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Biomedical subjects

S Lee

Publications and source records attributed to S Lee.

At least 523 records · Page 29Linked to original sources
PubMed1999

The prevalence of osteoporosis in the Hong Kong Chinese female population.

OBJECTIVES: This paper aims to present population-based age-related bone mass values in the Hong Kong Chinese female population, and to assess the number and proportion of Chinese women considered osteoporotic according to the WHO diagnostic guidelines. METHODS: A total of 769 community-based female subjects were recruited. Social demographic characteristics of these subjects were similar to the Hong Kong general population. All bone mass measurements were performed by means of a dual energy X-ray densitometry (Norland XR 26) at two sites: lumbar vertebrae L2-L4 and left hip. These values were expressed as T-scores, with reference to the mean bone mineral density (BMD) values of the group aged 21-40 years. RESULTS: The study revealed that, in women aged 60 years and above, their mean BMD values are 30% lower than the young normal mean. The prevalence of osteoporosis at the spine increased dramatically from about 10% in the age group 50-59 to 45% in the group aged 60-69. In women aged 70 onwards, over half have osteoporosis at the hip. The prevalence of osteoporosis at the spine is relatively stable in the age groups above 60, while that for osteoporosis at the hip increased exponentially with age. CONCLUSIONS: The prevalence of osteoporosis in Hong Kong women is comparable to that found in Caucasian populations. Prevention of osteoporosis, involving both immediate and long-term measures, and targeting at different age groups, are required to combat this serious public health problem in Hong Kong.

Absorptiometry, Photon↗
PubMed1999

Proteolytic processing of big endothelin-3 by the kell blood group protein.

Kell blood group protein shares a consensus sequence (H.E.X.X.H) with a large family of zinc-dependent endopeptidases. Kell has closest homology with neutral endopeptidase 24.11, endothelin converting enzyme-1 (ECE-1), and the PEX gene product that, as a group, comprise the M13 subfamily of mammalian neutral endopeptidases. The proteolytic activity of the M13 members, but not of Kell, has been previously demonstrated. A secreted form of wild-type Kell protein (s-Kell), devoid of the intracellular and transmembrane domains, was expressed in sf9 cells. As a negative control, an inactive mutant Kell protein (E582G) was expressed. As determined by N-terminal amino acid sequencing and mass spectrometry of the cleaved products, wild-type s-Kell, but not the control mutant protein, specifically cleaved big endothelin-3 (ET-3) at Trp(21)-Ile(22), yielding ET-3, and, to a much lesser extent, also cleaved big ET-1 and big ET-2 at Trp(21)-Val(22), yielding ET-1 and ET-2. Enzymatic activity was partially inhibited by phosphoramidon. s-Kell has an acidic pH optimum (pH 6.0 to 6.5). Like the recombinant protein, red blood cells of common Kell phenotype also preferentially process big ET-3, in contrast to Ko (null) cells that do not. These data demonstrate that the Kell blood group protein is a proteolytic enzyme that processes big ET-3, generating ET-3, a potent bioactive peptide with multiple biological roles.

Endothelin-3↗
PubMed1999

Identification of a defect in the intracellular trafficking of a Kell blood group variant.

Blood group polymorphisms have been used as tools to study the architecture of the red blood cell (RBC) membrane. Some blood group variants have reduced antigen expression at the cell surface. Understanding the underlying mechanism for this reduced expression can potentially provide structural information and help to elucidate protein trafficking pathways of membrane proteins. The Kp(a+) phenotype is a variant in the Kell blood group system that is associated with a single amino acid substitution (R281W) in the Kell glycoprotein and serologically associated with a weakened expression of other Kell system antigens by an unknown mechanism. We found by immunoblotting of RBCs that the weakening of Kell antigens in this variant is due to a reduced amount of total Kell glycoprotein at the cell surface rather than to the inaccessibility of the antigens to Kell antibodies. Using a heterologous expression system, we demonstrate that the Kpa mutation causes retention of most of the Kell glycoprotein in a pre-Golgi compartment due to differential processing, thereby suggesting aberrant transport of the Kell protein to the cell surface. Furthermore, we demonstrated that single nucleotide substitutions into the coding region of the common KEL allele, as predicted by the molecular genotyping studies, was sufficient to encode three clinically significant low incidence antigens. We found that two low incidence antigens can be expressed on a single Kell protein, thus showing that the historical failure to detect such a variant is not due to structural constraints in the Kell protein. These studies demonstrate the power of studying the molecular mechanisms of blood group variants for elucidating the intracellular transport pathways of membrane proteins and the requirements for cell surface expression.

Alleles↗
PubMed1999

Immune modulation by IL-10 gene transfer via viral vector and plasmid DNA: implication for gene therapy.

In the present report, we have evaluated and compared the modulatory effect of the cytokine interleukin (IL)-10 expressed via viral vector or plasmid DNA on viral antigen-induced cutaneous inflammatory lesions. Our data demonstrate the superior potency of both recombinant vaccinia virus and herpes simplex virus IL-10 expression vectors after single intramuscular administration, but the effects were only short term and only functioned in animals lacking immunity to the viral vectors used for modulation. In contrast, modulatory effects achieved by plasmid DNA expressing IL-10 were delayed in onset and milder in effect but were far more persistent than those achieved by viral vectors. Moreover, plasmid DNA expressing IL-10 provided effective modulation when given repeatedly to animals. Our data also showed that IL-10 gene delivery resulted in a systemic and durable modulatory effect while the effect caused by a single IL-10 protein treatment was transient and confined to the injected site. Our results imply that the viral vector system is superior for obtaining short-term effects, whereas the plasmid DNA approach represents a better strategy to achieve gene therapy to modulate chronic inflammatory lesions.

Animals↗
PubMed1999

Gating of transmission in climbing fibre paths to cerebellar cortical C1 and C3 zones in the rostral paramedian lobule during locomotion in the cat.

1. Climbing fibre field potentials evoked by low intensity (non-noxious) electrical stimulation of the ipsilateral superficial radial nerve have been recorded in the rostral paramedian lobule (PML) in awake cats. Chronically implanted microwires were used to monitor the responses at eight different C1 and C3 zone sites during quiet rest and during steady walking on a moving belt. The latency and other characteristics of the responses identified them as mediated mainly via the dorsal funiculus-spino-olivocerebellar path (DF-SOCP). 2. At each site, mean size of response (measured as the area under the field, in mV ms) varied systematically during the step cycle without parallel fluctuations in size of the peripheral nerve volley. Largest responses occurred overwhelmingly during the stance phase of the step cycle in the ipsilateral forelimb while smallest responses occurred most frequently during swing. 3. Simultaneous recording from pairs of C1 zone sites located in the anterior lobe (lobule V) and C1 or C3 zone sites in rostral PML revealed markedly different patterns of step-related modulation. 4. The findings shed light on the extent to which the SOCPs projecting to different parts of a given zone can be regarded as functionally uniform and have implications as to their reliability as channels for conveying peripheral signals to the cerebellum during locomotion.

Animals↗
PubMed1999

The AcbC protein from Actinoplanes species is a C7-cyclitol synthase related to 3-dehydroquinate synthases and is involved in the biosynthesis of the alpha-glucosidase inhibitor acarbose.

The putative biosynthetic gene cluster for the alpha-glucosidase inhibitor acarbose was identified in the producer Actinoplanes sp. 50/110 by cloning a DNA segment containing the conserved gene for dTDP-D-glucose 4,6-dehydratase, acbB. The two flanking genes were acbA (dTDP-D-glucose synthase) and acbC, encoding a protein with significant similarity to 3-dehydroquinate synthases (AroB proteins). The acbC gene was overexpressed heterologously in Streptomyces lividans 66, and the product was shown to be a C7-cyclitol synthase using sedo-heptulose 7-phosphate, but not ido-heptulose 7-phosphate, as its substrate. The cyclization product, 2-epi-5-epi-valiolone ((2S,3S,4S,5R)-5-(hydroxymethyl)cyclohexanon-2,3,4,5-tetrol), is a precursor of the valienamine moiety of acarbose. A possible five-step reaction mechanism is proposed for the cyclization reaction catalyzed by AcbC based on the recent analysis of the three-dimensional structure of a eukaryotic 3-dehydroquinate synthase domain (Carpenter, E. P., Hawkins, A. R., Frost, J. W., and Brown, K. A. (1998) Nature 394, 299-302).

Acarbose↗
PubMed1999

Chemoattractant-mediated transient activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in Dictyostelium.

Chemotaxis-competent cells respond to a variety of ligands by activating second messenger pathways leading to changes in the actin/myosin cytoskeleton and directed cell movement. We demonstrate that Dictyostelium Akt/PKB, a homologue of mammalian Akt/PKB, is very rapidly and transiently activated by the chemoattractant cAMP. This activation takes place through G protein-coupled chemoattractant receptors via a pathway that requires homologues of mammalian p110 phosphoinositide-3 kinase. pkbA null cells exhibit aggregation-stage defects that include aberrant chemotaxis, a failure to polarize properly in a chemoattractant gradient and aggregation at low densities. Mechanistically, we demonstrate that the PH domain of Akt/PKB fused to GFP transiently translocates to the plasma membrane in response to cAMP with kinetics similar to those of Akt/PKB kinase activation and is localized to the leading edge of chemotaxing cells in vivo. Our results indicate Akt/PKB is part of the regulatory network required for sensing and responding to the chemoattractant gradient that mediates chemotaxis and aggregation.

Amino Acid Sequence↗
PubMed1999

A Smad transcriptional corepressor.

Following TGFbeta receptor-mediated phosphorylation and association with Smad4, Smad2 moves into the nucleus, binds to target promoters in association with DNA-binding cofactors, and recruits coactivators such as p300/CBP to activate transcription. We identified the homeodomain protein TGIF as a Smad2-binding protein and a repressor of transcription. A TGFbeta-activated Smad complex can recruit TGIF and histone deacetylases (HDACs) to a Smad target promoter, repressing transcription. Thus, upon entering the nucleus, a Smad2-Smad4 complex may interact with coactivators, forming a transcriptional activation complex, or with TGIF and HDACs, forming a transcriptional repressor complex. Formation of one of these two mutually exclusive complexes is determined by the relative levels of Smad corepressors and coactivators within the cell.

Animals↗
PubMed1999

Identification of the block in targeted retroviral-mediated gene transfer.

A chimeric retroviral vector (33E67) containing a CD33-specific single-chain antibody was generated in an attempt to target cells displaying the CD33 surface antigen. The chimeric envelope protein was translated, processed, and incorporated into viral particles as efficiently as wild-type envelope protein. The viral particles carrying the 33E67 envelope protein could bind efficiently to the CD33 receptor on target cells and were internalized, but no gene transfer occurred. A unique experimental approach was used to examine the basis for this postbinding block. Our data indicate that the chimeric envelope protein itself cannot participate in the fusion process, the most reasonable explanation being that this chimeric protein cannot undergo the appropriate conformational change that is thought to be triggered by receptor binding, a suggested prerequisite to subsequent fusion and core entry. These results indicate that the block to gene transfer in this system, and probably in most of the current chimeric retroviral vectors to date, is the inability of the chimeric envelope protein to undergo this obligatory conformational change.

3T3 Cells↗
PubMed1999

Phospholipase D1 in caveolae: regulation by protein kinase Calpha and caveolin-1.

Caveolae are small plasma membrane invaginations that have been implicated in cell signaling, and caveolin is a principal structural component of the caveolar membrane. Previously we have demonstrated that protein kinase Calpha (PKCalpha) directly interacts with phospholipase D1 (PLD1), activating the enzymatic activity of PLD1 in the presence of phorbol 12-myristate 13-acetate (PMA) [Lee, T. G., et al. (1997) Biochim. Biophys. Acta 1347, 199-204]. In this study, using a detergent-free procedure for the purification of a caveolin-enriched membrane fraction (CEM) and immunoblot analysis, we show that PLD1 is enriched in the CEMs of 3Y1 rat fibroblasts. Purified PLD1 directly bound to a glutathione S-transferase-caveolin-1 fusion protein in in vitro binding assays. The association of PLD1 with caveolin-1 could be completely eliminated by preincubation of PLD1 with an oligopeptide corresponding to the scaffolding domain (amino acids 82-101) of caveolin-1, indicating that caveolin-1 interacts with PLD1 through the scaffolding domain. The peptide also inhibited PKCalpha-stimulated PLD1 activity and the interaction between PLD1 and PKCalpha with an IC50 of 0.5 microM. PMA elicits translocation of PKCalpha to the CEMs, inducing PLD activation through the interaction of PKCalpha with PLD1 in the CEMs. Caveolin-1 also coimmunoprecipitated with PLD1 in the absence of PMA, and the amounts of coimmunoprecipitated caveolin-1 decreased in response to treatment with PMA. Taken together, our results suggest a new mechanism for the regulation of the PKCalpha-dependent PLD activity through the molecular interaction between PLD1, PKCalpha, and caveolin-1 in caveolae.

Animals↗
PubMed1999

'Saccharomyces cerevisiae MSH2/6 complex interacts with Holliday junctions and facilitates their cleavage by phage resolution enzymes.

Genetic and biochemical studies have indicated that mismatch repair proteins can interact with recombination intermediates. In this study, gel shift assays and electron microscopic analysis were used to show that the Saccharomyces cerevisiae MSH2/6 complex binds to Holliday junctions and has an affinity and specificity for them that is at least as high as it has as for mispaired bases. Under equilibrium binding conditions, the MSH2/6 complex had a Kd of binding to Holliday junctions of 0.5 nM. The MSH2/6 complex enhanced the cleavage of Holliday junctions by T4 endonuclease VII and T7 endonuclease I. This is consistent with the view that the MSH2/6 complex can function in both mismatch repair and the resolution of recombination intermediates as predicted by genetic studies.

Base Sequence↗
PubMed1999

Isolation of human anti-branched chain alpha-oxo acid dehydrogenase-E2 recombinant antibodies by Ig repertoire cloning in idiopathic dilated cardiomyopathy.

The generation of human monoclonal autoantibodies is critical for understanding humoral immune response in autoimmunity. In this study, Ig gene repertoire cloning was performed from a regional lymph node of a patient with idiopathic dilated cardiomyopathy (IDCM), and the resulting combinatorial IgG library was screened with bovine branched chain alpha-oxo acid dehydrogenase-E-2 (BCOADC-E2), one of the autoantigens in IDCM. After three rounds of affinity selection, we isolated three human recombinant IgG Fab molecules, named BC1, BC2 and BC3, that specifically react with BCOADC-E2 by ELISA. Interestingly, BC2 showed weak cross-reactivity to pyruvate dehydrogenase complex-E2 (PDC-E2), another mitochondrial autoantigen found in primary biliary cirrhosis (PBC), and their kappa light chain genes have 95% homology with a light chain of the human anti-DNA antibody. Although the exact pathogenic effect of anti-BCOADC-E2 autoantibodies is still unknown in IDCM, the potential binding specificity and limited light chain gene usage of our recombinant IgG molecules may shed light on the initial mechanism as to how autoantibodies start developing in IDCM.

Acyltransferases↗
PubMed1999

Crystallographic and kinetic investigations on the mechanism of 6-pyruvoyl tetrahydropterin synthase.

The enzyme 6-pyruvoyl tetrahydropterin synthase (PTPS) catalyses the second step in the de novo biosynthesis of tetrahydrobiopterin, the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin. The Zn and Mg-dependent reaction includes a triphosphate elimination, a stereospecific reduction of the N5-C6 double bond and the oxidation of both side-chain hydroxyl groups. The crystal structure of the inactive mutant Cys42Ala of PTPS in complex with its natural substrate dihydroneopterinetriphosphate was determined at 1.9 A resolution. Additionally, the uncomplexed enzyme was refined to 2.0 A resolution. The active site of PTPS consists of the pterin-anchoring Glu A107 neighboured by two catalytic motifs: a Zn(II) binding site and an intersubunit catalytic triad formed by Cys A42, Asp B88 and His B89. In the free enzyme the Zn(II) is in tetravalent co-ordination with three histidine ligands and a water molecule. In the complex the water is replaced by the two substrate side-chain hydroxyl groups yielding a penta-co-ordinated Zn(II) ion. The Zn(II) ion plays a crucial role in catalysis. It activates the protons of the substrate, stabilizes the intermediates and disfavours the breaking of the C1'C2' bond in the pyruvoyl side-chain. Cys A42 is activated by His B89 and Asp B88 for proton abstraction from the two different substrate side-chain atoms C1', and C2'. Replacing Ala A42 in the mutant structure by the wild-type Cys by modelling shows that the C1' and C2' substrate side-chain protons are at equal distances to Cys A42 Sgamma. The basicity of Cys A42 may be increased by a catalytic triad His B89 and Asp B88. The active site of PTPS seems to be optimised to carry out proton abstractions from two different side-chain C1' and C2' atoms, with no obvious preference for one of them. Kinetic studies with dihydroneopterin monophosphate reveal that the triphosphate moiety of the substrate is necessary for enzyme specifity.

Animals↗
PubMed1999

Mitoxantrone, etoposide, and cytarabine plus cyclosporine for patients with relapsed or refractory acute myeloid leukemia: an Eastern Cooperative Oncology Group pilot study.

BACKGROUND: One potential mechanism of drug resistance to chemotherapy is the overexpression of multidrug resistance (MDR) genes coding for P-glycoprotein (P-gp), which leads to reduced intracellular retention of chemotherapy. This study tested the efficacy and toxicity of mitoxantrone, etoposide, and intermediate dose cytarabine (MEC) with cyclosporine (CSP) as an MDR modulator in patients with recurrent and refractory acute myeloid leukemia, and also correlated P-gp expression in leukemia cells with response. METHODS: Thirty-eight eligible patients who were in first recurrence after < 6 months of complete remission (CR) (11 patients), refractory to initial induction therapy or to one attempt at reinduction after recurrence (18 patients), in second recurrence (4 patients), or in recurrence after either allogeneic or autologous bone marrow transplantation (5 patients) received either MEC alone (13 patients) or MEC-CSP (25 patients). CSP was given as a loading dose of 6 mg/kg for 2 hours intravenously (i.v.) starting 2 hours before the first dose of etoposide, followed by a continuous i.v. infusion of 18 mg/kg/day for 98 hours. RESULTS: Three of the 13 patients (23%) who received MEC achieved CR, as did 6 of the 25 patients (24%) who received MEC-CSP. The median remission duration for all patients who achieved CR was 149 days (range, 26-466 days), 91 days (range, 81-172 days) for the 3 patients who received MEC, and 189.5 days (range, 26-466 days) for the patients treated with MEC-CSP. The median survival for the patients treated with MEC and MEC-CSP was 104 and 72 days, respectively. CONCLUSIONS: No significant association was found between P-gp expression and response. No apparent benefit in the CR rate, remission duration, or survival was observed with the addition of CSP to MEC.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Neutron radiotherapy for the treatment of locally advanced major salivary gland tumors.

BACKGROUND: Malignant salivary gland tumors are rare tumors of the head and neck region. The treatment of these tumors has generally consisted of surgical extirpation, with postoperative radiotherapy improving locoregional control and survival in patients with high risk tumors. Neutron radiotherapy has been found to be more efficacious than conventional radiotherapy in the setting of inoperable or subtotally resected salivary gland tumors. METHODS: One hundred forty-eight patients with malignant salivary tumors of major salivary gland origin were treated at the University of Washington Medical Center with fast neutron radiotherapy between the years 1984 and 1995. One hundred twenty-eight patients were treated with curative intent, and of these, 120 patients had evidence of gross residual disease at the time of treatment. These patients constitute the main analysis of this paper. Of these patients, 19% had recurrent disease, 39% were initially seen with positive lymph nodes, and 11% had previously received full dose conventional radiotherapy. At the time of analysis, the median period at risk of survivors was 26 months. RESULTS: The 5-year actuarial locoregional control rate for all patients with gross tumor treated with curative intent was 59%. A tumor size < or =4 cm was associated with an excellent locoregional control rate (80%), and cause-specific survival (73%) at 5 years compared with patients with larger tumors (35% and 22%, respectively, p<.001 in both cases). On univariate analysis, there appeared to be an advantage in locoregional control for patients with smaller sized tumors (< or =4 cm) who underwent an attempted surgical extirpation. Locoregional control was excellent (100%) in patients having a complete surgical resection of their tumors and undergoing postoperative neutron radiotherapy because of the presence of other high risk factors. Lymph node status at the time of treatment, base of skull involvement, and male sex were associated with the development of distant metastasis, with 52% of node positive patients developing distant metastases by 5 years, compared with 32% of node negative patients (p = .04). CONCLUSIONS: Neutron radiotherapy is an effective form of treatment for patients with high risk, locally advanced tumors of major salivary gland origin. An initial surgical resection appears beneficial in patients for whom such an approach is feasible.

Adult↗
PubMed1999

Mouse-to-rat testicle transplantation.

This report details mouse-to-rat testicular transplantation with immediate revascularization. Donor preparation involved grafting a long segment of aorta and inferior vena cava (IVC) containing the testicular artery and vein. The graft aorta and IVC were anastomosed to the rat aorta and IVC, respectively. Vasovasostomy was completed and the scrotal epithelia were anastomosed to draw the graft toward the host scrotal sac. Twenty-nine of 53 transplants were determined to be viable. Histologically, 6- to 18-hr-old grafts displayed moderate to minimal polymorphonuclear neutrophil (PMN) infiltrates. Ischemia set in somewhere between 18-24 hr postoperatively. Beyond 24 hr the grafts displayed progressive infiltration of PMN and perivascular and intertubular lymphocytes, disorganization of the germinal epithelium, and cessation of spermatogenesis.

Animals↗