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Biomedical subjects

S C Li

Publications and source records attributed to S C Li.

At least 127 records · Page 7Linked to original sources
PubMed1992

Ceramide glycanase from the earthworm, Lumbricus terrestris.

Ceramide glycanase (CGase) is an enzyme that cleaves the linkage between the sugar chain and the ceramide. To make this enzyme readily available, we have developed a simple method for preparing it from the earthworm, Lumbricus terrestris. The method involves Bio-Gel A-0.5m, octyl-Sepharose and p-aminophenylthiogalactoside-agarose column chromatography. By gel filtration, the molecular mass of earthworm CGase was found to be 43.7 kDa. With ganglioside GM1 as substrate, the optimal pH of this enzyme was found to be between pH 3.5 and 4.0. Earthworm CGase hydrolyses glycolipids only in the presence of a detergent. Among various bile salts tested, sodium cholate was found to be the most effective in stimulating the hydrolysis of GM1 by this enzyme. Earthworm CGase released intact glycan chains from various glycosphingolipids in which the glycan chain is linked to the ceramide through a beta-glucosyl linkage. It also detached glycan chains from lactosyldialkylglycerol and alkyl-beta-lactosides.

Animals↗
PubMed1992

Evidence for two cDNA clones encoding human GM2-activator protein.

Two cDNAs encoding GM2 activator, pGM2A (648 bp) and GAP (1093 bp), were isolated from human placenta lambda gt11 libraries. The DNA sequence of pGM2A from 1 to 302 was almost identical with GAP, but diverged from 303-648. PCR was used to demonstrate the presence of both species of GM2 activator in placental RNA. Both cDNAs hybridized to mRNAs of approximately 2.3 kb and to identical single bands on genomic Southern blots.

Amino Acid Sequence↗
PubMed1992

Thermostable, salt tolerant, wide pH range novel chitobiase from Vibrio parahemolyticus: isolation, characterization, molecular cloning, and expression.

A chitobiase gene from Vibrio parahemolyticus was cloned into plasmid pUC18 in Escherichia coli strain DH5 alpha. The plasmid construct, pC120, contained a 6.4 kb Vibrio DNA insert. The recombinant gene expressed chitobiase [EC 3.2.1.30] activity similar to that found in the native Vibrio. The enzyme was purified by ion exchange, hydroxylapatite and gel permeation chromatographies, and exhibited an apparent molecular weight of 80 kDa on SDS-polyacrylamide gel electrophoresis. Chitobiose and 6 more substrates, including beta-N-acetyl galactosamine glycosides, were hydrolyzed by the recombinant chitobiase, indicating its putative classification as an hexosaminidase [EC 3.2.1.52]. The enzyme was resistant to denaturation by 2 M NaCl, thermostable at 45 degrees C and active over a very unusual (for glycosyl hydrolases) pH range, from 4 to 10. The purified cloned chitobiase gave 4 closely focussed bands on an isoelectric focusing gel, at pH 4 to 6.5. The N-terminal 43 amino acid sequence shows no homology with other proteins in commercial databanks or in the literature, and from its N-terminal sequence, appears to be a novel protein, unrelated in sequence to chitobiases from other Vibrios reported and unrelated to hexosaminidases from other organisms.

Acetylglucosaminidase↗
PubMed1991

Molecular basis of an adult form of beta-hexosaminidase B deficiency with motor neuron disease.

A patient (KL) with progressive motor neuron disease associated with partial Hex A (alpha beta) and no Hex B (beta beta) activity, synthesized beta-chains which only associated with alpha-chains. To identify the molecular basis of this inability of beta-chains to self associate, RNA from cultured fibroblasts was reverse transcribed, the cDNA encoding the beta-chain amplified by polymerase chain reaction, subcloned, and sequenced to reveal two types of single missense mutation. The first mutation, (Type I) 619A----G, was paternally inherited and converted a 207IIe----Val in a highly conserved region believed to be associated with catalytic activity and activator protein binding. Biochemical evidence for impaired activator protein binding was obtained by purifying Hex A from KL urine and demonstrating a greater than 50% reduction of in vitro GM2 hydrolysis compared to normal urinary Hex A. In other cDNA species (Type II), a maternally inherited 1367A----C mutation converted 456Tyr----Ser in another highly conserved region of the beta-chain and we propose that this mutation leads to the inability of the beta-chains to self associate and thus reach maturity. These same cDNA species contained a second 362A----G mutation which converted 121Lys----Arg, but is apparently a polymorphism since it also occurs in some normal subjects. We propose that the patient is a compound heterozygote in which a combination of no self-association of the mutant beta-chains and impaired activator protein binding to alpha-beta (mutant) (Hex A) required for GM2 hydrolysis result in total beta-Hex B deficiency and slow accumulation of GM2 ganglioside, primarily in motor neurons.

Adult↗
PubMed1991

Synthesis of neoglycoconjugates using oligosaccharide transferring activity of ceramide glycanase.

Ceramide glycanase (CGase) isolated from the leech Macrobdella decora was found to transfer the oligosaccharide en bloc from various glycosphingolipids to suitable acceptors. For example, CGase transferred the intact II3NeuAcGgOse4 from GM1 to 4-phenyl-1-butanol, 1,8-octanediol and various 1-alkanols having a chain length of six or more carbons. Among various 1-alkanols, 1-octanol was found to be the best acceptor. In an incubation mixture of 50 microliters containing 30 nmol of GM1, 50 micrograms of sodium cholate, 20 microliters of 1-octanol, and 0.1 unit of CGase, the ratio between hydrolysis and transglycosylation was approximately 3:1. Negative fast atom bombardment-mass spectral analysis of the enzymatically synthesized octyl-II3NeuAcGgOse4 showed a mass ion at m/z 1109.7 for the parent ion, consistent with its expected mass. NMR analysis of the enzymatically synthesized octyl-II3NeuAcGgOse4 showed that the Glc residue is linked to the octanol through a beta-linkage. Vicinal coupling constants of the ring protons of the sugar residues indicate that their pyranose ring geometries are not affected by the transferase activity. CGase also transferred the oligosaccharide from GM1 to CF3CO-NH(CH2)5CH2OH, (CH3)3CO-CO-NH(CH2)5CH2OH, (HOCH2)3C-NHCO-(CH2)4-COOMe, CH2 = CH-(CH2)7CH2OH and 1,2:3,4-di-O-isopropylidene-D-galactopyranose. The oligosaccharide transferring reaction carried out by CGase should become useful for the synthesis of neoglycoconjugates to study the biological functions expressed by glycan chains in glycosphingolipids.

Animals↗
PubMed1991

Horizontal transmission of hepatitis B virus from siblings and intramuscular injection among preschool children in a familial cohort.

The authors followed 147 children from 113 families who were susceptible to hepatitis B virus infection for a total of 275 person-years. Among these children, 19 became infected with the hepatitis B virus and thus became new subclinical cases. In this cohort study, parents played a minor role in hepatitis B virus horizontal transmission. On the other hand, the estimated incidence rate ratio of hepatitis B virus infection for siblings of a hepatitis B e antigen (HBeAg) carrier was 2.8 (95% confidence interval 1.1-7.4) when compared with those children without a HBeAg sibling carrier as analyzed by multiple logistic regression. The adjusted incidence rate ratio among siblings increased with increased number of HBeAg carriers. In addition, intramuscular injections played an important role in hepatitis B virus transmission in children. It was found that 61.8% (170/275 person-years) of the children had received intramuscular injections. Most of the injections were administered at private clinics over a 2-year period. Hepatitis B virus infection showed a correlation with injection (adjusted incidence rate ratio = 3.3, 95% confidence interval 1.1-9.5) and with frequency of injections. The authors concluded that HBeAg was a valuable marker for infectivity in hepatitis B virus horizontal transmission. Furthermore, the authors found that hepatitis B virus infection is independently transmitted from sibling to sibling, and by iatrogenic injections because improperly sterilized syringes were shared in areas where hepatitis B is prevalent and intramuscular injection is common.

Adult↗
PubMed1991

Dosage adjustment and clinical outcomes of long-term use of high-dose tobramycin in adult cystic fibrosis patients.

A two-phase study was undertaken designed to investigate the impact of computer-aided drug monitoring on tobramycin concentrations and clinical outcomes in adult patients with cystic fibrosis. In phase one, a baseline (historical control) study of drug use patterns was performed. During the second phase, patients admitted for intravenous treatment with tobramycin for acute exacerbations of pseudomonal pulmonary infections were randomly allocated to one of two schedules. Group A patients had tobramycin dosage regimens decided by clinicians based on pre-existing protocols using serum tobramycin assay data determined three times weekly. Group B patients had dosage regimens determined by a computerized pharmacokinetic predictive program using both population-based pharmacokinetic parameter estimation and fitting of serum concentration-time data using Bayesian regression. The agreed therapeutic target was a peak serum tobramycin concentration of 8-10 mg/L and a trough concentration of 1-2 mg/L. There was a major difference between the two groups comparing the number of paired trough and peak concentrations within the target concentration ranges (group A-14%; group B-34.7%, chi 2 test, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Identification of a conserved lipopolysaccharide-plus-interleukin-4-responsive element located at the promoter of germ line epsilon transcripts.

Treatment of splenic B lymphocytes and certain B-lineage cell lines with the mitogen lipopolysaccharide (LPS) and the lymphokine interleukin-4 (IL-4) induces expression of germ line immunoglobulin C epsilon transcripts and class switching to the C epsilon gene. We show that LPS-plus-IL-4 induction of germ line epsilon transcripts (termed I epsilon transcripts) occurs at the transcriptional level in an Abelson murine leukemia virus-transformed pre-B-cell line. A 1.1-kb region of DNA surrounding the I epsilon promoter endows inducible transcription to a heterologous reporter gene stably transfected into these cells; such inducible expression depends on combined treatment with LPS and IL-4. Analyses of constructs transiently introduced into a B-cell lymphoma line demonstrated that LPS-plus-IL-4-inducible expression can be conferred by a 179-bp segment of DNA spanning the I epsilon transcriptional initiation site. Mutational analyses demonstrated that this expression depended on DNA sequences within a conserved region directly upstream from the I epsilon transcriptional initiation region. One nuclear protein that is constitutively expressed in normal B cells binds to the downstream end of the conserved sequence; its binding specificity correlates with the functional effect of several mutations. Two additional proteins, which are induced by IL-4 treatment of splenic B cells, bind to the transcription initiation sites of I epsilon. These proteins are indistinguishable in binding assays from proteins previously shown to bind an enhancer region of the class II major histocompatibility complex gene A alpha.

Abelson murine leukemia virus↗
PubMed1991

[Case control study on risk factors of cerebral thrombosis].

In order to explore the pathogenic risk factors or protective factors of cerebral thrombosis, 1:1 matched case control study was done in 110 cases diagnosed by computerized tomography (CT). Both cases and controls were selected from several provincial and municipal hospitals in Jinan, Shandong Province, China. Every case was matched with one control on sex, race, age, occupation, residential area, educational level and economic status. 31 factors were analysed and 9 risk or protective factors were found by univariate analysis and multiple stepwise regression. Using the same 9 factors as independent variables, conditional logistic regression was performed and 4 factors were confirmed as pathogenic risk factors or protective factor of cerebral thrombosis (alpha = 0.01). They are high blood pressure (beta = 3.46, OR = 7.57), abdominal skinfold thickness (beta = 3.21, OR = 3.77), familial aggregation of stroke (beta = 2.25, OR = 12.64) and high level HDL2-C (beta = -2.99, OR = 0.16). Moreover, reliability of collective data and control o: bias were evaluated and discussed.

Adult↗
PubMed1990

A novel sialidase which releases 2,7-anhydro-alpha-N-acetylneuraminic acid from sialoglycoconjugates.

The leech (Macrobdella decora) was found to contain two sialic acid-cleaving enzymes: an ordinary sialidase and a novel sialic acid-cleaving enzyme. This novel enzyme released 2,7-anhydro-alpha-N-acetylneuraminic acid (Neu2,7-anhydro5Ac) instead of alpha-N-acetylneuraminic acid (Neu5Ac) from 4-methylumbelliferyl-Neu5Ac, glycoproteins, and gangliosides. We have partially purified this novel sialidase from M. decora. We have also isolated Neu2,7-anhydro5Ac released from 4-methylumelliferyl-Neu5Ac and whale nasal keratan sulfate in pure form. The novel sialidase produced Neu2,7-anhydro5Ac only from sialoglycoconjugates, but not from free Neu5Ac. The structure of Neu2,7-anhydro5Ac produced by the novel sialidase was established by chemical analysis, mass spectrometry, and NMR spectroscopy. NMR analysis showed that instead of the original 2C5 conformation, the pyranose ring of Neu2,7-anhydro5Ac was in the 5C2 conformation, which makes the formation of the 2,7-anhydro bridge possible.

Animals↗
PubMed1990

Normal recombination substrate VH to DJH rearrangements in pre-B cell lines from scid mice.

To further analyze the VDJ recombination defect in lymphoid pre-B cells from mice with severe combined immune deficiency (scid mice), we have assayed the ability of Abelson murine leukemia virus (A-MuLV) transformed pre-B cells from scid mice to rearrange a recombination substrate in which inverted VH to DJH joins activate a selectable (gpt) gene. In unselected populations, substrate rearrangements occurred frequently, but were aberrant and probably analogous to the aberrant rearrangements observed at endogenous scid Ig gene loci. In contrast, populations of scid pre-B lines selected for gpt activity within the substrate contained mostly "normal" VH to DJH joins within the introduced substrate. These findings demonstrate that scid pre-B cells can make normal joins at low efficiency and are discussed with respect to the potential mechanism of the scid defect and the occurrence of Igs in leaky scid mice.

Abelson murine leukemia virus↗
PubMed1990

Development of a new radioimmunoassay for erythropoietin using recombinant erythropoietin.

The development of a 24 hour radioimmunoassay for erythropoietin (EPO) using EPO derived from recombinant DNA as both immunogen and ligand is described in the present paper. Mixed breed rabbits immunized with 10 micrograms/kg of EPO derived from a stably transfected cell line (Elanex Pharmaceuticals Inc., Bothel, Washington, USA, through McDonnell Douglas Corp., St. Louis, Missouri, USA; "MD") produced antibodies to EPO with high titer (up to 1:896,000 final dilution in the tube), high affinity (8.4 x 10(11) liter/M), and good specificity. Purified EPO from the above source or from AmGen Biologicals (Thousand Oaks, California, USA; "AG") were successfully radioiodinated with the chloramine-T method and used as ligand in the radioimmunoassay. Standard dose-response curves prepared with EPO from both commercial sources were not significantly different and showed a sensitivity of 0.75 to 0.96 mU/tube. The dose-response curves in both systems also showed parallelism with serially diluted serum from a patient with aplastic anemia. Within-assay and between-assay precision were determined by assaying multiple replicates of a serum pool. Recovery of exogenous EPO added to a serum pool averaged 97% for both systems. The range of normal human serum EPO was determined by assaying the sera of 153 hematologically-normal adult subjects and was found to be 1.1 to 27.3 mU/ml for MD EPO and 0.5 to 16.7 mU/ml for AG EPO. Sera from several patients with hematologic abnormalities were also assayed, including those of 36 patients with anemia of end-stage renal disease (mean +/- SEM, 29.5 +/- 4.0 mU/ml; P less than 0.01). In conclusion, this new, more rapid and sensitive radioimmunoassay system can be used to measure EPO levels in sera from normal human subjects and patients with several types of anemia, and should also be very useful in therapeutic drug monitoring of patients receiving EPO from various commercial sources.

Anemia↗
PubMed1990

Structure and expression of germ line immunoglobulin heavy-chain epsilon transcripts: interleukin-4 plus lipopolysaccharide-directed switching to C epsilon.

We have isolated cDNA clones complementary to a truncated immunoglobulin heavy-chain C epsilon RNA transcript previously found to be induced in B lymphoid cells by treatment with lipopolysaccharide (LPS) combined with interleukin-4 (IL-4). We demonstrate that this transcript initiates from a promoter upstream of the germ line epsilon class-switch recombination region (S epsilon region). The major germ line C epsilon transcript contains a small 5' exon contributed by sequences upstream of the S epsilon region spliced to the normal C epsilon exons. Treatment of splenic B lymphoid cells with LPS plus IL-4 induces the expression of transcripts from the germ line epsilon transcription unit followed by expression of normal immunoglobulin epsilon heavy-chain mRNA. Furthermore, we demonstrate that similar treatment of transformed precursor B cell lines induces the expression of germ line epsilon transcripts followed by class switching to epsilon expression in these lines. This is the first demonstration of switching to epsilon in cells of the pre-B stage. The general structure of the germ line epsilon transcript and transcription unit is similar to that previously characterized for germ line gamma 2b transcripts. However, expression of these two germ line transcription units in B-lineage cells is inversely regulated by IL-4 (plus LPS) treatment, correlating with the effects of these treatments on switching to these loci.

Animals↗
PubMed1990

A novel fluorescence-based system for assaying and separating live cells according to VDJ recombinase activity.

We describe two retroviral vector-based recombination substrate systems designed to assay for lymphoid VDJ recombinase activity in cultured cells. Both substrates incorporate a constitutive dominant marker gene (the simian virus promoter-driven neo gene) to allow selection of cells that stably integrate the substrate. Both substrates also include a second marker gene that becomes transcriptionally active only when inverted by a site-specific recombination event between flanking immunoglobulin variable-region gene segments. The first vector, similar in structure to previous retrovirus-based recombination substrates, utilizes the bacterial guanine-xanthine phosphoribosyltransferase gene (gpt) as its activatable marker; detection of inversion (VDJ recombinase activity) involves drug selection and Southern blotting analyses. We have used this vector to make a more extensive and quantitative survey of VDJ recombinase activity in B-lineage cell lines than has previously been performed with stable substrates, and we have compared our results with those of other studies that use transient recombination substrates. In the second vector, the activatable gene is the bacterial beta-galactosidase gene (lacZ). Detection for inversional activation of this gene is achieved by a fluorogenic assay, termed FACS-Gal, that detects beta-galactosidase activity in viable cells. The latter assay has the unique advantage of rapidly detecting cells that undergo recombination and also allows viable sorting of cells on the basis of the presence or absence of VDJ recombinase activity. We have used the lacZ vector to rapidly quantitate VDJ recombinase activity in B-lineage cell lines and compared the results with those obtained with the gpt vector. We have also used the lacZ vector to isolate variant pre-B-cell lines with low and high levels of VDJ recombinase activity.

Animals↗
PubMed1990

Risk factors for stroke in rural areas of the People's Republic of China: results of a case-control study.

A case-control study on stroke was carried out in 1985 in conjunction with a prevalence survey on neurologic disorders in 22 rural communities of the People's Republic of China. 585 cases of stroke, detected from the survey, together with an equal number of matched controls were investigated. The statistically significant factors associated with stroke in this study were: hypertension (history, findings at examination); high salt intake; family member(s) with cerebrovascular disease and/or hypertension; heart disease (history, abnormal findings at examination); history of transient ischemic attacks, and arteriosclerotic funduscopic findings. These factors were analyzed separately for hemorrhagic and ischemic strokes.

Case-Control Studies↗