Diagnosis of triple negative breast cancer using expression data with several machine learning tools.

Breast cancer is one of the top three commonly caused cancers worldwide. Triple Negative Breast Cancer (TNBC), a subtype of breast cancer, lacks expression of the oestrogen receptor, progesterone receptor, and HER2. This makes the prognosis poor and early detection hard. Therefore, AI based neural models such as Binary Logistic Regression, Multi-Layer Perceptron and Radial Basis Functions were used for differential diagnosis of normal samples and TNBC samples collected from signal intensity data of microarray experiment. Genes that were significantly upregulated in TNBC were compared with healthy controls. The MLP model classified TNBC and normal cells with anaccuracy of 93.4%. However, RBF gave 74% accuracy and binary Logistic Regression model showed an accuracy of 90.0% in identifying TNBC cases.

Strategies for smoking cessation (pharmacologic intervention versus enhanced motivation vs. standard motivation) in TB patients under treatment in the RNTCP, India - A cluster - Randomized trial.

BACKGROUND: Tuberculosis burden is still high and smoking prevalence among males has increased in India. It is found that increased morbidity, mortality and relapse among TB smokers. METHOD: Setting: Patients from two Revised National Tuberculosis Control Program Centres of Tamilnadu form the study population. OBJECTIVE: To compare the effectiveness of Bupropion therapy along with standard counselling versus enhanced counseling versus standard counseling for smoking cessation among TB patients. STUDY DESIGN: Cluster randomized effectiveness trial. PROCEDURE: Patients from each of the thirty-six Designated Microscopic Centres were randomly allocated to receive one of the three interventions using cluster randomization. Smoking cessation was assessed by self-reporting and confirmed by Carbon monoxide(CO) monitors, done at three-time points and TB treatment outcome at the end of ATT. RESULTS: Out of 517 male patients enrolled to the study, the smoking status is available only to 381 subjects. The proportion of patients who have quit smoking in drug, enhanced and standard arms at the end of treatment was 67%, 83% and 52% (P= < 0.001). There was no statistical significance in response to TB treatment between those who quit and those who did not (Favourable response 99.2% vs 97.6%). CONCLUSION: Both enhanced counselling arm and drug arm are effective strategies for smoking cessation among TB patients and their implementation in the TB programs are recommended.

Physician's advice on quitting smoking in HIV and TB patients in south India: a randomised clinical trial.

Setting: National Institute for Research in Tuberculosis, Madurai, India. Objective: To determine the efficacy of physician's advice on quitting smoking compared with standard counselling in patients with tuberculosis (TB) and patients with human immunodeficiency virus (HIV) infection. Design/Methods: This was a clinical trial conducted in Madurai, south India, among 160 male patients (80 with TB and 80 with HIV), randomised and stratified by nicotine dependence (low/high according to the Fagerström scale), who received physician's advice with standard counselling or standard counselling alone for smoking cessation. Abstinence at 1 month was assessed by self-report and carbon monoxide breath analysis. Results: The patients' mean age was 39.4 years (SD 8.5). Overall, 35% of the patients had high nicotine dependence. Most patients (41%) smoked both cigarettes and bidis. In a combined analysis including both the HIV and the TB groups, quit rates were 41% of the 68 patients in the physician group and 35% of the 68 patients in the standard counselling arm. Conclusions: Physician's advice to quit smoking delivered to patients with TB or HIV is feasible and acceptable. Smoking cessation could easily be initiated in TB patients in programme settings. Future studies should assess long-term abstinence rates with a larger sample size to demonstrate the efficacy of physician's advice.

Contexte : National Institute for Research in Tuberculosis (NIRT), Madurai, Inde.Objectif : Déterminer l'efficacité des conseils d'un médecin comparés aux conseils standard d'arrêt du tabac chez des patients atteints de tuberculose (TB) et chez des patients infectés par le virus de l'immunodéficience humaine (VIH).Schéma/Méthodes : Cet essai clinique a été réalisé à Madurai, dans le sud de l'Inde. Cent soixante patients masculins (80 TB et 80 VIH) ont été randomisés, stratifiés en fonction de leur dépendance à la nicotine (faible/élevée selon l'échelle de Fagerström) pour bénéficier soit des conseils d'un médecin avec des conseils standard ou des conseils standard seuls pour l'arrêt du tabac. L'abstinence à un mois a été évaluée par déclaration des patients et analyse du monoxyde de carbone dans l'air expiré.Résultats : L'âge moyen (DS) a été de 39,4 (±8,5) ans. Dans l'ensemble, 35% des patients avaient une dépendance élevée à la nicotine. La majorité des patients (41%) fumait à la fois des cigarettes et des bidis (cigarettes indiennes). En analyse combinée (à la fois le groupe VIH et TB), les taux de cessation ont été de 41% sur 68 patients dans le groupe « médecin ¼ et de 35% de 68 patients dans le bras « conseil standard ¼ (non significatif).Conclusions: Les conseils d'un médecin en matière d'arrêt de tabac délivrés aux patients atteints de TB ou de VIH sont faisables et acceptables. L'arrêt du tabac pourrait bien être mis en œuvre dans le contexte des programmes chez les patients TB. De futures études devraient évaluer le taux d'abstinence à long terme avec des échantillons de plus grande taille afin de démontrer l'efficacité des conseils d'un médecin.

Marco de referencia: El Instituto Nacional de Investigación en Tuberculosis de Madurai, en la India.Objetivo: Comparar la eficacia de los consejos que da el médico con el asesoramiento corriente sobre el abandono del tabaco, en pacientes aquejados de tuberculosis (TB) o infección por el virus de la inmunodeficiencia humana (VIH).Métodos: El ensayo clínico inicial se llevó a cabo en Madurai, en el sur de la India. Se escogieron de manera aleatoria 160 pacientes de sexo masculino (80 con diagnóstico de TB y 80 de infección por el VIH), se estratificaron en función de la dependencia de la nicotina (baja y alta, según la escala de Fagerström) y se asignaron a un grupo que recibiría consejos del médico además del asesoramiento corriente sobre el abandono del tabaco o un grupo que solo recibiría el asesoramiento corriente. Al cabo de un mes, se evaluó la abstinencia a partir de la información proporcionada por los pacientes y la medición de monóxido de carbono en el aire espirado.Resultados: El promedio de edad de los participantes fue 39,4 años (desviación estándar 8,5 años). En general, el 35% de los participantes exhibía una alta dependencia de la nicotina. La mayoría fumaba cigarrillos y también bidis (41%). En el análisis conjunto (ambos grupos: TB y VIH), las tasas de abandono fueron 41% en los 68 pacientes del grupo que recibió consejo médico y 35% en los 68 pacientes del grupo que obtuvo asesoramiento corriente (diferencia no significativa).Conclusión: El consejo sobre el abandono del tabaco ofrecido por el médico a los pacientes con diagnóstico de TB o infección por el VIH es factible y bien aceptado. Es posible iniciar intervenciones de abandono del tabaco con los pacientes TB en el marco programático. Futuros estudios podrían evaluar las tasas de abstinencia a largo plazo con muestras más grandes, a fin de demostrar la eficacia del consejo dado por los médicos.

Sequential release of drugs from hollow manganese ferrite nanocarriers for breast cancer therapy.

Single drug therapies for cancer are often suboptimal and may not provide long term clinical benefits. To overcome this obstacle for effective treatment the applications of two or more drugs are preferable. A limitation of multidrug use is the varying pharmacokinetics of different drugs. To overcome these impediments, we designed and synthesized multi-layered polyvinyl alcohol tethered hollow manganese ferrite nanocarriers capable of encapsulating two drugs with unique attributes of sensitivity towards tumor acidic milieu, mono-dispersive, compactness and high encapsulation efficiency. We encapsulated tamoxifen and diosgenin in the peripheral and subsequent inner layers of multilayered nanocarriers. In vitro and in vivo studies evaluated the nanocarrier uptake and retention ability of the tumor through magnetic saturation studies and elucidated the molecular mechanisms mediating drug(s)-induced apoptosis. The acidity of the tumor environment triggers extracellular dissociation of the peripheral coats resulting in release of tamoxifen blocking the estrogen receptor. The partially degraded nanocarriers localize intracellularly through endosomal escape and release diosgenin. Nanocarrier treatment reduced the cellular levels of Bcl2 and p53, while increasing the levels of Bim. This delivery system successfully embodies the sequential release of drugs and may provide a therapeutic strategy for sequentially affecting multiple targets in advanced cancers.

Are we justified in treating for multidrug-resistant tuberculosis based on positive follow-up smear results?

SETTING: National Institute for Research in Tuberculosis, India. OBJECTIVE: To assess, among new culture-confirmed smear-positive pulmonary tuberculosis (TB) patients, the proportion of follow-up smear-positives that were culture-negative (S+C-) by month of follow-up examination, human immunodeficiency virus (HIV) status, pre-treatment drug susceptibility status and smear grading. DESIGN: We extracted follow-up smear (fluorescence microscopy) and culture (Löwenstein-Jensen) results of patients enrolled in clinical trials from January 2000 to August 2012 and treated with the WHO Category I regimen (2EHRZ3/4HR3). RESULTS: Of 520 patients, including 176 who were HIV-infected, respectively 199, 81, 47 and 43 were smear-positive at months 2, 4, 5 and 6; of these, respectively 138 (69%), 62 (75%), 32 (68%) and 27 (63%) were culture-negative. The S+C- phenomenon was more pronounced among '1+ positive' patients than in 2+ or 3+ positive patients and in 'pan-susceptible' patients than in those with any resistance, and did not vary by HIV status. CONCLUSION: Nearly two thirds of patients with follow-up smears positive at months 5 and 6 were culture-negative. Starting multidrug-resistant TB (MDR-TB) treatment empirically based on smear results, even in resource-limited settings, is incorrect and can have hazardous consequences. There is an urgent need to revisit the WHO recommendation concerning empirical MDR-TB treatment.

AEE788 potentiates celecoxib-induced growth inhibition and apoptosis in human colon cancer cells.

AIMS: Combinatorial therapies that target multiple signaling pathways may provide improved therapeutic responses over monotherapies. In the present study, we evaluated the effect of celecoxib and AEE788 alone and in combination on cell proliferation, invasion, migration, angiogenesis, morphological changes, actin filament organization and apoptosis induction in the human colon cancer cell lines. MAIN METHODS: Effect of celecoxib and AEE788 alone and in combination on colon cancer cell lines was evaluated by cell proliferation assay, morphological analysis, cell cycle analysis, scratch-wound healing and chorioallantoic membrane assays, zymography, nuclear fragmentation and western blot analyses. KEY FINDINGS: Either drug alone or in combination inhibited human colonic adenocarcinoma cell lines HCT 15 and HT 29 in a dose-dependent manner. Microscopic analysis revealed inhibition of cell membrane extensions, cell shrinkage, and disorganization of actin filaments. Additionally, either drug alone or in combination inhibited HCT 15 migration, invasion and angiogenesis by suppressing matrix metalloproteinase-2 and -9 activities. Increased reactive oxygen generation, loss of mitochondrial membrane potential, cleavage of PARP, caspase-3 activation and DNA ladder formation characterized the induction of apoptosis by celecoxib and/or AEE788 treatment. Either drug individually induced apoptosis via down-regulation of the anti-apoptotic proteins Bcl(2) and Bcl-x(L), and up-regulation of pro-apoptotic protein Bax, cleavage of PARP, activation of caspase-3 and inhibition of vascular endothelial growth factor receptor signaling pathways. SIGNIFICANCE: Results indicate that AEE788 potentiates celecoxib-mediated inhibition of proliferation and angiogenesis in HCT 15 colon cancer cells and may prove useful for developing a combinatorial therapy for colon cancer.

Survival analysis of women with breast cancer under adjuvant therapy in South India.

While there has been much research in identifying risk factors and prognostic factor for breast cancer for breast cancer survival, the research specific to South Indian population is limited: Most of the association studies between breast cancer and risk factor have been widely studied in developed countries. This study attempts to explore the survival experience of breast cancer patients treated under adjuvant and neo-adjuvant therapy. The data were obtained from a Government Cancer Hospital, Tamil Nadu, South India and included 522 women diagnosed and treated with adjuvant and neo-adjuvant therapy between January 2000 to December 2008 and follow up to May 2010. The survival experiences under two treatments are presented using Kaplan-Meier survival curves. The important prognostic variables for response to treatment survival were identified using Cox regression with and without time-dependent covariates. Of the 522 cases, 248(47.5%) were of stage 2 (A and B), 249 (47.7%) were of stage 3 (A and B). About 90% received neo-adjuvant therapy. About 94% of the patients had response to treatment. The Cox model showed that apart from the chemotherapy, number of children, child birth status and stage 3B and 4 turn out to be significant predictors for response to treatment survival. This is the first study to evaluate adjuvant therapy effects under hospital setup in South India. The results show that response to treatment survival is related poor in advanced stage patients under treatment.

The potential of celecoxib-loaded hydroxyapatite-chitosan nanocomposite for the treatment of colon cancer.

Celecoxib has shown potential anticancer activity against most carcinomas, especially in patients with familial adenomatous polyposis and precancerous disease of the colon. However, serious side effects of celecoxib restrict its generalized use for cancer therapy. In order to resolve these issues and develop an alternative strategy/preliminary approach, chitosan modified hydroxyapatite nanocarriers-mediated celecoxib delivery represents a viable strategy. We characterized the nanoparticle for morphology, particle size, zeta potential, crystalinity, functional group analysis, entrapment efficiency, drug release and hemocompatibility. The effects of celecoxib-loaded nanoparticles on colon cancer cell proliferation, morphology, cytoskeleton, cellular uptake and apoptosis were analysed in vitro. Further, we evaluated the antiproliferative, apoptotic and tumor inhibitory efficacy of celecoxib-loaded nanocarriers in a nude mouse human xenograft model. Nanoparticles exhibited small, narrow hydrodynamic size distributions, hemocompatibility, high entrapment efficiencies and sustained release profiles. In vitro studies showed significant antiproliferation, apoptosis and time-dependent cytoplasmic uptake of celecoxib-loaded Hap-Cht nanoparticles in HCT 15 and HT 29 colon cancer cells. Additional in vivo studies demonstrated significantly greater inhibition of tumor growth following treatment with this modified nanoparticle system. The present study indicates a promising, effective and safe means of using celecoxib, and potentially other therapeutic agents for colon cancer therapy.

Effect of AEE788 and/or Celecoxib on colon cancer cell morphology using advanced microscopic techniques.

Analysis of changes in cancer cell morphology and cytoskeletal element induced by external stimuli is focus of current cancer chemotherapeutic studies. Cancer cell cytoskeleton is complex network of interwoven protein fibers composed of microtubules, microfilaments and intermediate filaments. These interwoven protein fibers are responsible for maintaining cell morphology, movement, adhesion and transmembrane signal transmission. In this study, morphological and cytoskeletal changes induced by AEE788 and/or Celecoxib on colon cancer cell HCT 15 were analyzed using advanced microscopic techniques. Cell proliferation assay was used for determining IC(50) of AEE788 and/or Celecoxib on HCT 15. Confocal microscopic analysis of AEE788 and/or Celecoxib treated HCT 15 was performed using Rhodamine-Phalloidin (actin stain) and Hoechst 33342 (nuclear stain). Atomic force (AFM) and scanning electron microscopic (SEM) studies were also performed to analyze cell morphology and cell wall extension (filopodia and lamellipodia). In addition, quantitative analysis of morphological parameters was studied using cellular image processing technique. This is the first report that combination of AEE788 and Celecoxib additively increase growth inhibition and cell death on human colon cancer cell HCT 15 as estimated by cell proliferation assay. Morphological analysis of AEE788 or Celecoxib treated HCT 15 cell for 24h have not revealed significant change in morphology under phase contrast microscopy. But, slight morphological changes were observed in combination (AEE788+Celecoxib) treated HCT 15 for 24h. In contrast, high resolution confocal laser fluorescence and atomic force microscopic studies have revealed cell shrinkage, disorganized actin filament and, loss of filopodia and lamellipodia. These changes were more prominent in combination of AEE788 and Celecoxib treated HCT 15 than either drug alone. These results may suggest antiproliferative and antimetastatic activity of AEE788 and/or Celecoxib. Quantitative analysis of morphological parameters using cellular image processing technique have shown decrease in mean area, perimeter, compactness and eccentricity of combination drug treated cells than either drug alone. These results further support the confocal and AFM study. Scanning electron microscopic study of AEE788 and/or Celecoxib treated HCT 15 has also shown morphological changes and loss of filopodia and lamellipodia. In conclusion, this investigation of morphological and cytoskeletal changes using advanced microscopic techniques present a significant foundation for evaluating anticancer activity of a drug and form a new strategy for evaluating effect of AEE788 and/or Celecoxib on colon cancer.

Defective solubilization of immune complexes and activation of the complement system in patients with pulmonary tuberculosis.

INTRODUCTION: Association between inherited deficiencies of the complement components and immune complex disease indicates the importance of the complement system in the handling of circulating immune complexes. High levels of circulating immune complexes are seen in pulmonary tuberculosis. This study is, therefore, aimed to look at the concentration of circulating immune complexes, the status of complement-mediated immune complex handling, and the extent of complement activation in untreated pulmonary tuberculosis compared to treated pulmonary tuberculosis patients and healthy controls. RESULTS: High immune complex levels, decreased complement-mediated solubilization, and increased activation of the complement system were observed in untreated tuberculosis patients. CONCLUSIONS: The results obtained from the present study suggest that complement mediated solubilization is less in patients with tuberculosis, and this defective solubilization is likely to take part in a vicious cycle involving immune complex deposition and complement activation and, thus, may lead to disease progression depending on the nature of the defect.

Combination therapeutic effect of cisplatin along with Solanum trilobatum on benzo(a)pyrene induced experimental lung carcinogenesis.

Lung cancer is one of the leading causes of cancer death in the world and is notoriously difficult to treat effectively. In the present study, male Swiss albino mice were divided into five groups of six animals each: group I animals received corn oil orally and served as a control; group II cancer-induced animals received benzo(a)pyrene (B[a]P) (50 mg kg(-1) bodyweight dissolved in corn oil, orally) twice weekly for four successive weeks; group III cancer-bearing animals (after 12 weeks of induction) were treated with cisplatin (6 mg kg(-1) bodyweight, i.p.) once weekly for 4 weeks; group IV cancer-bearing animals were treated with cisplatin along with Solanum trilobatum (300 mg kg(-1) bodyweight) orally once weekly for 4 weeks; and group V animals constituted the drug control treated with cisplatin along with S. trilobatum. The serum, lung and liver were investigated biochemically for aryl hydrocarbon hydroxylase, gamma-glutamyl transpeptidase, 5'-nucleotidase, lactate dehydrogenase (LDH) and protein-bound carbohydrate components (hexose, hexosamine and sialic acid). These enzyme activities were increased significantly in cancer-bearing animals compared with control animals. The elevation of these in cancer-bearing animals was indicative of the persistent deteriorating effect of B[a]P in cancer-bearing animals. Our data suggest that cisplatin, administered with S. trilobatum, may extend its chemotherapeutic effect through modulating protein-bound carbohydrate levels and marker enzymes, as they are indicators of cancer. The combination of cisplatin with S. trilobatum could effectively treat the B[a]P-induced lung cancer in mice by offering protection from reactive oxygen species damage and also by suppressing cell proliferation.

Site-directed alkylation and the alternating access model for LacY.

In a functional lactose permease mutant from Escherichia coli (LacY) devoid of native Cys residues, almost every residue was replaced individually with Cys and tested for reactivity with the permeant alkylating agent N-ethylmaleimide in right-side-out membrane vesicles. Here we present the results in the context of the crystal structure of LacY. Engineered Cys replacements located near or within the inward-facing hydrophilic cavity or at other solvent-accessible positions in LacY react well with this alkylating agent. Cys residues facing the low dielectric of the membrane or located in tightly packed regions of the structure react poorly. Remarkably, in the presence of ligand, increased reactivity is observed with Cys replacements located predominantly on the periplasmic side of the sugar-binding site. In contrast, other Cys replacements largely on the cytoplasmic side of the binding site exhibit decreased reactivity. Furthermore, both sets of Cys replacements in the putative cavities are located at the periplasmic (increased reactivity) and cytoplasmic (decreased reactivity) ends of the same helices and distributed in a pseudosymmetrical manner. The results are consistent with a model in which the single sugar-binding site in the approximate middle of the molecule is alternately exposed to either side of the membrane due to opening and closing of cytoplasmic and periplasmic hydrophilic cavities.

HIV alters plasma and M. tuberculosis-induced cytokine production in patients with tuberculosis.

To test the hypothesis that HIV infection brings about an alteration in the immune response to tuberculosis (TB), mycobacterial antigen-induced production and plasma levels of the inflammatory cytokine interferon-gamma (IFN-gamma) and its regulatory cytokines interleukin-12 (IL-12), IL-18, and IL-10 were determined in patients infected dually with HIV and TB and compared with individuals with either disease and with healthy controls. Peripheral blood mononuclear cells (PBMCs) of TB patients with HIV infection produced lesser amounts of IFN-gamma and IL-12 compared with TB patients without HIV infection after in vitro stimulation with mycobacterial antigens. There was no difference in antigen-induced IL-18 production in TB patients with or without HIV infection. The in vivo cytokine pattern did not correlate with that seen in vitro. Higher levels of IFN-gamma, IL-12, and IL-18 were detected in the plasma of TB patients infected with HIV compared with TB patients without HIV infection. The presence of significantly higher plasma levels of proinflammatory cytokines suggests a greater degree of immune activation in individuals with HIV and TB, particularly those with low CD4 counts. In vitro IL-10 production by HIV-positive TB patients was similar to that of the HIV-negative TB group and higher than in HIV-positive individuals without TB, but the plasma levels were similar. HIV infection downregulates the in vitro Th1 cytokine response to TB and simultaneously increases systemic levels of these cytokines.

Native fluorescence spectroscopy of blood plasma in the characterization of oral malignancy.

Native fluorescence characteristics of blood plasma were studied in the visible spectral region, at two different excitation wavelengths, 405 and 420 nm, to discriminate patients with different stages of oral malignancy from healthy subjects. The fluorescence spectra of blood plasma of oral malignant subjects exhibit characteristic spectral differences with respect to normal subjects. Different ratios were calculated using the fluorescence intensity values at those emission wavelengths that give characteristic spectral features of each group of experimental subjects studied. These fluorescence intensity ratios were used as input variables for a multiple linear discriminant analysis across different groups. Leave-one out cross-validation was used to check the reliability of each discriminant analysis performed. The discriminant analysis performed across normal and oral cancerous subjects classified 94.7% of the original grouped cases and 93.7% of the cross-validated grouped cases. A classification algorithm was developed on the basis of the score of the discriminant functions (discriminant score) resulted in the analyses. The diagnostic potentiality of the present technique was also estimated in the discrimination of malignant subjects from normal and nonmalignant diseased subjects such as liver diseases. In the discriminant analysis performed across the three groups, normal, oral malignancy (including early and advanced stages) and liver diseases, 99% of the original grouped cases and 95.9% of the cross-validated grouped cases were correctly classified. Similar analysis performed across normal, early stage of oral malignancy, advanced oral malignancy and liver diseases correctly classified 94.9% of the original grouped cases and 91.8% of the cross-validated grouped cases.

Cytogenetic studies on spray painters in south India.

Studies on the frequencies of chromosomal aberrations were carried out on 104 spray painters working in automobile body reconditioning, steel furniture making and refrigerator repainting workshops in the metro city, Chennai, of south India. Randomly selected 50 male subjects not connected with this occupation were included as controls in the study. Chromosomal analysis was carried out in 48h lymphocyte (short duration) cultures representing the first mitotic division, on a subset of samples consisting of 50 spray painters, 20 controls and 72h (longer duration) cultures representing the second cell division, on all subjects. Baseline frequency of chromosomal aberrations was significantly higher among painters as compared to matched controls. Smoking and alcoholism as modulating factors had no added effect on the frequency of aberrant metaphases. Stepwise multiple linear regression analysis indicated that duration of service and age were significant factors that influence the frequency of chromosomal aberrations observed.

Site-directed sulfhydryl labeling of the lactose permease of Escherichia coli: helix VII.

Site-directed sulfhydryl modification in situ is employed to investigate structural and dynamic features of transmembrane helix VII and the beginning of the periplasmic loop between helices VII and VIII (loop VII/VIII). Essentially all of the Cys-replacement mutants in the periplasmic half of the helix and the portion of loop VII/VIII tested are labeled by N-[(14)C]ethylmaleimide (NEM). In contrast, with the exception of two mutants at the cytoplasmic end of helix VII, none of the mutants in the cytoplasmic half react with the alkylating agent. Labeling of most of the mutants is unaltered by ligand at 25 degrees C. However, at 4 degrees C, conformational changes induced by substrate binding become apparent. In the presence of ligand, permease mutants with a Cys residue at position 241, 242, 244, 245, 246, or 248 undergo a marked increase in labeling, while the reactivity of a Cys at position 238 is slightly decreased. Labeling of the remaining Cys-replacement mutants is unaffected by ligand. Studies with methanethiosulfonate ethylsulfonate (MTSES), a hydrophilic impermeant thiol reagent, show that most of the positions that react with NEM are accessible to MTSES; however, the two NEM-reactive mutants at the cytoplasmic end of helix VII and position 236 in the middle of the membrane-spanning domain are not. The findings demonstrate that positions in helix VII that reflect ligand-induced conformational changes are located in the periplasmic half and accessible to the aqueous phase from the periplasmic face of the membrane. In the following papers in this issue (Venkatesan, P., Lui, Z., Hu, Y., and Kaback H. R.; Venkatesan, P., Hu, Y., and Kaback H. R.), the approach is applied to helices II and X.

Site-directed sulfhydryl labeling of the lactose permease of Escherichia coli: N-ethylmaleimide-sensitive face of helix II.

Cys-scanning mutagenesis of helix II in the lactose permease of Escherichia coli [Frillingos, S., Sun, J. et al. (1997) Biochemistry 36, 269-273] indicates that one face contains positions where Cys replacement or Cys replacement followed by treatment with N-ethylmaleimide (NEM) significantly inactivates the protein. In this study, site-directed sulfhydryl modification is utilized in situ to study this face of helix II. [(14)C]NEM labeling of 13 single-Cys mutants, including the nine NEM-sensitive Cys replacements, in right-side-out membrane vesicles is examined. Permease mutants with a single-Cys residue in place of Gly46, Phe49, Gln60, Ser67, or Leu70 are alkylated by NEM at 25 degrees C in 10 min, and mutants with Cys in place of Thr45 and Ser53 are labeled only in the presence of ligand, while mutants with Cys in place of Ile52, Ser56, Leu57, Leu62, Phe63, or Leu65 do not react. Binding of substrate leads to a marked increase in labeling of Cys residues at positions 45, 49, or 53 in the periplasmic half of helix II and a slight decrease in labeling of Cys residues at positions 60 or 67 in the cytoplasmic half. Labeling studies with methanethiosulfonate ethylsulfonate (MTSES) show that positions 45 and 53 are accessible to solvent in the presence of ligand only, while positions 46, 49, 67, and 70 are accessible to solvent in the absence or presence of ligand. Position 60 is also exposed to solvent, and substrate binding causes a decrease in solvent accessibility. The findings demonstrate that the NEM-sensitive face of helix II participates in ligand-induced conformational changes. Remarkably, this membrane-spanning face is accessible to the aqueous phase from the periplasmic side of the membrane. In the following paper in this issue [Venkatesan, P., Hu, Y., and Kaback, H. R. (2000) Biochemistry 39, 10656-10661], the approach is applied to helix X.

Site-directed sulfhydryl labeling of the lactose permease of Escherichia coli: helix X.

Helix X in the lactose permease of Escherichia coli contains two residues that are irreplaceable with respect to active transport, His322 and Glu325, as well as Lys319, which is charge-paired with Asp240 in helix VII. Structural and dynamic features of transmembrane helix X are investigated here by site-directed thiol modification of 14 single-Cys replacement mutants with N-[(14)C]ethylmaleimide (NEM) in right-side-out membrane vesicles. Permease mutants with a Cys residue at position 326, 327, 329, 330, or 331 in the cytoplasmic half of the transmembrane domain are alkylated by NEM at 25 degrees C, a mutant with Cys at position 315 at the periplasmic surface is labeled in the presence of substrate exclusively, and mutants with Cys at positions 317, 318, 320, 321, 324, 328, 332, or 333 do not react with NEM under the conditions tested. Binding of substrate causes increased labeling of a Cys residue at position 315 and decreased labeling of Cys residues at positions 326, 327, and 329. Studies with methanethiosulfonate ethylsulfonate indicate that Cys residues at positions 326, 329, 330, and 331 in the cytoplasmic half are accessible to the aqueous phase from the periplasmic face of the membrane. Ligand binding results in clear attenuation of solvent accessibility of Cys at position 326 and a marginal increase in accessibility of Cys at position 327 to solvent. The findings indicate that the cytoplasmic half of helix X is more reactive/accessible to thiol reagents and more exposed to solvent than the periplasmic half. Furthermore, positions that reflect ligand-induced conformational changes are located on the same face of helix X as Lys319, His322, and Glu325.

Inhibition of radiation-induced lipid peroxidation by tetrahydrocurcumin: possible mechanisms by pulse radiolysis.

The antioxidant property of tetrahydrocurcumin (THC), a reduced derivative of curcumin, was examined by its ability to inhibit radiation-induced lipid peroxidation in rat liver microsomes and compared with curcumin. The lipid peroxidation caused by irradiation of N2O-purged and aerated buffered aqueous solutions was found to be inhibited by THC in a dose- and concentration-dependent manner. In order to understand the actual reaction mechanisms involved in the inhibition process, pulse radiolysis investigation of THC with radiolytically produced radicals like hydroxyl, model peroxyl radicals, and azide radicals were done and the transients were detected by kinetic spectrophotometry. The reaction of THC with hydroxyl and azide radicals gave rise to transient absorption in the region 200-400 nm with two peaks at 310 nm and 390 nm. From the spectral properties and kinetics of these radicals, a suitable mechanism is discussed to explain the antioxidant actions of THC.

Characterization of multidrug resistance and monitoring of tumor response by combined 31P and 1H nuclear magnetic resonance spectroscopic analysis.

A combined 31P and 1H nuclear magnetic resonance (NMR) spectroscopic study was carried out in drug-sensitive and adriamycin- and mitoxantrone-resistant P388 murine leukemic cells. Typical spectral changes characteristic of multidrug resistance were observed in resistant cells compared to drug-sensitive cells. Quantitative comparison of phosphate metabolites ATP, phosphocreatine, phosphomonoesters and phosphodiesters revealed a significant alteration in the metabolism of resistant cells. The elevated levels of energy metabolites supported the energy-dependent process of drug efflux in resistant cells. An increased rate of glycolysis in resistant cells as indicated by the elevated lactate level further supported this. The near total loss of energy metabolites and marked decrease in phospholipid metabolites in sensitive cells upon treatment with drug compared to unaltered metabolite levels in resistant cells suggested that the spectral changes can reveal the subtle differences in tumor response between drug-sensitive and -resistant cells. The results substantiate the potential of this non-invasive method to characterize the multidrug resistance phenotype and monitor tumor response.