Granzyme B-induced cell death involves induction of p53 tumor suppressor gene and its activation in tumor target cells

In this study we investigated the involvement of p53 in cytotoxic T-lymphocyte (CTL)-induced tumor target cell killing mediated by the perforin/granzymes pathway. For this purpose we used a human CTL clone (LT12) that kills its autologous melanoma target cells (T1), harboring a wild type p53. We demonstrated initially that LT12 kills its T1 target in a perforin/granzymes-dependent manner. Confocal microscopy and Western blot analysis indicated that conjugate formed between LT12 and T1 resulted in rapid cytoplasmic accumulation of p53 and its activation in T1 target cells. Cytotoxic assay using recombinant granzyme B (GrB) showed that this serine protease is the predominant factor inducing such accumulation. Furthermore, RNA interference-mediated lowering of the p53 protein in T1 cells or pifithrin-alpha-induced p53-specific inhibition activity significantly decreased CTL-induced target killing mediated by CTL or recombinant GrB. This emphasizes that p53 is an important determinant in granzyme B-induced apoptosis. Our data show furthermore that when T1 cells were treated with streptolysin-O/granzyme B, specific phosphorylation of p53 at Ser-15 and Ser-37 residues was observed subsequent to the activation of the stress kinases ataxia telangiectasia mutated (ATM) and p38K. Treatment of T1 cells with pifithrin-alpha resulted in inhibition of p53 phosphorylation at these residues and in a significant decrease in GrB-induced apoptotic T1 cell death. Furthermore, small interference RNAs targeting p53 was also accompanied by an inhibition of streptolysin-O/granzyme B-induced apoptotic T1 cell death. The present study supports p53 induction after CTL-induced stress in target cells. These findings provide new insight into a potential role of p53 as a component involved in the dynamic regulation of the major pathway of CTL-mediated cell death and may have therapeutic implications.     

Novel 3-(6-methylpyridin-2-yl)coumarin-based chalcones as selective inhibitors of cancer-related carbonic anhydrases IX and XII endowed with anti-proliferative activity

Carbonic anhydrases (CAs) are one of the promising targets for the development of anticancer agents. CA isoforms are implicated in various physiological processes and are expressed in both normal and cancerous cells. Thus, non-isoform selective inhibitors are associated with several side effects. Consequently, designing selective inhibitors towards cancer-related hCA IX/XII rather than the ubiquitous cytosolic isozymes hCA I and II is the main research objective in the field. Herein, a new series of 3-(6-methylpyridin-2-yl)coumarin derivatives 3 and 5a–o was designed and synthesised. The CA inhibition activities for the synthesised coumarins were analysed on isoforms hCA I, II, IX, and XII. Interestingly, both cancer-linked isoforms hCA IX/XII were inhibited by the prepared coumarins with inhibition constants ranging from sub- to low-micromolar range, whereas hCA I and II isoforms haven’t been inhibited up to 100 µM. Furthermore, the target coumarins were assessed for their antitumor activity on NCI-59 human cancer types.     

Biogas manufacture from co-digestion of untreated primary sludge with raw chicken manure under anaerobic mesophilic environmental conditions

This work aimed to co-digest various wastes to assess the best combination of all mixing ratio, also at choosing the best ratio between untreated primary sludge (UPS) singly from two sources, (South valley University (SUPS) and Abu tesht wastewater station (AUPS) and raw chicken manure (RCM) and comparing the results in either case. The co-digestions of untreated primary sludge from Abu tesht wastewater treatment stations with different levels of raw chicken manure (0:100, 10:90, 30:70, 50:50, 90:10, and 100:0) to obtain the best mixtures. Also, co-digestion of untreated primary sludge from south valley university with different levels of raw chicken manure at the same ratios, to obtain the best mixtures. Batch digestion tests were applied in 2.5 L digester with a working volume of 2.0 L. The samples in triplicates were separately loaded into the digesters locally fabricated and kept for 20 days as a retention period and diluted with the same amount of water. Mesophilic under 35 °C was adopted for untreated primary sludge as well as mixtures with raw chicken manure based on total solids (TS) and volatile solid (VS) proportions. The average biogas yields from AUPS/RCM mixture obtained ranged from 8570 to 5600 ml, by the following descending order, 10: 90 > 90:10 and so on >100:0, and the average biogas yields from SUPS/RCM obtained ranged from 6330 to 5635 ml, in the order of 90: 10 > 10:90 and so on >100:0. The results showed highest biogas yield from AUPS/RCM and SUPS/RCM mixtures with mixing ratio of 10:90 and 90:10, respectively, however, the lowest biogas production detected in separate digestion of AUPS and SUPS. The results indicated that co-digestion between the sludge and raw chicken manure could increase total biogas production volume, enhance sludge treatment process, and produce eco-friendly sludge because of co-digestion process than separate processing of each feedstock.     

Physiological compartmental analysis of alpha-linolenic acid metabolism in adult humans

A physiological compartmental model of alpha-linolenic acid metabolism was derived from the plasma concentration-time curves for d5-18:3n-3, d5-20:5n-3, d5-22:5n-3, and d5-22:6n-3 in eight healthy subjects. Subjects received a 1-g oral dose of an isotope tracer of alpha-linolenate (d5-18:3n-3 ethyl ester) while subsisting on a rigorously controlled beef-based diet. By utilizing the Windows Simulation and Analysis Modeling program, kinetic parameters were determined for each subject. Half-lives and mean transit times of the n-3 fatty acids in the plasma were also determined. The model predicted plasma values for the n-3 fatty acids in good accordance with the measured steady state concentrations and also predicted dietary linolenic acid intake for each subject in accordance with values determined by lipid analysis of the diet. Only about 0.2% of the plasma 18:3n-3 was destined for synthesis of 20:5n-3, approximately 63% of the plasma 20:5n-3 was accessible for production of 22:5n-3, and 37% of 22:5n-3 was available for synthesis of 22:6n-3. The inefficiency of the conversion of 18:3n-3 to 20:5n-3 indicates that the biosynthesis of long-chain n-3 PUFA from alpha-linolenic acid is limited in healthy individuals. In contrast, the much greater rate of transfer of mass from the plasma 20:5n-3 compartment to 22:5n-3 suggests that dietary eicosapentaenoic acid may be well utilized in the biosynthesis of 22:6n-3 in humans.     

Reversal of docosahexaenoic acid deficiency in the rat brain, retina, liver, and serum

The loss of docosahexaenoic acid (DHA) from the retina or brain has been associated with a loss in nervous-system function in experimental animals, as well as in human infants fed vegetable oil-based formulas. The reversibility of the loss of DHA and the compensation by an increase in the n-6 docosapentaenoic acid (DPAn-6) was studied in young adult rats. Long-Evans rats were subjected to a very low level of n-3 fatty acids through two generations. The F2 generation, n-3-deficient animals at 7 weeks of age were provided a repletion diet containing both alpha-linolenate and DHA. A separate group of F2 generation rats had been maintained on an n-3-adequate diet of the same composition. Tissues from the brain, retina, liver, and serum were collected on weeks 0, 1, 2, 4, and 8 from both groups of animals. The concentrations of DHA, DPAn-6, and other fatty acids were determined and the rate of recovery and length of time needed to complete DHA recovery were determined for each tissue. The DHA level in the brain at 1 and 2 weeks after diet reversal was only partially recovered, rising to approximately 20% and 35%, respectively, of the n-3-adequate group level. Full recovery was not obtained until 8 weeks after initiation of the repletion diet. Although the initial rate of retinal DHA accretion was greater than that of brain DHA, the half-time for DHA recovery was only marginally greater. On the other hand, the levels of DHA in the serum and liver were approximately 90% and 100% replaced, respectively, within 2 weeks of diet reversal. A consideration of the total amounts and time courses of DHA repleted in the nervous system compared with the liver and circulation suggests that transport-related processes may limit the rate of DHA repletion in the retina and brain.-- Moriguchi, T., J. Loewke, M. Garrison, J. N. Catalan, N. Salem, Jr. Reversal of docosahexaenoic acid deficiency in the rat brain, retina, liver, and serum. J. Lipid Res. 2001. 42: 419--427.     

Correlation of observed fold frequency with the occurrence of local structural motifs

It is well known that some protein folds (superfolds) occur very frequently. We show that compared to other folds, most superfold structures have a higher proportion of their alpha-helical or beta-strand residues in one of three basic units of supersecondary structure (alpha-hairpin, beta-hairpin or betaalphabeta-unit). Furthermore, by taking into consideration two more complex motifs, the four-stranded Greek-key (beta4) and the betaalpha-Greek key (betaalphabetabeta), we demonstrate that the remaining superfold structures contain many of these higher order units of three-dimensional packing. The implications of these results for folding are discussed.     

Lymphoplasmacytic sclerosing pancreato-cholangitis: a case report and review of the literature

Autoimmune pancreatitis is a rare but important cause of pancreatitis that is becoming increasingly recognized in the West. Lymphoplasmacytic sclerosing pancreatitis (LPSP) is a benign form of chronic pancreatitis characterized clinically by infrequent attacks of abdominal pain, jaundice, and weight loss, and pathologically by focal or diffuse chronic or lymphoplasmacytic inflammatory infiltrates centered around pancreatic ducts and ductules, accompanied by obliterative phlebitis, acinar atrophy, and interstitial fibrosis. It has been described alone or as a part of the spectrum of autoimmune gallbladder and biliary tract disease, with clinical, radiological, and pathological overlap reported with primary sclerosing cholangitis. It has been described as "primary sclerosing pancreatitis," "sclerosing cholangitis," "non-alcoholic duct destructive chronic pancreatitis," and "autoimmune pancreatitis." We report a case of LPSP that mimicked pancreatic adenocarcinoma and was subsequently treated with a pylorus-preserving Whipple procedure. This may point towards a primary biliary autoimmune process involving the pancreatic duct, causing a benign form of chronic pancreatitis that may be difficult to characterize pre-operatively to avoid surgery. This case typifies the growing awareness of this relatively recently characterized clinical entity, its similar presentation to pancreatic carcinoma, and the importance for LPSP to be included in the differential diagnosis of pancreaticobiliary disease. Finally, we review the literature.     

Antikinetoplastid activity of 3-aryl-5-thiocyanatomethyl-1,2,4-oxadiazoles

A series of 5-thiocyanatomethyl- and 5-alkyl-3-aryl-1,2,4-oxadiazoles were synthesized and evaluated for their activity against kinetoplastid parasites. Formation of the oxadiazole ring was accomplished through the reaction of benzamidoximes with acyl chlorides, while the thiocyanate group was inserted by reacting the appropriate 5-halomethyl oxadiazole with ammonium thiocyanate. The thiocyanate-containing compounds possessed low micromolar activity against Leishmania donovani and Trypanosoma brucei, while the 5-alkyl oxadiazoles were less active against these parasites. 3-(4-Chlorophenyl)-5-(thiocyanatomethyl)-1,2,4-oxadiazole (compound 4b) displayed modest selectivity for L. donovani axenic amastigote-like parasites over J774 macrophages, PC3 prostate cancer cells, and Vero cells (6.4-fold, 3.8-fold, and 9.1-fold, respectively), while 3-(3,4-dichlorophenyl)-5-(thiocyanatomethyl)-1,2,4-oxadiazole (compound 4 h) showed 30-fold selectivity against Vero cells but was not selective against PC3 cells. In a murine model of visceral leishmaniasis, compound 4b decreased liver parasitemia caused by L. donovani by 48% when given in five daily i.v. doses at 5mg/kg and by 61% when administered orally for 5 days at 50 mg/kg. These results indicate that aromatic thiocyanates hold promise for the treatment of leishmanial infections if the selectivity of these compounds can be improved.