Search PubMed⌕ Search

Biomedical subjects

L Jin

Publications and source records attributed to L Jin.

At least 397 records · Page 22Linked to original sources
PubMed1993

Genomic typing of BK virus in clinical specimens by direct sequencing of polymerase chain reaction products.

Two hundred and twelve urine specimens, from several clinical groups, were examined for BK virus (BKV) using the polymerase chain reaction (PCR) to detect the VP1 region of BKV DNA. Positive results were obtained on 14 specimens from 44 post-transplant patients (31.8%), 10 specimens from 39 pregnant women (25.6%), and 5 specimens from 100 children (5%) but not on any specimens from 29 laboratory staff. Twenty-eight of the amplified BKV genomes, 19 from urine specimens, eight from culture fluid of inoculated tissue, and also one from a throat washing were directly sequenced from single-stranded templates immobilized via a biotinylated primer; it was possible to assign all to one of the four subtypes of BKV which had previously been identified on the basis of variation in nucleotide sequence of the VP1 region. Serological subgroup classification correlated with the genomic subtyping results in 21 of the isolates. The distribution of the BKV subtypes and the clinical status of the infected individuals are discussed.

BK Virus↗
PubMed1993

Rapid genomic typing of BK virus directly from clinical specimens.

A simple and rapid method, PCR-restriction enzyme analysis (PCR-RE) for BK virus (BKV) typing was developed, based on the presence of type-specific restriction enzyme sites in a 327 bp PCR-generated fragment which partially encodes the VP1 protein. The enzymes, Alu I, Xmn I and Ava II, were used to digest the PCR products in two stages. Ethidium bromide banding patterns characteristic of each of four subtypes of BKV were visualized through gel electrophoresis. A total of 37 samples from clinical specimens and culture fluids were successfully subtyped using the PCR-RE. A second method, PCR-sequencing, was applied to samples that generated less than 100 ng of DNA. These were subjected to a second round of PCR and then sequenced from single stranded templates immobilised via a biotinylated primer. The subtypes were assigned on the basis of the type-specific sequences previously characterized.

BK Virus↗
PubMed1993

A unified approach to study hypervariable polymorphisms: statistical considerations of determining relatedness and population distances.

Relatedness between individuals as well as evolutionary relationships between populations can be studied by comparing genotypic similarities between individuals. When hypervariable loci are used to describe genotypes, it is shown that both of these problems can be approached with a unified theory based on allele sharing between individuals. The distributions of the number of shared alleles between individuals indicate their kin relationships. Extending this, we obtain statistics for genetic distances between populations based on average number of alleles shared between individuals within and between two different populations. Traditional statistical inferential procedure can be used to establish specific kinship relationships between individuals. We derive estimates of the number of hypervariable loci needed for a specified reliability of such an inference. Evolutionary dynamics of genetic distance statistics based on allele sharing is also studied. It shows that such measures of genetic distances remain linear with the time of divergence for a period comparable to that of the gene frequency-based measures of genetic distances. Statistical properties of measures based on allele sharing establish that for using such summary statistics it is not necessary to know the full characteristics of all loci used. It is enough to know the degree of heterozygosity per locus and the number of loci. Therefore, in principle, this approach can also be used for DNA fingerprinting data in the studies of relatedness between individuals as well as between populations. The possible compromising features of multilocus DNA fingerprinting data are also discussed.

Alleles↗
PubMed1993

Specificity of factor Xa in the cleavage of fusion proteins.

The precursor protein honey bee prepromelittin has been expressed as a fusion protein in Escherichia coli joined to the C-terminus of a truncated form of the bacteriophage gene 10 protein via an engineered recognition sequence for Factor Xa. Factor Xa was found to cleave poorly at the engineered site, giving a low yield of the required prepromelittin. In contrast, cleavage on the C-terminal side of the sequence VLGR at residue 67 in the gene 10 sequence proceeded in high yield. Factor Xa may be inhibited by adjacent hydrophobic sequences on the C-terminal side of a potential cleavage site.

Amino Acid Sequence↗
PubMed1993

On allele frequency computation from DNA typing data.

Forensic applications of DNA typing data require the estimation of the frequencies of all observed alleles, which is currently done by a fixed set of groupings (binning) of alleles in a database. Recently it's validity has been questioned on the ground that when a DNA fragment size is close to a bin boundary, the frequencies of all adjacent bins should be added. On the contrary, the current forensic database indicates that when the match window of a DNA fragment overlaps 2 bins, it is enough to consider the bin with the larger frequency, and this never underestimates the frequency within the match interval with the current choice of fixed-bin widths. On average, the current fixed-bin procedure yields an allele frequency at least 2-fold higher than that of a floating-bin.

Alleles↗
PubMed1993

VNTR allele frequency distributions under the stepwise mutation model: a computer simulation approach.

Variable numbers of tandem repeats (VNTRs) are a class of highly informative and widely dispersed genetic markers. Despite their wide application in biological science, little is known about their mutational mechanisms or population dynamics. The objective of this work was to investigate four summary measures of VNTR allele frequency distributions: number of alleles, number of modes, range in allele size and heterozygosity, using computer simulations of the one-step stepwise mutation model (SMM). We estimated these measures and their probability distributions for a wide range of mutation rates and compared the simulation results with predictions from analytical formulations of the one-step SMM. The average heterozygosity from the simulations agreed with the analytical expectation under the SMM. The average number of alleles, however, was larger in the simulations than the analytical expectation of the SMM. We then compared our simulation expectations with actual data reported in the literature. We used the sample size and observed heterozygosity to determine the expected value, 5th and 95th percentiles for the other three summary measures, allelic size range, number of modes and number of alleles. The loci analyzed were classified into three groups based on the size of the repeat unit: microsatellites (1-2 base pair (bp) repeat unit), short tandem repeats [(STR) 3-5 bp repeat unit], and minisatellites (15-70 bp repeat unit). In general, STR loci were most similar to the simulation results under the SMM for the three summary measures (number of alleles, number of modes and range in allele size), followed by the microsatellite loci and then by the minisatellite loci, which showed deviations in the direction of the infinite allele model (IAM). Based on these differences, we hypothesize that these three classes of loci are subject to different mutational forces.

Alleles↗
PubMed1993

A single polymorphic STR system in the human phenylalanine hydroxylase gene permits rapid prenatal diagnosis and carrier screening for phenylketonuria.

Phenylketonuria (PKU) is an autosomal recessive genetic disorder caused by phenylalanine hydroxylase (PAH) deficiency. Individuals afflicted with PKU develop irreversible mental retardation that can be largely prevented by the administration of a low-phenylalanine diet. A number of restriction fragment-length polymorphisms (RFLPs) have been identified in the PAH gene. Combinations of RFLPs constitute unique haplotypes that can be used to identify mutant PAH chromosomes for prenatal diagnostic purpose in PKU families. Unfortunately, the utility of haplotype analysis is limited in populations with a single predominant haplotype. We have identified a novel short tandem repeat (STR) within the PAH gene that has an average level of heterozygosity of about 75% in Orientals and about 80% in European Caucasian populations. This single marker is as informative as haplotype analysis in Europeans and nearly twice as informative as haplotype analysis in Orientals. Although there is statistically significant disequilibrium between STR alleles and RFLP-based haplotypes, there is a relatively low degree of disequilibrium between STR alleles and certain RFLP sites. Nevertheless, the combined use of the STR and RFLP haplotype systems increases the informativity of linkage-based tests for prenatal diagnosis and carrier screening in PKU families.

Alleles↗
PubMed1993

Differentiation of human pituitary adenomas determines the pattern of chromogranin/secretogranin messenger ribonucleic acid expression.

The distribution of chromogranin/secretogranin (Cg/Sg) mRNAs, determined by Northern and in situ hybridization, was analyzed in 14 cultured pituitary adenomas characterized by immunohistochemistry and hormone secretion in a defined medium in vitro. There were 5 functional GH adenomas, 1 silent GH adenoma, 7 null cell adenomas, and 1 oncocytoma. The null cell adenomas, oncocytoma, and silent GH adenomas were also analyzed by electron microscopy. Most null cell adenomas and the oncocytoma secreted FSH and LH into the culture medium. GH adenomas, which are examples of well differentiated tumors based on morphological examination, expressed significantly more SgIII mRNA compared to the null cell adenomas and oncocytoma (70 +/- 6% vs. 22 +/- 5%; P < 0.001). GH adenomas also expressed significantly less CgA mRNA compared to the less well differentiated null cell adenomas and oncocytoma (27 +/- 6% vs. 67 +/- 4%; P < 0.001), which could be considered less well differentiated based on ultrastructural morphological features. After treatment with phorbol 12-myristate 13-acetate (10(-7) M) for 7 days, there was an increase in the mRNA for CgB and SgII mRNAs in GH and null cell tumors, while dexamethasone treatment for 7 days increased CgA mRNA in GH and null cell adenomas. GnRH treatment for 7 days increased CgB mRNA in null cell adenomas. Phorbol 12-myristate 13-acetate also decreased the percentage of immunoreactive GH cells and GHm RNA, determined by in situ and Northern hybridization analyses. These results indicate that pituitary adenomas have a distinct pattern of Cg/Sg mRNA expression, which appears to be related to the degree of morphological differentiation of these neoplasms, and suggest that the effects of secretagogues on various Cg/Sg mRNA levels may be related to the stimulation of hormone secretion.

Adenoma↗
PubMed1993

Effects of estrogen and epidermal growth factor on prolactin and Pit-1 mRNA in GH3 cells.

The effects of epidermal growth factor (EGF) and 17 beta-estradiol (E2) on the expression of prolactin (PRL), the transcription factor Pit-1/GHF-1 (Pit-1), and on dopamine D2 receptor mRNA in GH3 cells were analyzed by immunocytochemistry, in situ hybridization, and Northern analysis in a defined serum-free cell culture medium. Radioimmunoassay was used to determine PRL secretion. Both EGF and E2 stimulated PRL mRNA and PRL secretion, although the effects of EGF were more rapid than those of E2. Pit-1 mRNA levels were not significantly changed in spite of the 2- to 8-fold increases in PRL mRNA levels and significant increases in PRL secretion. Analysis of dopamine D2 receptor mRNA by in situ hybridization and Northern hybridization detected expression of dopamine receptor in GH3 cells, but the receptor mRNA levels were not modified by EGF or E2 treatment in complete serum or in serum-free media. These observations suggest that EGF and E2 modulate PRL mRNA levels and PRL secretion significantly in vitro, while the mRNA levels of Pit-1 do not change significantly in GH3 cells cultured in a defined culture medium.

Animals↗
PubMed1993

Rat placental hepatocyte growth factor/scatter factor: purification, characterization, and developmental regulation.

In view of significant species-specific sequence differences between human and rat placental hepatocyte growth factor (HGF)/scatter factor (SF), the rat placental HGF/SF (rpSF) was purified, and its properties compared with human placental HGF/SF (hpSF). Like hpSF, rpSF scattered Madin-Darby canine kidney cells at 1-2 ng/ml and is composed of two subunits of 60 kDa and 30 kDa. Higher amounts (> 50%) of uncleaved 90-kDa form was present in the HGF/SF preparations from both human and rat placentas. Rat placental SF reacts with antibodies raised against hpSF in rabbits and chickens. The SF activity when expressed per gram rat placental tissue rises rapidly up to 9 days and then levels off. When expressed per milligram tissue protein it also increases rapidly up to 9 days and then declines. The expression of HGF/SF mRNA during development parallels that of HGF/SF activity. The specific activity of HGF/SF receptor (c-met) mRNA also appears to peak at 6 days. These findings suggest that (i) in spite of significant (> 10%) sequence differences between rpSF and hpSF, they exhibit similar structural, biologic, and immunologic characteristics and (ii) HGF/SF and its receptor are expressed in high amounts on Day 6 and then decline in developing placenta.

Animals↗
PubMed1993

Identification of repeat sequence heterogeneity at the polymorphic short tandem repeat locus HUMTH01[AATG]n and reassignment of alleles in population analysis by using a locus-specific allelic ladder.

An allelic ladder containing amplified sequences of seven alleles of the polymorphic human tyrosine hydroxylase locus, HUMTH01, was constructed and employed as a standard marker. Sequence analysis of each ladder component indicates that fragments differ by integral multiples of the AATG core repeat sequence characteristic of this locus. Individual alleles are designated "5" through "11," according to the number of complete reiterations of the core repeat contained within them. Comparison of the HUMTH01 allelic ladder with DNA samples amplified at this locus revealed core repeat length heterogeneity (i.e., deletions or insertions shorter than one core repeat) within the human population. In particular, a common allele was identified which migrates more quickly than allele 10, but more slowly than allele 9, on electrophoresis through a denaturing polyacrylamide gel. Sequence analysis of this allele, designated "10-1," reveals lack of a single adenine normally present in the seventh copy of the AATG. The allelic ladder was used to reevaluate previously published population data. Results of testing for Hardy-Weinberg equilibrium and population substructure were not altered significantly by these modifications.

Alleles↗
PubMed1993

Pituitary specific transcription factor messenger ribonucleic expression in adenomatous and nontumorous human pituitary tissues.

BACKGROUND: The pituitary-specific transcription factor, Pit-1/GHF-1 (Pit-1) is a member of the POU family of homeobox genes. This transacting factor has a role in the development and expression of selected anterior pituitary cells including prolactin, growth hormone (GH), and thyrotropin, cell types in rodent pituitary. The expression of Pit-1 mRNA in human pituitaries has not been previously analyzed and the role of specific secretagogues in the regulation of Pit-1 transcripts in human pituitary adenomas has not been examined. Analysis of Pit-1 expression in normal and neoplastic human pituitaries may provide insight into the specificity of this transcription factor for pituitary cell types and the role of Pit-1 in human pituitary function. EXPERIMENTAL DESIGN: In situ and Northern hybridization analyses were used to study the expression of Pit-1 mRNA in surgically removed human pituitary adenomas and nontumorous pituitaries obtained within 6 hours postmortem. A cultured GH surgically removed adenoma was used to analyze the regulation of GH and Pit-1 in vitro. RESULTS: In situ hybridization localized Pit-1 mRNA in all pituitary adenomas, although the mRNA levels in GH and prolactin adenomas were significantly higher than those in ACTH, null cell and gonadotroph tumors. Northern hybridization analysis revealed a 2.4 and a 1.2 kb mRNA transcript for Pit-1. Although GH mRNA transcript was markedly changed by dexamethasone and phorbol 12-myristate 13-acetate in vitro, Pit-1 mRNA transcripts were not changed significantly by these secretagogues. CONCLUSIONS: Pit-1 mRNA transcripts are expressed mainly in prolactin and GH pituitary adenomas, but are also found in other adenomas and in the nontumorous adult human pituitary. Pit-1 mRNA transcript levels are not readily modulated in cultured GH adenoma cells by various secretagogues in vitro compared with the marked modulation of GH mRNA transcripts by these same secretagogues.

Adenoma↗
PubMed1993

[The effect of low dose aspirin on the platelet function in patients with acute myocardial infarction].

To assess the appropriate dose of aspirin in the treatment of patients with acute myocardial infarction (AMI), 60 cases of AMI were randomized into 2 groups, 30 cases each; one with conventional therapy, another with conventional therapy combined with daily oral aspirin 100mg. The second group was further divided into subgroup I with serum peak CK < 1000 U/L and subgroup II with serum peak CK > 1000U/L. The parameters of platelet function including plasma TXB2, TXB2/6-keto-PGF1 alpha, platelet aggregation induced by 5-HT and epinephrine were studied on different days for 3 weeks. The results revealed that there was a successful inhibition of platelet function as assessed by significant decrease of plasma TXB2 and TXB2/6-keto-PGF1 alpha ratio and inhibition of platelet aggregation in the subgroup I, but little effect on subgroup II. It is shown that in the treatment of AMI, a daily dose of 100mg of aspirin is insufficient for severe cases, and according to the observation in ISIS-2 study, a daily dose of aspirin around 160mg may be appropriate.

6-Ketoprostaglandin F1 alpha↗
PubMed1993

Determination of relatedness between individuals using DNA fingerprinting.

Relatedness between individuals is an important element of genetic-epidemiological and evolutionary investigations in the context of anthropological research. In general, data on relationships between individuals are gathered from personal interviews or from examination of vital records. When blood samples are collected, such information can be validated from genotypic similarities of individuals. Although genotype data may offer opportunities to exclude certain types of relationships, inclusionary statements are necessarily only probabilistic in nature. The limitations of such probabilistic statements depend on the number of segregating alleles and the extent of polymorphisms at the loci employed. With the advent of DNA technology, several hypervariable single-locus probes (SLPs) and multilocus probes (MLPs) are now available for many organisms. These can be used to circumvent limitations of unequivocal assignment of relationships from genotype data. In this article we describe analytical principles for such investigations. In particular, we propose summary measures of DNA fingerprinting data (e.g., number of different alleles and number of shared alleles) that can be used to describe kinship relationships between individuals. We derive the expected distributions of number of alleles in individuals and of number of shared alleles between individuals of known relationships in a population. These distributions can be used in hypothesis testing to determine relatedness between individuals. We also derive the number of SLPs, each detecting a hypervariable polymorphism, needed to determine a specified relationship for given ranges of errors of prediction. Illustrations of the theory with data on several short tandem repeat loci and variable number of tandem repeat (VNTR) loci indicate that with 6 to 12 SLPs the parent-offspring pairs can be reliably distinguished from random pairs of individuals. This theory also serves the purpose of detecting inbreeding levels in a natural population.

Alleles↗
PubMed1993

HGF-SF: effects on motility and morphology of normal and tumor cells.

HGF-SF is a recently discovered cytokine which has both mitogenic and motogenic effects on a wide variety of cells. We used a computerized digital imaging system to measure motility and morphology of isolated cells. In this chapter we will describe the effect of HGF-SF and of other growth factors on the velocity, area, circularity, and flatness of normal and tumor cells. We will then discuss the possible mechanism of HGF-SF induced motility and the possible role of this factor in biological and pathological processes.

Animals↗
PubMed1993

Protein kinase C (PKC) activity and PKC messenger RNAs in human pituitary adenomas.

Protein kinase C (PKC) is involved in the differentiation and growth regulation of a variety of tissues including anterior pituitary gland cells. To determine the distribution of PKC in different types of adenomas, PKC activity was analyzed in human pituitary tumors and the effects of hypothalamic hormone stimulation on PKC activity were examined in cultured adenoma cells. Gonadotroph (LH/FSH) and null cell adenomas had significantly higher levels of particulate, soluble, and total PKC activity compared with growth hormone (GH) adenomas (P < 0.05). Chronic stimulation of null cell adenomas with gonadotropin hormone-releasing hormone or of one GH adenoma with GH-releasing hormone for 7 days did not significantly alter total PKC activity in pituitary cells cultured in serum-free medium. Localization of the calcium-dependent PKC isozymes (alpha, beta and gamma) by immunohistochemistry and in situ hybridization revealed predominantly PKC alpha in all adenomas and variable expression of PKC beta and gamma in some tumors. When the calcium-independent PKC isozymes (delta, epsilon, and zeta) were localized by in situ hybridization, normal and neoplastic pituitaries expressed abundant mRNA for PKC epsilon, whereas some tumors and one normal pituitary had a few cells positive for PKC zeta mRNA as evaluated by grain density and the number of cells labeled. These results indicate that there is a variable distribution of PKC mRNA isozymes in human pituitary adenomas and that normal pituitaries and pituitary adenoma cells express the mRNA for both the calcium-dependent and some of the calcium-independent PKC isozymes. Chronic treatment with the hypothalamic gonadotropin hormone-releasing hormone and GH-releasing hormone, which increased LH/FSH and GH secretion, respectively, did not increase PKC activity in cultured adenoma cells. The presence of calcium-dependent and calcium-independent PKC isozymes in normal and neoplastic pituitary cells indicates that PKC probably plays a major role in signal transduction in the human pituitary adenomas examined in this study.

Adenoma↗