The elusive transporters with a high affinity for glutamate

Removal of glutamate from the synaptic cleft is an essential component of the transmission process at glutamatergic synapses. This requirement is fulfilled by transporters that have a high affinity for glutamate and exhibit a unique coupling to Na+, K+ and OH- ions. Independently, three groups have succeeded in cloning cDNAs encoding high-affinity Na(+)-dependent glutamate transporters. These transporters are structurally distinct from previously characterized neurotransmitter transporters and show sequence identity with prokaryotic glutamate and dicarboxylate transporters. In addition, they exhibit significant differences in their structure, function and tissue distribution. This review compares and contrasts these differences, and incorporates into the existing body of knowledge these new breakthroughs.     

Calmodulin as a versatile tag for antibody fragments

Calmodulin is a highly acidic protein (net charge -24 at pH 8.0 in the absence of calcium) that binds to peptide and organic ligands with high affinity (Ka > 10(9) M-1) in a calcium-dependent manner. We have exploited these properties to develop calmodulin as a versatile tag for antibody fragments. Fusions of calmodulin with single chain Fv fragments (scFv) could be expressed by secretion from bacteria in good yield (5-15 mg/l in shaker flasks), and purified from periplasmic lysates or broth to homogeneity in a single step, either by binding to anion-exchange resin (DEAE-Sephadex), or to an organic ligand of calmodulin (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide-agarose). The antibody fusions could be detected by binding of fluorescently labeled peptide ligands, as illustrated by their use in confocal microscopy, fluorescent activated cell sorting and "band shift" gel electrophoresis. Moreover, the interaction between calmodulin and peptide ligands could provide a means of heterodimerization of proteins, as illustrated by the assembly of an antibody-calmodulin fusion with maltose binding protein tagged with a peptide ligand of calmodulin.     

Dendritic cells transduced with an adenoviral vector encoding a model tumor-associated antigen for tumor vaccination

Evaluation of the potential role of dendritic cells (DCs) as adjuvants for tumor vaccination has focused primarily on techniques that load DCs with peptide tumor antigens. Our aim has been to optimize the induction of antitumor immunity by enhancing the ability of DCs to present tumor-associated antigens endogenously to the afferent lymphatic system in the appropriate major histocompatibility complex (MHC)-restricted context. We have used replication-defective adenovirus vectors (Ads) to transduce DCs with various genes, including tumor antigen genes. We found that 90% of murine bone marrow derived-DCs could be infected with an Ad vector expressing the beta-galactosidase gene and still retain their physiologic and phenotypic characteristics. Furthermore, we demonstrated that transgene expression was detectable in the spleen for at least 3 days following intravenous injection of Ad-transduced DCs. Using a polyoma middle T (PymT) transgenic murine mammary carcinoma model, we have shown that a single injection of 10(5)-4 x 10(6) DCs transduced with an Ad vector expressing PymT provided complete and specific protection against tumor cell challenge in 100% of vaccinated animals. Immunization against the PymT tumor by injection with the PymT expressing Ad vector alone resulted in varying degrees of effectiveness, was highly dependent upon the route of administration, and led to significant hepatic toxicity that was not seen in mice immunized with DC transduced with the Ad vector. Our results suggest that: (i) DCs can be very efficiently modified by ex vivo Ad transduction to express tumor-specific antigens, (ii) such modified DCs appear nontoxic and stimulate a potent antitumor response.     

The high affinity state of inositol 1,4,5-trisphosphate receptor is a functional state

Inositol 1,4,5-trisphosphate (InsP3) is a second messenger responsible for the rapid and discontinuous release of Ca2+ from intracellular stores. In this study, the effects of the sulfhydryl reagent thimerosal were investigated on Ca2+ mobilization and on InsP3 binding. Thimerosal was shown to release Ca2+, in a dose-dependent manner, with an EC50 of 135.8 +/- 5.2 microM, from bovine adrenal cortex microsomes. Thimerosal-induced Ca2+ release was not prevented by heparin (250 micrograms/ml), ruling out a participation of InsP3 receptor in that effect. The slow rate of thimerosal-induced Ca2+ release rather suggested an inhibition of microsomal Ca2+ ATPase. At submaximal concentration, thimerosal (100 microM) was also shown to potentiate the release of Ca2+ induced by InsP3. Dose-response experiments revealed that thimerosal enhanced the apparent affinity of InsP3 by a factor 2.21 +/- 0.28, without modifying the maximal amount of Ca2+ released by InsP3. Thimerosal also enhanced, in a dose-dependent manner, [3H]InsP3 binding to adrenal cortex microsomes (EC50 = 43.3 +/- 7.6 microM). A similar effect was also observed on [3H]InsP3 binding to solubilized receptors, suggesting a direct modification of the receptor protein by thimerosal. The effects of thimerosal on Ca2+ release and [3H]InsP3 binding were abolished in the presence of the reducing agent dithiothreitol (1 mM), suggesting a modification by thimerosal of specific thiol groups on these microsomal proteins. Scatchard analysis revealed that thimerosal (100 microM) increased InsP3 receptor affinity by 1.87 +/- 0.26-fold. Kinetic analysis indicated that this increased affinity was due to an enhancement of InsP3 association rate constant. The concomitant increases of binding affinity and Ca2+ releasing potency suggest that the high affinity state of InsP3 receptor is a functional state.     

Neuroblastoma in an adult with a high serum level of carbohydrate antigen, CA125: report of a case

OBJECTIVE: The clinical characteristics and outcomes of endometrial cancer patients 45 years of age and younger were compared with those of patients older than 45 years of age. METHODS: We performed a cross-sectional study of 301 consecutive endometrial cancer patients referred to our center from 1989 to 1994. Of the 289 patients eligible for study, 40 were 45 years of age or younger (group A) and 249 were older than 45 years of age (group B). RESULTS: The majority of patients in both groups presented with stage I disease. Of the women with stage I disease, patients in group A were more likely than those in group B to have low-grade disease localized to the endometrium (P < .001; relative prevalence 3.39; confidence interval [CI] 1.88, 6.12). However, the distribution of stages I to IV overall was the same for the two groups (P = .269). Although univariate analysis revealed that 11% of the patients in group A and 2% in group B had synchronous ovarian malignancies (P = .007; relative prevalence 5.42; CI 1.39, 21.14), multivariate logistic regression found that nulliparity, not age, was an independent risk factor for synchronous ovarian malignancy (P = .017; relative prevalence 6.15; CI 1.52, 25.61). There were no statistically significant differences by age in the prevalence of high-risk endometrial histology (serous and clear cell carcinoma) or in survival. CONCLUSION: The overall distribution of tumor stage and survival were the same for the younger and older women; this finding contradicts previous reports that suggest that young women with endometrial cancer are at lower risk. Additionally, nulliparity, which occurs with a higher prevalence in younger women who develop endometrial cancer, is associated statistically with the development of synchronous ovarian malignancies.     

Characterization of human anti-high molecular weight-melanoma-associated antigen single-chain Fv fragments isolated from a phage display antibody library

The human high molecular weight-melanoma-associated antigen (HMW-MAA) meets the criteria to be used as an immunogen for immunotherapy of malignant melanoma, because it is expressed by a large percentage of melanoma lesions with limited heterogeneity and has a restricted distribution in normal tissues. The high immunogenicity of the HMW-MAA in BALB/c mice has resulted in the development of a large number of anti-HMW-MAA monoclonal antibodies (mAbs). In contrast, no human anti-HMW-MAA mAbs have been described. Because the latter may serve as useful probes to characterize the antigenic profile of the HMW-MAA, human anti-HMW-MAA single-chain fragments of the variable region (scFvs) were isolated by panning synthetic scFv library 1 on purified HMW-MAA. Colony hybridization studies and nucleotide sequence analysis revealed that scFv 19, 44, 56, and 61 belong to the V(H)3 gene family and use the DP-38 germ-line gene segment but have a diverse third complementarity-determining region. The human scFvs share some characteristics with mouse anti-HMW-MAA mAb but also display some distinct features. Like mouse mAbs, human scFvs recognize determinants of HMW-MAA with a heterogeneous cellular and molecular distribution in human melanoma cells. Furthermore, like some mouse mAbs, human scFvs react with rat neural cells expressing the chondroitin sulfate proteoglycan NG2, which shows 81% homology to the HMW-MAA. However, at variance with mouse mAbs, the human scFvs show poor reactivity with guinea pig melanoma cells. Lastly, human scFv 61 stains both benign and malignant lesions of melanocytic origin, although with a lower frequency than mouse mAbs. Analysis of the clinical significance of the differential expression of the scFv 61-defined determinant in melanoma lesions will be facilitated by its reactivity with formalin-fixed melanoma lesions. In contrast to mouse mAb, scFv 61 immunoprecipitates the >450-kDa chondroitin sulfate proteoglycan component of the HMW-MAA, but not its 250-kDa subunit from melanoma cells. Thus, contrary to the current view about the structure of HMW-MAA, our results demonstrate that the two components are not associated. The described scFv antibodies, which represent the first example of human anti-HMW-MAA antibodies, have provided novel information about the structure of this antigen. Future studies will assess the impact of these in vitro-assembled antibody fragments on the identification of antigenic determinants of the HMW-MAA that can be recognized by the human immune system.     

Expression of carbohydrate antigen sialyl Le(a): a new functional prognostic factor in gastric cancer

PURPOSE: The prognostic value of the altered expression of carbohydrate antigens sialyl Le(a) (sLe(a)) and sialyl Le(x) (sLe(x)), which have been implicated as functional ligands in heterotypic-cell-adhesion systems in the multistep process of tumor metastasis, were evaluated. PATIENTS AND METHODS: The level of expression of sLe(a) and sLe(x) antigens was examined immunohistochemically in paraffin-embedded tumor samples from 137 patients who underwent resection for gastric cancer. Correlation between the antigens' expression, various established clinicopathologic factors, and prognosis were studied by univariate and multivariate analysis. RESULTS: Tumors that were positive for the sLe(a) antigen were significantly more likely to be large (P = .035), to be localized at the proximal third of the stomach (P = .018), to have an infiltrate appearance (P = .013), to have an invasive mode both in depth of invasion (P = .028) and in lymphatic invasion (P = .002), and to be classified as late stage (P = .011) than those that were negative for sLe(a), whereas the sLe(x) antigen status was not correlated with any clinicopathologic factors. The overall survival of patients with an sLe(a)-antigen-positive tumor was significantly poorer than that of those with an sLe(a)-antigen-negative tumor (P = .0001). Survival within each pathologic stage differed also (stage I, P = .030; stage II, P = .046; stage III, P = .026, respectively). A Cox regression analysis with multiple covariates showed that positive sLe(a) antigen status was an independent prognostic factor for a worse outcome in patients with gastric cancer. According to the mode of recurrence, increased sLe(a) antigen expression significantly affected both peritoneal dissemination and liver metastasis. CONCLUSION: Increased expression of the sLe(a) antigen may serve as a potent prognostic indicator for recurrence in patients with gastric cancer. Careful follow-up and intensive therapy are required for patients with an sLe(a)-antigen-positive gastric cancer.     

Three-dimensional structure of a human Fab with high affinity for tetanus toxoid

BACKGROUND: The wide range of antibody specificity and affinity results from the differing shapes and chemical compositions of their binding sites. These shapes range from discrete grooves in antibodies elicited by linear oligomers of nucleotides and carbohydrates to shallow depressions or flat surfaces for accommodation of proteins, peptides and large organic compounds. OBJECTIVES: To determine the Fab structure of a high-affinity human antitoxin antibody. To explore structural features which enable the antibody to bind to intact tetanus toxoid, peptides derived from the sequence of the natural immunogen and antigenic mimics identified by combinatorial chemistry. To explain why this Fab shows a remarkable tendency to produce crystals consistently diffracting to d spacings of 1.7-1.8 A. To use this information to engineer a strong tendency to crystallize into the design of other Fabs. STUDY DESIGN: The protein was crystallized in hanging or sitting drops by a microseeding technique in polyethylene glycol (PEG) 8000. Crystals were subjected to X-ray analysis and the three-dimensional structure of the Fab was determined by the molecular replacement method. Interactive computer graphics were employed to fit models to electron density maps, survey the structure in multiple views and discover the crystal packing motif of the protein. RESULTS: Exceptionally large single crystals of this protein have been obtained, one measuring 5 x 3 x 2 mm (l x w x d). The latter was cut into six irregular pieces, each retaining the features of the original in diffracting to high resolution (1.8 A) with little decay in the X-ray beam. In an individual Fab, the active site is relatively flat and it seems likely that the protein antigen and derivative peptides are tightly held on the outer surface without significant penetration into the interior. There is no free space to accommodate even a dipeptide between VH and VL. One of the unique features of the B7-15A2 Fab is a large aliphatic ridge dominating the center of the active site. The CDR3 of the H chain contributes significantly to this ridge, as well as to adjoining regions projected to be important for the docking of the antigen. Both the ease of crystallization and the favorable diffraction properties are mainly attributable to the tight packing of the protein molecules in the crystal lattice. DISCUSSION: The B7-15A2 active site provides a stable and well defined platform for high affinity docking of proteins, peptides and their mimotopes. The advantages for future developments are suggested by the analysis of the crystal properties. It should be possible to incorporate the features promoting crystallization, close packing and resistance to radiation damage into engineered human antibodies without altering the desired specificities and affinities of their active sites.     

Increased binding affinity and valence of recombinant antibody fragments lead to improved targeting of tumoral angiogenesis

The formation of new blood vessels (angiogenesis) is an important step in tumor progression. Molecules capable of selectively targeting markers of angiogenesis may offer opportunities for the in vivo imaging of aggressive tumors and for the delivery of toxic agents to the tumoral vasculature. Using antibody phage display libraries and combinatorial mutagenesis, we isolated single-chain Fv antibody fragments, which recognize with different affinities the same epitope of the ED-B domain of fibronectin, a marker of angiogenesis. Two single-chain Fv fragments, E1 and L19, with dissociation constants of 41 nM and 0.054 nM, respectively, were investigated for their ability to target F9 murine teratocarcinoma grafted s.c. in nude mice when injected i.v. in either monomeric or homodimeric form (Mr 27,000 and 54,000, respectively). Biodistribution studies, performed at two time points (4 h and 24 h) with radiolabeled samples, showed that the higher affinity antibody targets the tumor significantly better than the lower affinity one, in terms both of tumor:organ ratios and of the amounts of antibody delivered to the tumor. In particular, more than 20% of the injected dose of dimeric L19 accumulated per gram of tumor at 4 h; the tumor:organ ratios at 4 h and 24 h were in the (2.1-8.6):1 and (10.3-29.4):1 range, respectively. This study demonstrates that, although vasculature represents only a small fraction of the total tumor mass, anti-ED-B antibodies can selectively target tumors in vivo and that this process is particularly efficient if very high-affinity binders are used.     

Functional characterization of the somatic hypermutation process leading to antibody D1.3, a high affinity antibody directed against lysozyme

The impact of somatic hypermutation on the affinity of Abs directed against protein Ags remains poorly understood. We chose as a model the secondary response Ab D1.3 directed against hen egg lysozyme. During the maturation process leading to this Ab, five replacement somatic mutations occurred. After reconstituting the germline Ab from which D1.3 originated, we assessed the energetic and kinetic importance of each of the somatic mutations, individually or combined, using the BIAcore apparatus. We found that the mutations induced an overall 60-fold improvement of affinity, principally due to a decrease in the kinetic rate of dissociation. We showed that their effects were additive and context independent; therefore, in the case of D1.3, the order in which somatic mutations were introduced and selected is unimportant. Interestingly, most of the affinity improvement was due to a single somatic mutation (Asn50-->Tyr in VL), involving a residue that belongs to the functional interface between Ab D1.3 and lysozyme. This replacement could either establish new Van der Waals contacts between the Ab and the Ag or help stabilize the conformation of a closely situated crucial residue of the Ab paratope. The four other mutations played only a marginal part in affinity maturation; potential reasons for which these mutations were nevertheless selected are discussed.     

Antitumor effect of diphtheria toxin A-chain gene-containing cationic liposomes conjugated with monoclonal antibody directed to tumor-associated antigen of bovine leukemia cells

Monoclonal antibody c143 against tumor-associated antigen (TAA) expressed on bovine leukemia cells was conjugated to cationic liposomes carrying a plasmid pLTR-DT which contained a gene for diphtheria toxin A-chain (DT-A) under the control of the long terminal repeat (LTR) of bovine leukemia virus (BLV) in the multicloning site of pUC-18. The specificity and antitumor effects of the conjugates were examined in vitro and in vivo using TAA-positive bovine B-cell lymphoma line as the target tumor. In vitro studies with the TAA-positive cell line indicated that luciferase gene-containing cationic liposomes associated with the c143 anti-TAA monoclonal antibody caused about 2-fold increase in luciferase activity compared with cationic liposomes having no antibody, and also that the c143-conjugated cationic liposomes containing pLTR-DT exerted selective growth-inhibitory effects on the TAA-positive B-cell line. Three injections of pLTR-DT-containing cationic liposomes coupled with c143 into tumor-bearing nude mice resulted in significant inhibition of the tumor growth. The antitumor potency of the c143-conjugated cationic liposomes containing pLTR-DT was far greater than that of normal mouse IgG-coupled cationic liposomes containing pLTR-DT as assessed in terms of tumor size. These results suggest that cationic liposomes bearing c143 are an efficient transfection reagent for BLV-infected B-cells lymphoma cells, and that the delivery of the pLTR-DT gene into BLV-infected B-cells by the use of such liposomes may become a useful technique for gene therapy of bovine leukosis.     

An N-substituted polyurea coating with high affinity for heparin

A new N-substituted polyurea with tertiary amino groups in the polycarbamidic chain (NPUTA) has been synthesized. The polymer is soluble in C1-C4 alcohols, has high adhesion to polar molds, and has high H2O uptake (130-150%). The material can be coated on many biomaterials (polyurethanes, charcoal hemosorbents, cellulosic hemodialysis membranes), and high amounts of heparin can be adsorbed onto treated surfaces. NPUTA cast from 0.5-3.5% ethanol solutions can absorb large amounts of heparin from anti-coagulant solution (40-60 micrograms/cm2) and heparinized plasma. Heparin release into phosphate buffered saline (PBS) solution or plasma is minimal. The influence of NPUTA solution concentration and pre-absorbed heparin on the protein adsorption, platelet adhesion, surface induced hemolysis, and complement activation of these films has been investigated. Radiolabeled protein assays, radiolabeled platelet assays, and other methods were used. It was shown that modified surfaces for the listed materials, with heparinization, demonstrate improved in vitro blood compatibility without any changes in functional properties. For example, treatment with NPUTA/heparin does not reduce sorption of middle molecules by activated charcoal hemosorbent, while markedly and significantly decreasing platelet adhesion and complement activation. NPUTA/heparin modified, glutaraldehyde treated bovine pericardium exhibited significantly reduced calcification in a rat subcutaneous implant model. Other ex vivo circulation experiments also confirm the blood compatibility of different NPUTA treated surfaces.     

The impact of antigen density and antibody affinity on antibody-dependent cellular cytotoxicity: relevance for immunotherapy of carcinomas

We studied the sensitivity of human melanoma (Bro strain) xenografts to drugs of the nitrosoalkylurea (NAU) class: nitrosomethylurea (NMM), karmustin (BCNU), nimustin (ACNU), nitrulin, and ADEKO. High antitumor activity of NAM was shown when the drugs were applied not only at the early, but also at the late stages of tumor progression (tumor mass 400 and 1200 mg, respectively). The therapeutic effect of the drugs was estimated with the use of criteria characterizing the kinetics of tumor regression, increased life span, and survival of treated animals. After early administration of the drugs (Day 4 after tumor transplantation), 67% and 50% of animals survive under the influence of nitrulin and ACNU, respectively, while the rate of tumor regression increased in the sequence nitrulin < karmustin < NMM < ACNU. After late administration (11 days after tumor transplantation), NMM was most effective at increasing survival (35% of survived animals by 35 days of observation), while the rate of tumor regression increased in the sequence ADEKO < NMM < karmustin < nitrulin < ACNU.     

IgE antibody up-regulates high affinity IgE binding on murine bone marrow-derived mast cells

We have examined 3-week-old alcian blue positive cells (putatively mast cells) derived from mouse bone marrow for their expression of Fc epsilon RI. Using an indirect method of sensitizing the cells with immunoglobulin E (IgE) antibody (anti-DNP IgE) and detecting the level of bound IgE antibody by flow cytometry, we found that prolonged culture (1-5 days) with IgE, but not IgG, increased the total receptor density 6 +/- 1.9 fold. During the same period, histamine release in response to antigen (DNP-HSA) increased approximately 6-fold while the cell's response to either thrombin or ionomycin remained constant. The greatest up-regulation occurred in the first 2 days of culture. Using 2.4G2 to detect Fc epsilon RII RIII, we could not detect any up-regulation of this receptor. Culturing the cells for 1 h after sensitization did not result in any loss of cell surface IgE, suggesting a reasonably high affinity binding similar to that expected for Fc epsilon RI. This up-regulation was completely inhibited by co-culture with 2 micrograms/ml cycloheximide. These data suggest that IgE is capable of inducing a significant, protein synthesis, dependent up-regulation of its own high affinity receptor on mast cells/basophils.     

A humanized antibody with specificity for hepatitis B surface antigen

A murine monoclonal antibody H67 was characterized for the binding specificity, which showed that H67 recognizes a disulfide-bond-dependent conformational epitope of common a antigenic determinant on the hepatitis B surface antigen. The result suggested that this antibody may have the potential of replacing hepatitis B immune globulin in the prevention of hepatitis B virus (HBV) infection. Therefore, we have constructed the humanized antibody HuS10 by grafting the complementarity determining regions and some framework amino acid residues of H67 onto the most homologous human antibody variable regions, 21/28 for heavy chain variable region and B1 and J kappa 2 for light chain variable region, followed by combining with human constant regions C gamma 1 and C kappa. The affinity of the HuS10 was the same as that of the H67, 8 x 10(8) x 10(8)M-1, and the HuS10 neutralized the in vitro infection of adult human hepatocyte primary culture by adr or ayw subtype of HBV. The neutralization assay showed that the HuS10 had approximately 2,000-times higher specific activity than commercially available polyclonal HBIG. These results suggest that the humanized antibody will be useful in the prevention or treatment of HBV infection.     

Aminopyrimidines with high affinity for both serotonin and dopamine receptors

A series of [4-[2(4-arylpiperazin-1-yl)alkyl]cyclohexyl]pyrimidin-2-ylamine s was prepared and found to have receptor binding affinity for D2 and D3 dopamine (DA) receptors and serotonin 5-HT1A receptors. The structural contributions to D2/D3 and 5-HT1A receptor binding of the aminopyrimidine, cycloalkyl, and phenylpiperazine portions of the molecule were examined. From these studies compounds 14, 39, 42, 43, having potent affinity for both DA D2 and 5-HT1A receptors, were evaluated for intrinsic activity at these receptors, in vitro and in vivo. Compound 14 (PD 158771) had a profile indicative of partial agonist activity at both D2 and 5-HT1A receptors causing partially decreased synthesis of the neurotransmitters DA and 5-HT and their metabolites. This compound has a profile in behavioral tests that is predictive of antipsychotic activity, suggesting that mixed partial agonists such as 14 may have utility as antipsychotic agents with increased efficacy and decreased side effects.     

Recurrent digoxin overdose and treatment with digoxin-specific Fab antibody fragments

A case of overdose with digoxin on three separate occasions is presented. The patient received digoxin-specific Fab antibody fragments on each presentation without adverse effects. A discussion of digoxin toxicity and specific treatment follows. Immunologic aspects of therapy are emphasized.     

Weight-loss with low or high carbohydrate diet?

OBJECTIVE: With obesity being recognized as an important cardiovascular risk factor, it is important to determine the optimal hypocaloric diet for decreasing that risk. The goal of this study was to compare the effects of two hypocaloric diets of similar caloric value, but differing in carbohydrate content (25% and 45%). SUBJECTS: Sixty-eight out-patients were followed for 12 w. DESIGN: The patients were assigned to one of two groups that received either a low (25% CHO, n = 31) or a high (45% CHO, n = 37) carbohydrate hypocaloric diet (5.0 MJ/d, 1200 Kcal/d). RESULTS: After 12 w, the mean weight loss was similar and did not differ significantly between the two groups: 10.2 +/- 0.7 kg (25% CHO) and 8.6 +/- 0.8 kg (45% CHO). Furthermore, loss of adipose tissue was similar, 8.1 +/- 0.5 kg (25% CHO) and 7.1 +/- 0.7 kg (45% CHO). Despite a high protein intake (1.4 g/kg/ideal body weight) there was loss of lean body mass: 2.2 +/- 0.4 kg (25% CHO) and 1.4 +/- 0.3 kg (45% CHO). The waist/hip ratio diminished significantly (P < 0.001) and identically in both groups. The fasting blood glucose (even though normal, along with cholesterol and triglyceride concentrations, were significantly decreased after weight loss. The fasting blood insulin which was mildly elevated before weight loss decreased more markedly with the 25% CHO diet compared to the 45% CHO diet (P < 0.003). The glucose/insulin ratio improved significantly (P < 0.05) after weight loss with both diets (0.17 +/- 0.04 mmol/mU (25% CHO) vs 0.10 +/- 0.03 mmol/mU (45% CHO). CONCLUSIONS: Neither diet offered a significant advantage when comparing weight loss or other, metabolic parameters over a 12 w period. However, considering the greater improvement of fasting blood insulin, the glucose/insulin ratio and blood triglyceride, the low carbohydrate diet (25%) could be more favourable in the long-term. The improvement of fasting blood insulin could be explained by the differences in monounsaturated fat composition in the low carbohydrate diet.     

Azepinoindole derivatives with high affinity for brain dopamine and serotonin receptors

We synthesized 20 and 21 as conformationally constrained analogues of the dopamine receptor antagonist SKF-83742, as well as analogues 6-9, 16, and 18-22. Although 20 and 21 were inactive, 7, 9, and 19 showed strong binding to D-1, D-2, S-2, and alpha-1 receptors, as well as antipsychotic activity in vivo.