Identification of threonine 66 as a functionally critical residue of the interleukin-1 receptor-associated kinase

We have mutated a conserved residue of the death domain of the interleukin-1 (IL-1) receptor-associated kinase (IRAK), threonine 66. The substitution of Thr-66 with alanine or glutamate prevented spontaneous activation of NF-kappaB by overexpressed IRAK but enhanced IL-1-induced activation of the factor. Like the kinase-inactivating mutation, K239S, the T66A and T66E mutations interfered with the ability of IRAK to autophosphorylate and facilitated the interactions of IRAK with TRAF6 and with the IL-1 receptor accessory protein, AcP. Wild-type IRAK constructs tagged with fluorescent proteins formed complexes that adopted a punctate distribution in the cytoplasm. The Thr-66 mutations prevented the formation of these complexes. Measurements of fluorescence resonance energy transfer among fluorescent constructs showed that the Thr-66 mutations abolished the capacity of IRAK to dimerize. In contrast, the K239S mutation did not inhibit dimerization of IRAK as evidenced by fluorescence resonance energy transfer measurements, even though microscopy showed that it prevented the formation of punctate complexes. Our results show that Thr-66 plays a crucial role in the ability of IRAK to form homodimers and that its kinase activity regulates its ability to form high molecular weight complexes. These properties in turn determine key aspects of the signaling function of IRAK.     

Evaluation of hepatitis C virus serotypes and IgM anti-HCV antibodies in two groups of anti-HCV positive subjects in south-east Italy

We investigated the relation between the presence of IgM antibodies to hepatitis C virus (HCV) and serotypes of HCV, in particular when a high level of IgM antibodies are present in patients infected with HCV serotype I and may be associated with the source, duration and evolution of infection. The study involved two anti-HCV positive groups with chronic liver disease from the same area, one of which was a group of haemodialysis patients attending the same haemodialysis centre.     

Evidence for male XO sex-chromosome system in Pentodon bidens punctatum (Coleoptera Scarabaeoidea: Scarabaeidae) with X-linked 18S-28S rDNA clusters

In scarab beetle species of the genus Pentodon, the lack of analysis of sex chromosomes in females along with the poor characterization of sex chromosomes in the males, prevented all previous investigations from conclusively stating sex determination system. In this study, somatic chromosomes from females and spermatogonial chromosomes from males of Pentodon bidens punctatum (Coleoptera: Scarabaeoidea: Scarabaeidae) from Sicily have been analyzed using non-differential Giemsa staining. Two modal numbers of chromosomes were obtained: 2n = 20 and 19 in females and males, respectively. This finding along with other karyological characteristics such as the occurrence of one unpaired, heterotypic chromosome at metaphase-I and two types of metaphase-II spreads in spermatocytes demonstrate that a XO male/XX female sex determining mechanism - quite unusual among Scarabaeoidea - operates in the species investigated here. Spermatocyte chromosomes have also been examined after a number of banding techniques and fluorescent in situ hybridization with ribosomal sequences as a probe (rDNA FISH). The results obtained showed that silver and CMA(3) staining were inadequate to localize the chromosome sites of nucleolus organizer regions (NORs) due to the over-all stainability of both constitutive heterochromatin and heterochromatin associated to the NORs. This suggests that heterochromatic DNA of P. b. punctatum is peculiar as compared with other types of heterochromatin studied so far in other invertebrate taxa. By rDNA FISH major ribosomal genes were mapped on the X chromosome.     

High-throughput approaches to catalyst discovery

High-throughput synthesis and screening approaches to catalyst discovery and optimization are systematically changing the way in which catalyst research is conducted. Increased rates of innovation, cost effectiveness, improved intellectual property, reduced time to market and an improved probability of success are some of the attractive features that demand consideration. Advances made over the past few years reveal that any initial skepticism is waning, and high-throughput approaches to catalyst discovery are now being implemented broadly in industrial and academic laboratories.     

Microglial Phagocytosis of Dopamine Neurons at Early Phases of Apoptosis

Transection of the medial forebrain bundle caused apoptosis of dopamine neurons in the rat substantia nigra. Immunohistochemical localization of activated microglia and tyrosine hydroxylase in the axotomized substantia nigra showed that activation of microglia was rapid and OX-6 (MHC-II marker)-positive and ED1 (lysosomal phagocytic marker)-positive microglia were apposed to structurally intact tyrosine hydroxylase-positive dopamine neurons, indicating microglial phagocytosis of degenerating dopamine neurons. The occurrence of microglial phagocytosis at early stages of apoptosis may indicate the evolution of apoptosis into an irreversible state. Alternatively, interventions that suppress early activation of microglia might lead to novel mechanisms for neuron protection.     

Non-random chromosomal distribution of SSLPs: systematic assessment using a novel genetic linkage map between two closely related rat strains

Simple sequence length polymorphisms (SSLPs) are a widely used tool for genetic studies in humans and model animals. Experimental crosses among closely related strains that differ primarily in the trait that is to be mapped carry the advantage of avoiding co-segregation of potentially confounding traits. However, their realization is encumbered by the limited availability of newly arisen informative SSLPs among such strains. Here we report the establishment of a genome-wide SSLP panel for the spontaneously hypertensive rat (SHR) and its close relative, the stroke-prone SHR (SHRSP), consisting of a total of 273 polymorphic markers that were found among 2,734 rat SSLPs screened. In addition to limitations in numbers, we also found the distribution of informative markers to be heterogeneous, with clustering and paucity of informative markers, respectively, in particular regions. Notably, the majority of regions thus identified was also seen when we examined an unrelated set of strains from the literature, indicating, on a more generic level, the presence of mutagenically more and less stable genomic regions.     

Characterization of caveolae from rat heart: localization of postreceptor signal transduction molecules and their rearrangement after norepinephrine stimulation

Caveolae are plasma membrane subcompartments that have been implicated in signal transduction. In many cellular systems, caveolae are rich in signal transduction molecules such as G proteins and receptor-associated tyrosine kinases. An important structural component of the caveolae is caveolin. Recent evidence show that among the caveolin gene family, caveolin-3 is expressed in skeletal and cardiac muscle and caveolae are present in cardiac myocyte cells. Both the ANP receptor as well as the muscarinic receptor have been localized to the caveolae of cardiac myocytes in culture. These findings prompted us to conduct a further analysis of cardiac caveolae. In order to improve our understanding of the mechanisms of signal transduction regulation in cardiac myocytes, we isolated cardiac caveolae by discontinuous sucrose density gradient centrifugation from rat ventricles and rat neonatal cardiocytes. Our analysis of caveolar content demonstrates that heterotrimeric G proteins, p21ras and receptor-associated tyrosine kinases are concentrated within these structures. We also show that adrenergic stimulation induces an increase in the amount of diverse alpha- and beta-subunits of G proteins, as well as p21ras, in both in vivo and in vitro experimental settings. Our data show that cardiac caveolae are an important site of signal transduction regulation. This finding suggests a potential role for these structures in physiological and pathological states.     

Site-directed mutagenesis and deletion of three phosphorylation sites of calsequestrin of skeletal muscle sarcoplasmic reticulum. Effects on intracellular targeting

Calsequestrin (CS) is segregated to the junctional sarcoplasmic reticulum (jSR) of skeletal muscle fibers and is responsible for intraluminal Ca(2+) binding. A chimeric CS-HA1, obtained by adding the nine-amino-acid viral epitope hemagglutinin (HA1) to the carboxy-terminal of CS and shown to be correctly segregated to skeletal muscle jSR in vivo (A. Nori, K. A. Nadalini, A. Martini, R. Rizzuto, A. Villa, and P. Volpe, 1997, Am. J. Physiol. 272, C1420-C1428), is mutagenized in order to identify domains of CS involved in targeting. Since a putative targeting mechanism of CS implies phosphorylation-dependent steps in the endoplasmic reticulum (ER) and/or Golgi complex, five CS-HA1 mutants disrupting the three phosphorylation sites of CS (Thr(189), Thr(229), and Thr(353)) were engineered by either site-directed mutagenesis or deletion: CS-HA1DeltaP1 (Thr(189) --> Ile); CS-HA1DeltaP2 (Thr(229) --> Asn); CS-HA1DeltaP1,2; in which Thr(189) and Thr(229) were changed to Ile and Asn, respectively; and CS-HA1Delta14(COOH) and CS-HA1Delta49 (COOH), in which 14 residues (Glu(354)-Asp(367)) and 49 residues (Asp(319)-Asp(367)), respectively, were deleted at the carboxy-terminal. Mutant cDNAs were transiently transfected in either HeLa cells, cultured myoblasts of rat skeletal muscle, or regenerating soleus muscle fibers of adult rats. Each CS-HA1 mutant was identified by Western blot as a single polypeptide of the predicted molecular weight. The intracellular localization of CS-HA1 mutants was studied by immunofluorescence using specific antibodies against either CS or HA1. CS-HA1 mutants colocalized with ER markers, e.g., calreticulin, and partially overlapped with Golgi complex markers, e.g., alpha-mannosidase II, in HeLa cells and myotubes. CS-HA1 mutants were expressed and retained in ER and ER/SR of HeLa cells and myotubes, respectively, and correctly segregated to jSR of regenerating soleus muscle fibers. Thus, the targeting mechanism of CS in vivo is not affected by phosphorylation(s); i.e., sorting and segregation of CS appear to be independent of posttranslational phosphorylation(s).