Synthesis of Lanostane‐Type Triterpenoid N‐Glycosides and Their Cytotoxicity against Human Cancer Cell Lines

Seventeen lanostane‐type triterpenoid derivatives ( 2 – 18 ), including 11N‐glycosides ( 8 – 18 ), were synthesized from the natural triterpenoid, lanosterol ( 1 ), and were evaluated for their cytotoxicity against the human cancer cell lines, HL‐60, A549, and MKN45, as well as the normal human lung cells, WI‐38. Among them, N‐β‐d ‐2‐acetamido‐2‐deoxyglucoside ( 10 ) showed cytotoxicity against HL‐60, A549, MKN45, and WI‐38 cells (IC₅₀ 0.0078 – 2.8 μm ). However, N‐β‐d ‐galactoside ( 12 ) showed cytotoxicity against HL‐60 and MKN45 cells (IC₅₀ 0.0021 – 4.0 μm ), but not the normal WI‐38 cells. Furthermore, Western blot analysis suggested that 12 induces apoptosis by activation of caspases‐3, 8, and 9. These results will be useful for the synthesis of other tetracyclic triterpenoids or steroid N‐glycosides to increase their cytotoxicity and apoptosis‐inducing activities.     

Estrogenic modulation of CYP3A38, CYP3A40, and CYP19 in mature male medaka (Oryzias latipes)

We examined cytochrome P450 production and activity and circulating hormone concentrations in male medaka exposed to 17beta-estradiol (E2) or 17alpha-ethinylestradiol (EE2). Intraperitoneal injection of E2 at 1, 10, or 100 microg/g-fish completely suppressed CYP3A38 protein production and suppressed CYP3A40 protein levels by 89%, 52%, or 47%, respectively. CYP3A38 and CYP3A40 mRNA expression was unaltered, and CYP3A enzymatic activity initially increased and then decreased with increasing E2 dose. Males co-cultured with females were exposed to a markedly high concentration (43 ng/L) of E2 secreted by females. CYP3A protein levels in co-cultured males were suppressed. Serum testosterone (TE) and 11keto-testosterone levels in co-cultured males were downregulated to 40% of pre-exposure levels. Serum E2 levels increased in co-cultured males or males exposed to EE2. Testicular CYP19, which converts TE to E2, increased by 9.5 times in males exposed to 50 ng/L EE2 and by 21.5 times in those exposed to 100 ng/L EE2. Male medaka exposed to EE2 showed increased serum Vtg levels. Estrogenic exposure induced Vtg production, suppressed CYP3A protein production, downregulated TE metabolism, and enhanced CYP19 activity. Serum E2 endogenously induced by CYP19 could contribute to Vtg induction in male medaka.     

Homogeneous fluorescence assays for RNA diagnosis by pyrene-conjugated 2'-O-methyloligoribonucleotides

We developed a bispyrene-conjugated 2'-O-methyloligoribonucleotide as an RNA-specific RNA-probe. The probe hybridized with the complementary RNA, greatly enhancing fluorescence and discriminating RNA from DNA. The assay was carried out in homogeneous aqueous media without removing the unbound probe from the detection solution. This homogeneous fluorescence assay also discriminated mismatch sequences in the target RNA. These pyrene probes could possess high potential to detect RNA in biological specimens simply.     

Phenylimidazole derivatives as specific inhibitors of bacterial enoyl-acyl carrier protein reductase FabK

Bacterial enoyl-acyl carrier protein (ACP) reductases (FabI and FabK) catalyze the final step in each cycle of bacterial fatty acid biosynthesis and are attractive targets for the development of new antibacterial agents. Here, we report the development of novel FabK inhibitors with antibacterial activity against Streptococcus pneumoniae. Based on structure-activity relationship (SAR) studies of our screening hits, we have developed novel phenylimidazole derivatives as potent FabK inhibitors.     

Sialyl oligosaccharides of human colostrum: changes in concentration during the first three days of lactation

Sialyl oligosaccharides of human milk/colostrum are generally believed to be of biological significance, for example with respect to anti-adhesion of pathogenic organism, providing precursors for biosynthesis of brain, and so on. However, the levels of each of the sialyl oligosaccharides in human colostrum have not so far been determined. The present study was designed to determine the concentrations of nine major sialyl oligosaccharides in human colostrum, collected during the first 3 d (days 1-3) from the start of lactation. We found that the concentration of 3'-sialyllactose was significantly higher on day 1 than on day 2 and 3, but the levels of 6'-sialyllactose and sialyllacto-N-tetraose a were higher on day 3 than on day 1. These results are consistent with the view that during the first 3 d of lactation, the concentration of sialyl oligosaccharides in human colostrum change in accordance with the physiological demands of newborn infants.     

In vivo distribution and localization of chorein

Chorea-acanthocytosis (ChAc) is a hereditary neurodegenerative disorder caused by loss of function mutations in the VPS13A gene encoding chorein. In this study, we produced an antibody against chorein and examined its protein-level expression and localization in mouse. Immunoblot analysis revealed that chorein was expressed in a gene dose-dependent manner in the VPS13A deletion-mice that we recently developed, which confirms the sensitivity of the antibody. Chorein was highly expressed in testis, kidney, spleen, and brain, and was expressed ubiquitously in various brain regions. Subcellular analysis of the brain showed high levels of chorein in microsomal and synaptosomal fractions. Immunohistochemically, chorein-like immunoreactivity was ubiquitously observed in the brain in the neuronal perinuclear region, cytoplasm and fibers. In testis and kidney, clear cell-specific patterns of chorein-like immunoreactivity were detected. Our findings provide basic information on chorein in vivo and may contribute to taking the first step toward understanding molecular pathogenesis of ChAc.     

Curculin exhibits sweet-tasting and taste-modifying activities through its distinct molecular surfaces

Curculin isolated from Curculigo latifolia, a plant grown in Malaysia, has an intriguing property of modifying sour taste into sweet taste. In addition to this taste-modifying activity, curculin itself elicits a sweet taste. Although these activities have been attributed to the heterodimeric isoform and not homodimers of curculin, the underlying mechanisms for the dual action of this protein have been largely unknown. To identify critical sites for these activities, we performed a mutational and structural study of recombinant curculin. Based on the comparison of crystal structures of curculin homo- and heterodimers, a series of mutants was designed and subjected to tasting assays. Mapping of amino acid residues on the three-dimensional structure according to their mutational effects revealed that the curculin heterodimer exhibits sweet-tasting and taste-modifying activities through its partially overlapping but distinct molecular surfaces. These findings suggest that the two activities of the curculin heterodimer are expressed through its two different modes of interactions with the T1R2-T1R3 heterodimeric sweet taste receptor.     

Methyladeninylcobamide functions as the cofactor of methionine synthase in a Cyanobacterium, Spirulina platensis NIES-39

To clarify the physiological function of pseudovitamin B₁₂ (or adeninylcobamide; AdeCba) in Spirulina platensis NIES-39, cobalamin-dependent methionine synthase (MS) was characterized. We cloned the full-length Spirulina MS. The clone contained an open reading frame encoding a protein of 1183 amino acids with a molecular mass of 132 kDa. Deduced amino acid sequences of the Spirulina MS contained critical residues identical to cobalamin-, zinc-, S-adenosylmethionine-, and homocysteine-binding motifs. The recombinant Spirulina enzyme showed higher affinity for methyladeninylcobamide than methylcobalamin as a cofactor. These results indicate that Spirulina cells can utilize AdeCba synthesized as the cofactor for MS.     

Constitutively Active BMP Type I Receptors Transduce BMP-2 Signals without the Ligand in C2C12 Myoblasts

Bone morphogenetic protein-2 (BMP-2), a member of transforming growth factor-β superfamily, inhibits the terminal differentiation of C2C12 myoblasts and changes their differentiation pathway into cells expressing osteoblast phenotypes such as alkaline phosphatase (ALP) activity and osteocalcin production (Katagiriet al.,1994,J. Cell Biol.127, 1755–1766). Two type I receptors for BMP-2 (BMPR-IA and BMPR-IB) have been cloned, but the role of the respective receptors in signal transduction is not clear. In the present study, we examined the signal transduction of BMP-2 in C2C12 cells using constitutively activated mutant BMPR-IA and BMPR-IB. C2C12 cells expressed BMPR-IA and BMPR-II mRNAs, but not BMPR-IB mRNA at detectable levels in Northern blotting. When mutated BMPR-IA and BMPR-IB were transiently transfected into C2C12 cells, both BMPR-IA and BMPR-IB similarly induced ALP activity in the absence of BMP-2. We also established subclonal cell lines of C2C12 cells by stably transfecting mutated BMPR-IB. When the mutated BMPR-IB-transfected cells were cultured in medium with low serum (differentiation medium) without BMP-2, the cells differentiated into ALP-positive mononuclear cells and not into myosin heavy chain-positive myotubes. These mutated BMPR-IB-transfected cells expressed ALP activity and osteocalcin mRNA in a time-dependent manner, but neither muscle creatine kinase nor myogenin mRNAs. These results indicate that the mutated BMP-2 type I receptors can constitutively transduce BMP-2 signals in the absence of the ligand in C2C12 cells.