Nonthermal Plasma‐Induced Degradation of Morin and Enhancement of Biological Activities

In the present study, non‐thermal dielectric barrier discharge (DBD) plasma of induced structural changes of morin resulted in the isolation of one previously undescribed benzofuranone derivative, along with two known compounds. The chemical structures of these degradation products were elucidated by UV, NMR and FAB‐MS spectroscopic analyses. The isolated three compounds showed potent antioxidative activities in two different tests, with IC₅₀ values in the range of 12.9–41.8 μm in the 2,2′‐azino‐bis (3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS⁺) radical scavenging activity, 19.0–71.9 μm for hydroxyl radical scavenging activity test. Furthermore, the new methoxylated benzofuranone exhibited enhancement of inhibitory effects against pancreatic lipase with an IC₅₀ value of 90.7±1.6 μm , when compared to the parent morin. These results suggested that the degradation products isolated from plasma exposed morin might be beneficial for prevention of obesity and related diseases.     

Interleukin-1-β and Tumor Necrosis Factor-α Increase Peripheral-Type Benzodiazepine Binding Sites in Cultured Polygonal Astrocytes

Peripheral-type benzodiazepine binding sites (PTBBS) are markedly increased in the injured CNS. Astrocytes appear to be the primary cell type which express increased PTBBS. Because certain cytokines within the injured CNS are potent mitogens for astrocytes, we examined the effects of two such cytokines, interleukin (IL)-1 beta and tumor necrosis factor (TNF), on PTBBS in cultured astrocytes using [3H]Ro 5-4864 as the specific ligand. Purified cultures of either polygonal or process-bearing astrocytes were prepared from neonatal rat cerebral hemispheres. At a concentration of 1.8 nM, specific binding of the radioactive ligand to polygonal astrocytes reached equilibrium within 60 min and was half-maximal by 5-10 min. By contrast, specific binding to process-bearing astrocytes barely exceeded background levels. IL-1 and TNF increased PTBBS within polygonal astrocytes in both dose- and time-dependent manners. At 10-50 ng/ml, IL-1 beta and TNF-alpha elevated [3H]Ro 5-4864 binding in polygonal astrocyte cultures 65 and 87%, respectively, above the level in control cultures. However, no changes in PTBBS were seen within polygonal astrocytes after IL-2 treatment. Scatchard analysis of saturation binding experiments suggested that the increase in PTBBS promoted by TNF was due to an increased number of binding sites present in polygonal astrocytes and not due to an increase in receptor affinity. Binding data suggested that PTBBS within cultures of process-bearing astrocytes were virtually absent irrespective of the treatment. These in vitro data suggest that certain cytokines found in the injured brain may be involved in up-regulating PTBBS within a particular subtype of astrocyte.     

Modulation of Intracellular Calcium Levels by Calcium Lactate Affects Colon Cancer Cell Motility through Calcium-Dependent Calpain

Cancer cell motility is a key phenomenon regulating invasion and metastasis. Focal adhesion kinase (FAK) plays a major role in cellular adhesion and metastasis of various cancers. The relationship between dietary supplementation of calcium and colon cancer has been extensively investigated. However, the effect of calcium (Ca²⁺) supplementation on calpain-FAK-motility is not clearly understood. We sought to identify the mechanism of FAK cleavage through Ca²⁺ bound lactate (CaLa), its downstream signaling and role in the motility of human colon cancer cells. We found that treating HCT116 and HT-29 cells with CaLa immediately increased the intracellular Ca²⁺ (iCa²⁺) levels for a prolonged period of time. Ca²⁺ influx induced cleavage of FAK into an N-terminal FAK (FERM domain) in a dose-dependent manner. Phosphorylated FAK (p-FAK) was also cleaved in to its p-N-terminal FAK. CaLa increased colon cancer cells motility. Calpeptin, a calpain inhibitor, reversed the effects of CaLa on FAK and pFAK cleavage in both cancer cell lines. The cleaved FAK translocates into the nucleus and modulates p53 stability through MDM2-associated ubiquitination. CaLa-induced Ca²⁺ influx increased the motility of colon cancer cells was mediated by calpain activity through FAK and pFAK protein destabilization. In conclusion, these results suggest that careful consideration may be given in deciding dietary Ca²⁺ supplementation to patient undergoing treatment for metastatic cancer.     

Age-related changes in oxidized proteins

We have previously described the oxidative inactivation of several key metabolic enzymes by a variety of mixed function oxidation systems. Because many of the enzymes which are inactivated have been shown by others to accumulate as inactive or less active forms during cellular aging, we have examined the levels of oxidatively modified proteins in two model systems used for studies on aging. The results show that levels of oxidatively modified proteins increase with age in circulating erythrocytes, and this change is correlated with the loss of marker enzyme activity. Our studies also show that in cultured fibroblasts from normal donors the levels of oxidatively modified proteins increase only after the age of 60. However, the levels of oxidatively modified proteins in fibroblasts from individuals with progeria or Werner's syndrome are significantly higher than age-matched controls. Moreover, treatment of glucose-6-phosphate dehydrogenase with a mixed function oxidation system leads to oxidative modification and increased heat lability of the enzyme. Taken together these results suggest that loss of functional enzyme activity and increased heat lability of enzymes during aging may be due in part to oxidative modification by mixed function oxidation systems.     

Activation of dopamine beta-monooxygenase by external and internal electron donors in resealed chromaffin granule ghosts

Membrane ghosts derived from chromaffin vesicles of bovine adrenal medullas have been used to examine the mechanism of reduction of dopamine beta-monooxygenase in its compartmentalized state. The rate of the dopamine beta-monooxygenase-catalyzed conversion of dopamine to norepinephrine is greatly stimulated by the presence of ATP, reflecting substrate hydroxylation on the ghost interior subsequent to the active transport of dopamine. We demonstrate a 2-3-fold increase in the turnover rate for ghosts resealed with 0.2-2 mM potassium ferrocyanide, conditions leading to a slight decrease in the rate of dopamine transport. These data provide the first evidence that an intravesicular pool of reductant can activate dopamine beta-monooxygenase, as required by models in which vesicular ascorbate behaves as enzyme reductant. Although there is sufficient catecholamine (endogenous plus substrate) to keep internal ferrocyanide reduced in these experiments, an additional 2-3-fold increase in turnover occurs in the presence of 0.2-2 mM ascorbate on the ghost exterior. The magnitude of this activation is found to be constant at all concentrations of internal ferrocyanide (both below and above saturation), implying that reductants on opposite sides of the membrane behave independently. Replacement of ascorbate by potassium ferrocyanide as external reductant leads to almost identical results, and we are able to rule out an inward transport of dehydroascorbate as the source of activation by external ascorbate. We conclude that external reductants are capable of reducing membrane-bound dopamine beta-monooxygenase from the exterior face of the vesicle, either by direct reduction or through a membrane-bound mediator. It appears that two viable modes for reduction of dopamine beta-monooxygenase may exist in vivo, involving the reduction of membrane-bound enzyme by cytosolic ascorbate as well as the reduction of soluble enzyme by the pool of intravesicular ascorbate present in chromaffin vesicles.     

Cloning of a Vero toxin (VT2) gene from a VT2-converting phage isolated from Escherichia coli 0157: H7

Abstract A Vero toxin (VT2 or Shiga-like toxin II)-converting phage was isolated from Escherichia coli 0157: H7 strain J-2. Nontoxigenic E. coli C600 produced VT2 when lysogenized with the toxin-converting phage. Eco RI fragments of the phage DNA were ligated with Eco RI-digested pBR322 or pUC118 and were transformed into E. coli MC1061 or MV1184. Transformants exhibiting VT2 production commonly contained a 4.6 kb Eco RI fragment. It was found that a 2.3 kb Kpn I- Sph I fragment coded VT2 production and that this fragment hybridized weakly with the 2.1 kb fragment encoding VT1.     

Ultrastructure of poplar cortical cells during the transition from growing to wintering stages and vice versa

Summary Electron microscopic studies revealed that major cytological changes in the cortical cells of poplar (Populus euramericana cv. gelrica) began to occur in early September in conjunction with the metabolic transition from the growing to the wintering stage. During this transition, the cells became temporarily rich in endoplasmic reticulum, polysomes and vesicles. As the conspicuous formation of organelles progressed, the large vacuoles became smaller and filled with osmiophilic materials. Undefined organelles (protein-lipid bodies) also increased in number. From late October until March, organelles involved in protein synthesis were sparsely distributed in the cells, indicating that the number of these organelles is probably linked to the seasonal cycle of protein synthesis. In early February, after release from dormancy, fusion of vacuoles proceeded in the cells. The inclusion of organelles and a gradual decrease in the amount of osmiophilic materials in the vacuoles occurred at this stage. Subsequently, the structure of the cells continued to undergo changes to accommodate growth, which occurred in early May.