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Biomedical subjects

K Yu

Publications and source records attributed to K Yu.

At least 19 recordsLinked to original sources
PubMed2002

Development and characterization of simple sequence repeat (SSR) markers and their use in determining relationships among Lycopersicon esculentum cultivars.

The simple sequence repeat (SSR) or microsatellite marker is currently the preferred molecular marker due to its highly desirable properties. The aim of this study was to develop and characterize more SSR markers because the number of SSR markers currently available in tomato is very limited. Five hundred DNA sequences of tomato were searched for SSRs and analyzed for the design of PCR primers. Of the 158 pairs of SSR primers screened against a set of 19 diverse tomato cultivars, 129 pairs produced the expected DNA fragments in their PCR products, and 65 of them were polymorphic with the polymorphism information content (PIC) ranging from 0.09 to 0.67. Among the polymorphic loci, 2-6 SSR alleles were detected for each locus with an average of 2.7 alleles per locus; 49.2% of these loci had two alleles and 33.8% had three alleles. The vast majority (93.8%) of the microsatellite loci contained di- or tri-nucleotide repeats and only 6.2% had tetra- and penta-nucleotide repeats. It was also found that TA/AT was the most frequent type of repeat, and the polymorphism information content (PIC) was positively correlated with the number of repeats. The set of 19 tomato cultivars were clustered based on the banding patterns generated by the 65 polymorphic SSR loci. Since the markers developed in this study are primarily from expressed sequences, they can be used not only for molecular mapping, cultivar identification and marker-assisted selection, but for identifying gene-trait relations in tomato.

Alleles↗
PubMed2002

Solution structure of a cathelicidin-derived antimicrobial peptide, CRAMP as determined by NMR spectroscopy.

CRAMP was identified from a cDNA clone derived from mouse femoral marrow cells as a member of cathelicidin-derived antimicrobial peptides. This peptide shows potent antimicrobial activity against gram-positive and gram-negative bacteria but no hemolytic activity against human erythrocytes. CRAMP was known to cause rapid permeabilization of the inner membrane of Escherichia coli. In this study, the structure of CRAMP in TFE/H2O (1 : 1, v/v) solution was determined by CD and NMR spectroscopy. CD spectra showed that CRAMP adopts a mainly alpha-helical conformation in TFE/H2O solution, DPC micelles, SDS micelles and liposomes, whereas it has a random structure in aqueous solution. The tertiary structure of CRAMP in TFE/H2O (1 : 1, v/v), as determined by NMR spectroscopy, consists of two amphipathic alpha-helices from Leu4 to Lys10 and from Gly16 to Leu33. These two helices are connected by a flexible region from Gly11 to Gly16. Previous analysis of series of fragments composed of various portion of CRAMP revealed that an 18-residue fragment with the sequence from Gly16 to Leu33 was found to retain antibacterial activity. Therefore, the amphipathic alpha-helical region from Gly16 to Leu33 of CRAMP plays important roles in spanning the lipid bilayers as well as its antibiotic activity. Based on this structure, novel antibiotic peptides having strong antibiotic activity, with no hemolytic effect will be developed.

Amino Acid Sequence↗
PubMed2002

Three electrophysiological phenotypes of cultured human umbilical vein endothelial cells.

The conventional whole cell patch-clamp technique was used to measure the resting membrane conductance and membrane currents of nonstimulated cultured human umbilical vein endothelial cells (HUVECs) in different ionic conditions. Three electrophysiological phenotypes of cultured HUVECs (n = 122) were determined: first, 20% of cells as type I mainly displaying the inwardly rectifying potassium current (IKi); second, 38% of cells as type II in which IKi was super-posed on a TEA-sensitive, delayed rectifying current; third, 27% of cells as type III predominantly displaying the outwardly rectifying current which was sensitive to TEA and slightly inhibited by a chloride channel blocker niflumic acid (N.A.). In cells of type I, the mean zero-current potential (V0) was dependent on extracellular K+ ([K+]o) but not on Cl-, indicating major permeability to K+. Whereas V0 of type II was also affected by extracellular Cl- ([Cl-]o), indicating the contribution of an outward Cl- current in setting V0. The cells of type III were not sensitive to decrease of [Cl-]o and the outward current was activated in a relative stable voltage range. This varying phenotypic expression and multipotential behavior of HUVECs suggests that the electrical features of HUVEC may be primarily determined by embryonic origin and local effect of the microenvironment. This research provided the detailed electrophysiological knowledge of the endothelial cells.

Cells, Cultured↗
PubMed2001

RGS18 is a myeloerythroid lineage-specific regulator of G-protein-signalling molecule highly expressed in megakaryocytes.

Myelopoiesis and lymphopoiesis are controlled by haematopoietic growth factors, including cytokines, and chemokines that bind to G-protein-coupled receptors (GPCRs). Regulators of G-protein signalling (RGSs) are a protein family that can act as GTPase-activating proteins for G(alphai)- and G(alphaq)-class proteins. We have identified a new member of the R4 subfamily of RGS proteins, RGS18. RGS18 contains clusters of hydrophobic and basic residues, which are characteristic of an amphipathic helix within its first 33 amino acids. RGS18 mRNA was most highly abundant in megakaryocytes, and was also detected specifically in haematopoietic progenitor and myeloerythroid lineage cells. RGS18 mRNA was not detected in cells of the lymphoid lineage. RGS18 was also highly expressed in mouse embryonic 15-day livers, livers being the principal organ for haematopoiesis at this stage of fetal development. RGS1, RGS2 and RGS16, other members of the R4 subfamily, were expressed in distinct progenitor and mature myeloerythroid and lymphoid lineage blood cells. RGS18 was shown to interact specifically with the G(alphai-3) subunit in membranes from K562 cells. Furthermore, overexpression of RGS18 inhibited mitogen-activated-protein kinase activation in HEK-293/chemokine receptor 2 cells treated with monocyte chemotactic protein-1. In yeast cells, RGS18 overexpression complemented a pheromone-sensitive phenotype caused by mutations in the endogeneous yeast RGS gene, SST2. These data demonstrated that RGS18 was expressed most highly in megakaryocytes, and can modulate GPCR pathways in both mammalian and yeast cells in vitro. Hence RGS18 might have an important role in the regulation of megakaryocyte differentiation and chemotaxis.

Amino Acid Sequence↗
PubMed2001

Neurophysiological evidence of spared upper motor conduction fibers in clinically complete spinal cord injury: discomplete SCI in rats.

Motor evoked potentials (MEP) were recorded and characterized by epidural electrodes (scMEP) and extracellular microelectrodes (exMEP) on T(13) level from 10 normal rats and 40 rats with chronic spinal cord injury (SCI). The spinal cord of 40 anesthetized rats were injured with various severity (sham, 35, 70, and 100 g/cm impact injury) at T(8)-T(9) cord using Allen's drop model. The incline plane and Tarlov techniques were investigated to assess clinical neurological function. MEPs in the normal rats elicited by applying transcortical suprathreshold stimulation consisted of 3-4 early negative peaks (N(1), N(2), N(3), N(4)) followed by several late waves. The N(1) and N(2) peaks had their maximal amplitudes in the anterior and ventrolateral funiculus, respectively, irrespective of the polarity of stimulation, which indicated that these impulses were conducted mostly through the extrapyramidal pathways. The 100 g/cm impact injury or transection of the cord caused abolishment of the MEP signals distal to the lesion, whereas the 35 g/cm injury resulted in a latency shift and amplitude decrement of the MEP peaks. Out of 20 rats with 70 g/cm injuries, 18 showed clinically paraplegia. Among them, seven had neurophysiological evidence of residual conduction pathways through the injured cord segment, such as the presence of N(1) and N(2) peaks in scMEP or exMEP. After 4-aminopyridine (4-AP) administration (1 mg/kg), the amplitude of spared exMEP increased significantly and spread more widely. These results suggest that MEPs evoked by transcortical stimulation travel mostly in the extrapyramid tract. The present study provides further direct and objective electrophysiological evidences of spared functional axons after discomplete SCI, since many other studies on this field have achieved similar results previously. Furthermore, pharmaceutical treatment with 4-AP or other K(+) channel blocking agents proved to be a potential therapeutic strategy for patient with chronic SCI.

Animals↗
PubMed2001

Structural basis for fibroblast growth factor receptor 2 activation in Apert syndrome.

Apert syndrome (AS) is characterized by craniosynostosis (premature fusion of cranial sutures) and severe syndactyly of the hands and feet. Two activating mutations, Ser-252 --> Trp and Pro-253 --> Arg, in fibroblast growth factor receptor 2 (FGFR2) account for nearly all known cases of AS. To elucidate the mechanism by which these substitutions cause AS, we determined the crystal structures of these two FGFR2 mutants in complex with fibroblast growth factor 2 (FGF2). These structures demonstrate that both mutations introduce additional interactions between FGFR2 and FGF2, thereby augmenting FGFR2-FGF2 affinity. Moreover, based on these structures and sequence alignment of the FGF family, we propose that the Pro-253 --> Arg mutation will indiscriminately increase the affinity of FGFR2 toward any FGF. In contrast, the Ser-252 --> Trp mutation will selectively enhance the affinity of FGFR2 toward a limited subset of FGFs. These predictions are consistent with previous biochemical data describing the effects of AS mutations on FGF binding. Alterations in FGFR2 ligand affinity and specificity may allow inappropriate autocrine or paracrine activation of FGFR2. Furthermore, the distinct gain-of-function interactions observed in each crystal structure provide a model to explain the phenotypic variability among AS patients.

Acrocephalosyndactylia↗
PubMed2001

Preparation and functional properties of polyclonal and monoclonal antibodies to murine MD-1.

Rabbits, rats and hamsters were immunized with KLH-coupled synthetic peptide sequences of the murine MD-1 molecule. Serum from immunized animals bound in Western gels to a 25 KDa protein extracted from LPS stimulated mouse spleen cells, as did a rat hybridoma (SH1.2.47) prepared from peptide-immunized rats. CHO cells transfected with a plasmid cDNA construct encoding murine MD-1, the target antigen for the antibodies in question, were also stained (in FACS) by the same antibodies. Patching and capping of the antigen(s) detected by any one of these sera abolished binding of all antibodies in subsequent FACS analysis, consistent with the hypothesis that they all detected the same antigen. In a final study to assess the possible involvement of MD-1 in regulation of cell activation for cytokine production following allostimulation, we found that all of the antibodies inhibited IL-2 and IFNgamma production, while enhancing IL-4 and IL-10 production, in mixed leukocyte reactions (MLR) in vitro.

Amino Acid Sequence↗
PubMed2001

Carboxy ester hydrolysis promoted by a zinc(II) 2-[bis(2-aminomethyl)amino]ethanol complex: a new model for indirect activation on the serine nucleophile by zinc(II) in zinc enzymes.

A complexation study on the new Zn(II) complexes of asymmetric tripodal ligand 2-[bis(2-aminoethyl)amino]ethanol (L) has revealed that the alcoholic OH group of complex ZnL exhibits remarkable acidity with a very low pK(a) value of 7.7 at 25 degrees C. Both the monomeric complex [ZnH(-0.25)L(H(2)O)](ClO(4))(1.75) (I) and the dimeric alkoxide-coordinating complex [Zn(2)(H(-1)L)(2)](ClO(4))(2) (II) were synthesized, and their structures were determined by X-ray diffraction. The Zn(II)-bound alkoxide, as the reactive nucleophile toward the hydrolysis of esters, has shown a second-order rate constant of 0.13 +/- 0.01 M(-1) s(-1) in 10% (v/v) CH(3)CN at 25 degrees C in 4-nitrophenyl acetate (NA) hydrolysis, which is almost the same as the corresponding value for the very reactive alcohol-dependent [12]aneN(3)-Zn complex. Present work shows for the first time that Zn(II) complexes of the asymmetric tripodal polyamine bearing an ethoxyl pod can also serve as good models of Zn(II)-containing enzymes.

Chelating Agents↗
PubMed2001

A novel fibroblast growth factor receptor-5 preferentially expressed in the pancreas(1).

Using the polymerase chain reaction on human embryonic cDNAs, we isolated a cDNA encoding a novel 504 amino acid protein, termed fibroblast growth factor receptor (FGFR)-5, which is highly homologous to known FGFRs. The NH(2)-terminal portion of FGFR5 contains a putative secretory signal sequence, three typical immunoglobulin-like domains, six cysteines, and an acidic box, but no HAV motif. The COOH-terminal portion of FGFR5 contains one transmembrane domain but no intracellular kinase domain. Recombinant FGFR5 expressed in COS-7 cells is not secreted, but recombinant truncated FGFR5 lacking the predicted transmembrane domain is secreted. Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) do not bind to FGFR5. Among 23 adult human tissues, FGFR5 mRNA is preferentially expressed in the pancreas. These results suggest that FGFR5 may provide a binding site for some other fibroblast growth factors and may regulate some pancreatic function.

Amino Acid Sequence↗
PubMed2001

Activation of Gadd34 by diverse apoptotic signals and suppression of its growth inhibitory effects by apoptotic inhibitors.

DNA damage has many cellular consequences including, in some cases, apoptosis. Expression of Gadd34 was shown to be increased by ionizing radiation only in cells that undergo rapid apoptosis following this treatment. The effects of various other apoptosis-inducing agents as well as apoptosis-inhibiting genes on regulation of Gadd34 were investigated. In many cell types, agents which have been reported to lead to increased intracellular ceramide levels led to an increase in Gadd34 transcript levels. These included TNFalpha, the ceramide analog C-2 ceramide, dimethyl sphingosine and anti-Fas antibody as well as ionizing radiation. Induction of Gadd34 by ionizing radiation was coincident with the onset of apoptosis and increased as apoptosis progressed. In a short-term transfection assay, more than 30% of Gadd34-transfected cells exhibited nuclear fragmentation by 48 hours. Apoptosis, as well as induction of Gadd34 by apoptotic stimuli, was attenuated by the apoptosis inhibitors, Bcl-2, cowpox virus CrmA and herpes simplex virus ICP34.5. Thus, activation of Gadd34 is a downstream event in apoptotic signaling pathways and may directly contribute to the apoptotic process.

Antigens, Differentiation↗
PubMed2001

CD200 immunoadhesin suppresses collagen-induced arthritis in mice.

DBA/1 mice immunized with 100 microg bovine collagen type II emulsified in Freund's adjuvant, followed by booster injection in incomplete adjuvant at 18 days, develop profound arthritis (>50% of animals) by 30 days postinjection. The molecule CD200 (previously called OX2), associated with, among others, follicular dendritic cells, is implicated in delivery of immunosuppressive signals to the immune system, and an immunoadhesin in which the extracellular domains of CD200 were linked to a mouse IgG2a Fc region has been shown to promote renal allograft survival. DBA/1 mice receiving 15 microg/mouse CD200Fc at 3-day intervals following immunization with collagen did not develop arthritis in this model. Lymphocytes taken from CD200Fc-treated, collagen-immunized mice produced significantly lower levels of TNFalpha and IFN-gamma in culture supernatants after restimulation in vitro with collagen, in contrast to cells taken from control mice treated with PBS or normal mouse Ig. Serum from CD200Fc-treated mice contained less anti-collagen IgG (approximately 50% reduction), with relatively more IgG2b and IgG3, and lower levels of TNFalpha and IFN-gamma, than control mice. These data indicate that this immunoadhesin may have a potent role to play in the regulation of autoimmune disorders.

Animals↗
PubMed2001

Effects of Fe(2+) on ion channels: Na(+) channel, delayed rectified and transient outward K(+) channels.

The effects of Fe(2+) on the properties of three types of ion channels were studied in acutely dissociated rat hippocampal pyramidal neurons from area CA1 at postnatal ages of 7-14 days using the whole cell patch clamp technique. The results indicated that: (1) in the existence of Fe(2+), the activation voltage threshold of transient outward K(+) currents (I(A)) was decreased. The normalized current-voltage curves of activation were well fitted with a single Boltzmann function, and the V(1/2) was 2.44+/-1.14 mV (n=15) in control, whereas 1.79+/-1.53 (n=15), -2.96+/-0.92 (n=14), -5.11+/-1.31 (n=13), -9.05+/-1.64 mV (n=12) in 1, 10, 100 and 1000 microM Fe(2+), respectively. Differences between two groups were significant (P<0.05, n=12-15), except for that between the control and 1 microM (P>0.05, n=15). (2) Fe(2+) caused a left shift of the current-voltage curves of steady-state inactivation of I(A) in a concentration-dependent manner. The curves were well fitted with a single Boltzmann function with similar slope (P>0.05, n=10-13). The V(1/2) were -70.71+/-1.23 (n=13), -71.14+/-1.37 (n=13), -78.21+/-1.17 (n=11), -84.61+/-1.34 (n=12), and -89.68+/-2.59 mV (n=10) in control, 1, 10, 100 and 1000 microM Fe(2+), respectively. Fe(2+) also shifted the current-voltage curves of Na(+) channel steady-state inactivation to more negative depolarization potentials in parallel, with V(1/2), -67.37+/-1.33 mV (n=12) in control, and -67.52+/-1.28 mV (n=12), -68.24+/-1.61 mV (n=10), -71.58+/-1.45 mV (n=10), -76.65+/-1.76 mV (n=9) in 1, 10, 100 and 1000 microM Fe(2+) solutions, respectively. (3) In Fe(2+) solutions, the recovery from inactivation of I(A) was slowed. (4) With application of different concentrations of Fe(2+), the voltage threshold of activation of delayed rectified outward K(+) currents (I(K)) was decreased, while Fe(2+) showed a little inhibition at more positive depolarization. Briefly, the results demonstrated that Fe(2+) is a dose- and voltage-dependent, reversible modulator of I(A), I(K) and Na(+) channels. The results will be helpful to explain the mechanism of Fe(2+) physiological function and Fe(2+) intoxication in the central nervous system.

Animals↗
PubMed2001

Disparate cleavage of poly-(ADP-ribose)-polymerase (PARP) and a synthetic tetrapeptide, DEVD, by apoptotic cells.

In the present investigations, we have shown differential cleavage of cellular PARP and a caspase 3-selective synthetic tetrapeptide substrate, Z-DEVD-AFC or Ac-DEVD-AMC using a T lymphoblastoid cell line Jurkat, and its variant clone E6.1(J-E6). Anti-Fas antibody-mediated apoptosis resulted in DNA fragmentation and PARP cleavage in both Jurkat and J-E6 cells. However, unlike Jurkat, J-E6 cells did not cleave a synthetic tetrapeptide substrate efficiently. The failure to cleave the DEVD tetrapeptide by apoptotic J-E6 cells was not due to insufficient expression or processing of caspase 3 in J-E6 cells. Interestingly, when the J-E6 cells were transiently transfected with a cDNA encoding caspase 3, efficient cleavage of Z-DEVD-AFC was achieved. The observations that apoptotic J-E6 cells barely cleaved a synthetic DEVD tetrapeptide, but efficiently cleaved endogenous PARP, potentially at the most preferred DEVD site, suggest that active caspases may have disparate characteristics to recognize substrates presented in different context.

Apoptosis↗
PubMed2001

Interactions between mastoparan B and the membrane studied by 1H NMR spectroscopy.

Mastoparan B (MP-B) is an antimicrobial cationic tetradecapeptide amide isolated from the venom of the hornet Vespa basalis. NMR spectroscopy was used to study the membrane associated structures of MP-B in various model membrane systems such as 120 mM DPC micelles, 200 mM SDS micelles, and 3%(w/v) DMPC/DHPC (1:2) bicelles. In all systems, MP-B has an amphiphilic alpha-helical structure from Lys2 to Leu14. NOESY experiments performed on MP-B in nondeuterated SDS micelles show that protons in the indole ring of Trp9 are in close contact with methylene protons of SDS micelles. T1 relaxation data and NOE data revealed that the bound form of MP-B may be dominant in SDS micelles. The interactions between MP-B and zwitterionic DPC micelles were much weaker than those between MP-B and anionic SDS micelles. By substitution of Trp9 with Ala9, the pore-forming activity of MP-B was decreased dramatically. All of these results imply that strong electrostatic interactions between the positively charged Lys residues in MP-B and the anionic phospholipid head groups must be the primary factor for MP-B binding to the cell membrane. Then, insertion of the indole ring of Trp9 into the membrane, as well as the amphiphilic alpha-helical structures of MP-B may allow MP-B to span the lipid bilayer through the C-terminal portion. These structural features are crucial for the potent antibiotic activities of MP-B.

Alanine↗
PubMed2001

Estimating the frequency distribution of crossovers during meiosis from recombination data.

Estimation of tetrad crossover frequency distributions from genetic recombination data is a classic problem dating back to Weinstein (1936, Genetics 21, 155-199). But a number of important issues, such as how to specify the maximum number of crossovers, how to construct confidence intervals for crossover probabilities, and how to obtain correct p-values for hypothesis tests, have never been adequately addressed. In this article, we obtain some properties of the maximum likelihood estimate (MLE) for crossover probabilities that imply guidelines for choosing the maximum number of crossovers. We give these results for both normal meiosis and meiosis with nondisjunction. We also develop an accelerated EM algorithm to find the MLE more efficiently. We propose bootstrap-based methods to find confidence intervals and p-values and conduct simulation studies to check the validity of the bootstrap approach.

Algorithms↗
PubMed2001

Effects of tumor necrosis factor alpha on host immune response in chronic persistent tuberculosis: possible role for limiting pathology.

Reactivation of latent tuberculosis contributes significantly to the incidence of disease caused by Mycobacterium tuberculosis. The mechanisms involved in the containment of latent tuberculosis are poorly understood. Using the low-dose model of persistent murine tuberculosis in conjunction with MP6-XT22, a monoclonal antibody that functionally neutralizes tumor necrosis factor alpha (TNF-alpha), we examined the effects of TNF-alpha on the immunological response of the host in both persistent and reactivated tuberculous infections. The results confirm an essential role for TNF-alpha in the containment of persistent tuberculosis. TNF-alpha neutralization resulted in fatal reactivation of persistent tuberculosis characterized by a moderately increased tissue bacillary burden and severe pulmonic histopathological deterioration that was associated with changes indicative of squamous metaplasia and fluid accumulation in the alveolar space. Analysis of pulmonic gene and protein expression of mice in the low-dose model revealed that nitric oxide synthase was attenuated during MP6-XT22-induced reactivation, but was not totally suppressed. Interleukin-12p40 and gamma interferon gene expression in TNF-alpha-neutralized mice was similar to that in control mice. In contrast, interleukin-10 expression was augmented in the TNF-alpha-neutralized mice. In summary, results of this study suggest that TNF-alpha plays an essential role in preventing reactivation of persistent tuberculosis, modulates the pulmonic expression of specific immunologic factors, and limits the pathological response of the host.

Animals↗