Heparan sulfate deficiency leads to hypertrophic chondrocytes by increasing bone morphogenetic protein signaling.

OBJECTIVE: Exostosin-1 (EXT1) and EXT2 are the major genetic etiologies of multiple hereditary exostoses and are essential for heparan sulfate (HS) biosynthesis. Previous studies investigating HS in several mouse models of multiple hereditary exostoses have reported that aberrant bone morphogenetic protein (BMP) signaling promotes osteochondroma formation in Ext1-deficient mice. This study examined the mechanism underlying the effects of HS deficiency on BMP/Smad signaling in articular cartilage in a cartilage-specific Ext-/- mouse model. METHOD: We generated mice with a conditional Ext1 knockout in cartilage tissue (Ext1-cKO mice) using Prg4-Cre transgenic mice. Structural cartilage alterations were histologically evaluated and phospho-Smad1/5/9 (pSmad1/5/9) expression in mouse chondrocytes was analyzed. The effect of pharmacological intervention of BMP signaling using a specific inhibitor was assessed in the articular cartilage of Ext1-cKO mice. RESULTS: Hypertrophic chondrocytes were significantly more abundant (P = 0.021) and cartilage thickness was greater in Ext1-cKO mice at 3 months postnatal than in control littermates (P = 0.036 for femur; and P < 0.001 for tibia). However, osteoarthritis did not spontaneously occur before the 1-year follow-up. matrix metalloproteinase (MMP)-13 and adamalysin-like metalloproteinases with thrombospondin motifs(ADAMTS)-5 were upregulated in hypertrophic chondrocytes of transgenic mice. Immunostaining and western blotting revealed that pSmad1/5/9-positive chondrocytes were more abundant in the articular cartilage of Ext1-cKO mice than in control littermates. Furthermore, the BMP inhibitor significantly decreased the number of hypertrophic chondrocytes in Ext1-cKO mice (P = 0.007). CONCLUSIONS: HS deficiency in articular chondrocytes causes chondrocyte hypertrophy, wherein upregulated BMP/Smad signaling partially contributes to this phenotype. HS might play an important role in maintaining the cartilaginous matrix by regulating BMP signaling.

Dynamics of a polymer chain confined in a membrane.

We present a Brownian dynamics theory with full hydrodynamics (Stokesian dynamics) for a Gaussian polymer chain embedded in a liquid membrane which is surrounded by bulk solvent and walls. The mobility tensors are derived in Fourier space for the two geometries, namely, a free membrane embedded in a bulk fluid, and a membrane sandwiched by the two walls. Within the preaveraging approximation, a new expression for the diffusion coefficient of the polymer is obtained for the free-membrane geometry. We also carry out a Rouse normal mode analysis to obtain the relaxation time and the dynamical structure factor. For large polymer size, both quantities show Zimm-like behavior in the free-membrane case, whereas they are Rouse-like for the sandwiched membrane geometry. We use the scaling argument to discuss the effect of excluded-volume interactions on the polymer relaxation time.

Drag coefficient of a liquid domain in a two-dimensional membrane.

Using a hydrodynamic theory that incorporates a momentum decay mechanism, we calculate the drag coefficient of a circular liquid domain of finite viscosity moving in a two-dimensional membrane. We derive an analytical expression for the drag coefficient which covers the whole range of domain sizes. Several limiting expressions are discussed. The obtained drag coefficient decreases as the domain viscosity becomes smaller with respect to the outer membrane viscosity. This is because the flow induced in the domain acts to transport the fluid in the surrounding matrix more efficiently.

Morphological transition and emulsification failure in globular microemulsions.

We consider the condensation transition of microemulsion droplets of oil which are dispersed in water in the presence of surfactant. Since a macroscopic oil phase is formed due to this transition, it is called "emulsification failure." Based on the free energy approach, we determine the transition lines between the spherical and the cylindrical droplet phases as well as the phase boundary lines of the emulsification failure. The phase diagrams are calculated by changing the physical properties of the surfactant monolayer such as the saddle-splay modulus and the spontaneous curvature. For a negative saddle-splay modulus, the spherical droplet phase coexists with the excess oil phase. In some cases, a re-entrant transition (sphere-->cylinder-->sphere) is expected to take place. For a positive saddle-splay modulus, the system undergoes a direct transition from the cylindrical droplet phase to the macroscopically phase separated state. The sphere-to-cylinder transition line approaches the emulsification failure boundary as the saddle-splay modulus becomes larger.

Concentration fluctuations in binary fluid membranes.

We investigate the dynamics of critical fluctuations in binary fluid membranes using a two-dimensional hydrodynamic model with momentum decay to the surrounding water. In particular, the decay rate of concentration fluctuations is obtained analytically. In the limit of small wavenumber q with respect to the correlation length, the decay rate is proportional to q², as usual. In the large-q limit, however, the effective diffusion coefficient increases only logarithmically with q.

Uptake and oxidation of acetaldehyde by intact human erythrocytes.

Human erythrocytes were examined for acetaldehyde-oxidizing capacity of intact cells, in comparison with its uptake under various conditions. The oxidation was found to be predominant over other possible acetaldehyde-metabolizing pathways, particularly with low concentrations of acetaldehyde. Further investigation using three media which differed in the initial pyruvate level showed an independence of acetaldehyde oxidation rate up to the high cellular lactate/pyruvate concentration ratio. The apparent non-oxidative acetaldehyde uptake obtained by subtraction of oxidation from the total uptake, was reduced when pretreating the erythrocytes with pyridoxal but not with pyridoxal 5'-phosphate. Though N-ethylmaleimide also inhibited the non-oxidative uptake, another sulfhydryl blocking reagent, p-chloromercuribenzoate was without effect. Thus, in addition to the oxidation, acetaldehyde may be involved in a reaction such as binding to the red cell components, as the minor metabolizing pathway in the erythrocytes.

Possible dominant-negative mutation of the SHIP gene in acute myeloid leukemia.

The SH2 domain-containing inositol 5'-phosphatase (SHIP) is crucial in hematopoietic development. To evaluate the possible tumor suppressor role of the SHIP gene in myeloid leukemogenesis, we examined primary leukemia cells from 30 acute myeloid leukemia (AML) patients, together with eight myeloid leukemia cell lines. A somatic mutation at codon 684, replacing Val with Glu, was detected in one patient, lying within the signature motif 2, which is the phosphatase active site. The results of an in vitro inositol 5'-phosphatase assay revealed that the mutation reduced catalytic activity of SHIP. Leukemia cells with the mutation showed enhanced Akt phosphorylation following IL-3 stimulation. K562 cells transfected with the mutated SHIP-V684E cDNA showed a growth advantage even at lower serum concentrations and resistance to apoptosis induced by serum deprivation and exposure to etoposide. These results suggest a possible role of the mutated SHIP gene in the development of acute leukemia and chemotherapy resistance through the deregulation of the phosphatidylinositol-3,4,5-triphosphate (PI(3,4,5)P3)/Akt signaling pathway. This is the first report of a mutation in the SHIP gene in any given human cancer, and indicates the need for more attention to be paid to this gene with respect to cancer pathogenesis.

Atherogenic change in the thoracic aorta of the senescence-accelerated mouse.

Biochemical and histological changes in the thoracic aorta of the senescence-accelerated prone mouse were examined in comparison with those of the senescence-accelerated resistant mouse. At 3 and 5 months of age, the lipid peroxide level in the thoracic aorta was higher in the former than in the latter. At 5 months of age, levels of both total cholesterol and triglycerides in the aorta were higher in the former than in the latter, and vice versa for the level of aortic phospholipids. At 5 months of age, the prone mice showed macrophage invasion beneath the endothelial cells of the thoracic aorta, whereas the resistant ones did not.

The metabolism of acetaldehyde and not acetaldehyde itself is responsible for in vivo ethanol-induced lipid peroxidation in rats.

A single oral administration of ethanol (5 g/kg) to rats induced a marked increase in lipid peroxidation, in the liver and kidney within 9 hr, as assessed by malondialdehyde accumulation. The pretreatment with alcohol dehydrogenase (ADH) inhibitor, 4-methylpyrazole (1 mmol/kg) caused approximately 50% inhibition of the hepatic ADH activity and abolished this ethanol-induced lipid peroxidation. The disulfiram treatment (100 mg/kg) significantly inhibited 63% of the hepatic low Km aldehyde dehydrogenase (ALDH) but not the high Km ALDH. The cyanamide treatment (15 mg/kg) effectively decreased 83% of the low Km and 70% of the high Km ALDH in the liver. Although there was more than a 20-fold elevation of acetaldehyde levels by the inhibition of acetaldehyde metabolism with disulfiram or cyanamide, the ethanol-induced lipid peroxidation was significantly suppressed by pretreatment with these drugs. More than 90% inhibition of xanthine oxidase and dehydrogenase by the pretreatment with allopurinol (100 mg/kg), with no effect on the hepatic ADH and ALDH activities, did not alter the enhancement of lipid peroxidation following ethanol administration. We propose that the metabolism of acetaldehyde (probably via the low Km ALDH) and not acetaldehyde itself is responsible for the ethanol-induced lipid peroxidation in vivo and that the contribution of xanthine oxidase, as an initiator of lipid peroxidation through acetaldehyde oxidation is minute during acute intoxication.

Biosynthesis of polymyxin E by a cell-free enzyme system. IV. Acylation of enzyme-bound L-2,4-diaminobutyric acid.

An enzyme fraction, which catalyzes the ATP-PPi exchange reaction dependent on the three constituent amino acids of polymyxin E, was partially purified from crude extracts of Aerobacillus polyaerogenes. The approximate molecular weight was estimated to be 640,000 by Sepharose 4B gel filtration. Incubation of the enzyme with octanoyl coenzyme A and diaminobutyric acid in the presence of ATP and an ammonium sulfate fraction yielded octanoyldiaminobutyric acid thioesterified to the enzyme protein. On mild alkali treatment, octanoyldiaminobutyric acid, identified by paper chromatography, was released from the enzyme protein. From its acid hydrolyzate, diaminobutyric acid and octanoic acid were recovered in a molar ratio of 1 to 0.7. An ammonium sulfate fraction was required as the source of an acyltransferase for acylation of the enzyme-bound diaminobutyric acid. When [14C]-threonine was incubated with L-2,4-diaminobutyric acid in the presence of octanoyl coenzyme A, octanoyldiaminobutyrylthreonine bound to the enzyme protein was formed. These results suggest that acyldiaminobutyric acid bound to the enzyme protein is a possible initiation complex in the biosynthesis of polymyxin E.

Biosynthesis of polymyxin E by a cell-free enzyme system. II. Synthesis of enzyme-bound octanoyldiaminobutyric acid.

A partially purified enzyme of Aerobacillus polyaerogenes, which produces polymyxin E, activates L-2,4-diaminobutyric acid and binds it as a thioester. The incubation of the enzyme preparation with octanoyl coenzyme A and L-2,4-diaminobutyric acid in the presence of ATP and an ammonium sulfate fraction yields octanoyldiaminobutyric acid thioesterified to the enzyme.

Partial purification and properties of L-2,4-diaminobutyric acid activating enzyme from a polymyxin E producing organism.

An L-2,4-diaminobutyric acid activating enzyme was found in crude extracts of Aerobacillus polyaerogenes, which produces polymyxin E1 and E2. The enzyme was partially purified by sonication of the cells, followed by ultracentrifugation, ammonium sulfate fractionation, and DEAE-cellulose column chromatography. In addition to L-2,4-diaminobutyric acid, the enzyme activated L-leucine and L-threonine, which are constituent amino acids of polymyxin E. All three amino acids were bound to the enzyme as thioesters. These results suggest that polymyxin is synthesized by a multienzyme thiotemplate mechanism, in the same way as gramicidin S, tyrocidines, bacitracins, and gramicidin A.

Effects of interferon-beta in combination with 5-fluorouracil on the growth of esophageal cancer cells in vitro.

The possible antiproliferative potency of human recombinant interferon-beta (hIFN-beta) towards ten human esophageal cancer cell lines was examined in comparison with the activity of the factor towards human malignant melanoma cell lines. The cell growth of esophageal cancer cell lines was inhibited by hIFN-beta in a dose- and time- dependent manner. The 50% inhibitory concentrations (IC50) of hIFN-beta on nine cell lines out of ten ranged between 23 to 332 IU/ml of culture medium. The remaining cell line, T.Tn, was less sensitive to the interferon (IC50, 611 IU/ml). Under the same culture conditions, the melanoma cell lines tested differed markedly in their sensitivity to hIFN-beta. When the esophageal cancer cells were treated with 5-fluorouracil (5-FU) in the presence of a low concentration of hIFN-beta, the effectiveness of 5-FU was markedly enhanced. In particular, the rate of growth inhibition of T.Tn cells was more than the added potencies of 5-FU and hIFN-beta indicating that the interferon is an effective biomodulator of 5-FU. All these data suggest that combination therapy with hIFN-beta and the anticancer drug 5-FU would be beneficial for the treatment of carcinoma of the esophagus.

Reexamination of the relationship between alcohol preference and brain monoamines in inbred strains of mice including senescence-accelerated mice.

The relationship between voluntary alcohol consumption and brain monoamine levels was studied in the inbred strains of C57BL/6N, C57BL/6J, A/J, BALB/cA, CBA/N, C3H/He and DBA/2cr mice; the congeneric mouse strain, B10.Br/Sg, and the senescence accelerated mouse (SAM P1, SAM P2). The C57BL strains exhibited a high alcohol preference whereas the other strains exhibited a low alcohol preference. A clear positive relationship was found between alcohol intake (g/kg/day) and brain norepinephrine level (r = 0.683, p less than 0.05), and a clear negative relationship between alcohol intake and brain serotonin level (r = -0.628, p less than 0.05). The content of brain dopamine was not clearly correlated with alcohol intake (r = -0.206, p greater than 0.05). These findings suggest that in mice voluntary alcohol preference is influenced by brain norepinephrine and serotonin levels genetically.

Dynamical fluctuation of the mesoscopic structure in ternary C12E5-water-n-octane amphiphilic system.

Dynamical fluctuations of the bicontinuous microemulsion and lamellar structures in ternary C12E5-water-n-octane amphiphilic system are studied by means of neutron spin echo (NSE) spectrometry. The decay rates of the time correlation of the concentration were analyzed in terms of three theories: (1) A. G. Zilman and R. Granek, Phys. Rev. Lett. 77, 4788 (1996), (2) M. Nonomura and T. Ohta, J. Chem. Phys. 110, 7516 (1999), and (3) R. Granek and M. E. Cates, Phys. Rev. A 46, 3319 (1992), in the first of which a Langevin equation for membrane plaquettes and in the latter two of which time-dependent Ginzburg-Landau equations for the order parameters are considered. The result shows that the intermediate correlation functions I(q,t) for the ranges of 0

Fundamental studies on alcohol dependence and disposition.

This article reviews some recent studies on alcohol preference, dependence, metabolism and pharmacokinetics which were mainly carried out in our department. The inbred strains of mice with genetically different alcohol drinking behavior and alcohol animal model treated with the neurotoxins, 6-hydroxydopamine and 5,7-dihydroxytryptamine, are useful for a behavioral and pharmacological approach to evaluate the contribution of specific neural systems to alcohol, drug dependence mechanism and alcohol drinking behavior. The relations between alcohol preference and some physiological conditions are reviewed. On the drug-alcohol interaction, some drugs containing the chemical group = CHONO2, antimony and methamphetamine are addressed. This article also deals with recent topics in the pharmacokinetics and pharmacodynamics of alcohol. The dose-dependency of the alcohol elimination rate, the first-pass metabolism during alcohol drinking, and the pharmacodynamic model for describing pulse rate reaction to plasma acetaldehyde are discussed.

Rapid postmortem changes of rat striatum dopamine, serotonin, and their metabolites as monitored by brain microdialysis.

Brain microdialysis was used to monitor changes in extracellular dopamine (DA), serotonin (5-HT), and their metabolite levels in the rat striatum at death by cervical dislocation. Maximum respective 450-fold and 150-fold increases in the extracellular output of DA and 5-HT were observed within the first 30 min of death. DA and 5-HT outputs remained elevated over the following 2 h at levels about 100-fold and 50-fold above pre-death values, respectively. In contrast with monoamine outputs, the outputs of the DA metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), and the 5-HT metabolite, 5-hydroxyindoleacetic acid (5-HIAA), rapidly decreased by 10% and 20%, respectively 1 h after death. 5-Hydroxytryptophol (5-HTOL) gradually decreased after death. Before death both the extracellular DOPAC/DA and 5-HIAA/5-HT ratios were about 400; after death these ratios dropped to 0.56 and 4.0, respectively at 30 min. These observations suggested that regulation of neurotransmitter releases through the neuronal membrane and metabolisms in the rat striatum were seriously disrupted at death. This finding may be helpful in the determination of death in the field of forensic medicine.

Effects of ion channel blockers on rapid postmortem changes in extracellular dopamine and serotonin levels in the rat nucleus accumbens.

In the present study, we used in vivo brain microdialysis to examine the effects of ion channel blockers tetrodotoxin (TTX), EGTA-free Ca2+ and verapamil on rapid postmortem changes in extracellular levels of dopamine (DA), serotonin (5-HT) and their metabolites dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) in the ACC of freely moving rats. Extracellular ACC DA levels decreased following the perfusion of the three ion channel blockers in freely moving rats, and then, at death by cervical dislocation, maximum respective 220-, 60- and 90-fold increases were observed in the extracellular output of DA in animals treated with EGTA, verapamil and TTX, respectively. Also, ACC 5-HT decreased following perfusion with the three blockers in the freely moving rats, and then maximum increases of 80-, 30- and 45-fold in the extracellular output of 5-HT were observed at death in animals treated with EGTA, verapamil and TTX, respectively, compared to the baseline. Cervical dislocation-induced rapid postmortem changes were inhibited markedly by perfusion with CSF containing the CA2+ entry blocker verapamil. These observations suggested that rapid postmortem changes in ACC DA and 5-HT release were associated with the action of calcium ion channels and/or voltage gated channels in the CNS.

Immunohistochemical investigation of pulmonary surfactant-associated protein A in fatal poisoning.

To evaluate the immunohistochemical distribution of pulmonary surfactant-associated protein A (SP-A) in fatal poisoning in relation to the effects of drugs and poisons on respiratory function, 42 forensic autopsy cases were examined by scoring the staining intensity. The highest scores of SP-A staining, with dense granular deposits (aggregates) in the intra-alveolar space, were observed in fatalities from pancuronium bromide (muscle relaxant) injection and petroleum (butane) gas inhalation. Poisoning with organophosphate pesticides and arsenic (ingestion) showed a second grade SP-A score. However, The SP-A scores were relatively low in ethanol and sedative-hypnotic intoxication. Carbon monoxide intoxication showed a varied degree of SP-A score, and the aggregated SP-A score tended to be higher in cases of lower blood carboxyhemoglobin concentration. A varied SP-A score was also observed in methamphetamine fatalities, in which the score was relatively low in cases with a higher serum drug level. Increase of SP-A was not always associated with the intra-alveolar effusion or hemorrhages. The above-described observations suggested that the immunohistochemical score of SP-A may be a possible indication for intensity and duration of drug/poison-dependent respiratory distress.

Effects of repeated cocaine administration on alcohol consumption.

OBJECTIVE: Alcohol consumption (alcohol preference or alcohol intake) in animals is an index of human drinking behavior. Cocaine is the most frequently abused drug at present. Therefore, an increasing number of cases demonstrating concurrent use of alcohol and cocaine is being noted. We examined whether cocaine affects alcohol consumption and studied the mechanism of change in alcohol consumption following cocaine administration. METHOD: We measured alcohol consumption in inbred mice, C57BL/6J and C3H/HeJ, when 10 or 50 mg/kg cocaine was administered intraperitoneally once a day for 1 week. Then the rate of blood ethanol disappearance from C57BL/6J mice in vivo was measured. Also, liver alcohol dehydrogenase(ADH) and aldehyde dehydrogenase(ALDH) activity in vitro were measured in the C57BL/6J mice. RESULTS: Following 50 mg/kg cocaine administration, alcohol consumption was reduced in C57BL/6J mice, but there was no clear change in C3H/HeJ mice. The rate of blood ethanol disappearance was not changed by pretreatment with cocaine. Neither liver ADH nor ALDH activity was changed by repeated cocaine administration. CONCLUSIONS: The present study showed that repeated cocaine administration decreased alcohol consumption in C57BL/6J mice without altering the metabolism of ethanol.