Naoko Kajimura

In the present study, we examined the changes in the morphofunction of astrocytes in rat hippocampus under different circulating corticosteroid conditions by immunohistochemistry analysis of glial fibrillary acidic protein (GFAP) and ultra-high-voltage electron microscopy. Each GFAP-immunoreactive cell showed a hypertrophic appearance with well-developed thicker fibrous processes, and the number and the density of GFAP-immunoreactive cells were increased 4 weeks after adrenalectomy, whereas the changes were restored to the sham-control level with corticosterone replacement. The morphometric changes were observed in particular around the pyramidal neurons of CA1 and in the subgranular layer of dentate gyrus. The quantitative analysis clearly showed a significant increase in the number and the density of GFAP-immunoreactive cells in the adrenalectomy group; following corticosterone replacement, these increases were returned to the sham-control level. These changes were also specifical...

Recently we have identified a 15-mer peptide, SNP-1, by a random phage library that can bind to bone marrow stromal cell antigen-1 (BST-1)/CD157 [Sato, A., Yamamoto, S., Ishihara, K., Hirano, T. & Jingami, H. (1999) Biochem. J. 337, 491-496]. SNP-1 inhibits BST-1 ADP-ribosyl cyclase activity uncompetitively with a Ki value of 180 +/- 40 nM. In this study we analysed biophysically the SNP-1 binding to a soluble form of BST-1 (sBST-1). Equilibrium binding data of wild-type SNP-1 from surface plasmon resonance studies gave a Kd value of 500 +/- 35 nM. Titration calorimetry analysis showed that the binding reaction is exothermic at 20 degrees C. The values of Kd = 211 nM, enthalpy change, DeltaH = -18.68 kcal.mol-1, and saturated molar ratio of bound SNP-1 per sBST-1, N = 0.8 mol.mol-1 were obtained. On the basis of the molecular masses of SNP-1 and sBST-1 calculated by analytical ultracentrifugation, the stoichiometry of the binding was determined to be 2 : 2. Electron microscopy also revealed the dimer form of sBST-1. To delineate the core residue of SNP-1 responsible for binding, each amino acid residue has been replaced by alanine. A region from amino acid residues 7-12 appeared to be critical for the SNP-1 binding to sBST-1. The substitution of the first residue, His, to Ala led to a reduction in binding, suggesting that the N-terminal residue is also crucial.

Ghrelin is a novel brain-gut peptide that stimulates food intake and body weight gain. We studied the anabolic effect of ghrelin in a cancer cachexia mouse model. SEKI, a human melanoma cell line, was inoculated into nude mice to examine the effects of ghrelin on food intake and body weight. The intraperitoneal administration of ghrelin twice a day (6 nmol/mice/day) for 6 days suppressed weight loss in SEKI-inoculated mice and increased the rate of weight gain in vehicle-treated nude mice. Ghrelin administration also increased food intake in both SEKI- and vehicle-treated mice. Both the weight of white adipose tissue and the plasma leptin concentration were reduced in tumor-inoculated mice compared with vehicle-treated mice; these factors increased following ghrelin administration. The levels of both ghrelin peptide and mRNA in the stomach were upregulated in tumor-inoculated mice. The anabolic effect of ghrelin efficiently reverses the cachexia in mice bearing SEKI human melanoma. Ghrelin therefore may have a therapeutic ability to ameliorate cancer cachexia.

Spinocerebellar degenerations (SCDs) are a large class of sporadic or hereditary neurodegenerative disorders characterized by progressive motion defects and degenerative changes in the cerebellum and other parts of the CNS. Here we report the identification and establishment from a C57BL/6J mouse colony of a novel mouse line developing spontaneous progressive ataxia, which we refer to as ts3. Frequency of the phenotypic expression was consistent with an autosomal recessive Mendelian trait of inheritance, suggesting that a single gene mutation is responsible for the ataxic phenotype of this line. The onset of ataxia was observed at about three weeks of age, which slowly progressed until the hind limbs became entirely paralyzed in many cases. Micro-MRI study revealed significant cerebellar atrophy in all the ataxic mice, although individual variations were observed. Detailed histological analyses demonstrated significant atrophy of the anterior folia with reduced granule cells (GC) and abnormal morphology of cerebellar Purkinje cells (PC). Study by ultra-high voltage electron microscopy (UHVEM) further indicated aberrant morphology of PC dendrites and their spines, suggesting both morphological and functional abnormalities of the PC in the mutants. Immunohistochemical studies also revealed defects in parallel fiber (PF)-PC synapse formation and abnormal distal extension of climbing fibers (CF). Based on the phenotypic similarities of the ts3 mutant with other known ataxic mutants, we performed immunohistological analyses and found that expression levels of two genes and their products, glutamate receptor delta2 (grid2) and its ligand, cerebellin1 (Cbln1), are significantly reduced or undetectable. Finally, we sequenced the candidate genes and detected a large deletion in the coding region of the grid2 gene. Our present study suggests that ts3 is a new allele of the grid2 gene, which causes similar but different phenotypes as compared to other grid2 mutants.

We previously reported that the human melanoma cell line, SEKI, induces severe weight loss in nude mice. In the present study, we examined the expression of weight-regulating neuropeptide mRNAs in the hypothalamus of this cancer cachectic model by using a sensitive quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method and in situ hybridization. mRNA levels of neuropeptide Y (NPY) and corticotropin-releasing hormone (CRH) in the whole hypothalamus were elevated significantly in the SEKI mice as compared with control mice. In situ hybridization showed that NPY and CRH mRNA were upregulated in the arcuate nucleus and the paraventricular nucleus, respectively. There were no significant differences in melanin-concentrating hormone (MCH), orexin (OX), and cholecystokinin mRNA levels between the SEKI and control mice. These results suggest that the NPYergic system is functioning in the rodent model of cancer cachexia; however, the role of the CRHergic system in energy homeostasis remains to be elucidated. This is the first report of the hypothalamic neuropeptide response to cachexia-inducing human cells.

Leukemia inhibitory factor (LIF) is known to be a causative factor for cachexia and thrombocytosis in nude mice bearing human cancer cells. In the present study, we investigated whether recombinant human (rh) LIF can induce these biological activities in a primate model. rhLIF was synthesized by the expression of LIF protein in Escherichia coli. rhLIF (5, 20, or 80 micrograms/kg) was administered subcutaneously twice daily to cynomolgus monkeys for 14 consecutive days. A remarkable decrease of body weight (10%) was observed in the 80 micrograms/kg/day group. Approximately two-fold increases in platelet counts were observed at doses higher than 5 micrograms/kg/day when compared with control counts. These biological effects disappeared soon after the cessation of rhLIF treatment. Macroscopically, a remarkable reduction in subcutaneous fatty tissues and severe splenomegaly were observed. The results of this study demonstrate that rhLIF induces weight loss and thrombocytosis in a primate model.

Toxohormones are tumor-derived factors that induce cancer cachexia syndrome in tumor-bearing animals. Nude mice bearing tumors induced by eight human cancer cell lines with this activity were studied for cytokine production and expression of a newly identified gene, ob, which has the ability to control body weight. A melanoma cell line, SEKI, and a neuroepithelioma cell line, NAGAI, produced a large amount of the cytokine, leukemia-inhibitory factor (LIF). A uterine carcinoma cell line, Yumoto, produced a large amount of interleukin 6 (IL-6), and an oral cavity carcinoma cell line, OCC-1C, concomitantly produced LIF, IL-6, and IL-11. Reverse transcription polymerase chain reaction studies revealed that ob gene mRNA was not expressed in any of these cell lines, suggesting that the gene does not have a role as a tumor product responsible for cancer cachexia in this model. These findings suggest that in four of eight animal models in which cancer cachexia syndrome developed, LIF, IL-6, or possibly IL-11 produced by cancer cells may be toxohormones, but in the remaining four cancer cell lines the mechanism responsible for cachexia syndrome remains unknown.

Ghrelin is a novel brain-gut peptide that stimulates food intake and body weight gain. We studied the anabolic effect of ghrelin in a cancer cachexia mouse model. SEKI, a human melanoma cell line, was inoculated into nude mice to examine the effects of ghrelin on food intake and body weight. The intraperitoneal administration of ghrelin twice a day (6 nmol/mice/day) for 6 days suppressed weight loss in SEKI-inoculated mice and increased the rate of weight gain in vehicle-treated nude mice. Ghrelin administration also increased food intake in both SEKI- and vehicle-treated mice. Both the weight of white adipose tissue and the plasma leptin concentration were reduced in tumor-inoculated mice compared with vehicle-treated mice; these factors increased following ghrelin administration. The levels of both ghrelin peptide and mRNA in the stomach were upregulated in tumor-inoculated mice. The anabolic effect of ghrelin efficiently reverses the cachexia in mice bearing SEKI human melanoma. Ghrelin therefore may have a therapeutic ability to ameliorate cancer cachexia.