The rapid mode of calcium uptake into heart mitochondria (RaM): comparison to RaM in liver mitochondria

A mechanism of Ca(2+) uptake, capable of sequestering significant amounts of Ca(2+) from cytosolic Ca(2+) pulses, has previously been identified in liver mitochondria. This mechanism, the Rapid Mode of Ca(2+) uptake (RaM), was shown to sequester Ca(2+) very rapidly at the beginning of each pulse in a sequence [Sparagna et al. (1995) J. Biol. Chem. 270, 27510-27515]. The existence and properties of RaM in heart mitochondria, however, are unknown and are the basis for this study. We show that RaM functions in heart mitochondria with some of the characteristics of RaM in liver, but its activation and inhibition are quite different. It is feasible that these differences represent different physiological adaptations in these two tissues. In both tissues, RaM is highly conductive at the beginning of a Ca(2+) pulse, but is inhibited by the rising [Ca(2+)] of the pulse itself. In heart mitochondria, the time required at low [Ca(2+)] to reestablish high Ca(2+) conductivity via RaM i.e. the 'resetting time' of RaM is much longer than in liver. RaM in liver mitochondria is strongly activated by spermine, activated by ATP or GTP and unaffected by ADP and AMP. In heart, RaM is activated much less strongly by spermine and unaffected by ATP or GTP. RaM in heart is strongly inhibited by AMP and has a biphasic response to ADP; it is activated at low concentrations and inhibited at high concentrations. Finally, an hypothesis consistent with the data and characteristics of liver and heart is presented to explain how RaM may function to control the rate of oxidative phosphorylation in each tissue. Under this hypothesis, RaM functions to create a brief, high free Ca(2+) concentration inside mitochondria which may activate intramitochondrial metabolic reactions with relatively small amounts of Ca(2+) uptake. This hypothesis is consistent with the view that intramitochondrial [Ca(2+)] may be used to control the rate of ADP phosphorylation in such a way as to minimize the probability of activating the Ca(2+)-induced mitochondrial membrane permeability transition (MPT).     

Stability of interleukin 6, soluble interleukin 6 receptor,interleukin 10 and CC16 in human serum

The stability of interleukin 6 (IL-6), its soluble receptor (sIL-6R), IL-10 and CC16 or uteroglobin (an endogenous cytokine inhibitor) in human serum was examined using an accelerated stability testing protocol according to the Arrhenius equation. Further, the effect of time delay between blood sampling and sample processing, clotting temperature and repeated freeze-thaw cycles on serum levels of these proteins were determined. Paired serum samples were stored at 4 degrees C, 20 degrees C, 30 degrees C and 40 degrees C for 1 to 21 days. We found that IL-6 and CC16 concentrations did not change at 4 degrees C, 20 degrees C and 30 degrees C. Interleukin-6 concentrations significantly declined after 11 days at 40 degrees C. The concentrations of sIL-6R and IL-10 did not change at 4 degrees C but significantly decreased at 20 degrees C (after 21 and 14 days respectively), 30 degrees C and 40 degrees C (after 1 day at both temperatures for both cytokines). Arrhenius-plots indicated that sIL-6R and IL-10 are stable for at least several years at -20 degrees C and -70 degrees C, respectively. Since their relative stability, no Arrhenius-plot could be calculated for IL-6 and CC16. The concentrations of the proteins examined were not significantly altered by repeated freeze-thaw cycles, nor by extended clotting times at 4 degrees C or 20 degrees C. We conclude that serum samples for the determination of IL-6, sIL-6R and CC16 can be stored at -20 degrees C for several years, but for IL-10 determinations, storage at -70 degrees C is recommended.     

SH2-B is required for growth hormone-induced actin reorganization

The Src homology-2 (SH2) domain-containing protein SH2-Bbeta is a substrate of the growth hormone (GH) receptor-associated tyrosine kinase JAK2. Here we tested whether SH2-Bbeta is involved in GH regulation of the actin cytoskeleton. Based on cell fractionation and confocal microscopy, we find SH2-Bbeta present at the plasma membrane and in the cytosol. SH2-Bbeta colocalized with filamentous actin in GH and platelet-derived growth factor (PDGF)-induced membrane ruffles. To test if SH2-Bbeta is required for actin reorganization, we transiently overexpressed wild-type or mutant SH2-Bbeta in 3T3-F442A cells and assayed for GH- and PDGF-induced membrane ruffling and fluid phase pinocytosis. Overexpression of wild-type SH2-Bbeta enhanced ruffling and pinocytosis produced by submaximal GH but not submaximal PDGF. Point mutant SH2-Bbeta (R555E) and truncation mutant DeltaC555, both lacking a functional SH2 domain, inhibited membrane ruffling and pinocytosis induced by GH and PDGF. Mutant DeltaN504, which possesses a functional SH2 domain and enhances JAK2 kinase activity in overexpression systems, also inhibited GH-stimulated membrane ruffling. DeltaN504 failed to inhibit GH-induced nuclear localization of Stat5B, indicating JAK2 is active in these cells. Taken together, these results show that SH2-Bbeta is required for GH-induced actin reorganization by a mechanism discrete from the action of SH2-Bbeta as a stimulator of JAK2 kinase activity.     

Isolation and genetic characterization of a relapsing fever spirochete isolated from Ornithodoros puertoricensis collected in central Panama

Tick-borne relapsing fever (TBRF) spirochetes are likely an overlooked cause of disease in Latin America. In Panama, the pathogens were first reported to cause human disease in the early 1900s. Recent collections of Ornithodoros puertoricensis from human dwellings in Panama prompted our interest to determine whether spirochetes still circulate in the country. Ornithodoros puertoricensis ticks were collected at field sites around the City of Panama. In the laboratory, the ticks were determined to be infected with TBRF spirochetes by transmission to mice, and we report the laboratory isolation and genetic characterization of a species of TBRF spirochete from Panama. Since this was the first isolation of a species of TBRF spirochete from Central America, we propose to designate the bacteria as Borrelia puertoricensis sp. nov. This is consistent with TBRF spirochete species nomenclature from North America that are designated after their tick vector. These findings warrant further investigations to assess the threat B. puertoricensis sp. nov. may impose on human health.     

Thermodynamic characterization of the interaction of human cyclophilin 18 with cyclosporin A

Isothermal titration calorimetry (ITC) was used to investigate thermodynamic parameters of the cyclosporin A (CsA)-cyclophilin 18 (hCyp18) association reaction. We have calculated the thermodynamic parameters (enthalpy, entropy, heat capacity, and free energy of binding) of the CsA/hCyp18 complexation. All but two methods described in the literature underestimate the affinity to hCyp18 of CsA. We found that the association constant (1.1·10⁸ M⁻¹ at 10 °C) of CsA to hCyp18 is in close agreement with the reciprocal of the reported inhibitory constant of the peptidylprolyl cis/trans isomerase activity of hCyp18. Interpretation of the thermodynamic parameters in buffered solution of water, 30% glycerol and D₂O leads to the conclusion that the highly specific binding of CsA to hCyp18 is mainly mediated through hydrogen bonding and to a lesser degree through hydrophobic interaction. Furthermore, the pH dependence of the association constant was determined and analyzed according to a single proton linkage model, resulting in a pK_a value of 5.7 in free hCyp18 and below 4.5 in the CsA complexed form. Titration experiments using different single component buffers possessing different heats of ionization allowed us to estimate that statistically half a proton is transferred upon CsA binding from the binding interface of hCyp18 to the buffer at pH 5.5. No proton transfer was detected at pH 7.5. The thermodynamic results are discussed in relation to the published X-ray and NMR structure of the free and CsA complexed hCyp18.     

A new mutation in MT-ND1 m.3928G>C p.V208L causes Leigh disease with infantile spasms

New mutations in mitochondrial DNA encoded genes of complex I are rarely reported. An infant developed Leigh disease with infantile spasms. Complex I enzyme activity was deficient and response to increasing coenzyme Q concentrations was reduced. Complex I assembly was intact. A new mutation in MT-ND1 m.3928G>C p.V208L, affecting a conserved amino acid in a critical domain, part of the coenzyme Q binding pocket, was present at high heteroplasmy. The unaffected mother did not carry measurable mutant mitochondrial DNA, but concern remained for gonadal mosaicism. Prenatal testing was possible for a subsequent sibling. The ND1 p.V208L mutation causes Leigh disease.     

Bromoperoxidase activity and vanadium level of the brown alga Ascophyllum nodosum

Vanadium-dependent peroxidase activity in extracts of Ascophyllum nodosum growing in the intertidal region close to Roscoff/France, and algal vanadium levels, followed approximately similar seasonal variation, as deduced from a study lasting from April 2005 to March 2006. High peroxidase (PO) activity was found in extracts obtained from algae collected in between midwinter to spring [∼100-190 U per g dry mass (dm), triiodide assay] with a maximum in April. Periods of reduced PO activity lasted from summer to early winter (∼50-90 U per g dm). High vanadium levels (1.5-2.2 mg kg⁻¹ dm) were found in algae collected from midwinter to spring, whereas reduced levels (0.6-1.4 mg kg⁻¹ dm) were found in summer to early winter.     

Cloning and Characterization of Yeast LEU4, One of Two Genes Responsible for α-Isopropylmalate Synthesis

By complementation of an alpha-isopropylmalate synthase-negative mutant of Saccharomyces cerevisiae (leu4 leu5), a plasmid was isolated that carried a structural gene for alpha-isopropylmalate synthase. Restriction mapping and subcloning showed that sequences sufficient for complementation of the leu4 leu5 strain were located within a 2.2-kilobase SalI-PvuII segment. Southern transfer hybridization indicated that the cloned DNA was derived intact from the yeast genome. The cloned gene was identified as LEU4 by integrative transformation that caused gene disruption at the LEU4 locus. When this transformation was performed with a LEU4fbr LEU5 strain, the resulting transformants had lost the 5',5',5'-trifluoro-D,L-leucine resistance of the recipient strain but were still Leu+. When it was performed with a LEU4 leu5 recipient, the resulting transformants were Leu-. The alpha-isopropylmalate synthase of a transformant that carried the LEU4 gene on a multicopy plasmid (in a leu5 background) was characterized biochemically. The transformant contained about 20 times as much alpha-isopropylmalate synthase as wild type. The enzyme was sensitive to inhibition by leucine and coenzyme A, was inactivated by antibody generated against alpha-isopropylmalate synthase purified from wild type and was largely confined to the mitochondria. The subunit molecular weight was 65,000-67,000. Limited proteolysis generated two fragments with molecular weights of about 45,000 and 23,000. Northern transfer hybridization showed that the transformant produced large amounts of LEU4-specific RNA with a length of about 2.1 kilonucleotides. The properties of the plasmid-encoded enzyme resemble those of a previously characterized alpha-isopropylmalate synthase that is predominant in wild-type cells. The existence in yeast of a second alpha-isopropylmalate synthase activity that depends on the presence of an intact LEU5 gene is discussed.