Synthesis, and functional properties of a modified human insulin A-chain: implication in a 'mini-insulin' structure determination

The design and total synthesis of a novel insulin A-chain mutant, analogue 3, is reported. In this compound, the cysteines implied in the two insulin inter-chain disulfide bridges are replaced by two serines (residues Ser(A7) and Ser(A20)) and the intra-A-chain disulfide bridge (residues Cys(A6) and Cys(A11)) is conserved. This A-chain analogue (3) has been tested in three in vitro cell culture assays, using insulin as a reference. The data clearly showed that analogue 3 mimics insulin effects on DNA synthesis, glucose uptake and glycogen synthesis without loss of potency as compared to insulin. To our knowledge, these are the first results showing that an isolated insulin chain displays functional properties similar to those of insulin. The implication of these new findings in insulin structure-function relationships and in a 'mini-insulin' structure determination is discussed.     

Intra-tissue Characteristics of Chloroplasts in the Lamina and Petiole of Mature Winter Leaf of Arum italicum Miller

Abstract: Laminae and petioles from mature winter leaves of Arum italicum were studied in order to obtain information on the sun—shade intra-tissue properties of chloroplasts. This inference was based on the: (1) micro- and submicroscopic characteristics of the chloroplasts, (2) cytochemical localizations of functional PS I and PS II, (3) pigment patterns and compositions, (4) immunolocalization of Rubisco, and (5) net photosynthesis. It was inferred that all the chloroplasts across the lamina had adaptations to intermediate shade conditions, without a sun-shade dimorphism between the palisade and the spongy tissues. In the petiole, where normally-structured chloroplasts were surprisingly present in the entire thickness of the organ, a structural and chemical dimorphism was found between the outer chlorenchyma and the inner aerenchyma where intermediate shade-type and extreme shade-type chloroplasts were present, respectively. However, some anomalies in the pigment composition were noted chiefly in the inner aerenchyma (low concentrations of β-carotene and lutein, absence of zeaxanthin, presence of unusual pigments, for instance lutein epoxide, lutein cis-isomer, and chlorophyllide a). The Rubisco immunolabelling in the outer chlorenchyma of the petiole was similar to that in the lamina, while it was very scant in the inner aerenchyma. Net photosynthesis in the petiole was about 75% of that recorded in the lamina. These data suggest that the petiole of the mature winter leaf of A. italicum closely co-operates with the lamina for enhancing light capture and utilization.     

Delayed development of interstitial cells of Cajal in the ileum of a human case of gastroschisis

The Interstitial Cells of Cajal (ICC) are responsible for rhythmic electrical activity. A paralytic ileus is present in gastroschisis (GS), a malformation due to a defective closure of the abdominal wall through which part of the intestine herniates during pregnancy. In experimental GS, ICC morphological immaturity was shown in the rat foetus at-term but it could not be demonstrated whether differentiation is accomplished post-natally. For this purpose we morphologically investigated ICC, as well as enteric neurons and smooth muscle cells, in a case of human GS at birth and 1 month later when peristaltic activity had initiated. A 36 weeks gestation female was born by c/section with prenatal diagnosis of GS and possible volvulus of the herniated intestine. At birth, the necrotic intestine was resected and both ileostomy and colostomy were performed. The intestine continuity was restored after 4 weeks. Intestinal specimens, taken during both operations at the level of the proximal stoma, were immunostained with c-kit, neuron-specific-enolase and alpha-smooth-muscle-actin antibodies and some processed for electron microscopy. ICC were present at the myenteric plexus only. At birth, these cells were rare and ultrastructurally immature; 1 month later, when partial enteral feeding was tolerated, they formed rows or groups and many of them were ultrastructurally differentiated. Neurons and smooth muscle cells, immature at birth, had developed after 1 month. Therefore, ICC differentiation, as well as that of neurons and smooth muscle cells, is delayed at birth and this might explain the paralytic ileus in GS. One month later, differentiation quickly proceeded at all cellular levels paralleling the increasing tolerance of enteral nutrition.     

The impact of mitochondrial DNA on human lifespan: a view from studies on centenarians

The role of inherited and somatic mutations of mitochondrial DNA (mtDNA) in aging and longevity is complex and highly controversial, owing to its peculiar genetics, including the phenomenon of heteroplasmy. Most of the data on mtDNA and longevity have been obtained on humans and particularly on centenarians, i. e., people who escaped or delayed the major age-related pathologies and reached the extreme limit of human lifespan. In this review we summarize the most recent advances in this field that suggest a consistent role in human longevity of both germ-line inherited and somatically acquired mutations. The particular case of the association with longevity of the somatic C150T mutation is extensively discussed, challenging the tenet that mtDNA mutations are basically detrimental. We also stress several limitations of our present knowledge, regarding the difficulty in extrapolating to humans the results obtained in animal models, owing to a variety of biological differences, including the very limited genetic variability of mtDNA in the strains used in laboratory experiments. The use of high-throughput technologies and the extensive analysis, possibly at the single cell level, of different tissues and cell types derived from the same individual will help in disentangling the complexity of mtDNA in aging and longevity.     

Double-strand break DNA repair genotype predictive of later mortality and cancer incidence in a cohort of non-smokers

We followed-up for mortality and cancer incidence 1088 healthy non-smokers from a population-based study, who were characterized for 22 variants in 16 genes involved in DNA repair pathways. Follow-up was 100% complete. The association between polymorphism and mortality or cancer incidence was analyzed using Cox Proportional Hazard regression models. Ninety-five subjects had died in a median follow-up time of 78 months (inter-quartile range 59-93 months). None of the genotypes was clearly associated with total mortality, except variants for two Double-Strand Break DNA repair genes, XRCC3 18067 C>T (rs#861539) and XRCC2 31479 G>A (rs#3218536). Adjusted hazard ratios were 2.25 (1.32-3.83) for the XRCC3 C/T genotype and 2.04 (1.00-4.13) for the T/T genotype (reference C/C), and 2.12 (1.14-3.97) for the XRCC2 G/A genotype (reference G/G). For total cancer mortality, the adjusted hazard ratios were 3.29 (1.23-7.82) for XRCC3 C/T, 2.84 (0.81-9.90) for XRCC3 T/T and 3.17 (1.21-8.30) for XRCC2 G/A. With combinations of three or more adverse alleles, the adjusted hazard ratio for all cause mortality was 17.29 (95% C.I. 8.13-36.74), and for all incident cancers the HR was 5.28 (95% C.I. 2.17-12.85). Observations from this prospective study suggest that polymorphisms of genes involved in the repair of DNA double-strand breaks significantly influence the risk of cancer and non-cancer disease, and can influence mortality.     

Spontaneous Cell Fusion in Macrophage Cultures Expressing High Levels of the P2Z/P2X7 Receptor

Mouse and human macrophages express a plasma membrane receptor for extracellular ATP named P2Z/P2X₇. This molecule, recently cloned, is endowed with the intriguing property of forming an aqueous pore that allows transmembrane fluxes of hydrophylic molecules of molecular weight below 900. The physiological function of this receptor is unknown. In a previous study we reported experiments suggesting that the P2Z/P2X₇ receptor is involved in the formation of macrophage-derived multinucleated giant cells (MGCs; Falzoni, S., M. Munerati, D. Ferrari, S. Spisani, S. Moretti, and F. Di Virgilio. 1995. J. Clin. Invest. 95:1207– 1216). We have selected several clones of mouse J774 macrophages that are characterized by either high or low expression of the P2Z/P2X₇ receptor and named these clones P2Zhyper or P2Zhypo, respectively. P2Zhyper, but not P2Zhypo, cells grown to confluence in culture spontaneously fuse to form MGCs. As previously shown for human macrophages, fusion is inhibited by the P2Z/P2X₇ blocker oxidized ATP. MGCs die shortly after fusion through a dramatic process of cytoplasmic sepimentation followed by fragmentation. These observations support our previous hypothesis that the P2Z/P2X₇ receptor is involved in macrophage fusion.     

Early defect of transforming growth factor β1 formation in Huntington’s disease

A defective expression or activity of neurotrophic factors, such as brain‐ and glial‐derived neurotrophic factors, contributes to neuronal damage in Huntington’s disease (HD). Here, we focused on transforming growth factor‐β (TGF‐β₁), a pleiotropic cytokine with an established role in mechanisms of neuroprotection. Asymptomatic HD patients showed a reduction in TGF‐β₁ levels in the peripheral blood, which was related to trinucleotide mutation length and glucose hypometabolism in the caudate nucleus. Immunohistochemical analysis in post‐mortem brain tissues showed that TGF‐β₁ was reduced in cortical neurons of asymptomatic and symptomatic HD patients. Both YAC128 and R6/2 HD mutant mice showed a reduced expression of TGF‐β₁ in the cerebral cortex, localized in neurons, but not in astrocytes. We examined the pharmacological regulation of TGF‐β₁ formation in asymptomatic R6/2 mice, where blood TGF‐β₁ levels were also reduced. In these R6/2 mice, both the mGlu2/3 metabotropic glutamate receptor agonist, LY379268, and riluzole failed to increase TGF‐β₁ formation in the cerebral cortex and corpus striatum, suggesting that a defect in the regulation of TGF‐β₁ production is associated with HD. Accordingly, reduced TGF‐β₁ mRNA and protein levels were found in cultured astrocytes transfected with mutated exon 1 of the human huntingtin gene, and in striatal knock‐in cell lines expressing full‐length huntingtin with an expanded glutamine repeat. Taken together, our data suggest that serum TGF‐β₁ levels are potential biomarkers of HD development during the asymptomatic phase of the disease, and raise the possibility that strategies aimed at rescuing TGF‐β₁ levels in the brain may influence the progression of HD.     

A distinct basic fibroblast growth factor (FGF-2)/FGF receptor interaction distinguishes urokinase-type plasminogen activator induction from mitogenicity in endothelial cells.

Basic fibroblast growth factor (FGF-2) induces cell proliferation and urokinase-type plasminogen activator (uPA) production in fetal bovine aortic endothelial GM 7373 cells. In the present paper we investigated the role of the interaction of FGF-2 with tyrosine-kinase (TK) FGF receptors (FGFRs) in mediating uPA up-regulation in these cells. The results show that FGF-2 antagonists suramin, protamine, heparin, the synthetic peptide FGF-2(112-155), and a soluble form of FGFR-1 do not inhibit FGF-2-mediated uPA up-regulation at concentrations that affect growth factor binding to cell surface receptors and mitogenic activity. In contrast, tyrosine phosphorylation inhibitors and overexpression of a dominant negative TK- mutant of FGFR-1 abolish the uPA-inducing activity of FGF-2, indicating that FGFR and its TK activity are essential in mediating uPA induction. Accordingly, FGF-2 induces uPA up-regulation in Chinese hamster ovary cells transfected with wild-type FGFR-1, -2, -3, or -4 but not with TK- FGFR-1 mutant. Small unilamellar phosphatidyl choline:cholesterol vesicles loaded with FGF-2 increased uPA production in GM 7373 cells in the absence of a mitogenic response. Liposome-encapsulated FGF-2 showed a limited but significant capacity, relative to free FGF-2, to interact with FGFR both at 4 degrees C and 37 degrees C and to be internalized within the cell. uPA up-regulation by liposome-encapsulated FGF-2 was quenched by neutralizing anti-FGF-2 antibodies, indicating that the activity of liposome-delivered FGF-2 is mediated by an extracellular action of the growth factor. Taken together, the data indicate that a distinct interaction of FGF-2 with FGFR, quantitatively and/or qualitatively different from the one that leads to mitogenicity, is responsible for the uPA-inducing activity of the growth factor.