Immobilized E-cadherin model can enhance cell attachment and differentiation of primary hepatocytes but not proliferation

E-Cadherin is an intercellular adhesion molecule that regulates cell functions such as differentiation and proliferation of cells. To clarify the potential of E-cadherin-mediated adhesion to induce differentiation, we constructed an adsorbable recombinant E-cadherin molecule by fusing with an immunoglobulin G (IgG) Fc region (E-cad-Fc). Hepatocytes could adhere to the fusion protein-coated surface by a homophilic interaction of E-cadherins and showed differentiated phenotypes such as low DNA synthesizing activity and maintenance of tryptophan oxygenase expression, similar to those of spheroid-formed hepatocytes that are known as a highly differentiated tissue-like cell aggregation. These results suggest that E-cadherin is a key molecule for maintaining differentiation of primary hepatocytes.     

Production of interferon-β by fibroblast cells on membranes prepared by extracellular matrix proteins

The fibroblast cells from normal human skin were cultured on Langmuir-Blodgett (LB) and cast membranes prepared using extracellular matrix proteins (e.g., collagen, fibronectin, laminin and vitronectin). The cell density of the fibroblast cells cultured on the cast membranes was found to be higher than that on the cast membranes made of fibronectin, vitronectin and collagen-blended membranes. This indicates that not only the primary structure of proteins but the preparation methods of the membranes, i.e., casting and LB methods, are a strong factor affecting cell growth. The concentration and production of interferon-β per unit cell were found to be higher on the LB membranes than on the cast membranes made of the same proteins except in the case of collagen. However, the cell density on the cast membranes was higher than that on the LB membranes. These results appear to result from the suppressed growth of NB1-RGB cells on the LB membranes leading to the enhanced production of interferon-β on the LB membranes. The highest production of interferon-β per unit cell was observed for the NB1-RGB cells on the collagen-blended membranes with fibronectin and vitronectin. The collagen-blended membranes appear to offer a more natural and appropriate environment for NB1-RGB cells to produce interferon-β. This revised version was published online in September 2006 with corrections to the Cover Date.     

Biased reorientation in the chemotaxis of peritrichous bacteria Salmonella enterica serovar Typhimurium

Many kinds of peritrichous bacteria that repeat runs and tumbles by using multiple flagella exhibit chemotaxis by sensing a difference in the concentration of the attractant or repellent between two adjacent time points. If a cell senses that the concentration of an attractant has increased, their flagellar motors decrease the switching frequency from counterclockwise to clockwise direction of rotation, which causes a longer run in swimming up the concentration gradient than swimming down. We investigated the turn angle in tumbles of peritrichous bacteria swimming across the concentration gradient of a chemoattractant because the change in the switching frequency in the rotational direction may affect the way tumbles. We tracked several hundreds of runs and tumbles of single cells of Salmonella enterica serovar Typhimurium in the concentration gradient of L-serine and found that the turn angle depends on the concentration gradient that the cell senses just before the tumble. The turn angle is biased toward a smaller value when the cells swim up the concentration gradient, whereas the distribution of the angle is almost uniform (random direction) when the cells swim down the gradient. The effect of the observed bias in the turn angle on the degree of chemotaxis was investigated by random walk simulation. In the concentration field where attractants diffuse concentrically from the point source, we found that this angular distribution clearly affects the reduction of the mean-square displacement of the cell that has started at the attractant source, that is, the bias in the turn angle distribution contributes to chemotaxis in peritrichous bacteria.     

Glucose Intolerance and Hyperlipidemia Prior to Diabetes Onset in Female Spontaneously Diabetic Torii (SDT) Rats

The Spontaneously Diabetic Torii (SDT) rat, a newly established animal model for diabetes mellitus, presents nonobese type 2 diabetes with ocular complications. In the present study, oral glucose tolerance tests and biochemical and histopathological examinations were performed in female SDT rats at 16 and/or 25 weeks of age, before the onset of diabetes. At 25 weeks of age, glucose tolerance was significantly impaired, and plasma immunoreactive insulin levels at 120 min after glucose loading were significantly higher (P < 0.05). Body weight and fasting levels of plasma triglycerides and nonesterified fatty acids were significantly higher than those in control animals. Histopathologically, inflammatory cell infiltration and fibrosis were observed in and around the pancreatic islets. These results strongly suggest that female SDT rats are useful as a model to investigate impairment of glucose tolerance and hyperlipidemia prior to the onset of diabetes.     

Interaction between the M2- and M3-Receptor Subtypes in Muscarinic Electrical and Mechanical Responses of Intestinal Smooth Muscles

In various gastrointestinal smooth muscles, two different muscarinic receptor subtypes, M₂ and M₃, are expressed; these receptors are the target for the parasympathetic neurotransmitter acetylcholine. Although the number of M₂ receptors is much greater than that of M₃ receptors, the functional role of the former receptor subtype has yet to be fully defined, since pharmacological analyses of the contractile responses to acetylcholine and other muscarinic agonists have revealed that such responses are mediated extensively by the minor M₃ subtype. The M₃ receptor links to Ca²⁺ store release, and the released Ca²⁺ ions may contribute to the contraction. However, many studies indicated the importance of Ca²⁺ influx through voltage-gated Ca²⁺ channels, rather than Ca²⁺ release, in muscarinic contractions, since the contractile responses are markedly inhibited by Ca²⁺ channel blockers. The major M₂ receptors link to the opening of cationic channels leading to the membrane depolarization, which in turn activates voltage-gated Ca²⁺ channels. Thus, there should be somewhere a point of contact between the M₃- and M₂-mediated signal transductions, as if M₃ receptor stimulation is connected with membrane depolarization. Our electrophysiological and pharmacological findings suggest that the M₂-mediated cationic channel opening and a resulting increase in the membrane electrical activity are the primary mechanism for mediating the contractile response to muscarinic agonists. An allosteric interaction between M₂ and M₃ receptors such that M₃ activation intensifies the M₂/cation channel pathway may account at least in part for the failure of many previous analyses to detect M₂ participation in the contractile responses to full agonists.     

Involvement of heat shock proteins in the healing of acetic acid-induced gastric ulcers in rats.

The present study examined the expression of 73-kDa of heat shock cognate protein (HSC70), 72-kDa of heat shock protein (HSP70) and 47-kDa of HSP (HSP47) observed in the ulcer healing process in rats. Gastric ulcers were induced by a luminal application of acetic acid in male Donryu rats. During the ulcer healing process, the expression of HSPs in the ulcerated tissue was determined. A high level of HSC70 expression was observed both in the normal mucosa and ulcerated tissue, but the level did not change upon ulceration and ulcer healing. While HSP70 and HSP47 were markedly expressed in the ulcer base during ulceration, and decreased with ulcer healing. HSP70 expression in the ulcer margin was gradually increased with ulcer healing. Omeprazole accelerated the healing of gastric ulcers with strong inhibition of gastric acid secretion, while indomethactin delayed in ulcer healing despite slight inhibition of gastric acid secretion. Omperazole enhanced the expression of HSP70 both in the ulcer margin and base, but it reduced HSP47 expression in the ulcer base Indomethacin markedly enhanced HSP47 expression only in the ulcer base. In conclusion, the expression of HSP70 and HSP47 is changed during ulcer healing. Furthermore, it was suggested that the enhanced expression of HSP70 is involved in acceleration of ulcer healing, but overexpression of HSP47 is involved in delayed ulcer healing.     

Does bilirubin play a role in the pathogenesis of both cholesterol and pigment gallstone formation? Direct and indirect influences of bilirubin on bile lithogenicity.

Bilirubin is found in the center of cholesterol gallstones, but its pathogenic role in their formation is unknown. Bilirubin causes a disproportionate reduction of biliary lipid secretion without affecting bile salt secretion in association with a change of biliary lecithin species, which modulates the cholesterol crystallization process. Therefore, the present study investigated whether bilirubin can influence the cholesterol crystallization procedure, and the mechanism(s) of any such action. Supersaturated model bile was prepared (taurocholate/lecithin/cholesterol at 71:18:11, a total lipid concentration of 9.0 g/dl, and cholesterol saturation index of 1.8), and cholesterol crystallization was monitored over time using a spectrophotometer and video-enhanced differential contrast microscopy in the absence or presence of bilirubin (at a final concentration of 10 microM, 20 microM, 40 microM, and 100 microM). Bilirubin enhanced the onset of cholesterol crystallization by 50%, whereas the crystal growth rate and final crystal mass were reduced at a high concentration of bilirubin. Taken together, these results suggest that bilirubin influences the cholesterol crystallization process, by either a direct interaction with biliary lipids that alters metastability, an indirect alteration of the bile salt-micellar lipid holding capacity, or both. Thus, bilirubin may play a role in the pathogenesis of both cholesterol and pigment gallstones.     

4-Hydroxybenzyl alcohol accumulates in suspension-cell cultures and inhibits somatic embryogenesis in carrot

Somatic embryogenesis in carrot ( Daucus carota L.) is strongly inhibited by certain factors that accumulate in culture medium of high-density cultures of embryogenic cells. We previously identified 4-hydroxybenzyl alcohol (4HBA) as one of the inhibitory factors. In this study, we analyzed the accumulation pattern of 4HBA in the cultures of carrot suspension cells. When somatic embryogenesis was induced by culturing embryogenic cells in phytohormone-free Murashige and Skoog medium at various initial cell densities, 4HBA accumulated in the culture medium. The concentration of 4HBA in high cell density cultures was higher than in low cell density cultures. The accumulation of 4HBA in high cell density cultures was rapid during the early days of culture. This rapid accumulation of 4HBA in high cell density cultures might result in the strong inhibition of somatic embryogenesis. The production of 4HBA decreased as the somatic embryos developed. In addition, embryogenic cells released larger amount of 4HBA into the culture medium compared with non-embryogenic cells. These results suggest that the production of 4HBA is both related to embryogenic competence and developmentally regulated during somatic embryogenesis.     

Protective effects of bacteriophage on experimental Lactococcus garvieae infection in yellowtail.

The present study describes the in vitro and in vivo survival of Lactococcus garvieae bacteriophages and the potential of the phage for controlling experimental L. garvieae infection in yellowtail. Anti-L. garvieae phages persisted well in various physicochemical (water temperature, salinity, pH) and biological (feed, serum and alimentary tract extracts of yellowtail) conditions, except for low acidity. In the in vivo, the phage PLgY-16 was detected in the spleens of yellowtail until 24 h after intraperitoneal (i.p.) injection, or the phage was recovered from the intestine of yellowtail 3 h after the oral administration of phage-impregnated feed but undetectable 10 h later. Simultaneous administration of live L. garvieae and phage enhanced recovery of the phage from the spleen or intestine. The survival rate was much higher in yellowtail that received i.p. injection of the phage after i.p. challenge with L. garvieae, compared with that of control fish without phage injection. When fish were i.p. injected with phage at different hours after L. garvieae challenge, higher protective effects were demonstrated in fish that received phage treatment at the earlier time. Protection was also obtained in yellowtail receiving phage-impregnated feed, in which fish were challenged by an anal intubation with L. garvieae. Anal-intubated L. garvieae were detected constantly in the spleens of the control fish, while they were detected sporadically and disappeared from the phage-treated fish 48 h later. On the other hand, orally administered phage was detected at high plaque-forming units from the intestines and spleens of the phage-treated fish until 48 h later. These results indicate that intraperitoneally or orally administered anti-L. garvieae phage prevented fish from experimental L. garvieae infection, suggesting potential use of the phage for controlling the disease.