Ming-Ko Chiang

Heterodimers of Placenta Growth Factor/Vascular Endothelial Growth Factor ENDOTHELIAL ACTIVITY, TUMOR CELL EXPRESSION, AND HIGH AFFINITY BINDING TO Flk-1/KDR

Here we show that the Escherichia coli expressed monomers of placenta growth factor (PLGF) and vascular endothelial growth factor (VEGF) can be re-folded in vitro to form PLGF/VEGF heterodimers. The purified recombinant PLGF/VEGF heterodimers and VEGF homodimers have potent mitogenic and chemotactic effects on endothelial cells. However, PLGF/VEGF heterodimers display 20-50-fold less mitogenic activity than VEGF homodimers. In contrast, PLGF homodimers have little or no effect in these in vitro assays. We also demonstrate the presence of natural PLGF/VEGF heterodimers in the conditioned media of various human tumor cell lines. While PLGF/VEGF heterodimers bind with high affinity to a soluble Flk-1/KDR receptor, PLGF homodimers fail to bind to this receptor. Cross-linking of I-ligands to human umbilical vein endothelial cells reveals that PLGF/VEGF heterodimers and VEGF homodimers, but not PLGF homodimers, form complexes with membrane receptors. VEGF homodimers and PLGF/VEGF heterodimers stimulate tyrosine phosphorylation of a 220-kDa protein, the expected size for the KDR receptor in human umbilical vein endothelial cells, whereas PLGF homodimers are unable to induce tyrosine phosphorylation of this protein. These data indicate that PLGF may modulate VEGF-induced angiogenesis by the formation of PLGF/VEGF heterodimers in cells producing both factors.

Single-Cell Transcript Analysis of Pancreas Development

DNA microarray analysis was combined with a modified single-cell PCR procedure to study gene expression profiles of single cells at different stages of pancreatic development. This method identifies distinct cell types at embryonic day 10.5, a stage when the pancreatic epithelium is morphologically uniform. Some cells express unexpected combinations of genes, and these expression patterns provide new insights into pancreas development. Following on these findings, we use PCR products from different cell types to identify novel pancreatic genes, some of which mark subtypes of developing pancreatic cells. By integrating these data with previous genetic and biochemical studies, we propose a pathway for pancreatic cell development. This form of single-cell transcriptional analysis can be applied to any developmental process or tissue to characterize distinct cell types.

Murine c-mpl : a member of the hematopoietic growth factor receptor superfamily that transduces a proliferative signal

The murine myeloproliferative leukemia virus has previously been shown to contain a fragment of the coding region of the c‐mpl gene, a member of the cytokine receptor superfamily. We have isolated cDNA and genomic clones encoding murine c‐mpl and localized the c‐mpl gene to mouse chromosome 4. Since some members of this superfamily function by transducing a proliferative signal and since the putative ligand of mpl is unknown, we have generated a chimeric receptor to test the functional potential of mpl. The chimera consists of the extracellular domain of the human interleukin‐4 receptor and the cytoplasmic domain of mpl. A mouse hematopoietic cell line transfected with this construct proliferates in response to human interleukin‐4, thereby demonstrating that the cytoplasmic domain of mpl contains all elements necessary to transmit a growth stimulatory signal. In addition, we show that 25–40% of mpl mRNA found in the spleen corresponds to a novel truncated and potentially soluble isoform of mpl and that both full‐length and truncated forms of mpl protein can be immunoprecipitated from lysates of transfected COS cells. Interestingly, however, although the truncated form of the receptor possesses a functional signal sequence and lacks a transmembrane domain, it is not detected in the culture media of transfected cells.

Ephrin-B3, a ligand for the receptor EphB3, expressed at the midline of the developing neural tube

The ephrins are a family of ligands that bind to Eph family receptor tyrosine kinases, and have been implicated in axon guidance and other patterning processes during vertebrate development. We describe here the identification and characterization of murine ephrin-B3. The cDNA encodes a 340 amino acid transmembrane molecule, most closely related to the two other known transmembrane ligands, ephrin-B1 and ephrin-B2. In addition to homology in their extracellular receptor binding domains, these transmembrane ligands share striking homology between their cytoplasmic domains, with 31 of the last 34 amino acids of ephrin-B3 being identical to ephrin-B2, suggesting functional interactions of the cytoplasmic tail. While most Eph family ligands are promiscuous in their interactions with Eph receptors, binding studies with the five receptors known to bind other transmembrane ligands only revealed a high affinity interaction of ephrin-B3 with EphB3, with a dissociation constant of approximately 1 nM. In situ hybridization of mouse embryos showed ephrin-B3 is expressed prominently at the dorsal and ventral midline of the neural tube, particularly in the floor plate, a structure with key functions in patterning the nervous system. The isolation of this ligand may help to elucidate the molecular basis of patterning activities at the neural tube midline.

Assessment of hypermucoviscosity as a virulence factor for experimental Klebsiella pneumoniae infections: comparative virulence analysis with hypermucoviscosity-negative strain

BackgroundKlebsiella pneumoniae displaying the hypermucoviscosity (HV) phenotype are considered more virulent than HV-negative strains. Nevertheless, the emergence of tissue-abscesses-associated HV-negative isolates motivated us to re-evaluate the role of HV-phenotype.ResultsInstead of genetically manipulating the HV-phenotype of K. pneumoniae, we selected two clinically isolated K1 strains, 1112 (HV-positive) and 1084 (HV-negative), to avoid possible interference from defects in the capsule. These well-encapsulated strains with similar genetic backgrounds were used for comparative analysis of bacterial virulence in a pneumoniae or a liver abscess model generated in either naïve or diabetic mice. In the pneumonia model, the HV-positive strain 1112 proliferated to higher loads in the lungs and blood of naïve mice, but was less prone to disseminate into the blood of diabetic mice compared to the HV-negative strain 1084. In the liver abscess model, 1084 was as potent as 1112 in inducing liver abscesses in both the naïve and diabetic mice. The 1084-infected diabetic mice were more inclined to develop bacteremia and had a higher mortality rate than those infected by 1112. A mini-Tn5 mutant of 1112, isolated due to its loss of HV-phenotype, was avirulent to mice.ConclusionThese results indicate that the HV-phenotype is required for the virulence of the clinically isolated HV-positive strain 1112. The superior ability of the HV-negative stain 1084 over 1112 to cause bacteremia in diabetic mice suggests that factors other than the HV phenotype were required for the systemic dissemination of K. pneumoniae in an immunocompromised setting.

Genetic Requirements for Klebsiella pneumoniae-Induced Liver Abscess in an Oral Infection Model

ABSTRACT Klebsiella pneumoniae is the predominant pathogen of primary liver abscess. However, our knowledge regarding the molecular basis of how K. pneumoniae causes primary infection in the liver is limited. We established an oral infection model that recapitulated the characteristics of liver abscess and conducted a genetic screen to identify the K. pneumoniae genes required for the development of liver abscess in mice. Twenty-eight mutants with attenuated growth in liver or spleen samples out of 2,880 signature-tagged mutants that produced the wild-type capsule were identified, and genetic loci which were disrupted in these mutants were identified to encode products with roles in cellular metabolism, adhesion, transportation, gene regulation, and unknown functions. We further evaluated the virulence attenuation of these mutants in independent infection experiments and categorized them accordingly into three classes. In particular, the class I and II mutant strains exhibited significantly reduced virulence in mice, and most of these strains were not detected in extraintestinal tissues at 48 h after oral inoculation. Interestingly, the mutated loci of about one-third of the class I and II mutant strains encode proteins with regulatory functions, and the transcript abundances of many other genes identified in the same screen were markedly changed in these regulatory mutant strains, suggesting a requirement for genetic regulatory networks for translocation of K. pneumoniae across the intestinal barrier. Furthermore, our finding that preimmunization with certain class I mutant strains protected mice against challenge with the wild-type strain implied a potential application for these strains in prophylaxis against K. pneumoniae infections.

Interactions between the Flk-1 receptor, vascular endothelial growth factor, and cell surface proteoglycan identified with a soluble receptor reagent.

Fetal liver kinase-1 (Flk-1) is a transmembrane tyrosine kinase that was identified in endothelial cells and populations of cells enriched in hematopoietic progenitors. To characterize the interaction of Flk-1 with potential ligands the receptor extracellular domain was genetically fused to an alkaline phosphatase (AP) tag. A soluble ligand for Flk-1 was identified in the supernatants of numerous mesenchymal cell lines by co-immunoprecipitation with the Flk1-AP fusion protein. This polypeptide was shown by N-terminal sequencing to be vascular endothelial growth factor (VEGF). Receptor-AP fusion proteins can thus be used to identify soluble ligands as well as transmembrane ligands, and this approach is therefore likely to be widely applicable to many types of orphan receptor. The Flk1-AP soluble receptor was also found to bind to cell surfaces, showing two apparent classes of binding site with different affinities. This interaction could be reconstructed by introducing a VEGF expression plasmid into cells. These results indicate that VEGF presented at the cell surface can bind to the Flk-1 receptor, and could mediate a direct cell-cell interaction. The Flk1-AP fusion protein was also found to bind heparin, implying that ligand binding by the Flk-1 receptor may involve a three way interaction between the Flk-1 receptor, VEGF, and heparin-like cell surface proteoglycans.

Correlation between Klebsiella pneumoniae carrying pLVPK-derived loci and abscess formation

Klebsiella pneumoniae-caused liver abscess (KLA) is an emerging infectious disease. However, factors other than K1-specific loci that contribute to the pathogenesis of this disease have not been identified. pLVPK is a 219,385-bp plasmid of K. pneumoniae CG43, an invasive K2 strain associated with KLA. We aimed in this study to evaluate the involvement of pLVPK in K. pneumoniae virulence and its clinical significance in abscess formation. A pLVPK-cured CG43 was isolated and its virulence was examined in a mouse model. The prevalence of pLVPK-derived loci terW, iutA, rmpA, silS, and repA was investigated in 207 clinical isolates by screening with specific primers. Loss of pLVPK abolished the ability of K. pneumoniae to disseminate into extraintestinal sites and, consequently, attenuated abscess formation in mice. Primary K. pneumoniae abscess isolates (n = 94) were more likely to be terW+-iutA+-rmpA+-silS+ than those related to non-abscess infections (n = 113) (62% vs. 27%; p < 0.0001). Logistic regression analysis indicated that the presence of the terW-rmpA-iutA-silS loci was a significant risk factor (odds ratio, 4.12; 95% confidence interval, 2.02–8.4; p < 0.0001) for abscess formation. pLVPK is a determinant for K. pneumoniae virulence and infection with strains carrying the pLVPK-derived terW-rmpA-iutA-silS loci may predispose patients to abscess formation.

Genotoxic Klebsiella pneumoniae in Taiwan.

Background Colibactin is a nonribosomal peptide-polyketide synthesized by multi-enzyme complexes encoded by the pks gene cluster. Colibactin-producing Escherichia coli have been demonstrated to induce host DNA damage and promote colorectal cancer (CRC) development. In Taiwan, the occurrence of pyogenic liver abscess (PLA) has been suggested to correlate with an increasing risk of CRC, and Klebsiella pneumoniae is the predominant PLA pathogen in Taiwan Methodology/Principal Findings At the asn tRNA loci of the newly sequenced K. pneumoniae 1084 genome, we identified a 208-kb genomic island, KPHPI208, of which a module identical to the E. coli pks colibactin gene cluster was recognized. KPHPI208 consists of eight modules, including the colibactin module and the modules predicted to be involved in integration, conjugation, yersiniabactin production, microcin production, and unknown functions. Transient infection of BALB/c normal liver cells with K. pneumoniae 1084 increased the phosphorylation of histone H2AX, indicating the induction of host DNA damage. Colibactin was required for the genotoxicity of K. pneumoniae 1084, as it was diminished by deletion of clbA gene and restored to the wild type level by trans-complementation with a clbA coding plasmid. Besides, BALB/c mice infected with K. pneumoniae 1084 exhibited enhanced DNA damage in the liver parenchymal cells when compared to the isogenic clbA deletion mutant. By PCR detection, the prevalence of pks-positive K. pneumoniae in Taiwan is 25.6%, which is higher than that reported in Europe (3.5%), and is significantly correlated with K1 type, which predominantly accounted for PLA in Taiwan. Conclusions Our knowledge regarding how bacteria contribute to carcinogenesis has just begun. The identification of genotoxic K. pneumoniae and its genetic components will facilitate future studies to elucidate the molecular basis underlying the link between K. pneumoniae, PLA, and CRC.

The high incidence of JC virus infection in urothelial carcinoma tissue in Taiwan.

Human polyomaviruses, JC virus (JCV) and BK virus (BKV), usually remain latent in kidney and urothelial tissue after primary infection. Infection with human polyomavirus has still not been correlated conclusively with malignancy of kidney and urothelial tissue. The present study investigated further the possible relationship between JCV/BKV infection and urothelial carcinoma. Tissue samples were examined from 33 urothelial carcinomas and 5 renal cell carcinomas for JCV/BKV infection, using nested PCR with primers common to both JCV and BKV. The viral genotypes were further verified by endonuclease digestion and DNA sequencing following the PCR. In addition, immunohistochemistry and Western blotting were also performed to detect viral large tumor protein (LT) and the late capsid protein (VP1) in the tissue samples. The results from nested PCR showed that 90.1% (30/33) of the urothelial carcinomas samples and all of the renal cell carcinomas samples (5/5) were JCV DNA positive. Both archetypal and re‐arranged JCV genotypes were detected. On the other hand, BKV DNA was detected in only one (3%) of the urothelial carcinoma tissue samples. The immunohistochemical results showed that 30% (10/33) of urothelial carcinoma tissues was stained positive for large tumor antigen (LT). However, the structural protein VP1 was not detectable in any of the tissue samples examined. The present study demonstrated that JCV is highly prevalent in urothelial carcinoma tissue as is the expression of large tumor antigen. Therefore, the findings support the hypothesis that JCV infection is associated with urothelial carcinoma. J. Med. Virol. 83:2191–2199, 2011.