Biomedical subjects

H Li

Publications and source records attributed to H Li.

At least 343 records · Page 19Linked to original sources

PubMed2001

A monoclonal antibody specific for human thymidine kinase 1.

Previous research has shown that thymidine kinase 1 (TK1), a nucleotide salvage pathway enzyme, is an accurate prognostic and diagnostic tumor marker. However, the current radioisotope assay for TK1 is cumbersome and has hampered the clinical application of this diagnostic technique in cancer management. To overcome the problems of the current radioisotope assay, we have produced monoclonal antibodies (MAbs) using purified TK1 from Raji cell extract. Production and confirmation of their specificity was confirmed using Western blot, immunohistochemical staining, TK1 activity inhibition assays, and enzyme-linked immunoadsorbent assay (ELISA) techniques. Thus, in the future, these antibodies may aid in the early detection of cancer and more accurate prognosis, as well as allowing for an increased ability to study the function of TK1 in basic cellular processes.

Antibodies, Monoclonal↗

PubMed2001

Oligoacetic acid characterization by isocratic and linear salt gradient anion-exchange chromatography.

A homologous series of four oligomers of acetic acid (namely acetic acid, succinic acid, tricarballylic acid, and tetracarboxylic acid) is characterized using isocratic and linear salt gradient anion-exchange chromatography. The double logarithmic plot of the isocratic retention factors versus the salt concentration gives straight lines for all samples. These straight lines (with the exception of the line for the strongly retained succinate peak) have a common intersection point--something which is proved to be a direct consequence of the stoichiometric mass-action ion-exchange model. The characteristic charge and the equilibrium ion-exchange constant (and the corresponding Gibbs free energy of ion exchange, or deltaG(exchange)) are determined from the isocratic experiments. The characteristic charge agrees satisfactorily with the number of carboxylic acid groups in the samples, and the deltaG(exchange) value decreases linearly with the characteristic charge. Succinic acid always gives two chromatographic peaks despite the proven chemical purity of the sample. The characteristic charge that is calculated for both of the succinic acid peaks is approximately two. The deltaG(exchange) value calculated for the weakly retained succinic acid peak falls in the free energy versus characteristic charge straight line defined by the other homologues. The deltaG(exchange) value of the strongly retained peak is lower than that of the weakly retained peak by 1.85 kJ/mol. The two succinic acid peaks are explained in terms of an equilibrium between two conformers in solution--one binding the solution counterions tightly and the other loosely. An analysis of all samples under a linear salt gradient provides retention times that increase linearly with the number of functional groups. Using an appropriate model (along with the isocratically determined characteristic charge and ion-exchange constant), we predict theoretically the linear gradient retention times, which agree reasonably well with the experimental ones.

Journal Article↗

PubMed2001

Column robustness case study for a liquid chromatographic method validated in compliance with ICH, VICH, and GMP guidelines.

This article presents a case study in dealing with robustness investigations and attempts by our analytical laboratory to address these issues without sacrificing valuable time in revamping the method validation prior to submission. A liquid chromatographic method is developed for the analysis of a novel triazinetrione anticoccidial product. The method effectively separates the active pharmaceutical ingredient (API), impurities, and preservatives in the API and product formulation. For much of the validation, the method holds up to the rigorous guidelines of the International Conference of Harmonization, the International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products, and the Good Manufacturing Practices. However, in analyzing a base-degraded sample one of the impurity peaks yields inconsistent retention times (RTs) during a series of injections. When switching the system to another analytical column from the same supplier, this impurity peak elutes at a different retention window and the remaining peaks in the chromatographic profile remain essentially the same. This RT variation of a single peak in the chromatographic profile is observed with additional columns from the same supplier and from different manufacturing lots. This suitability problem is not encountered with the columns used in the method development stage. The method no longer meets the robustness criteria established for pharmaceutical methods. An investigation is commenced and it is discovered that with the addition of tetrabutylammonium hydroxide to the mobile phases, the impurity peak gives a consistent RT in relation to the active peak. The peak shows comparable RTs relative to that of the API peak with columns of different silica lots and bond lots. All peaks, including the aforementioned impurity peak, are well-resolved under the revised high-performance liquid chromatographic conditions. This temporary solution enables continued submission work for FDA, but the robustness of this method is still a concern. After further investigation, it is determined that inhomogeneity of the active sites on the column's stationary phase is the likely culprit. Fortunately, a new column is found to be more suitable for this method and a column qualification study is initiated.

Journal Article↗

PubMed2001

Antimicrobial susceptibility of Neisseria gonorrhoeae isolates from three Caribbean countries: Trinidad, Guyana, and St. Vincent.

BACKGROUND: The percentage of Neisseria gonorrhoeae isolates resistant to antimicrobial agents commonly used for treatment is unknown in many Caribbean countries. GOAL: To determine the antimicrobial susceptibility of N gonorrhoeae isolates from Trinidad (144 isolates), Guyana (70 isolates), and St. Vincent (68 isolates) so baseline data can be established for further studies, and to assist in establishing effective treatment guidelines. STUDY DESIGN: Consecutive urethral and endocervical specimens from several clinics were collected and identified as N gonorrhoeae. Isolates of N gonorrhoeae were tested for their susceptibility to penicillin, tetracycline, ceftriaxone, ciprofloxacin, spectinomycin, and azithromycin. The presumptive identification of penicillinase-producing N gonorrhoeae and/or tetracycline-resistant N gonorrhoeae isolates based on MIC was confirmed by plasmid and tetM content analysis. RESULTS: High percentages of penicillin and/or tetracycline resistance were observed in N gonorrhoeae isolates from Guyana (92.9%), St. Vincent (44.1%), and Trinidad (42.4%). Isolates from all three countries were susceptible to ceftriaxone, ciprofloxacin, and spectinomycin. One penicillinase-producing N gonorrhoeae/tetracycline-resistant N gonorrhoeae from Guyana had an MIC of 0.5 microg/l to ciprofloxacin. This and nine other isolates from Guyana also were resistant to azithromycin (defined as MIC > or = 2.0 microg/ml) as well as penicillin and tetracycline. A reduced susceptibility to azithromycin was displayed by 16% of the isolates from St. Vincent and 72% of the isolates from Guyana (MIC, 0.25-1.0 microg/ml). Most penicillinase-producing N gonorrhoeae isolates carried Africa-type plasmids (61/90), with 28 of 90 having Toronto-type plasmids and a single isolate carrying an Asia-type plasmid. The tetM determinant in tetracycline-resistant N gonorrhoeae isolates was predominantly of the Dutch type (68/91). CONCLUSIONS: The high prevalence of N gonorrhoeae isolates from 3 of 21 English- and Dutch-speaking Caricom countries in the Caribbean with either plasmid-mediated or chromosomal resistance to penicillin and tetracycline supports international observations that these drugs should not be used to treat gonococcal infections. The detection of isolates with reduced susceptibility to drugs such as azithromycin, which currently are recommended for treatment in the region, attest to the importance of the continued monitoring of gonococcal antimicrobial susceptibility for the maintenance of effective treatment guidelines.

Anti-Bacterial Agents↗

PubMed2001

Reduced susceptibility to azithromycin and high percentages of penicillin and tetracycline resistance in Neisseria gonorrhoeae isolates from Manaus, Brazil, 1998.

BACKGROUND: The identification of Neisseria gonorrhoeae isolates resistant to antimicrobial agents currently recommended for the treatment of gonococcal infections continues to escalate globally. Thus, in some areas, resistance to fluoroquinolone drugs is commonplace; several reports document resistance to third-generation cephalosporins, and the sporadic isolation of spectinomycin-resistant isolates continues unabated. Gonococcal resistance to azithromycin, an antibiotic used for the primary treatment of gonococcal infections in some Latin American countries, also has been described. Because the prevalence of resistant isolates is insufficiently documented in many areas of Latin America, the efficacy of locally recommended therapies for gonococcal infections is often unknown. GOAL: To determine the antimicrobial susceptibility and strain types of N gonorrhoeae isolates collected in Manaus, Brazil. These data will establish antimicrobial susceptibility baseline data for the region as a reference point for future surveillance. STUDY DESIGN: Consecutive N gonorrhoeae isolates from urethral and endocervical specimens were collected and examined for identity, antimicrobial susceptibility, and strain type (plasmid content, tetM type, auxotype, and serovar). RESULTS: Most of the isolates (65/81; 85.2%) were resistant to tetracycline, penicillin, or both, with the majority (n = 62) carrying plasmid-mediated resistance to tetracycline (tetracycline-resistant N gonorrhoeae [TRNG]). All of the TRNG contained the Dutch-type tetM plasmid, and 18 were A/S class NR/IA-02. Penicillinase-producing N gonorrhoeae comprised 8.2% (7/81) of the isolates. Of these seven isolates, four also were TRNG, and two carried chromosomal resistance to tetracycline. The isolates were susceptible to ciprofloxacin, spectinomycin, and ceftriaxone. However, 23 isolates were characterized by reduced susceptibility to azithromycin (MIC, 0.25-0.5 microg/ml), and one isolate had reduced susceptibility to ciprofloxacin (MIC, 0.25 microg/ml). CONCLUSIONS: This study supports the continued use of third-generation cephalosporins, spectinomycin, and fluoroquinolone drugs for the primary treatment of gonococcal infections in Manaus. The occurrence of isolates with reduced susceptibility to azithromycin and ciprofloxacin underscores the importance of ongoing antimicrobial susceptibility monitoring to support decisions regarding appropriate drugs for the treatment of gonococcal infections.

Anti-Bacterial Agents↗

PubMed2001

The Rop GTPase switch controls multiple developmental processes in Arabidopsis.

G proteins are universal molecular switches in eukaryotic signal transduction. The Arabidopsis genome sequence reveals no RAS small GTPase and only one or a few heterotrimeric G proteins, two predominant classes of signaling G proteins found in animals. In contrast, Arabidopsis possesses a unique family of 11 Rop GTPases that belong to the Rho family of small GTPases. Previous studies indicate that Rop controls actin-dependent pollen tube growth and H(2)O(2)-dependent defense responses. In this study, we tested the hypothesis that the Rop GTPase acts as a versatile molecular switch in signaling to multiple developmental processes in Arabidopsis. Immunolocalization using a general antibody against the Rop family proteins revealed a ubiquitous distribution of Rop proteins in all vegetative and reproductive tissues and cells in Arabidopsis. The cauliflower mosaic virus 35S promoter-directed expression of constitutively active GTP-bound rop2 (CA-rop2) and dominant negative GDP-bound rop2 (DN-rop2) mutant genes impacted many aspects of plant growth and development, including embryo development, seed dormancy, seedling development, lateral root initiation, morphogenesis of lateral organs in the shoot, shoot apical dominance and growth, phyllotaxis, and lateral organ orientation. The rop2 transgenic plants also displayed altered responses to the exogenous application of several hormones, such as abscisic acid-mediated seed dormancy, auxin-dependent lateral shoot initiation, and brassinolide-mediated hypocotyl elongation. CA-rop2 and DN-rop2 expression had opposite effects on most of the affected processes, supporting a direct signaling role for Rop in regulating these processes. Based on these observations and previous results, we propose that Rop2 and other members of the Rop family participate in multiple distinct signaling pathways that control plant growth, development, and responses to the environment.

Arabidopsis↗

PubMed2001

Chloroplast protein translocon components atToc159 and atToc33 are not essential for chloroplast biogenesis in guard cells and root cells.

Protein import into chloroplasts is mediated by a protein import apparatus located in the chloroplast envelope. Previous results indicate that there may be multiple import complexes in Arabidopsis. To gain further insight into the nature of this multiplicity, we analyzed the Arabidopsis ppi1 and ppi2 mutants, which are null mutants of the atToc33 and atToc159 translocon proteins, respectively. In the ppi2 mutant, in contrast to the extremely defective plastids in mesophyll cells, chloroplasts in guard cells still contained starch granules and thylakoid membranes. The morphology of root plastids in both mutants was similar to that in wild type. After prolonged light treatments, root plastids of both mutants and the wild type differentiated into chloroplasts. Enzymatic assays indicated that the activity of a plastid enzyme was reduced only in leaves but not in roots. These results indicated that both the ppi1 and ppi2 mutants had functional root and guard cell plastids. Therefore, we propose that import complexes are cell type specific rather than substrate or plastid specific.

Arabidopsis↗

PubMed2001

Computerized radiographic mass detection--part I: Lesion site selection by morphological enhancement and contextual segmentation.

This paper presents a statistical model supported approach for enhanced segmentation and extraction of suspicious mass areas from mammographic images. With an appropriate statistical description of various discriminate characteristics of both true and false candidates from the localized areas, an improved mass detection may be achieved in computer-assisted diagnosis (CAD). In this study, one type of morphological operation is derived to enhance disease patterns of suspected masses by cleaning up unrelated background clutters, and a model-based image segmentation is performed to localize the suspected mass areas using stochastic relaxation labeling scheme. We discuss the importance of model selection when a finite generalized Gaussian mixture is employed, and use the information theoretic criteria to determine the optimal model structure and parameters. Examples are presented to show the effectiveness of the proposed methods on mass lesion enhancement and segmentation when applied to mammographical images. Experimental results demonstrate that the proposed method achieves a very satisfactory performance as a preprocessing procedure for mass detection in CAD.

Breast Neoplasms↗

PubMed2001

Computerized radiographic mass detection--part II: Decision support by featured database visualization and modular neural networks.

Based on the enhanced segmentation of suspicious mass areas, further development of computer-assisted mass detection may be decomposed into three distinctive machine learning tasks: 1) construction of the featured knowledge database; 2) mapping of the classified and/or unclassified data points in the database; and 3) development of an intelligent user interface. A decision support system may then be constructed as a complementary machine observer that should enhance the radiologists performance in mass detection. We adopt a mathematical feature extraction procedure to construct the featured knowledge database from all the suspicious mass sites localized by the enhanced segmentation. The optimal mapping of the data points is then obtained by learning the generalized normal mixtures and decision boundaries, where a is developed to carry out both soft and hard clustering. A visual explanation of the decision making is further invented as a decision support, based on an interactive visualization hierarchy through the probabilistic principal component projections of the knowledge database and the localized optimal displays of the retrieved raw data. A prototype system is developed and pilot tested to demonstrate the applicability of this framework to mammographic mass detection.

Artificial Intelligence↗

PubMed2001

Antisense display: a new method for functional gene screen and its application to angiogenesis-related gene isolation.

We have developed an antisense-oriented method for functional gene screening, which we termed "antisense display." After evaluation in a cell-free system, we applied this method to a cellular system for isolating angiogenesis-related genes and came across a gene previously not described in humans. A screen of 10 mer antisense repertoire led to the identification of a unique 'angiostatic' antisense, and the gene corresponding to it was found to be preferentially expressed in vascular endothelial cells, coding for a putative type-2 membrane protein containing Rab-GAP/TBC and EF-hand-like domains. We named it vascular Rab-GAP/TBC domain-containing protein (VRP). The results obtained thus indicate that the antisense display method is useful for the isolation of functional genes responsible for phenotypes concerned.

Genetic Testing↗

PubMed2001

An rpsL cassette, janus, for gene replacement through negative selection in Streptococcus pneumoniae.

Natural genetic transformation offers a direct route by which synthetic gene constructs can be placed into the single circular chromosome of Streptococcus pneumoniae. However, the lack of a general negative-selection marker has hampered the introduction of constructs that do not confer a selectable phenotype. A 1.3-kb cassette was constructed comprising a kanamycin (Kn) resistance marker (kan) and a counterselectable rpsL(+) marker. The cassette conferred dominant streptomycin (Sm) sensitivity in an Sm-resistant background in S. pneumoniae. It was demonstrated that it could be used in a two-step transformation procedure to place DNA of arbitrary sequence at a chosen target site. The first transformation into an Sm-resistant strain used the cassette to tag a target gene on the chromosome by homologous recombination while conferring Kn resistance but Sm sensitivity on the recombinant. Replacement of the cassette by an arbitrary segment of DNA during a second transformation restored Sm resistance (and Kn sensitivity), allowing construction of silent mutations and deletions or other gene replacements which lack a selectable phenotype. It was also shown that gene conversion occurred between the two rpsL alleles in a process that depended on recA and that was susceptible to correction by mismatch repair.

Anti-Bacterial Agents↗

PubMed2001

Characterization and mutational analysis of three allelic lsc genes encoding levansucrase in Pseudomonas syringae.

In the plant pathogen Pseudomonas syringae pv. glycinea PG4180 and other bacterial species, synthesis of the exopolysaccharide levan is catalyzed by the extracellular enzyme levansucrase. The results of Southern blotting and PCR analysis indicated the presence of three levansucrase-encoding genes in strain PG4180: lscA, lscB, and lscC. In this study, lscB and lscC were cloned from a genomic library of strain PG4180. Sequence analysis of the two lsc genes showed that they were virtually identical to each other and highly similar to the previously characterized lscA gene. lscA and lscC had a chromosomal location, whereas lscB resided on an indigenous plasmid of PG4180. Mutants with impaired expression of individual lsc genes and double mutants were generated by marker exchange mutagenesis. Determination of levansucrase activities in these mutants revealed that the lscB gene product was secreted but not that of lscA or lscC. Our results indicated that lscB and lscC but not lscA contributed to periplasmic levan synthesis of PG4180. The lscB lscC double mutant was completely defective in levan formation and could be complemented by either lscB or lscC. Our data suggested a compartment-specific localization of two lsc gene products, with LscB being the secreted, extracellular enzyme and LscC being the predominantly periplasmic levansucrase. Results of Western blot analyses indicated that lscA was not expressed and that lscA was not associated with levansucrase activities in any particular protein fraction. LscA could be detected in PG4180 only when transcribed from the vector-borne P(lac) promoter. PCR screening in various P. syringae strains with primers derived from the three characterized lsc genes demonstrated the presence of multiple Lsc isoenzymes in other P. syringae pathovars.

Alleles↗

PubMed2001

Detection of sporadic cases of hepatitis E virus (HEV) infection in China using immunoassays based on recombinant open reading frame 2 and 3 polypeptides from HEV genotype 4.

We reported previously on the complete sequence of hepatitis E virus (HEV) genotype 4, isolated from patients with sporadic cases of acute HEV infection in China. At least eight HEV genotypes have now been described worldwide, and further isolates await classification. Current immunoassays for the detection of anti-HEV antibodies are based on polypeptides from genotypes 1 and 2 only and may be inadequate for the reliable detection of other genotypes. Because genotypes 1 and 4 predominate in China, we wished to investigate the antigenic reactivities of HEV genotype 4 proteins. Four overlapping regions of open reading frame 2 (ORF2) (FB5, amino acids [aa] 1 to 130; E4, aa 67 to 308; F2-2, aa 288 to 461; E5, aa 414 to 672) and the entire ORF3 product were expressed in Escherichia coli as fusion proteins. Enzyme immunoassays based on each of the five purified polypeptides were evaluated with sera from patients with sporadic cases of acute HEV infection. Individual immunoassays derived from HEV genotype 4 detected more cases of acute hepatitis E than a commercial assay. Some serum samples, which were positive for anti-HEV immunoglobulin G only by assays based on HEV genotype 4, were positive for HEV RNA by reverse transcription-PCR. Polypeptide FB5, from the N terminus of ORF2, had the greatest immunoreactivity with sera from patients with acute hepatitis E. These data indicate that the N terminus of ORF2 may provide epitopes which are highly reactive with acute-phase sera and that assays based on genotypes 1 and 2 alone may be inadequate for the detection of HEV infection in China, where sporadic cases of HEV infection are caused predominantly by HEV genotypes 4 and 1.

Acute Disease↗

PubMed2001

Human herpesvirus 8 (HHV-8)-encoded cytokines induce expression of and autocrine signaling by vascular endothelial growth factor (VEGF) in HHV-8-infected primary-effusion lymphoma cell lines and mediate VEGF-independent antiapoptotic effects.

The potential roles of human herpesvirus 8 (HHV-8) cytokines in HHV-8 pathogenesis were investigated by determining the expression of the HHV-8 chemokines viral macrophage inflammatory protein 1A (vMIP-1A) and vMIP-1B in primary effusion lymphoma (PEL)-derived cell lines and examining the signaling activities of these chemokines and HHV-8-encoded vIL-6 in these cells. Secreted vMIP-1A and vMIP-1B were detected in biologically significant concentrations following tetradecanoyl phorbol acetate treatment, which induces productive replication. vIL-6 and vMIP-1A, added exogenously to cultures of four different PEL cell lines, induced the expression of vascular endothelial growth factor type B (VEGF-B) and VEGF-A, respectively. These cells were found to express VEGF receptor 1 (Flt-1) protein, and signaling by recombinant VEGF-A(165) was demonstrated for two of the PEL cell lines, indicating the potential for autocrine, as well as paracrine, effects of viral cytokine-induced VEGF. In addition, vMIP-1A and vMIP-1B, but not VEGF-A(165), were found to inhibit chemically induced apoptosis in PEL cells. Our data suggest that vIL-6 and vMIP-1A may influence PEL through VEGF autocrine and paracrine signaling that promotes PEL cell growth and extravascular effusion and that vMIP-1A and vMIP-1B can act independently of VEGF as antiapoptotic factors.

Animals↗

PubMed2001

Effect on polyomavirus T-antigen function of mutations in a conserved leucine-rich segment of the DnaJ domain.

The N-terminal part of the mouse polyomavirus T antigens contains a highly conserved segment (-LLELLKL-), including amino acid residues 13 to 19. The sequence motif is predicted to form alpha helix I in the DnaJ domain of the T antigens. Four mutants with conservative substitutions of amino acid residues 13 and 14 were constructed. Of the four substitutions, L13M, L13I, L13V, and L14V, only L13V resulted in a phenotypic change. In transfected mouse cells, L13V large T antigen showed a more than 100-fold-reduced viral DNA synthesis. The viral replication could not be rescued by cotransfection of the cells with DNA expressing small t antigen or a large T antigen truncated at the C terminus that would compensate for a defect in host cell stimulation. In contrast to the effect on DNA replication, the L13V substitution in large T antigen did not prevent complex formation with Hsc70 and the Rb protein. Also, the activity of the protein in transactivation of transcription from the adenovirus E2 promoter was unimpaired, showing that the transcription factor E2F was released from pRb. The L13V substitution also caused a defect in small t antigen. However, this phenotypic change was due to protein instability. In contrast, middle T antigen with the L13V substitution remained stable and functional in cellular transformation. Together, the data show that the effect of the L13V substitution did not abrogate the Hsc70 interaction of the DnaJ domain. However, it is possible that the substitution of amino acid residue 13 affected specific DnaJ functions of large T antigen.

3T3 Cells↗

PubMed2001

Detection of direct binding of human herpesvirus 8-encoded interleukin-6 (vIL-6) to both gp130 and IL-6 receptor (IL-6R) and identification of amino acid residues of vIL-6 important for IL-6R-dependent and -independent signaling.

Human herpesvirus 8 (HHV-8) is associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease; in all of these diseases, interleukin-6 (IL-6) has been implicated as a likely mitogenic and/or angiogenic factor. HHV-8 encodes a homologue of IL-6 (viral IL-6 [vIL-6]) that has been shown to be biologically active in several assays and whose activities mirror those of its mammalian counterparts. Like these proteins, vIL-6 mediates its effects through the gp130 signal transducer, but signaling is not dependent on the structurally related IL-6 receptor (IL-6R; gp80) subunit of the receptor-signal transducer complex. However, as we have shown previously, IL-6R can enhance vIL-6 signal transduction and can enable signaling through a gp130 variant (gp130.PM5) that is itself unable to support vIL-6 activity, indicating that IL-6R can form part of the signaling complex. Also, our analysis of a panel of vIL-6 mutants in transfection experiments in Hep3B cells (that express IL-6R and gp130) showed that most were able to function normally in this system. Here, we have used in vitro vIL-6-receptor binding assays to demonstrate direct binding of vIL-6 to both gp130 and IL-6R and vIL-6-induced gp130-IL-6R complex formation, and we have extended our functional analyses of the vIL-6 variants to identify residues important for IL-6R-independent and IL-6R-dependent signaling through native gp130 and gp130.PM5, respectively. These studies have identified residues in vIL-6 that are important for IL-6R-independent and IL-6R-mediated functional complex formation between vIL-6 and gp130 and that may be involved directly in binding to gp130 and IL-6R.

Antigens, CD↗

PubMed2001

Regulatory role of arginase I and II in nitric oxide, polyamine, and proline syntheses in endothelial cells.

Endothelial cells (EC) metabolize L-arginine mainly by arginase, which exists as two distinct isoforms, arginase I and II. To understand the roles of arginase isoforms in EC arginine metabolism, bovine coronary venular EC were stably transfected with the Escherichia coli lacZ gene (lacZ-EC, control), rat arginase I cDNA (AI-EC), or mouse arginase II cDNA (AII-EC). Western blots and enzymatic assays confirmed high-level expression of arginase I in the cytosol of AI-EC and of arginase II in mitochondria of AII-EC. For determining arginine catabolism, EC were cultured for 24 h in DMEM containing 0.4 mM L-arginine plus [1-(14)C]arginine. Urea formation, which accounted for nearly all arginine consumption by these cells, was enhanced by 616 and 157% in AI-EC and AII-EC, respectively, compared with lacZ-EC. Arginine uptake was 31-33% greater in AI-EC and AII-EC than in lacZ-EC. Intracellular arginine content was 25 and 11% lower in AI-EC and AII-EC, respectively, compared with lacZ-EC. Basal nitric oxide (NO) production was reduced by 60% in AI-EC and by 47% in AII-EC. Glutamate and proline production from arginine increased by 164 and 928% in AI-EC and by 79 and 295% in AII-EC, respectively, compared with lacZ-EC. Intracellular content of putrescine and spermidine was increased by 275 and 53% in AI-EC and by 158 and 43% in AII-EC, respectively, compared with lacZ-EC. Our results indicate that arginase expression can modulate NO synthesis in bovine venular EC and that basal levels of arginase I and II are limiting for endothelial syntheses of polyamines, proline, and glutamate and may have important implications for wound healing, angiogenesis, and cardiovascular function.

Animals↗

PubMed2001

Functional expression of a GFP-tagged Kv1.5 alpha-subunit in mouse ventricle.

The experiments here were undertaken to determine the feasibility of increasing the cell surface expression of voltage-gated ion channels in cardiac cells in vivo and to explore the functional consequences of ectopic channel expression. Transgenic mice expressing a green fluorescent protein (GFP)-tagged, voltage-gated K+ (Kv) channel alpha-subunit, Kv1.5-GFP, driven by the cardiac-specific alpha-MHC promoter, were generated. In recent studies, Kv1.5 has been shown to encode the micromolar 4-aminopyridine (4-AP)-sensitive delayed rectifier K+ current (I(K,slow)) in mouse myocardium. Unexpectedly, Kv1.5-GFP expression is heterogeneous in the ventricles of these animals. Although no electrocardiographic abnormalities were evident, expression of Kv1.5-GFP results in marked decreases in action potential durations in GFP-positive ventricular myocytes. In voltage-clamp recordings from GFP-positive ventricular myocytes, peak outward K+ currents are significantly higher, and their waveforms are distinct from those recorded from wild-type cells. Pharmacological experiments revealed a selective increase in a micromolar 4-AP-sensitive current, similar to the 4-AP-sensitive component of I(K,slow) in wild-type cells. The inactivation rate of the "overexpressed" current, however, is significantly slower than the Kv1.5-encoded component of I(K,slow) in wild-type cells, suggesting differences in association with accessory subunits and/or posttranslational processing.

Action Potentials↗