Prognostic significance and gene expression profiles of p53 mutations in microsatellite-stable stage III colorectal adenocarcinomas

Although the prognostic value of p53 abnormalities in Stage III microsatellite stable (MSS) colorectal cancers (CRCs) is known, the gene expression profiles specific to the p53 status in the MSS background are not known. Therefore, the current investigation has focused on identification and validation of the gene expression profiles associated with p53 mutant phenotypes in MSS Stage III CRCs. Genomic DNA extracted from 135 formalin-fixed paraffin-embedded tissues, was analyzed for microsatellite instability (MSI) and p53 mutations. Further, mRNA samples extracted from five p53-mutant and five p53-wild-type MSS-CRC snap-frozen tissues were profiled for differential gene expression by Affymetrix Human Genome U133 Plus 2.0 arrays. Differentially expressed genes were further validated by the high-throughput quantitative nuclease protection assay (qNPA), and confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and by immunohistochemistry (IHC). Survival rates were estimated by Kaplan-Meier and Cox regression analyses. A higher incidence of p53 mutations was found in MSS (58%) than in MSI (30%) phenotypes. Both univariate (log-rank, P = 0.025) and multivariate (hazard ratio, 2.52; 95% confidence interval, 1.25–5.08) analyses have demonstrated that patients with MSS-p53 mutant phenotypes had poor CRC-specific survival when compared to MSS-p53 wild-type phenotypes. Gene expression analyses identified 84 differentially expressed genes. Of 49 down-regulated genes, LPAR6, PDLIM3, and PLAT, and, of 35 up-regulated genes, TRIM29, FUT3, IQGAP3, and SLC6A8 were confirmed by qNPA, qRT-PCR, and IHC platforms. p53 mutations are associated with poor survival of patients with Stage III MSS CRCs and p53-mutant and wild-type phenotypes have distinct gene expression profiles that might be helpful in identifying aggressive subsets.     

Location of disorder in coiled coil proteins is influenced by its biological role and subcellular localization: a GO-based study on human proteome

Intrinsic disorder in proteins has been explored to study lack of structure-function aspects of many proteins. The current study focuses on coiled coils which are often linked to intrinsic disorder. We present a sequence level analysis of human coiled coils to find out if this is universally true for all coiled coils. When annotated coiled-coil regions were collected from UniProt and investigated with disorder prediction tools namely-IUPred and DISpro, three patterns were commonly observed-disordered coiled coils (DisCCs), ordered coiled coils (OCCs) and the last one having a disordered region outside the coiled-coil region (DOCCs). Differential enrichment in the gene ontology was seen in these three categories. We found that OCCs are enriched in structural components of the extracellular space including the fibrinogen complex and laminin complex. On the contrary, DisCCs were found to be exclusively over-represented in proteins involved in actin filament, lamellipodium, cell junction, macromolecule complexes, ciliary rootlet and nucleolus. DOCCs are found to be associated with many regulatory and adaptor functions including positive regulation of calcium ion transport via store-operated calcium channel activity, cytoskeletal adaptor activity etc. Other than the GO-based analysis, sequence level analysis showed that disordered coiled-coil regions bear a high proportion of low-complexity regions as compared to ordered coiled coils. The former also has a higher probability of forming a dimer as compared to the ordered counterpart. Our study shows that the in silico approach of mapping of disorder in or around coiled coils in other biological systems or organisms can be applied to understand and rationalize the mode of action of these dynamic motifs.     

Dramatic inhibition of osteoclast sealing ring formation and bone resorption in vitro by a WASP-peptide containing pTyr294 amino acid

Wiskott Aldrich Syndromeprotein (WASP) has a unique regulatory role in sealing ring formation and bone resorption in osteoclasts. Here, using the TAT-transduction method, we show the possible role of WASP domain(s) in sealing ring formation and bone resorption. Transduction of TAT-fused full-length WASP peptide induced Arp2/3 complex formation, F-actin content, sealing ring formation and bone resorption. Transduction of WASP peptides containing basic, verpolin-central, pTyr294, and proline-rich regions inhibited the processes listed above at various levels. The ability to resorb bone by WASP peptides containing basic, verpolin-central, and proline-rich regions was reduced and the resorbed area matched the size of the sealing ring. However, osteoclasts transduced with WASP peptide containing pTyr294aa demonstrated the following: a) a considerable decrease in the interaction and phosphorylation of c-Src with endogenous WASP; b) total loss of sealing ring-like structures; c) formation of actin-rich patches at the peripheral edge that contains filopodia-like projections; d) reduced capacity for bone resorption in vitro. These findings suggest that modulation of phosphorylation state of pTyr294aa assists in integrating multiple signaling molecule and pathways that partake in the assembly of sealing ring.     

Effects of NaCl on growth, ion accumulation, protein, proline contents and antioxidant enzymes activity in callus cultures of Jatropha curcas

Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel crop. The effect of NaCl stress on growth, ion accumulation, contents of protein, proline, and antioxidant enzymes activity in callus cultures of J. curcas was investigated. Exposure of callus to NaCl decreased growth in a concentration dependent manner. NaCl treated callus accumulated Na and declined in K, Ca and Mg contents. Na/K ratio increased steadily as a function of external NaCl treatment. NaCl induced significant differences in quality and quantity of proteins, whereas, proline accumulation remained more or less constant with treatment. NaCl stress enhanced the activity of superoxide dismutase (SOD; E.C. 1.15.1.1) and peroxidase (POX; E.C. 1.11.1.7). Further in the isoenzyme studies, four SOD isoenzymes (SOD 1, 2, 3, and 4) and two POX isoenzymes (POX 1 and 2) were detected with the treatment. NaCl strongly induced activity of SOD 4 isoenzyme in 40, 60, 80 mM and POX 2 isoenzyme in 40 and 80 mM NaCl concentrations. Increase in antioxidant enzymes activity could be a response to cellular damage induced by NaCl. This increase could not stop the deleterious effects of NaCl, but it reduced stress severity and thus allowed cell growth to occur.     

In vivo administration of calpeptin attenuates calpain activation and cardiomyocyte loss in pressure-overloaded feline myocardium

Calpain activation is linked to the cleavage of several cytoskeletal proteins and could be an important contributor to the loss of cardiomyocytes and contractile dysfunction during cardiac pressure overload (PO). Using a feline right ventricular (RV) PO model, we analyzed calpain activation during the early compensatory period of cardiac hypertrophy. Calpain enrichment and its increased activity with a reduced calpastatin level were observed in 24- to 48-h-PO myocardium, and these changes returned to basal level by 1 wk of PO. Histochemical studies in 24-h-PO myocardium revealed the presence of TdT-mediated dUTP nick-end label (TUNEL)-positive cardiomyocytes, which exhibited enrichment of calpain and gelsolin. Biochemical studies showed an increase in histone H2B phosphorylation and cytoskeletal binding and cleavage of gelsolin, which indicate programmed cardiomyocyte cell death. To test whether calpain inhibition could prevent these changes, we administered calpeptin (0.6 mg/kg iv) by bolus injections twice, 15 min before and 6 h after induction of 24-h PO. Calpeptin blocked the following PO-induced changes: calpain enrichment and activation, decreased calpastatin level, caspase-3 activation, enrichment and cleavage of gelsolin, TUNEL staining, and histone H2B phosphorylation. Although similar administration of a caspase inhibitor, N-benzoylcarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VD-fmk), blocked caspase-3 activation, it did not alleviate other aforementioned changes. These results indicate that biochemical markers of cardiomyocyte cell death, such as sarcomeric disarray, gelsolin cleavage, and TUNEL-positive nuclei, are mediated, at least in part, by calpain and that calpeptin may serve as a potential therapeutic agent to prevent cardiomyocyte loss and preserve myocardial structure and function during cardiac hypertrophy.     

Cytologic features of subependymal giant cell astrocytom: a review of 7 cases

OBJECTIVE: To describe the cytologic features of subependymal giant cell astrocytoma (SEGA) on smears and analyze cytomorphologic parameters that may help in reaching the diagnosis of SEGA. STUDY DESIGN: Cytologic smears of 7 cases of SEGA were reviewed and graded semi-quantitatively for 11 cytologic features: clustering, cytoplasmic fibrillary processes (fibrillarity), cellularity, small prominent nudcleoli, binucleation or multinucleation, "strap cells", spindle-shaped cells, mitoses, intranuclear inclusions, nuclear atypia and perivascular palisading/pseudorosettes. Corresponding histologic sections were also reviewed. RESULTS: The study included 5 male and 2 female patients with an average age of 8.3 years (range, 3-16) at surgery. Cytologic examination revealed loosely cohesive clusters of large cells possessing round to oval nuclei with no or minimal atypia; fine, evenly distributed chromatin; and abundant eosinophilic cytoplasm enmeshed in abundant thin, hairlike processes. Predominant features included hypercellularity, cell clustering, and fibrillarity. Binucleation or multinucleation; small, prominent nucleoli; and strap cells were often seen. Although common in histologic sections, perivascular palisading/pseudorosettes and spindled astrocytic cells were rarely noted on smears. CONCLUSION: The cytologic features of SEGA are highly characteristic and thus are of great use in supporting a diagnosis of SEGA and in excluding mimics, primarily gemistocytic astrocytoma and ependymoma.     

Rac Regulates Vascular Endothelial Growth Factor Stimulated Motility

During angiogenesis endothelial cells migrate towards a chemotactic stimulus. Understanding the mechanism of endothelial cell migration is critical to the therapeutic manipulation of angiogenesis and ultimately cancer prevention. Vascular endothelial growth factor (VEGF) is a potent chemotactic stimulus of endothelial cells during angiogenesis. The endothelial cell signal transduction pathway of VEGF represents a potential target for cancer therapy, but the mechanisms of post-receptor signal transduction including the roles of rho family GTPases in regulating the cytoskeletal effects of VEGF in endothelial cells are not understood.

Here we analyze the mechanisms of cell migration in the mouse brain endothelial cell line (bEND3). Stable transfectants containing a tetracycline repressible expression vector were used to induce expression of Rac mutants. Endothelial cell haptotaxis was stimulated by constitutively active V12Rac on collagen and vitronectin coated supports, and chemotaxis was further stimulated by VEGF. Osteopontin coated supports were the most stimulatory to bEND3 haptotaxis, but VEGF was not effective in further increasing migration on osteopontin coated supports. Haptotaxis on support coated with collagen, vitronectin, and to a lesser degree osteopontin was inhibited by N17 Rac. N17 Rac expression blocked stimulation of endothelial cell chemotaxis by VEGF. As part of the chemotactic stimulation, VEGF caused a loss of actin organization at areas of cell-cell contact and increased stress fiber expression in endothelial cells which were directed towards pores in the transwell membrane. N17 Rac prevented the stimulation of cell-cell contact disruption and the stress fiber stimulation by VEGF.

These data demonstrate two pathways of regulating endothelial cell motility, one in which Rac is activated by matrix/integrin stimulation and is a crucial modulator of endothelial cell haptotaxis. The other pathway, in the presence of osteopontin, is Rac independent. VEGF stimulated chemotaxis, is critically dependent on Rac activation. Osteopontin was a potent matrix activator of motility, and perhaps one explanation for the absence of a VEGF plus osteopontin effect is that osteopontin stimulated motility was inhibitory to the Rac pathway.     

Alkaline protease production by Basidiobolus (N.C.L. 97.1.1): effect of 'darmform' morphogenesis and cultural conditions on enzyme production and preliminary enzyme characterization

A strain of Basidiobolus (N.C.L. 97.1.1) was isolated from plant detritus which secreted alkaline protease optimally active at pH 10.0. It is the first report of a protease from Basidiobolus, which is stable to and active under high alkaline conditions. When incubated under stationary conditions in broth cultures containing salts such as ammonium chloride, 'darmform' morphogenesis was readily induced through enlargement and internal division of the hyphal segments. Secretion of high activity alkaline protease was obtained in cultures initiated with darmform morphogenesis whereas cultures initiated from mycelial inocula grew as large pellets in submerged cultures, with little or no protease secretion. Cultural conditions favoring alkaline protease secretion have been optimized and a preliminary characterization of the enzyme is presented. Compatibility of the alkaline protease with commercial detergents as well as its potential application in recovering silver from spent photographic films have also been investigated.     

Identification of a Live Attenuated Vaccine Candidate for Tularemia Prophylaxis

Francisella tularensis is the causative agent of a fatal human disease, tularemia. F. tularensis was used in bioweapon programs in the past and is now classified as a category A select agent owing to its possible use in bioterror attacks. Despite over a century since its discovery, an effective vaccine is yet to be developed. In this study four transposon insertion mutants of F. tularensis live vaccine strain (LVS) in Na/H antiporter (FTL_0304), aromatic amino acid transporter (FTL_0291), outer membrane protein A (OmpA)-like family protein (FTL_0325) and a conserved hypothetical membrane protein gene (FTL_0057) were evaluated for their attenuation and protective efficacy against F. tularensis SchuS4 strain. All four mutants were 100–1000 fold attenuated for virulence in mice than parental F. tularensis. Except for the FTL_0304, single intranasal immunization with the other three mutants provided 100% protection in BALB/c mice against intranasal challenge with virulent F. tularensis SchuS4. Differences in the protective ability of the FTL_0325 and FTL_0304 mutant which failed to provide protection against SchuS4 were investigated further. The results indicated that an early pro-inflammatory response and persistence in host tissues established a protective immunity against F. tularensis SchuS4 in the FTL_0325 immunized mice. No differences were observed in the levels of serum IgG antibodies amongst the two vaccinated groups. Recall response studies demonstrated that splenocytes from the FTL_0325 mutant immunized mice induced significantly higher levels of IFN-γ and IL-17 cytokines than the FTL_0304 immunized counterparts indicating development of an effective memory response. Collectively, this study demonstrates that persistence of the vaccine strain together with its ability to induce an early pro-inflammatory innate immune response and strong memory responses can discriminate between successful and failed vaccinations against tularemia. This study describes a live attenuated vaccine which may prove to be an ideal vaccine candidate for prevention of respiratory tularemia.