Bivalent IAP antagonists, but not monovalent IAP antagonists, inhibit TNF-mediated NF-κB signaling by degrading TRAF2-associated cIAP1 in cancer cells.

The inhibitor of apoptosis (IAP) proteins have pivotal roles in cell proliferation and differentiation, and antagonizing IAPs in certain cancer cell lines results in induction of cell death. A variety of IAP antagonist compounds targeting the baculovirus IAP protein repeat 3 (BIR3) domain of cIAP1have advanced into clinical trials. Here we sought to compare and contrast the biochemical activities of selected monovalent and bivalent IAP antagonists with the intent of identifying functional differences between these two classes of IAP antagonist drug candidates. The anti-cellular IAP1 (cIAP1) and pro-apoptotic activities of monovalent IAP antagonists were increased by using a single covalent bond to combine the monovalent moieties at the P4 position. In addition, regardless of drug concentration, treatment with monovalent compounds resulted in consistently higher levels of residual cIAP1 compared with that seen following bivalent compound treatment. We found that the remaining residual cIAP1 following monovalent compound treatment was predominantly tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2)-associated cIAP1. As a consequence, bivalent compounds were more effective at inhibiting TNF-induced activation of p65/NF-κB compared with monovalent compounds. Moreover, extension of the linker chain at the P4 position of bivalent compounds resulted in a decreased ability to degrade TRAF2-associated cIAP1 in a manner similar to monovalent compounds. This result implied that specific bivalent IAP antagonists but not monovalent compounds were capable of inducing formation of a cIAP1 E3 ubiquitin ligase complex with the capacity to effectively degrade TRAF2-associated cIAP1. These results further suggested that only certain bivalent IAP antagonists are preferred for the targeting of TNF-dependent signaling for the treatment of cancer or infectious diseases.

Differentiation between oligodendroglioma genotypes using dynamic susceptibility contrast perfusion-weighted imaging and proton MR spectroscopy.

BACKGROUND AND PURPOSE: Oligodendrogliomas with 1p/19q chromosome LOH are more sensitive to chemoradiation therapy than those with intact alleles. The usefulness of dynamic susceptibility contrast-PWI-guided ¹H-MRS in differentiating these 2 genotypes was tested in this study. MATERIALS AND METHODS: Forty patients with oligodendrogliomas, 1p/19q LOH (n = 23) and intact alleles (n = 17), underwent MR imaging and 2D-¹H-MRS. ¹H-MRS VOI was overlaid on FLAIR images to encompass the hyperintense abnormality on the largest cross-section of the neoplasm and then overlaid on CBV maps to coregister CBV maps with ¹H-MRS VOI. rCBVmax values were obtained by measuring the CBV from each of the selected ¹H-MRS voxels in the neoplasm and were normalized with respect to contralateral white matter. Metabolite ratios with respect to ipsilateral Cr were computed from the voxel corresponding to the rCBVmax value. Logistic regression and receiver operating characteristic analyses were performed to ascertain the best model to discriminate the 2 genotypes of oligodendrogliomas. Qualitative evaluation of conventional MR imaging characteristics (patterns of tumor border, signal intensity, contrast enhancement, and paramagnetic susceptibility effect) was also performed to distinguish the 2 groups of oligodendrogliomas. RESULTS: The incorporation of rCBVmax value and metabolite ratios (NAA/Cr, Cho/Cr, Glx/Cr, myo-inositol/Cr, and lipid + lactate/Cr) into the multivariate logistic regression model provided the best discriminatory classification with sensitivity (82.6%), specificity (64.7%), and accuracy (72%) in distinguishing 2 oligodendroglioma genotypes. Oligodendrogliomas with 1p/19q LOH were also more associated with paramagnetic susceptibility effect (P < .05). CONCLUSIONS: Our preliminary results indicate the potential of combing PWI and ¹H-MRS to distinguish oligodendroglial genotypes.

SIRPalpha1 receptors interfere with the EGFRvIII signalosome to inhibit glioblastoma cell transformation and migration.

EGFRvIII, a frequent genetic alteration of the epidermal growth factor receptor (EGFR), has been shown to increase the migratory potential of tumor cells and normal fibroblasts. Previously, we showed that signal regulatory protein alpha1 (SIRPalpha1) receptors interact with SHP-2 to inhibit wild-type (wt) EGFR-mediated tumor migration, survival and cell transformation. However, the effects of SIRPalpha1 inhibitory receptors on EGFRvIII-mediated phenotypes are unclear. The aim of this study was to investigate the effect of SIRPalpha1 receptor on the EGFRvIII signalosome and phenotypes. Overexpression of SIRPalpha1 in U87MG.EGFRvIII cells inhibited transformation and migration in a MAPK-dependent manner, and is independent of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. We observed reduced EGFRvIII/SHP-2/Gab1/Grb2/Sos-1 interaction and enhanced SIRP/SHP-2 association in U87MG.EGFRvIII/SIRPalpha1 cells when compared with empty vector control cells. Interestingly, SIRPalpha1 overexpression differentially modulated SHP-2 phosphorylation at tyrosyl 542 and 580 residues, which may regulate Erk1/2 activity and the EGFRvIII phenotype. In addition, SIRPalpha1-expressing cells exhibited reduced focal adhesion kinase (FAK) phosphorylation and its recruitment to the EGFRvIII/Grb2/Sos-1/Gab1/SHP-2 complex. Collectively, our data indicate that SIRPalpha1 specifically affects the SHP-2/FAK/Grb2/Sos-1/MAPK activation loop to downmodulate EGFRvIII-mediated migration and transformation. Further understanding of the molecular interactions between the SIRPalpha1 inhibitory receptor and the EGFRvIII signalosome may facilitate the identification of novel targets to inhibit the EGFRvIII glioblastoma phenotype.

One-way cross-talk between p38(MAPK) and p42/44(MAPK). Inhibition of p38(MAPK) induces low density lipoprotein receptor expression through activation of the p42/44(MAPK) cascade.

In this paper, we report that SB202190 alone, a specific inhibitor of p38(MAPK), induces low density lipoprotein (LDL) receptor expression (6-8-fold) in a sterol-sensitive manner in HepG2 cells. Consistent with this finding, selective activation of the p38(MAPK) signaling pathway by expression of MKK6b(E), a constitutive activator of p38(MAPK), significantly reduced LDL receptor promoter activity. Expression of the p38(MAPK) alpha-isoform had a similar effect, whereas expression of the p38(MAPK) betaII-isoform had no significant effect on LDL receptor promoter activity. SB202190-dependent increase in LDL receptor expression was accompanied by induction of p42/44(MAPK), and inhibition of this pathway completely prevented SB202190-induced LDL receptor expression, suggesting that p38(MAPK) negatively regulates the p42/44(MAPK) cascade and the responses mediated by this kinase. Cross-talk between these kinases appears to be one-way because modulation of p42/44(MAPK) activity did not affect p38(MAPK) activation by a variety of stress inducers. Taken together, these findings reveal a hitherto unrecognized one-way communication that exists between p38(MAPK) and p42/44(MAPK) and provide the first evidence that through the p42/44(MAPK) signaling cascade, the p38(MAPK) alpha-isoform negatively regulates LDL receptor expression, thus representing a novel mechanism of fine tuning cellular levels of cholesterol in response to a diverse set of environmental cues.

Diagnostic efficacy of polymerase chain reaction in granulomatous uveitis.

SETTING: The granulomatous uveitis, multifocal choroiditis and periphlebitis have been suspected to be of tubercular origin but no definitive reports about detection of etiological agents have been documented in the literature. Conventional bacteriological methods are not generally helpful in diagnosing ocular tuberculosis due to difficulty with potential morbidity associated with obtaining the biopsy material from the eye. Thus, the diagnosis of ocular tuberculosis is most often presumptive. OBJECTIVE: We evaluated the role of polymerase chain reaction (PCR) for detection of Mycobacterium tuberculosis in the aqueous humor samples obtained from eyes with active uveitis. METHODS: Aqueous samples from 53 patients having cellular reaction in the anterior chamber along with any one or more of the following: 1) active vasculitis; 2) anterior vitreous cells; 3) snowball opacities; 4) snow banking in the pars plana; 5) retinochoroiditis were withdrawn by anterior chamber paracentesis and subjected to PCR. Seventeen samples from patients with definite clinical diagnoses other than tuberculosis formed a disease control group. Fifteen aqueous samples obtained from healthy subjects undergoing routine cataract surgery served as healthy controls. PCR was performed using primers capable of amplifying a 150 b.p. segment from a conserved repetitive sequence in the genome of M. tuberculosis. RESULTS: Twenty out of the 53 samples (37.7%) in the study group were positive where as only one sample out of 17 in the disease control group (5.7%) showed a weakly positive band. No sample from the healthy control group showed a positive PCR. CONCLUSION: Our study shows that PCR can be effectively used for the diagnosis of intraocular tuberculosis in the presence of uveitis.

Heterogeneity in heat shock protein genes in Leishmania isolates.

Leishmaniasis, a group of visceral and cutaneous diseases, is caused by parasites belonging to one genus comprising approximately 13 different species. Many methods including serological, biochemical and molecular biological techniques have been used by various workers to characterize these different species and isolates of Leishmania, yet there is no single generally accepted criterion. We have identified certain cDNA clones from a library generated from the promastigotes of S1 strain of Leishmania donovani and used them as probes for identification of various isolates of L. donovani and Leishmania tropica. Two of the probes used, E2b (2.0 kb) and E1a (1.3 kb), sequence characterized to be hsp70 of Leishmania, were able to distinguish various isolates of L. donovani from different geographical origins as well as strains of L. donovani from those of L. tropica. Thus, by using recombinant hsp70 cDNA probes, the data indicated that there is a considerable degree of heterogeneity in the heat-shock genes of Leishmania.

Genetic polymorphism of Leishmania species using kinetoplast DNA restriction fragment length polymorphism and cDNA probe of Leishmania donovani.

Leishmaniasis represents a group of diseases that range from simple cutaneous lesions through metastasizing diffused cutaneous to severe systemic infection depending upon the taxon to which the causative parasite belongs. Therefore, it is important to identify the infecting Leishmania. Methods presently being used, including immunology, biochemistry and molecular biology have one or the other limitations, leaving scope for the search for newer probes. This study reports the characterization of leishmania isolates both by restriction fragment length polymorphism of kinetoplast DNA (kDNA) and genomic DNA. The genomic DNA was probed with a cDNA probe B2a1. Using a kDNA restriction pattern technique, different isolates of Leishmania donovani could be differentiated from the UR6 strain of L. tropica, but it was not possible to differentiate between newer local isolates of L. donovani with most of the restriction enzymes except AluI. However, the B2a1 cDNA probe was able to differentiate these isolates effectively. Both of these techniques could differentiate newer local isolates of L. donovani from the older isolates of L. donovani from India, i.e., DD8, RMRI and SS. The Indian isolates of L. donovani could also be differentiated from isolates of L. donovani from Jeddah and Germany using both techniques. The present study indicates that the cDNA probe B2a1 can be used as an important adjunct to kDNA restriction analysis for the characterization of Leishmania species.

A genetic analysis of HIV-1 from Punjab, India reveals the presence of multiple variants.

OBJECTIVE: To determine the extent of HIV-1 genetic variation in Indian patients. DESIGN: To avoid any bias in selecting viral variants, HIV-1 DNA was amplified directly from the peripheral blood mononuclear cells of patients and sequenced. Genetic similarity between Indian sequences and other geographic isolates was analysed by phylogenetic analysis algorithms. METHODS: A fragment encompassing the C2/V3-V5 regions of HIV-1 gp120 was amplified from the lymphocyte DNA of 12 Indian patients. Multiple clones from each patient were sequenced. Nucleotide sequences encompassing about 650 base pairs were aligned for the Indian and other geographically distinct isolates. Inter-isolate relationships were analysed by means of distance, parsimony and neighbour-joining algorithms. RESULTS: Nucleotide sequence comparisons showed low interpatient variation. Amino-acid comparisons revealed a high degree of homology between Indian sequences in this study and those studied earlier. On distance and parsimony trees, most of the Indian sequences clustered together as subtype C. However, sequences from three patients also showed significant homologies and phylogenetic clustering outside of subtype C. CONCLUSIONS: The predominant strain of HIV-1 in India belongs to subtype C and little interpatient nucleotide sequence divergence in the majority of cases suggests recent spread of HIV-1 in this region. This study also presents the first evidence for non-C subtypes in the Indian population with two epidemiologically linked samples remaining unclassified for any existing env subtype. The presence of variant subtypes in Indian patients sheds light on the transmission routes of HIV-1 to India and emphasizes the need to include these sequences in vaccine development strategies.

PIP: Health workers collected blood samples from 12 persons infected with HIV living in the Punjab in India to obtain peripheral blood mononuclear cells so researchers could determine the extent of HIV-1 genetic variation. They prepared multiple clones of the C2/V3-V5 regions of HIV-1 gp120 from the lymphocyte DNA of each patient. They used distance, parsimony, and neighbor-joining algorithms to analyze the inter-isolate relationships. They aligned nucleotide sequences of about 650 base pairs for the Indian and other geographically distinct isolates. All but two cases were males. The two females acquired HIV from their husbands. Based on the nucleotide sequence comparisons, there was low interpatient variation. Amino acid comparisons found a high degree of homology between Indian sequences in this study and those studied previously. Most Indian sequences clustered together as subtype C on the distant and parsimony trees. Three patients had significant homologies and phylogenetic clustering outside of subtype C. One patient had env gene homology to subtype B sequences prevalent in Europe and the Americas. The two others had env gene sequences that clustered away from all presently known subtypes of HIV-1. These three cases were the first sequences divergent from subtype C in India. All of these patients and one that clustered marginally with subtype C had possible contacts outside India. Variant subtypes in Indian patients provide clues on the transmission routes of HIV-1 to India. They also underscore the need for researchers to include these sequences as they develop an HIV/AIDS vaccine. Since the leading HIV-1 strain in India conforms to subtype C and there was limited nucleotide sequence variation in most cases, these findings indicate recent spread of HIV-1 in the Punjab.