of pneumonia (HR = 2.1; 95%CI, 1.2-3.6), while those complaining of 'sensitive teeth' had a lower risk of CVD (HR = 0.4; 95%CI, 0.2-0.9). Some oral symptoms may be predictors of mortality from pneumonia and CVD.

Galactosylation of N-linked oligosaccharides by human beta-1,4-galactosyltransferases I, II, III, IV, V, and VI expressed in Sf-9 cells.

Several studies showed that Sf-9 cells can synthesize the galactosylated N-linked oligosaccharides if beta-1,4-galactosyltransferase (beta-1,4-GalT) is supplied. The full-length human beta-1,4-GalT I, II, III, IV, V, and VI cDNAs were independently transfected into Sf-9 cells, and the galactosylation of endogenous membrane glycoproteins was examined by lectin blot analysis using Ricinus communis agglutinin-I (RCA-I), which preferentially interacts with oligosaccharides terminated with Galbeta1-->4GlcNAc group. Several RCA-I-reactive bands appeared in all of the gene-transfected cells, and disappeared on treatment of blots with beta-1,4-galactosidase or N-glycanase prior to incubation with lectin. Introduction of the antisense beta-1,4-GalT II and V cDNAs separately into human colorectal adenocarcinoma SW480 cells, in which beta-1,4-GalT I, II, and V genes were expressed, resulted in the reduction of RCA-I binding toward N-linked oligosaccharides of the membrane glycoproteins. Differences were found in their K(m) values toward UDP-Gal and GlcNAcbeta-S-pNP and in their acceptor specificities toward oligosaccharides with the GlcNAcbeta1-->4(GlcNAcbeta1-->2)Man branch and with the GlcNAcbeta1-->6(GlcNAcbeta1-->2)Man branch. These results indicate that beta-1,4-GalTs II, III, IV, V, and VI are involved in the N-linked oligosaccharide biosynthesis cooperatively but not in a redundant manner with beta-1,4-GalT I within cells.

Correlated gene expression between beta-1,4-galactosyltransferase V and N-acetylglucosaminyltransferase V in human cancer cell lines.

Since our previous study showed that the gene expression level of beta-1,4-galactosyltransferase (beta-1,4-GalT) V is only increased in mouse NIH3T3 transformant and that beta-1,4-GalT V preferentially galactosylates the GlcNAcbeta1 --> 6Man branch of oligosaccharides [Shirane et al. (1999) Biochem. Biophys. Res. Commun. 265, 434-438], whether its gene expression is correlated with malignant transformation was investigated. Northern blot analysis of beta-1, 4-GalTs I, II, III, IV, V, and VI and N-acetylglucosaminyltransferase (GlcNAcT)V in human cancer cell lines showed that the gene expression levels of beta-1,4-GalT V but not other beta-1,4-GalTs are strongly correlated with those of GlcNAcT V whose activity was shown to increase by malignant transformation. These results indicate that beta-1,4-GalT V is involved in the galactosylation of highly branched oligosaccharides characteristic of malignantly transformed cells.

Involvement of beta-1,4-galactosyltransferase V in malignant transformation-associated changes in glycosylation.

In spite of marked changes in the glycosylation upon malignant transformation of cells, no biological significance of beta-1, 4-galactosyltransferase (beta-1,4-GalT) activities has been elucidated. When beta-1,4-GalT activities toward 1 mM GlcNAcbeta-S-pNP were determined using homogenates of NIH3T3 and its transformant, MTAg, MTAg contained 1.3 times higher activities. Northern blot analysis, however, revealed that the beta-1,4-GalT V gene expression increases by three times with a decrease in that of beta-1,4-GalT II by one-fifth and without significant changes in those of other beta-1,4-GalTs in MTAg. Analysis of beta-1,4-GalT V acceptor-specificity showed that the GlcNAcbeta1-->6Man group of the GlcNAcbeta1-->6(GlcNAbeta1-->2)Manalpha1- branch is galactosylated. These results indicate that changes in beta-1,4-GalT II and V activities are important for the altered glycosylation.

Spin trapping of nitric oxide in aqueous solutions of cigarette smoke.

Nitric oxide, a gaseous free NO radical (.NO) generated in particulate-free gas-phase main-stream smoke of cigarettes, was observed with electrical spin resonance (ESR) using a spin trapping technique. N-Methyl-D-glucamine-dithiocarbamate (MGD)2-Fe2+ complex was used for the NO radical spin trapper in aqueous solution. The intensity of the ESR signal of the spin adduct formed by bubbling smoke from one cigarette increased gradually with time over 2 hours at about 20 degrees C and was constant for 2 days or longer. The time course of the production of the NO radical followed the rate equation y = 1520(1-e-0.018t) for the first-order reaction up to around 25 min after mixing of Fe2+ solution and then slowly approached the maximum value determined by the concentration of the spin adduct. These findings suggest that NO radical is produced slowly from NO radical donors such as amine .NO complexes, peroxinitrite (ONOO-), and other reactants such as nitrogen oxides (NOx), which are produced from the smoke of tobacco leaves, and suggest that its generation could be involved in the decomposition or cleavage of such substances.

Effects of N-3554S, a polyprenyl phosphate, on B16-F10 mouse melanoma cells.

N-3554S, an optically active S-isomer of alpha-dihydrodecaprenyl phosphate, reduced the tumorigenicity of cultured B16-F10 mouse melanoma cells probably by affecting protein N-glycosylation. Accordingly, membrane glycoprotein samples were prepared from the melanoma cells cultured with or without N-3554S, and amounts and structures of N-linked sugar chains were determined. Analyses of the N-linked oligosaccharides released by hydrazinolysis from these samples and reduced with NaB3H4 revealed that the N-3554S-treated cells contain 1.5-1.8 times as much oligosaccharides as the control cells, and the relative amounts of high-mannose-type and bi-, tri- and tetra-antennary complex-type sugar chains are almost the same between two samples. Western blot analysis, however, showed that binding of L-PHA, which binds to oligosaccharides with the GlcNAc beta 1-->6(GlcNAc beta 1-->2)Man structure, is significantly reduced in 90 K, 96 K, 140 K, 155 K and 180 K glycoproteins in N-3554S-treated cells. Immunoblot analysis showed that the 140 K glycoprotein could be a fibronectin receptor. It was also shown that N-3554S treatment enhances the adhesiveness of the cells to fibronectin. These results indicate that N-3554S affects N-glycosylation of membrane glycoproteins and alters the cell surface properties of B16-F10 cells.

Interspecies transmission of animal rotaviruses to humans as evidenced by phylogenetic analysis of the hypervariable region of the VP4 protein.

A phylogenetic tree constructed for the hypervariable region (aa 71-203) of the VP4 protein of 28 human and animal rotaviruses that were previously reported to belong to 13 distinct VP4 genotypes revealed unique positions of human rotavirus strains HCR3 and Ro1845, together with feline strain FRV64 and canine strains K9 and CU-1, in the animal rotavirus lineages, lending strong support to the view that both HCR3 and Ro1845 were of animal rotavirus origin.

Integrated optical-disk pickup that uses a focusing grating coupler with a high numerical aperture.

An integrated optical-disk pickup that uses a focusing grating coupler with a numerical aperture of 0.45 (a focal length of 1.0 mm and an aperture of 1.0 mm × 0.8 mm) was developed, and the readout signal on an International Standards Organization (ISO) -formatted 90-mm optical disk was detected by the pickup. The signal-amplitude ratio of the minimum-limit data pattern to the maximum was 0.53 on the innermost track. It meets the requirement on the signal resolution for optical-disk storage devices.

Asparaginyl endopeptidase of jack bean seeds. Purification, characterization, and high utility in protein sequence analysis.

Asparaginyl endopeptidase was highly purified from mature seeds of the jack bean (Canavalia ensiformis). The final enzyme preparation showed a single peak in high-performance liquid chromatography on a reversed-phase column, and the material in the peak gave the following NH2-terminal amino acid sequence on Edman degradation for 25 cycles: H-Glu-Val-Gly-Thr-Arg-Trp-Ala-Val-Leu-Val-Ala-Gly-Ser-Asn-Gly-Tyr-Gly-Asn-Tyr- Arg-His-Gln-Ala-Asp-Val-. Behavior of the enzyme toward various protease inhibitors suggested that it belongs to a family of cysteine proteases. Strict substrate specificity of this enzyme was verified by the use of 14 polypeptide substrates including those derived from proteins. Almost all the peptide bonds on the carboxyl side of Asn residues were susceptible to the enzyme. The exceptions were cases where the residue was at the NH2 terminus or the second position from the NH2 terminus of substrates and where it was N-glycosylated Asn. Peptide bonds on the carboxyl side of any other amino acid residues were not cleaved. These properties promise the high utility of this novel endopeptidase in protein sequence analysis. Identity of jack bean asparaginyl endopeptidase with a processing enzyme responsible for maturation of concanavalin A from its precursor is also discussed.

Chemical model of reaction cascades induced by activated enzymes or catalysts. Two-step cascades in visual transduction.

A dissipative system is approximated by a nonlinear rate equation: Z congruent to K1Z - K2Z3 (K2 greater than 0), in which the right side is derived from -delta G/delta Z of Taylor's series of the thermodynamic potential given by Gibbs' function G(Tc, Pc) (Z) at about the critical point C(Tc, Pc) of the control variables (parameters) T and P. The stability or instability of the system is treated by the changes in the control parameters. In the case that T not equal to P not equal to 0 in the steady state, Z = 0, and T and P pass the point C, K1 becomes negative. By this change, the G function is convex at Z = 0 and each product is created rapidly with concentration or number of the molecules Z = ([K1]/K2)1/2. This dynamic theory is applied to enzyme cascades. Based on cyclic GMP (cGMP) hypothesis in visual transduction, the cascade hydrolysis of cGMP of vertebrates is analyzed by dividing it into two-step reaction cascades: The initial process is that metarhodopsin II catalyzes the exchange of GDP for GTP by transducin (Gtd) and that GTP-Gtd complex is hydrolyzed to GDP-Gtd complex. In the following cascade cGMP is hydrolyzed with amplification of phosphodiesterase (PDE) activated by the removal of the small inhibitory subunit. The quantity of the hydrolysis of cGMP is estimated as approximately 5 x 10(4-5) molecules per photolyzed rhodopsin semiempirically, and this coincides well with experiments.

A self-organized chemical model and reaction cascade.

Some reaction cascades in biological systems are analyzed by a self-organized chemical model, an autocatalytic reaction. This model is described by the coupling of a primary system which stabilizes the initial stage of the reaction rapidly and a partial system which controls the primary system slowly. By the internal force caused by a trigger above the threshold, the coupled system in near-equilibrium is broken and changed into a new state. From the rate equation for the coupled system, a dimensionless nonlinear state equation, n = -n3 - un - v, is derived, where n is the concentration of intermediate, and u, v are dynamic variables of the system. This equation is similar to a nonequilibrium tri-molecular reaction. By using this chemical network theory, fibrin polymerization. F + F----fm----fp + X, where F is a fibrinogen molecule, fm is a fibrin monomer, fp is fibrin polymer, and X is small peptides released from fibrinogen, is discussed as an excellent example of the enzyme reaction cascade.

Network formation in negative charged membranes by two divalent cations and the catastrophe.

The ligands of Ca2+-Cu2+-phosphatidylserine (PS) complexes in membrane networks at the water-oil interface through the symmetry breaking instability and the head groups of PS molecules were changed into a solid-like state. A first step in this transition is described by the following scheme in one unit in which the molar ratio is Ca2+: Cu2+: PS = 1:2:4; [Oh]+2[Oh]*----3[Oh]*, where [Oh]* denotes a little distorted ligand structure [LnM2+...2H2O] from [LnM2+2H2O], where Ln is PS molecules (n = 2 to Cu2+ and 4 to Ca2+). All the ligands are changed to [D4h] by the unit-unit interaction due to the network formation; [Oh]*----[D4h]. The whole system is equivalent to Schlögl's scheme and is given by a cubic state equation for suitable variables transformations: x = -x3 - ux - v, where x corresponds to the concentration of [Oh]*, and u and v are related to rate constants in the first and the second steps, and they also depend on the initial [Oh] and the final [D4h] concentrations. This system is transferred into a new state with a cusp catastrophe.

Chemical dynamic approach to synergetics and instability in biological systems.

Based on Haken's theory, self-organization or synergetics is discussed using chemical dynamics to represent an autocatalytic reaction. In a simple case the changes in a self-organizing system are given by a set of two rate equations for a primary and a partial system. When these systems mutually form a feedback loop under the adiabatic condition, the rate equation of self-organization is described by a generalized Gibbs' free energy change delta U (delta x) followed by the reaction. The sign of the parameter k'3 (k0-kex; k0, kex: rate constants with or without an external stimulus) determines the instability of the coupled system in quasi-equilibrium (k'3 approximately greater than 0; k0 greater than kex). When the product exceeds the threshold (k'3 less than 0; k0 less than kex), the system transfers into a new state, or a phase transition appears. Considering the Boltzmann distribution, the transition parameter k'3 is evaluated by an average distribution of the states and the instability is discussed using the reaction velocities vqe and vqe in the quasi-equilibrium state. As an example of this model membrane excitation is discussed briefly.

Dynamic approach to thermotropic and inotropic phase transitions.

Thermotropic and inotropic phase transitions have been analysed with a dynamic theory on a self-organization. An equation of motion of a molecular assembly with strong interactions may be approximately described as: dQ/dt' congruent to -K1Q-K3Q3, where Q is a displacement from the equilibrium point Q0(identical to 0) in a vibrational state, K1 is a transition parameter. When the parameter K1 concerned with an internal driving force (partial system) changes from positive to negative through the potential bifurcation, the system transfers to a new stable state breaking down the symmetry. Such a sign change of K1 serves as a trigger to a phase transition. Using Weiss' approximation, we have evaluated the change of K1 by a function of temperature, kappa (T-TC), and have obtained the critical temperature TC of thermotropic phase transition. We have furthermore treated inotropic phase transition caused by the binding of divalent cations like Ca2+ using the function kappa (T-beta TC), where beta is a shift parameter of the critical temperature.

[Fundamental and clinical studies of cefotaxime in neonates and immature infants].

Cefotaxime (CTX) was used in the treatment and prophylaxis of infections in neonates and immature infants. The following results were obtained. 1. Mean serum concentrations (bioassay) 30 minutes after a single intravenous injection of about 20 mg/kg of CTX were 44.5 mcg/ml in neonates and 47.2 mcg/ml in immature infants aged 0-3 days, 45.8 mcg/ml in neonates and 56.4 mcg/ml in an immature infant aged 4-7 days and 40.6 mcg/ml in neonates and 38.1 mcg/ml in immature infants aged 8 or more days. Six hour values were respectively 10.9 mcg/ml, 17.0 mcg/ml, 4.6 mcg/ml, 13.4 mcg/ml, 3.8 mcg/ml and 2.7 mcg/ml. 2. Mean serum concentration half-lives were 3.0 hours in neonates and 3.2 hours in immature infants aged 0-3 days, 1.8 hours in neonates and 3.2 hours in an immature infant aged 4-7 days, and 1.5 hours in neonates and 1.6 hours in immature infants aged 8 or more days. 3. Urinary recovery rates were 0.8-78.0% for 0-6 hours after treatment. 4. Adequate clinical efficacy can be expected by the intravenous injection of CTX in doses of 20 mg/kg 2 times daily, every 12 hours, in neonates and immature infants aged 0-3 days, 20 mg/kg 3 times daily, every 8 hours, in neonates and immature infants aged 4-7 days, and 20 mg/kg 3 to 4 times daily, every 6-8 hours, in neonates and immature infants aged 8 or more days. 5. The clinical efficacy of CTX was good in all 4 cases of sepsis (including suspected case), excellent in 1 case of urinary tract infection, and good in all 4 cases of fever of unknown origin for a cure rate of 100%. 6. Adverse reactions were not noted in any cases.