Biomedical subjects

J Dong

Publications and source records attributed to J Dong.

At least 109 records · Page 6Linked to original sources

PubMed2000

[Clinical and experimental study on antiviral activity of reduqing against human cytomegalovirus].

OBJECTIVE: To investigate the therapeutic efficacy of traditional Chinese medicine Reduqing (RDQ) against human cytomegalovirus (HCMV) in clinical and its antiviral activity in vitro. METHODS: In clinical practice, HCMV antibody was detected to determine if the case was activity infected with HCMV. Fourteen patients were found and treated with RDQ. The drug was orally administered one dose, three times a day, for 18-30 days as one therapeutic course. And the efficacy was evaluated by ELISA, PCR and other methods. The in vitro inhibitory activity of RDQ against HCMV AD169 was carried out on human embryo lung fibroblasts (HEL) by cytopathic effect inhibition method. Ganciclovir (GCV) was used for positive control. RESULTS: Fourteen pre- or during pregnant women with HCMV infection were treated with RDQ. After 18-30 days of treatment, all of them showed HCMV-IgM negative conversion, HCMV DNA negative conversion in 7/10 cases, and virus excretion by urine and cervix secretion was inhibited in 4/4 and 1/1 case. Five women gave birth to 5 normal newborns at term after treatment, among them 2 were asymptomatic virus carrier, the other 3 were uninfected. Experimental study in vitro showed that the maximal tolerance dosage (TD0) of RDQ was 20 micrograms/L, the minimal therapeutic concentration (MTC) was 5 micrograms/L, 50% inhibitory concentration (IC50) was 5 micrograms/L and the therapeutic index (TI) was 4. It suggested that RDQ had anti-HCMV activity in vitro and the effect increased with its concentration. CONCLUSION: RDQ is a safe, valuable drug for inhibiting HCMV infection especially during pregnancy.

Adult↗

PubMed2000

[Cloning and sequencing of a Chinese complete TTV gene].

OBJECTIVE: To obtain the complete TT virus gene from a Chinese isolate. METHODS: Long-PCR was performed for amplifying the TTV gene from the serum of a Chinese with non-A to non-G hepatitis. Three overlapped DNA fragments were obtained, which contained 199 bp (1-199), 3 269 bp (38 approximately 3 306) and 434 bp (3 306 approximately 3 739) separately. These PCR products were sequenced by the standard method. RESULTS: The complete TTV gene (named as TTV CHN1) consisting of 3 739 nucleotides contained two ORFs. The first ORF located at 589 approximately 2 901 nt, coding 770 amino acids; the second located at 107 approximately 715 nt, coding 202 amino acids. In this genome, G is 22.25%, C 25.54%, A 31.26% and T 20.97%. The homology of Chinese and Japanese TTV genes was more than 94% at both nucleotide and amino acid levels. There exists a hyper-variation region (HVR) spanning at 1 380 approximately 1 800 bp. In this region, the HVR homology of Chinese and Japanese TTV genes was 88% and 81% at nucleotide and amino acid levels, respectively. CONCLUSION: Chinese and Japanese TTV isolates belong to the same genotype. There exists a hyper-variation region (HVR) spanning at 1 380 approximately 1 800 bp.

Amino Acid Sequence↗

PubMed2000

[Chemical-induced polycyst with renal tumor and expression of 8-OHdG in kidney tissue].

OBJECTIVE: To induce the rat model of polycyst with renal tumor and investigate the expression of 8-OHdG in the kidney tissues. METHODS: The rat model of polycyst with renal tumor, similar to human acquired cystic disease of the kidney (ACDK), was induced by oral administration of 2-amino-4, 5-diphenylthiazole (DPT) and N-nitrosomorpholine (NNM). Immunohistochemical method (LSAB) was used to assay the expression of 8-hydroxydeoxyguanosine (8-OHdG) in the rat kidney tissue. RESULTS: Three of 10 rats in the NNM group had renal solid adenomatous lesions. Bilateral polycysts were observed in all 9 rats of the DPT/NNM group. Seven of the 9 rats had cystic multistage renal tumor. In the DPT/NNM group, 4 rats had cystic adenomatous lesions, but none in the other groups showed this lesion. In the model, adenomatous lesions derived from polycysts in the rats were closely consistent to human ACDK in morphology. The significant expression of 8-OHdG was found on renal tubular cell, cystic epithelial cell, stromal cell and tumor cell in all rats of the NNM and DPT/NNM group. CONCLUSION: A rat model of polycysts with renal tumor, similar to human ACDK, induced by DPT and NNM provides evidences for further study on pathogenic mechanism in human ACDK with renal cell carcinoma. The expression of 8-OHdG, a DNA damage marker, in the renal tissues of rat model might help to explain the mechanism of cysticogenesis and carcinogenesis in human ACDK.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2000

[Repairing peripheral nerve defects by tissue engineering techniques:an experimental study].

OBJECTIVE: To develop a novel effective substitute material or technique to repair peripheral nerve gap. METHODS: We inoculated expanded Schwann cells (SCs) at re-arranged bio-absorbable polymer polyglycolic acid (PGA) fiber and incubated for two weeks, then we developed a novel tissue-engineered scaffolds. The scaffolds were used as cellular isografts to bridge 15 mm long gap of sciatic nerve in inbred strains of Wistar rats. In an autologous and pure PGA fiber control group, the same surgical procedure was used. Evaluation included general observation, electromyographic examination, muscle measurement, and histological observation of serial sections at 12 weeks after surgery. The total number and density of reinnervation and thickness of myelin sheath was measured by computerized image analysis. RESULTS: SCs put out a long and thin prominence and migrated along the PGA fibers in spirality or parallel when they divided and finally rank into a cell-chain formation similar to Büngner's band. TEM/SEM and immunohistochemical survey demonstrated that the SCs at PGA fiber also secreted a great deal of ECMs included laminin, which play a very important role in peripheral nerve regeneration. Non-tubular scaffold comprised SCs and laminin (LN) channel in three-dimensional longitudinal rank. Animal transplantation study indicated the sensory and motor functional results of hindlimbs of experimental group rats reached to similar level of those found in nerve autograft control group. The number of reinnervation in the experimental group rats was slightly fewer than that in nerve autograft control group; but the axonal density was just reversal between the two groups because of a large area ischemic necrosis in the center of nerve autografts. CONCLUSION: This new paradigm offers a potential solution to repairing a long gap of peripheral nerve.

Animals↗

PubMed2000

[Treatment of spondylolisthesis with pedicle screw instrumentation and anterior or posterior lumbar interbody fusion].

OBJECTIVE: To investigate the treatment of spondylolisthesis with pedicle screw instrumentation and anterior or posterior lumbar interbody fusion. METHODS: 26 patients, 14 males and 12 females, underwent spinal instrumentation with RF or SOCON instrumentation and anterior or posterior interbody fusion with TFC. Among them, 6 patients underwent RF instrumentation and posterior interbody fusion with TFC, 15 SOCON instrumentation and posterior interbody fusion with TFC, and 5 RF instrumentation and anterior interbody fusion with composite cortical ring. RESULTS: All the patients were followed up for an average of 22 months (range 6 - 18 months) after operation. The reduction of spondylolisthesis was 88%, and the fusion rate 89%. There was no recurrence of spondylolisthesis. CONCLUSIONS: High quality SOCON instrumentation may be reliable for the treatment of spondylolisthesis. Implantation of TFC into the interbody space can stabilize the fused segments, prevent the collapse of the interbody space, avoid bone resorption, remain or increase the heights of the interbody space, facilitate the union of the fused segments, and allow early mobilization.

Adult↗

PubMed2000

[Apoptosis and related gene expression in lacrimal gland of cases with Sjögren's syndrome].

OBJECTIVE: To assess the roles of apoptosis and expression of the related genes in lacrimal glandular destruction of patients with Sjögren's syndrome (SS). METHODS: Small pieces of palpebral lobes of lacrimal glands were obtained from 12 patients (10 with primary Sjögren's syndrome, 2 with secondary Sjögren's syndrome) and 7 normal controls. They had been investigated by the in situ end labeling and immunohistochemical staining to detect the apoptotic cells and the expression of Fas, FasL, bcl-2 and Bax. RESULTS: The number of epithelial apoptotic cells in lacrimal glands of patients with SS was significantly higher than that in normal glands, and SS epithelial cells showed increased expression of Fas, FasL and Bax. There was significant positive correlation between the number of the apoptotic cells and that of the cells expressing Fas, FasL and Bax, respectively, and there was significant negative correlation between the number of the apoptotic cells and that of the cells expressing bcl-2. CONCLUSIONS: The apoptosis of the epithelial cells in the lacrimal gland may be one of the mechanisms leading to the glandular destruction found in SS. In SS lacrimal glands, the expression of Fas, FasL and Bax may promote apoptosis, and the expression of bcl-2 may inhibit apoptosis.

Adolescent↗

PubMed2000

[A mutation of platelet glycoprotein I balpha results in defects in its interaction with immobilized vWF].

OBJECTIVE: To study the importance of glycoprotein (GP) I balpha mutation (A156V) in interaction between mutant expressing cell and immobilized vWF under fluid shear stress. METHODS: Mutant GP I balpha cDNA was cloned into the EcoR I site of the mammalian expression vector pDX, mutant cDNA was then transfected into CHO betaIX cells. Human vWF was purified from blood cryoprecipitate by glycine and NaCl precipitation and subsequent separation on sepharose 4B column. The purified vWF was immobilized onto a coverslip. Cell rolling was induced in a parallel-plate flow chamber and observed by phase-contrast video microscope. RESULTS: CHO cells expressing GP I b-IX-V complex could adhere to and roll on immobilized vWF. The A156V mutant cells retained the ability to adhere and roll on vWF matrix, but the rolling speed was significantly faster than that of wild type cells, indicating that the off-rate of the ligand-receptor bond between the mutant and vWF was impaired. Binding of monoclonal antibody AN51 to mutant GP I balpha decreased significantly, indicating that the A156V mutation altered the conformation of the N-terminal ligand-binding region of GP I balpha. CONCLUSION: The mutant GP I balpha has a faster off-rate for its interaction with immobilized vWF. The mutant polypeptide adopts an altered conformation in N-terminal ligand-binding region of GP I balpha. The parallel-plate flow chamber is an extremely useful system in evaluating interaction between GP I balpha and vWF.

Animals↗

PubMed2000

[The role of mtDNA deletion in the sensitivity to aminoglycoside antibiotic induced deafness].

OBJECTIVE: To establish an animal model of mitochondrial DNA (mtDNA) deletion and investigate the possible role of mtDNA deletion in aminoglycoside antibiotic induced deafness. METHODS: Thirty wistar rats (4 months) were randomly divided into group A and B. Doxorubicin (DOX) was subcutaneously injected at doses of 2 mg/kg twice per week for 3 months in group A and then kanamycin (KM) was intraperitoneally injected 500 mg/kg per day for 10 consecutive days. The treatments of group B were identical to group A, except normal saline was substituted for DOX. The thresholds of auditory brainstem response (ABR) were measured before and after the drug administrations. The inner ear membranous labyrinthine tissue was harvested and mtDNA was amplified to identify 4,834 bp deletion by PCR technique. RESULTS: The elevation of the mean ABR thresholds in group A(67.08 +/- 8.59) dB peSPL was significantly higher than that in group B (12.71 +/- 4.42) dB peSPL after KM administration (P < 0.001). In group A, 9 of the 15 rats demonstrated 4,834 bp mtDNA deletion. However, mtDNA 4,834 bp deletion was negative in group B animals. CONCLUSION: DOX can induce mtDNA deletion in the inner ear tissue of the rat. mtDNA deletion in the inner ear may play an important role in the hypersensitivity to aminoglycoside antibiotic ototoxicity.

Animals↗

PubMed1999

Using Raman spectroscopy to monitor the solvent-exposed and "buried" forms of flavin in p-hydroxybenzoate hydroxylase.

X-ray crystallographic studies of several complexes involving FAD bound to p-hydroxybenzoate hydroxylase (PHBH) have revealed that the isoalloxazine ring system of FAD is capable of adopting in two positions on the protein. In one, the "in" form, the ring is surrounded by protein groups and has little contact with solvent; in the second, "out" form, the ring is largely solvent exposed. Using Raman difference spectroscopy, it has been possible to obtain Raman spectra for the flavin ring in both conformational states for different complexes in solution. The spectra consist of a rich assortment of isoalloxazine ring modes whose normal mode origin can be assigned by using density functional theory and ab initio calculations. Further insight into the sensitivity of these modes to changes in environment is provided by the Raman spectra of lumiflavin in the solid state, in DMSO and in aqueous solution. For the protein complexes, the Raman difference spectra of flavin bound to wt PHBH and wt PHBH plus substrate, p-hydroxybenzoate, provided examples of the "in" conformation. These data are compared to those for flavin bound to wt PHBH plus 2,4-dihydroxybenzoate, where X-ray analysis show that the flavin is "out". There are several spectral regions where characteristic differences exist for flavin in the "in" or "out" conformation, these occur near 1700, 1500, 1410, 1350, 1235, and 1145 cm(-)(1). These spectral features can be used as empirical marker bands to determine the populations of "in" and "out" for any complex of PHBH and to monitor changes in those populations with perturbations to the system, e.g., by changing temperature or pH. Thus, it will now be possible to determine the conformational state of the flavin in PHBH for those complexes that have resisted X-ray crystallographic analysis. Raman difference data are also presented for the Tyr222Phe mutant. The Raman data show that the isoalloxazine ring is predominantly "out" for Tyr222Phe. However, in the presence of the substrate p-hydroxybenzoate there is clear evidence from the Raman marker bands that a mixed population of "in" and "out" exists with the majority being in the "out" state. This is consistent with the conclusions drawn from crystallographic studies on this complex (Gatti, D. L., Palfey, B. A., Lah, M. S., Entsch, B., Massey, V., Ballou, D. P., and Ludwig, M. L. (1994) Science, 266, 110-114).

4-Hydroxybenzoate-3-Monooxygenase↗

PubMed1999

Assessment of the serotonin reuptake blocking property of YM992: electrophysiological studies in the rat hippocampus and dorsal raphe.

YM992 is a selective serotonin (5-HT) reuptake inhibitor and a 5-HT(2A) antagonist with potential antidepressant activity. As expected from a 5-HT reuptake inhibitor, which induces an accumulation of 5-HT in the dorsal raphe, YM992 inhibited the firing activity of these 5-HT neurons (ED50: 2.0+/-0.2 mg/kg, i.v.). This effect was reversed by the 5-HT(1A) antagonist WAY 100635. YM992 also dose-dependently prolonged the time for CA3 neurons to recover 50% of their firing rate following microiontophoretic applications of 5-HT, a reliable index of the function of the 5-HT reuptake carrier. In a second series of experiments, the adaptative properties of 5-HT neurons were examined during sustained administration of YM992 (20 mg/kg/day, s.c., delivered by osmotic minipumps) after 2 days of treatment. YM992 decreased by more than 60% the firing activity of the 5-HT neurons. There was a partial recovery of firing after 7 days and a complete one after 14 days of treatment in the presence of the minipump still delivering the drug. In a third series of experiments, the sensitivity of pre- and postsynaptic 5-HT(1A) receptors in the dorsal raphe and the dorsal hippocampus were assessed. The results showed that YM992 attenuated the inhibitory effect of intravenous administration of LSD and the 5-HT(1A) agonist 8-OH-DPAT on the firing activity of 5-HT neurons. As did the selective 5-HT reuptake inhibitor fluvoxamine, YM992 markedly increased the effectiveness of the electrical stimulation of ascending 5-HT fibres on firing activity of the postsynaptic hippocampus pyramidal neurons. This enhancement of 5-HT neurotransmission by YM992 was attributable to a desensitization of the terminal 5-HT(1B) autoreceptors since the postsynaptic 5-HT(1A) receptors in the hippocampus remained normosensitive.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed1999

Inhibition of human tumor cell growth in vitro and in vivo by a specific inhibitor of human farnesyltransferase: BIM-46068.

Oncogenic mutations of the ras gene leading to constitutive activation of downstream effectors have been detected in a large spectrum of human cancers (pancreas, thyroid, colon and NSCLC). Membrane anchorage of Ras required for functional activity in signal transduction is facilitated by post-translational modifications resulting in covalent attachment of a farnesyl group to the cysteine in the C-terminal CAAX motif. This attachment is mediated by farnesyltransferase (FTase). Here, we report a novel series of potent FTase inhibitors, where the tetrapeptide CAAX motif has been modified by incorporation of a thiazolidine carboxylic acid moiety followed by reduction of the 1st and 2nd peptide bonds to a secondary and tertiary amine, respectively. The C-terminal carboxylate was converted to esters for improved cellular penetration. These compounds showed specific inhibition of purified human FTase enzyme, inhibition of proliferation in vitro in a large spectrum of human tumor cell lines and inhibition of growth of human tumor xenografts in athymic nude mice. In addition, in regard to a panel of cell lines, using the Compare analysis to determine the Pearson coefficient correlation, the anti-proliferative spectrum of BIM-46068 has been shown to be distinct from the profile of typical chemotherapeutic agents.

Alkyl and Aryl Transferases↗

PubMed1999

Kinetic and spectroscopic characterization of the gamma-carbonic anhydrase from the methanoarchaeon Methanosarcina thermophila.

The zinc and cobalt forms of the prototypic gamma-carbonic anhydrase from Methanosarcina thermophila were characterized by extended X-ray absorption fine structure (EXAFS) and the kinetics were investigated using steady-state spectrophotometric and (18)O exchange equilibrium assays. EXAFS results indicate that cobalt isomorphously replaces zinc and that the metals coordinate three histidines and two or three water molecules. The efficiency of either Zn-Cam or Co-Cam for CO(2) hydration (k(cat)/K(m)) was severalfold greater than HCO(3-) dehydration at physiological pH values, a result consistent with the proposed physiological function for Cam during growth on acetate. For both Zn- and Co-Cam, the steady-state parameter k(cat) for CO(2) hydration was pH-dependent with a pK(a) of 6.5-6.8, whereas k(cat)/K(m) was dependent on two ionizations with pK(a) values of 6.7-6.9 and 8.2-8.4. The (18)O exchange assay also identified two ionizable groups in the pH profile of k(cat)/K(m) with apparent pK(a) values of 6.0 and 8.1. The steady-state parameter k(cat) (CO(2) hydration) is buffer-dependent in a saturable manner at pH 8. 2, and the kinetic analysis suggested a ping-pong mechanism in which buffer is the second substrate. The calculated rate constant for intermolecular proton transfer is 3 x 10(7) M(-1) s(-1). At saturating buffer concentrations and pH 8.5, k(cat) is 2.6-fold higher in H(2)O than in D(2)O, suggesting that an intramolecular proton transfer step is at least partially rate-determining. At high pH (pH > 8), k(cat)/K(m) is not dependent on buffer and no solvent hydrogen isotope effect was observed, consistent with a zinc hydroxide mechanism. Therefore, at high pH the catalytic mechanism of Cam appears to resemble that of human CAII, despite significant structural differences in the active sites of these two unrelated enzymes.

Apoenzymes↗

PubMed1999

Molecular analysis of yeast and human type II topoisomerases. Enzyme-DNA and drug interactions.

The DNA sequence selectivity of topoisomerase II (top2)-DNA cleavage complexes was examined for the human (top2alpha), yeast, and Escherichia coli (i.e. gyrase) enzymes in the absence or presence of anticancer or antibacterial drugs. Species-specific differences were observed for calcium-promoted DNA cleavage. Similarities and differences in DNA cleavage patterns and nucleic acid sequence preferences were also observed between the human, yeast, and E. coli top2 enzymes in the presence of the non-intercalators fluoroquinolone CP-115,953, etoposide, and azatoxin and the intercalators amsacrine and mitoxantrone. Additional base preferences were generally observed for the yeast when compared with the human top2alpha enzyme. Preferences in the immediate flanks of the top2-mediated DNA cleavage sites are, however, consistent with the drug stacking model for both enzymes. We also analyzed and compared homologous mutations in yeast and human top2, i.e. Ser(740) --> Trp and Ser(763) --> Trp, respectively. Both mutations decreased the reversibility of the etoposide-stabilized cleavage sites and produced consistent base sequence preference changes. These data demonstrate similarities and differences between human and yeast top2 enzymes. They also indicate that the structure of the enzyme/DNA interface plays a key role in determining the specificity of top2 poisons and cleavage sites for both the intercalating and non-intercalating drugs.

Anti-Bacterial Agents↗

PubMed1999

Preparation of Cu Nanoparticles from Water-in-Oil Microemulsions.

Spherical Cu nanoparticles were synthesized in sodium dodecyl sulfate (SDS)/isopentanol/cyclohexane/water microemulsions with sodium borohydride as a reducing agent. Nanoparticles formed in microemulsions were characterized by transmission electron microscopy (TEM), x-ray diffractometry (XRD), and small-angle X-ray scattering (SAXS). The size and polydisperity of particles were greatly affected by the mole ratio of water to surfactant (w) and the concentration of precursor salt. The ideal size and monodispersity of nanoparticles can be obtained at the smaller w value and lower concentration of Cu(2+) solution. Copyright 1999 Academic Press.

Journal Article↗

PubMed1999

Metalloprotease-mediated ligand release regulates autocrine signaling through the epidermal growth factor receptor.

Ligands that activate the epidermal growth factor receptor (EGFR) are synthesized as membrane-anchored precursors that appear to be proteolytically released by members of the ADAM family of metalloproteases. Because membrane-anchored EGFR ligands are thought to be biologically active, the role of ligand release in the regulation of EGFR signaling is unclear. To investigate this question, we used metalloprotease inhibitors to block EGFR ligand release from human mammary epithelial cells. These cells express both transforming growth factor alpha and amphiregulin and require autocrine signaling through the EGFR for proliferation and migration. We found that metalloprotease inhibitors reduced cell proliferation in direct proportion to their effect on transforming growth factor alpha release. Metalloprotease inhibitors also reduced growth of EGF-responsive tumorigenic cell lines and were synergistic with the inhibitory effects of antagonistic EGFR antibodies. Blocking release of EGFR ligands also strongly inhibited autocrine activation of the EGFR and reduced both the rate and persistence of cell migration. The effects of metalloprotease inhibitors could be reversed by either adding exogenous EGF or by expressing an artificial gene for EGF that lacked a membrane-anchoring domain. Our results indicate that soluble rather than membrane-anchored forms of the ligands mediate most of the biological effects of EGFR ligands. Metalloprotease inhibitors have shown promise in preventing spread of metastatic disease. Many of their antimetastatic effects could be the result of their ability to inhibit autocrine signaling through the EGFR.

Antibodies, Monoclonal↗

PubMed1999

Modulating electron density in the bound product, 4-hydroxybenzoyl-CoA, by mutations in 4-chlorobenzoyl-CoA dehalogenase near the 4-hydroxy group.

The enzyme 4-chlorobenzoyl-CoA dehalogenase hydrolyzes 4-chlorobenzoyl-CoA (4-CBA-CoA) to 4-hydroxybenzoyl-CoA (4-HBA-CoA). Biochemical and crystallographic studies have identified a critical role for the dehalogenase residue Asp 145 in close proximity to the ligand's 4-hydroxy group in the structure of the product-enzyme complex. In the present study the effects of site selective mutations at Asp 145 on the product complex are explored by Raman spectroscopy. The spectral signatures of the WT-product complex, the large red shift in lambdamax, and the complete reorganization of the benzoyl ring modes in Raman data are absent for the D145E complex. The major spectral perturbations in the WT complex are brought about by strong electron "pull" at the benzoyl carbonyl and electron "push" by the side chain of Asp 145 near the 4-OH group. Acting in concert, these factors polarize the benzoyl's pi-electrons. Since the Raman data show that very strong electron pull occurs at the benzoyl's carbonyl in the D145E complex, it is apparent that the needed electron push near the benzoyl's 4-OH group is missing. Thus, very precise positioning of Asp 145's side chain near the benzoyl's 4-position is needed to bring about the dramatic electron reorganization seen in the WT complex, and this criterion cannot be met by the glutamate side chain with its additional CH2 group. For two other Asp145 mutants D145A and D145S that lack catalytic activity, Raman difference spectroscopic data for product complexes demonstrate the presence of a population of ionized product (i.e., 4-O-) in the active sites. The presence of the ionized phenolate form explains the observation that these complexes have highly red-shifted absorbance maxima with lambdamaxs near 400 nm. For the WT complex only the 4-OH form is seen, ionization being energetically expensive with the presence of the proximal negative charge on the Asp 145 side chain. Semiquantitative estimates of the pKa for the bound product in D145S and D145A indicate that this ionization lies in the pH 6.5-7.0 range. This is approximately 2 pH units below the pKa for the free product. The Raman spectrum of 4-dimethylaminobenzoyl-CoA undergoes major changes upon binding to dehalogenase. The bound form has two features near 1562 and 1529 cm-1 and therefore closely resembles the spectrum of product bound to wild-type enzyme, which underlines the quinonoid nature in these complexes. The use of a newly developed Raman system allowed us to obtain normal (nonresonance) Raman data for the dehalogenase complexes in the 100-300 microM range and heralds an important advance in the application of Raman spectroscopy to dilute solutions of macromolecules.

Acyl Coenzyme A↗

PubMed1999

Product catalyzes the deamidation of D145N dehalogenase to produce the wild-type enzyme.

Aspartate 145 plays an essential role in the active site of 4-chlorobenzoyl-CoA dehalogenase, forming a transient covalent link at the 4-position of the benzoate during the conversion of the substrate to 4-hydroxybenzoyl-CoA. Replacement of Asp 145 by residues such as alanine or serine results in total inactivation, and stable complexes can be formed with either substrate or product. The Raman spectroscopic characterization of some of the latter is described in the preceding publication (Dong et al.). The present work investigates complexes formed by D145N dehalogenase and substrate or product. Time-resolved absorption and Raman difference spectroscopic data show that these systems evolve rapidly with time. For the substrate complex, initially the absorption and Raman spectra show the signatures of the substrate bound in the active site of the asparagine 145 form of the enzyme but these signatures are accompanied by those for the ionized product. After several minutes these signatures disappear to be replaced with those closely resembling the un-ionized product in the active site of wild-type dehalogenase. Similarly, for the product complex, the absorption and Raman spectra initially show evidence for ionized product in the active site of D145N, but these are rapidly replaced by signatures closely resembling the un-ionized product bound to wild-type enzyme. It is proposed that product bound to the active site of asparagine 145 dehalogenase catalyzes the deamidation of the asparagine side chain to produce the wild-type aspartate 145. For the complexes involving substrate, the asparagine 145 enzyme population contains a small amount of the WT enzyme, formed by spontaneous deamidation, that produces product. In turn, these product molecules catalyze the deamidation of Asn 145 in the major enzyme population. Thus, conversions of substrate to product and of D145N to D145D dehalogenase go on simultaneously. The spontaneous deamidation of asparagine 145 has been characterized by allowing the enzyme to stand at RT in Hepes buffer at pH 7.5. Under these conditions deamidation occurs with a rate constant of 0.0024 h-1. The rate of product-catalyzed deamidation in Hepes buffer at 22 degrees C was measured by stopped-flow kinetics to be 0.024 s-1, 36000 times faster than the spontaneous process. A feature near 1570 cm-1 could be observed in the early Raman spectra of both substrate and product-enzyme complexes. This band is not associated with either substrate or product and is tentatively assigned to an ester-like species formed by the attack of the product's 4-O- group on the carbonyl of asparagine's side chain and the subsequent release of ammonia. A reaction scheme is proposed, incorporating these observations.

Amides↗

PubMed1999

Expression and function of leptin receptor isoforms in myeloid leukemia and myelodysplastic syndromes: proliferative and anti-apoptotic activities.

The receptor for the gene product of the obesity gene, leptin, was recently reported to be expressed on murine and human hematopoietic progenitor cells. Therefore, we studied the expression of the leptin receptor, OB-R, in normal myeloid precursors, human leukemia cell lines, and primary leukemic cells using reverse-transcriptase polymerase chain reaction. In normal hematopoiesis, OB-R was expressed in CD34(+) cells. Normal promyelocytes (CD34(-)33(+) and CD34(-)13(+)) expressed only very low levels of the short, presumably nonsignaling isoform. Both the long and short isoforms of OB-R were expressed in 10 of 22 samples from patients with newly diagnosed primary or secondary acute myeloid leukemia (AML), with a higher incidence of the long isoform in primary AML (87.6% v 28.6%; P =.01). The incidence of OB-R expression was higher in recurrent than in newly diagnosed AML (P <.001), and samples from four patients with refractory AML showed strong expression of both isoforms. Both OB-R isoforms were also expressed in newly diagnosed and recurrent acute promyelocytic leukemia cells but were essentially absent in samples of chronic or acute lymphocytic leukemia. In vitro growth of myeloid leukemic cell lines and of blasts from 14 primary AMLs demonstrated that recombinant human leptin alone induced low level proliferation, significantly (P <.05) increased proliferation induced by recombinant human granulocyte colony-stimulating factor, interleukin 3, and stem cell factor in a subset of AML and increased colony formation (P <.005). Also, leptin reduced apoptosis induced by cytokine withdrawal in MO7E and TF-1 cells. Serum leptin levels correlated only with body mass index (P <. 001) and gender (P =.03). Results confirm the reported expression of leptin receptor in normal CD34(+) cells and demonstrate the frequent expression of leptin receptors in AML blasts. While normal promyelocytes lack receptor expression, leukemic promyelocytes express both isoforms. We also demonstrate proliferative effects of leptin alone and in combination with other physiologic cytokines, and anti-apoptotic properties of leptin. These findings could have implications for the pathophysiology of AML.

Apoptosis↗