Biomedical subjects
Publications and source records attributed to H Han.
Solution syntheses of azapeptide pentamers 2, 3, and 4 were accomplished. The binding affinity of these azapeptides and azatide 1 were examined in the context of monoclonal antibody 3-E7 known to strongly bind the [Leu5]enkephalin sequence.
We have investigated the role of the hematopoietically expressed G16 GTP-binding protein on T cell activation. We constructed transfectants of Jurkat T cells that express a function-deficient mutant of G alpha 16 predicted to prevent activation of this G protein. Upon stimulation with anti-CD3 epsilon antibodies, mutant G alpha 16 transfectants display a profound defect in the production of IL-2 and IL-10, as well as in the expression of CD69. In contrast, the phorbol 12-myristate 13-acetate (PMA)-induced IL-10 production and CD69 expression, and the ionomycin plus PMA-induced IL-2 production are not affected. Consistent with the reduction in cytokine production is the inhibition of early signaling events in the mutant G alpha 16-expressing cells. There are significant reductions in anti-epsilon-induced tyrosine phosphorylation of zeta, epsilon, ZAP-70, and phospholipase C gamma 1, as well as in intracellular Ca2+ mobilization. In accordance with the effects on tyrosine phosphorylation is the reduction of TCR/CD3-mediated Fyn and Lck activities in G alpha 16 mutant cells. Even though the mechanism through which the G alpha 16 mutant mediates inhibition of T cell activation is not known, the data suggest a model where G proteins become activated upon TCR/CD3 engagement and regulate the activation of tyrosine kinases and subsequent downstream signaling events that lead to the activation of cytokine genes.
PURPOSE: General use of nucleoside analogues in the treatment of viral infections and cancer is often limited by poor oral absorption. Valacyclovir, a water soluble amino acid ester prodrug of acyclovir has been reported to increase the oral bioavailability of acyclovir but its absorption mechanism is unknown. This study characterized the intestinal absorption mechanism of 5' -amino acid ester prodrugs of the antiviral drugs and examined the potential of amino acid esters as an effective strategy for improving oral drug absorption. METHODS: Acyclovir (ACV) and Zidovudine (AZT) were selected as the different sugar-modified nucleoside antiviral agents and synthesized to L-valyl esters of ACV and AZT (L-Val-ACV and L-Val-AZT), D-valyl ester of ACV (D-Val-ACV) and glycly ester of ACV (Gly-ACV). The intestinal absorption mechanism of these 5' -amino acid ester prodrugs was characterized in three different experimental systems; in situ rat perfusion model, CHO/hPEPT1 cells and Caco-2 cells. RESULTS: Testing 5' -amino acid ester prodrugs of acyclovir and AZT, we found that the prodrugs increased the intestinal permeability of the parent nucleoside analogue 3- to 10-fold. The dose- dependent permeation enhancement was selective for L-amino acid esters. Competitive inhibition studies in rats and in CHO cells transfected with the human peptide transporter, hPEPT1, demonstrated that membrane transport of the prodrugs was mediated predominantly by the PEPT1 H+/dipeptide cotransporter even though these prodrugs did not possess a peptide bond. Finally, transport studies in Caco-2 cells confirmed that the 5' - amino acid ester prodrugs enhanced the transcellular transport of the parent drug. CONCLUSIONS: This study demonstrates that L-amino acid-nucleoside chimeras can serve as prodrugs to enhance intestinal absorption via the PEPT1 transporter, providing a novel strategy for improving oral therapy of nucleoside drugs.
Viral proteins E1 and E2 are essential for transient human papillomavirus (HPV) DNA replication. E1 is a multifunctional protein which can bind DNA and complex with E2, has ATPase and helicase activities, and interacts with DNA polymerase alpha-primase. E2 is a transactivator-repressor protein, playing an important role in replication and transcriptional regulation. A series of deletion mutants of HPV-11 E1 were constructed and tested in functional assays to define those domains of HPV-11 E1 which are important for binding to the origin DNA and E2. The domain of HPV-11 E1 involved in binding to the origin was located between aa 186 and 649, and that for binding to E2 was between aa 346 and 649. Since E1 binds to the origin more efficiently in the presence of E2, we also mapped the DNA binding domain of E1 in the presence of E2, and found that when binding was enhanced, the region of E1 involved in binding was similar to that observed with E1 alone. The same deletion mutation constructs of E1 were subcloned into an expression vector for use in transient replication assays to study the effect of the deletions on the replication of the origin DNA in vivo and the data suggest that the C-terminal domain contains important functions for replication.
The RBP-J/Su(H) DNA-binding protein plays a key role in transcriptional regulation by targeting Epstein-Barr virus nuclear antigen 2 (EBNA2) and the intracellular portions of Notch receptors to specific promoters. Using the yeast two-hybrid system, we isolated a LIM-only protein, KyoT, which physically interacts with RBP-J. Differential splicing gave rise to two transcripts of the KyoT gene, KyoT1 and KyoT2, that encoded proteins with four and two LIM domains, respectively. With differential splicing resulting in deletion of an exon, KyoT2 lacked two LIM domains from the C terminus and had a frameshift in the last exon, creating the RBP-J-binding region in the C terminus. KyoT1 had a negligible level of interaction with RBP-J. Strong expression of KyoT mRNAs was detected in skeletal muscle and lung, with a predominance of KyoT1 mRNA. When expressed in F9 embryonal carcinoma cells, KyoT1 and KyoT2 were localized in the cytoplasm and the nucleus, respectively. The binding site of KyoT2 on RBP-J overlaps those of EBNA2 and Notchl but is distinct from that of Hairless, the negative regulator of RBP-J-mediated transcription in Drosophila. KyoT2 but not KyoT1 repressed the RBP-J-mediated transcriptional activation by EBNA2 and Notch1 by competing with them for binding to RBP-J and by dislocating RBP-J from DNA. KyoT2 is a novel negative regulatory molecule for RBP-J-mediated transcription in mammalian systems.
We analyzed the genetic defect in a 67-year-old Japanese male patient with apolipoprotein (apo) A-I and high density lipoprotein (HDL) deficiencies, corneal opacities, and coronary artery disease. The plasma concentrations of apoA-I and HDL cholesterol were 2.9 to 7.3 mg/dL and 0.08 to 0.19 mmol/L, respectively. The lecithin:cholesterol acyltransferase (LCAT) activity and cholesterol esterification rate were <40% of normal control values. LCAT mass was 550% of normal control. Sequence analysis of polymerase chain reaction-amplified DNA of the proband's apoA-I gene showed a homozygous T-to-A transition resulting in the substitution of Val 156 with Glu (apoA-I Oita). Direct sequencing of samples obtained from other family members showed that the brother was homozygous, whereas the son was a heterozygous carrier of apoA-I Oita. The heterozygote for apo A-I Oita showed nearly 60% of normal apoA-I and normal HDL cholesterol levels. In vivo turnover studies in rabbits demonstrated that the variant apoA-I was rapidly cleared from plasma compared with normal human apoA-I. Our data suggest that the Val156Glu substitution is associated with apoA-I and HDL deficiency, partial LCAT deficiency, and corneal opacities and that Val156 of apoA-I may play an important role in apoA-I function.
Amethod for the determination of diuron and chlortoluron in beef and beef products with high performance liquid chromatography is described. The sample was extracted with a mixture of acetonitrile and methanol (50:50, V/V). After filtration, the filtrate was defatted with petroleum ether, then water was added and further extracted with chloroform. The chloroform collected was evaporated in a rotary evaporator(45 degrees C). The residue was dissolved in acetonitrile-methanol(50:50, V/V) mixture, poured into an Al2O3 column and eluted with the same mixture. The eluate was collected for HPLC analysis. The analytical column was Selectosil C18 (5 microns, 250 mm x 4.6 mm i.d.), mobile phase was methanol-water(60:40, V/V) and detection wavelength was UV-245 nm. The minimum amounts of detection were 0.4 ng for diuron and 0.5 ng for chlortoluron. Recoveries were 87.34%-87.64% for diuron and 88.78%-91.94% for chlortoluron.
The extracellular penicillin acylase from Bacillus megaterium was immobilized by coupling to derivatives of polyacrylonitrile fibres. The apparent activity of the immobilized penicillin acylase was about 2,000 u/g (dry weight). The optimal pH and temperature were 9.0 and 50 degrees C for hydrolysis of penicillin G, respectively. The immobilized enzyme was stable in the pH range of 5.5-10.3, and at the temperature below 50 degrees C. The apparent Michaelis constant Ka and Vm of the immobilization enzyme were 1.33 x 10(-2) mol/L and 2.564 mmol.min-1, respectively. The inhibition constant of phenylacetic acid acted as a competitive inhibitor for The immobilization enzyme was 0.16 mol/L. The remained activity was about 80% after operating 20 times.
This investigation was aimed at the fatigue fracture of tibia that often occurs in soldiers, as well as in dancers and athletes. The data of ground reaction force(GRF) of soldiers were measured using a force platform in three common gaits: marching, parading and running. The stress distribution in tibia under these GRFs were analyzed with a FE model. The results showed that the smallest GRF(744.8N) is in quick marching, the next (1156.4N) in parading and the largest (1391.6N) in running. The stress distribution under GRF was mot uniform in the tibia. The maximum stresses occurred at a site 1/3 tibia length below the knee where tibia fracture is often seen clinically. The maximum stresses were 110 MPa and 115 MPa in parading and running respectively. This means that the tibial tractures in soldiers are mostly associated with stress, i.e. the greater the stress is, the higher the incidence of stress fracture comes about.
Through cloning of functional p53-binding sites (p53-tagged sites) from the human genome, we isolated a novel gene inducible by wild-type p53. Its cDNA sequence contained an open reading frame encoding a 431-amino acid peptide that showed a significant homology with members of the P2X family. This protein also revealed a similarity to RP-2, a gene activated in thymocytes undergoing programmed cell death. Northern blot analysis showed that it was expressed predominantly in skeletal muscle. Hence, we designated the gene P2XM (P2X specifically expressed in skeletal muscle). P2XM was localized to chromosomal band 22q11, where frequent loss of heterozygosity has been observed in rhabdoid tumors. Although we detected no genetic alteration in the coding sequences, one of four rhabdomyosarcoma cell lines examined had completely lost expression of this gene. Furthermore, a minor splice variant lacking a part of exon 1 that would encode residues corresponding to transmembrane domain M1 was relatively more abundant in two of seven sarcoma cell lines, one of which was derived from a rhabdomyosarcoma, and the other was derived from an osteosarcoma. The results suggest that P2XM may play a significant role in the proliferation and/or differentiation of skeletal muscle cells and that its altered expression may be involved in the development of some sarcomas.
Previously, our laboratory reported that lactosylceramide (LacCer) stimulated human aortic smooth muscle cell proliferation via specific activation of p44 mitogen-activated protein kinase (MAPK) in the p21(ras)/Raf-1/MEK2 pathway and induced expression of the transcription factor c-fos downstream to the p44 MAPK signaling cascade (Bhunia A. K., Han, H., Snowden, A., and Chatterjee S. (1996) J. Biol. Chem. 271, 10660-10666). In the present study, we explored the role of free oxygen radicals in LacCer-mediated induction of cell proliferation. Superoxide levels were measured by the lucigenin chemiluminescence method, MAPK activity was measured by immunocomplex kinase assays, and Western blot analysis and c-fos expression were measured by Northern blot assay. We found that LacCer (10 microM) stimulates endogenous superoxide production (7-fold compared with control) in human aortic smooth muscle cells specifically by activating membrane-associated NADPH oxidase, but not NADH or xanthine oxidase. This process was inhibited by an inhibitor of NADPH oxidase, diphenylene iodonium (DPI), and by antioxidants, N-acetyl-L-cysteine (NAC) or pyrrolidine dithiocarbamate. NAC and DPI both abrogated individual steps in the signaling pathway leading to cell proliferation. For example, the p21(ras).GTP loading, p44 MAPK activity, and induction of transcription factor c-fos all were inhibited by NAC and DPI as well as an antioxidant pyrrolidine dithiocarbamate or reduced glutathione (GSH). In contrast, depletion of GSH by L-buthionine (S, R)-sulfoximine up-regulated the above described signaling cascade. In sum, LacCer, by virtue of activating NADPH oxidase, produces superoxide (a redox stress signaling molecule), which mediates cell proliferation via activation of the kinase cascade. Our findings may explain the potential role of LacCer in the pathogenesis of atherosclerosis involving the proliferation of aortic smooth muscle cells.
PURPOSE: To catalogue the presenting symptoms of patients with AIDS who are presumed to have primary central nervous system lymphoma (PCNSL). To document the palliative efficacy of cranial irradiation (RT) relative to the endpoints of complete and overall response for the respective symptoms. METHODS: An analysis of 163 patients with AIDS-related PCNSL who were evaluated at nine urban hospitals was performed. These patients were treated for PCNSL after the establishment of a tissue diagnosis or on a presumptive basis after failing empiric treatment for toxoplasmosis. All patients were treated between 1983 and 1995 with radiotherapy (median dose-fractionation scheme = 3 Gy x 10) and steroids (>90% dexamethasone). Because multiple fractionation schemes were used, prescriptions were converted to biologically effective doses according to the formula, Gy10 = Total Dose x (1 + fractional dose/alpha-beta); using an alpha-beta value of 10. RESULTS: The overall palliative response rate for the entire group was 53%. In univariate analysis, trends were present associating complete response rates with higher performance status (KPS > or = 70 vs. KPS < or = 60 = 17% vs. 5%), female gender (women vs. men = 29% vs. 8%), and the delivery of higher biologically effective doses (BED) of RT (Gy10 > 39 vs. < or = 39 = 20% vs. 5%). In multivariate analysis of factors predicting complete response, both higher KPS and higher BED retained independent significance. A separate univariate analysis identified high performance status (KPS > or = 70 vs. KPS < or = 60 = 71% vs. 47%), and young age (< or = 35 vs. > 35 = 61% vs. 40%) as factors significantly correlating with the endpoint of the overall response. In multivariate analysis, high performance status and the delivery of higher biologically effective doses of irradiation correlated significantly with higher overall response rates. CONCLUSION: Most AIDS patients who develop symptoms from primary lymphoma of the brain can achieve some palliation from a management program that includes cranial irradiation. Young patients with excellent performance status are most likely to respond to treatment. The delivery of higher biologically effective doses of irradiation also may increase the probability of achieving a palliative response.
To demonstrate interaction between the mitochondrial membrane proteins CPT I and Bcl-2 the yeast two-hybrid system was used. Full-length CPT I was required for binding to Bcl-2. Direct protein interaction was confirmed in a GST binding assay and in coimmunoprecipitations using two different kinds of anti-Bcl-2 antibodies.
Many NMR measurements of cardiac microcirculation (perfusion, intramyocardial blood volume) depend on some kind of assumption of intracapillary-extravascular water exchange rate, e.g., fast exchange. The magnitude of this water exchange rate, however, is still unknown. The intention of this study was to determine a lower limit for this exchange rate by investigating the effect of perfusion on relaxation time. Studies were performed in the isolated perfused cardioplegic rat heart. After slice-selective inversion, the spin lattice relaxation rate of myocardium within the slice was studied as a function of perfusion and compared with a mathematical model which predicts relaxation rate as a function of perfusion and intracapillary-extravascular exchange rate. A linear relationship was found between relaxation rate T(-1) and perfusion P normalized by perfusate/tissue partition coefficient of water, lambda: deltaT(-1) = m x deltaP/lambda with 0.82 < or = m < or = 1.06. Insertion of experimental data in the model revealed that a lower bound of the exchange rate from intra- to extravascular space is 6.6 s(-1) (4.5 s(-1), P < 0.05), i.e., the intracapillary lifetime of a water molecule is less than 150 ms (222 ms, P < 0.05). Based on this finding, the T1 mapping after slice-selective inversion could become a valuable noncontrast NMR method to measure variations of perfusion.
Rapsyn is a 43-kDa cytoplasmic protein that clusters nicotinic acetylcholine receptors (AChR) in the postsynaptic membrane. Here we examine the effect of rapsynmediated AChR clustering on the metabolic stability of the AChR. When transfected into QT-6 fibroblasts, cell surface AChRs (alpha, beta, epsilon, and delta subunit combination) pulse labeled with 125I-alpha-bungarotoxin were degraded with a half-life of 16.4 +/- 1.1 h (mean +/- SEM). Cotransfection of rapsyn with AChR caused extensive AChR clustering and increased AChR half-life to 20.5 +/- 1.0 h. Anti-AChR antibodies such as mab 35 cause an increased AChR degradation often associated with myasthenia gravis: 80.8 +/- 2.5% of AChRs labeled at zero time were degraded over a 12-h period. Contransfection of rapsyn reduced this AChR loss to 66.4 +/- 3.8%. Rapsyn also reduced normal AChR degradation, from 53.2 +/- 2.1 to 44.2 +/- 2.2%. Muscle cell lines from wild-type myotubes displayed few AChR clusters, but treatment with neural agrin increased the number of AChR clusters 30-fold. Clustering was accompanied by reductions in AChR degradation (both in the presence and absence of mab 35) similar in magnitude to those produced by overexpression of rapsyn in QT-6 cells. In rapsyn-deficient myotubes, treatment with neural agrin neither caused AChR clustering nor reduced AChR degradation. Thus neural agrin may slow AChR degradation by inducing the rapsyn-dependent clustering of AChRs.
BACKGROUND/PURPOSE: Reports on the late results of choledochal cyst excision with hepaticojejunostomy in children are relatively few. METHODS: Of the 84 patients who had choledochal cyst who came under our care, 79 have had definitive surgery, three are awaiting surgery, one is being observed with Caroli's disease, and the parents of one child have refused surgery. Thirty-eight patients treated decades ago had internal drainage procedures. Since 1972, 41 patients have had cyst excision with hepaticojejunostomy using a 40-cm Roux loop without an antireflux procedure. Early complications in those who underwent cyst excision with hepaticojejunostomy included anastomotic leak in three patients who required reoperation, cholangitis in two, and fluid collection in the gall-bladder bed that required no intervention in one. RESULTS: During a follow-up period ranging from 4 months to 17 years (mean, 8.5 years), anastomotic stricture, cholangitis, and intrahepatic stone formation developed in two children after being well for 8 years and over 11 years. These children required additional surgical procedures to overcome their problems. Asymptomatic intrahepatic stones 2 years after cyst excision with hepaticojejunostomy developed in a third child. There was no mortality in the entire group that underwent cyst excision and they are all enjoying a good quality of life. CONCLUSIONS: Careful, long-term follow-up is important in children who have choledochal cyst excision with hepaticojejunostomy.
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Previously, our laboratory has shown that oxidized low density lipoproteins (Ox-LDL) can exert a concentration-dependent stimulation in the proliferation of aortic smooth muscle cells, "a hallmark in the pathogenesis of atherosclerosis" (Chatterjee, S. (1992) Mol. Cell. Biochem., 111, 143-147). Here we report a novel aspect of Ox-LDL-mediated signal transduction. We demonstrate that in aortic smooth muscle cells, Ox-LDL stimulates the activity of a UDP-galactose:glucosylceramide beta1-->4 galactosyltransferase (GalT-2) and phosphorylation/activation of p44 mitogen-activated protein (MAP) kinase (p44 MAPK). The activity of GalT-2 increased about 2-fold within 2.5-5 min of incubation of cells with Ox-LDL (10 microg/ml). After 5 min of incubation of cells with Ox-LDL, but not LDL, there was a 2-fold increase in the activity of p44 MAPK. Phosphoamino acid analysis employing thin layer chromatography revealed that the tyrosine and threonine moieties of p44 MAPK was phosphorylated by Ox-LDL. D-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP; a potent inhibitor of GalT-2) impaired the Ox-LDL mediated induction of p44 MAPK activity and the phosphorylation of tyrosine and threonine residues in p44 MAPK. This phenomenon was bypassed by the simultaneous addition of lactosylceramide. The upstream and downstream parameters in MAP kinase signaling pathways were investigated next. We found that Ox-LDL stimulated (9-fold) the loading of GTP on Ras. Interestingly, Ox-LDL specifically induced c-fos mRNA expression (6.5-fold) in these cells, as compared to the control. Thus, one of the biochemical mechanisms in Ox-LDL mediated induction in the proliferation in aortic smooth muscle cells may involve GalT-2 activation, lactosylceramide production, Ras GTP loading, activation of the kinase cascade, and c-fos expression.