Biomedical subjects

L Tong

Publications and source records attributed to L Tong.

At least 37 records · Page 2Linked to original sources

PubMed2001

Binding site elucidation of hydantoin-based antagonists of LFA-1 using multidisciplinary technologies: evidence for the allosteric inhibition of a protein--protein interaction.

The binding site on the lymphocyte function-associated antigen-1 (LFA-1) of a class of hydantoin-based antagonists of leukocyte cell adhesion has been identified. This site resides in the inserted-domain (I-domain) of the CD11a chain at a location that is distal to residues known to be required for interactions with the intercellular adhesion molecules. This finding supports the hypothesis that the molecules are antagonizing cell adhesion via an allosteric modification of LFA-1. The binding site was identified using an integrated immunochemical, chemical, and molecular modeling approach. Antibodies that map to epitopes on the I-domain were blocked from binding to the purified protein by the hydantoins, indicating that the hydantoin-binding site resides on the I-domain. Photoaffinity labeling of the I-domain followed by LC/MS and LC/MS/MS analysis of the enzymatic digest identified proline 281 as the primary amino acid residue covalently attached to the photoprobe. Distance constraints derived from this study coupled with known SAR considerations allowed for the construction of a molecular model of the I-domain/inhibitor complex. The atomic details of the protein/antagonist interaction were accurately predicted by this model, as subsequently confirmed by the X-ray crystal structure of the complex.

Allosteric Site↗

PubMed2001

Investigating the role of histidine 157 in the catalytic activity of human cytomegalovirus protease.

Herpesvirus proteases belong to a new class of serine proteases and contain a novel Ser-His-His catalytic triad, while classical serine proteases have an acidic residue as the third member. To gain a better understanding of the molecular basis for the functional role of the third-member His residue, we have carried out structural and biochemical investigations of human cytomegalovirus (HCMV) protease that bears mutations of the His157 third member. Kinetic studies showed that all the mutants have reduced catalytic activity. Structural studies revealed that a solvent molecule is hydrogen-bonded to the His63 second member and Ser134 in the H157A mutant, partly rescuing the activity of this mutant. This is confirmed by our kinetic and structural observations on the S134A/H157A double mutant, which showed further reductions in the catalytic activity. The structure of the H157A mutant is also in complex with the PMSF inhibitor. The H157E mutant has the best catalytic activity among the mutants; its structure, however, showed conformational readjustments of the His63 and Ser132 residues. The Ser132-His63 diad of HCMV protease has similar activity as the diads in classical serine proteases, whereas the contribution of the His157 third member to the catalysis is much smaller. Finally, structural comparisons revealed the presence of two conserved structural water molecules at the bottom of the S(1) pocket, suggesting a possible new direction for the design of HCMV protease inhibitors.

Alanine↗

PubMed2001

Crystallization and preliminary X-ray crystallographic studies of wild-type human ornithine transcarbamylase and two naturally occurring mutants at position 277.

Wild-type human ornithine transcarbamylase (OTCase) and two mutants (R277Q and R277W) that cause 'late-onset' hyperammonemia were crystallized and a preliminary structure determination was carried out. The unliganded wild-type enzyme crystallizes in the cubic space group I23, with unit-cell parameters a = b = c = 203.4 A. R277Q crystallizes in two crystal forms under the same crystallization conditions. One crystal form is isomorphous to that of unliganded wild-type crystals, with unit-cell parameters a = b = c = 202.2 A. The second form also belongs to a cubic space group, P4(3)32, but has unit-cell parameters a = b = c = 139.8 A. R277W crystals are isomorphous to the second crystal form of R277Q, with unit-cell parameters a = b = c = 138.7 A. None of these crystal forms is isomorphous to other crystal forms of OTCase that have been studied. The structures in both crystal forms have been solved using molecular replacement. In the first crystal form there are two monomers in the asymmetric unit, corresponding to a solvent content of 75%. Because of its high molecular and crystal symmetry and the presence of non-crystallographic symmetry, this structure could not be solved with AMoRe or X-PLOR, but was solved successfully with COMO. There is only one monomer in the asymmetric unit in the second crystal form, corresponding to a solvent content of 62%. This structure was successfully solved with AMoRe.

Crystallization↗

PubMed2001

Human ornithine transcarbamylase: crystallographic insights into substrate recognition and conformational changes.

Two crystal structures of human ornithine transcarbamylase (OTCase) complexed with the substrate carbamoyl phosphate (CP) have been solved. One structure, whose crystals were prepared by substituting N-phosphonacetyl-L-ornithine (PALO) liganded crystals with CP, has been refined at 2.4 A (1 A=0.1 nm) resolution to a crystallographic R factor of 18.4%. The second structure, whose crystals were prepared by co-crystallization with CP, has been refined at 2.6 A resolution to a crystallographic R factor of 20.2%. These structures provide important new insights into substrate recognition and ligand-induced conformational changes. Comparison of these structures with the structures of OTCase complexed with the bisubstrate analogue PALO or CP and L-norvaline reveals that binding of the first substrate, CP, induces a global conformational change involving relative domain movement, whereas the binding of the second substrate brings the flexible SMG loop, which is equivalent to the 240s loop in aspartate transcarbamylase, into the active site. The model reveals structural features that define the substrate specificity of the enzyme and that regulate the order of binding and release of products.

Amino Acid Sequence↗

PubMed2001

Beta -amyloid-(1-42) impairs activity-dependent cAMP-response element-binding protein signaling in neurons at concentrations in which cell survival Is not compromised.

Cognitive impairment is a major feature of Alzheimer's disease and is accompanied by beta-amyloid (Abeta) deposition. Transgenic animal models that overexpress Abeta exhibit learning and memory impairments, but neuronal degeneration is not a consistent characteristic. We report that levels of Abeta-(1-42), which do not compromise the survival of cortical neurons, may indeed interfere with functions critical for neuronal plasticity. Pretreatment with Abeta-(1-42), at sublethal concentrations, resulted in a suppression of cAMP-response element-binding protein (CREB) phosphorylation, induced by exposure to either 30 mm KCl or 10 microm N-methyl-d-aspartate. The effects of Abeta-(1-42) seem to involve mechanisms unrelated to degenerative changes, since Abeta-(25-35), a toxic fragment of Abeta, at sublethal concentrations did not interfere with activity-dependent CREB phosphorylation. Furthermore, caspase inhibitors failed to counteract the Abeta-(1-42)-evoked suppression of CREB activation. Abeta-(1-42) also interfered with events downstream of activated CREB. The Abeta-(1-42) treatment suppressed the activation of the cAMP response element-containing brain-derived neurotrophic factor (BDNF) exon III promoter and the expression of BDNF exon IIII mRNA induced by neuronal depolarization. In view of the critical role of CREB and BDNF in neuronal plasticity, including learning and memory, the observations indicate a novel pathway through which Abeta may interfere with neuronal functions and contribute to cognitive deficit in Alzheimer's disease before the stage of massive neuronal degeneration.

Amino Acid Chloromethyl Ketones↗

PubMed2001

Effects of exercise on gene-expression profile in the rat hippocampus.

Exercise has beneficial effects on brain function, including the promotion of plasticity and the enhancement of learning and memory performance. Previously we found that exercise increases the expression of certain neurotrophic factors including brain derived neurotrophic factor in the rat hippocampus. To further explore the molecular mechanisms underlying these changes, we used high-density oligonucleotide microarrays containing probe sets representing approximately 5000 genes to analyze the level of gene transcripts in the hippocampus of rats voluntary running for 3 weeks in comparison with sedentary animals. An improved statistical approach for the analysis of DNA microarray data, Cyber-T, was utilized in data analysis. Here we show that exercise leads to changes in the level of a large number of gene transcripts, many of which are known to be associated with neuronal activity, synaptic structure, and neuronal plasticity. Our data indicate that exercise elicits a differential gene expression pattern with significant changes in genes of relevance for brain function.

Aging↗

PubMed2001

A simple and rapid in situ preconcentration method for the determination of phosphate in environmental waters by use of solid-phase extraction, and its applications to brackish lake waters.

A simple and rapid in situ preconcentration method for the determination of phosphate in environmental waters has been developed for field analysis. This method is based on solid-phase extraction on a zirconium-loaded Sep-Pack Accell CM cartridge (Zr-SP) and is applicable to studies in which sampling is performed by use of a graduated syringe to prevent contamination and to ensure easy operation at sampling sites. The Zr-SP cartridge was prepared by passing 0.1 mol L(-1) zirconium solution through a Sep-Pak Accell CM cartridge, packed with cation exchange sorbent based on a silica matrix. The adsorption of phosphate and its desorption depend only on the pH of the solution. A water sample containing phosphate was adjusted to pH 2 and passed through the Zr-SP cartridge to collect it. The retained phosphate was quantitatively eluted with 0.5 mol L(-1) sodium hydroxide solution. The phosphate retained in the Zr-SP cartridge was stable for at least one month. The established preconcentration method was successfully applied to brackish lake waters to investigate seasonal changes in the distribution and behavior of phosphate in a brackish lake.

Environmental Monitoring↗

PubMed2001

Materials selection for a dry atmospheric mercury deposits sampler.

In order to develop a sampler for measuring dry deposition rates for atmospheric mercury, suitable materials are needed that neither adsorb nor release mercury. In this paper, four materials (polyvinyl chloride (PVC), acrylonitrile-butadiene-styrene (ABS), Teflon and glass) were tested. Each of the materials was placed in a beaker containing mercuric solution under varied conditions for pH, preservative concentration, initial mercuric concentration, temperature, acid type and contact time of the material. The concentrations of the mercuric solutions were determined using the cold-vapor atomic absorption (CVAA) technique. The experimental results show that glass has the lowest absolute net adsorption rates (NARs) of 0.026-1.13 pg/m2 among the materials tested. Teflon, PVC and ABS have NARs ranging from 0.54 to 10.4 microg/M2 over an adsorption duration of one or two weeks. ABS has significantly higher adsorption rates for mercury than PVC and Teflon, indicating its inappropriateness as the material for building the sampler. Teflon and PVC will be the materials of choice for the sampler. The experimental results can also be used in establishing appropriate sampling conditions in the field.

Acrylic Resins↗

PubMed2001

Physical activity elicits sustained activation of the cyclic AMP response element-binding protein and mitogen-activated protein kinase in the rat hippocampus.

To elucidate molecular mechanisms involved in physical activity-induced beneficial effects on brain function, we studied in rats the influence of voluntary running on the activation in the hippocampus of cyclic AMP response element-binding protein (CREB) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK). These are signaling molecules that play critical roles in synaptic plasticity, including learning and memory. Exercise resulted in an increase in the level of the activated transcription factor, CREB phosphorylated at Ser-133. The amount of the activated transcription factor about doubled already after 1 night of running and remained elevated for at least a week, although control levels were restored after 1 month of exercise. In addition, binding activity in nuclear extracts to cyclic AMP response element (CRE) motif containing oligonucleotides increased significantly in the hippocampus after 3 nights of exercise, although the total amount of the immunochemically identified CREB remained unaltered. Electrophoretic mobility supershift assays indicated that the increased binding was due to the recruitment of members of this transcription factor family, in addition to the CREB proper. Voluntary running also resulted in an increase in the level of phosphorylated MAPK (both p42 and p44). The time-courses of the increases in the level of the phosphorylated protein kinase and the activated transcription factor were different. In comparison with the activated CREB, the increase in the phosphorylated MAPK was delayed, but lasted longer, being detectable even after 1 month of exercise. These observations are consistent with the view that the relatively long-lasting activation of these signaling molecules participates in the regulation of genes, such as the neurotrophin genes, and contributes to the beneficial effects of physical exercise on brain function.

Animals↗

PubMed2001

Does macular laser alter the refraction in a diabetic with maculopathy? A pilot study.

PURPOSE: To determine whether macular photocoagulation has any effect on refraction in diabetics. METHODS: This was a prospective study of 12 eyes of 12 diabetics with refraction at 2 weeks before and at 4 weeks and 4 months after argon laser. This was combined with a questionnaire survey of attitude among ophthalmologists on prescribing spectacles for diabetics awaiting macular laser. The main outcome measure was the spherical equivalents (SEQ) before and after treatment. RESULTS: Only 36% of ophthalmologists surveyed would prescribe corrective lenses in patients waiting for macular photocoagulation. The median change in magnitude of SEQ at 4-6 weeks post-laser compared to pre-laser was 0.36 D (interquartile range 0.18-0.50 D). The median change in SEQ at 4 months was 0.25 D (interquartile range 0.12-0.56 D). These changes in SEQ from pretreatment values were not statistically significant on Wilcoxon signed-rank test (p = 0.17 and 0.10, respectively). The correlation coefficient between difference and average SEQ was 0.02 (95% CI -0.14 to 0.19, p = 0.75) at 4 weeks and 0.11 (95% CI -0.06 to 0.275, p = 0.17) at 4 months, indicating no tendency for change in refraction with increasing ametropia. CONCLUSION: This pilot study implies that argon laser photocoagulation in diabetic maculopathy does not alter refraction. Prescription of corrective lenses can be performed prior to laser therapy, if required.

Aged↗

PubMed2001

Molecular mechanism for dimerization to regulate the catalytic activity of human cytomegalovirus protease.

Biochemical studies indicate that dimerization is required for the catalytic activity of herpesvirus proteases, whereas structural studies show a complete active site in each monomer, away from the dimer interface. Here we report kinetic, biophysical and crystallographic characterizations of structure-based mutants in the dimer interface of human cytomegalovirus (HCMV) protease. Such mutations can produce a 1,700-fold reduction in the kcat while having minimal effects on the K(m). Dimer stability is not affected by these mutations, suggesting that dimerization itself is insufficient for activity. There are large changes in monomer conformation and dimer organization of the apo S225Y mutant enzyme. However, binding of an activated peptidomimetic inhibitor induced a conformation remarkably similar to the wild type protease. Our studies suggest that appropriate dimer formation may be required to indirectly stabilize the protease oxyanion hole, revealing a novel mechanism for dimerization to regulate enzyme activity.

Amino Acid Sequence↗

PubMed2001

Association of macular involvement with proliferative retinopathy in Type 2 diabetes.

AIMS: To determine whether documented evidence of pre-existing maculopathy is present in Type 2 diabetic patients who require photocoagulation or vitrectomy for proliferative disease. This is a retrospective case notes review. METHODS: The study was performed at Queen's Medical Centre, UK. All patients listed in the laser register and operating theatre register over 5 years (March 1994 to March 1999) who had undergone pan retinal photocoagulation (PRP) for diabetic retinopathy, in one consultant firm, were studied. The medical records of patients who had undergone vitrectomy as their first treatment for proliferative diabetic retinopathy in a vitreo-retinal firm over the 5 years were examined. The proportion of patients with documented maculopathy before development of proliferative retinopathy was determined. RESULTS: All except 1/134 or 0.7% (95% confidence interval 0.1-4.5%) patients had documentation of macular signs in at least one eye prior to the patient's first session of PRP. Of the remaining 133, 104 (78%) had had focal or grid photocoagulation of the macula. The median time between the documentation of maculopathy before the first PRP treatment session was 16 months (interquartile range 7-36). Fourteen patients had vitrectomy and endolaser performed as the initial treatment for proliferative disease. All patients had documented maculopathy before the onset of proliferative disease. CONCLUSIONS: The results of this study suggest that in Type 2 diabetes, proliferative disease occurs relatively late compared with maculopathy. In such patients, maculopathy is almost invariably present when proliferative disease is detected.

Age of Onset↗

PubMed2001

Use of three free flaps based on a single vascular pedicle for complex hand reconstruction in an electrical burn injury: a case report.

The use of conjoint flaps based on the dorsalis pedis artery enabled a transfer of 3 free flaps-dorsalis pedis flap, fillet flap of the second toe, and trimmed large toe-to reconstruct a severely traumatized hand in a 12-year-old girl. High-voltage electrical burn injury had caused a large wound over the volar wrist and exposed the flexor tendons and median/ulnar nerves. In addition, she suffered a partial loss of the thumb and had an open wound at the base of the index finger. The application of the conjoint flaps restored hand function in a one-stage procedure.

Burns, Electric↗

PubMed2001

CAK-independent activation of CDK6 by a viral cyclin.

In normal cells, activation of cyclin-dependent kinases (cdks) requires binding to a cyclin and phosphorylation by the cdk-activating kinase (CAK). The Kaposi's sarcoma-associated herpesvirus encodes a protein with similarity to D-type cyclins. This KSHV-cyclin activates CDK6, alters its substrate specificity, and renders CDK6 insensitive to inhibition by the cdk inhibitor p16(INK4a). Here we investigate the regulation of the CDK6/KSHV-cyclin kinase with the use of purified proteins and a cell-based assay. We find that KSHV-cyclin can activate CDK6 independent of phosphorylation by CAK in vitro. In addition, CAK phosphorylation decreased the p16(INK4a) sensitivity of CDK6/KSHV-cyclin complexes. In cells, expression of CDK6 or to a lesser degree of a nonphosphorylatable CDK6(T177A) together with KSHV-cyclin induced apoptosis, indicating that CDK6 activation by KSHV-cyclin can proceed in the absence of phosphorylation by CAK in vivo. Coexpression of p16 partially protected cells from cell death. p16 and KSHV-cyclin can form a ternary complex with CDK6 that can be detected by binding assays as well as by conformational changes in CDK6. The Kaposi's sarcoma-associated herpesvirus has adopted a clever strategy to render cell cycle progression independent of mitogenic signals, cdk inhibition, or phosphorylation by CAK.

Apoptosis↗

PubMed2001

Corneal and refractive error astigmatism in Singaporean schoolchildren: a vector-based Javal's rule.

BACKGROUND: Traditional approaches to Javal's rule do not use data from subjects with oblique astigmatism and have not been used to make predictions about subjects with oblique astigmatism. Vector approaches to analyzing refractive error can circumvent these problems. METHODS: Subjects were 993 Singaporean schoolchildren. We performed linear regression of refractive error astigmatism on corneal astigmatism, using J0 vectors to describe with-the-rule and against-the-rule astigmatism and J45 vectors to describe oblique astigmatism. RESULTS: We obtained the following statistically significant regression relationships: RJ0 = 0.931 x CJ0 - 0.276 and RJ45 = 0.638 x CJ45 + 0.010, where R and C denote refractive error astigmatism and corneal astigmatism, respectively. CONCLUSION: Our vector-based Javal's rule gives closer predictions of refractive astigmatism than the original Javal's rule and the simplified Javal's rule and can be applied in cases of corneal oblique astigmatism.

Adolescent↗

PubMed2001

Function of Rta is essential for lytic replication of murine gammaherpesvirus 68.

Rta, encoded primarily by open reading frame 50, is well conserved among gammaherpesviruses. It has been shown that the Rta proteins of Epstein Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV, or HHV-8), and murine gammaherpesvirus 68 (MHV-68; also referred to as gamma HV68) play an important role in viral reactivation from latency. However, the role of Rta during productive de novo infection has not been characterized in gammaherpesviruses. Since there are cell lines that can support efficient productive de novo infection by MHV-68 but not EBV or KSHV, we examined whether MHV-68 Rta plays a role in initiating viral lytic replication in productively infected cells. Rta, functioning as a transcriptional activator, can activate the viral promoter of early lytic genes. The amino acid sequence alignments of the Rta homologues suggest that the organizations of their functional domains are similar, with the DNA binding and dimerization domains at the N terminus and the trans-activation domain at the C terminus. We constructed two mutants of MHV-68 Rta, Rd1 and Rd2, with deletions of 112 and 243 amino acids from the C terminus, respectively. Rd1 and Rd2 could no longer trans-activate the promoter of MHV-68 gene 57, consistent with the deletions of their trans-activation domains at the C terminus. Furthermore, Rd1 and Rd2 were able to function as dominant-negative mutants, inhibiting trans-activation of wild-type Rta. To study whether Rd1 and Rd2 blocked viral lytic replication, purified virion DNA was cotransfected with Rd1 or Rd2 into fibroblasts. Expression of viral lytic proteins was greatly suppressed, and the yield of infectious viruses was reduced up to 10(4)-fold. Stable cell lines constitutively expressing Rd2 were established and infected with MHV-68. Transcription of the immediate-early gene, rta, and the early gene, tk, of the virus was reduced in these cell lines. The presence of Rd2 also led to attenuation of viral lytic protein expression and virion production. The ability of Rta dominant-negative mutants to inhibit productive infection suggests that the trans-activation function of Rta is essential for MHV-68 lytic replication. We propose that a single viral protein, Rta, governs the initiation of MHV-68 lytic replication during both reactivation and productive de novo infection.

Animals↗

PubMed2001

Sensitivity and specificity of a new scoring system for diabetic macular oedema detection using a confocal laser imaging system.

AIM: To assess the use of the Heidelberg retina tomograph (HRT) in screening for sight threatening diabetic macular oedema in a hospital diabetic clinic, using a new subjective analysis system (SCORE). METHODS: 200 eyes of 100 consecutive diabetic patients attending a diabetologist's clinic were studied, all eyes had an acuity of 6/9 or better. All patients underwent clinical examination by an ophthalmologist. Using the HRT, one good scan was obtained for each eye centred on the fovea. A System for Classification and Ordering of Retinal Edema (SCORE) was developed using subjective assessment of the colour map and the reflectivity image. The interobserver agreement of using this method to detect macular oedema was assessed by two observers (ophthalmic trainees) who were familiarized with SCORE by studying standard pictures of eyes not in the study. All scans were graded from 0-6 and test positive cases were defined as having a SCORE value of 0-2. The sensitivity of SCORE was assessed by pooling the data with an additional 88 scans of 88 eyes in order to reduce the confidence interval of the index. RESULTS: 12 eyes in eight out of the 100 patients had macular oedema clinically. Three scans in three patients could not be analysed because of poor scan quality. In the additional group of scans 76 out of 88 eyes had macular oedema clinically. The scoring system had a specificity of 99% (95% CI 96-100) and sensitivity of 67% (95% CI 57-76). The predictive value of a negative test was 87% (95% CI 82-99), and that of a positive test was 95% (95% CI 86-99). The mean difference of the SCORE value between two observers was -0.2 (95% CI -0.5 to +0.07). CONCLUSIONS: These data suggest that SCORE is potentially useful for detecting diabetic macular oedema in hospital diabetic patients.

Adult↗

PubMed2001

Analysis of publication trends in two internationally renowned ophthalmology journals.

International journals represent a forum for exchange of current information with contributions from all over the world. High standards are essential. In this report, we compared the publishing trends of two internationally renowned ophthalmology journals--the British Journal of Ophthalmology (BJO) and the American Journal of Ophthalmology (AJO).

Bibliometrics↗