Role of intracellular reactive oxygen species and mitochondrial dysfunction in evening primrose extract-induced apoptosis in Ehrlich ascites tumor cells

Herbal medicines are increasingly being utilized to treat a wide variety of disease processes. Evening primrose extract (EPE) is extracted from Oenothera biennis L., one species of evening primroses, which has been shown to have several pharmacological effects. However, anti-tumor activity in the extract of defatted seeds of O. biennis L. has not been defined thus far. In this study, we identified the major biochemical changes upon EPE treatment and investigated the functional relationship between these changes. We found that EPE-induced apoptosis in Ehrlich ascites tumor cells as evidenced by morphological changes. Furthermore, our results demonstrated rapid increase of intracellular peroxides levels, loss of mitochondrial membrane potential and the release of cytochrome c from mitochondria to cytosol. These results suggest that the rapid increase of intracellular peroxides levels after addition of EPE triggers off induction of apoptosis.     

Asymmetric formation and possible function of the primary pore canal in plutei of Temnopleurus hardwicki

The development and possible function of the primary pore canal (PPC) in plutei of the sea urchin Temnopleurus hardwicki was examined by immunochemistry, electron microscopy and microsurgery. Left and right PPC that extended from coelomic sacs in plutei contained a bundle of cilia with a 9 + 2 structure that was initially detected as a group of anti-acetylated tubulin antibody-binding granules in the epithelium of coelomic sacs in 28 h postfertilization (PF) prism larvae. The granules extended to be a bundle of fibers toward the larval dorsal surface, concurrent with formation of the PPC on both sides, over the next 4 h. The cilia in both PPC beat actively. However, the PPC on the right side disappeared by approximately 55 h PF, establishing left-right asymmetry by 60 h PF (the four-arm pluteus stage). The numbers of cilia in the left and right PPC in 56 h PF plutei were five and eight, respectively. Microsurgical removal of the coelomic sac from both sides or the left side only from 26 h PF prism larvae decreased body width to 64 and 91% of normal width by 50 h PF pluteus stage, respectively, whereas that of the right PPC did not. These observations suggest that PPC contribute to the maintenance of normal body width, and that there is asymmetrical activity between the left and right PPC.     

Distinct enzymatic properties of recombinant mouse DNA methyltransferases Dnmt3a and Dnmt3b

Recombinant mouse Dnmt3a and Dnmt3b were expressed in sf9 cells and purified to near homogeneity. The purified Dnmt3a and Dnmt3b gave specific activities of 1.8 +/- 0.3 and 1.3 +/- 0.1 mol/h/mol enzyme towards poly(dGdC)-poly(dGdC), respectively, which were the highest among those reported. Dnmt3a or Dnmt3b showed similar K(m) values towards poly(dIdC)-poly(dIdC) and poly(dGdC)-poly(dGdC). The K(m) values for S-adenosyl-L-methionine were not affected by the methyl-group acceptors, poly(dI-dC)-poly(dIdC) and poly(dG-dC)-poly(dGdC). The results indicate that the enzymes are de novo-type DNA methyltransferases. Dnmt3a and Dnmt3b activities were inhibited by Mn(2+) and Ni(2+) and showed broad pH optima around neutral pH. Both enzymes were susceptible to sodium ions, which inhibited their activity at around physiological ionic strength. However, Dnmt3a was fully active at physiological potassium concentration, but Dnmt3b was not. Using designed oligonucleotides for the analysis of cytosine methylation, we demonstrated that, in addition to CpG, Dnmt3a methylated CpA but not CpT and CpC, and that Dnmt3b methylated CpA and CpT but scarcely CpC. The relative activity of Dnmt3b towards nonCpG sequences was higher than that of Dnmt3a. These differences in enzymatic properties of Dnmt3a and Dnmt3b may contribute to the distinct functions of these enzymes in vivo.     

An in vivo model for monitoring trans-differentiation of bone marrow cells into functional hepatocytes

The plasticity of bone marrow cells (BMCs) remains controversial. The present study found that persistent injury induces efficient trans-differentiation of BMCs into functional hepatocytes. Mice with liver cirrhosis induced by carbon tetrachloride were injected with 1 x 10(5) non-treated green fluorescent protein (GFP)-positive BMCs via the tail vein. In these mice, transplanted GFP-positive BMCs efficiently migrated into the peri-portal area of liver lobules after one day, repopulating 25% of the recipient liver by 4 weeks. In contrast, no GFP-positive BMCs were detected following transplantation into control mice with undamaged livers. BMCs trans-differentiated into functional mature hepatocytes via immature hepatoblasts. Serum albumin levels were significantly elevated to compensate for chronic liver failure in BMC transplantation. These results reveal that recipient conditions and microenvironments represent key factors for successful cell therapy using BMCs.     

Infertility observed in reproductive toxicity study of N-acetyl-L-cysteine in rats

The toxic effects of i.v. administration of N-acetyl-l-cysteine (NAC), a component of parenteral nutrition solutions, on fertility and embryonic development were investigated in SD male and female rats at doses of 100, 300, and 1000 mg kg-1 day-1. Infertility was observed in females in the 1000-mg/kg group throughout the period from before mating to embryogenesis. No effect of NAC on the reproductive ability of the male rats was seen. The oocytes and embryos were assessed morphologically to clarify the cause of the effects of NAC. The unfertilized oocytes (UO) recovered from the ampullae of the uterine tubes and Gestational Day (GD) 1 and 2 embryos recovered from the oviducts or uterus of the rats that received NAC i.v. at a dosage of 1000 mg kg-1 day-1 for more than 1 wk before mating were assessed morphologically by stereomicroscopy. In addition, the thickness of the zona pellucida (ZP) was calculated by morphometric evaluation of the UO. Fewer UO were collected in the NAC group than in the control (nontreatment) group. Interestingly, ZP-lacking or partially ZP-lacking oocytes were observed in the NAC group, and the morphometric evaluation of the UO showed thinning of the ZP. The number of embryos in each animal was markedly decreased on GD1, and no embryos were recovered on GD2 in the NAC group. The oocytes that had ZP affected by NAC treatment were abnormal or nonviable. The findings of the present study suggest that changes in the ZP are related to the infertility associated with NAC.     

Intraspecies polymorphism of Cryptosporidium parvum revealed by PCR-restriction fragment length polymorphism (RFLP) and RFLP-single-strand conformational polymorphism analyses

A glycoprotein (Cpgp40/15)-encoding gene of Cryptosporidium parvum was analyzed to reveal intraspecies polymorphism within C. parvum isolates. Forty-one isolates were collected from different geographical origins (Japan, Italy, and Nepal) and hosts (humans, calves, and a goat). These isolates were characterized by means of DNA sequencing, PCR-restriction fragment length polymorphism (PCR-RFLP), and RFLP-single-strand conformational polymorphism (RFLP-SSCP) analyses of the gene for Cpgp40/15. The sequence analysis indicated that there was DNA polymorphism between genotype I and II, as well as within genotype I, isolates. The DNA and amino acid sequence identities between genotypes I and II differed, depending on the isolates, ranging from 73.3 to 82.9% and 62.4 to 80.1%, respectively. Those among genotype I isolates differed, depending on the isolates, ranging from 69.0 to 85.4% and 54.8 to 79.2%, respectively. Because of the high resolution generated by PCR-RFLP and RFLP-SSCP, the isolates of genotype I could be subtyped as genotypes Ia1, Ia2, Ib, and Ie. The isolates of genotype II could be subtyped as genotypes IIa, IIb, and IIc. The isolates from calves, a goat, and one Japanese human were identified as genotype II. Within genotype II, the isolates from Japan were identified as genotype IIa, those from calves in Italy were identified as genotype IIb, and the goat isolate was identified as genotype IIc. All of the genotype I isolates were from humans. The Japanese isolate (code no. HJ3) and all of the Nepalese isolates were identified as genotypes Ia1 and Ia2, respectively. The Italian isolates were identified as genotype Ib, and the Japanese isolate (code no. HJ2) was identified as genotype Ie. Thus, the PCR-RFLP-SSCP analysis of this glycoprotein Cpgp40/15 gene generated a high resolution that has not been achieved by previous methods of genotypic differentiation of C. parvum.     

Comparison of binding and functional properties of two extrinsic components,Cyt c550 and a 12 kDa protein,in cyanobacterial PSII with those in red algal PSII

Cyt c550 and 12 kDa protein are two extrinsic proteins of photosystem II (PSII) found in cyanobacteria and some eukaryotic algae. The binding patterns of these two extrinsic proteins are different between cyanobacterial (Thermosynechococcus vulcanus) and red algal (Cyanidium caldarium) PSIIs [Shen and Inoue (1993) Biochemistry 32: 1825; Enami et al. (1998) Biochemistry 39: 2787]. In order to elucidate the possible causes responsible for these differences, we first cloned the psbV gene encoding Cyt c550 from a red alga, Cyanidium caldarium, which was compared with the homologous sequences from other organisms. Cross-reconstitution experiments were then performed with different combinations of the extrinsic proteins and the cyanobacterial or red algal PSII. (1). Both the cyanobacterial and red algal Cyt c550 bound directly to the cyanobacterial PSII, whereas none of them bound directly to the red algal PSII, indicating that direct binding of Cyt c550 to PSII principally depends on the structure of PSII intrinsic proteins but not that of Cyt c550 itself. (2). Cyt c550 was functionally exchangeable between the red algal and the cyanobacterial PSII, and the red algal 12 kDa protein functionally bound to the cyanobacterial PSII, whereas the cyanobacterial 12 kDa protein did not bind to the red algal PSII. (3). The antibody against the cyanobacterial or red algal 12 kDa protein reacted with its original one but not with the homologous protein from the other organism, whereas the antibody against the red algal Cyt c550 reacted with both cyanobacterial and red algal Cyt c550. These results imply that the structure and function of Cyt c550 have been largely conserved, whereas those of the 12 kDa protein have been changed, in the two organisms studied here.     

Reaction intermediate structures of 1-aminocyclopropane-1-carboxylate deaminase: insight into PLP-dependent cyclopropane ring-opening reaction

The pyridoxal 5'-phosphate-dependent enzymes have been evolved to catalyze diverse substrates and to cause the reaction to vary. 1-Aminocyclopropane-1-carboxylate deaminase catalyzes the cyclopropane ring-opening reaction followed by deamination specifically. Since it was discovered in 1978, the enzyme has been widely investigated from the mechanistic and physiological viewpoints because the substrate is a precursor of the plant hormone ethylene and the enzymatic reaction includes a cyclopropane ring-opening. We have previously reported the crystal structure of the native enzyme. Here we report the crystal structures of the two reaction intermediates created by the mutagenesis complexed with the substrate. The substrate was validated in the active site of two forms: 1). covalent-bonded external aldimine with the coenzyme in the K51T form and 2). the non-covalent interaction around the coenzyme in the Y295F form. The orientations of the substrate in both structures were quite different form each other. In concert with other site-specific mutation experiments, this experiment revealed the ingenious and unique strategies that are used to achieve the specific activity. The substrate incorporated into the active site is reactivated by a two-phenol charge relay system to lead to the formation of a Schiff base with the coenzyme. The catalytic Lys51 residue may play a novel role to abstract the methylene proton from the substrate in cooperation with other factors, the carboxylate group of the substrate and the electron-adjusting apparatuses of the coenzyme.     

Sphingosine kinase type 2 is a putative BH3-only protein that induces apoptosis

There are two isoforms of sphingosine kinase (SphK) that catalyze the formation of sphingosine 1-phosphate, a potent sphingolipid mediator. Whereas SphK1 stimulates growth and survival, here we show that SphK2 enhanced apoptosis in diverse cell types and also suppressed cellular proliferation. Apoptosis was preceded by cytochrome c release and activation of caspase-3. SphK2-induced apoptosis was independent of activation of sphingosine 1-phosphate receptors. Sequence analysis revealed that SphK2 contains a 9-amino acid motif similar to that present in BH3-only proteins, a pro-apoptotic subgroup of the Bcl-2 family. As with other BH3-only proteins, co-immunoprecipitation demonstrated that SphK2 interacted with Bcl-xL. Moreover, site-directed mutation of Leu-219, the conserved leucine residue present in all BH3 domains, markedly suppressed SphK2-induced apoptosis. Hence, the apoptotic effect of SphK2 might be because of its putative BH3 domain.     

Determination of free N-acetylneuraminic acid in urine by high-performance liquid chromatography using 3-[(1-[[4-(5,6-dimethoxy-1-oxoisoindolin-2-yl)-2-methoxyphenyl]sulfonyl]pyrrolidin-2-yl)carbonylamino]phenylboronic acid as a fluorescent labeling reagent

A highly sensitive high-performance liquid chromatography (HPLC) method for the determination of urinary N-acetylneuraminic acid (NeuAc) using 3-[(1-[[4-(5,6-dimethoxy-1-oxoisoindolin-2-yl)-2-methoxyphenyl]sulfonyl]pyrrolidin-2-yl)carbonylamino]phenylboronic acid as a fluorescent labeling reagent was developed. The labeling reaction was carried out at 30 degrees C for 30 min in the presence of pyridine. The derivative was monitored at Ex 314 nm and Em 388 nm. The detection limit of NeuAc was about 48 fmol per injection. The relative standard deviations of within-day and between-day precisions were 2.6-3.3 and 1.7-3.3%, respectively. Urine diluted 10 times with distilled water was analyzed by employing the standard-addition method. The concentrations were 8-89 nmol/mg creatinine (30+/-28 nmol/mg creatinine, n=9).