Search PubMed⌕ Search

Biomedical subjects

S Lin

Publications and source records attributed to S Lin.

At least 127 records · Page 7Linked to original sources
PubMed1999

A PSTTLRE-form of cdc2-like gene in the marine microalga Dunaliella tertiolecta.

To understand the genetic control of algal cell division cycle that pertains to phytoplankton bloom dynamics in the sea, we cloned and analyzed a gene coding for a cyclin-dependent kinase (CDK) for the chlorophyte Dunaliella tertiolecta. The cDNA cloned, 1061 bp long, contained an open reading frame of 314 amino acids. FASTA and GAP analyses showed that this sequence was most homologous to cdc2 out of all known cdks, with an identity of 54-68% and a similarity of 65-76% to cdc2 in higher plants, animals, and yeast. Several signature domains of cdc2 were identified from this sequence, although the PSTAIRE and GDSEID motifs were replaced with PSTTLRE and GDCELQ, respectively. Southern blot hybridization demonstrated that this gene occurred as a single copy in this species, and quantitative RT-PCR showed that the transcription of this gene was constitutive. The present results suggest that the universal cdc2 is conserved in the lower eukaryote with unique structural characteristics.

Algal Proteins↗
PubMed1999

Genetic organization of alpha-bungarotoxins from Bungarus multicinctus (Taiwan banded krait): evidence showing that the production of alpha-bungarotoxin isotoxins is not derived from edited mRNAs.

Two genomic DNAs with a size of approximately 2.8 kb, isolated from the liver of Bungarus multicinctus (Taiwan banded krait), encode the precursors of the long neurotoxins, alpha-Bgt(A31) and alpha-Bgt(V31), respectively. Both genes share virtually identical overall organization with three exons separated by two introns, which were inserted in the same positions in the coding regions of the genes. Moreover, their nucleotide sequences share approximately 98% identity. This result indicates that the two genes co-exist in the genome of B.multicinctus, and probably arose from gene duplication. The exon/intron structures of the alpha-Bgt genes were essentially the same as those reported for the short neurotoxins. This reflects that the long and short neurotoxins should share a common evolutionary origin. Comparative analyses on long neurotoxin and short neurotoxin genes showed that the protein coding regions of the exons were more diverse than the introns except for the signal peptide domain. This implies that the protein coding regions of the neurotoxins may have evolved via accelerated evolution. PCR amplification of venom gland cDNA mixtures revealed that only two amino acid sequences corresponding to alpha-Bgt(A31) and alpha-Bgt(V31) could be deduced from the cDNAs. The results of chromatographic analyses and protein sequencing again emphasized the view that, with the exception of alpha-Bgt(A31) and alpha-Bgt(V31), no other alpha-Bgt isotoxins with amino acid substitutions were present in B.multicinctus venom. In contrast to the proposition of Liu et al. ( Nucleic Acids Res., 1998,26, 5624-5629), our findings strongly suggest that each alpha-Bgt isotoxin is derived from the respective gene, and that alpha-Bgt RNA polymorphism does not originate from one single, intronless gene by the mechanism of RNA editing.

Animals↗
PubMed1999

Crucial role of the interleukin 1 receptor family member T1/ST2 in T helper cell type 2-mediated lung mucosal immune responses.

T1/ST2 is an orphan receptor of unknown function that is expressed on the surface of murine T helper cell type 2 (Th2), but not Th1 effector cells. In vitro blockade of T1/ST2 signaling with an immunoglobulin (Ig) fusion protein suppresses both differentiation to and activation of Th2, but not Th1 effector populations. In a nascent Th2-dominated response, anti-T1/ST2 monoclonal antibody (mAb) inhibited eosinophil infiltration, interleukin 5 secretion, and IgE production. To determine if these effects were mediated by a direct effect on Th2 cells, we next used a murine adoptive transfer model of Th1- and Th2-mediated lung mucosal immune responses. Administration of either T1/ST2 mAb or T1/ST2-Ig abrogated Th2 cytokine production in vivo and the induction of an eosinophilic inflammatory response, but failed to modify Th1-mediated inflammation. Taken together, our data demonstrate an important role of T1/ST2 in Th2-mediated inflammatory responses and suggest that T1/ST2 may prove to be a novel target for the selective suppression of Th2 immune responses.

Allergens↗
PubMed1999

[New advances in muscular dystrophy: an up-to-date diagnostic plan].

During the last few years new findings from molecular studies have much advanced the understanding of muscular dystrophies. A unifying concept has been put forward to explain many forms of such disorders. A complex of several proteins associated to dystrophin has been described which links the cytoskeleton to the extracellular matrix. The components of this complex can be examined at molecular and protein levels. This allows a search for an exact diagnosis in many such patients. Relevant new facts are reviewed in the frame of a traditional approach to the diagnosis of muscular dystrophies.

Diagnosis, Differential↗
PubMed1999

Human acyl-CoA:cholesterol acyltransferase-1 in the endoplasmic reticulum contains seven transmembrane domains.

Acyl-CoA:cholesterol acyltransferase (ACAT) plays important roles in cellular cholesterol homeostasis and is involved in atherosclerosis. ACAT-1 protein is located mainly in the ER. The hydropathy plot suggests that ACAT-1 protein contains multiple transmembrane segments. We inserted either the hemagglutinin tag or the HisT7 tag at various hydrophilic regions within the human ACAT-1 protein and used immunofluorescence microscopy to determine the topography of the tagged proteins expressed in mutant Chinese hamster ovary cells lacking endogenous ACAT. All of the tagged proteins are located mainly in the ER and retain full or partial enzyme activities. None of the tagged proteins produces detectable intracellular degradation intermediates. Treating cells with digitonin at 5 micrograms/ml permeabilizes the plasma membranes while leaving the ER membranes sealed; in contrast, treating cells with 0.25% Triton X-100 or with cold methanol permeabilizes both the plasma membranes and the ER membranes. After appropriate permeabilization, double immunostaining using antibodies against the N-terminal region and against the inserted tag were used to visualize various regions of the tagged protein. The results show that human ACAT-1 in the ER contains seven transmembrane domains.

Algorithms↗
PubMed1999

Glutamate decarboxylase isoforms in thalamic nuclei in lethargic mouse model of absence seizures.

To test the hypothesis that altered GABA synthesis within nucleus reticularis thalami (NRT) neurons regulates absence seizures, we analyzed and quantitated the distribution of GAD(67) and GAD(65), the rate-limiting enzymes of GABA synthesis, in thalamic nuclei from the Cacnb4lh model of absence seizures and non-epileptic (+/+) controls. In situ hybridization and Western blot results indicate a significant increase in GAD(67) expression (mRNA and protein) per cell but no change in GAD(65) in Cacnb4lh mice. These data suggest that GABA-synthesis is maintained or increased in NRT neurons in the Cacnb4lh mouse model.

Animals↗
PubMed1999

Surfactant protein B (SP-B) -/- mice are rescued by restoration of SP-B expression in alveolar type II cells but not Clara cells.

Surfactant protein B (SP-B) mRNA and protein are restricted to alveolar Type II and Clara cells in the respiratory epithelium. In order to investigate the function of SP-B in these distinct cell types, transgenic mice were generated in which SP-B expression was selectively restored in Type II cells or Clara cells of SP-B -/- mice. The 4.8-kilobase murine SP-C promoter was used to generate 3 transgenic lines which expressed human SP-B in Type II cells (mSP-C/hSP-B). Likewise, the 2.3-kilobase murine CCSP promoter was used to generate two transgenic lines which expressed human SP-B in Clara cells (mCCSP/hSP-B). mSP-C/hSP-B and mCCSP/hSP-B transgenic mice were subsequently bred to SP-B +/- mice in order to selectively express SP-B in Type II cells or Clara cells of SP-B -/- mice. Selective restoration of SP-B expression in Type II cells completely rescued the neonatal lethal phenotype in SP-B -/- mice. Expression of SP-B in some, but not all Type II cells of SP-B -/- mice, allowed postnatal survival, but resulted in significantly altered lung architecture and function. Selective restoration of SP-B expression in Clara cells of SP-B -/- mice resulted in respiratory dysfunction and invariable neonatal death, related to the complete absence of mature SP-B peptide in these mice. These results indicate that expression and processing of the SP-B proprotein to the mature peptide in Type II cells is absolutely required for lung function in vivo and that SP-B expression in Clara cells cannot substitute for this function.

Animals↗
PubMed1999

Lipid A modifications characteristic of Salmonella typhimurium are induced by NH4VO3 in Escherichia coli K12. Detection of 4-amino-4-deoxy-L-arabinose, phosphoethanolamine and palmitate.

Two-thirds of the lipid A in wild-type Escherichia coli K12 is a hexa-acylated disaccharide of glucosamine in which monophosphate groups are attached at positions 1 and 4'. The remaining lipid A contains a monophosphate substituent at position 4' and a pyrophosphate moiety at position 1. The biosynthesis of the 1-pyrophosphate unit is unknown. Its presence is associated with lipid A translocation to the outer membrane (Zhou, Z., White, K. A., Polissi, A., Georgopoulos, C., and Raetz, C. R. H. (1998) J. Biol. Chem. 273, 12466-12475). To determine if a phosphatase regulates the amount of the lipid A 1-pyrophosphate, we grew cells in broth containing nonspecific phosphatase inhibitors. Na2WO4 and sodium fluoride increased the relative amount of the 1-pyrophosphate slightly. Remarkably, NH4VO3-treated cells generated almost no 1-pyrophosphate, but made six major new lipid A derivatives (EV1 to EV6). Matrix-assisted laser desorption ionization/time of flight mass spectrometry of purified EV1 to EV6 indicated that these compounds were lipid A species substituted singly or in combination with palmitoyl, phosphoethanolamine, and/or aminodeoxypentose residues. The aminodeoxypentose residue was released by incubation in chloroform/methanol (4:1, v/v) at 25 degrees C, and was characterized by 1H NMR spectroscopy. The chemical shifts and vicinal coupling constants of the two anomers of the aminodeoxypentose released from EV3 closely resembled those of synthetic 4-amino-4-deoxy-L-arabinose. NH4VO3-induced lipid A modification did not require the PhoP/PhoQ two-component regulatory system, and also occurred in E. coli msbB or htrB mutants. The lipid A variants that accumulate in NH4VO3-treated E. coli K12 are the same as many of those normally found in untreated Salmonella typhimurium and Salmonella minnesota, demonstrating that E. coli K12 has latent enzyme systems for synthesizing these important derivatives.

Amino Sugars↗
PubMed1999

OHIO-1 beta-lactamase mutants: the Arg244Ser mutant and resistance to beta-lactams and beta-lactamase inhibitors.

Mutations at residue 244 (Ambler numbering system) in the class A TEM beta-lactamase confer resistance to inactivation by beta-lactamase inhibitors and result in diminished turnover of beta-lactam substrates. The Arg244Ser mutant of the OHIO-1 beta-lactamase, an SHV family enzyme, demonstrates variable susceptibilities to beta-lactamase inhibitors and has significantly reduced catalytic efficiency. The minimum inhibitory concentrations (MICs) for Escherichia coli DH5alpha expressing the Arg244Ser beta-lactamase were reduced when compared to the strain bearing the OHIO-1 beta-lactamase: ampicillin, 512 vs. 8192 micrograms ml-1; cephaloridine, 4 vs. 32 micrograms ml-1, respectively. The MICs for the beta-lactam beta-lactamase inhibitor combinations demonstrated resistance only to ampicillin-clavulanate, 16/8 vs. 8/4 micrograms ml-1 respectively. In contrast, there was increased susceptibility to ampicillin-sulbactam, ampicillin-tazobactam, and piperacillin-tazobactam. When compared to the OHIO-1 beta-lactamase homogenous preparations of the Arg244Ser beta-lactamase enzyme demonstrated increased Km and decreased kcat values for benzylpenicillin (Km=17 vs. 50 microM, kcat=345 vs. 234 s-1) and cephaloridine (Km=97 vs. 202 microM, kcat=1023 vs. 202 s-1). Although the Ki and IC50 values were increased for each inhibitor when compared to OHIO-1 beta-lactamase, the turnover numbers (tn) required for inactivation were increased only for clavulanate. For the Arg244Ser mutant enzyme of OHIO-1, the increased Ki, decreased tn for the sulfones, and different partition ratio (kcat/kinact) support the notion that not all class A enzymes are inactivated in the same manner, and that certain class A beta-lactamase enzymes may react differently with identical substitutions in structurally conserved amino acids. The resistance phenotype of a specific mutations can vary depending on the enzyme.

Drug Resistance↗
PubMed1999

Human acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) gene organization and evidence that the 4.3-kilobase ACAT-1 mRNA is produced from two different chromosomes.

Acyl-CoA:cholesterol acyltransferase (ACAT) plays important roles in cellular cholesterol homeostasis. Four human ACAT-1 mRNAs (7.0, 4.3, 3.6, and 2.8 kilobases (kb)) share the same short 5'-untranslated region (exon 1) and coding sequence (exons 2-15). The 4.3-kb mRNA contains an additional 5'-untranslated region (1289 nucleotides in length; exons Xa and Xb) immediately upstream from the exon 1 sequence. One ACAT-1 genomic DNA insert covers exons 1-16 and a promoter (the P1 promoter). A separate insert covers exon Xa (1277 base pairs) and a different promoter (the P7 promoter). Gene mapping shows that exons 1-16 and the P1 promoter sequences are located in chromosome 1, while exon Xa and the P7 promoter sequence are located in chromosome 7. RNase protection assays demonstrate three different protected fragments, corresponding to the 4.3-kb mRNA and the two other mRNAs transcribed from the two promoters. These results are consistent with the interpretation that the 4.3-kb mRNA is produced from two different chromosomes, by a novel RNA recombination mechanism involving trans-splicing of two discontinuous precursor RNAs.

5' Untranslated Regions↗
PubMed1999

A common pharmacophore for cytotoxic natural products that stabilize microtubules.

Taxol (paclitaxel), a complex diterpene obtained from the Pacific yew, Taxus brevifolia, is arguably the most important new drug in cancer chemotherapy. The mechanism of cytotoxic action for paclitaxel-i.e., the stabilization of microtubules leading to mitotic arrest-is now shared by four recently identified natural products, eleutherobin, epothilones A and B, and discodermolide. Their ability to competitively inhibit [3H]paclitaxel binding to microtubules strongly suggests the existence of a common binding site. Recently, we have developed nonaromatic analogues of paclitaxel that maintain high cytotoxicity and tubulin binding (e.g., nonataxel). We now propose a common pharmacophore that unites paclitaxel, nonataxel, the epothilones, eleutherobin, and discodermolide, and rationalizes the extensive structure-activity relationship data pertinent to these compounds. Insights from the common pharmacophore have enabled the development of a hybrid construct with demonstrated cytotoxic and tubulin-binding activity.

Alkaloids↗
PubMed1999

[The relation between alpha1-antichymotrypsin gene polymorphism and epsilon4 allele of apolipoprotein E gene in Alzheimer disease in Chinese]

OBJECTIVE: To detect the relation between alpha1-antichymotrpsin(AACT)gene polymorphism and epsilon4 allele of apolipoprotein E (ApoE) gene in Alzheimer disease (AD) in Chinese. METHODS: The gene polymorphisms of ApoE and AACT were genotyped in 125 AD cases and 140 controls with PCR methods and RFLP typing. Then the association between AACT polymorphism and ApoE epsilon4 was analysed. RESULTS: There was no association between AD and any allele or genotype of AACT polymorphism; AACT polymorphism was not associated with AD ApoE epsilon4 or without ApoE epsilon4. In AACT* AT and AACT* TT genotypes, ApoE epsilon4 allele was associated with AD, but no association was observed in AACT* AA genotype. CONCLUSION: AACT may not be associated with AD in Chinese, and this effect can not be influenced by ApoE epsilon4, but AACT gene polymorphism may affect the association between ApoE epsilon4 allele and AD.

Journal Article↗
PubMed1999

Differentially expressed genes in activin-induced apoptotic LNCaP cells.

Gene transcripts differentially expressed in activin-induced human prostatic LNCaP apoptotic cells have been discovered by an improved subtractive hybridization method, uracil-DNA subtraction assay (USA), which involves digestion with uracil-DNA glycosylase and mung-bean nuclease. Among the five up-regulated and seven down-regulated genes, we have identified six known (>95% homology and similar size; p16, p53, Siva, RHAMM, Pax2, and eIF-4a1), three homologues (>95% homology but different size; myosin, a helicase motif, and a kinase motif), and three novel genes (no homology). In addition, anti-sense knock-out of a resulting novel kinase-like gene was found to abolish the apoptotic DNA fragmentation in activin-treated LNCaP cells. These findings indicate a new potential mechanism in DNA fragmentation of activin-induced cell-cycle arresting and apoptosis.

Activins↗
PubMed1999

Substrate specificity and stereoselectivity of rat brain microsomal anandamide amidohydrolase.

Anandamide amidohydrolase (AAH) catalyzes the hydrolysis of arachidonylethanolamide (anandamide), an endogenous cannabinoid receptor ligand. To delineate the structural requirements of AAH substrates, rat brain microsomal AAH hydrolysis of a series of anandamide congeners was studied using two reverse-phase high-performance liquid chromatography (RP-HPLC) assays developed in our laboratory. Arachidonamide (1) was found to be the best substrate with an apparent Km of 2.34 mM and a Vmax of 2.89 nmol/min/mg of protein. Although anandamide (2) has a similar Km value, its Vmax is approximately one-half that of arachidonamide. N, N-Bis(2-hydroxyethyl)arachidonamide (3) was not hydrolyzed, suggesting specificity for unsubstituted or mono-N-substituted arachidonamides. Analogues with a methyl group at the 1'-position of the ethanolamido headgroup were also found to have greater resistance to enzymatic turnover and therefore increased metabolic stability. The enzyme exhibited high stereoselectivity as the rate of hydrolysis of (R)-alpha-methanandamide (2.4%) (anandamide = 100%) was about 10-fold lower than that of its (S)-enantiomer (23%). In contrast, (R)-beta-methanandamide was 6-times more susceptible (121%) than the (S)-beta-enantiomer (21%). Interestingly, an inverse correlation was shown between AAH stereoselectivity and the brain cannabinoid receptor affinity as the enantiomers with high receptor affinity displayed low susceptibility to hydrolysis by AAH. Metabolic stability is also imparted to analogues with a short hydrocarbon headgroup as well as to those possessing 2-monomethyl or 2,2-dimethyl substituents. 2-Arachidonylglycerol and racemic 1-arachidonylglycerol were shown to be excellent AAH substrates. To identify AAH inhibitors, hydrolysis of anandamide was also studied in the presence of a select group of cannabimimetics. Of these, (-)-Delta8-THC and SR141716A, a biarylpyrazole CB1 antagonist, were found to inhibit enzymatic activity. These newly defined enzyme recognition parameters should provide a foundation for the rational development of stable, therapeutically useful anandamide analogues with high receptor affinity.

Amidohydrolases↗
PubMed1999

Modulation of slow inactivation in human cardiac Kv1.5 channels by extra- and intracellular permeant cations.

1. The properties and regulation of slow inactivation by intracellular and extracellular cations in the human heart K+ channel hKv1.5 have been investigated. Extensive NH2- and COOH-terminal deletions outside the central core of transmembrane domains did not affect the degree of inactivation. 2. The voltage dependence of steady-state inactivation curves of hKv1.5 channels was unchanged in Rb+ and Cs+, compared with K+, but biexponential inactivation over 10 s was reduced from approximately 100 % of peak current in Na+ to approximately 65 % in K+, approximately 50 % in Rb+ and approximately 30 % in Cs+. This occurred as a result of a decrease in both fast and slow components of inactivation, with little change in inactivation time constants. 3. Changes in extracellular cation species and concentration (5-300 mM) had only small effects on the rates of inactivation and recovery from inactivation (tau recovery approximately 1 s). Mutation of residues at a putative regulatory site at R487 in the outer pore mouth did not affect slow inactivation or recovery from inactivation of hKv1.5, although sensitivity to extracellular TEA was conferred. 4. Symmetrical reduction of both intra- and extracellular cation concentrations accelerated and augmented both components of inactivation of K+ (Kd = 34.7 mM) and Cs+ (Kd = 20.5 mM) currents. These effects could be quantitatively accounted for by unilateral reduction of intracellular K+ (K+i) (Kd = 43.4 mM) or Cs+i with constant 135 mM external ion concentrations. 5. We conclude that inactivation and recovery from inactivation in hKv1.5 were not typically C-type in nature. However, the ion species dependence of inactivation was still closely coupled to ion permeation through the pore. Intracellular ion modulatory actions were more potent than extracellular actions, although still of relatively low affinity. These results suggest the presence of ion binding sites capable of regulating inactivation located on both intracellular and extracellular sides of the pore selectivity filter.

Cations↗
PubMed1999

Structure-activity relationships of pyrazole derivatives as cannabinoid receptor antagonists.

As a potent, specific antagonist for the brain cannabinoid receptor (CB1), the biarylpyrazole N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A; 1) was the lead compound for initiating studies designed to examine the structure-activity relationships of related compounds and to search for more selective and potent cannabimimetic ligands. A series of pyrazole derivatives was designed and synthesized to aid in the characterization of the cannabinoid receptor binding sites and also to serve as potentially useful pharmacological probes. Therapeutically, such compounds may have the ability to antagonize harmful side effects of cannabinoids and cannabimimetic agents. Structural requirements for potent and selective brain cannabinoid CB1 receptor antagonistic activity included (a) a para-substituted phenyl ring at the 5-position, (b) a carboxamido group at the 3-position, and (c) a 2,4-dichlorophenyl substituent at the 1-position of the pyrazole ring. The most potent compound of this series contained a p-iodophenyl group at the 5-position, a piperidinyl carboxamide at the 3-position, and a 2,4-dichlorophenyl group at the 1-position of the pyrazole ring. The iodinated nature of this compound offers additional utility as a gamma-enriching SPECT (single photon emission computed tomography) ligand that may be useful in characterizing brain CB1 receptor binding in vivo.

Animals↗