The neuron-specific Rai (ShcC) adaptor protein inhibits apoptosis by coupling Ret to the phosphatidylinositol 3-kinase/Akt signaling pathway

Rai is a recently identified member of the family of Shc-like proteins, which are cytoplasmic signal transducers characterized by the unique PTB-CH1-SH2 modular organization. Rai expression is restricted to neuronal cells and regulates in vivo the number of postmitotic sympathetic neurons. We report here that Rai is not a common substrate of receptor tyrosine kinases under physiological conditions and that among the analyzed receptors (Ret, epidermal growth factor receptor, and TrkA) it is activated specifically by Ret. Overexpression of Rai in neuronal cell lines promoted survival by reducing apoptosis both under conditions of limited availability of the Ret ligand glial cell line-derived neurotrophic factor (GDNF) and in the absence of Ret activation. Overexpressed Rai resulted in the potentiation of the Ret-dependent activation of phosphatidylinositol 3-kinase (PI3K) and Akt. Notably, increased Akt phosphorylation and PI3K activity were also found under basal conditions, e.g., in serum-starved neuronal cells. Phosphorylated and hypophosphorylated Rai proteins form a constitutive complex with the p85 subunit of PI3K: upon Ret triggering, the Rai-PI3K complex is recruited to the tyrosine-phosphorylated Ret receptor through the binding of the Rai PTB domain to tyrosine 1062 of Ret. In neurons treated with low concentrations of GDNF, the prosurvival effect of Rai depends on Rai phosphorylation and Ret activation. In the absence of Ret activation, the prosurvival effect of Rai is, instead, phosphorylation independent. Finally, we showed that overexpression of Rai, at variance with Shc, had no effects on the early peak of mitogen-activated protein kinase (MAPK) activation, whereas it increased its activation at later time points. Phosphorylated Rai, however, was not found in complexes with Grb2. We propose that Rai potentiates the MAPK and PI3K signaling pathways and regulates Ret-dependent and -independent survival signals.     

Stabilization of S-adenosyl-L-methionine promoted by trehalose

S-adenosyl-L-methionine (SAM), an important metabolic intermediate of mammals, is a well-known therapeutic agent. The molecule is chemically unstable, both in solution and in dry state, and forms different degradation products. Because the chemical instability represents a real problem during the preparation of therapeutic formulations, we investigated the capacity of some sugars to improve the SAM stability over time. In the present work, we demonstrated that the disaccharide trehalose exercises a protective effect towards the lyophilized SAM slackening its degradation (65% of SAM was detected after 50 days at 37 degrees C). A parallel study, performed to stabilize the SAM into lyophilized yeast cells enriched in the sulfonium compound, assessed the positive effect of trehalose also in whole cells, but in lesser measure.     

Caspase-1 levels in biological fluids from patients with multiple sclerosis and from patients with other neurological and non-neurological diseases

In multiple sclerosis (MS), pathological white matter damage in the central nervous system is sustained by immune-inflammatory response. Caspase-1 plays a pivotal role in immune-mediated inflammation, as it regulates the cellular export of IL-1beta and IL-18. We carried out a preliminary in vitro study of the kinetics of extracellular caspase-1 release. We then measured caspase-1 levels in paired serum and cerebrospinal fluid (CSF) samples of 75 MS patients, 15 healthy subjects, and patients with other neurological diseases. Paired synovial fluid and serum samples of patients with juvenile idiopathic arthritis, and paired sputum and serum samples of asthma patients were also studied. Mean serum caspase-1 concentrations did not differ between groups. Caspase-1 was detected in the CSF of patients with acute, but not stable, MS [7.5 +/- (SEM) 0.9 pg/ml; test's sensitivity, 56% and specificity, 100%]. Its levels correlated with pleocytosis. The highest mean caspase-1 levels were found in the arthritic synovial fluids (945.5 +/- 126.6 pg/ml, which correlated with erythrocyte sedimentation rate), and in the sputum samples (370.1 +/- 71.0 pg/ml, which correlated with the number of macrophages in the sputum). On condition that caspase-1 is determined in the fluids pertaining to the disease-specific inflammatory sites, its level is a reliable marker of ongoing immune-inflammatory response. The enzyme measurement in CSF can also help define state-trait in MS.     

A new RNase-based immunoconjugate selectively cytotoxic for ErbB2-overexpressing cells

We report a new tumor-directed immunoRNase, a chimeric protein made up of an antibody fragment (single-chain Fv fragment) directed to ErbB2, a cell surface receptor, and a non-toxic, human ribonuclease, which upon cell internalization becomes cytotoxic. The immunoRNase is active as a ribonuclease, specifically binds and selectively kills ErbB2-positive cells. ErbB2 is one of the most specific tumor-associated antigens identified so far, overexpressed on tumor cells of different origin. Its choice as target antigen and that of a non-toxic, human RNase as the killer moiety makes this immunoRNase a new, potentially attractive anticancer agent.     

Quaternary narcotic antagonists' relative ability to prevent antinociception and gastrointestinal transit inhibition in morphine-treated rats as an index of peripheral selectivity

Single doses of naloxone (0.025 to 0.5 mg/kg) or of one of four quaternary narcotic antagonists (i.e. nalorphine allobromide, nalorphine methobromide, naloxone methobromide or naltrexone methobromide, 1 to 60 mg/kg) were given s.c. to rats before morphine, 5 mg/kg i.v. In the absence of antagonists morphine reduced G.I. transit of a charcoal meal to about 15% of drug-free controls and consistently delayed nociceptive reactions (55°C hot plate) in all animals. Doses of antagonists slightly reducing morphine antinociception (centrally effective = A) and restoring G.I. transit to about 50% of drug-free rats (peripherally effective = B) were estimated. The A:B ratio, indicating peripheral selectivity, was at least 8 for any of the quaternary antagonists given 10 min before morphine, but prolonging this interval may have resulted in a lower figure (i.e. less peripheral selectivity) because of reduced A and increased B. This was definitely so for naltrexone methobromide (A:B, > 60 at 10 min, about 1 at 80 min) and was not apparent for nalorphine methobromide according to available data, which for nalorphine allobromide and to a lesser extent for naloxone methobromide showed only an increase in B at intervals longer than 10 min. Both morphine-induced antinociception and inhibition of G.I. transit were reduced by naloxone at the lower doses tested and were fully prevented at the higher. These findings indicate that, unlike naloxone, the investigated quaternary narcotic antagonists are interesting prototype drugs for selective blockade of opiate receptors outside the CNS, although certain critical aspects, possibly biological N-dealkylation to the corresponding tertiary antagonists, condition peripheral selectivity.     

IL-6 inhibits the tolerogenic function of CD8 alpha+ dendritic cells expressing indoleamine 2,3-dioxygenase.

The outcome of dendritic cell (DC) presentation of tumor and/or self peptides, including P815AB (a tumor peptide of murine mastocytoma cells) and NRP-A7 (a synthetic peptide mimotope recognized by diabetogenic T cells), may depend on a balance between the activities of immunogenic (CD8alpha(-)) and tolerogenic (CD8alpha(+)) DC. By virtue of their respective actions on CD8(-) and CD8(+) DC, IL-12 and IFN-gamma have functionally opposing effects on peptide presentation by the CD8(-) DC subset, and IFN-gamma-activated CD8(+) DC mediate tolerogenic effects that prevail over the adjuvant activity of IL-12 on CD8(-) DC. We have previously shown that CD40 ligation abrogates the tolerogenic potential of CD8(+) DC, an effect associated with an impaired capacity of the CD40-modulated and IFN-gamma-treated DC to degrade tryptophan and initiate T cell apoptosis in vitro. We report here that IL-6 may both replace (upon administration of the recombinant cytokine) and mediate (as assessed by the use of neutralizing Abs) the effect of CD40 ligation in ablating the tolerogenic activity of CD8(+) DC. The activity of IL-6 includes down-regulation of IFN-gammaR expression in the CD8(+) DC subset and correlates to a reduced ability of these cells to metabolize tryptophan and initiate T cell apoptosis in vitro.     

Waist Circumference and Cardiovascular Risk Factors in Prepubertal Children

Objective: Intra‐abdominal fat has been identified as being the most clinically relevant type of fat in humans. Therefore, an assessment of body‐fat distribution could possibly identify subjects with the highest risk of adverse lipid profile and hypertension. Few data on the relationship between body‐fat distribution and cardiovascular risk factors are available in children, especially before puberty. Research Methods and Procedures: This cross‐sectional study was undertaken to explore the relationship between anthropometric variables, lipid concentrations, and blood pressure (BP) in a sample of 818 prepubertal children (ages 3 to 11 years) and to assess the clinical relevance of waist circumference in identifying prepubertal children with higher cardiovascular risk. Height, weight, triceps and subscapular skinfolds, waist circumference, and BP were measured. Plasma levels for triacylglycerol, total cholesterol, high‐density lipoprotein (HDL) cholesterol, low‐density lipoprotein cholesterol, apolipoprotein A1 (ApoA1), and apolipoprotein B (ApoB) were determined. Results: Females were fatter than males (5.8 [3.5] vs. 4.8 [3.3] kg of fat mass; p < 0.01). Males had higher HDL cholesterol and ApoA1/ApoB plasma concentrations than females (p < 0.001 and p < 0.01, respectively). Waist circumference had a higher correlation with systolic and diastolic BP (r = 0.40 and 0.29, respectively; p < 0.001) than triceps (r = 0.35 and 0.21, respectively; p < 0.001) and subscapular (r = 0.28 and 0.16, respectively; p < 0.001) skinfolds and relative body weight (0.33 and 0.23, respectively; p < 0.001). Multivariate linear model analysis showed that ApoA1/ApoB, HDL cholesterol, total cholesterol/HDL cholesterol, and systolic as well as diastolic BP were significantly associated with waist circumference and triceps and subscapular skinfolds, independently of age, gender, and body mass index. Discussion: Waist circumference as well as subscapular and triceps skinfolds may be helpful parameters in identifying prepubertal children with an adverse blood‐lipids profile and hypertension. However, waist circumference, which is easy to measure and more easily reproducible than skinfolds, may be considered in clinical practice. Children with a waist circumference greater than the 90th percentile are more likely to have multiple risk factors than children with a waist circumference that is less than or equal to the 90th percentile.     

Reductive nitrosylation of ferric cyanide horse heart myoglobin is limited by cyanide dissociation

Cyanide binds to ferric heme-proteins with a very high affinity, reflecting the very low dissociation rate constant (k_off). Since no techniques are available to estimate k_off, we report herewith a method to determine k_off based on the irreversible reductive nitrosylation reaction to trap ferric myoglobin (Mb(III)). The k_off value for cyanide dissociation from ferric cyanide horse heart myoglobin (Mb(III)-cyanide) was determined at pH 9.2 and 20.0 °C. Mixing Mb(III)-cyanide and NO solutions brings about absorption spectral changes reflecting the disappearance of Mb(III)-cyanide with the concomitant formation of ferrous nitrosylated Mb. Since kinetics of reductive nitrosylation of Mb(III) is much faster than Mb(III)-cyanide dissociation, the k_off value, representing the rate-limiting step, can be directly determined. The k_off value obtained experimentally matches very well to that calculated from values of the second-order rate constant (k_on) and of the dissociation equilibrium constant (K) for cyanide binding to Mb(III) (k_off = k_on × K).