Homer proteins and InsP(3) receptors co-localise in the longitudinal sarcoplasmic reticulum of skeletal muscle fibres.

Striated muscle represents one of the best models for studies on Ca(2+) signalling. However, although much is known on the localisation and molecular interactions of the ryanodine receptors (RyRs), far less is known on the localisation and on the molecular interactions of the inositol trisphosphate receptors (InsP(3)Rs) in striated muscle cells. Recently, members of the Homer protein family have been shown to cluster type 1 metabotropic glutamate receptors (mGluR1) in the plasma membrane and to interact with InsP(3)R in the endoplasmic reticulum of neurons. Thus, these scaffolding proteins are good candidates for organising plasma membrane receptors and intracellular effector proteins in signalosomes involved in intracellular Ca(2+) signalling. Homer proteins are also expressed in skeletal muscle, and the type 1 ryanodine receptor (RyR1) contains a specific Homer-binding motif. We report here on the relative sub-cellular localisation of InsP(3)Rs and Homer proteins in skeletal muscle cells with respect to the localisation of RyRs. Immunofluorescence analysis showed that both Homer and InsP(3)R proteins present a staining pattern indicative of a localisation at the Z-line, clearly distinct from that of RyR1. Consistent herewith, in sub-cellular fractionation experiments, Homer proteins and InsP(3)R were both found in the fractions enriched in longitudinal sarcoplasmic reticulum (LSR) but not in fractions of terminal cisternae that are enriched in RyRs. Thus, in skeletal muscle, Homer proteins may play a role in the organisation of a second Ca(2+) signalling compartment containing the InsP(3)R, but are apparently not involved in the organisation of RyRs at triads.

Structure and mutation analysis of the glycogen storage disease type 1b gene.

Glycogen storage disease (GSD) 1b is the deficiency of endoplasmic reticulum glucose-6-phosphate (G6P) transport. We here report the structure of the gene encoding a protein likely to be responsible for G6P transport, and its mapping to human chromosome 11q23.3. The gene is composed of nine exons spanning a genomic region of approximately 4 kb. Primers based on the genomic sequence were used in single strand conformation polymorphism (SSCP) analysis and mutations were found in six out of seven GSD 1b patients analysed.

[Three cases of congenital cutaneous candidosis]. / Tre casi di Candidosi Cutanea Congenita.

We report three cases of term newborns with Congenital Cutaneous Candidosis (CCC) occured in a ten months period. Two of these showed respiratory distress that require mechanical ventilation and the administration of exogenous surfactant in one case. All the three cases recovered after therapy with fluconazole. Early onset of severe respiratory distress may require intubation and mechanical ventilation, and systemic involvement requires systemic antimichotic therapy. We did not find any predisposing factors of such a rare disease, in spite of the occurrence of three cases in a short period of time.

Relaxed enantioselectivity of human mitochondrial thymidine kinase and chemotherapeutic uses of L-nucleoside analogues.

Our discovery that Herpes virus thymidine kinase (TK) and cellular deoxycytidine kinase lack enantioselectivity, being able to phosphorylate both D- and L-enantiomers of the substrate, suggested the use of unnatural L-nucleoside analogues as antiviral drugs (Herpes, hepatitis and immunodeficiency viruses). Several L-nucleoside analogues have displayed a short-term cytotoxicity much lower than their corresponding D-counterpart. Since the delayed cytotoxicity of a drug often depends on its effects on mitochondrial metabolism, we have investigated the degree of enantioselectivity of human mitochondrial thymidine kinase (mt-TK). We demonstrate that mt-TK does not show an absolute enantioselectivity, being able to recognize, although with lower efficiency, the L-enantiomers of thymidine, deoxycytidine and modified deoxyuridines, such as (E)-5-(2-bromovinyl)-2'-deoxyuridine and 5-iodo-2'-deoxyuridine. Interestingly, the reported negative co-operativity of mt-TK phosphorylating beta-D-2'-deoxythymidine (D-Thd), disappears when the deoxyribose moiety has the inverted configuration, resulting in the preferential phosphorylation of d-Thd even in the presence of high concentrations of the L-enantiomer. This, coupled with the higher Km for beta-L-2'-deoxythymidine (L-Thd), makes mt-TK resistant to high concentrations of L-Thd and L-Thd analogues, minimizing the mitochondria-dependent delayed cytotoxicity that might be caused by the administration of L-nucleoside analogues as antivirals.

Lack of enantiospecificity of human 2'-deoxycytidine kinase: relevance for the activation of beta-L-deoxycytidine analogs as antineoplastic and antiviral agents.

We demonstrate that human 2'-deoxycytidine kinase (dCK) is a nonenantioselective enzyme because it phosphorylates beta-D-2'-deoxycytidine (D-dCyd), the natural substrate, and beta-L-2'-deoxycytidine (L-dCyd), its enantiomer, with the same efficiency. Kinetic studies showed that L-dCyd is a competitive inhibitor of the phosphorylation of D-dCyd with a Kl value of 0.12 microM, which is lower than the K(m) value for D-dCyd (1,2 microM). Chemical modifications of either the base or the pentose ring strongly decrease the inhibitory potency of L-dCyd, L-dCyd is resistant to cytidine deaminase and competes in cell cultures with the natural D-dCyd as substrate for dCK, thus reducing the incorporation of exogenous [3H]dCyd into DNA. L-dCyd had no effect on the pool of dTTP deriving from the salvage or from the de novo synthesis, does not inhibit short term RNA and protein syntheses, and shows little or no cytotoxicity. Our results indicate a catalytic similarity between human dCK and herpetic thymidine kinases, enzymes that also lack stereospecificity. This functional analogy underlines the potential role of dCK as activator of L-deoxycytidine analogs as antiviral and antineoplastic agents and lends support to the hypothesis that herpesvirus thymidine kinase might have evolved from a captured cellular dCK gene, developing the ability to phosphorylate thymidine and retaining that to phosphorylate deoxycytidine.