Hepatocyte growth factor-induced ectodomain shedding of cell adhesion molecule L1: role of the L1 cytoplasmic domain

The L1 cell adhesion molecule and its soluble form are tumor-associated proteins and potential markers for tumor staging as well as targets for therapeutic intervention. Soluble L1 is produced by metalloprotease-mediated ectodomain shedding of L1. We investigated effects of hepatocyte growth factor (HGF), a growth factor shown to increase invasiveness of renal carcinoma cells, on ectodomain shedding of L1 from these cells. All of the tested L1-positive renal carcinoma cell lines released a 180-kDa form of L1 into the medium. In the presence of serum, addition of HGF led to a dose-dependent increase in L1 shedding with a maximum reached at 5 ng/ml. In contrast, L1 shedding was inhibited by glial cell line-derived neurotrophic factor (GDNF). The tyrosine kinase inhibitor Genistein reduced basal and HGF-stimulated L1 shedding, indicating that protein phosphorylation is involved. To investigate the role of the L1 intracellular domain, two mutants of the L1 cytoplasmic part were constructed. L1trun lacking the complete intracellular domain showed enhanced basal shedding. In a L1YH mutant, containing the mutation tyrosine 1229 to histidine that deletes the ankyrin binding motif of L1, basal shedding was reduced. Disruption of actin assembly by cytochalasin D also reduced shedding of L1. These results indicate that the cytoplasmic domain regulates basal shedding of L1, and association with the cytoskeleton through the L1 ankyrin binding site is involved. HGF stimulated L1 shedding in both mutants, indicating that receptor-mediated phosphorylation in the L1 cytoplasmic domain is not required for HGF-stimulated shedding.     

Mutations of the ephrin-B1 gene cause craniofrontonasal syndrome

Craniofrontonasal syndrome (CFNS) is an X-linked craniofacial disorder with an unusual manifestation pattern, in which affected females show multiple skeletal malformations, whereas the genetic defect causes no or only mild abnormalities in male carriers. Recently, we have mapped a gene for CFNS in the pericentromeric region of the X chromosome that contains the EFNB1 gene, which encodes the ephrin-B1 ligand for Eph receptors. Since Efnb1 mutant mice display a spectrum of malformations and an unusual inheritance reminiscent of CFNS, we analyzed the EFNB1 gene in three families with CFNS. In one family, a deletion of exons 2-5 was identified in an obligate carrier male, his mildly affected brother, and in the affected females. In the two other families, missense mutations in EFNB1 were detected that lead to amino acid exchanges P54L and T111I. Both mutations are located in multimerization and receptor-interaction motifs found within the ephrin-B1 extracellular domain. In all cases, mutations were found consistently in obligate male carriers, clinically affected males, and affected heterozygous females. We conclude that mutations in EFNB1 cause CFNS.     

In vivo modulation of extracellular hippocampal glutamate and GABA levels and limbic seizures by group I and II metabotropic glutamate receptor ligands

The effects of several metabotropic receptor (mGluR) ligands on baseline hippocampal glutamate and GABA overflow in conscious rats and the modulation of limbic seizure activity by these ligands were investigated. Intrahippocampal mGluR group I agonist perfusion via a microdialysis probe [1 mm (R,S)-3,5-dihydroxyphenylglycine] induced seizures and concomitant augmentations in amino acid dialysate levels. The mGlu1a receptor antagonist LY367385 (1 mm) decreased baseline glutamate but not GABA concentrations, suggesting that mGlu1a receptors, which regulate hippocampal glutamate levels, are tonically activated by endogenous glutamate. This decrease in glutamate may contribute to the reported LY367385-mediated anticonvulsant effect. The mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl)-pyridine (50 mg/kg) also clearly abolished pilocarpine-induced seizures. Agonist-mediated actions at mGlu2/3 receptors by LY379268 (100 microm, 10 mg/kg intraperitoneally) decreased basal hippocampal GABA but not glutamate levels. This may partly explain the increased excitation following systemic LY379268 administration and the lack of complete anticonvulsant protection within our epilepsy model with the mGlu2/3 receptor agonist. Group II selective mGluR receptor blockade with LY341495 (1-10 microm) did not alter the rats' behaviour or hippocampal amino acid levels. These data provide a neurochemical basis for the full anticonvulsant effects of mGlu1a and mGlu5 antagonists and the partial effects observed with mGlu2/3 agonists in vivo.     

Trichlorfon predisposes to aneuploidy and interferes with spindle formation in in vitro maturing mouse oocytes

The pesticide trichlorfon (TCF) has been implicated in human trisomy 21, and in errors in chromosome segregation at male meiosis II in the mouse. We previously provided evidence that TCF interferes with spindle integrity and cell-cycle control during murine oogenesis. To assess the aneugenic activity of TCF in oogenesis, we presently analysed maturation, spindle assembly, and chromosome constitution in mouse oocytes maturing in vitro in the presence of 50 or 100 microg/ml TCF for 16 h or in pulse-chase experiments. TCF stimulated maturation to meiosis II at 50 microg/ml, but arrested meiosis in some oocytes at 100 microg/ml. TCF at 100 microg/ml was aneugenic causing non-disjunction of homologous chromosomes at meiosis I, a significant increase of the hyperploidy rate at metaphase II, and a significant rise in the numbers of oocytes that contained a 'diploid' set of metaphase II chromosomes (dyads). TCF elevated the rate of precocious chromatid segregation (predivision) at 50 and 100 microg/ml. Pulse-chase experiments with 100 microg/ml TCF present during the first 7 h or the last 9 h of maturation in vitro did not affect meiotic progression and induced intermediate levels of hyperploidy at metaphase II. Exposure to > or =50 microg/ml TCF throughout maturation in vitro induced severe spindle aberrations at metaphase II, and over one-third of the oocytes failed to align all chromosomes at the spindle equator (congression failure). These observations suggest that exposure to high concentrations of TCF induces non-disjunction at meiosis I of oogenesis, while lower doses may preferentially cause errors in chromosome segregation at meiosis II due to disturbances in spindle function, and chromosome congression as well as precocious separation of chromatids prior to anaphase II. The data support evidence from other studies that TCF has to be regarded as a germ cell aneugen.     

Macromolecular immunosuppressants

Recombinant immunosuppressants have come of age and represent a significant class of quite diverse drugs. They target extracellular molecules, and either label or inhibit them. Those targets are soluble factors or membrane proteins almost all of which are components of a very complex molecular network of communication and amplification. Notably, many recombinant immunosuppressants have been developed in a rather short period of time. This is due to the fact that developing the actual drug is left to nature, so to speak; either it is a human protein as is the case for Anakinra or it is an antibody or antibody derivative "developed" by the immune system of mice or rats following exposure to the antigen. The challenge for developing recombinant immunosuppressants is to identify relevant targets. It is no longer very difficult to generate proteins to targets, once those are identified. The clinical use of recombinant immunosuppressants has yet to show which targets are truly relevant and which drugs prove effective.     

Body mass effects of a physical activity and healthy food intervention in middle schools

Objective: To evaluate the effects of a 2‐year middle school physical activity and healthy food intervention, including an environmental and computer‐tailored component on BMI and BMI z‐score in boys and girls. Research Methods and Procedures: A random sample of 15 schools with seventh and eighth graders was randomly assigned to three conditions: an intervention with parental support group, an intervention‐alone group, and a control group. Weight and height were measured at the beginning and end of each school year to assess BMI and BMI z‐score. A physical activity and healthy food program was implemented over 2 school years. Results: In girls, BMI and BMI z‐score increased significantly less in the intervention with parental support group compared with the control group (p < 0.05) or the intervention‐alone group (p = 0.05). In boys, no significant positive intervention effects were found. Discussion: This was the first study evaluating the effectiveness of an intervention combining environmental changes with personal computer‐tailored feedback on BMI and BMI z‐score in middle school children. After 2 school years, BMI and BMI z‐score changed in a more positive direction in girls as a result of the intervention with parental support.     

Fasting-based estimates of insulin sensitivity in overweight and obesity: a critical appraisal

Objective: To identify simple methods to estimate the degree of insulin resistance. Research Methods and Procedures: The performance of a wide range of fasting‐based index estimates of insulin sensitivity was compared by receiver operating characteristic analysis (area under curves and their 95% confidence intervals) against the M value from euglycemic insulin clamp studies collected in the San Antonio (non‐Hispanic whites and Hispanic residents of San Antonio, TX) and European Group for the Study of Insulin Resistance (non‐diabetic white Europeans) databases (n = 638). Results: Insulin resistance differed substantially between lean (BMI < 25 kg/m²), overweight or obese (BMI ≥ 25 kg/m²), and type 2 diabetic individuals. Estimates of insulin resistance were, therefore, assessed in each group separately. In the overweight and obese subgroup (n = 302), the receiver operating characteristic performance of fasting‐based indices varied from 0.72 (0.62 to 0.82), in the case of the insulin/glucose ratio, to 0.80 (0.72 to 0.88) in the case of Belfiore free fatty acids. One superior method could not be identified; the confidence intervals overlapped, and no statistically significant differences emerged. All indices performed better when using the whole study population, with fasting plasma insulin, homeostatic model assessment, insulin/glucose ratio, quantitative insulin sensitivity check index, glucose/insulin ratio, Belfiore glycemia, revised quantitative insulin sensitivity check index, McAuley index, and Belfiore free fatty acids showing area under curves of 0.83, 0.90, 0.66, 0.90, 0.66, 0.90, 0.85, 0.83, and 0.86, respectively, because of the inclusion of very insulin sensitive (lean) and very insulin resistant cases (diabetic subjects). Discussion: In conclusion, a superior fasting‐based index estimate to distinguish between the presence and absence of insulin resistance in overweight and obesity could not be identified despite the use of the large datasets.     

Apocrine cysts of the breast: biomarkers, origin, enlargement, and relation with cancer phenotype

Up to one-third of women aged 30-50 years have cysts in their breasts and are presumed to be at increased risk of developing breast cancer. Here we present an extensive proteomic and immunohistochemistry (IHC) study of breast apocrine cystic lesions aimed at generating specific biomarkers and elucidating the relationship, if existent, of apocrine cysts with cancer phenotype. To this end we compared the expression profiles of apocrine macrocysts obtained from mastectomies from high risk cancer patients with those of cancerous and non-malignant mammary tissue biopsies collected from the same patients. We identified two biomarkers, 15-hydroxyprostaglandin dehydrogenase and 3-hydroxymethylglutaryl-CoA reductase, that were expressed specifically by apocrine type I cysts as well as by apocrine metaplastic cells in type II microcysts, terminal ducts, and intraductal papillary lesions. No expression of these markers was observed in non-malignant terminal ductal lobular units, type II flat cysts, stroma cells, or fat tissue as judged by IHC analysis of matched non-malignant tissue samples collected from 93 high risk patients enrolled in our cancer program. IHC analysis of the corresponding 93 primary tumors indicated that most apocrine changes have little intrinsic malignant potential, although some may progress to invasive apocrine cancer. None of the apocrine lesions examined, however, seemed to be a precursor of invasive ductal carcinomas, which accounted for 81% of the tumors analyzed. Our studies also provided some insight into the origin, development, and enlargement of apocrine cysts in mammary tissue. The successful identification of differentially expressed proteins that characterize specific steps in the progression from early benign lesions to apocrine cancer opens a window of opportunity for designing and testing new approaches for pharmacological intervention, not only in a therapeutic setting but also for chemoprevention, to inhibit cyst development as both 15-hydroxyprostaglandin dehydrogenase and 3-hydroxymethylglutaryl-CoA reductase are currently being targeted for chemoprevention strategies in various malignancies.     

Pharmacological and neurochemical characterization of the involvement of hippocampal adrenoreceptor subtypes in the modulation of acute limbic seizures

Noradrenaline exerts inhibitory effects on seizure susceptibility. Subtype selective agonists and antagonists were used to identify the anticonvulsant hippocampal adrenoreceptors. Intrahippocampal dialysis was used for administration of all compounds, including pilocarpine for limbic seizure induction, and as the neurotransmitter sampling tool. The noradrenaline reuptake inhibitor maprotiline mediated anticonvulsant effects, associated with dose-dependent increases in extracellular hippocampal noradrenaline, dopamine and GABA levels. At high concentrations, maprotiline produced proconvulsant effects associated with high levels of noradrenaline, dopamine and glutamate. Maprotiline's anticonvulsant effect was blocked by administration of either a selective α(2) - and β(2) -antagonist. α(2) -Antagonist administration with maprotiline was associated with a further increase in noradrenaline and dopamine from maprotiline alone; whereas β(2) -antagonist administered with maprotiline inhibited the dopamine increases produced by maprotiline. α(1A) -Antagonism blocked the GABA-ergic but not the anticonvulsive effect of maprotiline. These results were confirmed as combined but not separate α(2) - and β(2) -adrenoreceptor stimulation, using selective agonists, inhibited limbic seizures. Interestingly, α(1A) -receptor stimulation and α(1D) -antagonism alone also inhibited seizures associated with respectively significant hippocampal GABA increases and glutamate decreases. The main findings of this study are that (i) increased hippocampal noradrenergic neurotransmission inhibits limbic seizures via combined α(2) - and β(2) -receptor activation and (ii) α(1A) - and α(1D) -adrenoreceptors mediate opposite effects on hippocampal excitability.