Characterization of an endogenous substrate related to insulin and insulin-like growth factor-I receptors in lizard brain

Lizard insulin receptors are evolutionarily highly conserved. Wheat germ agglutinin-purified brain membranes demonstrate the presence of an endogenous substrate (pp 105) for both the insulin and insulin-like growth factor-I receptors. Both insulin and I-insulin-like growth factor-I stimulate the phosphorylation of this endogenous substrate in a dose-dependent manner. Following insulin-stimulated autophosphorylation of the beta subunit, there is a lag period of about 5 min prior to observable phosphorylation of the endogenous substrate. Phosphoamino acid analysis of both the beta subunit as well as pp 105 reveal primarily phosphotyrosine in both the basal as well as the stimulated state.     

ABO incompatibility and reproductive failure. I. Prenatal selection

An analysis of previous spontaneous abortions and the frequencies of blood-group combinations in mother-child pairs was carried out in 500 gravidae. The rate of previous spontaneous abortions in blood-group-O women whose latest child has blood group B is significantly higher than in all other women. On the other hand, the combination mother B/child AB is rarer than expected, but no increase in the rate of previous spontaneous abortions is obvious among these women. These discrepancies are interpreted as an indication that prenatal selection associated with ABO incompatibility may operate at various stages from fertilization through pregnancy, and that different incompatible combinations may be subject to selection at different stages.     

Methylation pattern of mouse mitochondrial DNA.

The pattern of methylation of mouse mitochondrial DNA (mtDNA) was studied using several techniques. By employing a sensitive analytical procedure it was possible to show that this DNA contains the modified base 5-methylcytosine (m5Cyt). This residue occurred exclusively at the dinucleotide sequence CpG at a frequency of 3 to 5%. The pattern of methylation was further investigated by determining the state of methylation of several MspI (HpaII) sites. Different sites were found to be methylated to a different extent, implying that methylation of mtDNA is nonrandom. Based on the known base composition and nucleotide sequence of mouse mtDNA, the dinucleotide sequence CpG was found to be underrepresented in this DNA. The features of mtDNA methylation (CpG methylation, partial methylation of specific sites and CpG underrepresentation) are also characteristic of vertebrate nuclear DNA. This resemblance may reflect functional relationship between the mitochondrial and nuclear genomes.     

Psychometric curves of lateral facilitation

Visibility of an oriented stimulus may be enhanced by nearby stimuli that are co-aligned with the target. However, the underlying mechanism governing this facilitation is controversial. Here we measured the dependence of percent correct detection on the target's contrast (psychometric curve) with and without flankers, where flankers were either collinear with or orthogonal to the target. We find that the effect of collinear flankers can be described as a translation of the psychometric function along the linear contrast axis. This behavior is consistent, within experimental error, with two types of models: (1) non-linear transduction of target contrast with collinear flankers having additive effects on contrast, and (2) uncertainty reduction (Pelli, 1985) by collinear flankers. We discuss properties of collinear facilitation that can help deciding between these two models.     

Extracellular matrix proteins modulate endocytosis of the insulin receptor

Internalization of the insulin receptor (IR) is a highly regulated multi-step process whose underlying molecular basis is not fully understood. Here we undertook to study the role of extracellular matrix (ECM) proteins in the modulation of IR internalization. Employing Chinese hamster ovary cells that overexpress IR (CHO-T cells), our results indicate that IR internalization proceeds unaffected even when Tyr phosphorylation of IR substrates, such as IRS-1, is impaired (e.g. in CHO-T cells overexpressing IRS-1 whose pleckstrin-homology domain has been deleted or in CHO-T cells that overexpress the PH/PTB domain of IRS-1). In contrast, IR internalization is affected by the context of the ECM proteins to which the cells adhere. Hence, IR internalization was inhibited 40-60% in CHO-T cells adherent onto galectin-8 (an ECM protein and an integrin ligand of the galectin family) when compared with cells adherent onto fibronectin, collagen, or laminin. Cells adherent to galectin-8 manifested a unique cytoskeletal organization, which involved formation of cortical actin and generation of F-actin microspikes that contrasted with the prominent stress-fibers formed when cells adhered to fibronectin. To better establish a role for actin filament organization in IR endocytosis, this process was assayed in CHO-T cells (adherent onto fibronectin), whose actin filaments were disrupted upon treatment with latrunculin B. Latrunculin B did not affect insulin-induced Tyr phosphorylation of IR or its ability to phosphorylate its substrates; still, a 30-50% reduction in the rate of IR internalization was observed in cells treated with latrunculin B. Treatment of cells with nocodazole, which disrupts formation of microtubules, did not affect IR internalization. These results indicate that proper actin, but not microtubular, organization is a critical requirement for IR internalization and suggest that integrin-mediated signaling pathways emitted upon cell adhesion to different extracellular matrices and the altered cytoskeletal organizations generated thereof affect the itinerary of the insulin receptor.     

The imprinting mechanism of the Prader-Willi/Angelman regional control center

The 2 Mb domain on chromosome 15q11-q13 that carries the imprinted genes involved in Prader-Willi (PWS) and Angelman (AS) syndromes is under the control of an imprinting center comprising two regulatory regions, the PWS-SRO located around the SNRPN promoter and the AS-SRO located 35 kb upstream. Here we describe the results of an analysis of the epigenetic features of these two sequences and their interaction. The AS-SRO is sensitive to DNase I, and packaged with acetylated histone H4 and methylated histone H3(K4) only on the maternal allele, and this imprinted epigenetic structure is maintained in dividing cells despite the absence of clearcut differential DNA methylation. Genetic analysis shows that the maternal AS-SRO is essential for setting up the DNA methylation state and closed chromatin structure of the neighboring PWS-SRO. In contrast, the PWS-SRO has no influence on the epigenetic features of the AS-SRO. These results suggest a stepwise, unidirectional program in which structural imprinting at the AS-SRO brings about allele-specific repression of the maternal PWS-SRO, thereby preventing regional activation of genes on this allele.     

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.