Doublecortin-like kinase 1 expression associates with breast cancer with neuroendocrine differentiation.

Doublecortin-like kinase 1 (DCLK1), a microtubule associated kinase, has recently been proposed to be a putative marker for stemness and adverse prognosis in gastrointestinal cancers. However, it is not clear whether the protein also plays similar roles in breast cancer. Here, the expression of DCLK1 was analyzed in a large cohort of invasive breast cancers (IBC) by immunohistochemistry. DCKL1 was associated with favorable clinico-pathologic features, namely lower histologic grade, absence of lymphovascular invasion, fibrotic focus, necrosis and lower pN stage (p≤0.045). Additionally, independent significant correlations were found with estrogen receptor and neuroendocrine markers (p ≤0.019), implicating its relationship with IBC with neuroendocrine differentiation (IBC-NED). In the current cohort, IBC-NED showed worse outcome than luminal cancers without NED (hazard ratio=1.756, p=0.041). Interestingly, within the IBC-NED group, DCLK1 was found to be a good prognostic factor (hazard ratio =0.288, p=0.011). These findings were in contrast to those in gastrointestinal cancers, suggesting different functional roles of DCLK1 in different types of cancers. In clinical practice, NED is not routinely assessed; thus IBC-NED are not well studied. Its poor outcome and significant heterogeneity warrants more attention. DCLK1 expression could aid in the prognostication and management of this special cancer subtype.

Cellular and molecular mechanism of ofloxacin induced apoptotic cell death under ambient UV-A and sunlight exposure.

Ofloxacin (OFLX) is a racemic mixture of levofloxacin which revealed phototoxicity in patients exposed with sunlight after medication. Here, we have been addressed the possible cellular and molecular mechanisms of OFLX induced apoptosis under ambient UV-A and sunlight exposure using HaCaT cell line as a model. The results showed that Photodegradation and three photo-products formation of OFLX by LC-MS/MS under ambient intensities of UV-A (1.5 and 2.2 mW/cm(2)) and sunlight. OFLX produced (1)O2, O2(.-), and OH radicals via type-II- and type-I-dependent reaction mechanism, which corroborated by its specific quenchers. 2'-dGua degradation in photochemical and % tail DNA formation in cell line using comet test advocated the genotoxic potential of OFLX. Photocytotoxic assays (MTT and NRU) revealed the considerable decline in cell viability by OFLX. OFLX triggered apoptosis, proved by cell cycle, Annexin V/PI double staining along with acridine orange (AO)/ethidium bromide (EB), and Hoechst staining as well as caspase-3 activity by colorimetric assay. OFLX induced lysosomal disruption and mitochondrial membrane destabilization confirmed through fluorescence staining with AO/JC-1. OFLX significantly upregulated the expression of p21 and bax genes. In conclusion, the study revealed that photosensitized OFLX induced apoptosis via ROS-mediated DNA damage, destabilization of lysosomal and mitochondrial membrane, and upregulation of p21, bax, and caspase-3 genes.

Fibrotic focus in breast carcinomas: relationship with prognostic parameters and biomarkers.

BACKGROUND: Fibrotic focus (FF) has been observed in breast cancers and is suggested to be an important prognostic marker. However, most of these observations were reported by the same group of investigators with similar sample cohort. The relationship of FF and molecular subtypes as well as its associated prognosis has not been elucidated. METHODS: In this study, 450 cases of breast carcinomas were evaluated for the presence of FF and its association with clinicopathologic parameters and biomarkers. RESULTS: FF was found in 18.7% of all consecutive cases. FF was associated positively with infiltrative margins (p=0.03) but negatively with extensive in situ component (p<0.001) and lymphocytic infiltration (p<0.001). It was positively associated with estrogen receptor (p=0.007) but negatively with human epidermal growth factor receptor 2 (HER2; p=0.001), epidermal growth factor receptor (p=0.021), Ki-67 (p=0.001), and c-kit (p=0.009). Concomitantly, FF was seen more commonly in luminal A cancers (p<0.001) but less so in luminal B (p=0.045) and HER2-overexpressing cancers (p=0.011). Analysis on patient outcome (median 41 months, range 1-69 months) indicated that FF was an independent poor prognostic factor for disease-free survival (hazard ratio=2.57; 95% confidence interval=1.267-5.214, p=0.009), particularly in the luminal B subtype. CONCLUSIONS: The findings suggested that FF is associated with specific tumor morphology of an infiltrative, stellate pattern (typical invasive ductal carcinoma-not otherwise specified) rather than round, cellular mass with intense lymphocytic infiltrate (basal-like breast cancers). The poor prognostic implication of FF is additional and independent of other adverse prognostic indicators.

High incidence of Epstein Barr virus infection in childhood acute lymphocytic leukemia: a preliminary study.

INTRODUCTION: Epstein Barr virus (EBV) has a unique association with several human malignancies, especially lymphoproliferative disorders, mainly lymphomas in adults. There is paucity of data pertaining to EBV association with various cancers in India . OBJECTIVE: The study aims to investigate the association of EBV in childhood leukemia. MATERIAL AND METHODS: Patients attending pediatric oncology services of the referral center have been included in the study. Twenty-five consecutive pediatric patients with acute lymphocytic lukemia (ALL) were subjected to EBV studies employing sensitive polymerase chain reaction followed by hybridization for presence of Bam H1-W region of EBV genome and detection of anti Z EBV replication activator (ZEBRA) antibodies using Western blot. Positive control included a case of Burkitt's lymphoma and infectious mononucleosis each. Raji cells were used as positive control with each test. RESULTS: The PCR for EBV was positive in 8/25 patients of ALL. Western blot test using anti ZEBRA antibodies was positive in 5/25(20%) cases of ALL. Considering PCR as the gold standard, 32% of the children with ALL had evidence of active EBV replication. The positive controls were consistently positive. None of the 30 healthy laboratory controls, 22 age matched disease controls, 12 cases of AML and 15 cases of multiple myeloma were positive either by PCR or Western blots assays (P < 0. 01). There was no statistically significant correlation between duration of therapy and EBV positivity (P > 0.05). CONCLUSION: These studies indicate that a significant number of patients with ALL show evidence of active EBV replication.

Structure and acetyl-lysine recognition of the bromodomain.

Histone lysine acetylation is central to epigenetic control of gene transcription. The bromodomain, found in chromatin-associated proteins and histone acetyltranferases, functions as the sole protein module known to bind acetyl-lysine motifs. Recent structural and functional analyses of bromodomains' recognition of lysine-acetylated peptides derived from major acetylation sites in histones and cellular proteins provide new insights into differences in ligand binding selectivity as well as unifying features of histone recognition by the bromodomains. These new findings highlight the functional importance of bromodomain/acetyl-lysine binding as a pivotal mechanism for regulating protein-protein interactions in histone-directed chromatin remodeling and gene transcription. These new studies also support the notion that functional diversity of a conserved bromodomain structural fold is achieved by evolutionary changes of structurally flexible amino-acid sequences in the ligand binding site such as the ZA and BC loops.

Leishmania donovani: identification of novel vaccine candidates using human reactive sera and cell lines.

Leishmaniasis is a complex of diseases caused by protozoan parasites belonging to the genus Leishmania. The development of specific resistance against re-infection after cure suggests that a vaccine approach is feasible. Various studies in humans and experimental animals strongly suggest that Th1 type of cell-mediated immune response is important for protection against the disease. A defined antigen that could elicit a specific T-cell-mediated immune response in the host would be an ideal candidate for the vaccine against this parasite. In order to select a candidate antigen, we established a screening system to identify the recombinant clone, expressing antigen having T-cell epitopes from a cDNA library. We screened the library using an established Leishmania specific cell line (LSCL) from a naive healthy human subject. The cell line with predominantly CD4+ cells behaved in a Leishmania specific manner. Fifty-two immuno-reactive clones were screened against the LSCL in vitro and we identified three cDNA clones expressing recombinant antigens that could induce proliferation of these cells to produce INFgamma. The protective efficacy of one of these recombinant proteins was investigated in a hamster model of experimental visceral leishmaniasis and showed protection against a virulent challenge. The identified antigens might be potential candidates for vaccine against Leishmania.

Coinfection with epstein barr virus in north Indian patients with HIV/AIDS.

Acquired immunodeficiency syndrome (AIDS) has emerged as a serious health problem in India. Although tuberculosis appears to be the commonest opportunistic infection, studies pertaining to opportunistic viruses are scant In the present study co infection with EBV was evaluated in patients with AIDS using a highly sensitive polymerase chain reaction besides anti Zebra antibody assays for diagnosis of an active EBV infection in 37 patients of full-blown AIDS and 32 healthy seropositives. Thirty healthy laboratory workers were used as controls. Out of 37 patients with AIDS, 12 were positive for anti Zebra antibodies and 23 were positive for EBV by the PCR reaction. Out of the 32 seropositives, 3 were positive for anti Zebra antibodies and 4 were positive by PCR assay. The difference between seropositives and AIDS was significant (p < .05). None of the controls were positive for an active EBV infection. It is concluded that active EBV infection is an important co infection in patients with AIDS and may contribute significantly to morbidity and mortality in these patients.

Cytomegalovirus co-infection in patients with HIV/AIDS in north India.

BACKGROUND & OBJECTIVES: Cytomegalovirus (CMV) is a frequent opportunistic infection in immunocompromised individuals particularly those receiving organ transplants and harbouring HIV infection. The classical CMV syndrome may be seen in only a small percentage of patients and tissue diagnosis is cumbersome, costly and requires hospitalization. Hence there is an urgent need to establish accurate and early diagnosis for proper institution of therapy. An attempt was made to detect active CMV co-infection in patients with HIV/AIDS using three assays and the positivity rates in the 2 groups compared. METHODS: In the present study, we used a highly sensitive polymerase chain reaction (PCR) for immediate early gene of CMV, pp65 antigenaemia assay and IgM ELISA assay to detect the presence of CMV co-infection in 37 patients with AIDS and 32 healthy HIV seropositives. Thirty healthy laboratory workers served as normal controls. RESULTS: Of the 37 patients with AIDS, 12 (32.4%) showed a positive reaction by PCR and only 4 patients were positive by the antigenaemia assay. Of the 32 HIV seropositives, only one was positive by PCR (3%), and all were negative for antigen assay. None of the controls showed positivity by any of the tests. The difference in PCR positivity rates between HIV seropositives and patients with AIDS was significant (P < 01). IgM antibodies were positive in four (10.3%) AIDS patients and only one (3%) HIV seropositive, the difference was insignificant. The difference in antigen positivity between IIIV seropositives and AIDS patients was also insignificant. INTERPRETATION & CONCLUSION: CMV appears to be an important co-infection in patients with AIDS in India and PCR is a powerful tool for detection of CMV in blood and is superior to the antigenaemia assay. PCR can be performed with a small volume of blood avoiding any invasive procedure, and can provide quick information for timely institution of therapy.

Cellular and serological markers of disease activity in Indian patients with HIV/AIDS.

There has been an exponential rise of HIV positive patients as observed at the surveillance center of Nehru Hospital. Most patients are poor and cannot afford repeated viral load assays. Therefore, there is a need to identify cost effective and reliable surrogate markers of disease activity. In the present study absolute number of CD4 cells, beta2 micro-globulin, circulating nucleosomes were studied in 30 patients of AIDS, 30 seropositives and 30 healthy controls. In addition viral load, P-24 assay, and TNFR-II assays were done in seropositive and AIDS patients. The mean CD4 cells in patients with AIDS were 69.66 +/- 68.25 mm3 while in seropositives values was 370 +/- 201.29 mm3. The mean CD4 cells in healthy controls were however 690 +/- 198 mm3. The differences in all the groups were highly significant (p<0.001). The mean CD4 values in Indians are significantly lower than reported from the west. The lower number of CD4 cells in healthy population is interpreted to be due to immune activation. The CD8 cell number in controls was 650 +/- 207 mm3 this figure is also higher than that observed in the west. P-24 assay failed to delineate between seropositives and patients with AIDS. Although, beta2 microglobulin levels were significantly higher in AIDS than in seropositives and higher in seropositives than in controls yet with the best possible cut off, it had a sensitivity of only 70% in delineating the two conditions. The correlation between CD4 cells and viral load was more significant when the CD4 cells were below 200 mm3. Five out of 30 patients with a CD4 of 300-600 mm3 had a viral load of over 1 x 10(5) cop/ml. The difference in TNF R-II levels between seropositives and AIDS was however more impressive. With a cut off of 550 pg/ml it had a sensitivity of 95% in delineating HIV from AIDS. It is concluded that a combination of absolute number of CD4 cells and TNF R-II assay along with clinical evaluation may be used to monitor therapy in resource poor countries where frequent viral load assay is unaffordable.

Solution structure of ERK2 binding domain of MAPK phosphatase MKP-3: structural insights into MKP-3 activation by ERK2.

MAP kinases (MAPKs), which control mitogenic signal transduction in all eukaryotic organisms, are inactivated by dual specificity MAPK phosphatases (MKPs). MKP-3, a prototypical MKP, achieves substrate specificity through its N-terminal domain binding to the MAPK ERK2, resulting in the activation of its C-terminal phosphatase domain. The solution structure and biochemical analysis of the ERK2 binding (EB) domain of MKP-3 show that regions that are essential for ERK2 binding partly overlap with its sites that interact with the C-terminal catalytic domain, and that these interactions are functionally coupled to the active site residues of MKP-3. Our findings suggest a novel mechanism by which the EB domain binding to ERK2 is transduced to cause a conformational change of the C-terminal catalytic domain, resulting in the enzymatic activation of MKP-3.

Structural basis of SNT PTB domain interactions with distinct neurotrophic receptors.

SNT adaptor proteins transduce activation of fibroblast growth factor receptors (FGFRs) and neurotrophin receptors (TRKs) to common signaling targets. The SNT-1 phosphotyrosine binding (PTB) domain recognizes activated TRKs at a canonical NPXpY motif and, atypically, binds to nonphosphorylated FGFRs in a region lacking tyrosine or asparagine. Here, using NMR and mutational analyses, we show that the PTB domain utilizes distinct sets of amino acid residues to interact with FGFRs or TRKs in a mutually exclusive manner. The FGFR1 peptide wraps around the beta sandwich structure of the PTB domain, and its binding is possibly regulated by conformational change of a unique C-terminal beta strand in the protein. Our results suggest mechanisms by which SNTs serve as molecular switches to mediate the essential interplay between FGFR and TRK signaling during neuronal differentiation.

Immunogenicity enhancement of recombinant rabbit 55-kilodalton zona pellucida protein expressed using the baculovirus expression system.

In the present study we have used a molecular approach to evaluate the immunogenicity and antigenicity of glycosylated and non-glycosylated recombinant rabbit 55-kDa zona pellucida (ZP) protein. The 55-kDa cDNA was expressed in insect (Sf9) cells through use of a baculovirus expression system to obtain nonfusion glycosylated recombinant ZP protein (BV-55). SDS-PAGE and immunoblot analysis demonstrated that the recombinant protein is expressed as two forms having relative molecular masses of 70 kDa and 80 kDa. Because cells treated with tunicamycin produce predominantly the 70-kDa form, this heterogeneity is presumed to be due to differential glycosylation. Further studies using lectin blot and immunoblot analyses showed that the BV-55 protein has both N-linked and O-linked oligosaccharides. However, this glycosylation is distinct from that of the native 55-kDa ZP protein, since it was not recognized by a monoclonal antibody associated with lactosaminoglycan-type carbohydrate epitopes in native ZP proteins. Immunogenicity studies demonstrated that antibodies against the BV-55 protein are developed by female rabbits and guinea pigs and that these antibodies recognize epitopes associated with native, enzyme-deglycosylated as well as nonglycosylated recombinant forms of the rabbit 55-kDa ZP protein. In contrast, recombinant protein expressed in bacteria did not elicit antibodies in either rabbits or guinea pigs. These results demonstrate that expression of the 55-kDa recombinant protein in the baculovirus expression system enhances its immunogenicity.

Malaria control and long-term periodicity of the disease in Pakistan.

The classic investigations of the malaria epidemics in the Punjab led to the conclusion that in this most populous and most malarious province of the present-day Pakistan, epidemics occurred regularly at intervals of approximately eight years. Against this background, the results of a Malaria Control Programme launched in 1975 are examined. The Programme, supported by USAID and WHO, represents in economic terms the greatest effort made against malaria in the country. Malathion, the main attack weapon of the Programme, was used on an unprecedented scale. This created logistic and--unexpectedly--toxicity problems among the spraying workers. Despite these difficulties, an over-all reduction of 76% in the slide positivity rate was observed in the first two years of operations of the Programme. The authors warn against measures which may curtail the activities of the Programme when, according to the cyclical periodicity of malaria in the Punjab, an epidemic wave can be expected in 1980-81, with inevitable repercussions all over the country.