Loss of the tumor suppressor Snf5 leads to aberrant activation of the Hedgehog-Gli pathway

Aberrant activation of the Hedgehog (Hh) pathway can drive tumorigenesis. To investigate the mechanism by which glioma-associated oncogene family zinc finger-1 (GLI1), a crucial effector of Hh signaling, regulates Hh pathway activation, we searched for GLI1-interacting proteins. We report that the chromatin remodeling protein SNF5 (encoded by SMARCB1, hereafter called SNF5), which is inactivated in human malignant rhabdoid tumors (MRTs), interacts with GLI1. We show that Snf5 localizes to Gli1-regulated promoters and that loss of Snf5 leads to activation of the Hh-Gli pathway. Conversely, re-expression of SNF5 in MRT cells represses GLI1. Consistent with this, we show the presence of a Hh-Gli-activated gene expression profile in primary MRTs and show that GLI1 drives the growth of SNF5-deficient MRT cells in vitro and in vivo. Therefore, our studies reveal that SNF5 is a key mediator of Hh signaling and that aberrant activation of GLI1 is a previously undescribed targetable mechanism contributing to the growth of MRT cells.     

A procedure for the purification of beta-lactoglobulin from bovine milk using gel filtration chromatography at low pH

A simple and rapid procedure for the purification of beta-lactoglobulin (β-LG) from bovine milk is described. The procedure exploits the major difference in molecular mass of β-LG and other whey components and the existence of the former in monomeric form at acidic pH. Gel filtration of whey was carried out using a Bio-Gel P10 column at pH 3.0. Residual caseins and other milk proteins were excluded from the gel and β-LG and alpha-lactalbumin (α-LA) emerged as two fully resolved peaks. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) suggested that β-LG was purified to apparent homogeneity, while absorption, fluorescence, and circular dichroism spectroscopy indicated the native-like conformation of the protein. Western blot analysis revealed that the antibodies raised against the purified β-LG in rabbits also readily react with the commercial bovine protein. This procedure requires only 4-5 hr for the purification of about 10 mg of β-LG from a single run while using a small column (2.3 cm x 83 cm) of Bio-Gel P10 and has the potential for scaling up.     

Accuracy of cytologic interpretation of pancreatic neoplasms by fine needle aspiration and pancreatic duct brushings

OBJECTIVE: To investigate the accuracy of fine needle aspiration (FNA) specimens and pancreatic duct brushings in the detection of pancreatic lesions and to compare the results with follow-up biopsy and/or surgical interpretation. STUDY DESIGN: We reviewed a total of 57 specimens (37/20), 37 FNA specimens and 20 pancreatic duct brushings, from 45 patients treated at Froedtert Memorial Lutheran Hospital, affiliated with the Medical College of Wisconsin, Milwaukee, over a 4-year period. The FNA and brushing samples were categorized as follows: positive for malignancy (21/3 = 24), suspicious for malignancy (8/7 = 15) and atypical (8/10 = 18). The results were then correlated with the tissue diagnosis. RESULTS: The 24 cytologic samples positive for malignancy included 23 (20/3) pancreatic ductal carcinoma (CA) and 1 (1/0) neuroendocrine CA; in the suspicious category, 11 (6/5) were pancreatic ductal CA; 2 (0/2) mucinous neoplasms and (2/0) neuroendocrine neoplasms; in the atypical category; 2 (2/0) suggestive of mucinous neoplasia, 1 (1/0) suggestive of serous neoplasia and 9 (2/7) favor reactive; and 6 (3/3) without further categorization. Tissue diagnoses were available in 26 cases: 12 (10/2) cases positive for malignancy, 8 (5/3) suspicious for malignancy and 6 (5/1) atypical. The 12 cytologically positive cases confirmed by histology showed 10 ductal CA, 1 neuroendocrine CA and 1 negative. All 8 cases (100%) suspicious for malignancy revealed positive results, including 5 ductal CA, 1 neuroendocrine neoplasm, 1 mucinous cystic neoplasm and 1 lymphoma. Of the 6 atypical lesions, 1 showed ductal CA, 2 mucinous cystic neoplasm and 3 chronic pancreatitis. CONCLUSION: Pancreatic FNA and duct brushings [table: see text] are accurate methods in identifying pancreatic lesions, particularly ductal CA. Accuracy can be improved in the case of mucinous and other lesions with adequate cellularity of the smear and recognizing the limitations of brush samples in the case of mucinous cystic lesions. False negative results may occur in cases of poor representation of malignant cells or poor sampling.     

Palmitate oxidation by the mitochondria from volume-overloaded rat hearts

In this work, an attempt was made to identify the reasons of impaired long-chain fatty acid utilization that waspreviously described in volume-overloaded rat hearts. The most significant data are the following: (1) The slowing down of long-chain fatty acid oxidation in severely hypertrophied hearts cannot be related to a feedback inhibition of carnitine palmitoyltransferase I from an excessive stimulation of glucose oxidation since, because of decreased tissue levels of L-carnitine, glucose oxidation also declines in volume-overloaded hearts. (2) While, in control hearts, the estimated intracellular concentrations of free carnitine are in the range of the respective K_m of mitochondrial CPT I, a kinetic limitation of this enzyme could occur in hypertrophied hearts due to a 40% decrease in free carnitine. (3) However, the impaired palmitate oxidation persists upon the isolation of the mitochondria from these hearts even in presence of saturating concentrations of L-carnitine. In contrast, the rates of the conversion of both palmitoyl-CoA and palmitoylcarnitine into acetyl-CoA are unchanged. (4) The kinetic analyses of palmitoyl-CoA synthase and carnitine palmitoyltransferase I reactions do not reveal any differences between the two mitochondrial populations studied. On the other hand, the conversion of palmitate into palmitoylcarnitine proves to be substrate inhibited already at physiological concentrations of exogenous palmitate. The data presented in this work demonstrate that, during the development of a severe cardiac hypertrophy, a fragilization of the mitochondrial outer membrane may occur. The functional integrity of this membrane seems to be further deteriorated by increasing concentrations of free fatty acids which gives rise to an impaired functional cooperation between palmitoyl-CoA synthase and carnitine palmitoyltransferase I. In intact myocardium, the utilization of the generated in situ palmitoyl-CoA can be further slowed down by decreased intracellular concentrations of free carnitine.     

Development of novel benzofuran-isatin conjugates as potential antiproliferative agents with apoptosis inducing mechanism in Colon cancer

In the current work, a new set of carbohydrazide linked benzofuran-isatin conjugates (5a–e and 7a–i) was designed and synthesised. The anticancer activity for compounds (5b–d, 7a, 7b, 7d and 7g) was measured against NCI-55 human cancer cell lines. Compound 5d was the most efficient, and thus subjected to the five-dose screen where it showed excellent broad activity against almost all tested cancer subpanels. Furthermore, all conjugates (5a–e and 7a–i) showed a good anti-proliferative activity towards colorectal cancer SW-620 and HT-29 cell lines, with an excellent inhibitory effect for compounds 5a and 5d (IC₅₀ = 8.7 and 9.4 µM (5a), and 6.5 and 9.8 µM for (5d), respectively). Both compounds displayed selective cytotoxicity with good safety profile. In addition, both compounds provoked apoptosis in a dose dependent manner in SW-620 cells. Also, they significantly inhibited the anti-apoptotic Bcl2 protein expression and increased the cleaved PARP level that resulted in SW-620 cells apoptosis.     

Synthesis of novel pyrroles and fused pyrroles as antifungal and antibacterial agents

Pyrroles and its fused forms possess antimicrobial activities, they can easily interact with biomolecules of living systems. A series of substituted pyrroles, and its fused pyrimidines and triazines forms have been synthesised, all newly synthesised compound structures were confirmed by spectroscopic analysis. Generally, the compounds inhibited growth of some important human pathogens, the best effect was given by: 2a, 3c, 4d on Gram-positive bacteria and was higher on yeast (C. albicans), by 5c on Gram-negative bacteria and by 5a then 3c on filamentous fungi (A. fumigatus and F. oxysporum). Such results present good antibacterial and antifungal potential candidates to help overcome the global problem of antibiotic resistance and opportunistic infections outbreak. Compound 3c gave the best anti-phytopathogenic effect at a 50-fold lower concentration than Kocide 2000, introducing a safe commercial candidate for agricultural use. The effect of the compounds on DNA was monitored to detect the mode of action.     

Continuous degradation of maltose by enzyme entrapment technology using calcium alginate beads as a matrix

Maltase from Bacillus licheniformis KIBGE-IB4 was immobilized within calcium alginate beads using entrapment technique. Immobilized maltase showed maximum immobilization yield with 4% sodium alginate and 0.2 M calcium chloride within 90.0 min of curing time. Entrapment increases the enzyme–substrate reaction time and temperature from 5.0 to 10.0 min and 45 °C to 50 °C, respectively as compared to its free counterpart. However, pH optima remained same for maltose hydrolysis. Diffusional limitation of substrate (maltose) caused a declined in V_max of immobilized enzyme from 8411.0 to 4919.0 U ml^?1 min^?1 whereas, K_m apparently increased from 1.71 to 3.17 mM ml^?1. Immobilization also increased the stability of free maltase against a broad temperature range and enzyme retained 45% and 32% activity at 55 °C and 60 °C, respectively after 90.0 min. Immobilized enzyme also exhibited recycling efficiency more than six cycles and retained 17% of its initial activity even after 6th cycles. Immobilized enzyme showed relatively better storage stability at 4 °C and 30 °C after 60.0 days as compared to free enzyme.     

Molecular identification of Campanulotes bidentatus Scopoli, 1763 (Phthiraptera,Philopteridae) infecting the domestic pigeon Columba livia from Saudi Arabia

The taxonomy of the order Phthiraptera is unstable and still problematic to researchers. Most of the current taxon classifications are mainly based on morphological features. Campanulotes bidentatus belongs to the chewing lice of the Philopteridae family that mostly parasitic on birds. There is a lack of sequence data and phylogenetic analyses on the family Philopteridae. In the current study, C. bidentatus was collected from the domestic pigeon Columba livia and identified morphologically and molecularly based on the mitochondrial cytochrome c oxidase subunit 1 gene (COI). The infection rate of the Campanulotes genus was approximately 58.82% in this study. Phylogenetic analysis based on the mt COI gene was informative for members of Philopteridae and the group taxon genera formed distinct clades. Future studies were recommended using the 16s rRNA to enhance the tree topology and obtain clear differentiation between genera.