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J Lim

Publications and source records attributed to J Lim.

At least 19 recordsLinked to original sources
PubMed1997

Evaluation of some fluorogenic substrates for continuous assay of aminopeptidase P.

Three potential fluorogenic substrates for assay of aminopeptidase P (AP-P) have been prepared and evaluated, using enzyme purified from porcine kidney. They are based on internal quenching of the synthetic, fluorescent amino acid (R, S)-2-amino-3-(7-methoxy4-coumaryl)propanoic acid ((R,S)-Amp) by a 2, 4dinitrophenyl (DNP) group. The compounds are X-Pro-Pro-(R, S)-Amp-NH2, where X is H-Lys(epsilon-DNP), H-Orn(delta-DNP), or L-2-amino-3-(DNP)aminopropionic acid. The first two were found to be excellent substrates for AP-P, with respective Km values of 4.8 and 5.2 microM. An advantageous feature is that under the conditions of assay, using 4-mm2 cells, the substrates are without noticeable quenching effect on the fluorescence of Pro-Pro-(R,S)-Amp-NH2 (the product liberated by the action of AP-P). At concentrations greater than about 30-50 microM, both substrates appear to inhibit the enzyme, but this has little practical consequence since assays can be carried out at substrate concentrations, giving up to approximately 80% of Vmax without this inhibitory effect being noticeable. The Lys derivative was found to be a very useful substrate for a continuous assay for AP-P and equally good in a discontinuous assay of multiple samples using microtiter plates. The racemic center at the Amp residue did not prevent total hydrolysis of the Lys derivative, suggesting that subsite specificity in AP-P does not extend as far as the P3' position.

Aminopeptidases↗
PubMed1997

A novel splice site mutation in the androgen receptor gene results in exon skipping and a non-functional truncated protein.

Mutations of the androgen receptor (AR) gene and protein are associated with complete androgen insensitivity syndromes (CAIS) in individuals with XY genotypes causing them to develop as phenotypic females. Splice site mutations of the AR gene are very rare and in this report we describe the consequences of a novel G --> A mutation at the exon 7/intron 7 splice junction of the AR gene that resulted in CAIS in two siblings. Reverse transcriptase-polymerase chain reaction (RT-PCR) of the AR transcript in patient's fibroblasts was performed and sequencing of the product showed omission of exon 7, with exon 6 being spliced directly to exon 8. This resulted in a shift of the reading frame and the introduction of a premature stop codon 10 amino acids into exon 8. Immunoblot analyses showed that the resultant AR protein was partially deleted in its C-terminal region and was approximately 1.5 kDa smaller than the wild type. This truncated AR was non-functional as it was unable to bind its physiological ligand (dihydrotestosterone) in androgen-binding assays. This is the first documentation of a point mutation in the AR gene which causes exon skipping and proves that the mutation is the cause of CAIS in our two subjects.

Androgens↗
PubMed1997

Synthesis and anti-HIV-1 activity of a series of 1-alkoxy-5-alkyl-6-(arylthio)uracils.

A series of 1-alkoxy-5-alkyl-6-(arylthio)uracils was synthesized and tested for their ability to inhibit HIV-1 replication. Treatment of 2-alkyl-3,3-bis(methylthio)acryloyl chlorides (5a-e) with AgOCN in benzene followed by reaction of the resulting isocyanates 6a-e with an appropriate alkoxyamine gave N-alkoxy-N'-((2-alkyl-3,3-bis(methylthio)acryloyl)ureas (10a-z) in good to excellent yields. Cyclization of 10a-z in AcOH containing a catalytic amount of p-TsOH produced 1-alkoxy-5-alkyl-6-(methylthio)uracils (11a-z). Oxidation of 11a-z with 3-chloroperoxybenzoic acid in CH2Cl2 resulted in high yields of 1-alkoxy-5-alkyl-6-(methylsulfonyl)uracils (12a-x and 12z) and 1-(benzyloxy)-6-(methylsulfinyl)thymine (12y), which were subsequently reacted with an appropriate arenethiol in ethanolic NaOH solution to afford 1-alkoxy-5-alkyl-6-(arylthio)uracils (14-49). Substitution at the 3- and 5-positions of the C-6-(phenylthio) ring by two methyl groups significantly increased its original anti-HIV-1 activity (EC50: 6-((3,5-dimethylphenyl)thio)-5-isopropyl-1-propoxyuracil (18), 0.064 microM; 6-((3,5-dimethylphenyl)thio)-1-(3-hydroxypropoxy)-5-isopropyluracil++ + (23), 0.19 microM). Among the various alkoxy substituents at the N-1, the propoxy group was the most beneficial for improving the anti-HIV-1 activity. The 1-propoxy derivative 18 proved to be the most potent inhibitor of HIV-1 replication, followed by the 1-(3-hydroxypropoxy) derivative 23. Introduction of an isopropyl group at C-5 of the uracil base also remarkably enhanced the activity. When compound 18 was incubated with a rat liver homogenate preparation, no metabolite was observed, thus confirming the metabolic stability of the N-O bond in these 1-alkoxyuracils.

Animals↗
PubMed1997

A second type of rod opsin cDNA from the common carp (Cyprinus carpio).

A second type of rhodopsin cDNA from carp (cRh-II) shared 97.2% polynucleotide identity with the previously reported cRh-I. The deduced amino acid sequences of cRh-I and cRh-II exhibited 98.6% identity. The key difference between these two types of cRh is that valine at position 169 of cRh-I was replaced by glutamic acid in cRh-II. Southern blot analysis of genomic DNA showed that there were two types of cRh gene. These two rod opsin genes were proven to be expressed in carp retinas by using RT-PCR with type-specific primers.

Amino Acid Sequence↗
PubMed1997

Isolation of murine and human homologues of the fission-yeast dis3+ gene encoding a mitotic-control protein and its overexpression in cancer cells with progressive phenotype.

To investigate genes involved in metastasis, we used a differential display method to compare the levels of gene expression in three cell lines derived from murine colon-adenocarcinoma 26 that show different metastatic potentials. The results, and subsequent Northern analyses, confirmed that one gene was expressed most strongly in NL17, the cell line with the highest experimentally metastatic potential to the lung; strongly in NL22, the line with moderately metastatic potential; and very weakly in NL4, which has no metastatic potential in recipient mice. Using this fragment as a probe, we isolated the murine cDNA as well as its human homologue and determined their DNA sequences. The cDNA sequences from both species contained open reading frames of 2874 nucleotides, encoding peptides of 958 amino acids with calculated molecular weights of approximately 109,000; the murine and human nucleotide sequences were 90% identical. The deduced amino acid sequences of these cDNAs revealed significant homology (45% identity) to the dis3+ gene product of Schizosaccharomyces pombe, a protein thought to be essential for mitotic control in the yeast. We therefore termed the murine and human genes hmc (homologue to the mitotic-control gene) and HMC, respectively. In 7 of 13 patients with colorectal cancers and liver metastases, expression of HMC was increased up to 38-fold in primary tumors and metastatic foci as compared to adjacent normal colorectal mucosa. An increase in expression of HMC, its novel product likely to belong to a structurally distinct family of mitotic-control proteins, may be associated with malignant phenotypes of some colorectal cancers.

Adenocarcinoma↗
PubMed1997

Association of csk-homologous kinase (CHK) (formerly MATK) with HER-2/ErbB-2 in breast cancer cells.

Protein-tyrosine kinases, such as HER-2/ErbB-2, have been specifically linked to breast cancer. The Csk-homologous kinase (CHK), formerly MATK, is a tyrosine kinase that contains the Src homology 2 and 3 (SH2 and SH3) domains and demonstrates homology ( approximately 50%) to the Csk tyrosine kinase. Like Csk, CHK is able to phosphorylate and inactivate Src family kinases. In this report, we investigated whether CHK is expressed in breast cancer tissues and whether it participates in the ErbB-2 signaling pathway in T47D and MCF-7 breast cancer cell lines. Immunostaining of the CHK protein in breast tissues demonstrated that primary invasive ductal carcinomas, stage II (13 of 15 cases) and stage I (8 of 15 cases), expressed the CHK protein, while this protein was not detected in the adjacent normal tissues from the same patients. To study the role of CHK in the ErbB-2 signaling pathway, glutathione S-transferase fusion proteins containing the SH2 and SH3 domains of CHK were generated. CHK-SH2 and CHK-SH3-SH2, but not CHK-SH3 or CHK-NH2-SH3, precipitated the tyrosine-phosphorylated ErbB-2 upon stimulation with heregulin. EGF or interleukin-6 stimulation of T47D cells failed to induce CHK-SH2 association with ErbB-2, the EGF-receptor, or the interleukin-6 receptor. In vivo association of the tyrosine-phosphorylated ErbB-2 with CHK was observed in co-immunoprecipitation studies with anti-CHK antibodies. EGF-R, ErbB-3, and ErbB-4 were not detected in the CHK immunoprecipitates or in the precipitates of the GST-SH2 fusion proteins of CHK, suggesting that the association of CHK with ErbB-2 upon heregulin stimulation is receptor-specific (ErbB-2) and ligand-specific (heregulin). These results indicate that CHK might participate in signaling in breast cancer cells by associating, via its SH2 domain, with ErbB-2 following heregulin stimulation.

Glycoproteins↗
PubMed1997

An Arabidopsis photolyase mutant is hypersensitive to ultraviolet-B radiation.

Photolyases are DNA repair enzymes that use energy from blue light to repair pyrimidine dimers. We report the isolation of an Arabidopsis thaliana mutant (uvr2-1) that is defective in photorepair of cyclobutylpyrimidine dimers (CPDs). Whereas uvr2-1 is indistinguishable from wild type in the absence of UV light, low UV-B levels inhibit growth and cause leaf necrosis. uvr2-1 is more sensitive to UV-B than wild type when placed under white light after UV-B treatment. In contrast, recovery in darkness or in light lacking photoreactivating blue light results in equal injury in uvr2-1 and wild type. The uvr2-1 mutant is unable to remove CPDs in vivo, and plant extracts lack detectable photolyase activity. This recessive mutation segregates as a single gene located near the top of chromosome 1, and is a structural gene mutation in the type II CPD photolyase PHR1. This mutant provides evidence that CPD photolyase is required for plant survival in the presence of UV-B light.

Apoenzymes↗
PubMed1997

Regulation of amelogenin gene expression.

The amelogenins are found uniquely in enamel, where they constitute the predominant class of secreted matrix proteins and where they play a fundamental role in normal enamel formation. To better understand the high level of tissue-specific expression, we cloned the bovine X and Y chromosomal amelogenin genes and the murine amelogenin gene and determined the DNA sequences for the regions upstream of the transcription start sites. We observed segments of strong homology among species, and identified consensus sequences for the binding of various transcription factors, including the glucocorticoid receptor, AP1, RXR and p53. Although specific sis-elements conferring enhanced transcription have not yet been identified, elements have been localized that have silencing effect in non-ameloblast cells. Conserved sequences are likely to be involved in tissue-specific expression. Transgenic mouse studies have shown that 3.5 kb of upstream region is sufficient but 900 bp is insufficient for specific expression in vivo. Alternative splicing of the primary transcript is an effective mechanism for generating molecular heterogeneity. Amelogenin genes contain seven exons, and exons 3, 4, 5 and most of 6 can be deleted by alternative splicing. However, the pattern of exon splicing varies according to the species, and skipping of bovine exon 3 appears to be developmentally regulated. It will be important to determine whether the relative amounts of translation products differ among species as do the mRNAs, and to correlate the various protein structures with function. These findings also suggest that the regulation of amelogenin gene expression is complex and takes place at several levels.

Alternative Splicing↗
PubMed1997

Role of testis exposure levels in the insensitivity of prepubertal rats to carbendazim-induced testicular toxicity.

Our recent studies have indicated that benomyl (BNL)-induced testicular toxicity is mediated by its major metabolite carbendazim (CBZ). The present study has used CBZ to investigate hypotheses that could explain prepubertal insensitivity to BNL. When CBZ (164 mg/kg intraperitoneally) was administered to postpubertal and prepubertal rats, it caused little testicular damage in prepubertal rats, but in adult rats, sloughing of the seminiferous epithelium resulted. When the inhibitory effect of CBZ on prepubertal testicular microtubule assembly was compared with that on postpubertal assembly, the IC50 values were very similar. Pharmacokinetic studies revealed that blood levels of CBZ were comparable in the two age groups; however, higher levels of CBZ were found in the adult testes (210.52 nmol/g wet wt) in comparison with young testes (67.77 nmol/g wet wt). These data suggest that delivery to and/or retention of CBZ in the testis may play a role in the age-dependent differences in susceptibility to CBZ toxicity. When CBZ was administered intratesticularly to reach levels sufficient to cause damage, the young animals did show an increased incidence of vacuolization and detachment of the seminiferous epithelium; however, in contrast to the older animals, sloughing of the seminiferous epithelium was not observed in the prepubertal animals. Overall, the low levels of CBZ measured in the testes of prepubertal animals offer a partial explanation for the insensitivity of young animals to CBZ-induced testicular toxicity following intraperitoneal administration. A differential responsiveness between the two age groups is also likely, however, since prepubertal animals lack elongated spermatids and it is sloughing of this cell type that characterizes CBZ-induced testicular toxicity in the adult.

Animals↗
PubMed1997

The role of the benomyl metabolite carbendazim in benomyl-induced testicular toxicity.

The present study has investigated the role of benomyl (BNL) vs carbendazim (CBZ) in BNL-induced testicular toxicity. Equivalent molar concentrations of BNL and CBZ were administered to rats intraperitoneally (859 mumol/kg) or by direct injection into the testis (1.37 mumol/testis). Whereas no significant testicular damage was observed both 1 and 2 hr after BNL administration by the ip route, CBZ administration resulted in sloughing of the seminiferous epithelium after 1 hr, which increased in severity at the 2-hr time point. Intratesticular treatment of BNL caused little testicular damage after 1 hr whereas an equimolar amount of CBZ elicited severe disruption of the seminiferous epithelium. Testicular levels of CBZ and BNL were measured at various times after both routes of administration. The AUC from the concentration of CBZ in the testis vs time plot showed an excellent relationship to the number of tubules which exhibited slouging. The BNL AUC also showed a straight-line relationship to severity of lesion. However, when the contribution of CBZ to the BNL response was subtracted, no effect of BNL was discernible. The effect of BNL and CBZ on testicular microtubule assembly was then investigated. IC50 for CBZ was 5 microM and that for BNL was 75 microM. Again, the effect of BNL on microtubule assembly could be largely accounted for by the presence of the CBZ breakdown product. These results strongly suggest that the BNL metabolite CBZ, and not BNL itself, is the mediator of BNL-induced testicular toxicity and inhibitor of testicular microtubule assembly.

Animals↗
PubMed1997

Uncommon lesions presenting as tumors of the internal auditory canal and cerebellopontine angle.

OBJECTIVE: The aim of this study was to identify distinguishing characteristics of uncommon lesions of the cerebellopontine angle (CPA) and internal auditory canal (IAC) in order to attain the correct diagnosis and thus formulate an appropriate therapeutic protocol. STUDY DESIGN: A retrospective chart analysis was performed on all patients with surgically managed lesions of the IAC and CPA referred to neuropathology from January 1985 to April 1996. SETTING: All patients were treated by New York University faculty at a tertiary referral center. PATIENTS: Among 426 surgical cases identified, 384 patients (90.1%) with acoustic neuromas and 18 patients (4.2%) with meningiomas were excluded. The remaining 24 cases, involving 17 women and seven men with a median age of 34 years, were analyzed. INTERVENTION: Most patients underwent audiovestibular evaluations, as well as magnetic resonance imaging (MRI) and computed tomographic (CT) scanning, and all patients underwent neurotologic surgery as part of their management protocol. MAIN OUTCOME MEASURES: Correlating patient presentation, preoperative imaging, and surgical findings often identified distinguishing characteristics of unusual CPA and IAC lesions. RESULTS: Unusual lesions identified at the CPA and IAC included: four epidermoids, four lipomas, two facial neuromas, two arachnoid cysts, two choroid plexus papillomas, two metastatic adenocarcinomas, one metastatic neuroblastoma, one ependymoma, one lymphoma, one cholesterol cyst, one angioleiomyoma, one venous hemangioma, one cavernous angioma, and one pontine glioma. CONCLUSIONS: Preoperative tumor differentiation based on the patient history, physical examination, audiovestibular testing, CT, and MRI help the surgeon to formulate an appropriate treatment protocol.

Adult↗
PubMed1996

Chemical modification of porcine kidney aminopeptidase P indicates the involvement of two critical histidine residues.

Aminopeptidase P (AP-P), purified to homogeneity from porcine kidney membranes, was completely inactivated by treatment with 0.2 mM diethylpyrocarbonate (DEP) at pH 7.0. Treatment of the modified enzyme with 20 mM hydroxylamine resulted in recovery of AP-P activity. The differential absorption of native and modified AP-P at 240 mm showed that DEP modified two histidyl residues per mol of AP-P. The substrates, bradykinin(1-5) and Gly-Pro-Hyp, and also the inhibitor, apstatin, could protect against DEP inactivation. These results suggest that histidine residues are critical for AP-P activity.

Aminopeptidases↗
PubMed1996

Effect of felbamate on the pharmacokinetics of lamotrigine.

To assess the possible interaction between lamotrigine and felbamate, a double-blind, randomized, placebo-controlled, two-way crossover study was conducted in 21 healthy male volunteers. Volunteers were given lamotrigine (100 mg every 12 hours) and felbamate (1,200 mg every 12 hours) or matching placebo for 10 days during each period of the crossover. After morning administration on day 10, blood samples were obtained over 12 hours for measurement of lamotrigine. Felbamate increased the maximum concentration (Cmax) and and area under the concentration-time curve from time 0 to 12 hours (AUC0-12) of lamotrigine by 13% and 14%, respectively, compared with placebo. The 90% confidence intervals of the log-transformed pharmacokinetic parameters were within the 80-125% bioequivalance limits, however. Felbamate had no significant effect on the urinary excretion of lamotrigine (total), unconjugated lamotrigine, or the N-glucuronide. One volunteer discontinued the study after developing a rash while taking lamotrigine and placebo. All other adverse events were primarily related to the central nervous system and gastrointestinal tract, with a higher incidence reported during coadministration of lamotrigine and felbamate than with placebo. Overall, felbamate appears to have no clinically relevant effects on the pharmacokinetics of lamotrigine.

Adult↗
PubMed1996

Dexamethasone-induced impairment in skeletal muscle glucose transport is not reversed by inhibition of free fatty acid oxidation.

Our previous studies suggested a possible role for the glucose-free fatty acid (FFA) cycle, ie, preferential utilization of FFA by muscle at the expense of glucose, in dexamethasone (DEX)-induced insulin resistance. To determine whether this resistance could be reversed by inhibiting FFA utilization, we used etomoxir, a potent inhibitor of mitochondrial FFA oxidation. Male Sprague-Dawley rats were injected subcutaneously with 1 mg/kg DEX or the vehicle every other day for 10 days, and half of each group was administered 10 mg/kg etomoxir by gavage once per day and 1 hour before the experiment. As expected, etomoxir treatment increased serum FFA levels and inhibited FFA oxidation by diaphragm in vitro. Administration of etomoxir decreased serum glucose and insulin concentrations under basal conditions in both control and DEX-treated animals, implying enhanced insulin sensitivity. DEX treatment significantly increased endogenous glucose production and decreased whole-body glucose disposal, as well as 2-deoxyglucose (2-DG) uptake by skeletal muscle during euglycemic-hyperinsulinemic clamps. Administration of etomoxir led to small but significant increases in glucose disposal rates of both control (14%) and DEX (23%) groups, but had no effect on residual endogenous glucose production. Thus, DEX-induced insulin resistance was marginally ameliorated but not completely reversed by etomoxir. Depressed 2-DG uptake by individual muscle tissues observed in the present study in conjunction with the absence of free intracellular glucose in muscle tissue following glucose-insulin infusion strongly suggests that the primary defect in glucose metabolism is at the level of transport. Neither overall abundance of the insulin-sensitive glucose transporter (GLUT-4) in skeletal muscle nor its distribution between intracellular stores and plasma membrane were modified by DEX treatment, either, under basal conditions or in response to acute insulin stimulus. These results suggest a defect(s) in the inherent activity of plasma membrane-bound GLUT-4 as the likely mechanism for DEX-induced insulin resistance.

Animals↗