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L Jin

Publications and source records attributed to L Jin.

At least 343 records · Page 19Linked to original sources

Crystal structure determination of an acidic neurotoxin (BmK M8) from scorpion Buthus martensii Karsch at 0.25 nm resolution.

The crystal structure of an acidic neurotoxin, BmK M8, from Chinese scorpion Buthus martensii Karsch was determined at 0.25 nm resolution. The X-ray diffraction data of BmK M8 crystals at 0.25 nm resolution were collected on a Siemens area detector. Using molecular replacement method with a basic scorpion toxin AaH II in a search model, the cross-rotation function, PC-refinement and translation function were calculated by X-PLOR program package. The correct orientation and position of BmK M8 molecule in crystal were determined in a resolution range of 1.5-0.35 nm. The crystallographic refinement was further performed by stereo-chemical restrict least-square technique, followed by simulated annealing, slow-cooling protocols. The final crystallographic R-factor at 0.8-0.25 nm is 0.171. The standard deviations of bond length and bond angle from ideality are 0.0017 nm and 2.24 degrees, respectively. The final model of BmK M8 structure is composed of a dense core of secondary structure elements by a stretch of alpha-helix with two and a half turns (residues 19-28) and a three-stranded antiparallel beta-sheet (residues 2-4, 32-37, 45-51). In addition, three loops protruded from the structural core. The general folding properties of BmK M8 molecule were described; a common structure motif which may appear in all scorpion neurotoxins was identified. The conserved aromatic residues and charged residues were found to be distributed on two roughly opposite surfaces of the molecule. The relationship between these two faces and receptor-binding sites are also discussed.

Animals↗

Bacterial lipoproteins may substitute for cytokines in the humoral immune response to T cell-independent type II antigens.

Bacterial lipoproteins share a common structural motif that has been shown to stimulate proliferation and Ig secretion of murine B cells, in a manner distinct from that mediated by LPSs. Studies of lipoprotein-mediated B cell activation utilized heterogeneous populations of lymphoid cells, leaving unresolved their ability to directly activate resting B cells, as well as their ability to interact with other B cell stimuli. Using highly enriched and/or sort-purified resting murine B cells, we demonstrate that, in contrast to previous reports, lipoproteins (lipoprotein-D, lipoprotein-OspA, and/or the synthetic analogue Pam3Cys) stimulate little, if any, proliferation or Ig secretion in resting B cells. However, when combined with a multivalent membrane (m)Ig-mediated cross-linking signal, dextran-conjugated anti-IgD Abs (alpha delta-dex), lipoproteins mediate up to 10,000-fold inductions in IgM secretion and up to 25-fold enhancements in cellular proliferation relative to that observed with alpha delta-dex alone, in the absence of added cytokines. This mIg-mediated enhancement of Ig secretion was not observed when B cells were stimulated with bivalent, unconjugated anti-Ig. CD40 ligand (CD40L), shows a similar, although somewhat more moderate, synergy with lipoproteins for induction of proliferation and IgM secretion. By contrast, lipoproteins by themselves are relatively ineffective at costimulating Ig secretion in the presence of various combinations of cytokines. These data suggest that bacteria may induce Ag-specific humoral immunity through the action of bacterial polysaccharides that mediate an Ag-specific multivalent mIg signal, in concert with bacterial lipoproteins that deliver ancillary signals, without a requirement for recruitment of non-B cell types.

Animals↗

Expression, isolation, and characterization of the hepatitis C virus ATPase/RNA helicase.

The genome of the hepatitis C virus directs the synthesis of a single polyprotein, which is proteolytically cleaved into at least nine functional proteins. The amino-terminal portion of the polyprotein forms the structural proteins, while the carboxy-terminal region constitutes a variety of viral enzymes. The nonstructural 3 (NS3) protein, consisting of amino acids 1027-1657 of the polyprotein, is believed to be a multifunctional protein with an amino-terminal serine protease domain, which is involved in polyprotein processing, and a carboxy-terminal ATPase/RNA helicase domain, presumably involved in viral replication. We have assembled an expression vector which directs the synthesis of residues 1207-1612 of the polyprotein with an amino-terminal polyhistidine purification tag. This portion of the NS3 protein contains the putative ATPase/helicase domain. The protein has been purified to yield 30-50 mg of enzymatically active protein per liter of culture. The purified NS3 protein has both NTPase and RNA helicase activities. ATP is the preferred substrate for the NTPase; GTP is also utilized; however, UTP is a very poor substrate and CTP is not utilized. The RNA helicase activity is dependent on ATP and divalent cation. Either manganese or magnesium can serve as the divalent cation.

Adenosine Triphosphatases↗

Use of alpha-N,N-bis[carboxymethyl]lysine-modified peroxidase in immunoassays.

Horseradish peroxidase was activated by periodate oxidation of the carbohydrate moiety and then modified by the covalent attachment of alpha-N,N-bis[carboxyethyl]lysine (CM-Lys) by reductive alkylation using sodium cyanoborohydride. The resultant CM-Lys peroxidase was charged with nickel ions and then used as a specific labeling reagent for histidine-tagged recombinant proteins. This labeling method was effective for proteins that are soluble or insoluble in the absence of chaotropic agents. The labeled proteins were very effective in direct sandwich enzyme-linked immunosorbent assay for detecting antibodies against the protein in sera as demonstrated by assays for antibodies to such diverse viral proteins as hepatitis B surface and core proteins, hepatitis C core and helicase protein (NS3), and retroviral core proteins.

Alkylation↗

Nephrotoxicity of sevoflurane compound A [fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether] in rats: evidence for glutathione and cysteine conjugate formation and the role of renal cysteine conjugate beta-lyase.

Compound A, which is a breakdown product of the volatile anesthetic sevoflurane, is nephrotoxic in rats, although the mechanism of this toxicity is unknown. In the present investigation, the role of glutathione conjugation, glutathione conjugate processing to cysteine conjugates, and renal cysteine conjugate beta-lyase in the pathogenesis of Compound A nephrotoxicity was investigated in the rat. Following intraperitoneal administration of Compound A (1 mmol/kg), the presence of bile of two types of Compound A-glutathione conjugates, and the urinary excretion of two types of Compound A-mercapturic acid conjugates, was demonstrated by ionspray-tandem mass spectrometry. Aminooxyacetic acid, a competitive inhibitor of renal cysteine conjugate beta-lyase, partially protected against Compound A-induced diuresis and proteinuria. These results suggest that glutathione conjugate formation, subsequent processing to cysteine conjugates, and cysteine conjugate metabolism by renal beta-lyase may be important factors in the pathogenesis of Compound A-mediated nephrotoxicity in rats.

Animals↗

Expression of scatter factor in human bladder carcinoma.

BACKGROUND: Scatter factor (SF) is a protein secreted by stromal (supporting) cells that induces disruption of intercellular junctions and stimulates motility and invasiveness of carcinoma cells. SF is also a potent inducer of angiogenesis (new blood vessel formation), a process required for tumor growth and dissemination. Invasion and angiogenesis are characteristics of biologically aggressive tumors, suggesting that the accumulation of SF within tumors might promote progression to a more malignant phenotype. PURPOSE: This study was designed to determine if SF is overexpressed in carcinoma of the bladder and to evaluate the potential mechanisms that might account for such overproduction. METHODS: We measured the SF content in urine from 20 patients with carcinoma of the bladder and various control groups. We also measured expression of SF in bladder tumor extracts, histologic sections of tumors, and cell culture models, using a variety of techniques, including enzyme-linked immunosorbent assays, immunohistochemistry, and Western and Northern blot analyses. Statistical comparisons were performed using two-tailed t tests. RESULTS: Urinary SF content was found to be significantly elevated in patients with bladder carcinoma as compared with normal control subjects (P < .001), patients with benign prostatic hypertrophy (P = .0055), and patients with prostate carcinoma, another genitourinary malignancy (P = .002). Extracts of bladder cancers, especially those from high-grade, invasive tumors, contained very high levels of SF. Both SF and its proto-oncogene (c-met)-encoded receptor were detected in bladder carcinoma tissue sections by immunostaining. Three different bladder carcinoma cell lines produced no detectable SF but produced very high titers of a high-molecular-weight (> 30 kd), heat-sensitive protein that stimulates SF production by stromal cell types. High titers of a similar SF-inducing activity were detected in vivo, in bladder carcinoma extracts, and in the urine of patients with bladder carcinoma. CONCLUSIONS: Our results suggest that SF is overproduced in bladder carcinomas and accumulates within the tumor and in the urine. Overproduction of SF may result from an abnormal urothelial-stromal interaction in which dysplastic or carcinomatous urothelium secretes factors that stimulate SF expression by bladder wall stromal cells. IMPLICATION: Quantitation of SF in the urine and tumor deserves further study as a possible marker of urothelial malignancy.

Blotting, Northern↗

Phylogeny of six Sciurus aberti subspecies based on nucleotide sequences of cytochrome b.

The tassel-eared squirrel, Sciurus aberti, is separated into six subspecies which occupy restricted and comparable habitats in ponderosa pine forests in the south-western United States and Mexico. These forests and squirrel populations are currently isolated by large arid areas and, as such, S. aberti appears to offer an example of incipient speciation. We sequenced the complete mitochondrial cytochrome b gene to construct a molecular phylogeny for S. aberti and to determine whether subspecific genetic structure and geographic patterns are correlative. Twenty alleles were identified among 612 squirrels throughout the species' range. Nucleotide divergence between alleles ranged from 0.009 to 0.0233, whereas average sequence divergence between S. aberti and an outgroup species, Sciurus niger, was 0.1823. Neighbor-joining and maximum parsimony analyses identified three major genetic assemblages composed of the following subspecies groups: (1) barberi and durangi; (2) aberti and kaibabensis; and (3) ferreus. The chuscensis samples were the only population with divergent sequences; one sequence was identical to an aberti sequence and a second unique sequence clustered with the ferreus sequences. The presence of divergent sequences in the chuscensis population, coupled with its central geographic position between aberti and ferreus, suggests a relatively recent influx of aberti mtDNA. Estimates of the times separating sequences in subspecies within different groups ranged from 0.94 to 1.52 x 10(6) years, based on a rate estimate of 7.15 x 10(-9) substitutions/year/site. The limited divergence observed between (1) aberti and kaibabensis as well as (2) barberi and durangi suggests relatively recent separations of subspecies within each assemblage. In fact, populations defined morphologically and geographically as ferreus exhibited greater sequence divergence than the aforementioned groups, identifying ferreus as the subspecies with the greatest genetic substructuring. The levels of cytochrome b divergence observed for the three distinct groups argues against a significant role for late Pleistocene glaciation in dispersal of this particular species; however, the proposed intermixing of aberti and chuscensis populations may well have been associated with such glacial events. Nucleotide diversity within subspecies ranked chuscensis >> aberti > barberi approximately kaibabensis approximately ferreus subpopulations; the relatively high level of diversity of chuscensis samples likely results from the apparent introgression of an aberti haplotype. The comparative levels of diversity in the aberti, barberi, kaibabensis, and ferreus sample populations do not correlate with respective habitat size (and presumably population size), suggesting that relatively recent forces, e.g., glaciation and inconsistent timber harvests, may have influenced diversity in these populations without apparent alterations in population size.

Amino Acid Sequence↗

Antiestrogenic activity of two 11 beta-estradiol derivatives on MCF-7 breast cancer cells.

Two 11 beta-derivatives of estradiol (E2) were tested for their potential antiestrogenic activity in the MCF-7 breast cancer model: one contained a phenoxydimethylaminoethyl side-chain (RU 39,411), the other a pentafluoropentylsulfinyl side-chain (RU 58,668). The former compound displayed mixed estrogenic/antiestrogenic properties, while the latter indicated only an antiestrogenic activity. Both the compounds produced a growth inhibition of MCF-7 cells at doses related to their binding affinity for the estrogen receptor (ER); E2 suppressed this inhibition. The compounds also down-regulated the estrogen binding capacity of the cells but failed to reduce ER mRNA levels, indicating that the grafting of their side-chains prevented this antagonistic effect usually observed with steroidal estrogens. Assessment of ER levels by enzyme immunoassay revealed a marked increase with RU 39,411 and a decrease with RU 58,668; different mechanisms of action should, therefore, be considered. Finally, the estrogenic activity of RU 39,411 was demonstrated by its strong ability to induce synthesis of the progesterone receptor; RU 58,668 failed to display this agonistic activity.

Antineoplastic Agents↗

Histochemical demonstration of heavy metals in mouse skin.

The localization of heavy metals in normal mouse skin was investigated light microscopically using the Neo-Timm staining method. Positive staining was found in the horny layer of epidermis, connective tissue of dermis and in hair roots. Chelation of heavy metals by pretreatment with diethyldithiocarbamate prevented this staining. The study demonstrates that heavy metals, mainly zinc, are localized in the mouse skin and that the Neo-Timm staining method is useful for the investigation of the role of zinc in this organ.

Animals↗

Prevalence and distribution of BK virus subtypes in healthy people and immunocompromised patients detected by PCR-restriction enzyme analysis.

BACKGROUND: Four antigenic subtypes of BK virus (BKV) have recently been characterised by both genomic subtyping and serological reactivity. OBJECTIVES: To study the prevalence and distribution of subtypes of BKV in different groups of patients. STUDY DESIGN: Urine specimens were collected from 33 bone marrow transplant (BMT) recipients, from 101 HIV-infected patients, from 15 children aged 2-5 and from 40 pregnant women were tested for BKV DNA by polymerase chain reaction (PCR) and subtyped using a PCR-sequencing (PCR-S) and a modified PCR-restriction enzyme analysis (PCR-RE) methods. RESULTS: BKV DNA was detected in 12/18 (67%) of BMT patients with haematuria and 5/15 (33%) without. Overall BKV DNA was detected in 45% of HIV-infected patients, the prevalence of BKV DNA increased with greater immunosuppression as defined by CD4 cell counts. BKV DNA was detected in urine samples from 27% of children and 47% of pregnant women. Four stable BKV subtypes were detected in these patient groups. Dual infections with more than one subtype were identified in urine samples from HIV-infected patients, children and pregnant women but not in the samples from bone marrow recipients. CONCLUSION: This study has confirmed the high prevalence of BKV infection in immunocompromised patients and suggests that stable BKV subtypes with conserved sequences are circulating in the human population. The techniques of PCR-S and PCR-RE described in this study are sufficiently sensitive for subtyping BKV direct from clinical specimens.

Journal Article↗

Population structure, stepwise mutations, heterozygote deficiency and their implications in DNA forensics.

In a substructured population the overall heterozygote deficiency can be predicted from the number of subpopulations (s), their time of divergence (t), and the nature of the mutations. At present the true mutational mechanisms at the hypervariable DNA loci are not known. However, the two existing mutation models (the infinite allele model (IAM) and the stepwise mutation model (SMM)) provide some guides to predictions from which the possible effect of population substructuring may be evaluated, assuming that the subpopulations do not exchange any genes among them during evolution. The theory predicts that the loci with larger mutation rate, and consequently showing greater heterozygosity within subpopulations, should exhibit a smaller proportional heterozygote deficiency (GST) and, hence, the effects of population substructuring should be minimal at the hypervariable DNA loci (an order of magnitude smaller than that at the blood group and protein loci). Applications of this theory to data on six Variable Number of Tandem Repeat (VNTR) loci and five short tandem repeat (STR) loci in the major cosmopolitan populations of the USA show that while the VNTR loci often exhibit a large significant heterozygote deficiency, the STR loci do not show a similar tendency. This discordant finding may be ascribed to the limitations, coalescence and nondetectability of alleles associated with the restriction fragment length polymorphism (RFLP) analysis through which the VNTR loci are scored. Such limitations do not apply to the polymerase chain reaction (PCR) method, through which the STR loci are scored. The implications of these results are discussed in the context of the forensic use of DNA typing data.

DNA↗

A novel measure of genetic distance for highly polymorphic tandem repeat loci.

Genetic distance measures are indicators of relatedness among populations or species and are useful for reconstructing the historic and phylogenetic relationships among such groups. Classical measures of genetic distance were developed to analyze biochemical and serological polymorphisms, systems which generally show limited variability. However, these traditional measures of genetic distance are inadequate for the analysis of certain classes of variable number tandem repeat (VNTR) loci, which have a larger number of alleles and higher levels of heterozygosity than traditional genetic markers. At the higher levels of heterozygosity observed at these loci, the standard measures of genetic distance are nonlinear and do not account for the mutational mechanisms of hypervariable loci. We have developed a measure of genetic distance, DSW, which is appropriate for the analysis of highly polymorphic DNA loci. Using computer simulations of diverging populations, we show that DSW conforms to linearity and that the variance is similar in magnitude to traditional measures of genetic distance. Comparisons of phylogenetic trees derived from the simulated divergence of human racial groups demonstrate that the branch lengths of trees prepared using DSW are more similar to the model tree than those generated using other measures. Finally, we demonstrate the applicability of DSW to evolutionary analysis by reconstructing the relationships among eight human populations using 14 microsatellite and STR loci. The phylogenetic trees generated using DSW are different from trees constructed with traditional measures and better reflect the well-documented ancient divergence of African and non-African populations.

Animals↗

In situ hybridization analysis of chromogranin A and B mRNAs in neuroendocrine tumors with digoxigenin-labeled oligonucleotide probe cocktails.

The chromogranin/secretogranin (Cg/Sg) molecules are a family of acidic proteins present in neuroendocrine cells and tumors with secretory granules. They have been frequently used to characterize neuroendocrine cells and tumors by immunohistochemical analyses. Immunoreactivity for CgA is related to the presence of secretory granules in these tumors, so immunohistochemical staining for CgA may be absent in neuroendocrine tumors with only a few secretory granules. RNA in situ hybridization with a series of oligonucleotide probes for CgA and CgB was used to detect the mRNA transcripts for CgA and CgB with digoxigenin-labeled probes in 31 neuroendocrine tumors. These results were compared to ISH with 35S-labeled probes and with immunohistochemical staining for CgA and synaptophysin in the same neoplasms. ISH with 35S-labeled probes for CgA and B detected mRNA transcripts in 31 of 31 tumors, whereas the digoxigenin-labeled probe cocktails for CgA and B were positive in 19 of 31 cases when used separately and in 24 of 31 cases when used together. Immunohistochemical staining for CgA was positive in 22 of 31 cases and for synaptophysin in 23 of 31 cases. The CgA and B oligonucleotide probe cocktails were highly specific, since nonneuroendocrine cells and tumors did not stain and the hybridization signal was abolished by ribonuclease A pretreatment. These results indicate that non-isotopic ISH with digoxigenin-labeled probe cocktails for CgA and B or with 35S-labeled probes can be used in characterizing neuroendocrine cells and tumors in formalin-fixed paraffin-embedded tissue sections even when the CgA protein is not detected by immunohistochemistry.

Base Sequence↗

Nitric oxide synthase in the human pituitary gland.

Nitric oxide (NO) is generated by the NO synthase family of isozymes, which is present in many mammalian cells. The constitutive NO synthase isozymes generate NO, which acts via signal transduction mechanisms in the regulation of many functions including vascular tone and blood pressure, and the inducible isozymes mediate immunological mechanisms by cytotoxic and cytostatic effects. To determine whether NO has a role in anterior pituitary cell function, immunohistochemistry and in situ hybridization analyses were used to study NO synthase expression in normal and neoplastic human pituitary tissues. Brain NO synthase was localized in the anterior pituitary in secretory and in folliculo-stellate cells and in the posterior pituitary. Pituitary adenomas had higher levels of brain NO synthase protein and mRNA compared with normal pituitaries. Endothelial NO synthase was also present in anterior and posterior pituitary cells and in endothelial cells of the pituitary. Immunoblotting studies with brain NO synthase antibodies detected a slowly migrating approximately 155-kd band and more rapidly migrating approximately 90-kd and approximately 60-kd bands. Endothelial NO synthase, but not macrophage NO synthase, was also detected in the pituitary by immunoblotting studies, confirming the immunohistochemical observations. These findings indicate that NO synthase is expressed in normal and neoplastic human pituitary tissues with increased levels of brain NO synthase protein and mRNA in adenomas compared with non-neoplastic pituitary cells and suggest that NO may play a regulatory role in hormone secretion in anterior pituitary cells.

Adenoma↗

Analysis of the chromogranin A post-translational cleavage product pancreastatin and the prohormone convertases PC2 and PC3 in normal and neoplastic human pituitaries.

Several members of the chromogranin/secretogranin (Cg/Sg) family are post-translationally processed in neuroendocrine cells and tumors to smaller peptides, some of which are biologically active. For example, CgA is processed to pancreastatin, parastatin, and other peptides. We analyzed the distribution of pancreastatin and CgA proteins in normal and neoplastic pituitaries as well as the prohormone convertases PC2 and PC3/1 (PC3), the putative processing enzymes for the Cg/Sg family, in 35 pituitary adenomas and 4 non-neoplastic pituitaries by immunohistochemistry and immunoblotting with highly specific antisera. CgA and CgB mRNAs were also examined. Pancreastatin was present in all subtypes of pituitary tumors, although prolactin-secreting adenomas expressed this peptide less frequently than did other tumor types. CgA protein and CgA mRNA expression were also restricted in prolactin adenomas and in normal prolactin cells, as shown by combined in situ hybridization and immunostaining. The prohormone convertases PC2 and PC3 were present in pituitary tumors and in non-neoplastic pituitaries. Immunoblot analysis and immunostaining showed a principal approximately 69-kd PC3 band and a approximately 68-kd PC2 band. Adrenocorticotrophic hormone-secreting adenomas expressed mainly PC3 as determined by immunoblotting and immunohistochemistry, whereas all other adenoma groups expressed predominantly PC2. These results indicate that the enzymes capable of processing CgA and other members of the Cg/Sg family to peptides with biological activity such as pancreastatin are widely expressed in human pituitary adenomas and in non-neoplastic pituitaries, with adrenocorticotrophic hormone tumors expressing predominantly PC3 and other adenomas expressing mainly PC2. The infrequent expression of CgA protein and pancreastatin peptides in normal and neoplastic prolactin cells suggests a unique role of CgA in these tumors.

Adult↗

Population genetics of dinucleotide (dC-dA)n.(dG-dT)n polymorphisms in world populations.

We have characterized eight dinucleotide (dC-dA)n.(dG-dT)n repeat loci located on human chromosome 13q in eight human populations and in a sample of chimpanzees. Even though there is substantial variation in allele frequencies at each locus, at a given locus the most frequent alleles are shared by all human populations. The level of heterozygosity is reduced in isolated or small populations, such as the Pehuenche Indians of Chile, the Dogrib of Canada, and the New Guinea highlanders. On the other hand, larger average heterozygosities are observed in large and cosmopolitan populations, such as the Sokoto population from Nigeria and German Caucasians. Conformity with Hardy-Weinberg equilibrium is generally observed at these loci, unless (a) a population is isolated or small or (b) the repeat motif of the locus is not perfect (e.g., D13S197). Multilocus genotype probabilities at these microsatellite loci do not show departure from the independence rule, unless the loci are closely linked. The allele size distributions at these (CA)n loci do not follow a strict single-step stepwise-mutation model. However, this features does not compromise the ability to detect population affinities, when these loci are used simultaneously. The microsatellite loci examined here are present and, with the exception of the locus D13S197, are polymorphic in the chimpanzees, showing an overlapping distribution of allele sizes with those observed in human populations.

Alleles↗

A comparative study of genetic variation at five VNTR loci in three ethnic groups of Houston, Texas.

Following the technique of Southern blot restriction fragment length polymorphisms (RFLP) analysis, we generated a database of DNA profiles at five Variable Number of Tandem Repeats loci (D1S7, D2S44, D4S139, D10S28, and D17S79) for 669 individuals of three major ethnic populations (Caucasians, Blacks, and Hispanics) of Houston, Texas. Analysis of fragment sizes at these loci within each sample, as well as their fixed-bin analyses, reveal that the assumptions of independence of allelic occurrences within and between loci are valid for this database. Fixed-bin allele frequency tables, therefore, are the best descriptors of this database for conservative forensic calculations. Finally, we demonstrate that this regional database from Houston, Texas, does not yield any meaningfully different forensic inference than the one obtained from the National database of the respective ethnic groups.

Alleles↗