Biomedical subjects

L Jiang

Publications and source records attributed to L Jiang.

At least 91 records · Page 5Linked to original sources

PubMed2000

Extracellular ATP-dependent suppression of proliferation and induction of differentiation of human HL-60 leukemia cells by distinct mechanisms.

Extracellular ATP suppressed the growth of HL-60 leukemia cells and induced their differentiation as revealed by N-formyl-methionyl-leucyl-phenylalanine-induced beta-glucuronidase release. ATP degraded to ADP, AMP, and adenosine, and the effect of ATP on cell growth was mimicked by these metabolites added to the cultures. The stable analog alpha,beta-methylene ATP, however, had only a weak inhibitory effect on cell growth. Adenine nucleotide-induced growth suppression was reversed by uridine, suggesting the involvement of intracellular pyrimidine starvation secondary to adenosine accumulation. Consistent with this, ATP induced intracellular starvation of pyrimidine nucleotides, and this effect was also prevented by pretreatment of cells with uridine. The order of effectiveness of ATP-induced differentiation of HL-60 cells, unlike that for growth suppression, was ATP > ADP > AMP, and adenosine had no effect. Furthermore, uridine had no effect and the stable analog, alpha,beta-methylene ATP also induced HL-60 cell differentiation, suggesting that differentiation was due to ATP per se. We tested the hypothesis that ATP-induced differentiation arises from activation of adenylyl cyclase by the novel P2Y(11) receptor using the cell-permeable inhibitor of protein kinase A, Rp-CPT-cAMPS (8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphorothioate, Rp isomer). Rp-CPT-cAMPS (1-100 microM) prevented ATP-induced differentiation of HL-60 cells as assessed by fMLP-induced beta-glucuronidase release. However, Rp-CPT-cAMPS did not prevent ATP-induced growth suppression. Taken together, the data indicate that extracellular ATP suppresses HL-60 growth and induces their differentiation by distinct mechanisms. Growth suppression arises from adenosine generation and consequent pyrimidine starvation. Differentiation arises, at least in part, from a distinct mechanism involving the activation of cell surface P2 receptors coupled to cAMP generation and activation of protein kinase A.

Adenosine↗

PubMed2000

Spatial regulation and activity modulation of plasmin by high affinity binding to the G domain of the alpha 3 subunit of laminin-5.

Cells in complex tissues contact extracellular matrix that interacts with integrin receptors to influence gene expression, proliferation, apoptosis, adhesion, and motility. During development, tissue remodeling, and tumorigenesis, matrix components are modified by enzymatic digestion with subsequent effects on integrin binding and signaling. We are interested in understanding the mechanisms by which broad spectrum proteinases such as plasmin are targeted to their extracellular matrix protein substrates. We have utilized plasmin-mediated cleavage of the epithelial basement membrane glycoprotein laminin-5 as a model to evaluate molecular events that direct plasmin activity to specific structural domains. We report that plasminogen and tissue plasminogen activator (tPA) exhibit high affinity, specific binding to the G(1) subdomain of the N terminus of the laminin-5 alpha(3) subunit, with equilibrium dissociation constants of 50 nm for plasminogen and 80 nm for tPA. No high affinity binding to the G(2), G(3), and G(4) subdomains was observed. As a result of binding to the G(1) subdomain, the catalytic efficiency of tPA-catalyzed plasminogen activation is enhanced 32-fold, leading to increased matrix-associated plasmin that is positioned favorably for cleavage within the G(4) subdomain as we have reported previously (Goldfinger, L. E., Stack, M. S., and Jones, J. C. R. (1998) J. Cell Biol. 141, 255-265). Thus, physical constraints dictated by interaction of proteinase and matrix macromolecule control not only enzymatic activity but may regulate substrate targeting of proteinases.

Basement Membrane↗

PubMed2000

The cytoplasmic C-terminal fragment of polycystin-1 regulates a Ca2+-permeable cation channel.

The cytoplasmic C-terminal portion of the polycystin-1 polypeptide (PKD1(1-226)) regulates several important cell signaling pathways, and its deletion suffices to cause autosomal dominant polycystic kidney disease. However, a functional link between PKD1 and the ion transport processes required to drive renal cyst enlargement has remained elusive. We report here that expression at the Xenopus oocyte surface of a transmembrane fusion protein encoding the C-terminal portion of the PKD1 cytoplasmic tail, PKD1(115-226), but not the N-terminal portion, induced a large, Ca(2+)-permeable cation current, which shifted oocyte reversal potential (E(rev)) by +33 mV. Whole cell currents were sensitive to inhibition by La(3+), Gd(3+), and Zn(2+), and partially inhibited by SKF96365 and amiloride. Currents were not activated by bath hypertonicity, but were inhibited by acid pH. Outside-out patches pulled from PKD1(115-226)-expressing oocytes exhibited a 5.1-fold increased NP(o) of endogenous 20-picosiemens cation channels of linear conductance. PKD1(115-226)-injected oocytes also exhibited elevated NP(o) of unitary calcium currents in outside-out and cell-attached patches, and elevated calcium permeability documented by fluorescence ratio and (45)Ca(2+) flux experiments. Both Ca(2+) conductance and influx were inhibited by La(3+). Mutation of candidate phosphorylation sites within PKD1(115-226) abolished the cation current. We conclude that the C-terminal cytoplasmic tail of PKD1 up-regulates inward current that includes a major contribution from Ca(2+)-permeable nonspecific cation channels. Dysregulation of these or similar channels in autosomal dominant polycystic kidney disease may contribute to cyst formation or expansion.

Amino Acid Sequence↗

PubMed2000

Thermal stable and oxidation-resistant variant of subtilisin E.

A remarkable thermal stable and oxidation-resistant mutant was obtained using the random mutagenesis PCR technique on the mutant M222A gene of subtilisin E. Sequencing analysis revealed an A was replaced by G at nucleotide 671 of the subtilisin E gene, converting the asparagine codon (AAT) to serine codon (AGT) at position 118. The half-life of M222A/N118S enzyme activity, when heated at 65 degrees C, was approximately 80 min while the half-life of M222A and wild-type subtilisin E were 13 min and 15 min, respectively. This suggested the stability of the M222A/N118S mutant was five times greater than that of the wild-type enzyme. The mutant was also as oxidation resistant as the mutant M222A of subtilisin E. These results indicated the M222A/N118S mutant is both an oxidation-resistant and a heat-stable variant of subtilisin E.

Amidohydrolases↗

PubMed2000

Biogenesis of the protein storage vacuole crystalloid.

We identify new organelles associated with the vacuolar system in plant cells. These organelles are defined biochemically by their internal content of three integral membrane proteins: a chimeric reporter protein that moves there directly from the ER; a specific tonoplast intrinsic protein; and a novel receptor-like RING-H2 protein that traffics through the Golgi apparatus. Highly conserved homologues of the latter are expressed in animal cells. In a developmentally regulated manner, the organelles are taken up into vacuoles where, in seed protein storage vacuoles, they form a membrane-containing crystalloid. The uptake and preservation of the contents of these organelles in vacuoles represents a unique mechanism for compartmentalization of protein and lipid for storage.

Amino Acid Sequence↗

PubMed2000

Study on colloidal Au-enhanced DNA sensing by quartz crystal microbalance.

Colloidal Au is reported for enhancement the immobilization capacity and ultimately detection limit of DNA using quartz crystal microbalance (QCM). Immobilization of approximately 12 nm-diameter colloidal Au on to an Au-coated QCM resulted in an easier attachment of oligonucleotide, with a mercaptohexyl group at the 5'-phosphate end and an increased capacity for nucleic acid detection. DNA immobilization and hybridization was monitored from QCM frequency changes. Hybridization was induced by exposure of the DNA-containing films to complementary DNA in solution. A much higher sensitivity was obtained for the analyte. The Au nanoparticle films on the Au plate provide a novel means for the fabrication of DNA sensor.

Animals↗

PubMed2000

Class I MHC is stabilized against thermal denaturation by physiological concentrations of NaCl.

Class I MHC molecules are ternary complexes composed of an allotype specific heavy chain, a noncovalently associated protein beta(2)-microglobulin (beta(2)m), and a peptide. The complexes are assembled in the endoplasmic reticulum by a complex series of chaperones and peptide-loading mechanisms. In the absence of beta(2)m or peptide, very little class I heavy chain is transported to the surface of the cell. Complexes that do not contain all three parts of the protein are not made productively in vivo and not at all in vitro. The ability of the complex to withstand thermal denaturation in vitro has been shown to be related to the binding affinity of the peptide. Paradoxically, some low-affinity peptide complexes denature at or below human basal body temperatures in vitro but are effective biological agents in vivo. Here we show that these complexes are stabilized against thermal denaturation by physiological cosolvents and maximally stabilized by 150 mM NaCl. While the degree of stabilization by 150 mM NaCl is greatest for low-affinity peptide/MHC complexes, the mechanism of stabilization is independent of peptide sequence. This effect is hypothesized to occur by multiple mechanisms including increasing the affinity of beta(2)m for the complex and charge screening.

Amino Acid Sequence↗

PubMed2000

[Contrast test using three kinds of air-microorganism samplers LWC-I, CA6 and CA2 in public places].

A sampling experiment was conducted using three kinds of air-samplers, i.e. LWC-1, CA6 and CA2, on bacteria and fungus in the air under the same conditions and at the same time yet in three different public places. The results showed that there was no significant difference in the total number of bacteria and fungus colony forming units in the air in different public places using the above mentioned three air-samplers. In comparison, the LWC-1 air-sampler demonstrated a feature of small size, light weight, simple structure, and little noise, therefore, it was easy in carrying and requires no external power source. Such being the case, the LWC-1 air-sampler was more suitable for monitoring microorganism in the air in public places.

Air Microbiology↗

PubMed2000

Paired-pulse modulation at individual GABAergic synapses in rat hippocampus.

1. Unitary inhibitory postsynaptic currents (uIPSCs) were recorded in synaptically coupled pairs of CA1 hippocampal interneurons and pyramidal neurons in rat brain slices by using dual patch-clamp techniques. Paired-pulse modulation of uIPSCs at individual GABAergic synapses was tested. 2. GABAergic synapses could be divided into two subgroups, high and low failure, depending on their failure rate. 3. The external Ca2+ levels modulate the failure rate of uIPSCs. In 0.51 mM Ca2+, low-failure pairs had a high-failure characteristic, whereas high-failure pairs had a low-failure characteristic in 8 mM Ca2+. The results suggest that uIPSC failures result from the Ca2+-dependent release mechanism rather than axon propagation failures. 4. Paired-pulse facilitation (PPF) occurred in high-failure pairs when the interspike interval was 20 ms. Paired-pulse depression (PPD) was not predominant in high-failure pairs. 5. Potency of uIPSCs, the average amplitude of non-failure events, was enhanced by PPF, suggesting that multiple synapses connect each pair. Differing numbers of activated synapses contributed to the variable amplitude of uIPSCs from a given pair. 6. PPD occurred in low-failure pairs at the tested range of interspike intervals (20-200 ms). The uIPSC2 after a large uIPSC1 was smaller than the uIPSC2 after a small uIPSC1, suggesting that PPD is use dependent and due to a decrease in the quantal content (m) after the first release. 7. In 8 mM Ca2+, PPD occurred in high-failure pairs and was larger in low-failure pairs, suggesting that the occurrence of PPF or PPD depends on the baseline release probability. 8. The GABAB receptor antagonist CGP 55845A (5 microM) decreased the baseline release probability of inhibitory synapses and attenuated PPD indirectly, rather than by blocking presynaptic GABAB autoreceptors.

Animals↗

PubMed2000

Development of multi-ESI-sprayer, multi-atmospheric-pressure-inlet mass spectrometry and its application to accurate mass measurement using time-of-flight mass spectrometry.

The atmospheric pressure sampling nozzle (orifice, heated capillary, or inlet) of a high mass accuracy time-of-flight mass spectrometer (TOF-MS) was modified by replacing its single nozzle with multiple atmospheric pressure nozzles. This allowed multiple streams of liquids to be introduced into the MS in parallel (an electrosprayer for each nozzle), with minimum analyte interactions between the streams. The chemical contents of all liquid streams were analyzed concurrently using a single mass spectrometer. To obtain a higher mass accuracy by providing internal reference on each scan (acquisition) and to evaluate the suitability of TOF-MS for molecular-formula confirmation, a dual-ESI-sprayer, dual-nozzle version of this design was used. The accurate masses of tens of organic compounds in the mass range of 200-3000 Da were measured, and the results were compared with those obtained using dual-sprayer, single-nozzle TOF-MS. A significant improvement in mass accuracy was observed when the former technique was used. Comparison between the mass accuracy using dual-ESI-sprayer, dual-nozzle TOF-MS and that obtained using a double-focusing mass spectrometer operating under chemical ionization (CI) and fast atom bombardment (FAB) shows the suitability of the technique for elemental-composition confirmation. Approximately 85% of samples analyzed had mass errors of less than 5 ppm, and the other 15% had mass errors less than 8 ppm. Using a high-performance liquid chromatography (HPLC) as a device for introduction of one liquid stream (sample) and a syringe pump as a device for introduction of the second liquid stream (reference standard), the accurate mass of a tryptic digest of cytochrome c was measured. The range of mass errors was from -6.1 ppm to +3.6 ppm, a significant improvement over our previously reported mass accuracy for this digest using single-nozzle TOF-MS. The interactions between analytes in the liquid streams also were investigated using a variety of sample-introduction and nozzle-design combinations, including single-ESI-sprayer, single-nozzle; dual-ESI-sprayer, single-nozzle; dual-ESI-sprayer, Y-shaped inlet; and dual-ESI-sprayer, dual-inlet. The results demonstrated that the dual-ESI-sprayer, dual-inlet design provides reference peaks on every acquisition with minimum analyte-reference interaction and, therefore, higher consistent mass accuracy.

Atmospheric Pressure↗

PubMed2000

Sensitivity-enhanced MQ-HCN-CCH-TOCSY and MQ-HCN-CCH-COSY pulse schemes for (13)C/(15)N labeled RNA oligonucleotides.

Sensitivity enhanced multiple-quantum 3D HCN-CCH-TOCSY and HCN-CCH-COSY experiments are presented for the ribose resonance assignment of (13)C/(15)N-labeled RNA sample. The experiments make use of the chemical shift dispersion of N1/N9 of pyrimidine/purine to distinguish the ribose spin systems. They provide a complementary approach for the assignment of ribose resonance to the currently used HCCH-COSY and HCCH-TOCSY type experiments in which either (13)C or (1)H is utilized to separate the different ribose spin systems. The pulse schemes have been demonstrated on a 23-mer (13)C/(15)N-labeled RNA aptamer complexed with neomycin and tested on a 32-mer RNA complexed with a 23-residue peptide.

Carbon Isotopes↗

PubMed2000

In vitro neurogenesis by progenitor cells isolated from the adult human hippocampus.

Neurogenesis persists in the adult mammalian hippocampus. To identify and isolate neuronal progenitor cells of the adult human hippocampus, we transfected ventricular zone-free dissociates of surgically-excised dentate gyrus with DNA encoding humanized green fluorescent protein (hGFP), placed under the control of either the nestin enhancer (E/nestin) or the Talpha1 tubulin promoter (P/Talpha1), two regulatory regions that direct transcription in neural progenitor cells. The resultant P/Talpha1:hGFP+ and E/nestin:enhanced (E)GFP+ cells expressed betaIII-tubulin or microtubule-associated protein-2; many incorporated bromodeoxyuridine, indicating their genesis in vitro. Using fluorescence-activated cell sorting, the E/nestin:EGFP+ and P/Talpha1:hGFP+ cells were isolated to near purity, and matured antigenically and physiologically as neurons. Thus, the adult human hippocampus contains mitotically competent neuronal progenitors that can be selectively extracted. The isolation of these cells may provide a cellular substrate for re-populating the damaged or degenerated adult hippocampus.

Adult↗

PubMed2000

Mosaic-like distribution of endothelial cell antigens in capillaries and juxta-alveolar microvessels in the normal human lung.

The distribution patterns of endothelial cell antigens, including thrombomodulin and von Willebrand factor (vWf), were studied in normal lung tissues obtained from distant areas of solitary nodules (seven adenocarcinomas and four hamartomas). By single immunoalkaline phosphatase and dual immunofluorescence stainings, the plasma membranes of alveolar capillary endothelium showed linear distribution of thrombomodulin, but their cytoplasm was rarely reactive for vWf (thrombomodulin-dominant pattern). Microvessels with a diameter larger than 10 microm located in the connective tissue zones demonstrated band-like reaction for vWf in their cytoplasm, and their plasma membranes often lacked reactivity for thrombomodulin (vWf-dominant pattern). The juxta-alveolar microvessels located along the borders between the alveolar- and connective-tissue zones showed mosaic-like pattern of distribution for these antigens. The pulmonary venules and peribronchial microvessels measuring up to 40 microm in diameter, demonstrated the expression of thrombomodulin along the plasma membrane, and that of vWf in the cytoplasm. Capillaries of the bronchial circulation were also characterized by mosaic-like pattern of distribution. Both antigens were often expressed in a single cytoplasmic segment. The heterogeneous distribution pattern of these antigens suggests topographic difference in endothelial cell function to maintain coagulatory and anticoagulatory balance in the normal human lung.

Adult↗

PubMed2000

Follicle-stimulating hormone mediated calcium signaling by the alternatively spliced growth factor type I receptor.

Ovarian granulosa cell and testicular Sertoli cell functions are regulated by the tropic action of the pituitary follicle-stimulating hormone (FSH), which may exert pleiotropic effects using a variety of signaling pathways. The effects of FSH on the mobilization of Ca(2+) into granulosa and Sertoli cells have been widely studied, but whether all the effects of the hormone are mediated by the single G-protein-coupled (G(s)) receptor with the seven-transmembrane structure (R1) has remained an enigma. With the object of resolving this mystery, we have compared the hormonal responses of HEK 293 cells transfected with three different cloned FSH receptor cDNAs of testis/ovary, designated R1 (G(s)), R2 (similar to R1 but having a shorter carboxyl terminus), and R3, a novel FSH receptor exhibiting a growth factor type I receptor motif. The latter two that use the same DNA segment for alternative splicing of the single large 80- to 100-kilobase gene create different structural motifs and carboxyl termini. Of the three receptors, only the FSH-R3 type induced a significant rise in intracellular free calcium concentration ([Ca(2+)](i)), as measured by single cell fluorescence digital imaging with the Ca(2+) sensitive dye fura-2AM. FSH induced a rapid [Ca(2+)](i) response that was concentration dependent. The response was hormone-specific, as neither its individual alpha/beta subunits nor the related glycoprotein hormone LH were effective. To determine whether the [Ca(2+)](i) response was due to Ca(2+) influx or to intracellular Ca(2+) mobilization, cells were exposed to Ca(2+)-free buffer and to the Ca(2+)-channel blocker diltiazem (10(-5) M). FSH-Induced [Ca(2+)](i) responses were inhibited in Ca(2+)-free buffer and abrogated in the presence of diltiazem. These novel data demonstrate that FSH can increase [Ca(2+)](i) through L-type voltage-dependent Ca(2+) channels via the growth factor type 1 receptor. Our findings support the concept that different receptor motifs act to integrate intracellular signaling events.

Alternative Splicing↗

PubMed2000

Potent and selective activity of a combination of thymidine and 1843U89, a folate-based thymidylate synthase inhibitor, against Plasmodium falciparum.

Unlike mammalian cells, malarial parasites are completely dependent on the de novo pyrimidine pathway and lack the enzymes to salvage preformed pyrimidines. In the present study, first, it is shown that 1843U89, even without polyglutamylation, is a potent folate-based inhibitor of purified malarial parasite thymidylate synthase. The binding was noncompetitive with respect to methylenetetrahydrofolate, and 1843U89 had a K(i) of 1 nM. The compound also had potent antimalarial activity in vitro. Plasmodium falciparum cells in culture were inhibited by 1843U89, with a 50% inhibitory concentration of about 70 nM. The compound was effective against drug-sensitive as well as drug-resistant clones of P. falciparum. As predicted by the biochemistry of the parasite, the potent inhibition of parasite proliferation by 1843U89 could not be reversed with 10 microM thymidine. In contrast, in the presence of 10 microM thymidine, mammalian cells were unaffected by 1843U89 even at concentrations as high as 0.1 mM, thus offering a selectivity window of more than 1,000-fold. On this basis, folate-based thymidylate synthase inhibitors may represent a powerful additional tool that can be used to combat drug-resistant malaria.

Animals↗

PubMed2000

Heterogeneous distribution of thrombomodulin and von Willebrand factor in endothelial cells in the human pulmonary microvessels.

Laser scanning confocal fluorescence microscopy techniques were used to study the localization of von Willebrand factor (vWf; Factor VIII-related antigen) and thrombomodulin (transmembrane receptor for thrombin) in the microvascular endothelial cells in the normal human lung. Tissues were obtained from lobectomy specimens resected for solitary nodules (7 adenocarcinomas and 4 hamartomas) from 11 patients. The plasma membranes of the capillary endothelial cells in the alveolar zones (A-zones) showed red linear fluorescence for thrombomodulin. However, their cytoplasm was mostly unreactive for vWf. The microvessels which were located in the connective tissue (C-zones), including peribronchial, and subpleural areas and large vascular walls, consistently demonstrated band-like green fluorescence for vWf in their cytoplasm, and their plasma membranes usually lacked reactivity for thrombomodulin. Only a limited number of peribronchial capillaries measuring <10 microm in diameter showed a mosaic-like appearance, in which red fluorescence along the plasma membranes was found together with green fluorescence in the subjacent cytoplasm. In the juxtaalveolar (J-zones) microvessels located along the borders between A- and C-zones, and measuring up to 40 microm in diameter, the endothelial cells showed a mosaic-like pattern of distribution of the two antigens. However, the localization of thrombomodulin in the J-zone microvessels was separate and independent from that of vWf. The thrombomodulin-reactive cells were directly connected to the alveolar capillary endothelial cells. Heterogeneous patterns of distribution of thrombomodulin and vWf suggest that topographic differences of endothelial function occur to maintain a balance of coagulation and anticoagulation in the normal human lung.

Adult↗

PubMed2000

Human renin mRNA expression and renin activity in transgenic mice.

The renin-angiotensin system (RAS) plays a very important role in the regulation of blood pressure and electrolyte homeostasis in mammals. It has also been hypothesized to regulate local tissue and organ blood supply in an autocrine/paracrine manner. To further study RAS physiology, transgenic mice carrying the human renin gene were produced by microinjection and were screened by PCR. The human renin transgene was found, by RT-PCR, to be expressed in the heart, kidney, and lung of transgenic mice, but not in the liver or skeletal muscle. The mean levels of renin activity in various tissues were determined by radioimmunoassay. The mean level of renin activity in the plasma and the heart/body weight ratio of the transgenic mice (n=7) were also found to be significantly higher (p< 0.05) than those of the control mice (n= 8). However, no significant differences were seen in the mean levels of renin activities in the kidney and heart between transgenic and control mice. Thus, the transcription of human renin gene in transgenic mice is regulated in a tissue-specific manner. This transgenic model will be useful in clarifying the regulatory mechanisms of the renin gene and the relationship between this gene and diseases.

Animals↗

PubMed2000

[Clinical manifestation and pathological change of autoimmune hepatitis].

OBJECTIVE: To determine clinical manifestation and pathological changes in autoimmune hepatitis (AIH) by liver biopsy. METHODS: The clinical manifestation and pathological changes by liver biopsy in 14 AIH patients were investigated retrospectively. RESULTS: Clinically, AIH affects females with a females/male ratio of 13/1, the age range was 28-66 year; there was extended elevation of ALT and AST, hyperglobulinemia, hypergammaglobulinemia with predominant elevation of IgG and different kinds of antibodies. Patients usually had autoimmune diseases with pathological change of typical chronic hepatitis. The levels of globulin (33 approximately 60 g/L), gammaglobulin (23.9% approximately 60.5%), IgG (8.2 approximately 39. 0 g/L), ds-DNA (1.5 approximately 58.0 g/L) in AIH were significantly higher than those of normal (t=9.7, 9.3, 6.2, 3.5, P<0.01). CONCLUSION: AIH has severe liver impairment with many other symptoms due to multi-systemic damage out of liver. AIH is a distinct autoimmune disease.

Adult↗