The mitochondrial ryanodine receptor in rat heart: A pharmaco-kinetic profile

A protein discovered within inner mitochondrial membranes (IMM), designated as the mitochondrial ryanodine receptor (mRyR), has been recognized recently as a modulator of Ca²⁺ fluxes in mitochondria. The present study provides fundamental pharmacological and electrophysiological properties of this mRyR. Rat cardiac IMM fused to lipid bilayers revealed the presence of a mitochondrial channel with gating characteristics similar to those of classical sarcoplasmic reticulum RyR (SR-RyR), but a variety of other mitochondrial channels obstructed clean recordings. Mitochondrial vesicles were thus solubilized and subjected to sucrose sedimentation to obtain mRyR-enriched fractions. Reconstitution of sucrose-purified fractions into lipid bilayers yielded Cs⁺-conducting, Ca²⁺-sensitive, large conductance (500-800 pS) channels with signature properties of SR-RyRs. Cytosolic Ca²⁺ increased the bursting frequency and mean open time of the channel. Micromolar concentrations of ryanodine induced the appearance of subconductance states or inhibited channel activity altogether, while Imperatoxin A (IpTx_a), a specific activator of RyRs, reversibly induced the appearance of distinct subconductance states. Remarkably, the cardiac mRyR displayed a Ca²⁺ dependence of [³H]ryanodine binding curve similar to skeletal RyR (RyR1), not cardiac RyR (RyR2). Overall, the mRyR displayed elemental attributes that are present in single channel lipid bilayer recordings of SR-RyRs, although some exquisite differences were also noted. These results therefore provide the first direct evidence that a unique RyR occurs in mitochondrial membranes.     

The Isolation and Characterization of The Lamellar Body of Bovine Lung

A novel procedure for preparing intact lamellar bodies from bovine ³ lung has been developed. The procedure consists of enzymatic digestion ⁴ of ground-lung tissue, with Nagarse, homogenization with a loosely-fitting pestle and then differential and density gradient separation of the various cellular fractions as the basis for selection of the lamellar body fraction.     

The yeast clathrin adaptor protein complex 1 is required for the efficient retention of a subset of late Golgi membrane proteins

In yeast, certain resident trans-Golgi network (TGN) proteins achieve steady-state localization by cycling through late endosomes. Here, we show that chitin synthase III (Chs3p), an enzyme involved in the assembly of the cell wall at the mother-bud junction, populates an intracellular reservoir that is maintained by a cycle of transport between the TGN and early endosomes. Traffic of Chs3p from the TGN/early endosome to the cell surface requires CHS5 and CHS6, mutant alleles of which trap Chs3p in the TGN/early endosome. Disruption of the clathrin adaptor protein complex 1 (AP-1) restores Chs3p transport to the plasma membrane. Similarly, in AP-1 deficient cells, the resident TGN/early endosome syntaxin, Tlg1p, is missorted. We propose that clathrin and AP-1 act to recycle Chs3p and Tlg1p from the early endosome to the TGN.     

An association between the acute phase response and patterns of antigen induced T cell proliferation in juvenile idiopathic arthritis

The aim of this research was to determine whether all memory T cells have the same propensity to migrate to the joint in patients with juvenile idiopathic arthritis. Paired synovial fluid and peripheral blood mononuclear cell proliferative responses to a panel of antigens were measured and the results correlated with a detailed set of laboratory and clinical data from 39 patients with juvenile idiopathic arthritis. Two distinct patterns of proliferative response were found in the majority of patients: a diverse pattern, in which synovial fluid responses were greater than peripheral blood responses for all antigens tested; and a restricted pattern, in which peripheral blood responses to some antigens were more vigorous than those in the synovial fluid compartment. The diverse pattern was generally found in patients with a high acute phase response, whereas patients without elevated acute phase proteins were more likely to demonstrate a restricted pattern. We propose that an association between the synovial fluid T cell repertoire and the acute phase response suggests that proinflammatory cytokines may influence recruitment of memory T cells to an inflammatory site, independent of their antigen specificity. Additionally, increased responses to enteric bacteria and the presence of αEβ7 T cells in synovial fluid may reflect accumulation of gut associated T cells in the synovial compartment, even in the absence of an elevated acute phase response. This is the first report of an association between the acute phase response and the T cell population recruited to an inflammatory site.     

Detection of cytomegalovirus in urine of HIV-infected patients by DNA-DNA hybridization comparison with virus isolation, immunofluorescence and immunoperoxidase.

Immunofluorescence and immunoperoxidase test directed against early viral antigens, and DNA-DNA hybridization were compared with viral isolation for their abilities to detect Cytomegalovirus (CMV) in the urine of 89 HIV infected patients. From the 100 urine samples collected, 70 were found positive by at least one method. Considering viral isolation as the "gold standard" technique, immunofluorescence and immunoperoxidase had a sensitivity of 92.3% and 88% respectively, with a specificity in both cases of 95%. DNA-DNA hybridization showed a sensitivity of 90% but with lower (60%) specificity. All of the three assays were effective in detecting CMV from urine and the technical advantage of each is discussed.     

Transfer of a plasmid determining bacteriocin Bc-48 production and immunity, and response to sexual pheromones in Enterococcus faecalis S-48.

Production of bacteriocin Bc-48 by Enterococcus faecalis S-48 is encoded by the conjugative plasmid pMB1, which is approximately 90 kb and also responds to sex pheromones of E. faecalis OG1X. Mutants harboring deleted forms of this plasmid (pMB1-del, 75 kb) have lost both the phenotype Bc-48 (production and immunity) and the clumping response. The conjugal transfer of pMB1 to E. faecalis OG1X results in the acquisition by this strain of both bacteriocin production and immunity and also the clumping response. In the transconjugants isolated, the bacteriocinogenic trait is associated with a smaller plasmid (52 kb), which we call pMB1-1. The relationship among plasmids pMB1, pMB1-del, and pMB1-1 has been demonstrated by DNA hybridization. Plasmid pMB1-1 has been transferred with high frequency to E. faecalis mutants cured of Bc-48 production (carrying pMB1-del), conferring to them the Bc-48 trait and clumping response. In the transconjugants from a second mating, pMB1-1 and pMB1-del coexist without appreciable segregation.     

Bacterial Production and Purification of the Fish Pituitary Hormone Somatolactin

Somatolactin, a pituitary hormone belonging to the growth hormone/prolactin family, is produced in the intermediate lobe of teleost pituitary. To date, the functions of this new hormone and the target tissues are unknown. ASolea senegalensissomatolactin (ssSL) cDNA has previously been cloned and isolated. Here we have inserted this cDNA into a pET-3a plasmid in order to produce recombinant ssSL inE. coliBL21 (DE3) cells. The protein induced was isolated from inclusion bodies by a solubilization–renaturation procedure originally developed to generate native disulfide bonds, to get putative active proteins. The recombinant somatolactin was further purified to homogeneity by gel filtration on FPLC. The estimated molecular weight of 26 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis agrees well with the molecular mass calculated from the translated cDNA sequence and with native somatolactin (SL). The recombinant protein showed electrophoretic mobility identical to that of one of the native forms of SL secretedin vitroby cultured pituitaries from sole. Another native SL expressed inS. senegalensisrepresented a glycosylated modified hormone as shown byN-glycosidase treatment. Further, recombinant SL was recognized by an anti-native SL antibody and used to generate polyclonal sera reactive with the native pituitary hormone. To date, this represents the first recombinant SL protein isolated in sufficient quantities for biophysical and biochemical investigation and for studies on its physiological actions.     

Compound kinetochores of the Indian muntjac

The chromosomes of the Indian muntjac (Muntiacus muntjak vaginalis) are unique among mammals due to their low diploid number (2N=6, 7) and large size. It has been proposed that the karyotype of this small Asiatic deer evolved from a related deer the Chinese muntjac (Muntiacus reevesi) with a diploid chromosome number of 2n= 46 consisting of small telocentric chromosomes. In this study we utilized a kinetochore-specific antiserum derived from human patients with the autoimmune disease scleroderma CREST as an immunofluorescent probe to examine kinetochores of the two muntjac species. Since CREST antiserum binds to kinetochores of mitotic chromosomes as well as prekinetochores in interphase nuclei, it was possible to identify and compare kinetochore morphology throughout the cell cycle. Our observations indicated that the kinetochores of the Indian muntjac are composed of a linear beadlike array of smaller subunits that become revealed during interphase. The kinetochores of the Chinese muntjac consisted of minute fluorescent dots located at the tips of the 46 telocentric chromosomes. During interphase, however, the kinetochores of the Chinese muntjac clustered into small aggregates reminiscent of the beadlike arrays seen in the Indian muntjac. Morphometric measurements of fluorescence indicated an equivalent amount of stained material in the two species. Our observations indicate that the kinetochores of the Indian muntjac are compound structures composed of linear arrays of smaller units the size of the individual kinetochores seen on metaphase chromosomes of the Chinese muntjac. Our study supports the notion that the kinetochores of the Indian muntjac evolved by linear fusion of unit kinetochores of the Chinese muntjac. Moreover, it is concluded that the evolution of compound kinetochores may have been facilitated by the nonrandom aggregation of interphase kinetochores in the nuclei of the ancestral species.