Biomedical subjects

M Lin

Publications and source records attributed to M Lin.

At least 163 records · Page 9Linked to original sources

PubMed1999

Comparison between micellar electrokinetic chromatography and HPLC for the determination of Betamethasone Dipropionate, Clotrimazole and their related substances.

The complete separation of a composite mixture that consisted of Betamethasone Dipropionate (BMD), Clotrimazole and their derivatives in a pharmaceutical dosage form was achieved within 15 min using sodium dodecyl sulfate (SDS) micellar electrokinetic chromatography (MEKC). For the MEKC separations, electrophoretic media consisting of SDS-phosphate buffer and various concentrations of alcohols or acetonitrile were used. The optimal condition for separating BMD, Clotrimazole and their analogues was found to be 50 mM SDS-15% acetonitrile-5% butanol at pH 7.2. The results demonstrated that the method was valid for the quantitation of BMD, Clotrimazole and analogues with selectivity and precision comparable to that of High-Performance Liquid Chromatography (HPLC).

Administration, Topical↗

PubMed1999

Affinity NMR: decoding DNA binding.

We have shown that affinity NMR can be used to edit a NMR spectrum so that ligands that have affinity to DNA can be observed in the presence of other nonbinding molecules. Diffusion encoded spectroscopy (DECODES) can be used to identify the binding ligands. We were able to identify Hoechst 33342 as binding to the Drew-Dickerson dodecamer d(CGCGAATTCGCG)2 in the presence of the nonbinding molecules adenine, adenosine, and thiamine. Affinity NMR appears to be readily applicable to DNA systems for the following reasons. (1) The relaxation rate of the DNA oligonucleotides is favorable, thus the signal intensity loss due to relaxation is not severe. (2) A comparison of the patterns of the DNA cross-peaks upon binding in the two-dimensional total correlation spectroscopy and correlation spectroscopy spectrum are easily performed, and the ligand signals in the two-dimensional DECODES spectrum can be readily identified. (3) The aromatic part of the DNA spectrum is devoid of 2D cross-peaks in these correlation spectra, greatly facilitating the interpretation of the bound ligand in the DECODES spectrum.

Adenine↗

PubMed1999

Benzylisoquinoline alkaloids from gnetum parvifolium

The investigation of the chemical constituents from the lianas of Gnetum parvifolium resulted in the isolation of three new benzylisoquinoline alkaloids, (+/-)-N-methylhigenamine (1), (-)-N-methylhigenamine N-oxide (2), and (+/-)-8-(p-hydroxybenzyl)-2, 3,10,11-tetrahydroxyprotoberberine (3), together with two known alkaloids, higenamine and trigonelline. The structures of 1-3 were determined by chemical methods and spectral analysis including 2D NMR and NOE studies.

Journal Article↗

PubMed1999

Acrosome formation during sperm transit through the epididymis in two marsupials, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula).

In certain Australian marsupials including the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula), formation of the acrosome is not completed in the testis but during a complex differentiation process as spermatozoa pass through the epididymis. Using transmission and scanning electron microscopy this paper defined the process of acrosome formation in the epididymis, providing temporal and spatial information on the striking reorganisation of the acrosomal membranes and matrix and of the overlying sperm surface involved. On leaving the testis wallaby and possum spermatozoa had elongated 'scoop'-shaped acrosomes projecting from the dorsal surface of the head. During passage down the epididymis, this structure condensed into the compact button-like organelle found on ejaculated spermatozoa. This condensation was achieved by a complex process of infolding and fusion of the lateral projections of the 'scoop'. In the head of the epididymis the rims of the lateral scoop projections became shorter and thickened and folded inwards, to eventually meet midway along the longitudinal axis of the acrosome. As spermatozoa passed through the body of the epididymis the lateral projections fused together. Evidence of this fusion of the immature outer acrosomal membrane is the presence of vesicles within the acrosomal matrix which persist even in ejaculated spermatozoa. When spermatozoa have reached the tail of the epididymis the acrosome condenses into its mature form, as a small button-like structure contained within the depression on the anterior end of the nucleus. During the infolding process, the membranes associated with the immature acrosome are either engulfed into the acrosomal matrix (outer acrosomal membrane), or eliminated from the sperm head as tubular membrane elements (cytoplasmic membrane). Thus the surface and organelles of the testicular sperm head are transient structures in those marsupials with posttesticular acrosome formation and this must be taken into consideration in attempts to dissect the cell and molecular biology of fertilisation.

Acrosome↗

PubMed1999

The ultrastructure and metabolism of ejaculated tammar wallaby sperm are impaired by swim-up procedures when compared with sperm from the cauda epididymidis.

The metabolism, rate of intracellular accumulation of sugars, motility and ultrastructure of ejaculated tammar sperm were impaired by swim-up into artificial media, particularly when the cells were subsequently exposed to N-acetyl-D-glucosamine (NAG). The inclusion of hyaluronate, serum albumin, catalase or Desferal in swim-up media helped prevent deterioration of sperm motility, but failed to prevent detrimental NAG-induced metabolic and ultrastructural changes. However, the sperm were unavoidably diluted during swim-up into artificial media and their behavioural properties were modified by dilution. Thus, sperm collected from the cauda epididymidis were immotile and their rate of oxygen uptake was low in undiluted caudal epididymal semen (CES). Nevertheless, these sperm were viable, and vigorous motility was induced by 5- to 50-fold dilution in Krebs-Ringer phosphate (KRP). Sperm respiration also dramatically increased with moderate dilution (5- or 15-fold) in KRP, but decreased again at higher rates (50-fold). This suggested that motility and the metabolic properties of tammar sperm are modified both by dilution and on leaving the suppressing conditions of the epididymis. Diluted tammar epididymal sperm also displayed a Pasteur effect, but rapidly lost capacity for motility in an oxygen-depleted atmosphere. It was concluded that swim-up procedures compromise ejaculated tammar sperm by promoting dilution-induced changes. This may alter the permeability of the membrane with loss of the enzymes that process the ammonia generated during the metabolism of NAG in seminal plasma. Subsequent exposure to NAG further promotes ultrastructural damage culminating in loss of viability.

Acetylglucosamine↗

PubMed1999

Oligostilbenes from the roots of Vitis amurensis.

Two new oligostilbenes, amurensin A (1) and amurensin B (2), were isolated from the roots of Vitis amurensis Rupr. together with five known oligostilbenes. Their structures were elucidated by means of spectroscopic evidence, and amurensin B was the first naturally occurring stilbene trimer with a cis dihydrobenzofuran moiety.

Magnoliopsida↗

PubMed1999

High polymorphism at the human melanocortin 1 receptor locus.

Variation in human skin/hair pigmentation is due to varied amounts of eumelanin (brown/black melanins) and phaeomelanin (red/yellow melanins) produced by the melanocytes. The melanocortin 1 receptor (MC1R) is a regulator of eu- and phaeomelanin production in the melanocytes, and MC1R mutations causing coat color changes are known in many mammals. We have sequenced the MC1R gene in 121 individuals sampled from world populations with an emphasis on Asian populations. We found variation at five nonsynonymous sites (resulting in the variants Arg67Gln, Asp84Glu, Val92Met, Arg151Cys, and Arg163Gln), but at only one synonymous site (A942G). Interestingly, the human consensus protein sequence is observed in all 25 African individuals studied, but at lower frequencies in the other populations examined, especially in East and Southeast Asians. The Arg163Gln variant is absent in the Africans studied, almost absent in Europeans, and at a low frequency (7%) in Indians, but is at an exceptionally high frequency (70%) in East and Southeast Asians. The MC1R gene in common and pygmy chimpanzees, gorilla, orangutan, and baboon was sequenced to study the evolution of MC1R. The ancestral human MC1R sequence is identical to the human consensus protein sequence, while MC1R varies considerably among higher primates. A comparison of the rates of substitution in genes in the melanocortin receptor family indicates that MC1R has evolved the fastest. In addition, the nucleotide diversity at the MC1R locus is shown to be several times higher than the average nucleotide diversity in human populations, possibly due to diversifying selection.

Alleles↗

PubMed1999

Relationship between iron concentration and hepatitis C virus RNA level in liver tissue.

Patients with chronic hepatitis C virus (HCV) infection frequently have increased hepatic iron stores. The role of hepatitis C in hepatic iron deposition is unknown. The authors examined whether there is a relation between hepatitis C virus level in liver tissue and hepatic iron concentration. Forty-two paired samples obtained from the liver explants of five patients who underwent transplantation for liver disease due to hepatitis C were studied. Hepatitis C virus levels were measured at multiple sites within each liver by a branched deoxyribonucleic assay. Measurements of hepatic iron concentration were made at adjacent sites by a colorimetric assay. Random effects modeling showed wide intrahepatic variation in hepatic HCV ribonucleic acid (RNA) concentration (variance = 1.2 x 10(4) [mEq/g]2) and hepatic iron concentration (variance = 1.3 x 10(6) [microg/g]2). There was, however, a trend toward an association between the mean HCV level and the mean hepatic iron concentration for each liver (r = 0.30, p = 0.05). In conclusion, HCV level and iron concentration varied within and between cirrhotic livers. Variability in intrahepatic iron concentration was not related to variability in intrahepatic HCV RNA concentration. More studies are needed to determine the cause of variability in hepatic iron and HCV RNA concentration within and between livers in patients with chronic hepatitis C.

Hepacivirus↗

PubMed1999

Functional effects of mutations in KvLQT1 that cause long QT syndrome.

INTRODUCTION: The long QT syndrome (LQT) is caused by mutations in genes encoding ion channels that modulate the duration of ventricular action potentials. One of these genes, KVLQT1, encodes an alpha subunit that coassembles with another subunit, hminK, to form the cardiac slow delayed rectifier (I(Ks)) K+ channel. METHODS AND RESULTS: The functional effects of seven mutations in KVLQT1 were assessed using two-microelectrode voltage clamp and the Xenopus oocyte expression system. Most mutations in KVLQT1 caused loss of function when expressed alone. Oocytes were also injected with equal amounts of wild-type (WT) KVLQT1 and mutant KVLQT1 cRNA (with or without coinjection of hminK) and the resulting currents compared to currents induced by WT KvLQT1 alone. A341V, R190Q, or G189R KVLQT1 subunits did not affect expression of WT KvLQT1. The other mutations in KVLQT1 caused a variable degree of dominant-negative suppression of I(Ks). The order of potency for this effect was G345E > G306R = V254M > A341E. CONCLUSIONS: LQT1-associated mutations in KVLQT1 caused a spectrum of dysfunction in I(Ks) and KvLQT1 channels. The degree of I(Ks) dysfunction did not correlate with the QTc interval or the presence of symptoms in the respective gene carriers. In contrast to previous reports, we found that loss of function mutations are not exclusive to recessively inherited LQT.

Animals↗

PubMed1999

Use of recombinant flagellin protein as a tracer antigen in a fluorescence polarization assay for diagnosis of leptospirosis.

The objective of the present study was to investigate the usefulness of a recombinant flagellar protein, FlaB, of Leptospira interrogans serovar pomona in the serodiagnosis of leptospirosis by the fluorescence polarization assay (FPA). The recombinant protein FlaB was purified to homogeneity by a combination of nickel-nitriloacetic acid agarose chromatography, electrophoresis, and electroelution. Purified FlaB was labeled with fluorescein isothiocyanate (FITC). Western blotting was performed by using bovine sera with microscopic agglutination test (MAT) titers of antibodies against L. interrogans serovar pomona and L. bergpetersenii serovars hardjo and sejroe to confirm the antigenicity of FlaB. Western blot analysis demonstrated that labeled as well as unlabeled FlaB was recognized by the positive sera tested, indicating the broad serovar cross-reactivity of this protein. It also indicated that labeling with FITC did not affect the antigenicity. By using FITC-labeled FlaB as a tracer antigen, a homogeneous FPA was developed to detect antileptospiral antibodies in bovine sera. A population of 208 MAT-positive and 208 MAT-negative serum samples was tested by FPA. The FPA cutoff was determined by receiver operating characteristic analysis. By FPA, 83. 7% of the MAT-positive serum samples were positive and 81.2% of the MAT-negative serum samples were negative. Compared to the results of MAT, the positive predictive value of FPA was 81.7% and the negative predictive value of FPA was 83.3%. The FPA is a simple and rapid technique for the detection of anti-Leptospira antibodies.

Animals↗

PubMed1999

Iron primes hepatic macrophages for NF-kappaB activation in alcoholic liver injury.

NF-kappaB activation induced by lipopolysaccharide (LPS) in cultured hepatic macrophages (HM) may be abrogated by pretreatment of cells with a lipophilic iron chelator, 1,2-dimethyl-3-hydroxypyrid-4-one (L1, deferiprone), suggesting a role for iron in this molecular event [M. Lin, M., R. A. Rippe, O. Niemelä, G. Brittenham, and H. Tsukamoto, Am. J. Physiol. 272 (Gastrointest. Liver Physiol. 35): G1355-G1364, 1997]. To ascertain the relevance in vivo of this hypothesis, HM from an experimental model of alcoholic liver injury were examined for the relationship between nuclear factor (NF)-kappaB activation and iron storage. HM showed a significant increase in nonheme iron concentration (+70%), accompanied by enhanced generation of electron paramagnetic resonance-detected radicals (+200%), NF-kappaB activation (+100%), and tumor necrosis factor-alpha (+150%) and macrophage inflammatory protein-1 (+280%) mRNA induction. Treatment of the cells ex vivo with L1 normalized all these parameters. HM content of ferritin protein, ferritin L chain mRNA, and hemeoxygenase-1 mRNA and splenic content of nonheme iron were increased, suggesting enhanced heme turnover as a cause of the increased iron storage and NF-kappaB activation. To test this possibility, increased iron content in HM was reproduced in vitro by phagocytosis of heat-treated red blood cells. Treatment caused a 40% increase in nonheme iron concentration and accentuated LPS-induced NF-kappaB activation twofold. Both effects could be abolished by pretreatment of cells with zinc protoporphyrin, a hemeoxygenase inhibitor. To extend this observation, animals were splenectomized before 9-wk alcohol feeding. Splenectomy resulted in further increments in HM nonheme iron storage (+60%) and NF-kappaB activation (+90%) and mononuclear cell infiltration (+450%), particularly around the iron-loaded HM in alcohol-fed animals. These results support the pivotal role of heme-derived iron in priming HM for NF-kappaB activation and expression of proinflammatory genes in alcoholic liver injury.

Animals↗

PubMed1999

A newly identified nonsecretor allele of the human histo-blood group alpha(1,2)fucosyltransferase gene (FUT2).

BACKGROUND AND OBJECTIVES: The human Secretor alpha(1, 2)fucosyltransferase gene determines the ABH secretor status and influences the Lewis phenotype of an individual. Studies were carried out on the Lewis (a+b-) nonsecretors of different groups indigenous to Taiwan to demonstrate their se genotypes. METHODS: The Lewis phenotype of the blood samples was determined by a microplate method. The se genotypes of the individuals with the Lewis (a+b-) phenotype were analyzed by a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method designed for the se alleles reported previously. PCR and cloning techniques were used to determine the coding sequence of the novel se gene. RESULTS: A new se allele, se685, with a three-nucleotide deletion of GTGGT to GT in the coding region of nucleotides 685 through 689 was identified in a Le (a+b-) nonsecretor from the Ami tribe indigenous to Taiwan. The deletion predicts the loss of the amino acid Val230 in the corresponding secretor enzyme's C-terminal segment. The distribution of the se685 allele in the Ami tribe was further verified by PCR-RFLP analysis. CONCLUSION: The Se gene exhibits heterogeneity with some Se alleles being common but others displaying a unique distribution in different ethnic populations. The newly identified se685 allele seems to exist only in the Ami tribe indigenous to Taiwan.

ABO Blood-Group System↗

PubMed1999

Targeted mutation of plasma phospholipid transfer protein gene markedly reduces high-density lipoprotein levels.

It has been proposed that the plasma phospholipid transfer protein (PLTP) facilitates the transfer of phospholipids and cholesterol from triglyceride-rich lipoproteins (TRL) into high-density lipoproteins (HDL). To evaluate the in vivo role of PLTP in lipoprotein metabolism, we used homologous recombination in embryonic stem cells and produced mice with no PLTP gene expression. Analysis of plasma of F2 homozygous PLTP-/- mice showed complete loss of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, and partial loss of free cholesterol transfer activities. Moreover, the in vivo transfer of [3H]phosphatidylcholine ether from very-low-density proteins (VLDL) to HDL was abolished in PLTP-/- mice. On a chow diet, PLTP-/- mice showed marked decreases in HDL phospholipid (60%), cholesterol (65%), and apo AI (85%), but no significant change in non-HDL lipid or apo B levels, compared with wild-type littermates. On a high-fat diet, HDL levels were similarly decreased, but there was also an increase in VLDL and LDL phospholipids (210%), free cholesterol (60%), and cholesteryl ester (40%) without change in apo B levels, suggesting accumulation of surface components of TRL. Vesicular lipoproteins were shown by negative-stain electron microscopy of the free cholesterol- and phospholipid-enriched IDL/LDL fraction. Thus, PLTP is the major factor facilitating transfer of VLDL phospholipid into HDL. Reduced plasma PLTP activity causes markedly decreased HDL lipid and apoprotein, demonstrating the importance of transfer of surface components of TRL in the maintenance of HDL levels. Vesicular lipoproteins accumulating in PLTP-/- mice on a high-fat diet could influence the development of atherosclerosis.

Animals↗

PubMed1999

Complementary deoxyribonucleic acid cloning and enzymatic characterization of a novel 17beta/3alpha-hydroxysteroid/retinoid short chain dehydrogenase/reductase.

17Beta-hydroxysteroid dehydrogenases (17betaHSDs) convert androgens and estrogens between their active and inactive forms, whereas retinol dehydrogenases catalyze the conversion between retinol and retinal. Retinol dehydrogenases function in the visual cycle, in the generation of the hormone retinoic acid, and some also act on androgens. Here we report cloning and expression of a complementary DNA that encodes a new mouse liver microsomal member of the short chain dehydrogenase/reductase (SDR) superfamily and its enzymatic characterization, i.e. 17betaHSD9. Although 17betaHSD9 shares 88% amino acid identity with rat 17betaHSD6, its closest homolog, the two differ in substrate specificity. In contrast to other 17betaHSD, 17betaHSD9 has nearly equivalent activities as a 17betaHSD (with estradiol approximately = adiol) and as a 3alphaHSD (with adiol approximately = androsterone). It also recognizes retinol as substrate and represents in part the NAD+-dependent liver microsomal dehydrogenase that uses unbound retinol, but not retinol complexed with cellular retinol-binding protein. Thus, this enzyme has catalytic properties that overlap with two subgroups of SDR, 17betaHSD and retinol dehydrogenases. Inactivation of estrogen and a variety of androgens seems to be its most probable function. Because of its apparent inability to access retinol bound with cellular retinol-binding protein, a function in the pathway of retinoic acid biosynthesis seems less obvious. These data provide additional insight into the enzymology of estrogen, androgen, and retinoid metabolism and illustrate how closely related members of the SDR superfamily can have strikingly different substrate specificities.

17-Hydroxysteroid Dehydrogenases↗

PubMed1999

A 33 kDa molecular marker of sperm acrosome differentiation and maturation in the tammar wallaby (Macropus eugenii).

This study was undertaken to identify potential molecular markers of acrosomal biogenesis and post-testicular maturation in marsupials, using the tammar wallaby as a model species. A two-step sperm extraction procedure yielded two protein extracts of apparent acrosomal origin and a tail extract. The extracts were analysed by SDS-PAGE under reducing conditions. Several prominent polypeptide bands (45, 38 and 33 kDa) appeared common to both acrosomal extracts. Antiserum raised against the 33 kDa polypeptide from the inner acrosomal membrane matrix (IAMM) extract showed immunoreactivity with 45, 38 and 33 kDa polypeptides in both acrosomal extracts, indicating that the 33 kDa polypeptide was related to the proteins in the 45 and 38 kDa bands. Therefore, the antiserum was used as a molecular probe. Indirect immuno-fluorescence indicated that the acrosome was the major location of the 33 kDa polypeptide. This contention was confirmed by ultrastructural study: immunogold labelling indicated that the 33 kDa polypeptide associated with acrosomal matrix components throughout acrosomal development in the testes and throughout post-testicular maturation in the epididymis. The label clearly delineated the changing morphology of the maturing marsupial acrosome. This is the first study to use immunocytochemical techniques to chart testicular and post-testicular development of any sperm organelle in a marsupial. As a result of this study, a 33 kDa molecular marker of marsupial acrosome differentiation and maturation has been identified. It may be possible to chart similar events in other marsupial species and identify opportunities for manipulating fertility.

Acrosome↗

PubMed1999

Quantitative study of the variability of hepatic iron concentrations.

BACKGROUND: The hepatic iron concentration (HIC) is widely used in clinical practice and in research; however, data on the variability of HIC among biopsy sites are limited. One aim of the present study was to determine the variability of HIC within both healthy and cirrhotic livers. METHODS: Using colorimetric methods, we determined HIC in multiple large (microtome) and small (biopsy-sized) paraffin-embedded samples in 11 resected livers with end-stage cirrhosis. HIC was also measured in multiple fresh samples taken within 5 mm of each other ("local" samples) and taken at sites 3-5 cm apart ("remote" samples) from six livers with end-stage cirrhosis and two healthy autopsy livers. RESULTS: The within-organ SD of HIC was 13-1553 microg/g (CV, 3.6-55%) for microtome samples and 60-2851 microg/g (CV, 15-73%) for biopsy-sized samples. High variability of HIC was associated with mild to moderate iron overload, because the HIC SD increased with increasing mean HIC (P <0.002). Livers with mean HIC >1000 microg/g exhibited significant biological variability in HIC between sites separated by 3-5 cm (remote sites; P <0.05). The SD was larger for biopsy-sized samples than for microtome samples (P = 0.02). CONCLUSION: Ideally, multiple hepatic sites would be sampled to obtain a representative mean HIC.

Biopsy↗

PubMed1999

[Missense mutations of exons 14 and 18 of Wilson's disease gene in Chinese patients].

OBJECTIVE: To investigate the characteristics of mutations of exons 14 and 18 of Wilson's disease (WD) gene in Chinese patients. METHODS: The subjects of study included 60 unrelated normal controls and 44 unrelated WD patients. Genomic DNA was prepared from peripheral blood leukocytes by a salt-out method. Mutations of exons 14 and 18 in these subjects were screened by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and further confirmed by sequencing. RESULTS: One patient was homozygous for Arg1041Pro mutation of exon 14 and another patient was heterozygous for Asn1270Ser mutation of exon 18. CONCLUSION: The mutations of exons 14 and 18 of WD gene in Chinese patients were confirmed by sequencing for the first time in China. Arg1041Pro was identified as a novel missense mutation. In addition, an Asn1270Ser, previously described mutation, was detected in this study. But exons 14 and 18 are not the hot point mutations of WD gene in Chinese patients.

Adolescent↗

PubMed1999

UV Resonance Raman Spectroscopic Identification of Titanium Atoms in the Framework of TS-1 Zeolite.

Framework titanium atoms in titanium-substituted silicalite (TS-1) can be identified by UV resonance Raman spectroscopy since the associated Raman bands at 1125, 530, and 490 cm(-1) (see figure) are observed only when the charge transfer transition associated with the framework Ti atoms is excited by a UV laser. Thus, framework Ti atoms can be distinguished from nonframework Ti atoms and other defect sites. This method can be applicable to identifying transition metal atoms in the frameworks of other molecular sieves.

Journal Article↗