Kaoru Ichimura

Effect of Perinatal Hypothyroidism on Expression of Cytochrome C Oxidase Subunit I Gene, which is Cloned by Differential Plaque Screening from the Cerebellum of Newborn Rat

Early development of the central nervous system is influenced by several hormones including thyroid hormone. This study was designed to clone the gene whose expression is changed in association with perinatal hypothyroidism in the rat cerebellum. Rats were sacrificed at 15 day‐old postnatal age (P15) and their cerebella were removed. Poly (A)+ RNA was extracted to construct a cDNA library using λgt 10 cloning vector. Differential plaque screening was then performed using 32P‐labeled antisense cDNA synthesized from poly (A)+ RNA of the methimazole‐treated (hypothyroid) P15 rat cerebellum (hypothyroid probe), and of the euthyroid P15 rat cerebellum (euthyroid probe). The clones, which hybridized strongly to the euthyroid probe and weakly or not at all to the hypothyroid probe, were isolated. Sequence analysis of these clones revealed that all isolated clones encode cytochrome c oxidase subunit I (COX I), which is located in the mitochondria1 DNA. The decrease in COX I gene expression was not seen in the animals, which received methimazole treatment and daily replacement of thyroid hormone. In situ hybridization detection showed not only overall decrease in COX I gene expression but also change in distribution of hybridization signal in the cerebellar cortex of hypothyroid rat. Such change was not observed in the T4‐replaced animals. Based on the evidence that thyroid hormone greatly influences brain development, the results of the present study indicate that the terminal enzyme of mitochondria1 respiratory chain, COX I is one of the important target molecules regulated by thyroid hormone in the newborn rat cerebellum.

Direct detection of the protein quaternary structure and denatured entity by small-angle scattering: guanidine hydrochloride denaturation of chaperonin protein GroEL

A change in the higher-order structure of an oligomeric protein is directly detectable by small-angle scattering. A small-angle X-ray scattering (SAXS) study of the denaturation process of the chaperonin protein GroEL by guanidine hydrochloride (GdnHCl) showed that the disappearance of the quaternary structure can be monitored by using a Kratky plot of the scattered intensities, demonstrating the advantage of the SAXS method over other indirect methods, such as light scattering, circular dichroism (CD), fluorescence and sedimentation. The collapse of the quaternary structure was detected at a GdnHCl concentration of 0.8 M for a solution containing ADP (adenosine diphosphate)/Mg2+(2 mM)/K+. From pairwise plots of the change in forward scattering intensity J(0)/C (weight-average molecular weight) and the z-average (root mean square) radius of gyration against the GdnHCl concentration, the stability and nature of the denatured protein can be determined. The SAXS results suggest that the GroEL tetradecamer directly dissociates to the unfolded coil without going through a globular monomer state. The denatured ensemble is not a single unfolded monomer coil particle, but some mixture of entangled aggregates and a monomer of the coil molecules. Small-angle scattering is a powerful method for the detection and study of changes in quaternary and higher-order structures of oligomeric proteins.

Solution X-ray scattering study on the chaperonin GroEL from Escherichia coli

The molecular architecture of native GroEL has been studied by solution X-ray scattering. The radius of gyration for the native molecule was estimated to be 66.0 A in 50 mM Tris-HCl, 100 mM KCl at pH 7.5 and 25 degrees C. The maximum dimension was estimated to be 170 A, based on the pair distance distribution function. A cylindrical structure or two heptameric rings was found to be the best for native GroEL among structures examined by using a multi-sphere model analysis in which the radius of constituent sphere was 6 A. The results of the model analysis show that the radius of GroEL is 68.0 A and the height is 150.7 A. Unexpectedly, the central penetrating hole through GroEL was not confirmed in the best-fit structure.

Upregulation of histidine decarboxylase mRNA expression in scleroderma skin

Abstract The involvement of histamine in the pathogenesis of systemic sclerosis (SSc) has been suggested. Possible involvement of histamine and histidine decarboxylase (HDC), the synthesizing enzyme for histamine, in the formation of the skin abnormalities in SSc was studied. Skin histamine concentrations in SSc were significantly lower than in normal controls ( P < 0.02). In situ hybridization with an HDC probe revealed that the expression of the HDC gene in SSc was greater than in normal controls. The number of cells and the mean grain number per cell expressing HDC mRNA were both significantly greater in SSc than in normal controls (both P < 0.01). These results show a reduction in histamine concentration and an elevated HDC gene expression in SSc skin, indicating an increase in both histamine release and HDC gene expression. The upregulation of histamine turnover appears to be involved in the formation of the skin abnormalities of SSc.