A natural BH3 mimetic induces autophagy in apoptosis-resistant prostate cancer via modulating Bcl-2-Beclin1 interaction at endoplasmic reticulum.

A natural BH3-mimetic, small-molecule inhibitor of Bcl-2, (-)-gossypol, shows promise in ongoing phase II and III clinical trials for human prostate cancer. In this study we show that (-)-gossypol preferentially induces autophagy in androgen-independent (AI) prostate cancer cells that have high levels of Bcl-2 and are resistant to apoptosis, both in vitro and in vivo, but not in androgen-dependent (AD) cells with low Bcl-2 and sensitive to apoptosis. The Bcl-2 inhibitor induces autophagy through blocking Bcl-2-Beclin1 interaction, together with downregulating Bcl-2, upregulating Beclin1, and activating the autophagic pathway. The (-)-gossypol-induced autophagy is dependent on Beclin1 and Atg5. Our results show for the first time that (-)-gossypol can also interrupt the interactions between Beclin1 and Bcl-2/Bcl-xL at endoplasmic reticulum, thus releasing the BH3-only pro-autophagic protein Beclin1, which in turn triggers the autophagic cascade. Oral administration of (-)-gossypol significantly inhibited the growth of AI prostate cancer xenografts, representing a promising new regimen for the treatment of human hormone-refractory prostate cancer with Bcl-2 overexpression. Our data provide new insights into the mode of cell death induced by Bcl-2 inhibitors, which will facilitate the rational design of clinical trials by selecting patients who are most likely to benefit from the Bcl-2-targeted molecular therapy.

Clues for the differential diagnosis of hypersensitivity pneumonitis as an expectant variant of diffuse parenchymal lung disease.

Hypersensitivity pneumonitis, also called extrinsic allergic alveolitis, a type of diffuse parenchymal lung disease (DPLD), is an immunologically mediated pulmonary disease induced by inhalation of various antigens. As data on the frequency of hypersensitivity pneumonitis are lacking in Turkey, a retrospective analyses was performed in 43 patients with DPLD, followed up over seven years. The objective was to discover cases fulfilling the diagnostic criteria for hypersensitivity pneumonitis, to determine the frequency and/or the new characteristics of the disease, and to pick up clues for differentiating it from other DPLDs. The four subjects with hypersensitivity pneumonitis (9%) who lived in an urban area were studied in detail. The most common symptoms were dry cough and dyspnoea. According to the symptom duration, clinical features, radiological and pathological findings, three were diagnosed with chronic and one with subacute hypersensitivity pneumonitis. Patients with hypersensitivity pneumonitis and those with DPLD were compared by means of age, sex, smoking status, symptom duration, haematology, erythrocyte sedimentation rate, peripheral cell count, spirometric parameters, blood gases, and diffusion capacity. No statistically significant difference was detected in these parameters except for forced expiratory volume in one second (FEV(1)) and forced vital capacity (FVC). In conclusion, patients with a history of antigen exposure, with mild symptoms such as dry cough and dyspnoea, and who have diffuse interstitial lung involvement on radiology should be carefully evaluated for hypersensitivity pneumonitis. Moreover, among other DPLDs, stable FEV(1) or FVC values may be the clues for establishing the diagnosis of hypersensitivity pneumonitis. However, further studies are needed in larger series of patients.

Cellular profile of bronchoalveolar lavage fluid in Turkish miners.

Pneumoconiosis is still a health problem in Turkey and has a relatively high incidence. Retired underground miners were investigated to document alveolitis, and to observe the difference in the cellular profiles of bronchoalveolar lavage (BAL) fluid with or without pneumoconiosis. Twenty nine retired male miners and 17 controls, eight non-smokers (four male, four female) and nine smokers (six male, three female), without any dust exposure were evaluated. According to the International Labor Office 1980 classification system, the miners were allocated to three subgroups: eight without pneumoconiosis, 11 with simple pneumoconiosis, and 10 with progressive massive fibrosis (PMF). Spirometric tests and arterial blood gases analysis were done and fibreoptic bronchoscopy and BAL were performed in all subjects. The study and the control subjects were comparable in respect to age, smoking habits, except the non-smoker controls, and the duration of dust exposure, except the controls. The amount of recovered BAL fluid was lower in all miners compared with the non-smoker controls (p<0.05). The amount of recovered BAL fluid and the total cell count correlated significantly (r = 0.48, p<0.01). The percentage of lymphocytes in the BAL fluid of miners without pneumoconiosis and with PMF (p<0.05) and that of simple pneumoconiosis (p<0.01) was significantly lower compared with the non-smoker controls. Alveolitis was not a representative feature of Turkish subjects with an occupational history of underground mining, and BAL fluid cellular profile did not seem to be different in miners with or without pneumoconiosis.

Adenosine deaminase activity in bronchoalveolar lavage in Turkish patients with smear negative pulmonary tuberculosis.

The sputum smear-negative patients have been a diagnostic challenge for health professionals. Adenosine deaminase (ADA) activity has been shown to rise in various body fluids of patients with tuberculosis (Tb). A prospective clinical trial was conducted to determine the diagnostic value of ADA activity in bronchoalveolar lavage (BAL) in sputum smear-negative subjects highly suggestive for pulmonary Tb. Nineteen (M/F: 15/4, mean age 46.8 +/- 16.5 years) sputum smear-negative patients highly suggestive for pulmonary Tb constituted Group I. Acid fast bacilli (AFB) grew on sputum and/or BAL culture of all subjects in this group. Twenty-nine patients (M/F: 19/10, mean age 55.7 +/- 8.0 years) with non-tuberculous pulmonary diseases constituted Group II. Ten of them had interstitial lung disease, nine lung cancer, five pneumonia and five COPD. Twelve subjects (M/F: 7/5, mean age 48.4 +/- 12.8 years) constituted the controls (Group III) undergoing fiberoptic bronchoscopy (FOB) for various indications and the lungs were found to be normal eventually. Albumin and ADA activity levels were measured in plasma and BAL in all the subjects. LocalADA was calculated. PlasmaADA and BALADA of Group I was significantly higher (P < 0.001) than that of the other groups. LocalADA was also the highest in Group I when compared with the others (P < 0.001) but that of Group II was also higher (P < 0.01) when compared with controls. With a cut-off value derived from the control subjects, sensitivity of BALADA was 100% and specificity 85.3%. Sputum PCR results are available in a couple of days whereas that of BALADA are available in a couple of hours and BALADA costs cheaper than PCR in our country. Therefore, we conclude that BALADA may be a useful, cheaper and faster diagnostic test in sputum smear-negative patients highly suggestive for pulmonary Tb. LocalADA need not be calculated as it is also significantly higher in Group II subjects and thus not as reliable as BALADA.

Chlamydia pneumoniae infection and acute exacerbation of chronic obstructive pulmonary disease (COPD).

The objective of the study was to investigate the possible association of Chlamydia pneumoniae (Cpn) in acute exacerbations of chronic obstructive pulmonary disease (COPD) patients. Thirty-eight acutely exacerbated COPD patients and 17 healthy smokers were enrolled in the study, as the study and control groups respectively. Nasopharyngeal swabs and paired serum samples for antibody testing of Cpn (microimmunofluorescence--MIF) were obtained from all subjects. Sputum cultures of COPD patients were also performed. No pathogenic bacteria were isolated from nasopharyngeal swabs in any subject. Serologic evidence of recent Cpn infection was observed in 13 (34%) COPD patients and in one (5%) control subject. The prevalence of Cpn IgG and IgM antibodies representing acute infection were significantly higher in COPD patients than in control subjects (P < 0.05 and P < 0.01 respectively). Prevalence of IgA antibodies and IgG pre-existing antibodies did not show any difference (P > 0.05). Microbiologic culture of the sputa yielded potentially pathogenic micro-organisms in 23 of 38 (60%) COPD patients. Alpha-haemolytic streptococcus (35%), Niesseria spp. (31%) and Candida spp. (9.5%) were most prominent micro-organisms in positive cultures. Although a high prevalence of IgG antibodies against Cpn was detected, it was the sole causative agent in only four (10%) patients. We conclude that a remarkable number of COPD patients (34%) are acutely infected with Cpn and it may either be the sole causative agent or frequently a co-agent in acute exacerbations.

Evaluation of Cyfra 21-1: a potential tumor marker for non-small cell lung carcinomas.

Cyfra 21-1 is a tumor marker based on the determination of water-soluble cytokeratin 19 which is secreted by normal or malignantly transformed epithelial cells. It is suggested to be a valuable marker in patients with non-small cell lung carcinoma (NSCLC). A prospective clinical study was conducted to investigate the value of Cyfra 21-1 for diagnosis, determination of subtypes, staging, and evaluation of therapy response in patients with lung carcinoma (Ca). Sixty-nine patients (mean age: 60.9 +/- 9.2 years, M/F:12.8) treated between 1994 and 1998 inclusive, and 13 healthy smokers (mean age:50.9 +/- 4.8 years, M/F:1.6) constituted the study group and control group, respectively. Venous blood samples (10 ml) were obtained from all subjects. Posttreatment blood samples were also obtained from 14 NSCLC patients. Cyfra 21-1 levels (cutoff value 3.3 ng/ml) were determined by ELSA-Cyfra 21-1 kit (CIS bio international, France) through immunoradiometric assay (IRMA). Cyfra 21-1 levels did not differ between smoking and non-smoking subjects within each group (p > 0.05). Cyfra 21-1 was significantly elevated in lung Ca cases irrespective of the cell type (p < 0.05). It was significantly elevated in squamous cell and adenocarcinoma varieties with the most prominent elevation in squamous cell type (p < 0.05). In lung Ca, the specificity and sensitivity of Cyfra 21-1 was 92.3% and 52.2%, respectively. Sensitivity was 65.5% for NSCLC, 70.5% for squamous cell, and 45.5% for adenocarcinoma varieties, with highest sensitivity rates in Stage IIIA + IIIB (87.5%) and Stage IV (75%) of squamous cell lung Ca. Cyfra 21-1 level was significantly decreased after treatment in NSCLC patients (n 14) (p < 0.01). Cyfra 21-1 is a tumor marker that helps to establish the diagnosis and differentiation of cell type and evaluation of response to therapy in patients with NSCLC.

Interaction with mLin-7 alters the targeting of endocytosed transmembrane proteins in mammalian epithelial cells.

To investigate the targeting mechanism for proteins bound to the mammalian Lin-7 (mLin-7) PDZ domain, we created receptor protein chimeras composed of the carboxyl-terminal amino acids of LET-23 fused to truncated nerve growth factor receptor/P75. mLin-7 bound to the chimera with a wild-type LET-23 carboxyl-terminal tail (P75t-Let23WT), but not a mutant tail (P75t-Let23MUT). In Madin-Darby canine kidney (MDCK) cells, P75t-Let23WT localized to the basolateral plasma membrane domain, whereas P75t-Let23MUT remained apical. Furthermore, mutant mLin-7 constructs acted as dominant interfering proteins and inhibited the basolateral localization of P75t-Let23WT. The mechanisms for this differential localization were examined further, and, initially, we found that P75t-Let23WT and P75t-Let23MUT were delivered equally to the apical and basolateral plasma membrane domains. Although basolateral retention of P75t-Let23WT, but not P75t-Let23MUT, was observed, the greatest difference in receptor localization was seen in the rapid trafficking of P75t-Let23WT to the basolateral plasma membrane domain after endocytosis, whereas P75t-Let23MUT was degraded in lysosomes, indicating that mLin-7 binding can alter the fate of endocytosed proteins. Altogether, these data support a model for basolateral protein targeting in mammalian epithelial cells dependent on protein-protein interactions with mLin-7, and also suggest a dynamic role for mLin-7 in endosomal sorting.

Neurophysiological changes in COPD patients with chronic respiratory insufficiency.

Chronic hypoxemia is known to cause peripheral neuropathy (PNP) in chronic obstructive pulmonary disease (COPD) patients. We aimed to know how often PNP is encountered in such patients and the changes in the central nervous system (CNS) if any. We enrolled 32 patients (30 M, 2 F; mean age +/- SD: 61.5 +/- 8.8 years) with COPD into the study. PaO2 > or = 55 mmHg was considered as the cut-off value designating tissue hypoxia. According to this cut-off value the subjects were divided into two groups: Group I, n: 19, PaO2 < 55 mmHg and Group II, n: 13, PaO2 > or = 55 mmHg. All subjects were evaluated with motor and sensory nerve conduction studies (MNCV and SNCV, respectively), electromyography, visual and brainstem evoked potentials (VER and BAER, respectively). We detected PNP in 93.8% of the study subjects. Distal latency of sural nerve correlated significantly with cigarette consumption and reduction in PEFR. SNCV of median nerve was reduced as PaCO2 was elevated and pH was lowered. BAER wave III latency showed significant inverse correlation with PEFR, FEF25 and FEF25-75. Interpeak latency (IPL) of BAER I-III was also significantly and inversely correlated with FEV1/FVC and FEF25-75. IPL of BAER III-V too showed significant correlations with PaCO2, HCO3- and pH of the arterial blood. As BAER III and IPLs of it represent the pontomedullary portion of the brain, cigarette smoking and airways obstruction may not only cause peripheral neuropathy but also a delay in evoked responses of the brain stem by inducing chronic hypercapnia and respiratory acidosis in patients with COPD.

Mycobacterium tuberculosis drug resistance in Turkey, 1976-97.

Drug-resistant tuberculosis is increasing day by day and is a significant threat to tuberculosis control because there are few drugs effective against Mycobacterium tuberculosis. This study evaluates the resistance of the microorganism to primary anti-tuberculosis drugs over the 21-y period 1976-97. Records from the bacteriology laboratory of the Department of Chest Diseases and Tuberculosis, Ankara University Medical Faculty were evaluated retrospectively. Among 3,418 mycobacteria strains, 3,319 (97.1%,) M. tuberculosis were isolated and their susceptibility was examined by the proportion method in Loewenstein-Jensen medium. It was found that 60.8% of isolated strains were susceptible, whereas 39.2%, were resistant to at least one drug. Multi-drug resistant tuberculosis (MDR-TB) was found in 194 (5.8%) materials. Over the 21-y period studied, total resistance to isoniazid (INH), rifampicin (RF) and streptomycin (SM) were determined as 10.5, 6.9 and 7.0%, respectively. It was also observed that the resistance rates to INH or SM increased, whereas resistance to RF was not changed within this period. While resistance to the 2-drug combination RF+SM increased, resistance to INH+SM decreased significantly. There was no change in resistance to the 2-drug INH + RF or 3-drug INH + RF + SM combinations in the same period. In conclusion, combined therapy is still useful and available for the treatment of resistant tuberculosis, and INH should be included in the chemotherapeutic regimen even if high resistance rates are shown to exist.

mLin-7 is localized to the basolateral surface of renal epithelia via its NH(2) terminus.

In Caenorhabditis elegans, the basolateral localization of the Let-23 growth factor receptor tyrosine kinase requires the expression of three genes: lin-2, lin-7, and lin-10. Mammalian homologs of these three genes have been identified, and a complex of their protein products exists in mammalian neurons. In this paper, we examine the interaction of these mammalian proteins in renal epithelia. Coprecipitation experiments demonstrated that mLin-2/CASK binds to mLin-7, and immunofluorescent labeling showed that these proteins colocalized at the basolateral surface of Madin-Darby canine kidney cells and renal epithelia. Although labeling intensity varied markedly among different renal epithelial cells, those cells strongly expressing mLin-7 also showed intense mLin-2/CASK labeling. We have also demonstrated that mLin-2/CASK binding requires amino acids 12-32 of mLin-7 and have shown that this region of mLin-7 is also necessary for the targeting of mLin-7 to the basolateral surface. Furthermore, the overexpression of mLin-2/CASK mutants in Madin-Darby canine kidney cells caused endogenous mLin-7 to mislocalize. In summary, the NH(2) terminus of mLin-7 is crucial for its basolateral localization, likely through its interaction with mLin-2/CASK.

Molecular cloning and characterization of Pals, proteins associated with mLin-7.

In Caenorhabditis elegans, three PDZ domain proteins, Lin-2, Lin-7, and Lin-10, are necessary for the proper targeting of the Let-23 growth factor receptor to the basolateral surface of epithelial cells. It has been demonstrated that homologues of Lin-2, Lin-7, and Lin-10 form a heterotrimeric complex in mammalian brain. Using Far Western overlay assay, we have identified additional proteins that can bind to the amino terminus of mLin-7 and cloned the genes encoding these proteins using bacterial expression cloning. We call these proteins Pals, for proteins associated with Lin-7. These proteins, which include mammalian Lin-2, contain a conserved mLin-7 binding domain in addition to guanylate kinase, PDZ (postsynaptic density 95/discs large/zona occludens-1), and Src homology 3 domains. Using site-directed mutagenesis, we have identified the conserved residues among these proteins crucial for mLin-7 binding. Two of these proteins, Pals1 and Pals2, are newly described. Pals1 consists of 675 amino acids and maps to mouse chromosome 12. Pals2 was found to exist in two splice forms of 539 and 553 amino acids and maps to mouse chromosome 6. Like mLin-2, Pals1 and Pals2 localize to the lateral membrane in Madin-Darby canine kidney cells. Pals proteins represent a new subfamily of membrane-associated guanylate kinases that allow for multiple targeting complexes containing mLin-7.

Identification of an evolutionarily conserved heterotrimeric protein complex involved in protein targeting.

In Caenorhabditis elegans, lin-2, lin-7, and lin-10 genetically interact to control the trafficking of the Let-23 growth factor receptor to the basolateral surface of body epithelia. The human homologue of the lin-10 gene has recently been identified as a member of the X11 gene family. The X11 proteins contain one phosphotyrosine binding (PTB) and two PSD-95.Dlg.ZO-1 (PDZ) domains as well as an extended amino terminus. We have previously shown that the PTB domain of X11alpha (also known as Mint1) can bind to the amyloid precursor protein (APP) in a phosphotyrosine-independent fashion and can markedly inhibit the processing of APP to the amyloid beta (Abeta) peptide. Here, we report that X11alpha directly binds to the mammalian homologue of Lin-2 (mLin-2), also known as CASK. This binding is mediated by direct interaction between the Calmodulin Kinase II (CKII)-like domain of mLin-2 and the amino terminus of X11alpha. Furthermore, we can detect direct interactions between mLin-2 and mammalian Lin-7 (mLin-7). In mouse brain, we have identified a heterotrimeric complex that contains mLin-2, mLin-7, and X11alpha and that is likely important for the localization of proteins in polarized cells. This complex may play an important role in the trafficking and processing of APP in neurons.