Structure of human holocarboxylase synthetase gene and mutation spectrum of holocarboxylase synthetase deficiency

Holocarboxylase synthetase (HLCS) is an enzyme that catalyzes the incorporation of biotin into apo-carboxylases, and its deficiency causes biotin-responsive multiple carboxylase deficiency. The reported sequences of cDNA for human HLCS from liver, lymphocyte, and KG-1 myeloid cell lines differ at their 5' regions. To elucidate variations of the human HLCS mRNA and longer 5' cDNA ends, we performed screening of the human liver cDNA library and rapid amplification of the cDNA ends (RACE). Our results suggest the existence of three types of HLCS mRNA that start at different exons. The first type starts at exon 1, and the second type starts at exon 3, and both are found in various human tissues. The third type, corresponding to the cDNA from the KG-1 cell, starts at exon 2 of the HLCS gene. Various splicing patterns from exons 3-6 were also observed. None of the variations of cDNA found created a new initiation codon. Mutation screening from exons 6-14, therefore, was sufficient to detect amino acid changes in HLCS in patients. Our direct sequencing strategy for screening mutations in the HLCS gene revealed mutations in five Japanese patients and seven non-Japanese patients. Our analyses involving 12 Japanese and 13 non-Japanese patients and studies by others indicate that (1) there is no panethnically prevalent mutation; (2) the Arg508Trp, Gly581Ser, and Val550Met mutations are found in both Japanese and non-Japanese populations; (3) the IVS10+5G-->A mutation is predominant and probably a founder mutation in European patients; (4) the 655-656insA, Leu237Pro, and 780delG mutations are unique in Japanese patients; (5) the spectrum of the mutations in the HLCS gene may vary substantially among different ethnic groups.     

Examination of the factor structure of center of foot pressure movement and cross-validity

This study aimed to determine the factor structure of the center of foot pressure (CFP) movement during static upright posture, and to objectively categorize and summarize parameters to evaluate CFP movement. The subjects were 220 healthy young males and females. The measurement of CFP was carried out 3 times with 1 min rest and the mean of trials 2 and 3 was used for the analysis. The measurement device was an Anima's stabilometer G5500. The data sampling frequency was 20 Hz. Thirty-four parameters with high reliability were selected from the following 6 domains except for the center position which is a fundamental attribute: distance, distribution of amplitude, area, velocity, power spectrum, and body sway vector. Factor analysis (principal factor method and promax rotation) was applied to a correlation matrix consisting of 32 parameters. Four factors abstracted were interpreted as follows; unit time sway, front and back sway, left and right sway and high frequency band of power spectrum. The reliability coefficient (ICC=0.89-0.95) and the congruence coefficient (phi=0.80-0.97) between factors abstracted from the original and the cross-validity groups were very high. It was considered that the CFP movement consists of the above 4 factors that evaluate the amount of body sway and can be synthetically evaluated by them.     

Synthesis and characterization of novel phenylindoles as potential probes for imaging of β-amyloid plaques in the brain

We synthesized a novel series of phenylindole (PI) derivatives and evaluated their biological activities as probes for imaging Aβ plaques in vivo. The affinity for Aβ plaques was assessed by an in vitro-binding assay using pre-formed synthetic Aβ aggregates. 2-Phenyl-1H-indole (2-PI) derivatives showed high affinity for Aβ42 aggregates with K_i values ranging from 4 to 32 nM. 2-PI derivatives clearly stained Aβ plaques in an animal model of AD. In biodistribution experiments using normal mice, 2-PI derivatives displayed sufficient uptake for imaging, ranging from 1.1% to 2.6% ID/g. Although additional modifications are necessary to improve uptake by and clearance from the brain, 2-PI derivatives may be useful as a backbone structure to develop novel Aβ imaging agents.     

Decomposition Analysis of Waste Generation From Stocks in a Dynamic System

We conducted a decomposition analysis of material flows in a dynamic system, focusing on factors in the generation of waste consumer durables. A methodology for the analysis of consumer durables was developed and applied to three common consumer durables: cathode ray tube TVs, refrigerators, and passenger cars. The methodology decomposed changes in the numbers of waste products into three factors: changes in lifespan distribution, past trends in replacement sales, and past trends in sales for additional purchases. The decomposed equation clearly showed that the number of waste products would not necessarily be reduced by lifespan extension alone. This is because the number of waste products generated is affected not only by current lifespan distribution but also by past trends in sales for replacement and in additional purchases. The results show that changes in past replacement sales influence the current generation of waste, even if current replacement sales are declining. To reduce the generation of waste products on a short‐term basis, lifespan must be extended until the waste‐reducing effect of lifespan extension exceeds the waste‐increasing effect of the other two factors. From a long‐term perspective, controlling current replacement and additional purchases can be used to prevent future waste product generation.     

Characteristics and Efficiency of Glutamine Production by Coupling of a Bacterial Glutamine Synthetase Reaction with the Alcoholic Fermentation System of Baker’s Yeast

Glutamine production with bacterial glutamine synthetase (GS) and the sugar-fermenting system of baker’s yeast for ATP regeneration was investigated by determining the product yield obtained with the energy source for ATP regeneration (i.e., glucose) for yeast fermentation. Fructose 1,6-bisphosphate was accumulated temporarily prior to the formation of glutamine in mixtures which consisted of dried yeast cells, GS, their substrate (glucose and glutamate and ammonia), inorganic phosphate, and cofactors. By an increase in the amounts of GS and inorganic phosphate, the amounts of glutamine formed increased to 19 to 54 g/liter, with a yield increase of 69 to 72% based on the energy source (glucose) for ATP regeneration. The analyses of sugar fermentation of the yeast in the glutamine-producing mixtures suggested that the apparent hydrolysis of ATP by a futile cycle(s) at the early stage of glycolysis in the yeast cells reduces the efficiency of ATP utilization. Inorganic phosphate inhibits phosphatase(s) and thus improves glutamine yield. However, the analyses of GS activity in the glutamine-producing mixtures suggested that the higher concentration of inorganic phosphate as well as the limited amount of ATP-ADP caused the low reactivity of GS in the glutamine-producing mixtures. A result suggestive of improved glutamine yield under the conditions with lower concentrations of inorganic phosphate was obtained by using a yeast mutant strain that had low assimilating ability for glycerol and ethanol. In the mutant, the activity of the enzymes involved in gluconeogenesis, especially fructose 1,6-bisphosphatase, was lower than that in the wild-type strain.     

Regulation of Toll-like receptor 4 expression in mouse colon by macrophage migration inhibitory factor

Recent studies have indicated that macrophage migration inhibitory factor (MIF) and Toll-like receptor (TLR) play an important role in the regulation of innate immune responses. In this study, we investigated the effect of MIF on the expression of TLR4, a receptor that recognizes lipopolysaccharide, in colon using MIF-deficient mice. TLR4 mRNA expression in the colon tissues was determined by northern blot analysis. Western blot analysis and immunohistochemistry in the colon tissues were performed to evaluate the expression of TLR4 protein. The expressions of TLR4 mRNA and protein were remarkably down-regulated in colon tissues of MIF-deficient mice compared with wild-type mice and up-regulated by treatment with recombinant MIF. Immunohistochemical study revealed the presence of TLR4–positive staining in mononuclear cells in the lamina propria and intraepithelial mononuclear cells as well as weak staining in epithelial cells and crypts in colon tissues of wild-type mice. In contrast, MIF-deficient mice did not show TLR4-positive staining in the colonic mucosa. In MIF-deficient mice injected with recombinant mouse MIF (rMIF), TLR4-positive staining cells were observed in colon tissues similar to the findings in wild-type mice. Administration of dextran sulfate sodium (DSS) up-regulated the expression of TLR4 in the colons of WT mice but not in those of MIF-deficient mice. Furthermore, pretreatment with rMIF up-regulated the expression of TLR4 in response to DSS in MIF-deficient mice. Our results suggest that MIF affects the expression of TLR4 in mouse colon under both normal and colitic conditions.An erratum to this article can be found at     

Mating system ofTokunagayusurika akamusi (Diptera: Chironomidae): I. Copulation in the air by swarming and on the ground by searching

Observations on the mating system of the midge,Tokunagayusurika akamusi, revealed mating to occur both in the air by swarming and on the ground by searching. At the shores of Lake Biwa, midges appeared from November to early December. Newly emerged adults arrived at the resting place, lakeside vegetation, in the morning, during which time a number of males also walked about in search of mates. Many copulating pairs were observed at the resting place. Huge swarms occurred chiefly before sunset but the frequency of copulation observed in the swarm was extremely low. It is likely that, in the Lake Biwa population, the proportion of females inseminated by searching males at the resting place was much larger than that by swarming males in the air. Furthermore, by searching, males copulated with younger females than by swarming. The differences between the searching and swarming tactics are discussed.     

Dual regulation of phospholipase D1 by protein kinase C alpha in vivo

The regulation of phospholipase D1 (PLD1), which has been shown to be activated by protein kinase C (PKC) alpha, was investigated in the human melanoma cell lines. In G361 cell line, which lacks PKCalpha, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced PLD activation was potentiated by introducing PKCalpha by the adenovirus vector. The kinase-negative PKCalpha elevated TPA-induced PLD activity less significantly than the wild type. A PKC specific inhibitor GF109203X lowered PLD activation in the cells expressing PKCalpha, but did not prevent PLD potentiation induced by the kinase-negative PKCalpha. Expression of PKCbetaII and the kinase-negative PKCbetaII enhanced TPA-stimulated PLD activity moderately in MeWo cell line, in which PKCbetaII is absent. Furthermore, the TPA treatment increased the association of PKCalpha, PKCbetaII, and their kinase-negative mutants with PLD1 in melanoma cells. These results indicate that PLD1 is dually regulated through phosphorylation as well as through the protein-protein interaction by PKCalpha, and probably by PKCbetaII, in vivo.