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Biomedical subjects

K Lim

Publications and source records attributed to K Lim.

At least 73 records · Page 4Linked to original sources
PubMed1994

Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp41 from HIV.

The 3-dimensional crystal structure of glutathione S-transferase (GST) of Schistosoma japonicum (Sj) fused with a conserved neutralizing epitope on gp41 (glycoprotein, 41 kDa) of human immunodeficiency virus type 1 (HIV-1) (Muster T et al., 1993, J Virol 67:6642-6647) was determined at 2.5 A resolution. The structure of the 3-3 isozyme rat GST of the mu gene class (Ji X, Zhang P, Armstrong RN, Gilliland GL, 1992, Biochemistry 31:10169-10184) was used as a molecular replacement model. The structure consists of a 4-stranded beta-sheet and 3 alpha-helices in domain 1 and 5 alpha-helices in domain 2. The space group of the Sj GST crystal is P4(3)2(1)2, with unit cell dimensions of a = b = 94.7 A, and c = 58.1 A. The crystal has 1 GST monomer per asymmetric unit, and 2 monomers that form an active dimer are related by crystallographic 2-fold symmetry. In the binding site, the ordered structure of reduced glutathione is observed. The gp41 peptide (Glu-Leu-Asp-Lys-Trp-Ala) fused to the C-terminus of Sj GST forms a loop stabilized by symmetry-related GSTs. The Sj GST structure is compared with previously determined GST structures of mammalian gene classes mu, alpha, and pi. Conserved amino acid residues among the 4 GSTs that are important for hydrophobic and hydrophilic interactions for dimer association and glutathione binding are discussed.

Amino Acid Sequence↗
PubMed1994

Selective antagonism of dopamine D1 and D2 receptors does not block the development of behavioral sensitization to cocaine.

The objective of this study was to determine whether the development of behavioral sensitization to cocaine could be prevented by either D1 or D2 selective dopamine receptor antagonists. Male Wistar rats were treated daily for 7 days with either cocaine (15 mg/kg, IP) or vehicle in combination with the D1 dopamine antagonist SCH 23390 (0.3 mg/kg, SC), the D2 dopamine antagonist sulpiride (100 mg/kg, IP), or vehicle. After the daily injections, the rats were tested for locomotor activity in photocell arenas. Twenty-four hours after the last pre-exposure test session, all rats were given a challenge injection of cocaine (15 mg/kg, IP) and tested for activity. Cocaine treatments produced a greater relative increase in locomotor activity with repeated exposure (i.e. sensitization). Moreover, this increase in cocaine-induced locomotor activity was attenuated by both SCH 23390 and sulpiride. In contrast, neither sulpiride nor SCH 23390 blocked the development of behavioral sensitization to cocaine. That is, rats pretreated with sulpiride or SCH 23390 and cocaine did not differ from rats pre-exposed only to cocaine when given a cocaine challenge injection. These results suggest that behavioral sensitization to cocaine may develop through either D1 or D2 dopamine receptor stimulation or possibly through stimulation of some non-dopaminergic receptor.

Animals↗
PubMed1994

Osteoarthritis of the scapho-trapezial joint.

In a cohort of 199 patients with OA we studied the prevalence of radiographic scapho-trapezial joint osteoarthritis (STJ OA) and its clinical features. The prevalence of STJ OA was 38%. Lone STJ OA occurs but is uncommon (11%). In contrast, STJ OA in combination with first carpo-metacarpal joint OA was found in 27%. There appears to be an age-related progression from those without disease to lone disease to combined disease. The most frequent radiological features seen were joint space narrowing, subchondral sclerosis and osteophytes. There was a weak correlation between STJ OA and palpable swelling at the thumb base (phi = 0.231, P = 0.01) but no association was found between STJ OA and tenderness at the thumb base.

Adult↗
PubMed1994

Acute inflammatory polyarthritis in association with tamoxifen.

The antioestrogen drug tamoxifen is widely used to treat breast carcinoma. We report three cases in which its use was temporally related to the development of an acute inflammatory polyarthritis resembling RA; a side-effect not previously reported. Possible mechanisms for such an effect are discussed.

Acute Disease↗
PubMed1993

Regenerating liver-specific transacting factors of H2B histone gene are newly synthesized during liver regeneration.

We have investigated DNA synthesis and levels of H2B histone mRNA, and the binding pattern of nuclear proteins to various elements in the rat H2B histone gene upstream region with DNase I footprinting assay. Both DNA synthesis and H2B histone mRNA level were increased with maximal stimulation reached at 24 hrs and 36 hrs after partial hepatectomy, respectively. In DNase I footprinting analysis, the nuclear factors interacting with the three elements, TATA at -19 bp (site AR), site B at -29 bp, and CAAT at -69 bp (site C) were required during maximal increase of H2B histone mRNA level after partial hepatectomy. The DNase I protection pattern by nuclear extract of the cycloheximide-treated regenerating liver showed the same results with normal liver. These results suggest that transcriptional regulation of H2B histone gene during liver regeneration may be mediated by nuclear factors that are newly induced by partial hepatectomy.

Animals↗
PubMed1993

An immunohistochemical study of opsin in photoreceptor cells following light-induced retinal degeneration in the rat.

Light-induced retinal degeneration has been hypothesized to be rhodopsin-mediated. However, the alterations induced in the opsin moiety of the rhodopsin molecule and its distribution in the rod cell after a photic insult have not been definitively established. We used light and electron immunohistochemistry to study the alterations in retinal opsin immunoreactivity in a rat model of retinal photic injury. In normal unexposed rat retinas, opsin immunoreactivity was restricted to the rod outer segments. At 6 h after a 24-h light exposure, opsin immunoreactivity was present in the rod outer segments in both the superior and inferior retina, but in addition marked immunoreactivity was present in the inner segments in the superior quadrant of the light-damaged retina. At 6 days after exposure, intense immunoreactivity was noted around the severely degenerating rod nuclei and inner segments. However, at 21 days following light exposure, opsin immunoreactivity in areas of recovery was again restricted to the short regenerated rod outer segments. It appears that, despite severe light-mediated retinal degeneration, anti-opsin immunoreactivity persisted in the photoreceptor cells but with an altered pattern in damaged rod outer segments and photoreceptor perikarya. However, opsin immunoreactivity relocated to the regenerated rod outer segments in the recovery phase.

Animals↗
PubMed1993

Solubilization of thiazolobenzimidazole using a combination of pH adjustment and complexation with 2-hydroxypropyl-beta-cyclodextrin.

The thiazolobenzimidazole 1-(2,6-difluorophenyl)-1H,3H-thiazolo[3,4-a] benzimidazole, TBI, is an experimental drug for the treatment of AIDS which exhibits a low water solubility (11 micrograms/mL) and is therefore difficult to administer in an injectable solution dosage form at a target solution concentration of 10 mg/mL. The compound has a single ionizable functional group and exhibits an increase in solubility with decreasing pH consistent with a pKa of 3.55, but the maximum solubility attainable by pH adjustment has been shown to be only 0.4 mg/mL (at pH 2). TBI has been found to form inclusion complexes in either its neutral or its protonated form with 2-hydroxypropyl-beta-cyclodextrin (HPCD). The equilibrium constants for 1:1 complex formation were found to be 81 and 1033 M-1 for the protonated and neutral species, respectively. Although the formation of protonated complex is less favored in comparison to the neutral complex, the contribution of this species to the overall solubility of TBI predominates at low pH. Thus, using a combined approach of pH adjustment and complexation with HPCD, a solubility enhancement of 3 orders of magnitude is possible. NMR proton spectroscopy and molecular modeling studies, conducted to understand the orientation of TBI in the complex and the effect of protonation, are described.

2-Hydroxypropyl-beta-cyclodextrin↗
PubMed1992

Presence of a repressor protein for testis-specific H2B (TH2B) histone gene in early stages of spermatogenesis.

The rat testis-specific TH2B histone gene assumes a hypomethylated chromatin structure at all stages of spermatogenesis. However, the TH2B mRNA level is very low in pre-meiotic spermatogenic cells and rises sharply in meiotic pachytene spermatocytes. The low level of TH2B mRNA in pre-meiotic spermatogenic cells appears to be a result of transcriptional repression of the gene by a pre-meiotic cell-specific protein which binds to a site between the TATA element and the transcription initiation site of TH2B gene.

Animals↗
PubMed1992

Quenching of fluorescein-conjugated lipids by antibodies. Quantitative recognition and binding of lipid-bound haptens in biomembrane models, formation of two-dimensional protein domains and molecular dynamics simulations.

Three model biomembrane systems, monolayers, micelles, and vesicles, have been used to study the influence of chemical and physical variables of hapten presentation at membrane interfaces on antibody binding. Hapten recognition and binding were monitored for the anti-fluorescein monoclonal antibody 4-4-20 generated against the hapten, fluorescein, in these membrane models as a function of fluorescein-conjugated lipid architecture. Specific recognition and binding in this system are conveniently monitored by quenching of fluorescein emission upon penetration of fluorescein into the antibody's active site. Lipid structure was shown to play a large role in affecting antibody quenching. Interestingly, the observed degrees of quenching were nearly independent of the lipid membrane model studied, but directly correlated with the chemical structure of the lipids. In all cases, the antibody recognized and quenched most efficiently a lipid based on dioctadecylamine where fluorescein is attached to the headgroup via a long, flexible hydrophilic spacer. Dipalmitoyl phosphatidylethanolamine containing a fluorescein headgroup demonstrated only partial binding/quenching. Egg phosphatidylethanolamine with a fluorescein headgroup showed no susceptibility to antibody recognition, binding, or quenching. Formation of two-dimensional protein domains upon antibody binding to the fluorescein-lipids in monolayers is also presented. Chemical and physical requirements for these antibody-hapten complexes at membrane surfaces have been discussed in terms of molecular dynamics simulations based on recent crystallographic models for this antibody-hapten complex (Herron et al., 1989. Proteins Struct. Funct. Genet. 5:271-280).

Animals↗
PubMed1991

Presence of a bi-directional S phase-specific transcription regulatory element in the promoter shared by testis-specific TH2A and TH2B histone genes.

During mammalian spermatogenesis, somatic histones are replaced by testis-specific variants. The synthesis of the variants occurs primarily in the germ cells undergoing meiosis in the absence of DNA replication. We have cloned the genes encoding rat somatic and testis-specific H2A (TH2A) histones. The two genes share 300 bp of 5' upstream region with respective H2B genes: somatic H2A with somatic H2B and testis-specific TH2A with testis-specific TH2B gene. The deduced amino acid sequences show that H2A and TH2A histones have eight amino acid differences in the first half of the molecules and three consecutive changes in the C-terminal region. TH2A gene is expressed only in testis. Although synthesis of TH2A and TH2B histones is independent of DNA replication and insensitive to inhibitors of DNA synthesis in testis, the regulatory region shared by the two genes contain a bi-directional S phase-specific transcription regulatory element. In addition, TH2A gene, like TH2B gene, contains the consensus sequence element in the 3' non-coding region which is involved in the S phase-specific stabilization of histone mRNA.

Amino Acid Sequence↗
PubMed1991

Different responses between thyroxine 5'-monodeiodinase and protein disulfide isomerase activities to dietary fat and thyroid status in rat liver.

On the hypothesis that rat hepatic microsomal type I iodothyronine 5'-monodeiodinase (MDI) would be identical to hepatic protein disulfide isomerase (PDI) (Boado et al. Biochem. Biophys. Res. Commun. 155 1297-1304 (1988)), we examined the responses of these enzyme activities to dietary fat and thyroid status in rats. The hepatic MDI activity was higher in rats fed high fat diet than in rats fed low fat diet, but the hepatic PDI activity showed the reverse responses to the diets. Propylthiouracil administration (hypothyroidism) lowered the MDI activity, but elevated the PDI activity. Thyroxine administration (hyperthyroidism) elevated the MDI activity but had no effect on the PDI activity. These results indicate that the two enzyme activities are regulated by different mechanisms in vivo, suggesting that MDI and PDI are not identical enzymes.

Administration, Oral↗
PubMed1990

Reliability of colposcopy and directed punch biopsy.

A group of 118 women underwent laser cone biopsy. Data were collected routinely on proforma case notes and entered into a computerized database. The histology of the cone biopsies was compared with that of previous, colposcopically directed punch biopsies, with the cytology of smears taken in the clinic and with the colposcopic diagnosis. The punch biopsy had a 54% false negative rate and neither of the two microinvasive carcinomas biopsied in this way were detected by the biopsy. Ten of 24 women with negative punch biopsies had CIN III in the cone. When the punch biopsy showed CIN II or worse, the cone biopsy confirmed the presence of CIN in 86%. There was some evidence of false negative cone biopsies. The data suggest that management should not be based solely upon the punch biopsy result but should include consideration of the cytology and colposcopy findings. Excisional methods of treatment are more likely to reveal early invasion and adenocarcinoma-in-situ.

Biopsy↗
PubMed1990

Characterization of the S-phase-specific transcription regulatory elements in a DNA replication-independent testis-specific H2B (TH2B) histone gene.

The testis-specific H2B histone (TH2B) gene is expressed in pachytene spermatocytes of meiotic prophase I during rat spermatogenesis. The TH2B RNA and histones are not synthesized in any other tissues, and the synthesis is independent of DNA replication. However, the cloned TH2B gene has two DNA sequence elements which stimulate transcription of the cloned gene in an S-phase-dependent manner when introduced into somatic cells. The factors interacting with the two elements, CCAAT at -127 base pairs and octamer ATTTGCAT at -93 base pairs, interact with each other to bring about a maximum stimulation of S-phase-dependent transcription. The level of CCAAT and octamer-binding proteins is unchanged during the cell cycle, and the S-phase-dependent transcription of TH2B and endogenous mouse H2B genes does not require synthesis of new proteins during the S phase. Cell cycle-specific posttranslational modification of regulatory proteins may be responsible for the S-phase-dependent transcription of H2B histone genes. The biological significance of the presence of S-phase-specific transcription regulatory elements in the DNA replication-independent and tissue-specific TH2B gene is not known.

Animals↗
PubMed1990

Utilization of labeled thymidine in DNA synthesis: studies for PET.

Carbon-111-thymidine and positron emission tomography (PET) have the potential for noninvasively measuring DNA synthesis in tumors and tissues. In order to validate this potential, one needs to construct accurate biochemical models that reflect the metabolism of thymidine, including its uptake and degradation as well as its incorporation into DNA. Our studies employed thymidine labeled in the methyl position using 11C, 14C, and 3H. Studies with rapidly proliferating tissues of mice demonstrated that most of the activity, 60 min after injection of labeled thymidine, was present in DNA, with smaller amounts found in metabolites. Studies in dogs, however, reveal that more activity was present in metabolites, rather than in the DNA of tumors and spleen tissue. HPLC analysis of canine blood after the injection of thymidine revealed rapid conversion of thymidine to thymine and other metabolites. We have found significant differences in the metabolism of [3H]thymidine versus thymidine labeled with radioactive carbon. These differences, which were not found when comparing 14C and 11C, indicate that [3H]thymidine is not an appropriate standard for comparison with PET studies employing [11C] thymidine. To accurately interpret images of [11C]thymidine as representations of DNA synthesis we are developing kinetic models that take into account the metabolism of thymidine and the contribution of degradation products to the 11C images.

Animals↗
PubMed1989

A simple assay for DNA transfection by incubation of the cells in culture dishes with substrates for beta-galactosidase.

Transfection efficiency of different cell types as well as promoter strength of cloned genes can be easily determined by direct assay of beta-galactosidase activity encoded from recombinant genes containing the E. coli beta-galactosidase gene. A substrate for beta-galactosidase, o-nitrophenyl-beta-D-galactopyranoside (ONPG), can be added to dishes containing the transfected cells, and the intensity of the colored enzyme product released from either the intact cell or cells lysed in the dishes can be determined. The results obtained by this assay are a reliable measure of transfection efficiency as well as promotor strength of the genes introduced into the cells. In addition, cells expressing the transfected gene can be identified and quantitated under a light microscope after incubation with X-gal. Thus, it is more convenient to use the E. coli beta-galactosidase gene than the chloramphenicol acetyltransferase gene as a reporter gene in the evaluation of DNA transfection.

Animals↗
PubMed1988

Human placenta S-adenosylmethionine: protein carboxyl O-methyltransferase (protein methylase II). Purification and characterization.

1. Protein methylase II was purified from human placenta approx. 8700-fold with a yield of 14%. 2. Unlike protein methylase II from other sources, the activity of human placenta enzyme was completely inhibited by 2 mM Cu2+. Other divalent ions were without effect. 3. Human chorionic gonadotropin (HCG), immunoglobulin A and calf thymus histones served as good in vitro substrates for the enzyme, particularly HCG. 4. The Km for S-adenosyl-L-methionine and Ki for S-adenosyl-L-homocysteine were 2.08 x 10(-6) and 5.8 x 10(-7) M, respectively. 5. The protein methylase II activity in human placenta changed with gestational age, the activity at 1st and 2nd trimester being approximately twice that of term placenta.

Cations, Divalent↗