Human neuroblastoma cell lines having smooth muscle cell markers]

The neural crest gives rise to a variety of tissues, including peripheral neurons, Schwann cells, melanocytes and ectomesenchymal cells, which include the smooth muscle cells of large arteries. Cell lines derived from neuroblastoma (a neural crest tumor) have at least two distinct morphological cell types, a neuroblastic phenotype (N-type) and an epithelial-like phenotype (S-type) with characteristics of substrate-adhesiveness. We have analyzed 17 human neuroblastoma cell lines using a panel of monoclonal antibodies against cytoskeletal proteins. Three neuroblastoma cell lines (KP-N-SI, KP-N-YN and SMS-KCN) bound an alpha -smooth muscle actin antibody. In addition, one of these cell lines (KP-N-SI) bound anti-desmin monoclonal antibodies as determined by indirect immunofluorescence. A total of eight cloned cell lines were obtained from the above parent cell lines. These were composed of either N- or S-type cells and were confirmed to be the common neuroblastoma origin from each parent cell line by chromosomal analysis. Alpha-smooth muscle actin and desmin were demonstrated in the S-type cloned cells by indirect immunofluorescence, as well as by two-dimensional Western blot analysis. These results were confirmed by Northern blot analysis using a specific probe (pSH alpha SMA-3'UT) to human alpha-smooth muscle actin mRNA. This is the first report of the presence of alpha-smooth muscle actin and desmin in neuroblastoma cell lines. These data show that in addition to giving rise to cells with neural, Schwann cell and melanocyte markers, neuroblastoma can also give rise to the cells expressing smooth muscle cell markers.     

Preference for vegetable oils in the two-bottle choice test in mice

We evaluated in mice their preference for different vegetable oils using the two-bottle choice test. Mice showed preference for all vegetable oils (at 1% concentration in emulsion) tested, including corn oil, canola oil, and mixed vegetable oil, compared to a control fluid without oil. Mice also significantly preferred highly palatable sucrose solutions (5-20%) in this test. One percent corn oil was selected to the same extent as were these sucrose solutions. Mixtures of 1% corn oil and several concentrations of sucrose were preferentially selected as compared to corn oil or sucrose alone. The concentration-response curves of oil-sucrose mixtures vs sucrose (0.5-20% concentrations) were similar to those of sucrose vs control solutions. These results suggest that 1% corn oil and sucrose at the concentrations tested did not affect each other's preference. Chronic intake of 20% sucrose solution, but not 100% corn oil, changed the preference for lower concentrations of each, and both induced remarkable increases in body weight of mice. In conclusion, we showed that mice preferred vegetable oils, and this preference compared favorably with highly palatable sucrose solutions and was not changed by chronic intake of corn oil.     

Granzyme A and thrombin differentially promote the release of interleukin-8 from alveolar epithelial A549 cells

Some of extracellular serine proteases with trypsin-like specificity of cleavage have been known to increase the release of inflammatory mediators from various cell types. For instance, two well-known trypsin-like serine proteases circulating in blood, granzyme A (GrA) and thrombin, have been found to promote interleukin (IL)-8 release from an alveolar epithelial A549 cell line. However, the mechanisms by which the proteases promote IL-8 release from the cells are not fully understood. In the present study, using A549 cells we found that (1) thrombin promoted IL-8 release from the cells via a mechanism partially involving activation of protease-activated receptor-1, a G-protein coupled receptor, whereas a recombinant form of GrA (rGrA) did it via a mechanism that does not involve the receptor activation; that (2) unlike rGrA, thrombin did not cause detachment and microtubule disruption of the cells; and that (3) the release of IL-8 induced by rGrA was inhibited in the presence of taxol, a microtubule-stabilizing reagent, whereas that induced by thrombin was not. These findings suggest that rGrA and thrombin promote the release of IL-8 from A549 cells through distinct mechanisms.     

Effects of CH-19 sweet, a non-pungent cultivar of red pepper, in decreasing the body weight and suppressing body fat accumulation by sympathetic nerve activation in humans

'CH-19 Sweet' is a non-pungent red pepper and enhances the energy expenditure in humans in like manner to the pungent red pepper. We investigated in this study the effects of a repeated intake of CH-19 Sweet for two weeks on the body weight and body fat in humans. Changes in the autonomic nervous activity after ingesting CH-19 Sweet were also measured by a power spectral analysis. We established a new protocol which allows the precise detection of weight change in humans by using fewer subjects. These methods were used to show that the repeated intake of CH-19 Sweet reduced the body weight and suppressed body fat accumulation. Furthermore, the body weight loss due to the repeated intake of CH-19 Sweet was significantly correlated with the sympathetic nervous response after its ingestion. We propose that the repeated intake of CH-19 Sweet reduced the body weight and suppressed body fat accumulation by sympathetic nervous activation in humans.     

Development of an in vitro system for screening the ligands of a membrane glycoprotein CD36

It has well been known that human and rodents exhibit a preference for fats. This suggests the existence of an orosensory system responsible for the detection of dietary fats. A plasma membrane glycoprotein CD36, besides the role in the uptake of long-chain fatty acids (LCFAs) as well as oxidized low-density lipoprotein (OxLDL) in a variety of cells, has been postulated to be a candidate fat taste receptor on the tongue. Therefore, molecules that bind with CD36 to cause intracellular signaling but have fewer calories could be substitutes for dietary fats. In the present study, we developed an in vitro system for the screening of CD36 ligands using Chinese hamster ovary-K1 cells (CHO-K1) stably transfected with human or mouse CD36. When incubated with OxLDL labeled with fluorescence dye, the fluorescence was much higher in the transfected CHO-K1 cells than in non-transfected CHO-K1 cells. Incubation of the transfected cells with OxLDL caused a rapid phosphorylation of extracellular signal regulated kinase, and the degree was significantly higher compared with that in non-transfected CHO-K1 cells. The expression system using CHO-K1 cells could be a convenient tool to screen the novel ligands of CD36.     

A novel marker for terminal Schwann cells,homocysteine-responsive ER-resident protein,as isolated by a single cell PCR-differential display

Terminal Schwann cells (TSCs) that cover motor neuron terminals are known to play important roles in maintaining neuromuscular junctions, as well as in the repair process after nerve injury. However, molecular characteristics of TSCs remain unknown, because of the difficulties in analyzing them due to their paucity. We have established a method of selectively and efficiently collecting TSCs so that cDNA analysis can be done properly. The expression of 1-2% of whole mRNAs was compared between myelinating Schwann cells (MSCs) and TSCs, and it turned out that approximately one-third of the bands could be categorized as cell-type-specific bands. TSCs thus constitute a distinct entity from the viewpoint of gene expression. As one of the cDNA clones belonging to TSC-specific bands was identified homocysteine-responsive ER-resident protein (Herp), and in situ hybridization confirmed that Herp mRNA is expressed in TSCs on motor nerve terminals but not in MSCs, both in developing and adult rats. In conclusion, we have been able to identify Herp as a novel molecular marker for TSCs.     

Mercaptoacetate inhibition of fatty acid beta-oxidation attenuates the oral acceptance of fat in BALB/c mice

We investigated the effect of beta-oxidation inhibition on the fat ingestive behavior of BALB/c mice. Intraperitoneal administration to mice of mercaptoacetate, an inhibitor of fatty acid oxidation, significantly suppressed intake of corn oil but not intake of sucrose solution or laboratory chow. To further examine the effect of mercaptoacetate on the acceptability of corn oil in the oral cavity, we examined short-term licking behavior. Mercaptoacetate significantly and specifically decreased the number of licks of corn oil within a 60-s period but did not affect those of a sucrose solution, a monosodium glutamate solution, or mineral oil. In contrast, the administration of 2-deoxyglucose, an inhibitor of glucose metabolism, did not affect the intake or short-term licking counts of any of the tasted solutions. These findings suggest that fat metabolism is involved in the mechanism underlying the oral acceptance of fat as an energy source.     

Carrageenan inhibits granzyme A-induced detachment of and interleukin-8 release from alveolar epithelial A549 cells

Granzyme A (GrA) is a lymphocyte serine protease that is believed to enter virus-infected cells and growing tumors and induce apoptosis. We found recently that recombinant rat GrA (rGrA) promotes detachment of and interleukin (IL)-8 release from alveolar epithelial A549 cells and suggested that this protease is involved in the pathogenesis of certain inflammatory lung diseases. In the present study, we found that λ-carrageenan (a sulfated oligosaccharide constituting the cell walls of seaweeds) potently inhibits rGrA-induced detachment and IL-8 release of A549 cells. This sulfated oligosaccharide might be useful for suppressing the development of inflammatory lung diseases in which GrA is thought to be involved.     

Preparation of a verifiable peptide-protein immunogen: direction-controlled conjugation of a synthetic fragment of the monitor peptide with myoglobin and application for sequence analysis

A useful method for preparing a synthetic peptide-carrying protein for specific antibody production was established. The monitor peptide is a trypsin-sensitive cholecystokinin-releasing peptide purified from rat pancreatic juice on the basis of its stimulatory activity toward pancreatic enzyme secretion. The NH2-terminus fragment of the monitor peptide (residues 1-14) was synthesized by a solid phase method. Cysteine at the COOH terminus of the fragment was conjugated with amino groups of myoglobin using a hetero-bifunctional reagent. Sequence analysis of the fragment-myoglobin conjugate indicated that the peptide/myoglobin conjugation ratio was about 1/1 (mol/mol). Antiserum against the conjugate from a rabbit effectively abolished the stimulatory activity of the monitor peptide in the rat small intestine.     

Capsinoids, non-pungent capsaicin analogs, reduce body fat accumulation without weight rebound unlike dietary restriction in mice

Enhancing energy expenditure and reducing energy intake are both crucial for weight control. Capsinoids, which are non-pungent capsaicin analogs, are known to suppress body fat accumulation and reduce body weight by enhancing energy expenditure in both mice and humans. However, it is poorly understood whether the suppression of body fat accumulation by capsinoids has an advantage over dietary restriction. This study shows that the oxygen consumption was increased in mice administered with capsinoids but not in dietary-restricted mice, although there was a similar suppression of body fat accumulation in both groups. The weight rebound was more notable in the dietary-restricted mice than in the mice administered with capsinoids. These results indicate that suppressing body fat accumulation by capsinoids was more beneficial than a restricted diet for maintaining body weight.