Serine regulates phosphatidylethanolamine biosynthesis in the hamster heart.

The role of serine as a precursor and metabolic regulator for phosphatidylethanolamine biosynthesis in the hamster heart was investigated. Hearts were perfused with 50 microM [1-3H]ethanolamine in the presence or absence of serine for up to 60 min. Ethanolamine uptake was attenuated by 0.05-10 mM serine in a noncompetitive manner, and the incorporation of labeled ethanolamine into phosphatidylethanolamine was also inhibited by serine. Analysis of the ethanolamine-containing metabolites in the CDP-ethanolamine pathway revealed that the conversion of ethanolamine to phosphoethanolamine was reduced. The reduction was a result of an inhibition of ethanolamine kinase activity by an elevated pool of intracellular serine. Perfusion of the heart with 1 mM serine caused a 5-fold increase in intracellular serine pool. In order to examine the action of serine on other phosphatidylethanolamine metabolic pathways, hearts were perfused with [1-3H]glycerol in the presence and absence of serine. Serine did not cause any enhancement of phosphatidylethanolamine hydrolysis. The base-exchange reaction for phosphatidylserine formation or the decarboxylation of phosphatidylserine was not affected by serine perfusion. We conclude that circulating serine plays an important role in the modulation of phosphatidylethanolamine biosynthesis via the CDP-ethanolamine pathway in the hamster heart but does not affect the contribution of the decarboxylase pathway for phosphatidylethanolamine formation.     

Trichinella spiralis: light microscope monoclonal antibody localization and immunochemical characterization of phosphorylcholine and other antigens in the muscle larva

A panel of monoclonal antibodies was used to examine the structure of the muscle larva of Trichinella spiralis under the light microscope. Immunofluorescence and, in some cases, immunoperoxidase staining were used. All four antibodies reacted with the cuticle of the organism, although differences in the staining pattern were observed for some of these. Interestingly, all the antibodies also reacted with the stichosome. One of the antibodies (Ts2Ab) is specific for the hapten, phosphorylcholine. In a binding assay, this antibody also reacted with extracts of Trichuris suis, Ascaris suum, and Fasciolopsis buski, but not with extracts derived from Cysticercus cellulosae, Candida albicans, Salmonella typhi, or Escherichia coli. This crossreactivity was confirmed microscopically in which the cuticle, oviduct and eggs of T. suis, the cuticle, muscle cells, and eggs of A. suum, and the cuticle and vitelline glands of F. buski were seen to be clearly stained by the antibody. In addition, Ts2Ab also reacted with the cuticle and stichosome of the adult T. spiralis worm. In Western blot analysis, Ts2Ab recognized a 43-kDa antigen from T. spiralis muscle larvae extracts, while a previously studied antibody (7C2C5Ab) identified four major antigens (48.5, 47, 43, and 39 kDa) in this preparation. Similar results were obtained when the 24-hr excretory-secretory (ES) antigens of T. spiralis were immunoblotted with the antibodies, although the reactivity shown by Ts2Ab was relatively weak. With the 72-hr ES material, on the other hand, major antigens of lower mol wt (44, 28, and 25 kDa) were revealed by 7C2C5Ab, and no reactivity was seen with Ts2Ab. However, this antigen preparation reacted well with both antibodies in an enzyme-linked immunoassay. Taken together, the findings suggest that the 72-hr ES antigens probably result from extensive degradation of material originally secreted or excreted by the worm. Similar binding studies on the 24-hr ES preparation indicated that this source may be relatively rich in 7C2C5Ab-reactive epitopes and relatively poor in the antigen identified by Ts2Ab. Other studies performed demonstrated that the antigens recognized by these two antibodies were distinct and physically unassociated.     

Modulation of phosphatidylethanolamine biosynthesis by exogenous ethanolamine and analogues in the hamster heart

In the hamster heart, exogenous ethanolamine is taken up by the heart and utilized for the biosynthesis of phosphatidylethanolamine. The role of the exogenous supply of ethanolamine on phosphatidylethanolamine biosynthesis was examined by perfusing hamster heart with various concentrations of labelled ethanolamine. Analysis of the radioactivity distributed in the ethanolamine-containing metabolites indicated that at low exogenous ethanolamine concentrations ( 0.1 M), the conversion of phosphoethanolamine to CDP-ethanolamine was rate-limiting for phosphatidylethanolamine biosynthesis. However, perfusion with higher concentrations of ethanolamine ( 0.4 M) resulted in the phosphorylation of ethanolamine becoming rate-limiting. Since the intracellular ethanolamine levels remained unchanged, the alterations in radioactivity distribution suggested that the newly imported ethanolamine was preferentially utilized for phosphatidylethanolamine biosynthesis. The effects of ethanolamine analogues on ethanolamine uptake and subsequent conversion to phosphatidylethanolamine at physiological concentrations of exogenous ethanolamine were examined. Monomethylethanolamine was found to inhibit ethanolamine uptake, the conversion of ethanolamine to phosphoethanolamine and incorporation of radioactivity into phosphatidylethanolamine.The accumulation of radioactivity in the ethanolamine fraction by monomethylethanolamine, despite of the inhibition of ethanolamine uptake, further confirms the rate-limiting role of ethanolamine kinase in the biosynthesis of phosphatidylethanolamine. (Mol Cell Biochem116: 69–73, 1992)     

Pex17p is required for import of both peroxisome membrane and lumenal proteins and interacts with Pex19p and the peroxisome targeting signal-receptor docking complex in Pichia pastoris

Pichia pastoris PEX17 was cloned by complementation of a peroxisome-deficient strain obtained from a novel screen for mutants disrupted in the localization of a peroxisomal membrane protein (PMP) reporter. PEX17 encodes a 267-amino-acid protein with low identity (18%) to the previously characterized Saccharomyces cerevisiae Pex17p. Like ScPex17p, PpPex17p contains a putative transmembrane domain near the amino terminus and two carboxyl-terminal coiled-coil regions. PpPex17p behaves as an integral PMP with a cytosolic carboxyl-terminal domain. pex17Delta mutants accumulate peroxisomal matrix proteins and certain integral PMPs in the cytosol, suggesting a critical role for Pex17p in their localization. Peroxisome remnants were observed in the pex17Delta mutant by morphological and biochemical means, suggesting that Pex17p is not absolutely required for remnant formation. Yeast two-hybrid analysis demonstrated that the carboxyl terminus of Pex19p was required for interaction with Pex17p lacking the carboxyl-terminal coiled-coil domains. Biochemical evidence confirmed the interaction between Pex19p and Pex17p. Additionally, Pex17p cross-linked to components of the peroxisome targeting signal-receptor docking complex, which unexpectedly contained Pex3p. Our evidence suggests the existence of distinct subcomplexes that contain separable pools of Pex3p, Pex19p, Pex17p, Pex14p, and the peroxisome targeting signal receptors. These distinct pools may serve different purposes for the import of matrix proteins or PMPs.     

Chemical Constituents and Biological Activities of Piper as Anticancer Agents: A Review

Cancer has become the primary cause of death worldwide, and anticancer drugs are used to combat this disease. Synthesis of anticancer drugs has limited success due to adverse side effects has made compounds from natural products with minimal toxicity gain much popularity. Piper species are known to have a biological effect on human health. The biological activity is due to Piper species rich with active secondary metabolites that can combat most diseases, including cancer. This review will discuss the phytochemistry of Piper species and their anticancer activity. The identification and characterization of ten active metabolites isolated from Piper species were discussed in detail and their anticancer mechanism. These metabolites were mainly found could inhibit anticancer through caspase and P38/JNK pathways. The findings discussed in this review support the therapeutic potential of Piper species against cancer due to their rich source of active metabolites with demonstrated anticancer activity.     

Simultaneous saccharification and fermentation of cellulose for lactic acid production

Lactic acid production from α-cellulose by simultaneous saccharification and fermentation (SSF) was studied. The cellulose was converted in a batch SSF using cellulase enzyme Cytolase CL to produce glucose sugar andLactobacillus delbrueckii to ferment the glucose to lactic acid. The effects of temperature, pH, yeast extract loading, and lactic acid inhibition were studied to determine the optimum conditions for the batch processing. Cellulose was converted efficiently to lactic acid, and enzymatic hydrolysis was the rate controlling step in the SSF. The highest conversion rate was obtained at 46°C and pH 5.0. The observed yield of lactic acid from α-cellulose was 0.90 at 72 hours. The optimum pH of the SSF was coincident with that of enzymatic hydrolysis. The optimum temperature of the SSF was chosen as the highest temperature the microorganism could withstand. The optimum yeast extract loading was found to be 2.5 g/L. Lactic acid was observed to be inhibitory to the microorganisms’ activity.     

Exact inbreeding coefficients in populations with overlapping generations

A theory is given that allows inbreeding coefficients to be calculated exactly for populations with overlapping generations. Emphasis is placed on providing equations well suited for computer iteration. Both monoecious and dioecious populations are considered and family size is not restricted to being Poisson. One-locus and two-locus inbreeding coefficients are evaluated, although the reader may omit the two-locus sections. The exact treatment is shown to be preferable to approximate treatments in that it applies to both early and late generations for all populations sizes. Inbreeding effective numbers found by the exact treatment are compared to various approximate numbers, and the approximate values are found to be generally very good.     

Collagen/annexin V interactions regulate chondrocyte mineralization

Physiological mineralization in growth plate cartilage is highly regulated and restricted to terminally differentiated chondrocytes. Because mineralization occurs in the extracellular matrix, we asked whether major extracellular matrix components (collagens) of growth plate cartilage are directly involved in regulating the mineralization process. Our findings show that types II and X collagen interacted with cell surface-expressed annexin V. These interactions led to a stimulation of annexin V-mediated Ca(2+) influx resulting in an increased intracellular Ca(2+) concentration, [Ca(2+)](i), and ultimately increased alkaline phosphatase activity and mineralization of growth plate chondrocytes. Consequently, stimulation of these interactions (ascorbate to stimulate collagen synthesis, culturing cells on type II collagen-coated dishes, or overexpression of full-length annexin V) resulted in increase of [Ca(2+)](i), alkaline phosphatase activity, and mineralization of growth plate chondrocytes, whereas inhibition of these interactions (3,4-dehydro-l-proline to inhibit collagen secretion, K-201, a specific annexin channel blocker, overexpression of N terminus-deleted mutant annexin V that does not bind to type II collagen and shows reduced Ca(2+) channel activities) decreased [Ca(2+)](i), alkaline phosphatase activity, and mineralization. In conclusion, the interactions between collagen and annexin V regulate mineralization of growth plate cartilage. Because annexin V is up-regulated during pathological mineralization events of articular cartilage, it is possible that these interactions also regulate pathological mineralization.     

Sustained metaphase arrest in response to ionizing radiation in a non-small cell lung cancer cell line

Kodym, E., Kodym, R., Choy, H. and Saha, D. Sustained Metaphase Arrest in Response to Ionizing Radiation in a Non-small Cell Lung Cancer Cell Line. Radiat. Res. 169, 46-58 (2008). In solid tumors, non-apoptotic forms of tumor cell inactivation such as mitotic catastrophe appear to be predominant in the response to DNA-damaging agents. Despite its importance, the underlying molecular mechanisms of mitotic catastrophe have been only partially elucidated. We found that a large fraction of HCC2279 non-small cell lung cancer cells underwent mitotic catastrophe after irradiation. Cells were arrested in metaphase with chromosomal damage indicated by DNA fragments displaced from the metaphase plate and considerable numbers of residual gamma-H2AX foci. Although TP53 was nonfunctional, we detected a prompt radiation response on the level of checkpoint kinases. In contrast, CDC25A was the only checkpoint phosphatase that was responsive to radiation. CDC25B was not detectable, and CDC25C was constitutively phosphorylated at serine 216, leading to its cytoplasmic sequestration and functional inactivation. Therefore, radiation-induced mitotic catastrophe in HCC2279 cells appears to be induced by a combination of relative insufficiencies in the p53-mediated and checkpoint kinase-mediated pathways leading to premature entry into mitosis. Displaced chromosome fragments triggering an intra-M checkpoint in cells entering mitosis presumably result in a sustained metaphase arrest. The phenomenon found in these cells, which were derived directly from a human patient, might be responsible for therapy-induced genetic instability of tumors.