Constituents from the stems of Actinodaphne lancifolia

Two C(16)-lactonic compounds, actinolides A-B (1-2), were isolated from the stems of Actinodaphne lancifolia, together with five known lactones (3-7) and three known lignans (8-10). Their structures were determined spectroscopically, which included 2D NMR spectroscopic analysis.     

Sphingoid bases and ceramide induce apoptosis in HT-29 and HCT-116 human colon cancer cells

Complex dietary sphingolipids such as sphingomyelin and glycosphingolipids have been reported to inhibit development of colon cancer. This protective role may be the result of turnover to bioactive metabolites including sphingoid bases (sphingosine and sphinganine) and ceramide, which inhibit proliferation and stimulate apoptosis. The purpose of the present study was to investigate the effects of sphingoid bases and ceramides on the growth, death, and cell cycle of HT-29 and HCT-116 human colon cancer cells. The importance of the 4,5-trans double bond present in both sphingosine and C(2)-ceramide (a short chain analog of ceramide) was evaluated by comparing the effects of these lipids with those of sphinganine and C(2)-dihydroceramide (a short chain analog of dihydroceramide), which lack this structural feature. Sphingosine, sphinganine, and C(2)-ceramide inhibited growth and caused death of colon cancer cells in time- and concentration-dependent manners, whereas C(2)-dihydroceramide had no effect. These findings suggest that the 4,5-trans double bond is necessary for the inhibitory effects of C(2)-ceramide, but not for sphingoid bases. Evaluation of cellular morphology via fluorescence microscopy and quantitation of fragmented low-molecular weight DNA using the diphenylamine assay demonstrated that sphingoid bases and C(2)-ceramide cause chromatin and nuclear condensation as well as fragmentation of DNA, suggesting these lipids kill colon cancer cells by inducing apoptosis. Flow cytometric analyses confirmed that sphingoid bases and C(2)-ceramide increased the number of cells in the A(0) peak indicative of apoptosis and demonstrated that sphingoid bases arrest the cell cycle at G(2)/M phase and cause accumulation in the S phase. These findings establish that sphingoid bases and ceramide induce apoptosis in colon cancer cells and implicate them as potential mediators of the protective role of more complex dietary sphingolipids in colon carcinogenesis.     

Reaction equilibrium of penicillin G with amberlite LA-2 in a nonpolar organic solvent

Studies have been made of the reactive extraction of penicillin G by Amberlite LA-2, a secondary amine, dissolved in kerosene. On the basis of the previous works about extraction equilibria of monocarboxylic acids by some secondary amines in low polar organic solvents, four equilibrium models were suggested to describe the reaction equilibrium of penicillin G in the liquid-liquid extraction system. The calculated results from the models were compared with the experimental data of 96 runs, and only two equilibrium models seemed to be probable. Ultimately, the most reasonable extraction equilibrium model was chosen through spectroscopic studies on organic solutions obtained by five specific extraction equilibrium experiments.     

Founder mutation R245H of Sanfilippo syndrome type A in the Cayman Islands

Sanfilippo A syndrome is an autosomal recessive lysosomal storage disease. This disease was reported in the Cayman Islands population with carrier frequency of 1/7 to 1/10 in the West Bay district of Grand Cayman. The carrier testing of Sanfilippo A disease for families at risk was carried out using the thermal characteristics of sulfamidase activity. In the present study, a search for mutations in the sulfamidase gene in an index family was performed. In addition, 77 individuals, relatives of children with Sanfilippo A syndrome, were also studied by single-strand conformation polymorphism (SSCP), restriction fragment-length polymorphism (RFLP) analyses, and sequencing. A single mutation, G746A (R245H), was found in the family, with the patient being homozygous and both parents and 1 of the 3 siblings being carriers. Among the 77 family members of the patient with Sanfilippo syndrome, the same mutation was found among carriers of the disease. The finding of a single mutation supports the idea of a founder effect, which facilitates accurate carrier identification of Sanfilippo A syndrome in the population of Cayman Islands.     

An alternate targeting pathway for procathepsin L in mouse fibroblasts

In transformed mouse fibroblasts, a significant proportion of the lysosomal cysteine protease cathepsin L remains in cells as an inactive precursor which associates with membranes by a mannose phosphate-independent interaction. When microsomes prepared from these cells were resolved on sucrose gradients, this procathepsin L was localized in dense vesicles distinct from those enriched for growth hormone, which is secreted constitutively when expressed in fibroblasts. Ultrastructural studies using antibodies directed against the propeptide to avoid detection of the mature enzyme in lysosomes revealed that the proenzyme was concentrated in dense cores within small vesicles and multivesicular endosomes which labeled with antibodies specific for CD63. Consistent with the resemblance of these cores to those of regulated secretory granules, secretion of procathepsin L from fibroblasts was modestly stimulated by phorbol, 12-myristate, 13-acetate. When protein synthesis was blocked with cycloheximide and lysosomal proteolysis inhibited with leupeptin, procathepsin L was found to gradually convert to the active single-chain protease. The data suggest that when synthesis levels are high, a portion of the procathepsin L is packaged in dense cores within multivesicular endosomes localized near the plasma membrane. Gradual activation of this proenzyme achieves targeting of the proenzyme to lysosomes by a mannose phosphate receptor-independent pathway.     

Hydrazinocurcumin,a novel synthetic curcumin derivative,is a potent inhibitor of endothelial cell proliferation

Curcumin and some of its derivatives were known as in vivo inhibitors of angiogenesis. In present study, a novel curcumin derivative, named hydrazinocurcumin (HC) was synthesized and examined for its biological activities. HC potently inhibited the proliferation of bovine aortic endothelial cells (BAECs) at a nanomolar concentration (IC(50)=520 nM) without cytotoxicity. In vivo and in vitro angiogenesis experiments showed HC as a new candidate for anti-angiogenic agent.     

Production of monoclonal antibodies and immunohistochemical studies of brain myo-inositol monophosphate phosphatase

Five monoclonal antibodies that recognize porcine brain myo-inositol monophosphate phosphatase (IMPase) have been selected and designated as mAb IMPP 9, IMPP 10, IMPP 11, IMPP 15, and IMPP 17. These antibodies recognize different epitopes of the enzyme and one of these inhibited the enzyme activity. When the total proteins of the porcine brain homogenate separated by SDS-PAGE were probed with monoclonal antibodies, a single reactive protein band of 29 kDa, co-migrating with the purified porcine brain IMPase, was detected. Using the anti-IMPase antibodies as probes, the cross reactivities of the brain IMPase from human and other mammalian tissues, as well as from avian sources, were investigated. Among the human and animal tissues tested, the immunoreactive bands on Western blots appeared to have the same molecular mass of 29 kDa. In addition, there was IMPase immunoreactivity in the various neuronal populations in the rat brain. These results indicate that mammalian brains contain only one major type of immunologically similar IMPase, although some properties of the enzymes that were previously reported differ from each another. The first demonstration of the IMPase localization in the brain may also provide useful data for future investigations on the function of this enzyme in relation to various neurological diseases.     

Backbone 1H, 15N, and 13C resonance assignments of the Helicobacter pylori acyl carrier protein

One of the small proteins from Helicobacter pylori, acyl carrier protein (ACP), was investigated by NMR. ACP is related to various cellular processes, especially with the biosynthesis of fatty acid. The basic NMR resonance assignment is a prerequisite for the validation of a heterologous protein interaction with ACP in H. pylori. Here, the results of the backbone (1)H, (15)N, and (13)C resonance assignments of the H. pylori ACP are reported using double- and triple-resonance techniques. About 97% of all of the (1)HN, (15)N, (13)CO, (13)Calpha, and (13)Cbeta resonances that cover 76 of the 78 non-proline residues are clarified through sequential- and specific- assignments. In addition, four helical regions were clearly identified on the basis of the resonance assignments.