Proteomic analysis of serum marker proteins in recipient mice with liver cirrhosis after bone marrow cell transplantation

We previously found that transplantation with bone marrow cells (BMCs) improves liver function and liver fibrosis in cirrhotic mice. In the presence of liver damage induced by carbon tetrachloride (CCl4), transplanted BMC migrated into the peri-portal region and trans-differentiated into hepatocytes that produce albumin. Thus under these conditions, BMC transplantation induces liver regeneration. Detecting serum marker proteins is important to monitor the recovery of liver function of cirrhotic mice after BMC transplantation. We therefore initially resolved proteins extracted from serum samples at 48 h after BMC transplantation by 2-DE and compared spot intensity between control and BMC groups of mice. Six protein spots increased in the BMC group compared with the control group. MS revealed that these spots comprised apolipoprotein A1 (apoA1), apolipoprotein C3 (apoC3), vitamin D-binding protein, alpha-1-antitrypsin and proteasome subunit alpha type 1. We subsequently confirmed the levels of apoA1 in serum and liver samples by immunoblotting. ApoA1 increased at early stage (48 h and 1 wk) after BMC transplantation in this mouse model of liver cirrhosis. The early elevation of apoA1 might be useful to predict liver regeneration in cirrhotic mice after BMC transplantation.     

Cerasome as an infusible and cell-friendly gene carrier: synthesis of cerasome-forming lipids and transfection using cerasome

Sonication of a pre-agitated aqueous solution of cationic lipid having a (EtO)3SiCH2CH2CH2 group on the quarternized ammonium nitrogen results in partially silica- or ceramic-coated liposome (cerasome), which can be used as an excellent transfection agent. Non-silylated reference lipid, which may represent cationic lipids that are used in conventional lipofection experiments, form a compact liposome, which undergoes DNA-induced fusion to provide transfection-irrelevant and larger (100-300 nm), more toxic particles. The surface-rigidified cerasome is infusible and the monomeric cerasome complex of DNA is of viral size (approximately 70 nm) and exhibits a remarkable transfection performance with a 10(2)-10(3)-fold higher efficiency (relative to the non-silylated reference lipid), minimized cytotoxicity and serum compatibility. The cerasome lipid is obtained by the reaction of 3-bromopropyltriethoxysilane with a tertiary amine derivative of the lipid. Preparation of an aqueous cerasome solution takes 1-2 h. The cerasome-DNA complex and the transfection takes about 3 d to complete.     

Dispersal of yellow phase Japanese eels <Emphasis Type="Italic">Anguilla japonica</Emphasis> after recruitment in the Kojima Bay-Asahi River system,Japan

The density, size and age distribution were investigated for 233 eels, Anguilla japonica, sampled in fresh and brackish water areas of the Kojima Bay-Asahi River system, Okayama, Japan, to evaluate the possible patterns of dispersal of eels that recruit to this area. Migratory histories of 183 eels were categorized into 5 types depending on the Sr and Ca concentrations in their otoliths: (1) brackish water residents (74 fish, 40.4%), which settled in saline water and remained until capture; (2) freshwater residents (46 fish, 25.1%), which settled in freshwater and remained until capture; (3) upstream shifters (3 fish, 1.6%), which settled in saline water and moved upstream into freshwater; (4) downstream shifters (53 fish, 29.0%), which settled in freshwater and moved downstream into saline water; (5) multiple habitat shifters (7 fish, 3.8%), which shifted their habitats between freshwater and saline water more than twice. For eels captured in the brackish water area, fish density decreased with distance in the downstream direction, while the size and age of eels increased. For eels captured in the freshwater area, size and age were greater than those in the upper-most brackish site. These observations suggest that eels in this system initially accumulate in the lower reaches of the river and then disperse in both upstream and downstream directions following their growth.     

Effect of copper on the activation of the acid phosphatase from the green algae <Emphasis Type="Italic">Pseudokirchneriella subcapitata</Emphasis>

The presence of copper in water environment may have detrimental effects on aquatic organisms, including algae, where different enzymatic systems can be affected. Algae acid phosphatase plays important roles in metabolic processes such as decomposition of organic phosphate, autophagic digestive process, recycling cellular materials and zygote formation during reproduction. This work describes an in vitro activation effect of copper on the acid phosphatase of the green algae Pseudokirchneriella subcapitata (formely Selenastrum capricornutum) under preincubation condition. Apparent Michaelis constant values of 1.21 and 0.37 mM, and activation energy values of 26.8 and 13.6 kJ mol⁻¹ were determined in the absence and in the presence of 0.2 mM Cu²⁺, respectively. The dissociation constant value for Cu²⁺ binding to the enzyme was determined to be 22.04 μM. The decrease of the apparent Michaelis constant (Km) and activation energy values in the presence of Cu²⁺ correlates well with its activating effect on the acid phosphatase activity. This propriety could be used as a sensitive bioindicator for copper in environmental samples.     

A rostrocaudal muscular dystrophy caused by a defect in choline kinase beta, the first enzyme in phosphatidylcholine biosynthesis

Muscular dystrophies include a diverse group of genetically heterogeneous disorders that together affect 1 in 2000 births worldwide. The diseases are characterized by progressive muscle weakness and wasting that lead to severe disability and often premature death. Rostrocaudal muscular dystrophy (rmd) is a new recessive mouse mutation that causes a rapidly progressive muscular dystrophy and a neonatal forelimb bone deformity. The rmd mutation is a 1.6-kb intragenic deletion within the choline kinase beta (Chkb) gene, resulting in a complete loss of CHKB protein and enzymatic activity. CHKB is one of two mammalian choline kinase (CHK) enzymes (alpha and beta) that catalyze the phosphorylation of choline to phosphocholine in the biosynthesis of the major membrane phospholipid phosphatidylcholine. While mutant rmd mice show a dramatic decrease of CHK activity in all tissues, the dystrophy is only evident in skeletal muscle tissues in an unusual rostral-to-caudal gradient. Minor membrane disruption similar to dysferlinopathies suggest that membrane fusion defects may underlie this dystrophy, because severe membrane disruptions are not evident as determined by creatine kinase levels, Evans Blue infiltration, and unaltered levels of proteins in the dystrophin-glycoprotein complex. The rmd mutant mouse offers the first demonstration of a defect in a phospholipid biosynthetic enzyme causing muscular dystrophy, representing a unique model for understanding mechanisms of muscle degeneration.     

Spawning site of the fluvial eight-barbel loach, Lefua sp., in the natural environment

The spawning site of the fluvial eight-barbel loach, Lefua sp. (sensu Hosoya, 1993; Japanese name: nagare-hotoke-dojo), an endangered species, was investigated by searching for fertilized eggs in June and July 1995–1997 in an upper reach of the Kako River, Hyogo Prefecture, Japan. We obtained one egg each by a random sampling with agitating the river bottom (80 trials) and by collecting drift samples (89 trials). This finding indicates that the eggs were not scattered on the bottom or into the water column after being spawned. Two individual males were observed to patrol around particular interstices occurring under the buried cobbles and under the boulders that formed the riverbank. Males pecked other individuals that approached around the interstices. We obtained 9 and 15 eggs, respectively, from two such interstices in only two collection trials. The interstices were suggested to function as the spawning sites of this endangered fish in nature.     

Deletion and alanine mutation analyses for the formation of active homo- or hetero-dimer complexes of mouse choline kinase-alpha and -beta

Choline kinase (CK) is the first-step regulatory enzyme for the biosynthesis of phosphatidylcholine in all mammalian cells. It exists as at least three isoforms (alpha1, alpha2 and beta) that are encoded by two separate genes termed ck-alpha and ck-beta. The active enzyme has been proposed to consist of either their homo- or hetero-dimeric forms. Here, we report on the identification of several essential domains and amino acid residues involved in their active dimer formation. Full-length cDNAs or their truncated or alanine-mutated versions for mouse CK-alpha1 and CK-beta tagged with either HA or Myc at their N-termini were expressed in COS-7 cells. Each dimer formation was analyzed by immuno-precipitation followed by Western blotting. Kinetic analysis for CK reaction was performed with different expression products. Both the N-terminal domain-1 and C-terminal portions (E424-K430 for CK-alpha1 and Q379-K385 for CK-beta) were shown to be critical for the formation of active homo- or hetero-dimer complex. Interestingly, D320 in the CK-motif of CK-alpha1 was found to be essential for alpha1/alpha1 homo-dimerization but not for alpha1/beta hetero-dimerization. A mutation of the corresponding D276 of CK-beta to A276 did not show any effect on either its homo- or hetero-dimerization but it caused a strong inhibition of CK activity in either case.     

Cytotoxicity effect of algal polysaccharides on HL60 cells

The present study shows the cytotoxic effect of three different classes of algal polysaccharides on HL60 cells. Three galactofucans, fucoidan, and glucan were the polysaccharides utilized in this analysis. The parameters used for evaluating cell viability were [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) reduction, protein content, and phosphatase activity. We demonstrated stimulation of phosphatase activity, MTT reduction, and protein content in relation to three types of galactofucans (1, 2, and 3) with different molecular weights (1600, 1200, and 360 kD). However, when HL60 cells were treated with galactofucan type 3, the total protein remained unchanged. Under the same experimental conditions, an expressed increase in the phosphatase activity was detected when galactofucan 3 was utilized. In relation to the mitochondrial function, the stimulation was higher in cells treated with galactofucan type 1. Fucoidan did not have a significant effect on MTT reduction, but protein content was decreased (IC₅₀ around 30 μg/ml). Glucan also activated all the parameters that were analyzed, and this effect was more expressed in the phosphatase activity and in the protein content. This study provides new insights into the cytotoxic action of polysaccharides on HL60 cells and suggests for the first time the possible involvement of phosphatases in this process. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 12, pp. 1613–1617.     

Protective role of metallothionein against copper depletion

Copper (Cu) is one of the essential metals and its homeostasis is strictly regulated. Metallothionein (MT) is induced by excess Cu to mask the Cu toxicity. Although the role of MT in Cu toxicity has been explained in terms of Cu sequestration, its role under Cu-deficient conditions is not known. This study was carried out to determine the role of MT in Cu depletion by a Cu(I)-specific chelator, bathocuproine sulfonate (BCS), in cultured cells established from MT-knockout mouse and its wild type. Viability was decreased more severely in MT-null cells than in wild-type cells by BCS treatment. The expression levels of both MT isoforms were increased by BCS treatment in wild-type cells. Thus, MT was shown to be induced under Cu-deficient conditions to maintain the activities of intracellular cuproenzymes such as cytochrome c oxidase and Cu,zinc-superoxide dismutase.     

Helicobacter pylori eradication therapy on histologic change in the distal esophagus

BACKGROUND: Although cases of reflux esophagitis (RE) developing after treatment to eradicate Helicobacter pylori have been discussed in some detail, no reports are available concerning the histologic examination of RE both before and after eradication therapy. MATERIALS AND METHODS: Sixty-one patients and 111 specimens were investigated using endoscopic and histologic techniques. The histologic findings including basal zone height, papillar height, Ki-67 labeling index, and COX-2 expression before and after treatment for H. pylori infection were compared with those in normal controls and patients with endoscopic RE. RESULTS: Twelve months after eradication therapy, the incidence of newly developed endoscopic RE was 20% (5/25). Basal zone height and papillar height had increased at 1 month, but had returned to pretreatment levels after 12 months of eradication therapy. The Ki-67 labeling index was significantly increased 1 and 12 months after eradication therapy compared to values before treatment. COX-2 expression gradually increased after the treatment. The phenomena linked to esophagitis appeared after eradication therapy. However, the severity and extent of these signs were not so high after the treatment of H. pylori than those in patients with overt reflux esophagitis. Focusing on the patients with hiatal hernia, papillar height and Ki-67 labeling index increased significantly after eradication therapy, values being almost the same as those in the patients with endoscopic RE. CONCLUSIONS: Hiatal hernia plays an important role in the possible occurrence of hidden RE after treatment for a H. pylori infection.