Analysis of internal structure changes in black human hair keratin fibers with aging using Raman spectroscopy

To investigate the internal structure changes in virgin black human hair keratin fibers due to aging, the structure of cross-sections at various depths of virgin black human hair (sections of new growth hair: 2 mm from the scalp) from a group of eight Japanese females in their twenties and another group of eight Japanese females in their fifties were analyzed using Raman spectroscopy. For the first time, we have succeeded in recording the Raman spectra of virgin black human hair, which had been impossible due to high melanin granule content. The key points of this method are to cross-section hair samples to a thickness of 1.50-microm, to select points at various depths of the cortex with the fewest possible melanin granules, and to optimize laser power, cross slit width as well as total acquisition time. The reproducibility of the Raman bands, namely the alpha-helix (alpha) content, the beta-sheet and/or random coil (beta/R) content, the disulfide (--SS--) content, and random coil content of two adjoining cross-sections of a single hair keratin fiber was clearly good. The --SS-- content of virgin black human hair from the Japanese females in their fifties for the cortex region decreased compared with that of the Japanese females in their twenties. On the other hand, the beta/R and alpha contents of the cortex region did not change.     

Mutant analyses reveal different functions of fgfr1 in medaka and zebrafish despite conserved ligand-receptor relationships

Medaka (Oryzias latipes) is a small freshwater teleost that provides an excellent developmental genetic model complementary to zebrafish. Our recent mutagenesis screening using medaka identified headfish (hdf) which is characterized by the absence of trunk and tail structures with nearly normal head including the midbrain-hindbrain boundary (MHB). Positional-candidate cloning revealed that the hdf mutation causes a functionally null form of Fgfr1. The fgfr1hdf is thus the first fgf receptor mutant in fish. Although FGF signaling has been implicated in mesoderm induction, mesoderm is induced normally in the fgfr1hdf mutant, but subsequently, mutant embryos fail to maintain the mesoderm, leading to defects in mesoderm derivatives, especially in trunk and tail. Furthermore, we found that morpholino knockdown of medaka fgf8 resulted in a phenotype identical to the fgfr1hdf mutant, suggesting that like its mouse counterpart, Fgf8 is a major ligand for Fgfr1 in medaka early embryogenesis. Intriguingly, Fgf8 and Fgfr1 in zebrafish are also suggested to form a major ligand-receptor pair, but their function is much diverged, as the zebrafish fgfr1 morphant and zebrafish fgf8 mutant acerebellar (ace) only fail to develop the MHB, but develop nearly unaffected trunk and tail. These results provide evidence that teleost fish have evolved divergent functions of Fgf8-Fgfr1 while maintaining the ligand-receptor relationships. Comparative analysis using different fish is thus invaluable for shedding light on evolutionary diversification of gene function.     

Population dynamics of the harmful diatom Eucampia zodiacus Ehrenberg causing bleachings of Porphyra thalli in aquaculture in Harima-Nada, the Seto Inland Sea, Japan

The diatom Eucampia zodiacus Ehrenberg is one of the harmful diatom species which indirectly cause bleachings of Nori (Porphyra thalli) in aquaculture through competitive utilizing of nutrients (especially nitrogen) and resultant nutrient depletion in water columns during the bloom events. The seasonal changes in environmental factors, cell density and cell size of E. zodiacus were investigated for 4 years (April 2002–December 2005) to understand the population ecology of this diatom in Harima-Nada, the eastern part of the Seto Inland Sea, Japan. Vegetative cells of E. zodiacus were usually detected year-round. Total cell densities of E. zodiacus annually peaked from mid-February to early April, and high cell densities were observed in the whole water columns during the bloom-period. Nutrient concentrations decreased with the increase of cell density of E. zodiacus, and low nutrients concentrations continued throughout the E. zodiacus bloom-period. The average cell size (length of apical axis) of E. zodiacus populations ranged from 10.8 μm to 81.2 μm, and the restoration of cell size occurred once in autumn every year just after reaching the minimum cell size. In addition, its great seasonal regularity was confirmed by the decrease and restoration of its cell size through 4-year study period. Temperature and nutrients were suitable in autumn for the growth of E. zodiacus, its blooms never occur in that season. These results strongly suggest that E. zodiacus did not have a resting stage, and it spends autumn for size restoration and starts to bloom thereafter in Harima-Nada in winter and spring, causing fishery damage to Nori aquaculture by resulting nutrient deprivation.     

Glycomic mapping of pseudomucinous human ovarian cyst glycoproteins: identification of Lewis and sialyl Lewis glycotopes

Expression of sialyl Lewis x (sLe(x)) and sialyl Lewis a (sLe(a)) on cell-surface glycoproteins endows cells with the ability to adhere to E-, P-, and L-selectins present on endothelia, platelets, or leukocytes. Special arrangements of these glycotopes in cancers are thought to play a key role in metastasis. Previous studies have mostly described membrane-bound sLe(x) and sLe(a) activities. In this report, the major O-glycans of the secreted human ovarian cyst sialoglycoproteins from a Le(a+) nonsecretor individual (human ovarian cyst sample 350) were characterized by MS/MS analyses and immuno-/lectin-chemical assays. The results showed that HOC 350 carries a large number of epitopes for sLe(x), sLe(a), and Le(a) reactive antibodies. Advanced MS/MS sequencing coupled with mild periodate oxidation and exoglycosidase digestions further revealed that the O-glycans from HOC 350 are mostly of core 1 and 2 structures, extended and branched on the 3-arm with both type I and type II chains, complete with variable degrees of terminal sialylation and/or fucosylation to yield the sLe(x) or sLe(a) epitopes. Thus, the underlying core and peripheral backbone structures are similar to that of a previously proposed composite structural model for nonsialylated human ovarian cysts O-glycans, but with some notable distinguishing structural features in addition to sialylation.     

Blocking LC3 lipidation and ATG12 conjugation reactions by ATG7 mutant protein containing C572S

Autophagy, a system for the bulk degradation of intracellular components, is essential for homeostasis and the healthy physiology and development of cells and tissues. Its deregulation is associated with human disease. Thus, methods to modulate autophagic activity are critical for analysis of its role in mammalian cells and tissues. Here we report a method to inhibit autophagy using a mutant variant of the protein ATG7, a ubiquitin E1-like enzyme essential for autophagosome formation. During autophagy, ATG7 activates the conjugation of LC3 (ATG8) with phosphatidylethanolamine (PE) and ATG12 with ATG5. Human ATG7 interactions with LC3 or ATG12 require a thioester bond involving the ATG7 cysteine residue at position 572. We generated TetOff cells expressing mutant ATG7 protein carrying a serine substitution of this critical cysteine residue (ATG7C572S). Because ATG7C572S forms stable intermediate complexes with LC3 or ATG12, its expression resulted in a strong blockage of the ATG-conjugation system and suppression of autophagosome formation. Consequently, ATG7C572S mutant protein can be used as an inhibitor of autophagy.     

Effect of arylidene-cyclopentenedione radiosensitizers on ATP synthesis in mitochondria: action as potent inhibitors of phosphate transport

The effects of the arylidene-cyclopentenedione radiosensitizers, KIH-200, 201 and 202 on ATP synthesis in mitochondria were examined. In spite of the close similarity of their chemical structure to that of the most potent known weakly acidic uncoupler, SF 6847, they did not show any uncoupling activity at concentrations of up to 50 microM. However, these three compounds were found to have very potent inhibitory effects on Pi-transport into mitochondria, all causing 50% inhibition (I50%) at about 7 microM. Thus they are much more potent than the commonly used Pi-transport inhibitors N-ethylmaleimide (I50% = about 40 microM), and mersalyl (I50% = about 30 microM). They may act as SH-reagents, and inhibit Pi-transport by modifying an SH-group(s) in the Pi-transporter.     

Hypogonadism in liver cirrhosis: implication in altered amino acid metabolism in muscle

To investigate the relationship between hypogonadism and altered amino acid metabolism in patients with liver cirrhosis, we measured the basal levels of plasma testosterone, estradiol, and free amino acids, plus urinary 3-methylhistidine excretion, in 16 control and 19 cirrhotic patients. The concentration of plasma testosterone correlated significantly with that of plasma branched-chain amino acids, and inversely with urinary 3-methylhistidine excretion. This suggests that hypogonadism causes a disturbance in amino acid metabolism at least partly related to an augmented muscle protein turnover.     

Characterization of a new phosphonocerebroside, N-methyl-2-aminoethylphosphonylglucosylceramide, from the antarctic krill, Euphausia superba

A novel phosphonglycosphingolipid was purified from the whole tissue of the antarctic krill, Euphausia superba by successive column chromatography on DEAE- and QAE-Sephadex and silicic acid (Iatrobeads). The structure was elucidated by means of IR, FAB-MS, 1H-NMR, GC and GC-MS analyses of the water-soluble products after complete and partial acid hydrolysis, and methylation analysis of a product of hydrogen fluoride degradation; it was identified to be a phosphonocerebroside, 6'-O-(N-methyl-2-aminoethylphosphonyl)Glcp beta 1----1ceramide. The ceramide moiety was composed of tetradecasphingenine and octadecasphingatriene as the main sphingoids, and monounsaturated C22- and C24-acids and their 2-hydroxy homologues as the major fatty acids.     

Synthetic inhibitors of the processing of pretransfer RNA by the ribonuclease P ribozyme: enzyme inhibitors which act by binding to substrate

2,2'-p-Phenylene bis[6-(4-methyl-1-piperazinyl)]benzimidazole, 2,2'-bis(3,5-dihydroxyphenyl)-6,6'-bis benzimidazole, and 2,2'-bis(4-hydroxyphenyl)-6,6'-bis benzimidazole are shown by UV-visible and fluorescence spectrophotometry to be strong ligands for tRNA, giving simple, hyperbolic binding isotherms with apparent dissociation constants in the micromolar range. Hydroxyl radical footprinting indicates that they may bind in the D and T loops. On the basis of this tRNA recognition as a rationale, they were tested as inhibitors of the processing of precursor tRNAs by the RNA subunit of Escherichia coli RNase P (M1 RNA). Preliminary studies show that inhibition of the processing of Drosophila tRNA precursor molecules by phosphodiester bond cleavage, releasing the extraneous 5'-portion of RNA and the mature tRNA molecule, was dependent on both the structure of the inhibitor and the structure of the particular tRNA precursor substrate for tRNA(Ala), tRNA(Val), and tRNA(His). In more detailed followup using the tRNA(His) precursor as the substrate, experiments to determine the concentration dependence of the reaction showed that inhibition took time to reach its maximum extent. I(50) values (concentrations for 50% inhibition) were between 5.3 and 20.8 microM, making these compounds among the strongest known inhibitors of this ribozyme, and the first inhibitors of it not based on natural products. These compounds effect their inhibition by binding to the substrate of the enzyme reaction, making them examples of an unusual class of enzyme inhibitors. They provide novel, small-molecule, inhibitor frameworks for this endoribonuclease ribozyme.