Prevalence of and risk factors for depressive symptoms in non-tuberculous mycobacterial pulmonary disease.

BACKGROUND: The presence of depressive symptoms in patients with non-tuberculous mycobacterial pulmonary disease (NTM-PD) is an important research topic; however, the prevalence of depressive symptoms and the factors that influence their development are unclear.OBJECTIVE: To analyse the association between CES-D (Center for Epidemiological Studies Depression Scale) scores and clinical parameters such as age, disease duration, pulmonary function, imaging findings, blood data, physical functions, sleep disturbances, respiratory symptoms and health-related quality of life (HRQOL).METHODS: We conducted a cross-sectional retrospective study of 114 patients with NTM-PD at a single centre from March 2016 to January 2021 to evaluate the relationship between CES-D scores and clinical parameters.RESULTS: Participants had a median age of 64 years; 32.5% of them had depressive symptoms. Disease duration, albumin, C-reactive protein, pulmonary function, dyspnoea, exercise capacity, respiratory symptoms, cough-related HRQOL and sleep disturbances were associated with depressive symptoms. Binomial logistic regression analyses indicated that the CES-D score was significantly associated with cough-related HRQOL and sleep disturbances.CONCLUSION: A high percentage of NTM-PD patients in this study experienced depressive symptoms, and these patients had abnormalities of various clinical parameters. Cough-related HRQOL and sleep disturbance had a strong influence on the development of depressive symptoms.

Health-related quality of life associates with clinical parameters in patients with NTM pulmonary disease.

BACKGROUND: Previous studies have shown a reduction in health-related quality of life (HRQoL) in patients with non-tuberculous mycobacterial pulmonary disease (NTM-PD). However, the causes of this decline and the factors that contribute to it are unknown. This study was conducted to analyse the association between the St George´s Respiratory Questionnaire (SGRQ) and clinical parameters, including age, disease duration, body composition, pulmonary function, chest X-ray findings, blood data and physical function.METHODS: We performed a single-centre, cross-sectional, retrospective study of 101 patients with NTM-PD from December 2016 to October 2019. The relationship between the SGRQ scores and clinical parameters was evaluated.RESULTS: The median patient age was 67.0 years. Pulmonary function, radiological score, albumin levels, C-reactive protein levels and incremental shuttle walk test distance (ISWD) were significantly correlated with the total and component scores on the SGRQ. Multiple regression analysis showed that the SGRQ score was significantly associated with radiological score, pulmonary function and ISWD.CONCLUSION: This study was the first to assess the effect of clinical parameters on the SGRQ in patients with NTM-PD. HRQoL as determined using the SGRQ was associated with the radiological score, pulmonary function and ISWD in patients with NTM-PD.

Clinical significance of Aspergillus species isolated from respiratory specimens in patients with Mycobacterium avium complex lung disease.

Chronic pulmonary aspergillosis (CPA) is associated with mortality in patients with Mycobacterium avium complex lung disease (MAC-LD). An Aspergillus-positive respiratory specimen often reflects colonization, and thus the clinical significance of Aspergillus isolation in MAC-LD patients is not well understood. The objective of this study was to investigate the clinical characteristics and outcomes of MAC-LD patients in whom Aspergillus was isolated from respiratory specimens. We performed a retrospective review of the medical records of 329 MAC-LD patients. We compared the characteristics and mortality rates between patients with Aspergillus isolation and those without. All Aspergillus species detected from respiratory specimens within the follow-up period were reviewed. Aspergillus was detected in 40 (12.2%) of the 329 patients. There were no significant differences in the clinical characteristics and mortality rates between patients with and without Aspergillus isolation. Among the 40 patients with Aspergillus isolation, 9 (22.5%) developed CPA. CPA was most often caused by A. fumigatus. In the 40 Aspergillus-positive patients, patients with A. fumigatus isolation had a significantly higher mortality rate than those without (P < 0.001). The multivariate Cox proportional hazards model showed older age (P = 0.050), presence of respiratory comorbidities (P = 0.008), hypoalbuminemia (P < 0.001), and isolation of A. fumigatus (P = 0.005) to be prognostic factors for mortality in MAC-LD patients. There was no significant difference in the mortality rates between patients with Aspergillus isolation and those without. However, isolation of A. fumigatus may be associated with poor prognosis in MAC-LD patients.

Clinical significance of the radiological severity score in Mycobacterium avium complex lung disease patients.

SETTING: Practical methods for assessing the radiographic findings of Mycobacterium avium complex lung disease (MAC-LD) have not been established. OBJECTIVE: To identify a correlation between the radiological score and semi-quantitative culture results of respiratory samples, and to assess the prognostic impact of this radiological score in MAC-LD patients. DESIGN: We retrospectively studied 218 MAC-LD patients. Radiographic findings were classified as nodule (N), infiltration shadow (I), cavity (C) and bronchiectasis (E), scored individually according to the area occupied on six lung field divisions, and added to give the radiological severity score. RESULTS: The radiological score positively correlated with the semi-quantitative culture score (P = 0.003). In univariate analysis, the radiological score was a significant negative prognostic factor for overall survival. On multivariate analysis, factors I, C and E were independent negative prognostic factors for overall survival. We compared the prognostic value of the total score of all four factors and the three significant factors (I, C and E) using receiver operating characteristic curve analysis; the corresponding areas under the curves were respectively 0.628 and 0.763 (P < 0.001). CONCLUSIONS: The radiological score correlates with prognosis. The combined score of factors I, C and E may more accurately predict prognosis in MAC-LD patients.

Centrosomal proteins Nde1 and Su48 form a complex regulated by phosphorylation.

The centrosome modulates spindle formation and plays a critical role in guiding proper segregation of chromosomes during cell division. Centrosome aberrations, frequently seen in human tumors, may cause abnormal chromosome segregation and contribute to malignant transformation. To explore the components of the centrosomes, we previously identified a novel centrosomal protein called Su48. To further characterize the Su48-containing protein ensemble in the centrosome, we performed yeast two-hybrid screens and isolated a number of Su48-interacting molecules, including the centrosomal protein Nde1. Here, we demonstrate that Su48 can associate with Nde1. Moreover, we found that Nde1 is subjected to phosphorylation in vivo. In particular, we identified six putative Cdc2 phosphorylation sites in Nde1 and found that alteration of these sites diminishes phosphorylation by Cdc2 in vitro and affects the stability of Su48-Nde1 interactions and the centrosomal localization of Nde1. Ablation of Nde1 by gene specific small interfering RNA causes mitotic delay and cell death, coupled with a modest decrease in the incidence of the cells that harbor excessive centrosomes. Collectively, our findings indicate that Nde1 can form a protein complex with Su48 in the centrosome and plays an important role for successful mitosis.

Development of a yeast stop codon assay readily and generally applicable to human genes.

We established a yeast-based method to screen chain-terminating mutations that is readily applicable to any gene of interest. Based on the finding that 18- to 24-base-long homologous sequences are sufficient for gap repair in vivo in yeast, we used a strategy to amplify a test-gene fragment with addition of 24-bp sequences homologous to both cut-ends of a yeast expression vector, pMT18. After co-transformation with the amplified fragment and the linearized pMT18, each yeast (Saccharomyces cerevisiae) cell automatically forms a single-copy circular plasmid (because of CEN/ARS), which expresses a test-gene::ADE2 chimera protein. When the reading frame of the test-gene contains a nonsense or frameshift mutation, truncation of the chimera protein results in lack of ADE2 activity, leading to formation of a red colony. By using a nested polymerase chain reaction using proofreading Pfu polymerase to ensure specificity of the product, the assay achieved a low background (false positivity). We applied the assay to BRCA1, APC, hMSH6, and E-cadherin genes, and successfully detected mutations in mRNA and genomic DNA. Because this method--universal stop codon assay--requires only 4 to 5 days to screen a number of samples for any target gene, it may serve as a high-throughput screening system of general utility for chain-terminating mutations that are most prevalent in human genetic diseases.

Overexpression of homeobox gene HOXD3 induces coordinate expression of metastasis-related genes in human lung cancer cells.

Homeobox-containing genes are expressed in spatiotemporal fashion during embryogenesis and act as master transcription-regulating factors which control the expression of a variety of genes involved in morphogenesis. They are also expressed in a tissue-specific manner in normal adult tissues and appear to give cells spatial information in the maintenance of their architectural integrity. We transfected a HOXD3 class I homeobox-containing gene into human lung cancer A549 cells and investigated alterations in gene expressions and phenotypes related to the maintenance of tissue architecture in HOXD3-overexpressing A549 cells. In the HOXD3-overexpressing cell lines, expression of E-cadherin was lost and plakoglobin was strongly repressed, whereas integrin alpha3 and beta3 were up-regulated and N-cadherin and integrin alpha4 were newly expressed. Compared with parental and control transfectant lines, the HOXD3-overexpressing cell lines showed highly motile and invasive activity. Blocking experiments using anti-integrin beta1 and beta3 suggested that the increased haptotaxis of the HOXD3-overexpressing cells to vitronectin resulted from increased expression and activation of integrin alphavbeta3, and that overexpression of the HOXD3 gene converted the integrin beta1-dependent haptotaxis to fibronectin into both integrin beta1- and beta3-dependent one. HOXD3 overexpression increased production of matrix-degrative enzymes including matrix metalloproteinase-2 and urokinase-plasminogen activator. When the tumor cells were intravenously injected into the tail veins of nude mice, HOXD3 transfectants formed a significantly large number of metastatic foci in lungs compared with the control transfectants. These findings suggest that HOXD3 can act as a metastasis-promoting gene in human lung cancer A549 cells.

Interaction of MCC2, a novel homologue of MCC tumor suppressor, with PDZ-domain Protein AIE-75.

AIE-75 is a protein identified as an autoantigen in patients with autoimmune enteropathy and as a colon cancer-related antigen. It has recently been assigned to be a causative gene for Usher type 1C congenital syndromic hearing loss. The novel protein has three PSD-95/Dlg/ZO-1 (PDZ) protein-protein interaction domains and is therefore implicated to function as a molecular anchor or sorter. We have identified a novel protein that binds to AIE-75 by yeast two-hybrid screening. The protein has a high homology to the tumor suppressor MCC (mutated in colon cancer; or MCC1 hereafter) and was named MCC2. MCC2 protein binds the first PDZ domain of AIE-75 with its C-terminal amino acids -DTFL. Since the MCC1 does not bind to AIE-75 and the MCC2 displays different expression patterns in various organs compared to MCC1, they appear to play distinct roles in cells. The MCC2 gene is located on chromosome 19p13 in the vicinity of APCL gene, while MCC1 maps near to APC tumor suppressor gene. Because of negative expression of MCC2 in a panel of cancer cell-lines compared to the corresponding normal tissues, we suggest that further study is necessary to investigate a possible role of MCC2 as a tumor suppressor.

Frequent deletions and mutations of the beta-catenin gene are associated with overexpression of cyclin D1 and fibronectin and poorly differentiated histology in childhood hepatoblastoma.

Hepatoblastoma (HBL) is the most common malignant liver tumor in young children. Recent reports have shown that the beta-catenin gene was frequently mutated or deleted in HBLS: To elucidate the role of beta-catenin abnormalities in HBLs, we searched for mutations of beta-catenin and APC as well as expression of the target genes, cyclin D1, c-myc, and fibronectin, in 68 primary HBLS: The mutation analysis revealed that 44 (65%) tumors carried missense mutations or deletions of beta-catenin, all of which were somatic and targeted to the exon 3 encoding the amino acid residues involved in its degradation. However, no loss of function mutation of the APC gene was detected by the yeast functional assay. Of interest, beta-catenin mutation was significantly correlated with overexpression of the target genes, cyclin D1 and fibronectin, but not with that of c-myc in HBLs as measured by quantitative real-time reverse transcription-PCR. The immunohistochemical studies in 15 HBLs demonstrated that the nuclear/cytoplasmic accumulation of beta-catenin was positive in 13 tumors, 9 of which had the deletion or mutation of the gene. The significant correlation between the beta-catenin gene abnormality and the positive staining of cyclin D1 was also confirmed. Furthermore, the nuclear accumulation of beta-catenin was strongly associated with the poorly differentiated tumor cell components as well as with the positive staining of cyclin D1 within the tumor. Thus, our present results suggested that the gain of function mutation of beta-catenin played a crucial role in the malignant progression of HBL in vivo.

Inactivate the remaining p53 allele or the alternate p73? Preferential selection of the Arg72 polymorphism in cancers with recessive p53 mutants but not transdominant mutants.

Several reports have noted epidemiological differences in the prevalence or prognostic significance of p53 mutants with arginine (R) or proline (P) at the codon 72 polymorphism (R72/P72) in certain cancer types, but the biological significance of these variants is unclear. The ability of p53 mutants to interact with and inactivate the p53 homolog p73 was recently reported to depend on the conformational state of the p53 protein and the residue at codon 72. Since the conformation of p53 mutants may influence their ability to transdominantly inhibit wild-type p53, we tested whether there was a correlation between the amino acid at codon 72 and the transdominance of p53 alleles found in tumors. The transdominance test was performed using a simple yeast transcription assay, and the amino acid at codon 72 was determined by sequencing. A total of 100 p53 mutants were tested. Compared with the germline frequency (R:P = 427:297), an extreme bias in favor of the R72 allele was observed with recessive mutants (R:P = 50:7, P < 0.0002), whereas no selection for the R72 allele was seen with transdominant mutants (R:P = 23:20). p53 and p73 are known to transactivate overlapping sets of target genes. We interpret the R72 bias with recessive mutants as evidence that decreased activation of p53 target genes provides a selective growth advantage to tumor cells during the stage of tumorigenesis in which a wild-type and mutant p53 allele coexist. We suggest that transdominant p53 mutants achieve this by inactivation of the remaining wild-type p53 allele, whereas recessive p53 mutants achieve it through inactivation of p73.

Somatic mutations of the APC gene in primary breast cancers.

APC gene mutations play an important role in the initiation step of colorectal carcinogenesis in both familial adenomatous polyposis (FAP) patients and non-FAP patients. Although the APC gene is expressed in most tissues, including the lung, liver, kidney, and mammary gland, its somatic mutations have rarely been found in primary tumors affecting these organs. We have developed a sensitive yeast-based assay for screening almost the entire coding region of the APC gene. By this method, we have been able to detect somatic mutations of the APC gene in 57% of colorectal cancers and none in non-small cell lung cancers. Interestingly, the assay detected somatic APC gene mutations in 18% of breast cancers, in which APC gene mutation was previously considered rare. In the breast cancers, most of the APC mutations were distributed outside the mutation cluster region that has been advocated for colorectal cancers. We also noted a difference in the mutation pattern of the APC between colorectal and breast cancers. In colorectal cancers, all base substitutions were observed at C residues (5 of 5), whereas in breast cancers the majority of them were found at G residues (4 of 5). Furthermore, APC mutations were observed at a significantly high frequency in advanced stages of primary breast cancers (TNM classification, P < 0.05; T category, P < 0.01). Our data suggest that the etiology of the APC mutations and their biological role in carcinogenesis may differ between colorectal and breast cancers.

Distinct prognostic values of p53 mutations and loss of estrogen receptor and their cumulative effect in primary breast cancers.

A total of 76 primary breast cancers were screened for p53 mutations using the yeast p53 functional assay, and the mutations were determined by DNA sequencing. Clonal mutations of p53 were detected in 30 tumors (39%). Immunohistochemical staining for nuclear p53 accumulation performed on the yeast assay-positive cases clearly differentiated missense mutations in the DNA binding domain (contact mutant; 17 cases) as positive stain and nonsense-type mutations or missense mutations that may affect 3D-structure of p53 protein (structural mutant; 13 cases) as negative stain. Enzyme immunoassay revealed loss of estrogen receptor in 36 tumors (50%). Prognostic values of p53 mutation and loss of estrogen receptor were evaluated after a median follow-up period of 44 months. p53 mutations were associated with a short overall survival (log rank test, p = 0.0319), whereas it was not related to disease-free (recurrence-free) survival. Contact mutants were associated with slightly shorter survival compared with structural mutants. Inversely, loss of estrogen receptor was associated with early recurrence (p = 0.0461) but not with short overall survival. The patients with tumors harboring both p53 mutation and loss of estrogen receptor had the poorest outcome (p = 0.0019 and 0.0075 for overall and disease-free survivals, respectively), suggesting independent and additive effects of the 2 factors. The independent role of the 2 factors was confirmed by a multivariate analysis using the Cox proportional hazard model stratified according to clinical tumor stages. Although preliminary, due to the small number of patients studied and the relatively short follow-up time, our results suggest that p53 mutations and loss of estrogen receptor cooperatively affect the prognosis of primary breast cancer patients.

[Development of a detection system (APC yeast color assay) of APC mutations by color change of yeast].

The author developed a sensitive yeast-based color assay which expresses APC-ADE2 (reporter) fusion protein in yeast and can screen almost the entire coding region of the APC gene. In this assay, the wild-type APC coding sequence of 8.5 kb is divided into 5 overlapping regions which are respectively ligated in-frame with an ADE2 open reading frame. The resulting five constructs containing a part of wild-type APC gene preserve the ADE2(+) phenotype (white yeast colony) when introduced into the yeast, whereas the yeast transfected with plasmids containing frameshift mutations of the APC gene shows an ADE2(-) phenotype (red yeast colony). Six human colon cancer cell lines were analyzed by this yeast color assay. HCT116 cells with wild-type APC and normal colonic mucosa gave low percentages of red colonies (0-9.9%) in all the regions. On the other hand, more than 96% red colonies were observed in one of the five regions in SW480, Colo201 and Colo320DM cells. Sequence analysis demonstrated the clonal APC mutations at codon 1,338 in SW480, 1,554 in Colo201 and 811 in Colo320DM. Moreover, the assay detected a germline mutation of the APC gene in polyps of a familial adenomatous polyposis (FAP) patient which gave about 50% red colonies. For testing the assay for clinical utilization, 18 colon cancer tissues were subjected to the assay. Eleven cancers (61%) gave more than 10% red colonies (17-57%) and clonal mutations were detected in all these samples. The same mutations were demonstrated in both DNA and RNA samples derived from idendical tissues. These results suggest that this APC yeast color assay is powerful means for detection of APC mutations in clinical samples.

A functional and quantitative mutational analysis of p53 mutations in yeast indicates strand biases and different roles of mutations in DMBA- and BBN-induced tumors in rats.

In order to analyze the mutational events and to understand the biological significance of the p53 gene in chemical carcinogenesis, we applied a new yeast-based p53 functional assay to ovarian tumors induced by 7, 12-dimethylbenz[a]anthracene (DMBA), as well as to transitional cell carcinomas of the urinary bladder induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in rats. The assay demonstrated that 15 of 19 DMBA induced tumors harbored clonal p53 mutations, which is consistent with the expectations of the "clonal expansion" hypothesis. The majority of the mutations were purine (AG) to pyrimidine (CT) transversions (12/19) on the non-transcribed (sense) strand (NTS), which is likely to be due to depurination created by DMBA adduct formation on the NTS. In contrast, we found no pyrimidine to purine [corrected] transversion on the NTS. After cessation of BBN treatment, BBN-induced multifocal lesions in the bladder contained heterogeneous p53 mutations at an early stage. In the later stage, however, clonal p53 mutations were identified in 4 out of 7 bladders analyzed, conforming with the concept of "field cancerization". The observed base substitutions were G-->A (1/6) or C -->T transitions (2/6), and mutations at T (3/6) on the NTS in clonal mutations, together with non-clonal mutations, showing a preference of C-->T to G-->A (17 vs. 0). Thus, preferential repair was found in the transcribed strand of the p53 gene, whether modified by DMBA or by BBN carcinogens. Very similar mutation patterns were observed between clonal and non-clonal mutations in the DMBA- and BBN-induced tumors, indicating that the rat yeast p53 functional assay can be a potential tool for the characterization of in vivo mutation patterns of p53, when modified by chemical carcinogens.

A novel A-kinase anchoring protein in the heart interacts with G alpha 13.

A cDNA of a tentative A-kinase anchoring protein, presumably coupled with heterotrimeric GTP binding protein alpha 13 subunit (G alpha 13), was cloned from a human heart cDNA library. It was approximately 650 bases and its mRNA was expressed in the heart. Homology search of DNA sequences revealed that it was a novel cDNA with 84% homology with the partial sequence of rabbit cDNA of AKAP 120 without a stop codon. 3'-Rapid Amplification of cDNA Ends (3'-RACE) and yeast functional assay were performed to determine the 3'-end of the cDNA and ribosomal frameshifting was suggested as a translational mechanism. Here we report that a protein encoded by the cDNA may be involved in intracellular signal transduction via the G alpha 13 and PKA in hearts.

Glial cell line-derived neurotrophic factor induces barrier function of endothelial cells forming the blood-brain barrier.

Since a deep involvement of astrocytes, a kind of glial cells, in differentiation of the blood-brain barrier (BBB) has been suggested, we examined the relation of glial cell line-derived neurotrophic factor (GDNF) to the BBB. First, immunohistochemical examination of the cerebral cortex of rats revealed that glial cell line-derived neurotrophic factor receptor (GFRalpha1) was preferentially expressed on the cell membranes of capillary endothelial cells. Second, to elucidate the effects of GDNF on the BBB, capillary endothelial cells isolated from the porcine cerebral cortex were cultured and then changes in tight junction function of the endothelial cells were examined after addition of GDNF, in terms of transendothelial electrical resistance (TER) and permeability. GDNF at concentrations of 0.1 and 1 ng/ml significantly activated the barrier function of the endothelial cells in the presence of cAMP. Since GDNF is secreted from astrocytes sheathing capillary endothelial cells in the brain cortex, our results strongly suggest that GDNF enhances the barrier function of tight junctions of the BBB on the one hand, and also supports the survival of neurons on the other hand.

Suppression of pancreatic cancer by the dominant negative ras mutant, N116Y.

N116Y, H-ras mutant, possesses dominant negative activity to Ras function. The aim of this study is to assess whether N116Y can inhibit the proliferation of pancreatic cancer cell lines carrying K-ras mutations and cause reversion of the malignant phenotype. We transfected an expression vector of N116Y, pZIP-N116Y, into eight human pancreatic cancer cell lines with K-ras mutations (PCI 10, 19, 24, 35, 43, 55, 64, and 66) by using a lipofection procedure. The growth inhibition activity of N116Y was evaluated by the colony-forming efficiency in selection medium. In order to examine the effect of N116Y on the neoplastic phenotype, we established N116Y-expressing clones and analyzed their growth ability in soft agar and tumorigenicity in nude mice. The growth of the eight pancreatic cancer cell lines was strongly inhibited by the transfection of pZIP-N116Y. Moreover, the N116Y-expressing clones became less spread and lost their anchorage-independent growth ability. Furthermore, they were nontumorigenic in vivo. N116Y significantly inhibits the growth of pancreatic cancer cell lines and causes reversion of the malignant phenotypes. These results suggest that N116Y may be a candidate gene for use in the gene therapy of pancreatic cancer.

Gelsolin: a candidate for suppressor of human bladder cancer.

Human transitional cell carcinomas of the bladder frequently reveal chromosomal abnormalities that span a range between chromosome 9p12 and 9qter, even at early stages of bladder carcinogenesis. Because the gene that encodes an actin-regulatory protein, gelsolin, is localized in chromosome 9q33, we examined the expression of gelsolin in a number of human bladder cancer cell lines and tissues. In all 6 cell lines and in 14 of the 18 tumor tissues (77.8%), gelsolin expression was undetectable or extremely low in comparison with its expression in normal bladder epithelial cells. Furthermore, upon the introduction of the exogenous human or mouse authentic gelsolin cDNA into a human bladder cancer cell line, UMUC-2, gelsolin transfectants of UMUC-2 greatly reduced the colony-forming ability and the tumorigenicity in vivo. These results suggest that gelsolin plays a key role as a tumor suppressor in human urinary bladder carcinogenesis.