The effect of age on the expression of mineralized tissue progenitors in the periodontium--the effect of bFGF.
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Biomedical subjects
Publications and source records attributed to H W Liu.
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In an effort to characterize the diversity of mechanisms involved in cellular self-protection against the antitumor antibiotic mitomycin C (MC), DNA fragments from the producing organism (Streptomyces lavendulae) were introduced into Streptomyces lividans and transformants were selected for resistance to the drug. Subcloning of a 4.0-kb BclI fragment revealed the presence of an MC resistance determinant, mrd. Nucleotide sequence analysis identified an open reading frame consisting of 130 amino acids with a predicted molecular weight of 14,364. Transcriptional analysis revealed that mrd is expressed constitutively, with increased transcription in the presence of MC. Expression of mrd in Escherichia coli resulted in the synthesis of a soluble protein with an Mr of 14,400 that conferred high-level cellular resistance to MC and a series of structurally related natural products. Purified MRD was shown to function as a drug-binding protein that provides protection against cross-linking of DNA by preventing reductive activation of MC.
Nitric oxide (NO) is a short-lived free radical that is secreted by pulmonary macrophages (Mø). An inducible isoform of NO synthase (iNOS) catalyses the production of NO and is activated by lipopolysaccharide and certain T-helper(h) 1 cytokines, including interferon-gamma and TNF-alpha. In the present study, iNOS+ interstitial cells were demonstrated in the alveolar wall of normal Lewis rat lung. Enzymatic digests of normal lung showed that approximately one third of pulmonary ED1+ interstitial Mø (IM) were iNOS+ and secreted modest amounts of NO without ex vivo stimulation, whereas normal alveolar macrophages (AM) were iNOS- and showed no basal NO secretion. When incubated with heat-killed Listeria monocytogenes (HKL) in vitro, AM secreted larger amounts of NO than did IM. Recombinant murine GM-CSF stimulated production of NO by AM but not by IM. However, when IM were costimulated with GM-CSF and IFN-gamma, they expressed a marked increase in NO production. Intratracheal challenge with HKL yielded decreased NO production by IM. We conclude that iNOS+ IM are present in normal rat lung, where they regulate the pulmonary cell-mediated immune response to antigen.
The periodontal ligament (PDL) is a fibrous and cellular connective tissue that mediates tooth attachment to bone, and it comprises fibroblastic and mineralized tissue-forming (MTF) progenitors. The MTF progenitors are believed to give rise to the cementoblastic and osteoblastic lineages. Cementum attachment protein (CAP) is a collagenous cementum-derived protein which binds strongly to osteoblasts, moderately to PDL cells, and weakly to gingival fibroblasts. The aim of the present study was to determine the relationship between the capacity of PDL progenitors to bind CAP and their potential to express alkaline phosphatase (ALP) and form mineralized-like tissue in culture. Cloned human PDL progenitor populations obtained from nine human donors were assayed for their constitutive capacity to bind CAP and express ALP, and for the dexamethasone-induced potential to form mineralized-like tissue in culture in the presence of ascorbic acid and beta-glycerophosphate. Forty percent of the progenitor clones produced mineralized-like tissue. Two patterns of mineralization were observed: a spread and flat pattern similar to that produced by human bone cells in culture and a nodular ridge-like type resembling that formed by human cementoma-derived cells. A direct correlation was found between the percentage of ALP positive cells in each progenitor clone and the amount of mineralized-like tissue formed (r = 0.565). Similar correlations were found between the number of ALP positive cells and the binding capacity of each clone (r = 0.392) and between the CAP binding capacity and mineralized-like tissue formation (r = 0.584). Multiple regression analysis indicated that the constitutive capacity of a clone to bind CAP and express ALP is directly correlated to its dexamethasone-induced potential to form mineralized tissue (r = 0.675). These results indicate that CAP binding and ALP expression can serve as markers for the identification of MTF progenitors in the heterogeneous cultured population of the human periodontal ligament. These data show for the first time that binding capacity to extracellular components of mineralized tissues can be a marker for mineralized tissue-forming progenitors.
A convenient method for the quantitative analysis of adenosine and thymidine in Fritillaria bulbs was developed by means of dual wavelength ultraviolet spectrophotometry. This method has good linear relationship and the interrelated coefficient of standard curve for adenosine and thymidine were all found to be 0.9999. The methanol extracts of four species of Fritillaria have been analyzed with this method. The results show that the the bulbs of four species contain different quantities of adenosine and thymidine, which indicates that the nucleosides may be responsible for the anti-coagulation activity of Fritillaria.
OBJECTIVE: To investigate the relation of HLA-DMA and DMB alleles to the development of rheumatoid arthritis (RA). METHODS: We studied HLA-DMA and DMB alleles in 153 patients with RA and 138 healthy controls using the amplification created restriction site method and digestion with restriction enzymes. RESULTS: There was no significant difference in frequencies of HLA-DMA and DMB alleles between patients with RA and controls. The HLA-DMA and DMB alleles were not related to extraarticular involvement, seropositivity for rheumatoid factor, or bone erosion. There was also no significant difference in frequencies of HLA-DMA and DMB alleles among DR4-positive and DR4-negative patients and controls. CONCLUSION: HLA-DMA and DMB do not appear to be associated with the pathogenesis of RA.
CDP-6-deoxy-L-threo-D-glycero-4-hexulose 3-dehydrase (E1), together with its reductase (E3), catalyzes a novel deoxygenation reaction essential for the biosynthesis of 3,6-dideoxyhexoses. In an attempt to gain evidence substantiating the E1.E3 complex formation as a prerequisite for the C-3 deoxygenation activity, we have carried out experiments to study the interaction between these two proteins. The detection of a new species when a mixture of E1 and E3 was analyzed by size-exclusion chromatography was the initial indication supporting the proposed complex formation. Additional evidence for the expected complex formation was provided by the change of the CD spectrum of E1 upon its coupling with E3. The fact that the catalytic efficiency of this system is limited by the quantity of one enzyme, which becomes catalytically competent only after coupling with the second enzyme, further illustrated the importance of such a complex formation to the deoxygenation activity. By using the two-hybrid system which scores for interactions between two proteins coexpressed in yeast, the E1.E3 complex formation in vivo was also firmly established. These results, when considered with the incompatibility of other electron transfer proteins as replacements for E3 in this electron relay, nicely demonstrated the specificity of the E1-E3 recognition. The apparent dissociation constant of the E1.E3 complex formed in rapid equilibrium was estimated to be 288 +/- 22 nM from the correlation between the initial rate of the overall reaction and the concentration of one protein component, and the stoichiometry between E3 and E1 of this complex was deduced as 1.7. Interestingly, while the conformation of the E1.E3 complex was sensitive to the salt concentration in the buffer, the decrease in the catalytic activity at high ionic strength was most likely due to the retardation of the electron transfer mediated by E3. In conjunction with early mechanistic studies, the present data establish the significance of the E1.E3 complex formation for catalysis and, consequently, corroborate the mechanism proposed for the overall deoxygenation process.
The lipopolysaccharide of Yersinia pseudotuberculosis V includes a 3,6-dideoxyhexose, ascarylose, as the nonreducing end of the O-antigen tetrasaccharide. The C-3 deoxygenation of CDP-6-deoxy-L-threo-D-glycero-4-hexulose is a critical reaction in the biosynthesis of ascarylose. The first half of the reaction is a dehydration catalyzed by CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1), which is PMP-dependent and contains a redox-active [2Fe-2S] center. The second half is a reduction that requires an additional enzyme, CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase (E3, formerly known as CDP-6-deoxy-delta 3,4-glucoseen reductase), which has a FAD and a [2Fe-2S] center in the active site. Using NADH as the reductant in the coupled E1-E3 reaction, we have monitored the kinetics of a radical intermediate using both stopped-flow spectrophotometry and rapid freeze-quench EPR under aerobic and hypoxic conditions. In the EPR studies, a sharp signal at g = 2.003 was found to appear at a rate which is kinetically competent, reaching its maximum intensity at approximately 150 ms. Stopped-flow UV-vis analysis of the reaction elucidated a minimum of six optically distinguishable states in the mechanism of electron transfer from NADH to substrate. Interestingly, one of the detected intermediates has a time course nearly identical to that of the radical detected by rapid freeze-quench EPR. The difference UV-vis spectrum of this intermediate displays a maximum at 456 nm with a shoulder at 425 nm. Overall, these results are consistent with an electron transfer pathway that includes a radical intermediate with the unpaired spin localized on the substrate-cofactor complex. Evidence in support of this mechanism is presented in this report. These studies add the PMP-glucoseen radical to the growing list of mechanistically important bioorganic radical intermediates that have recently been discovered.
The conversion of CDP-4-keto-6-deoxy-D-glucose to CDP-4-keto-3,6-dideoxy-D-glucose is a key step in biosynthesis of ascarylose, the terminal dideoxyhexose of the O-antigen tetrasaccharide of the lipopolysaccharide from Yersinia pseudotuberculosis V. This transformation is catalyzed by two enzymes: CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1), which contains a pyridoxamine and a [2Fe-2S] center, and an NADH-dependent CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase (E3), which contains both an FAD and a [2Fe-2S] center. E1 reacts to form a Schiff base with CDP-4-keto-6-deoxy-D-glucose and catalyzes the elimination of the hydroxyl at position 3 of the glucose moiety, resulting in the formation of a covalently bound CDP-6-deoxy-delta(3,4)-glucoseen intermediate. E3 transfers electrons from NADH to E1, which uses these to reduce the delta(3,4)-glucoseen bond to produce CDP-4-keto-3,6-dideoxy-D-glucose. In this work, we have investigated the reductive half-reaction of E3 using both single wavelength and diode array stopped flow absorbance spectroscopy. We find that NADH binds to both oxidized (Kd = 52.5 +/- 2 microM) and two-electron-reduced (Kd = 12.1 +/- 1 microM) forms of E3. Hydride transfer from NADH to the FAD moiety occurs at 107.5 +/- 3 s-1 and exhibits a 10-fold deuterium isotope effect when (4R)-[2H]NADH is substituted for NADH. Following the hydride transfer reaction, NAD+ is released at 42.5 +/- 1 s-1 and electron transfer from the reduced FAD to the [2Fe-2S] center occurs rapidly. The extent of the intramolecular electron transfer reaction is pH-dependent with a pKa of 7.3 +/- 0.1, which may represent the ionization state of the N-1 position of the FAD hydroquinone of E3. Finally, E3 is converted to the three-electron-reduced state in a slow disproportionation reaction that consumes NADH: The [2Fe-2S] center of E3 was selectively disassembled by titration with mersalyl to give E3(apoFeS). The properties of this form of the enzyme are compared to those of the holoenzyme. Similarities and differences of the reductive half-reactions of E3 and related iron-sulfur flavoenzymes are discussed.
Studies of the biosynthesis of ascarylose, a 3,6-dideoxyhexose found in the lipopolysaccharide of Yersinia pseudotuberculosis V, have shown that the C-3 deoxygenation is a process consisting of two enzymatic steps. The first enzyme involved in this transformation is CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1), which is a pyridoxamine 5'-phosphate dependent iron-sulfur protein. The second catalyst, CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase, formally called CDP-6-deoxy-delta(3,4)-glucoseen reductase (E3), is an NADH dependent plant type [2Fe-2S] containing flavoenzyme. To better understand the electron transfer carried out by these two enzymes, the potentials of the E1 and E3 redox cofactors were determined spectroelectrochemically. At pH 7.5, the midpoint potential of the E3 FAD was found to be -212 mV, with the FADox/FADsq couple (E1o') and the FADsq/FADhq couple (E2o') calculated to be -231 and -192 mV, respectively. However, the E1o' and E2o' of the FAD in E3(apoFeS) at pH 7.5 were estimated to be -215 and -240 mV, respectively, which are quite different from those of the holo-E3, suggesting a significant effect of the iron-sulfur center on the redox properties of the flavin coenzyme. Our data also showed that the midpoint potential of the E3 iron-sulfur is -257 mV and that of the E1 [2Fe-2S] center is -209 mV. These values indicated a thermodynamic barrier to the proposed electron transfer of NADH->FAD=>E3[2Fe-2S]->E1[2Fe-2S] at pH 7.5. Regulation of electron transfer by several mechanisms is possible and experiments were performed to examine ways of overcoming the unfavorable electron transfer energetics in the E1/E3 system. It was found that both binding of E3 with NAD+ and complex formation between E3 and E1 showed no effect on the midpoint potentials of the E3 FAD and iron-sulfur center. Interestingly, the midpoint potential of the E3 FAD shifts dramatically to -273 mV (E1o' approximately -345 mV and E2o' approximately -200 mV) at pH 8.4, with very little semiquinone stabilization (< 5%). The potential of the E3 [2Fe-2S] center at pH 8.4 was also found to undergo a negative shift to -279 mV, and that of the E1 iron sulfur center remained essentially the same at -206 mV. These data indicated that the redox properties of this system may be regulated by pH and the electron transfer between the E3 redox centers may be prototropically controlled. These results also demonstrated that E3 is unique among this class of enzymes.
Increased plasma osmolality results in increased central as well as peripheral release of vasopressin. Experiments were carried out to determine whether, in this circumstance, vasopressin can act centrally to modulate its peripheral release. Prior to the start of a thirty-min i.v. infusion of 2.5 M or 0.15 M NaCl, the rats were given an intracerebroventricular (i.c.v.) injection of a peptide V1/V2 vasopressin antagonist (2 micrograms), OPC-31260 (60 micrograms), a non-peptide V2 antagonist, or 1-desamino-8-D-arginine vasopressin (dDAVP, 5 ng), a V2 agonist. Experiments with the peptide antagonist were carried out in male and non-estrous female rats. Since there were no differences between males and females in the measured responses, experiments with the other two drugs were carried out only in males. Pretreatment with either the V1/V2 antagonist or the V2 antagonist enhanced the increase in plasma vasopressin levels in response to the hypertonic saline infusion by about 50% at the end of 30 min. dDAVP, on the other hand, had no effect. None of the i.c.v. drugs had an affect on either the pressor or bradycardic responses to hypertonic saline infusion. These observations suggest that vasopressin can act centrally in a negative feedback fashion to attenuate its own release into the peripheral circulation in response to increased plasma osmolality.
NAD+-dependent nucleotidyl diphosphohexose 4,6-dehydratases which transform nucleotidyl diphosphohexoses into corresponding 4-keto-6-deoxy sugar derivatives are essential to the formation of all 6-deoxyhexoses. Studies of the CDP-D-glucose 4,6-dehydratase (Eod) from Yersinia had shown that this dimeric protein binds only 1 equiv of NAD+/mol of enzyme and, unlike other enzymes of the same class, displays a unique NAD+ requirement for full catalytic activity. Analysis of the primary sequence revealed an extended ADP-binding fold (GHTGFKG) which deviates from the common Rossman consensus (GXGXXG) and thus may have contributed to Eod's limited NAD+ affinity. In particular, the presence of His17 in the beta-turn region and that of Lys21 in a position typically occupied by a small hydrophobic residue may impose electronic or steric perturbations to this essential binding motif. To better understand the correlation between the binding properties and primary sequence, mutants (H17G and K21I) were constructed to provide enzymes containing an ADP binding region which more closely resembles the Rossman-type fold. Analysis of the cofactor and substrate binding characteristics of the wild-type and mutant enzymes helped define the presence of two binding sites for both CDP-d_glucose and NAD+ per enzyme molecule. While both mutants displayed enhanced NAD+ affinity, the H17G mutation resulted in an enzyme with slightly higher kcat and a 3-fold increase in catalytic efficiency (kcat/Km). The large anticooperativity found for NAD+ binding (K1=40.3 + or - 0.4 nM, K2=539.8 + or - 4.8 nM) may explain why the cofactor binding sites of wild-type Eod are only half-occupied. Further examination also revealed the purified Eod to contain sequestered NADH and that the affinity of Eod for NADH(K1=0.21 + or - 0.01 nM, K2= 7.46 + or -0.25 nM) is much higher than that for NAD+. Thus, it is possible that Eod's half-site saturation of NAD+ per enzyme dimer may also be attributed to a significant portion of the cofactor binding sites being occupied by NADH. Interestingly, the sequestered NADH is released upon binding with CDP-D-glucose. These results implicate a new kinetic mechanism for Eod catalysis.
Tadpoles of Rana catesbeiana at TK stages X-XII, about 9 months old, were laparotomized, and the males were implanted intraperitoneally with silastic tubes containing estradiol (E2) for various periods. Male tadpoles implanted with empty tubes served as the controls. Histology, secretions of E2, and testosterone (T) in the gonads were investigated. A rough estimate of estradiol released from silastic tubes suggested that about 90 micrograms per tadpole in 6 months. Histological observation revealed various degrees of transformation from testes toward ovaries in E2-treated testes. Ten in thirteen (77%), the testes were transformed into ovaries 6 months after the treatment. The testes of the controls, however, displayed normal histology. Radioimmunoassay showed that E2 level was increased while T level was decreased in E2-treated testes. These results indicate that a low dose of exogenous E2 may induce transformation of the testes into ovaries.
The fifth child of a Hong Kong Chinese mother developed moderate jaundice, attributable to antibodies (anti-Mi) against antigenic determinants in GP.Mur (Miltenberger, class III) red cells. Both the father and the eldest sister were of the phenotype GP.Mur. Testing of maternal serum against a red cell panel including cells known to carry the antigenic determinants of some Miltenberger phenotypes revealed the presence of anti-Mur. This report documents the first case of haemolytic disease of the newborn (HDN) due to anti-Mur in Hong Kong.
A series of xanthone derivatives was synthesized and tested in-vitro for their ability to inhibit aggregation of rabbit washed platelets and human platelet-rich plasma (PRP) induced by various inducers. 2-Prenyloxyxanthone showed the most potent inhibition of rabbit washed platelet aggregation induced by arachidonic acid (1C50 = 10.2 microM). Of the compounds tested in human PRP, 2-[3 (propylamino)-2-hydroxypropoxy]xanthone (4) hydrochloride salt exhibited the most potent inhibition of platelet aggregation induced by adrenaline (IC50 = 4.4 microM), whereas in evaluation of mouse antithrombotic activity, compound 4 exhibited the most potent protection of mice from thrombotic challenge. Compound 4, 2-[3-(isopropylamino)-2-hydroxypropoxylxanthone hydrochloride salt and 2,5 dihydroxyxanthone suppressed the secondary aggregation induced by adrenaline in human PRP. We conclude that the antiplatelet effects of these compounds are mainly due to an inhibitory effect on thromboxane formation.
The GP.Mur (Miltenberger class III) phenotype was found to occur in about 6.3 percent of Hong Kong (HK) Chinese blood donors. The incidence of antibodies directed against antigenic determinants of GP.Mur cells (anti-Mi) among patients was 0.34 percent, similar to that in Taiwan Chinese. A case of hydrops fetalis probably attributable to maternal anti-Mi was encountered in an HK Chinese woman during her sixth pregnancy. The anti-Mi was potent (titer 512, score 99). It fixed complement and was a mixture of IgG1 and IgG3. Two biological assays, the monocyte monolayer assay and the chemiluminescence test, were strongly positive. The father was found to be heterozygous for the GP.Mur gene.
Dendritic cells (DCs) have emerged as the dominant antigen-presenting cells (APCs) of the lung, playing a vital role in the induction of cell-mediated immunity to inhaled antigens. We have previously demonstrated that an airway challenge with the soluble antigen hen egg lysozyme yields rapid acquisition of specific antigen-presenting cell activity by purified pulmonary DCs and a cell-mediated immune response in the lung upon secondary challenge. To examine how a particulate antigen leads to a cell-mediated response in vivo, graded concentrations of heat-killed Listeria (HKL) were injected intratracheally into Lewis rats. The bacteria were rapidly ingested by lung macrophages and polymorphonuclear leukocytes. The ability of purified pulmonary DCs pulsed in vivo by an airway challenge with HKL to subsequently stimulate HKL-specific responses ex vivo showed a threshold response, requiring a dose in excess of 10(9) organisms/rat. By contrast, all dosages of HKL yielded specific sensitization of lymphocytes in the draining bilar nodes. Pulmonary DCs purified from rats after a secondary in vivo airway challenge with HKL at day 14 were ineffective antigen-presenting cells except at high dosages of antigen. The generation of cell-mediated pulmonary inflammation paralleled the antigen-presenting cell activity of pulmonary DCs and was observed only at high antigen dosages. Hen egg lysozyme immobilized onto polystyrene beads and injected intratracheally yielded comparable results to those observed with HKL. We suggest that a pulmonary cellular immune response is generated to an inhaled particulate antigen when the protective phagocytic capacities of the lung are exceeded and antigen is able to interact directly with interstitial DCs. The diversion of particulate antigens by pulmonary phagocytes may help to limit undesirable pulmonary inflammation while allowing the generation of antigen-specific immune lymphocytes in vivo.
The purpose of this study was to investigate the prevalence of hypercholesterolemia among subjects having diabetes and glucose intolerance, according to the guidelines of the National Cholesterol Education Program (Adult Treatment Panel II, ATP II). This survey consisted of 2090 subjects (856 men, 1234 women) aged 30 years or more from the Sun-Ming district of Kaohsiung city. Glucose tolerance status was ascertained for both medical history and a 75-g oral glucose tolerance test according to World Health Organization criteria. Frequency of elevated total cholesterol in female subjects with abnormal glucose tolerance is significantly greater than in those with normal glucose tolerance (NGT). However, only male subjects with undiagnosed NIDDM (UDDM) had a statistically higher rate of hypercholesterolemia than those with NGT. Of UDDM individuals, 68% have total cholesterol level between 200 and 239 mg/dl and two or more risk factors for heart disease or evidence of coronary heart disease or total cholesterol > or = 240 mg/dl or high-density lipoprotein (HDL) cholesterol < or = 35 mg/dl. Such individuals should have their low-density lipoprotein (LDL) cholesterol measured. Using the ATP II, LDL cholesterol levels warranting dietary treatment for hypercholesterolemia would be expected in 76% of UDDM. Due to the high prevalence of coronary heart disease in diabetic patients, investigation of blood lipid levels and coronary heart disease risk factors should be routine in these patients, and treatment strategies should include management of lipid disorders and the many other risk factors. A high frequency of dyslipidemia was found among UDDM group in our study. Early detection of undiagnosed diabetic patients is also very important in decreasing the prevalence of coronary heart disease.