Biomedical subjects

D Jin

Publications and source records attributed to D Jin.

At least 37 records · Page 2Linked to original sources

PubMed2000

Identification of a Drosophila vitamin K-dependent gamma-glutamyl carboxylase.

Using reduced vitamin K, oxygen, and carbon dioxide, gamma-glutamyl carboxylase post-translationally modifies certain glutamates by adding carbon dioxide to the gamma position of those amino acids. In vertebrates, the modification of glutamate residues of target proteins is facilitated by an interaction between a propeptide present on target proteins and the gamma-glutamyl carboxylase. Previously, the gastropod Conus was the only known invertebrate with a demonstrated vitamin K-dependent carboxylase. We report here the discovery of a gamma-glutamyl carboxylase in Drosophila. This Drosophila enzyme is remarkably similar in amino acid sequence to the known mammalian carboxylases; it has 33% sequence identity and 45% sequence similarity to human gamma-glutamyl carboxylase. The Drosophila carboxylase is vitamin K-dependent, and it has a K(m) toward a model pentapeptide substrate, FLEEL, of about 4 mm. However, unlike the human gamma-glutamyl carboxylase, it is not stimulated by human blood coagulation factor IX propeptides. We found the mRNA for Drosophila gamma-glutamyl carboxylase in virtually every embryonic and adult stage that we investigated, with the highest concentration evident in the adult head.

Adult↗

PubMed2000

Expression of bone morphogenetic protein receptors type-IA, -IB and -II correlates with tumor grade in human prostate cancer tissues.

Bone morphogenetic proteins (BMPs) are potential regulators of prostate cancer cell growth and metastasis that signal through an interaction with BMP membrane receptors (BMPRs) type I and type II. In the present study, Western blot and immunohistochemical analysis of BMPRs were carried out in benign and malignant human prostate tissues to explain the loss of BMP response in human prostate cancer cells. The results demonstrated that the benign prostate specimens expressed high levels of all three BMPRs. In normal prostate, BMPRs were localized predominantly to epithelial cells. Among prostate cancer specimens, well-differentiated cancers were positive for the expression of BMPR-II, BMPR-IA, and BMPR-IB, for the most part. In contrast, only 1 of 10 poorly differentiated prostate cancer cases was positive for each of the three BMPRs (P < 0.005 for all three receptors). Taken together, these results indicate that human prostate cancer cells frequently exhibit loss of expression of BMPRs and suggest that loss of BMPRs may play an important role during the progression of prostate cancer.

Blotting, Western↗

PubMed2000

Inhibition of chymase reduces vascular proliferation in dog grafted veins.

We investigated the effect of a chymase inhibitor Suc-Val-Pro-Phe(P)(OPh)(2) on the proliferation of the grafted vein in dog. By 28 days after the operation, the mean intimal area of the grafted vein in the placebo group was 3.24+/-0.32 mm(2). The intimal area of the grafted vein in the chymase inhibitor-treated group was reduced to 63.9%. In the placebo group, the activities of chymase and angiotensin-converting enzyme in grafted vein were significantly increased 15- and 2-fold, respectively. In the chymase inhibitor-treated group, chymase activity in the grafted veins was decreased significantly. These findings suggest that inhibition of chymase appears useful for preventing vascular proliferation.

Animals↗

PubMed2000

Platelet-derived growth factor (PDGF)-AA: a self-imposed cytokine in the proliferation of human fetal osteoblasts.

PDGF, a multiple mitogen, stimulates osteoblast replication and bone collagen degradation, and it has been described as a key factor in bone metabolism. Recently, PDGF was disclosed to be the autocrine cytokine of human osteoblasts. PDGF's effect on human bone development needs elucidation, and the mechanism of PDGF-AA autocrining remains unclear. In this work, osteoblasts were isolated from human fetal calvaria which resemble the cells in developing bone tissue. Using an in vitro model, the effect of PDGF-AA on the cells was investigated with the results showing that (i) after PDGF-AA was added into the medium for 24 h, PCI of the cells increased by 48.2% (P<0.05), and PCI decreased after the removal of PDGF-AA. (ii) 24 h after treatment with PDGF-AA, the percentage of osteoblasts in G(0)/G(1)phase decreased and that in S phase increased. (iii) The expression of PDGF-A mRNA was upregulated by PDGF-AA, PDGF-BB, TGFbeta,and bFGF respectively. These results proved that: (i) PDGF-AA enhances cell replication by accelerating cell recycle and inducing the quiescent cells into the proliferation portion of cell cycle. (ii) PDGF-AA is a self-imposed cytokine in human fetal osteoblast replication.

Bone Development↗

PubMed2000

A new method for simultaneous measurements of mast cell proteases in human vascular tissue.

1. Human mast cells contain carboxypeptidase A, chymase and tryptase. In the present study, in order to analyse the mast cell proteases simultaneously, we investigated a method for the measurement of carboxypeptidase A, chymase and tryptase in human vascular tissues. 2. Human vascular tissues were homogenized in 10 mmol/L phosphate buffer containing 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 or 1.8 mol/L KCl and 0.1% non-idet P-40 and samples were then extracted. Because carboxypeptidase A and chymase convert angiotensin (Ang)I to Ang-(1-9) and AngII, respectively, the extracts were incubated with AngI in the presence of an angiotensin-converting enzyme (ACE) inhibitor. The extract prepared in buffer with over 0.8 mol/L KCl converted AngI to Ang-(1-9) and AngII. Formation of Ang-(1-9) and AngII plateaued in extracts with 1.0 and 1.2 mol/L KCl, respectively. 3. The formation of Ang-(1-9) and AngII in the extract with 1.2 mol/L KCl was inhibited by inhibitors of carboxypeptidase A and chymase, respectively, suggesting that Ang-(1-9) and AngII were generated from AngI by carboxypeptidase A and chymase, respectively. 4. Using a specific tryptase substrate, tryptase activity was detected in extract in buffer with over 0.8 mol/L KCl and reached a plateau at concentrations of KCl over 1.0 mol/L. 5. These findings show that the maximum activity of carboxypeptidase A, chymase and tryptase was detected in extracts of human homogenized vascular tissues in buffer at 1.2 mol/L KCl. The present study demonstrates a new method for the simultaneous measurement of proteases derived from mast cells in humans.

Angiotensin I↗

PubMed2000

Characterization of recombinant human chymase expressed in Escherichia coli.

We compared recombinant human chymase expressed in Escherichia coli with human chymase purified from vascular tissues. The recombinant chymase, the structure of which was NH2-enterokinase cleavage site-chymase-COOH, was expressed in Escherichia coli and then was solubilized and renatured. The protein did not have a chymase activity, but gained this activity after the cleavage of the N-terminal site by enterokinase. The enzyme was purified by heparin affinity and gel filtration columns. The N-terminal sequence of the protein was identical to the sequence for human chymase. The molecular weights of the recombinant chymase and chymase purified from human vascular tissues were 26 and 30 kDa, respectively, and the 4 kDa difference was thought to be due to the presence or absence of glycan. The optimum pH of the recombinant enzyme activity was between 7.5 and 9.0. The activity of the recombinant enzyme was inhibited by chymostatin, soybean trypsin inhibitor and phenylmethylsulfonyl fluoride, but not by ethylenediaminetetraacetic acid and aprotinin. This enzyme cleaved specifically the Phe8-His9 bond of angiotensin (Ang) I to form Ang II and that of big endothelin (ET)-1 to form ET-1-(1-31). These findings demonstrated that the enzymatic characteristics of the recombinant enzyme were identical to that of native human chymase.

Amino Acid Sequence↗

PubMed2000

Characteristics of monkey tryptase purified from cheek pouch vascular tissues.

Tryptase purified from rat and dog tissues has been reported, although the characteristics of these enzymes are different from human tryptase. For pathophysiological studies of human tryptase, studies on species that have a similar tryptase to humans is needed. In this study, we purified monkey tryptase from cheek pouch vascular tissues using heparin affinity and gel filtration columns. The monkey tryptase, which had a molecular weight of 130 kDa by gel filtration, consisted of a tetramer of 33 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The N-terminal sequence showed high homology with tryptases from other species. The optimum pH and temperature were 7.5-9.0 and 25-40 degrees C, respectively. The enzyme was labile in high-KCl buffer, and the optimum KCl concentration was 0.1 M. The enzyme activity was completely inhibited by diisopropyl phosphorofluoridate and leupeptin but not by soybean trypsin inhibitor and alpha-antitrypsin. The enzyme hydrolyzed vasoactive intestinal peptide but did not affect angiotensin I, somatostatin and bradykinin. In the present study, we first isolated monkey tryptase from cheek pouch vascular tissues and showed that the characteristics of monkey tryptase are very similar to those of human tryptase.

Animals↗

PubMed2000

The functional ratio of chymase and angiotensin converting enzyme in angiotensin I-induced vascular contraction in monkeys, dogs and rats.

Recently, a chymase-dependent angiotensin (Ang) II-forming pathway was found in human cardiovascular tissues, and the significance of this pathway in the pathogenesis of some cardiovascular diseases was suggested. The present study examined the ratio of angiotensin converting enzyme (ACE) to chymase-dependent Ang II formation in various isolated vessels from monkeys, dogs and rats. In all of the examined vessels, the addition of KCl at a concentration of 50 mM could induce a maximal contraction. Except for monkey coronary artery and rat renal and femoral artery, the addition of Ang I could induce transitory contractions, whereas the force of contractions in these vessels was quite different. The sensitivity to Ang II in these vessels was similar to that for Ang I. In monkey gastroepiploic and mesenteric arteries, about 70% of the Ang I-induced contraction was suppressed by chymase inhibition, while it was suppressed about 50% in monkey renal, femoral and carotid arteries. In dog renal arteries, about 65% of the Ang I-induced contraction was suppressed by chymase inhibition, while it was suppressed by about 30% in other dog arteries. In contrast, in all rat arteries, Ang I-induced contractions were completely suppressed by treatment with ACE inhibitor alone. We concluded that regional differences in the response to Ang I exist in vascular tissues, and the ratio of ACE- to chymase-dependent Ang II formation is different in the various vessels.

Angiotensin I↗

PubMed2000

Modulation of transforming growth factor beta to platelet-derived growth factor receptor-alpha of human osteoblasts.

OBJECTIVE: To investigate the mechanism and the significance of TGF beta in modulating the expression of Platelet-Derived Growth Factor Receptor-alpha (PDGFR-alpha) in human osteoblasts. METHODS: The osteoblasts were isolated from human fetal calvaria. The percentage of cell increase (PCI) in every 4 hours was calculated to demonstrate the proliferation of osteoblasts affected by PDGF-AA and TGF beta. The osteoblasts were cultured with TGF beta for 24 hours and with PDGF-AA for another 24 hours, and the cells proliferation was shown by PCI too. The osteoblasts were cultured with TGF beta for 24 h, and the PDGFR-alpha of the cells were measured by immunofluorescent analysis. RESULTS: PCI was increased by 48.2% and 22.4% after PDGF-AA and TGF beta were added into the medium for 24 hours respectively (P < 0.05), and PCI decreased after the removal of the two cytokines. Preincubated with TGF beta for 24 hours and then stimulated with PDGF-AA, PCI grew slowly. TGF beta downregulated the expression of the PDGFR-alpha. CONCLUSION: TGF beta can downregulate the mitogenesis of PDGF-AA by lowering the number of PDGFR-alpha.

Cell Division↗

PubMed2000

[The effects of platelet-derived growth factor-AA on the osteoclasts].

OBJECTIVE: To study the effects of platelet-derived growth factor (PDGF)-AA on the osteoclasts. METHODS: The PDGF receptor alpha (PDGFR-alpha) was detected on the osteoclastic membrane with immunoelectronmicroscopy. Acid phosphatase (ACP) and tartrate-resistant acid phosphatase (TRAP) activities were measured kinetically and the introcellular hydrogen ions were calculated on the confocal laser scanning microscope (CLSM) stained with SNAFL. Stained by toluidine blue, the area and number of the resorption pits were determined with the Leica Quantimet 500 system. RESULTS: The colloidal gold precipitated on the cellular membrane, and the TRAP activity had no difference (P > 0.05). Hydrogen ions rose significantly but their release could not be promoted by PDGF-AA. The changes of the resorption pits were not significant. CONCLUSIONS: The PDGF-AA could promote hydrogen ions yielding but not stimulate osteoclastic bone resorption due to the significant TRAP changes of TRAP activities.

Acid Phosphatase↗

PubMed2000

[Mitogenesis of platelet-derived growth factors to human osteoblasts modulated by basic fibroblast growth factor].

OBJECTIVES: To investigate the mechanism by which basic fibroblast growth factor modulates the mitogenesis of platelet-derived growth factor to human osteoblasts. METHODS: The osteoblasts isolated from human fetal calvaria were incubated with PDGF-AB (100 ng/ml) or bFGF(10 ng/ml) combined with PDGF-AB (100 ng/ml); the growth curve was plotted. The(3)H-TdR incorporation of the osteoblasts was measured after the cells were incubated with different combination of bFGF and PDGF-AA or PDGF-BB. After incubated with bFGF (10 ng/ml) for 24 hours, the number of PDGFR-alpha and PDGFR-beta on the membrane of the osteoblasts was detected by fluoroimmunoassay. RESULTS: Four days after PDGF-AB added into the medium, the population of the osteoblasts was larger than that of the control (P < 0.05). The number of the osteoblasts incubated with PDGF-AB (12.1 x 10(4)) was 1.8 times as large as the control (6.8 x 10(4)) in the 10th day (P < 0.05), and that of the osteoblasts incubated with both bFGF and PDGF-AB increased more quickly than the cells only incubated with PDGF-AB. The incorporation of (3)H-TdR into the osteoblasts cultured with bFGF combined with PDGF-AA (533.6 +/- 13.1) was more than that cultured only with PDGF-AA (435.4 +/- 14.8, P < 0.01), so was the incorporation of (3)H-TdR of those cells cultured with bFGF and then PDGF-AA (633.8 +/- 51.5). bFGF up-regulated PDGFR-alpha and down-regulated PDGFR-beta on the surface of the human osteoblasts. CONCLUSION: bFGF elevates the mitogenesis of PDGF-AA or -AB to human osteoblasts by up-regulating PDGFR-alpha.

Cell Proliferation↗

PubMed2000

[Thoracolumbar injuries: pathomorphology of and a new classification].

OBJECTIVE: To introduce a new classification of thoracolumbar injuries. METHODS: Three modes were used to classify the injuries according to the mechanisms of injuries: Type A, flexion-compression fractures; Type B, vertical compression fractures; Type C, distraction injuries. Two groups were used to distinguish the fractures in one type according to the pathomorphological differences and the clinical incidence: Group A, with a more clinical incidence, and Group B, with a less clinical incidence. Three subgroups were used to divide the injuries in one group in consideration of the severity of the fracture instability and the fracture displacement: fracture, subluxation, and dislocation. RESULTS: A new classification of thoracolumbar fractures was established. CONCLUSION: The new classification is more systemic and more convenient.

Humans↗

PubMed2000

[Anterior interbody autografting and internal fixation for primary surgical management of thoracolumbar spinal tuberculosis].

OBJECTIVES: To verify the importance of early reconstruction of spinal stability and to evaluate the safety of anterior instrumentation in surgical management of spinal tuberculosis. METHODS: Eleven cases of thoracolumbar spinal tuberculosis were treated with primary anterior debridement, interbody autografting and Z-plate fixation from October 1997 to January 1999. RESULTS: All the 11 cases healed without any recurrence after follow-up for an average of 16 months. Spinal fusion occurred in 3.8 months on average after surgery. 18 degrees of kyphosis correction was achieved with no associated complication. CONCLUSIONS: Early reconstruction of spinal stability plays an important role in surgical management of spinal tuberculosis, and it is safe to implant anterior titanium spinal instrument in tuberculosis foci.

Adolescent↗

PubMed2000

[Biocompatibility studies on bioactive glass ceramics and polylactic acid combined with cultured bone marrow stromal cells in vitro].

OBJECTIVE: To study the biocompatibility on bioactive glass ceramics (BGC) and polylactic acid (PLA) combined with cultured bone marrow stromal cells (BMSCs) in bone tissue engineering. METHODS: BMSCs were cultured combined with BGC and PLA in vitro, and the morphological characters, cell proliferation, protein content, and alkaline phosphatase activity were detected. RESULTS: BMSCs could be attached to and extended on both BGC and PLA, and normally grown, proliferated, had active function. BGC could promote cell proliferation. CONCLUSION: The results show that both BGC and PLA have good biocompatibility with BMSCs, they can be used as biomaterials for cell transplantation in tissue engineering.

Animals↗

PubMed1999

Chymase-dependent angiotensin II formation in human vascular tissue.

BACKGROUND: Some reports have suggested that, in vitro, human heart chymase in homogenates contributes little to angiotensin (Ang) II formation in the presence of natural protease inhibitors such as alpha-antitrypsin. We studied whether chymase bound to heparin, resembling an in vivo form, could contribute to Ang II formation in the presence of natural protease inhibitors. METHODS AND RESULTS: The Ang II formation was increased time-dependently after incubation in an extract (1 mg of protein/mL) of human vascular tissues containing Ang I. The concentration of Ang II in the extract after incubation for 30 minutes was 1.67+/-0.06 nmol/mL, and we regarded this quantity of Ang II as 100%. The Ang II formation was inhibited 10%, 95%, and 96% by 1 micromol/L lisinopril, 100 micromol/L chymostatin, and 0.1 g/L alpha-antitrypsin, respectively. The extract was applied to a heparin affinity column. After the column was washed with PBS, the eluted PBS contained a weak Ang II-forming activity, which was completely inhibited by lisinopril. The eluted PBS, to which >0.8 mol/L NaCl had been added, showed a strong Ang II-forming activity which was inhibited by chymostatin and alpha-antitrypsin. After the application of the extract, the column was washed with PBS and then an Ang I solution in PBS was applied to the column. The Ang II formation in the PBS eluted from the incubated column was increased time-dependently. The concentration of Ang II in the PBS (1 mL) eluted from the column after incubation for 30 minutes was 2.56+/-0.28 nmol/mL, and we regarded this quantity of Ang II as 100%. To study the effects of inhibitors, the extract (1 mg of protein/mL) was applied to a heparin affinity column (1 mL) which was preequilibrated with PBS (3 mL); 100 micromol/L chymostatin or 0.1 g/L alpha-antitrypsin in PBS (1 mL) was then applied to the column. After the column was washed with PBS (3 mL), Ang I solution (1 mg/mL) in PBS was applied to the column, and the column was incubated for 30 minutes. The Ang II formation in the PBS eluted from the column was suppressed up to 5% by application of chymostatin, although this was not affected by application of alpha-antitrypsin. CONCLUSIONS: These findings suggest that human chymase bound to heparin plays a functional role in Ang II formation in the presence of natural protease inhibitors such as alpha-antitrypsin.

Adult↗

PubMed1999

Osteocalcin binds tightly to the gamma-glutamylcarboxylase at a site distinct from that of the other known vitamin K-dependent proteins.

Vitamin K-dependent proteins contain a propeptide that is required for recognition by the enzyme gamma-glutamylcarboxylase. Substrates used in vitro for carboxylation studies lacking a prosequence are characterized by Km values in the millimolar range, whereas the Km for peptides containing a prosequence is three or four orders of magnitude smaller. Here we report that descarboxy-osteocalcin is an exception in this respect. With descarboxy-osteocalcin in purified propeptide-free recombinant carboxylase, the Km was 1.8 microM. Furthermore, osteocalcin was an inhibitor of descarboxy-osteocalcin carboxylation with a Ki of 76 microM. In contrast with the other vitamin K-dependent proteins, free propeptides do not inhibit descarboxy-osteocalcin carboxylation. Moreover, propeptide-containing substrates were inhibited neither by osteocalcin nor by its propeptide. From our studies we conclude that descarboxy-osteocalcin must have an internal recognition sequence that binds to gamma-glutamylcarboxylase at a site different from the propeptide-recognition site.

Amino Acid Sequence↗

PubMed1999

Determination of D-amino acids labeled with fluorescent chiral reagents, R(-)- and S(+)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazoles, in biological and food samples by liquid chromatography.

D-Amino acids in food and biological samples labeled with R(-)- and S(+)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazoles (DBD-PyNCS) were separated by reversed-phase chromatography and detected fluorometrically at 550 nm (excitation at 460 nm). DL-Amino acids were efficiently labeled at 55 degrees C for 20 min in basic medium. The resulting thiocarbamoyl-amino acids were resolved by an isocratic elution using water:30% methanol in acetonitrile (72:28) containing 0.1% trifluoracetic acid as mobile phase for hydrophilic amino acids and gradient elutions using sodium acetate buffer (pH 5. 2)/acetonitrile as gradient solvent mixture for hydrophobic amino acids, respectively. The detection limits (S/N = 3) of DL-amino acids tested were in the range of 0.16-0.75 pmol. The proposed method was applied to determine the D-amino acid(s) in milk, cream, fermented dairy products (yogurt and yakult), tomato products (juice, puree, and catchup), fermented beverages (beer and red wine), and human urine. The existence of D-amino acid(s) was demonstrated in all the samples tested. Furthermore, the identification of the D-amino acid(s) was performed using both isomers of DBD-PyNCS and by on-line HPLC-electrospray ionization-MS.

Amino Acids↗

PubMed1999

Chromosomal localization of three human genes encoding bone morphogenetic protein receptors.

Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily that play a pivotal role in bone formation during embryogenesis and fracture repair. BMP signaling occurs via hetero-oligomeric serine/threonine kinase complexes of BMP type I (BMPR-IA or BMPR-IB) and type II receptors (BMPR-II). BMPR-IA and IB are closely related receptors, with sequence differences conserved between different species, suggesting that they serve distinct functions. Here we report the cDNA cloning of human BMPR1B and the chromosomal localization of all three BMPR genes. Using somatic cell hybrid and FISH analyses, the BMPR1A, BMPR1B, and BMPR2 genes were assigned to 10q23, 4q22-24, and 2q33-34, respectively. A processed BMPR1A pseudogene was mapped to 6q23.

Bone Morphogenetic Protein Receptors↗