Functional Proteomics Screen Enables Enrichment of Distinct Cell Types from Human Pancreatic Islets

The current world-wide epidemic of diabetes has prompted attempts to generate new sources of insulin-producing cells for cell replacement therapy. An inherent challenge in many of these strategies is the lack of cell-surface markers permitting isolation and characterization of specific cell types from differentiating stem cell populations. Here we introduce an iterative proteomics procedure allowing tag-free isolation of cell types based on their function. Our method detects and associates specific cell-surface markers with particular cell functionality by coupling cell capture on antibody arrays with immunofluorescent labeling. Using this approach in an iterative manner, we discovered marker combinations capable of enriching for discrete pancreatic cell subtypes from human islets of Langerhans: insulin-producing beta cells (CD9^high/CD56⁺), glucagon-producing alpha cells (CD9^- /CD56⁺) and trypsin-producing acinar cells (CD9^- /CD56^-). This strategy may assist future beta cell research and the development of diagnostic tools for diabetes. It can also be applied more generally for function-based purification of desired cell types from other limited and heterogeneous biological samples.     

The type I membrane protein EFF-1 is essential for developmental cell fusion

Multinucleate cells are widespread in nature, yet the mechanism by which cells fuse their plasma membranes is poorly understood. To identify animal fusogens, we performed new screens for mutations that abolish cell fusion within tissues of C. elegans throughout development. We identified the gene eff-1, which is expressed as cells acquire fusion competence and encodes a novel integral membrane protein. EFF-1 sequence motifs suggest physicochemical actions that could cause adjacent bilayers to fuse. Mutations in the extracellular domain of EFF-1 completely block epithelial cell membrane fusion without affecting other perfusion events such as cell generation, patterning, differentiation, and adhesion. Thus, EFF-1 is a key component in the mechanism of cell fusion, a process essential to normal animal development.     

New horizons in schizophrenia treatment: autophagy protection is coupled with behavioral improvements in a mouse model of schizophrenia

Autophagy plays a key role in the pathophysiology of schizophrenia as manifested by a 40% decrease in BECN1/Beclin 1 mRNA in postmortem hippocampal tissues relative to controls. This decrease was coupled with the deregulation of the essential ADNP (activity-dependent neuroprotector homeobox), a binding partner of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β) another major constituent of autophagy. The drug candidate NAP (davunetide), a peptide fragment from ADNP, enhanced the ADNP-LC3B interaction. Parallel genetic studies have linked allelic variation in the gene encoding MAP6/STOP (microtubule-associated protein 6) to schizophrenia, along with altered MAP6/STOP protein expression in the schizophrenic brain and schizophrenic-like behaviors in Map6-deficient mice. In this study, for the first time, we reveal significant decreases in hippocampal Becn1 mRNA and reversal by NAP but not by the antipsychotic clozapine (CLZ) in Map6-deficient (Map6^+/−) mice. Normalization of Becn1 expression by NAP was coupled with behavioral protection against hyperlocomotion and cognitive deficits measured in the object recognition test. CLZ reduced hyperlocomotion below control levels and did not significantly affect object recognition. The combination of CLZ and NAP resulted in normalized outcome behaviors. Phase II clinical studies have shown NAP-dependent augmentation of functional activities of daily living coupled with brain protection. The current studies provide a new mechanistic pathway and a novel avenue for drug development.     

Size at birth,maternal nutritional status in pregnancy,and blood pressure at age 17: population based analysis.

OBJECTIVE: To assess the effect of size at birth, maternal nutrition, and body mass index on blood pressure in late adolescence. DESIGN: Population based analysis of birth weight corrected for gestational age, mother''s weight before pregnancy and weight gain in pregnancy, obtained from the Jerusalem perinatal study, and blood pressure and body mass index at age 17, available from military draft records. SETTING: Jerusalem, Israel. SUBJECTS: 10,883 subjects (6684 men and 4199 women) born in Jerusalem during 1974-6 and subsequently drafted to the army. MAIN OUTCOME MEASURES: Systolic and diastolic blood pressures measured at age 17 and their correlation with birth weight, size at birth, mother''s body mass index and weight gain during pregnancy, and height and weight at age 17. RESULTS: Systolic and diastolic blood pressures were significantly and positively correlated with body weight, height, body mass index at age 17, and with mother''s body weight and body mass index before pregnancy, but not with birth weight or mother''s weight gain in pregnancy. CONCLUSION: Variables reflecting poor intrauterine nutrition, including low maternal body mass index before pregnancy, poor maternal weight gain in pregnancy, and being born small for gestational age, were not associated with a higher blood pressure in late adolescence.     

Insulin-like growth factor I receptors in neuronal and glial cells. Characterization and biological effects in primary culture

Primary cultures of neuronal and glial cells from 1-day-old neonatal rats contain high affinity receptors for insulin-like growth factor I (IGF-I). The IC50 for displacement of 125I-IGF-I binding by unlabeled IGF-I was 3 nM for neuronal cells and 4 nM for glial cells. Unlabeled insulin was 20-50 times less potent. Apparent molecular mass of the alpha subunits of the IGF-I receptor was 125 kDa in neuronal and 135 kDa in glial cells. IGF-I induced autophosphorylation of the IGF-I receptor beta subunit in lectin-purified membrane preparations in a dose-dependent manner. The major phosphoamino acid of the beta subunit in both cell types was tyrosine in the IGF-I-stimulated state and serine in the basal state. Apparent molecular mass of the beta subunits of the IGF-I receptors was 91 kDa for neuronal and 95 kDa for glial cells. Tyrosine kinase activity of the IGF-I receptors was demonstrated by IGF-I-induced phosphorylation of the exogenous substrate poly(Glu, Tyr) 4:1 in both cell types. IGF-I had no effect on 2-deoxyglucose uptake in neuronal cells. In contrast, in glial cells, IGF-I stimulated 2-deoxyglucose uptake at very high doses, presumably acting via the insulin receptor. The effect of IGF-I as a neurotrophic growth factor in both neuronal and glial cells was demonstrated by its stimulation of [3H]thymidine incorporation. These findings suggest the IGF-I is an important growth factor in nervous tissue-derived cells.     

Insulin-related materials in the nervous system of vertebrates and non-vertebrates: possible extrapancreatic production

Studies from multiple laboratories with a range of methods raised the possibility that insulin production occurs naturally at extrapancreatic sites. Part A covers the presence of insulin-related materials in organisms that do not have an endocrine pancreas, including unicellular prokaryotes and eukaryotes as well as multicellular non-vertebrate animals (insects et al.) and plants. Part B covers possible production of insulin by extrapancreatic tissues of vertebrates that are remote from a source of pancreatic insulin e.g. early chick embryos and mammalian cells in culture. Part C covers possible extrapancreatic insulin production in mammals in vivo. Each section ends with an outline summary with evidence in favor of and against the hypothesis.     

Insulin and insulin-like growth factor-I stimulate a common endogenous phosphoprotein substrate (pp185) in intact neuroblastoma cells

Mouse neuroblastoma N18 cells contain specific high affinity insulin and insulin-like growth factor-I (IGF-I) receptors. Insulin and IGF-I induce phosphorylation, in intact cells, of their respective receptor beta subunits. The insulin receptor beta subunit is represented by a 95-kDa phosphoprotein that is recognized by a specific antiserum (B10). The IGF-I receptor beta subunit is represented by two phosphoproteins of molecular mass 95 and 105 kDa. The hormone-induced phosphorylation was rapid and dose-dependent occurring on both phosphoserine and phosphotyrosine residues. In addition, both insulin and IGF-I induced phosphorylation of an endogenous protein of molecular mass 185 kDa (pp185). The rapidity and dose dependency of the phosphorylation of pp185 suggested that it may represent a common endogenous substrate for the insulin and IGF-I receptors in these neural-derived cells. Phosphorylation was primarily on phosphoserine and phosphotyrosine residues. pp185 did not absorb to wheat germ agglutinin-agarose and was not stimulated by either epidermal growth factor or platelet-derived growth factor. The finding of pp185 in these neural-related cells as well as in non-neural tissues suggests that it may represent a ubiquitous endogenous substrate for both the insulin and IGF-I receptor kinases.