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Various neuropeptides play an essential role in the nutrient sensing mechanism and related homeostasis. Nesfatin-1 is a newly identified neuropeptide having anorectic activity, and nesfatin-1-containing neurons are widely distributed in the brain, including the hypothalamus and brain stem. Our previous study showed that dehydration-induced anorectic effects are mediated via the central nesfatin-1 pathway in rats. Our recent studies have also shown that peripheral anorectic peptides (cholecystokinin-8, glucagon-like peptide-1, and leptin) and an antineoplastic agent (cisplatin) caused inhibition of feeding via the central nesfatin-1 pathway in rats. Nesfatin-1-containing neurons in the central nervous system, in particular the hypothalamus and the brain stem, may mediate peripheral nutrient signals and regulate feeding behavior.

Calpains are Ca(2 +) -dependent cysteine proteases. Fifteen gene products of calpains are expressed in mammals. Among them, Calpain 1 and Calpain 2 are ubiquitously expressed and have been investigated extensively. Under the physiological conditions, calpain activity is strictly regulated by endogenous inhibitory protein, Calpastatin. Calpains are activated in the various cardiovascular diseases and implicated in their pathogenesis by degrading numerous target proteins. Here we briefly summarize the physiological and pathological role of calpains in the cardiovascular diseases.

Vitamin K is well known for its role in the synthesis of a number of blood coagulation factors. Vitamin K is also an important factor for bone metabolism via gamma-carboxylation of vitamin K-dependent proteins such as osteocalcin, matrix Gla protein, and protein S. Recent studies suggest that there is potential vitamin K insufficiency in bone, even in sufficient vitamin K status for blood coagulation. In the present review, the studies concerning relationship between vitamin K status and fracture are reviewed.

Vitamin K(2) has dual actions, stimulates osteoblastic functions, for synthesis of osteocalcin, osteonectin and other matrix bone proteins, in addition, new finding, in stem cell culture found osteoblast producing gene expression of collagen type 1, the other action, vitamin K(2) contains mild antiresorpion by inducing the osteoclastic apoptosis. Our study found that postmenopausal and elderly women have high risk of vitamin K(2) deficiency, comparing to the normal value of young, reproductive females. The efficacy of vitamin K(2) will be fulfill benefit after 6 months of administration, prolong use will enhance of bone quality that prevent fracture.

Vitamin K (VK) is well known for its role in the synthesis of a number of blood coagulation factors. VK is also an important factor for bone metabolism via gamma-carboxylation of VK-dependent proteins such as osteocalcin, matrix Gla protein, and protein S. Recently, it is rare that severe VK deficiency is observed. However, low dietary VK intake or low VK status has been shown to be associated with low bone mineral density and increased hip fracture risk. These studies suggest that there is potential VK insufficiency in bone, even in sufficient VK status for blood coagulation. In the present review, the studies concerning relationship between serum VK concentration and bone health, including pharmacokinetics of VK analogues (such as phylloquinone and menaquinone) and factors which affect on blood circulation of VK, are reviewed.

Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity.

Periodic changes in calcium (Ca(2+)) concentration in cardiomyocytes are essential for cardiac contraction and relaxation, and the intracellular Ca(2+)concentration is integrally regulated by proteins associated with the sarcoplasmic reticulum (SR), an extensive intracellular membrane system. The activity of the cardiac ryanodine receptor (RyR2) and the sarco/endoplasmic reticulum Ca(2+) ATPase 2a (SERCA2a) are known to be under fine-tuning by their intrinsic regulatory domains and associated SR proteins. A growing body of evidence, including studies using genetically engineered mouse models, has shown that Ca(2+)cycling and Ca(2+)-dependent signaling pathways play a pivotal role in heart failure. The improvement of the SR function has ameliorated effects on cardiac pump function and it has potential therapeutic value for heart failure.

Recent studies have demonstrated that human neuroblastoma (NB) cell lines have at least two morphological appearance of neuroblastic (N-type) and substrate-adhesive (S-type) cells. Our previous study revealed that S-type cells expressed alpha-smooth muscle actin and/or desmin, suggesting the smooth muscle cell characteristics of S-type cells. In the present study, a new human NB cell line, KP-N-YS, was established from bone marrow metastasis of a four-year-old boy with advanced NB, originating from the left adrenal gland. Subsequently N-type cell clone (YS1n) and S-type cell clone (YS2s) was isolated from the parent cell line. Parent and clones cell lines were identified as NB by surface membrane antigen and cytoskeletal protein analysis, and these cell lines were demonstrated as common progenitors by chromosomal analysis. Furthermore the presence of basic calponin were determined by indirect immunofluorescence, Western blot, as well as by RT-PCR (reverse transcriptase-polymerase chain reaction). Our demonstration of basic calponin not in N-type cells, but in S-type cells supports the plausible smooth muscle cell characteristics of this NB cell line.

The present study was undertaken to determine abnormalities which are responsible for the diminished functional responsiveness to beta-adrenoceptor stimulation in experimental diabetic rats. Rats were given an intravenous injection of 45mg/kg streptozotocin and hearts were removed from 4 to 6 weeks later. The positive inotropic effects of isoproterenol, norepinephrine and epinephrine were markedly depressed in diabetic rat papillary muscles. Diabetic cardiac muscles also exhibited a reduced maximum contractile responses to forskolin, 3-isobutyl-1-methylxanthine and dibutylic cyclic AMP. The density of beta-adrenoceptors in membranes prepared from diabetic hearts was decreased by 40%, with uniform decreases in the beta 1- and beta 2-subtypes. The affinity of the receptor for the radioligand antagonist [125I]-iodocyanopindolol remained unchanged. Competition binding studies with isoproterenol did not reveal a difference in the fraction of beta-adrenoceptors with high-affinity binding between control and diabetic cardiac membranes, suggesting that interaction between beta-adrenoceptors and Gs may be preserved well in the diabetic state. All measures of adenylate cyclase activity showed that beta-adrenoceptor-dependent and Gs-dependent cyclic AMP productions are well maintained in membranes from diabetic hearts. Thus, these data suggest that the decreased positive inotropic response to beta-adrenoceptor stimulation in diabetic hearts is not attributable to changes in beta-adrenoceptor-Gs-adenylate cyclase system but rather may be due to an alteration in cellular function beyond the level of cAMP generation. To test this hypothesis, incorporation of [32P]-inorganic phosphate into phospholamban in sarcoplasmic reticulum was examined in Langendorff-perfused hearts. Isoproterenol (100nM) increased phosphorylation of phospholamban threefold in control hearts, but did not cause a significant change in the phosphorylation state in diabetic hearts. From these findings it is concluded that the decreased functional responses to cyclic AMP-increasing agents like beta-adrenoceptor agonists in diabetic hearts may be associated with impaired phosphorylation of cardiac regulatory phosphoproteins including phospholamban.

Familial amyloidotic polyneuropathy type IV, one of the hereditary systemic amyloidoses with an autosomal dominant trait, is clinically characterized by cranial neuropathy and corneal lattice dystrophy. Recent biochemical studies have indicated that the amyloid fibril protein in FAP IV is related to gelsolin, an actin-modulating protein. Cases were clustered in the Finnish population and only a few cases have been reported from other populations. Here we described a large kindred with FAP IV as the first report in Japan. This family comprises 42 members in three generations with 14 affected individuals. We examined 7 patients at the age ranging from 43 to 80 years. All cases have corneal lattice dystrophy type II. The disease begins with slowly progressive facial weakness in the fifth or sixth decade of life and consequently the V, XII, IX and X cranial nerves become involved. Peripheral neuropathy of the extremities remained mild until late of life. Microscopy of skin biopsy samples showed deposits of amyloid around the eccrine glands, sebaceous glands, epidermal-dermal junction and blood vessel walls. Immunohistochemistry of the skin revealed the immunopositive material against a monoclonal antibody to gelsolin in the amyloid deposits. Molecular analysis of the gelsolin gene is now in progress.

Calcium ion acts as an intracellular messenger of various types of extracellular signals including hormones, neurotransmitters, cytokines and growth factors. Recent advances in the research of the calcium messenger system have provided informations as to the structure and function of many proteins involved in the messenger system. Also, technical advances in the measurement of the dynamics of calcium ion in a small cell have made it possible to better understand the role of calcium in the activation of the cell. In the present review, a picture of the calcium messenger system is presented.

To investigate the pathogenesis of hereditary pancreatitis we determined whether pancreatic stone protein (PSP) gene was structurally altered in two independent families diagnosed as hereditary pancreatitis. Because, it has been shown that a decrease in the activity of PSP which inhibits CaCO3 crystal formation in pancreatic juice is closely related to the development of chronic calcifying pancreatitis. Southern blot analysis revealed neither a rearrangement nor a gross deletion of PSP gene in genomic DNA of affected members of both families. Furthermore, six exons of PSP gene amplified by polymerase chain reaction from genomic DNA was directly sequenced, while no apparent base mutation was observed. The immunohistochemical study utilizing monoclonal antibody to PSP showed the presence of immunoreactive PSP in the section of pancreatic tissue obtained from a patient affected with hereditary pancreatitis. However, the level of immunoreactive PSP in the remaining acinar cells of the patient pancreas was not reduced when compared with that of normal pancreas. Results, therefore, indicate that genetic alteration of PSP gene may not be responsible for the pathogenesis of hereditary pancreatitis.

Calpain is a Ca2(+)-dependent cysteine endopeptidase and calpastatin is a calpain-specific endogenous inhibitor protein. Both calpain and calpastatin are very widely distributed in various animal tissues and cells. Low (microM) Ca2(+)-requiring calpain I and high (mM) Ca2(+)-requiring calpain II are known to exist. Calpain consists of one heavy (80 kDa) and one light (30 kDa) subunit. The heavy subunits of calpains I and II are different genetic products, whereas the light subunits are the same for both calpains I and II. Molecular cloning as well as protein sequencing revealed that the heavy subunit has four domains, while the light subunit has two domains. The carboxyl terminal domain of each subunit is a calmodulin-like domain, whereas the catalytic site is located in domain 2 of the heavy subunit. Calpastatin has four internally repetitive inhibitory domains. A single domain, or even a truncated 27-mer fragment thereof, possesses inhibitory activity against calpains. Calpain shows a rather broad substrate specificity. It can cleave various enzymes, and cytoskeletal, membrane and receptor proteins. Calpain-catalyzed activation of protein kinase C and transglutaminase may represent a few of the physiological functions of calpain, but a great many other functions can be assigned as well to calpain. Immunohistochemical studies revealed very wide but quite diverse distribution of calpains I and II and calpastatin among various tissues and cells. The expression of the genes for calpain and calpastatin is found to be modulated by retrovirus (HTLV-I) infection to T-lymphocytes. The physiological significance of the calpain and calpastatin system is yet to be elucidated, and accumulating information definitely suggested the role of calpain/calpastatin in health and disease.

We have identified the proteins specifically expressed in multidrug-resistant tumor cells and studied the functions of these proteins. The 170-to 180-kDa membrane glycoprotein (P-glycoprotein) is an ATPase which works as a pump molecule transporting chemotherapeutic drugs outside the resistant cells. The 22-kDa soluble protein (sorcin) is a calcium binding protein of unknown function. The 85-kDa membrane protein is specifically expressed in adriamycin-resistant cells and induced by treatment with adriamycin, suggesting a mechanism unique for adriamycin resistance. Our monoclonal antibodies to these proteins may well become useful tools for the diagnosis of clinical drug resistance.