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Background: Glucocorticoid-remediable aldosteronism (GRA) is a form of heritable hypertension caused by a chimeric fusion resulting from unequal crossing over between 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2), which are two genes with similar sequences. Different crossover patterns of the CYP11B1 and CYP11B2 chimeric genes may be associated with a variety of clinical presentations. It is therefore necessary to develop an efficient approach for identifying the differences between the hybrid genes of a patient with GRA., Results: We developed a long-read analysis pipeline named GRAde (GRA deciphering), which utilizes the nonidentical bases in the CYP11B1 and CYP11B2 genomic sequences to identify and visualize the chimeric form. We sequenced the polymerase chain reaction (PCR) products of the CYP11B1/CYP11B2 chimeric gene from 36 patients with GRA using the Nanopore MinION device and analyzed the sequences using GRAde. Crossover events were identified for 30 out of the 36 samples. The crossover sites appeared in the region exhibiting high sequence similarity between CYP11B1 and CYP11B2, and 53.3% of the cases were identified as having a gene conversion in intron 2. More importantly, there were six cases for whom the PCR products indicated a chimeric gene, but the GRAde results revealed no crossover pattern. The crossover regions were further verified by Sanger sequencing analysis., Conclusions: PCR-based target enrichment followed by long-read sequencing is an efficient and precise approach to dissecting complex genomic regions, such as those involved in GRA mutations, which could be directly applied to clinical diagnosis. The scripts of GRAde are available at https://github.com/hsu-binfo/GRAde ., (© 2022. The Author(s).)

Aims: Angiotensin-converting enzyme 2 (ACE2) is a key negative regulator of the renin-angiotensin system and also a major receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we reveal a role for NF-κB in human lung cell expression of ACE2, and we further explore the potential utility of repurposing NF-κB inhibitors to downregulate ACE2., Main Methods: Expression of ACE2 was assessed by Western blotting and RT-qPCR in multiple human lung cell lines with or without NF-κB inhibitor treatment. Surface ACE2 expression and intracellular reactive oxygen species (ROS) levels were measured with flow cytometry. p50 was knocked down with siRNA. Cytotoxicity was monitored by PARP cleavage and MTS assay., Key Findings: Pyrrolidine dithiocarbamate (PDTC), an NF-κB inhibitor, suppressed endogenous ACE2 mRNA and protein expression in H322M and Calu-3 cells. The ROS level in H322M cells was increased after PDTC treatment, and pretreatment with N-acetyl-cysteine (NAC) reversed PDTC-induced ACE2 suppression. Meanwhile, treatment with hydrogen peroxide augmented ACE2 suppression in H322M cells with p50 knockdown. Two repurposed NF-κB inhibitors, the anthelmintic drug triclabendazole and the antiprotozoal drug emetine, also reduced ACE2 mRNA and protein levels. Moreover, zinc supplementation augmented the suppressive effects of triclabendazole and emetine on ACE2 expression in H322M and Calu-3 cells., Significance: These results suggest that ACE2 expression is modulated by ROS and NF-κB signaling in human lung cells, and the combination of zinc with triclabendazole or emetine shows promise for clinical treatment of ACE2-related disease., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

In articular cartilage-repair, grafts usually fuse unsatisfactorily with surrounding host cartilage. Enzymatic dissociation of cartilaginous matrix to free chondrocytes may benefit fusion. We tested such a hypothesis with human cartilage in vitro , and with porcine cartilage in vivo . Human articular cartilage was collected from knee surgeries, cut into disc-and-ring sets, and randomly distributed into three groups: disc-and-ring sets in Group 1 were left untreated; in Group 2 only discs, and in Group 3 both discs and rings were treated with enzyme. Each disc-and-ring reassembly was cultured in a perfusion system for 14 days; expression of cartilage marker proteins and genes was evaluated by immunohistochemistry and PCR. Porcine articular cartilage from knees was similarly fashioned into disc-and-ring combinations. Specimens were randomly distributed into a control group without further treatment, and an experimental group with both disc and ring treated with enzyme. Each disc-and-ring reassembly was transplanted into subcutaneous space of a nude mouse for 30 days, and retrieved to examine disc-ring interface. In in vitro study with human cartilage, a visible gap remained at disc-ring interfaces in Group 1, yet became indiscernible in Group 2 and 3. Marker genes, including type II collagen, aggrecan and Sox 9, were well expressed by chondrocytes in all specimens, indicating that chondrocytes' phenotype retained regardless of enzymatic treatment. Similar results were found in in vivo study with porcine cartilage. Enzymatic dissociation of cartilaginous matrix promotes fusion of adjacent cartilage. The clinical relevance may be a novel method to facilitate integration of repaired cartilage in joints., Competing Interests: The authors have declared that they have no conflict of interest., (© 2020 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.)

D-amino acid oxidase (DAO) has been reported to be associated with schizophrenia. This study aimed to search for genetic variants associated with this gene. The genomic regions of all exons, highly conserved regions of introns, and promoters of this gene were sequenced. Potentially meaningful single-nucleotide polymorphisms (SNPs) obtained from direct sequencing were selected for genotyping in 600 controls and 912 patients with schizophrenia and in a replicated sample consisting of 388 patients with schizophrenia. Genetic associations were examined using single-locus and haplotype association analyses. In single-locus analyses, the frequency of the C allele of a novel SNP rs55944529 located at intron 8 was found to be significantly higher in the original large patient sample (p = 0.016). This allele was associated with a higher level of DAO mRNA expression in the Epstein-Barr virus-transformed lymphocytes. The haplotype distribution of a haplotype block composed of rs11114083-rs2070586-rs2070587-rs55944529 across intron 1 and intron 8 was significantly different between the patients and controls and the haplotype frequencies of AAGC were significantly higher in patients, in both the original (corrected p < 0.0001) and replicated samples (corrected p = 0.0003). The CGTC haplotype was specifically associated with the subgroup with deficits in sustained attention and executive function and the AAGC haplotype was associated with the subgroup without such deficits. The DAO gene was a susceptibility gene for schizophrenia and the genomic region between intron 1 and intron 8 may harbor functional genetic variants, which may influence the mRNA expression of DAO and neurocognitive functions in schizophrenia.

Background: Mycobacterium tuberculosis is a vicious microbe co-existing with the infected host. This pathogen exploited opportunities to spread during periods of urbanization and social upheaval, and got retreated with improved hygiene., Objectives: This investigation was designed to clone and characterize M. tuberculosis mutT gene, a homologue of a DNA repair protein in Escherichia coli. The aim was to depict the possible role of this homologue in the virulent microbe., Materials and Methods: A DNA fragment of the mutT gene was amplified with PCR from the genomic DNA of strain H37Rv M. tuberculosis. The expression vector was transformed into E. coli strains BL21 (DE3) and MK602 (DE3) (mutT-). The protein activity assay was performed by biochemical methods., Results: M. tuberculosis MutT shares 23% identity with the E. coli MutT protein. The mutT gene DNA fragment was subcloned into the expression vector pET28a(+) and the recombinant plasmid was overexpressed in E. coli. Purified and refolded M. tuberculosis MutT possesses a dGTPase activity, which is one of the most well-known preference nucleotidase activities of MutT in E. coli. This study also showed that the dGTPase activity of M. tuberculosis MutT was enhanced by magnesium and inhibited by Ni(2+) or EDTA. Endogenous MutT protein in M. tuberculosis lysate displayed a smear pattern in the Western blot, suggesting instability of this protein in the bacteria similar to the important proteins, such as P53 protein, tightly regulated by protein degradation., Conclusions: The cloned M. tuberculosis mutT gene and MutT protein were characterized. M. tuberculosis MutT has a dGTPase activity, which is one of the most well-known preference nucleotidase activities of MutT in E. coli. These findings provide further understanding about the vicious bacterium.

Majority of the heroin-dependent patients smoke cigarettes. Although it has been reported that the OPRM1 genetic polymorphism is associated with the brain mu-opioid receptor binding potential in cigarette smokers, there is no direct evidence showing the impact of plasma cotinine, a nicotine metabolite, on treatment responses to methadone maintenance treatment (MMT). In this study, we aimed to test the hypothesis that the genetic polymorphisms in the OPRM1 are associated with the methadone treatment responses and the severity of cigarette smoking directly measured by the plasma concentration of cotinine in a Taiwanese MMT cohort. Fifteen OPRM1 single-nucleotide polymorphisms (SNPs) were selected and genotyped on DNA samples of 366 MMT patients. Plasma concentrations of cotinine were measured by cotinine enzyme-linked immunosorbent assay. The plasma cotinine concentration had positive correlation with concentrations of methadone (P = 0.042) and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine (P = 0.037). Methadone treatment non-responders, defined by a positive urine morphine test, had a higher plasma concentration of cotinine (P = 0.005), but a lower plasma concentration-to-dose ratio of both R- and S-methadone (P = 0.001 and 0.012, respectively) than the responders. OPRM1 genetic variants, rs1074287, rs6912029, rs1799971, rs12209447, rs510769, rs3798676, rs553202, rs7748401, rs495491, rs10457090, rs589046, rs3778152 and rs563649, were significantly associated with the plasma concentration of cotinine when using recessive model for genotypes (general linear model (GLM), P<0.038; false discovery rate (FDR)<0.035) and additive model for allele types (GLM, P<0.03; FDR<0.049) in association analyses. The G allele carriers of SNP rs1799971 (A118G) on exon 1 of OPRM1 gene had a lower plasma cotinine concentration than the A allele carriers (GLM, P = 0.029). OPRM1 genetic polymorphisms are associated with the plasma concentration of cotinine in a Taiwanese MMT cohort. Carriers with the major allele of SNP rs1799971 had a higher plasma cotinine concentration.

Implantation of autologous chondrogenic cells has become the mainstay strategy for repairing articular cartilage defects. Because the availability of autologous chondrocytes is extremely limited, many recent studies have used artificially induced mesenchymal stem cells (iMSCs) as substitutes for chondrocytes. In this study, we analyzed the differences between the iMSCs and chondrocytes, including their molecular biological and mechanical properties. Human bone marrow-derived MSCs were collected and induced to exhibit the chondrogenic phenotype by culturing the pelleted MSCs in a chemically defined culture medium supplemented with transforming growth factor-beta 1. The molecular biological properties of iMSCs and culture-expanded chondrocytes, including their mRNA profiles and surface proteomics, were analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry, respectively. The biomechanical properties of iMSCs and native chondrocytes, including their surface topology, adhesion force, and membrane stiffness, were analyzed using atomic force microscopy (AFM). Both iMSCs and chondrocytes presented type II collagen and glycosaminoglycan, whereas only chondrocytes presented type X collagen. Flow cytometric assays showed that the expression of type II collagen and integrin-1 was higher in the chondrocytes than in the iMSCs. AFM revealed that the MSCs, iMSCs, and chondrocytes greatly differed in their shape. The MSCs were spindle shaped and easily distinguishable from the spherical chondrocytes. The iMSCs appeared round and resembled the spherical chondrocytes; however, the iMSCs were flatter with a central hump of condensed mass and a surrounding thin and broad pleat. The mean adhesion force and mean surface stiffness were significantly lower for the iMSCs (4.54 nN and 0.109 N/m, respectively) than for the chondrocytes (6.86 nN and 0.134 N/m, respectively). To conclude, although the iMSCs exhibited the chondrogenic phenotype, they differed from the chondrocytes in their molecular biological and mechanical properties.

To explore the effects of deregulated expression of the EBNA1 binding protein 2 (EBP2) on cell growth, we generated human HEK293 stable clones constitutively expressing an EBP2-EGFP fusion protein. We found both RNA and protein levels of cyclin E1, a dominant oncoprotein, were elevated in the EBP2- EGFP stable clones. These findings were confirmed by flow cytometry bivariate analysis of cyclin expression versus DNA content. Moreover, the increase in p21 expression and the specific phosphorylation at Ser1981 of ATM and Ser15 of p53 were also observed in these stable clones, and these observations may explain the failure to observe an increase in Cdk2 kinase activity. In addition, after one year of passage culture, the EBP2-EGFP stable clones tended to lose 4 to 5 chromosomes per cell when compared to that of control cells. All of these findings provide a possible link between deregulated expression of EBP2 and tumor development.

In vitro activation of macrophages by LPS induces rapid release of vascular endothelial growth factor (VEGF) and soluble fms-like tyrosine kinase-1 receptor (sFlt-1), which are thought to be the effectors to cause sepsis. However, the signal pathway that controls the VEGF and sFlt-1 expressions in LPS-activated macrophages remains unclear. In this study, we demonstrated that phosphorylation of protein kinase C (PKC)delta played a key role in the VEGF and sFlt-1 signaling pathway of LPS-activated macrophages. PKC is a family of serine-threonine kinases, which are classified into three major groups based on homology and cofactor requirements: conventional PKCs, novel PKCs, and atypical PKCs. In the murine RAW264.7 cells, as well as in primary human monocytes/macrophages, pretreatment with a general PKC inhibitor GF109203X or with a novel PKCdelta inhibitor rottlerin or overexpression of a kinase-inactive form of PKCdelta (K376R) eliminated LPS-induced sFlt-1 expression and augmented LPS-induced VEGF expression at the protein and the transcription levels. In contrast, Gö6976, an inhibitor for the conventional PKCs, or myristoylated PKCzeta pseudosubstrate peptide, an inhibitor for the atypical PKCs, failed to exert the same effects. These data suggest that PKCdelta signaling is involved in LPS-induced sFlt-1 expression and serves as a negative mediator in LPS-induced VEGF expression in macrophages. A novel strategy controlling the LPS-induced PKC pathways, especially the PKCdelta isoform, may be developed based on this study.

Objective: The mechanical properties of chondrocytes influence maintenance of the articular cartilage extracellular matrix. To differentiate the mechanical properties of chondrocytes between a young, normal modulus and an old, osteoarthritic (OA) modulus, we used an atomic force microscope (AFM) to probe the surface ultrastructure and to measure their adhesion force and stiffness., Methods: We directly visualized a single chondrocyte cell by using AFM and quantitatively measured the dimensions of the cells., Results: Profiles displayed heights of 1026+/-203 and 1668+/-352 nm for old and young cells, respectively. Contour maps revealed differences in the sizes and structures of the two groups. Mean calculated adhesion forces differed between normal and OA chondrocytes (7.06+/-3.35 and 2.97+/-1.82 nN, respectively), as did calculated stiffness values (0.0960+/-0.009 and 0.0347+/-0.005 N/m, respectively)., Conclusion: These findings suggested that the mechanical properties of normal chondrocytes substantially differed from those of OA chondrocytes. We believe this study represents the first direct characterization of the surface ultrastructure and mechanical measurements of human chondrocytes between normal and OA stages. This new approach could be a useful technique for investigating age-related changes in the properties of human chondrocytes.

Objective: To assess how magnetic fields (MFs), with or without concurrent radio frequency (RF), influence chondrocytes and knee joint repair, we applied an MF strength via magnetic resonance imaging (MRI) slightly greater than the frequently used dosage in the clinics and examined the effects of these treatments in vitro on human chondrocytes and in vivo in pigs., Methods: Human chondrocytes were directly exposed to a 3-tesla (T) magnetic field (MF group) or a 3-T static magnetic field plus 125.3 MHz radio frequency (MF+RF group), and cell proliferation, apoptosis, cytosolic Ca2+ ([Ca2+]i) fluxes and expression of the apoptosis-related proteins of the treated cells were examined to assess the effects of the treatments. In the pig study, we examined the effects of the treatments on the recovery of surgically damaged pig knees., Results: A 3-T static MF and RF suppressed cell growth and induced apoptosis through p53, p21, p27 and Bax protein expression. In the pig model, we found that MRI surveillance had a deleterious effect on the recovery of the damaged knee cartilage., Conclusion: Magnetic strength, with or without concurrent RF, suppressed chondrocyte growth in vitro and affected recovery of damaged knee cartilage in vivo in the pig model. These results may be specific to the parameters used in this study and may not apply to other situations, field strengths, forms of cartilage injury, or animal species.

Background and Purpose: This study analyzed rpoB gene mutation and its correlation with demographic and clinical data, and the drug resistance profile in 41 consecutive patients with rifampin (RIF)-resistant Mycobacterium tuberculosis isolated at National Taiwan University Hospital from 2000 to 2002., Methods: The 411-bp fragment of the rpoB gene from 94 M. tuberculosis isolates (including 41 RIF-resistant and 53 RIF-susceptible isolates) was amplified and sequenced., Results: Of the 41 RIF-resistant isolates, 87.8% (36/41) showed mutations in rpoB. The following mutations were identified: Ser531 (68.3%), His526 (9.8%), Ser522 (4.9%) and Gln513 (4.9%). No silent substitutions were observed. No mutation within the entire 411-bp fragment was found in 12.2% (5/41) of the RIF-resistant isolates and 100% (53/53) of the RIF-susceptible isolates. Patients whose RIF-resistant isolates did not have rpoB mutation had higher frequencies of the following characteristics: elderly, no previous history of tuberculosis, human immunodeficiency virus-negative, no extrapulmonary tuberculosis involvement and favorable prognosis. Drug resistance patterns in RIF-resistant M. tuberculosis strains were significantly correlated with isoniazid resistance, i.e., multidrug-resistant strains (90.2%)., Conclusions: RIF-resistant M. tuberculosis isolates with rpoB mutation were clustered in the 69-bp core region in this study. Rapid detection of RIF resistance could be achieved by testing for rpoB mutation in Taiwan.

Background: Overexpression of vascular endothelial growth factor (VEGF) correlates with vascularity, metastasis, and proliferation in colorectal cancer but the role of its homologue, placenta growth factor (PlGF), is unknown. The aim of this study was to evaluate expression and clinical implications of PlGF in colorectal cancer., Methods: We investigated 74 tumour/non-tumour pairs of colorectal cryosections. Clinical staging was based on the UICC-TNM classification. Expression levels of mRNA for PlGF and VEGF were analysed with quantitative real time reverse transcription-polymerase chain reaction. Proteins were analysed by immunohistochemical staining and enzyme linked immunoabsorbant assay. Analysis of the differences in PlGF and VEGF levels between tumour and non-tumour tissues in the same patient were performed by paired t test; differences between localised and advanced disease patients by the Mann-Whitney, chi(2), and Fisher's exact tests and survival curves by the Kaplan-Meier method., Results: Expression levels for both growth factors were significantly higher in tumour than in non-tumour tissues (p

Background and Purpose: The mutation rate of hMSH2 and hMLH1 (20%) in Taiwanese hereditary nonpolyposis colorectal cancer (HNPCC) is lower than that reported in other countries. This study aimed to examine the microsatellite instability (MSI) status and gene expression pattern of Taiwanese HNPCC in an effort to establish correlation between these data and results of prior genetic screening., Methods: The "Bethesda markers" were used for the MSI analysis. Tumor and neighboring tissues were obtained from 10-mm sections of neutral formalin-fixed, paraffin-embedded, hematoxylin and eosin-stained specimens with a PixCell laser-capture microdissector. Four-mm sections were used for the immunohistochemical analysis by avidin-biotin complex method and final coloring with diaminobenzidine. A pathologist performed scoring of the pathological specimens twice, using a double-blinded methodology. Thirteen tissue blocks from 8 HNPCC families (Amsterdam's criteria) were included in this study., Results: Although the majority of the HNPCC tissues displayed a MSI-high phenotype (10/13, 76.9%), lack of expression of MSH2 and MLH1 was infrequent. Furthermore, only 1 germ-line mutation was detected in the peripheral blood leukocytes of the patients whose tumors had lost protein expression of MSH2 or MLH1., Conclusions: Our results indicate that the pathogenesis of Taiwanese HNPCC is different from that in other countries. Rather than immunohistochemical analysis, MSI status, and genetic screening, clinical history remains a reliable method for diagnosis of HNPCC in Taiwanese the population.

Colorectal cancer has become the third leading cause of death from cancer in Taiwan. Familial adenomatous polyposis (FAP) is an autosomal dominant inherited disease characterized by the presence of multiple adenomatous polyps in the colon and rectum. The gene responsible for FAP (APC) was cloned in 1991. Extensive analyses of the mutation spectra in FAP kindreds have been performed in different countries, but the results have been highly variable (30-80%). In this study, we used denaturing high-performance liquid chromatography (DHPLC) followed by automatic sequencing in an effort to establish the mutation spectrum of APC from DNA of peripheral blood cells. Among the 6 FAP probands analyzed, mutations were detected in 3 (50%), 2 of which were novel. The first novel mutation was at codon 2166, with a C to T transition, resulting in a stop codon. The second novel mutation was at codon 1971, with a C to G transversion, resulting in an amino acid change from serine to cysteine. The third mutation involved an A insertion in the sequence of -AAAAAA- at codons 1554-1556, which created a downstream stop codon (codon 1558). This study is the first to report mutation analysis in Taiwanese FAP probands., (Copyright 2004 National Science Council, ROC and S. Karger AG, Basel)

Hereditary non-polyposis colorectal cancer (HNPCC), the most common type of hereditary colorectal cancer, is thought to be a simple Mendelian disease involving DNA mismatch repair genes. The majority of mutations associated with HNPCC occur in the hMSH2 and hMLH1 genes. The reported incidence of mismatch repair gene mutations in HNPCC kindreds varies considerably (from 22 to 86%), and most mutations are unique. This study aimed to determine the genetic basis of Taiwanese HNPCC kindreds, focusing on the two major genes involved in this disease. A total of 15 Taiwanese HNPCC kindreds meeting the Amsterdam criteria, including 72 affected individuals among a total of 266 individuals, were analyzed using both RNA- and DNA-based methods. The mutation rate of hMSH2 and hMLH1 in these 15 kindreds was 0% and 20%, respectively, which is lower than that reported in other countries. Two novel mutations were discovered in hMLH1: one was an allelic loss of a 5.2-kb genomic fragment causing exon 16 deletion; and the other was a two-nucleotide deletion that resulted in a frameshift mutation of exon 3. We also identified one hMLH1 exon 4 mutation (a C to T transition in codon 117), which had been reported previously in western countries. This is the first genetic study of HNPCC from Taiwan.

Setting: National Taiwan University Hospital, Taipei, Taiwan., Objective: To study the variations in the NRAMP1 gene using five genotypes (274C/T, 577-18G/A, A318V, D543N and 3' untranslated region [UTR]), and the susceptibility of tuberculosis and HIV infection in Taiwanese., Design: The study sample included 49 patients with tuberculosis, 48 healthy control subjects and 60 HIV-infected patients. The polymerase chain reaction (PCR) products amplified from their genomic DNA were subjected to restriction enzyme digestion and were analysed using agarose gel electrophoresis., Results: A318V was not polymorphic in the studied population. Only D543N and 3'UTR were more heterozygous. In 274 C/T and 577-18G/A, the allele frequencies showed the predominant type to be the homozygous patterns C/C (94%) and G/G (94%), respectively. There were no statistically significant differences between the tuberculosis patients and the healthy control subjects. Despite the high susceptibility to Mycobacterium tuberculosis in HIV-infected patients, genotypic frequencies in the HIV-positive patients were not significantly different between tuberculous (n = 29) and non-tuberculous patients (n = 31). In comparison with previous studies, there were significant differences between different ethnic groups in allele frequencies for 274C/T, D543N and 3'UTR., Conclusion: The allele and genotype of NRAMPI polymorphism among Taiwanese differed from those of Caucasians, Africans and Hispanics. No allelic associations were identified between the NRAMP1 alleles and tuberculosis susceptibility.

The cDNA of the human mutY homologue (hMYH) was cloned from the total RNA of the tumor cell line SU-DHL-1 by reverse transcription-polymerase chain reaction (RT-PCR). Expression of hMYH in a plasmid can partially revert the mutator phenotype of the Escherichia coli mutY mutant MK609(DE3). The majority of the recombinant hMYH protein in E. coli was precipitated in the inclusion bodies. A minor fraction of the soluble recombinant protein was concentrated as the source of the protein in the activity assay. Recombinant hMYH displayed both glycosylase and AP lyase activity. Three independent rabbit antisera against an N-terminal peptide, HY90, a recombinant C-terminal fragment, and the full-length hMYH recombinant protein were prepared and affinity-purified, and these antisera recognized the 59 kDa endogenous hMYH protein in HeLa cells. Immunofluorescent staining experiments with these three antisera showed a consistent nuclear distribution of hMYH, excluding the nucleoli. This nuclear staining pattern was abolished if the antisera were incubated with specific peptide/protein competitors, whereas the staining pattern was unaffected if the antisera were incubated with nonspecific peptide competitors. Consistent with the immunofluorescent staining results, a flag-tagged transfected hMYH also showed a nuclear staining pattern excluding the nucleoli. These results suggest that hMYH is indeed a functional homologue of E. coli MutY and is localized in the nuclei of mammalian cells., (Copyright 2000 Wiley-Liss, Inc.)

Periplasmic copper, zinc superoxide dismutases (Cu,ZnSOD) of several Gram-negative pathogens have been shown to play an important role in protection against exogenous superoxide radicals and in determining virulence of the pathogens. Here we report the cloning and characterization of the sodC gene, encoding Cu,ZnSOD, from the Gram-positive Mycobacterium tuberculosis. The predicted protein sequence contains 240 amino acids with a putative signal peptide at the N-terminus and shows approximately 25% identity to other bacterial sodC. Recombinant proteins of a full-length sodC and a truncated form lacking the putative signal peptide were overexpressed in Escherichia coli and affinity purified. Renatured recombinant M. tuberculosis sodC protein possessed characteristics of a Cu,ZnSOD. Immunoblotting with an antiserum against the recombinant M. tuberculosis Cu,ZnSOD allowed detection of a single polypeptide in the lysate of M. tuberculosis. This polypeptide has a similar size as the recombinant protein without the putative signal peptide indicating that the endogenous Cu,ZnSOD in M. tuberculosis might be processed and secreted. Furthermore, immunogold electron microscopic image showed that Cu,ZnSOD is located in the periphery of M. tuberculosis. The enzymatic activity and subcellular localization of this novel Cu,ZnSOD suggest that it may play a role in determining virulence of M. tuberculosis.

We have selected a series of cell lines from the parental Syrian hamster smooth muscle cell line DDT1-MF2that are resistant to thapsigargin (TG), a specific inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+transport ATPases (SERCAs). Cells were selected for resistance to TG in the presence or absence of cyclosporin (CSA), which is a competitive inhibitor of the multidrug transporter p-glycoprotein (pgp). Since TG is a known substrate for pgp, selection for TG resistance was carried out in the presence of CSA in an attempt to minimize the contribution of pgp, and to identify the potential range of adaptive responses of the SERCA pump itself, during the development of the TG-resistant phenotype. Irrespective of whether the selection is carried out in the presence or absence of CSA, pgp is overexpressed in the TG-resistant DDT1-MF2cells. SERCA protein is also overproduced in the TG-resistant cell lines, which occurs through one of several mechanisms. Included among these, is amplification of the SERCA gene and enhanced transcription of the gene. Enhanced transcription is observed only upon long-term selection and occurs through the SERCA gene proximal promoter elements. Although SERCA transcription in wild-type cells is dependent upon the -284 to -72 bp region of the SERCA promoter, the TG-resistant cells utilize both the -284 to -72 bp and the -72 to +80 bp promoter regions for enhanced SERCA transcription. That is, additional elements within the -72 to +80 bp region are recruited in the TG-resistant cells to allow for increased SERCA expression. A post-transcriptional step may also be recruited by the TG-resistant cells in their overall strategy to produce increased amounts of the SERCA protein. These studies demonstrate that the DDT1-MF2cells can utilize different mechanisms which lead to increased levels of SERCA protein as the cells adapt to inhibition of the ATPase by TG.

Potential DNA contacts involved in the specific interaction between the Escherichia coli MutY protein and a 40-mer oligonucleotide containing an A/G mismatch have been examined by alkylation interference techniques. Ethylation interference patterns suggest that more than five phosphates are involved in electrostatic interactions between MutY and DNA. Interestingly, MutY has more contacts on the G-strand than on the A-strand. Methylation at both the N-7 position of the mismatched G and the N-3 position of the mispaired A interfere with MutY binding. In addition to these mismatched bases, MutY also contacts purines on both sides of the mismatch. Binding and endonuclease activities of MutY were assayed with 20-mer oligonucleotides containing A/G, A/C, A/7,8-dihydro-8-oxo-guanine (A/GO), A/inosine (A/I), A/2-aminopurine (A/2AP), nebularine/G (N/G), inosine/G (I/G), 2AP/G, and 7-deaza-adenosine/G (Z/G) mispairs. The C-8 keto group of GO in A/GO contributes to a much tighter binding but weaker endonuclease activity than is seen with A/G. Because A/I is not specifically well recognized by MutY, the 2-amino group of G in A/G is essential for recognition. The C-6 keto group present in A/G but absent in A/2AP is also important for recognition. The 6-amino group of adenine appears not to be required for either binding or endonuclease activity because N/G is as good a substrate as A/G. The 2AP/G mispair is bound and cleaved weaker than is the A/G mispair. Binding and endonuclease activities are abolished when the N-7 group of A is replaced by C-7 as in the Z/G mispair. When a C-6 keto group is present as in the I/G pair, its binding by MutY is as good as for A/G, but no endonuclease activity is observed. Taken together, our data suggest that DNA sequences proximal to and specific functional groups of mismatched bases are necessary for recognition and catalysis by MutY protein.

Thapsigargin (TG), a specific inhibitor of intracellular Ca2+ transport ATPases (SERCA), inhibits cell proliferation when added to culture media in the nanomolar concentration range. However, long term exposure to gradually increasing concentrations of TG induces resistance to TG inhibition in both the parental Chinese hamster lung fibroblast DC-3F and a subline derived from it via transfection and stable expression of a full-length cDNA encoding avian SERCA1 ATPase (DC-3F/Ca cells). TG resistance develops in parallel with selection of cells expressing higher levels of the endogenous SERCA2 as well as of the exogenous transfected SERCA1 ATPase, whose Ca2+ transport function can be studied in situ by imaging techniques and following isolation in microsomal fractions. Microsomes isolated from resistant cells contain two functionally distinct populations of ATPases: a population that is inhibited by stoichiometric titration with TG, and a population displaying resistance to inhibition even when TG exceeds the enzyme stoichiometry. It is apparent that resistance to TG develops in parallel with (a) selection of cells expressing high levels of SERCA ATPases, and (b) selection of an ATPase that is resistant to TG.

Repair of heteroduplex DNA containing an A/G mismatch in a mutL background requires the Escherichia coli mutY gene function. The mutY-dependent in vitro repair of A/G mismatches is accompanied by repair DNA synthesis on the DNA strand bearing mispaired adenines. The size of the mutY-dependent repair tract was measured by the specific incorporation of alpha-[32P]dCTP into different restriction fragments of the repaired DNA. The repair tract is shorter than 12 nucleotides and longer than 5 nucleotides and is localized to the 3' side of the mismatched adenine. This repair synthesis is carried out by DNA polymerase I.

In Escherichia coli the mutY (or micA)-dependent DNA mismatch repair pathway can convert A degrees G and A degrees C mismatches to C.G and G.C base pairs, respectively, through a short repair-tract mechanism. The MutY protein has been purified to near homogeneity from an E. coli overproducer strain. Purified MutY has been shown to contain both N-glycosylase and 3' apurinic/apyrimidinic (AP) endonuclease activities. The N-glycosylase removes the mispaired adenines of A degrees G and A degrees C mismatches, and the AP endonuclease acts on the first phosphodiester bond 3' to the AP sites. The N-glycosylase and the nicking (combined N-glycosylase and AP endonuclease) activities copurified through multiple chromatographic steps without a change in relative specific activities. Furthermore, both N-glycosylase and AP endonuclease activities can be recovered by renaturation of a single polypeptide band from an SDS/polyacrylamide gel. Renaturation required the presence of iron and sulfide. These findings suggest that the MutY protein, like endonuclease III, is an iron-sulfur protein. DNA fragments with A degrees C mismatches were 20-fold less active than DNA with A degrees G mispairs as a substrate for purified MutY.

The Escherichia coli methylation-independent repair pathway specific for A/G mismatches has been shown to require the gene product of micA. Extracts prepared from micA mutants do not form an A/G mismatch-specific DNA-protein complex and do not contain an A/G mismatch-specific nicking activity. Moreover, a partially purified protein fraction containing both A/G mismatch-specific nicking and binding activities restores repair activity in micA mutant extracts. The DNA sequence of a 2.3-kb fragment containing the micA gene has been determined. There are two open reading frames (ORF) in this DNA fragment: one ORF encodes a 25.7-kDa protein whose function is still unknown, the other ORF codes for a protein with an Mr of 39,147, but this ORF can be transcribed and the mRNA can be translated to yield a protein with an apparent Mr of 36 kDa on a sodium dodecyl sulfate-polyacrylamide gel. Deletion analysis showed that this 39.1-kDa ORF is the micA gene as judged by the capacity of the encoded protein to restore the A/G mismatch-specific nicking activity of micA mutant extracts. Furthermore, our results suggest that micA is the same gene as the closely mapped mutY, which encodes the A/G mismatch-specific glycosylase.