Effect of the antipsychotic agent amisulpride on glucose lowering and insulin secretion.

AIMS: To investigate the effects of the second generation antipsychotic (R/S)-amisulpride, and the chirally purified enantiomers, on glucose homeostasis in diet-induced obese (DIO) mice. METHODS: Normal and DIO mice were treated with pharmacologically relevant doses of amisulpride prior to oral glucose tolerance tests (OGTTs). Blood glucose, insulin, glucagon-like peptide-1, prolactin and amisulpride drug levels were determined. RESULTS: Racemic amisulpride significantly reduced glucose excursions during OGTT in both normal and DIO mice. This potent effect was preserved with the 'off-isomer', R-amisulpride (ED(50) 1 mg/kg). Insulin secretion was significantly increased with R-amisulpride with only a minor increase in prolactin levels. CONCLUSIONS: Amisulpride has antidiabetic actions in DIO mice resulting from increased insulin secretion. This provides some explanation for why amisulpride, unlike other atypical antipsychotics, is not diabetogenic in man. Furthermore, the observation that R-amisulpride is also antidiabetic and has minimal impact on prolactin levels presents the opportunity for development of this isomer as an antidiabetic agent.

Intermittent exposure to reduced oxygen levels affects prey size selection and consumption in swimming crab Thalamita danae Stimpson.

Portunid crabs Thalamita danae (carapace width: 46-56 mm) were exposed to low oxygen level (4.0 mg O2 l(-1)) and hypoxia (1.5 mg O2 l(-1)) for 6 h each day with three size classes (large: 15.0-19.9 mm, medium: 10.0-14.9 mm, small: 5.0-9.9 mm) of mussels Brachidontes variabilis offered as food. Consumption rate, prey size preference, and prey handling including breaking time, handling time, eating time and prey value, were studied during the time the crabs were exposed to reduced oxygen levels and results were compared with the crabs maintained at high oxygen level (8.0 mg O2 l(-1)) throughout the experiment. Consumption of mussels from all size classes was significantly higher at high oxygen level than at reduced oxygen levels. No mussel size preference was observed for crabs exposed to 4.0 or 8.0 mg O2 l(-1) but those crabs exposed to 1.5 mg O2 l(-1) preferred medium mussels. Both breaking time and handling time increased with mussel size but did not vary with oxygen level. Prey value of each mussel consumed (mg dry wt eaten crab(-1) s(-1)) was calculated by dividing the estimated dry weight of the mussel by the observed handling time. Mean prey value varied significantly with mussel size, with values obtained for large mussels being higher than small mussels at 4.0 and 8.0 mg O2 l(-1); the effect of oxygen level, however, was insignificant. In view of portunid crabs as major predators of mussels, results may help explain dominance of mussels in eutrophic harbours in Hong Kong.

Rapid generation of homologous internal standards and evaluation of data for quantitation of messenger RNA by competitive polymerase chain reaction.

Sensitive and quantitative measurement of messenger RNA (mRNA) is important for accurate assessment of gene expression. Conventional methods of mRNA measurement frequently lack the sensitivity required to detect mRNA expressed at low level, such as mRNA encoding receptors and intracellar signaling molecules. Thus, the extremely sensitive RT-PCR has become the method of choice for examination of gene expression. However, quantitation of mRNA by PCR is difficult because small variations in amplification efficiencies among sample tubes can lead to substantial differences in product yield, thereby rendering direct comparisons between samples invalid. Development of protocols for quantitative RT-PCR has relied on internal standards to monitor the efficiency of the RT-PCR in different reaction tubes. Technically, the two most serious limitations to routine successful application of competitive quantitative PCR is ready access to competitive internal standards and efficient methods for accurate quantitative analysis of the data. In the present manuscript, application and validation of a simple approach to generate homologous internal competitive standards and to quantitate data for rapid, accurate determination of the expression level of genes by quantitative PCR is described. Generation of the competitive standard from a previously amplified PCR product by the methods described requires only one additional primer pair, and an additional two-step reaction; it can be completed in 1-2 days. Analyzing the results of the competitive PCR reaction via phosphoimager analysis provides a simple, rapid method for accurate quantitation of results. Data presented here clearly illustrate that the methods described have been successfully applied, and that they should have wide application for competitive quantitative PCR analysis of gene expression.

Structure and function of C-CAM1. The first immunoglobulin domain is required for intercellular adhesion.

Cell-CAM105 proteins (also called C-CAM) are epithelial cell adhesion molecules of the immunoglobulin (Ig) superfamily. The sequences of C-CAM are highly homologous to those of human carcinoembryonic antigen (CEA)-family proteins. In previous studies using baculoviral vectors, we showed that expression of the L-form cell-CAM105 (also called C-CAM1) in insect cells resulted in cell aggregation (Cheung, P. H., Thompson, N. L., Earley, K., Culic, O., Hixson, D., and Lin, S. H. (1993) J. Biol. Chem. 268, 6139-6146). This result indicates that the insect-cell system is suitable for studying the adhesion function of C-CAM. Since C-CAM1 contains four extracellular Ig-domains, the structural features directly responsible for C-CAM1 adhesion function were investigated by site-directed deletion and expression in the baculovirus/insect cell system. Results from these studies indicated that the first Ig domain located in the NH2-terminal of C-CAM plays a crucial role in intercellular adhesion. Site-directed deletion producing mutants lacking the second, third, or fourth Ig domains had no effect on the adhesion function. In addition, adhesion function was retained when both the third and fourth Ig domains were deleted, although the adhesion activity was reduced to half that in control cells. However, simultaneous deletion of the second, third, and fourth domains abolished adhesion, suggesting that these domains affect the accessibility of the binding site localized in the first domain. In our previous studies, we showed that the cytoplasmic domains of C-CAM play a significant role in the isoforms' adhesion activity since expression of a C-CAM isoform containing only 6 instead of 71 amino acids intracellularly failed to show the adhesion phenotype (Cheung, P. H., Culic, O., Qiu, Y., Earley, K., Thompson, N., Hixson, D. C., and Lin, S.-H. (1993) Biochem. J. 295, in press). These results together suggest that both the cytoplasmic domain and the first N-terminal Ig-like domain are required for C-CAM-mediated cell adhesion activity.

The cytoplasmic domain of C-CAM is required for C-CAM-mediated adhesion function: studies of a C-CAM transcript containing an unspliced intron.

Cell-CAM105 (also named C-CAM) is a cell surface glycoprotein involved in intercellular adhesion of rat hepatocytes. It has four extracellular immunoglobulin (Ig) domains, a transmembrane domain and a cytoplasmic domain and therefore is a member of the Ig supergene family. We have characterized multiple cDNAs of the C-CAM genes in rat intestine. Sequence analyses showed that rat intestine contained not only the previously reported L-form and S-form C-CAMs (renamed C-CAM1 and C-CAM2 respectively) but also a new isoform, C-CAM3. The C-CAM3 transcript codes for a polypeptide with a truncated C-terminus that lacks 65 amino acids from the previously reported C-CAM1 cytoplasmic domain. Unlike C-CAM1, C-CAM3 did not mediate cell adhesion when expressed in insect cells using the baculoviral expression system. Thus the extra 65 amino acids in the cytoplasmic domain of C-CAM1 are important for adhesion phenotype when expressed in insect cells. Although C-CAM1 and C-CAM2 are encoded by different genes, sequence analysis suggests that C-CAM3 is probably derived from alternative splicing of the C-CAM1 gene. To examine this possibility, we have determined the exon organization of the C-CAM1 gene. C-CAM3 differed from C-CAM1 by the presence of a single unspliced intron which contained a stop codon immediately after the regular splice junction. As a result, translation of C-CAM3 terminates at the point where C-CAM1 and C-CAM3 sequences diverge. To investigate the expression of C-CAM1, C-CAM2 and C-CAM3 in different tissues, we used an RNAase-protection assay to simultaneously assess the levels of expression of these transcripts. Using total RNA prepared from various tissues, we showed that expression of C-CAM3 was tissue-specific, and the C-CAM3 transcript accounted for about 25% of the transcripts derived from the C-CAM1 gene. However, further analysis revealed that C-CAM3 transcript was not present in cytosolic RNA, rather it was enriched in nuclear RNA prepared from hepatocytes. Although C-CAM3 cDNA contains the polyadenylation signal and is polyadenylated, these results indicate that C-CAM3 is probably an incomplete spliced product of C-CAM1 gene.

Conformation dependence of antipeptide antibodies: characterization of cell-CAM105 isoform-specific antipeptide antibodies using proteins expressed in insect cells with baculoviral vectors.

Cell-CAM105 proteins are hepatocyte adhesion molecules of the immunoglobulin superfamily. The two isoforms, L-form and S-form, are highly homologous. In their extracellular domains, only 16 amino acid substitutions are found scattered in the first immunoglobulin domain of 105 amino acids. Peptide sequences containing these differences are selected for production of antibodies. Isoform specificities of these antibodies were evaluated with proteins expressed in the baculovirus-insect cell system. In immunoblot and immunoprecipitation experiments, anti-C1 antibody, which was generated against a peptide sequence found only in the cytoplasmic domain of the L-form, reacted only with the L-form cell-CAM105 protein. Anti-N antibody, which was generated against a pentadecapeptide of the L-form, reacted with both the S- and L-isoforms. The lack of isoform specificity of anti-N is not surprising because there is only one amino acid substitution in this pentadecapeptide sequence. In contrast, S3, a S-form-specific pentadecapeptide with four amino acid substitutions was able to elicit antibody, anti-S3, specific for the S-isoform. With the commonly used immunoprecipitation procedures, only anti-C1 was able to precipitate cell-CAM105 from the liver membrane. Anti-N and anti-S3 could precipitate the proteins only after the liver membrane samples had been boiled in the presence of denaturing agents. Hydropathy analysis of these peptides revealed that both peptides N and S3 are more hydrophobic than peptide C1, suggesting that the peptide fragments N and S3 are probably not located on the surface of the protein. This may explain why boiling of the protein sample was necessary before anti-N and anti-S3 could precipitate the protein. The present study demonstrates that it is possible to produce isoform-specific antibodies for highly homologous proteins. Furthermore, we show that special sample treatment may be required to expose the antigenic sites.

Arrhythmia models in the rat.

The rat has been used extensively for arrhythmia studies, and a large number of models have been developed, which include chemical, electrical, and pathological procedures for including arrhythmias. Such models have been used to investigate mechanisms for inducing arrhythmias as well as the actions of antiarrhythmic drugs. Arrhythmia models in rats are of particular importance in examination of the antiarrhythmic action of various drugs.

Cell-CAM105 isoforms with different adhesion functions are coexpressed in adult rat tissues and during liver development.

The rat hepatocyte cell adhesion molecule cell-CAM105 has recently been shown to be composed of at least two isoforms. Expression of the two isoforms in different tissues and during fetal liver development in rats was studied by RNase protection using a probe which could specifically and simultaneously detect both isoforms. This probe revealed protected fragments of expected lengths for the L-form and the S-form in RNA samples isolated from various adult rat tissues. High levels of the L-form and S-form messages were detected in liver and intestine, moderate levels were detected in lung, and weak signals were detected in muscle, kidney, and spleen. In liver development studies, the messages for cell-CAM105 showed a major increase on the first day after birth compared to the fetal stage, and both isoform messages were proportionally increased. These results indicate that both cell-CAM105 isoforms may have function(s) related to hepatocyte differentiation. To study the adhesion function of cell-CAM105 isoforms, full-length cDNAs for these isoforms were expressed in insect cells. The insect cells expressing the L-form cell-CAM105 were found to aggregate. However, expression of S-form cell-CAM105 did not support cell aggregation. These results indicate that L-form, but not S-form, cell-CAM105 directly mediates the cell adhesion function.

Localization and characterization of a parotid Ca(2+)-dependent ecto-ATPase.

Parotid acini were isolated and tested to further establish the presence of ecto-ATPase in the intact cells. Inhibitors were used to determine if the inhibitor profile of the ATPase was similar to that of a Ca(2+)-ATPase from parotid membranes identified previously as an ecto-ATPase. The Ca(2+)-ATPase of intact cells was insensitive to oligomycin (10 micrograms/ml), N-ethylmaleimide (NEM) (0.1 mM), ruthenium red (0.1 mM), sodium azide (1 mM), and was inhibited approximately 22% by sodium orthovanadate (Na3VO4) (1 mM). This profile was similar to the Ca(2+)-ATPase of intact cells. Trifluoperazine (TFP) (0.1 mM) inhibited the enzyme in intact cells by approximately 32%. The nucleotide substrate specificity of the enzyme also reflected very closely the pattern seen in isolated membranes.

A Ca(2+)-ATPase from rat parotid gland plasma membranes has the characteristics of an ecto-ATPase.

A Ca(2+)-ATPase with an apparent Km for free Ca2+ = 0.23 microM and Vmax = 44 nmol Pi/mg/min was detected in a rat parotid plasma membrane-enriched fraction. This Ca(2+)-ATPase could be stimulated without added Mg2+. However, the enzyme may require submicromolar concentrations of Mg2+ for its activation in the presence of Ca2+. On the other hand, Mg2+ could substitute for Ca2+. The lack of a requirement for added Mg2+ distinguished this Ca(2+)-ATPase from the Ca(2+)-transporter ATPase in the plasma membranes and the mitochondrial Ca(2+)-ATPase. The enzyme was not inhibited by several ATPase inhibitors and was not stimulated by calmodulin. An antibody which was raised against the rat liver plasma membrane ecto-ATPase, was able to deplete this Ca(2+)-ATPase activity from detergent solubilized rat parotid plasma membranes, in an antibody concentration-dependent manner. Immunoblotting analysis of the pellet with the ecto-ATPase antibody revealed the presence of a 100,000 molecular weight protein band, in agreement with the reported ecto-ATPase relative molecular mass. These data demonstrate the presence of a Ca(2+)-ATPase, with high affinity for Ca2+, in the rat parotid gland plasma membranes. It is distinct from the Ca(2+)-transporter, and immunologically indistinguishable from the plasma membrane ecto-ATPase.

Characteristics of anion-stimulated Mg-ATPase from rat parotid gland secretory granules.

Magnesium-dependent adenosine triphosphatase (Mg-ATPase) was assayed in highly purified secretory granules. The enzyme was stimulated by sulphite and isethionate, unaffected by chloride and inhibited by fluoride and thiocyanate. Inhibition was not related to the permeant properties of the anion, but the relative inhibitory potency of the anions was similar to that in some other studies of secretory granule ATPases. Maximum contribution to the anion-stimulated ATPase by contaminating mitochondria was estimated at 9.3%. The enzyme was inhibited by the stilbene disulphonic acid inhibitor, 4-acetamido-4'-isothiocyano-2,2'-stilbene disulphonic acid (SITS). The IC50 was 0.16 mM in the absence of sulphite and increased in the presence of sulphite. The relation of the inhibition by SITS to sulphite was complex. Both Vmax and Km parameters were changed by SITS. Furthermore the data are consistent with the presence of two anion-stimulated ATPases. The ATPase was sensitive to tributyltin, dicyclohexylcarbodiimide (DCCD) and oligomycin, only moderately sensitive to azide, probenecid and N-ethylmaleimide (NEM) and rather insensitive to carbonylcyanide m-chlorophenylhydrazone (CCCP) and sulphisoxazole. ATPase activity was stimulated by calcium both in the presence and absence of magnesium. These findings suggest that the ATPase(s) present in parotid secretory granules is unique among secretory granule ATPases.

Selective antitumor effect on L10 hepatocarcinoma cells of a potent immunoconjugate composed of the A chain of abrin and a monoclonal antibody to a hepatoma-associated antigen.

The toxic A chain of abrin was isolated by affinity chromatography and was demonstrated to be a potent inhibitor of protein synthesis in a cell-free rabbit reticulocyte system with a complete inhibitory dose at 1 X 10(-9) M. This A chain was coupled by a disulfide linkage to a purified monoclonal antibody directed against a tumor-associated antigen found on the line 10 hepatocarcinoma tumor in strain two guinea pigs. The immunoconjugate retained the functions of the individual components, i.e., antigen binding to the intact cell in vitro and inhibition of its protein synthesis. This conjugate was a selective antineoplastic agent with a cytocidal dose at 5 X 10(-9) M toward antigen-bearing cells in vitro. Several antigen-negative cells were much less susceptible to its cytotoxic effect. The cytotoxicity of the conjugate appeared to be by antibody-mediated delivery of toxic A chain into the target cell. When cells were pretreated with excess free antibody followed by a brief exposure to conjugate, there was a reversal of the cytotoxicity to antigen-positive cells but not to the antigen-negative cells. The therapeutic efficacy of the conjugate was assayed by injecting a single dose s.c. or i.v. into syngeneic guinea pigs bearing established line 10 tumors. These in vivo studies showed that (a) the conjugate was not toxic at a dosage of 60 to 1120 micrograms/guinea pig, (b) the conjugate decreased or abolished the growth of established solid tumors, (c) the conjugate delayed or inhibited tumor metastases to lymph nodes, and (d) 20 to 40% of the animals in selective groups had a long-term complete regression.

EPR and fluorescence depolarization studies on bovine cardiac myosin.

To test for possible differences in local conformation and S1 flexibility, bovine cardiac and rabbit skeletal myosins were labeled with a fluorophore (1,5-IAEDANS) and a spin label having iodoacetamide reactivity. The marked activation of the Ca2+-ATPase (6- to 8-fold) and inhibition of the K+ (EDTA)-ATPase (80-90%) by both labels indicated specific labeling of the fast-reacting thiols (SH1) of both myosins. Fluorescence depolarization studies of 1,5-IAEDANS-labeled cardiac myosin indicated that, like skeletal myosin, the SI moieties of cardiac myosin exhibit considerable segmental flexibility with respect to the rod portion of the molecule. This indicates that segmental flexibility may be a property of all myosins. Cardiac and skeletal myosins immobilized spin labels to approximately the same extent, indicating a similarity in steric restraints around the SH1 thiol of the two myosins. The magnitude of the changes in spin label mobility accompanying binding of MgADP and hydrolysis of MgATP was reduced in cardiac myosin relative to skeletal myosin. This suggests that the lower catalytic center activity of cardiac myosin is associated with more restricted conformational changes accompanying formation of M.ADP and M.ADP.Pi. From measurements of spin label mobility, the affinity of cardiac and skeletal myosin for ADP were similar: Kd (ADP) = 7 microM, n = 1.6. The EPR spectrum of spin labels attached to cardiac and skeletal myosin showed similar saturation effects upon actin binding indicating immobilization of myosin heads occurs with both proteins.