Identification of mitochondrial carriers in Saccharomyces cerevisiae by transport assay of reconstituted recombinant proteins

The inner membranes of mitochondria contain a family of carrier proteins that are responsible for the transport in and out of the mitochondrial matrix of substrates, products, co-factors and biosynthetic precursors that are essential for the function and activities of the organelle. This family of proteins is characterized by containing three tandem homologous sequence repeats of approximately 100 amino acids, each folded into two transmembrane alpha-helices linked by an extensive polar loop. Each repeat contains a characteristic conserved sequence. These features have been used to determine the extent of the family in genome sequences. The genome of Saccharomyces cerevisiae contains 34 members of the family. The identity of five of them was known before the determination of the genome sequence, but the functions of the remaining family members were not. This review describes how the functions of 15 of these previously unknown transport proteins have been determined by a strategy that consists of expressing the genes in Escherichia coli or Saccharomyces cerevisiae, reconstituting the gene products into liposomes and establishing their functions by transport assay. Genetic and biochemical evidence as well as phylogenetic considerations have guided the choice of substrates that were tested in the transport assays. The physiological roles of these carriers have been verified by genetic experiments. Various pieces of evidence point to the functions of six additional members of the family, but these proposals await confirmation by transport assay. The sequences of many of the newly identified yeast carriers have been used to characterize orthologs in other species, and in man five diseases are presently known to be caused by defects in specific mitochondrial carrier genes. The roles of eight yeast mitochondrial carriers remain to be established.     

Collective Electronic Excitations in Thin Ag Films on Ni(111)

The collective electronic excitations in thin Ag films deposited onto the Ni(111) surface were studied by high-resolution electron energy loss spectroscopy. A broad loss peak at 7.7 eV was assigned to the Ag multipole plasmon, in excellent agreement with calculations based on s-d polarization model. Ag multipole plasmon was excited only at grazing incidence. Furthermore, a strong dependence on the impinging energy exists. Multipole plasmon could be measured only for a very strict range of primary electron beam energies and it was excited by electrons scattered at a reflection plane located just underneath the jellium edge. Such mode was found to be dramatically more sensible to the state of the surface with respect to ordinary surface plasmon. Moreover, we report experimental evidence of interference effects in surface plasmon excitation.     

NMR solution structure of the acylphosphatase from Escherichia coli

The solution structure of Escherichia coli acylphosphatase (E. coli AcP), a small enzyme catalyzing the hydrolysis of acylphosphates, was determined by (1)H and (15)N NMR and restrained modelling calculation. In analogy with the other members of AcP family, E. coli AcP shows an alpha/beta sandwich domain composed of four antiparallel and one parallel beta-strand, assembled in a five-stranded beta-sheet facing two antiparallel alpha-helices. The pairwise RMSD values calculated for the backbone atoms of E. coli and Sulfolobus solfataricus AcP, Bovine common type AcP and Horse muscle AcP are 2.18, 5.31 and 5.12 A, respectively. No significant differences are present in the active site region and the catalytic residue side chains are consistently positioned in the structures.     

Biochemical Aspects of Chick Embryo Retina Development: The Effects of Glucocorticoids

In chick embryo retina during development, DNA synthesis and the activities of DNA polymerase, thymidine kinase, thymidylate synthetase, and ornithine decarboxylase (ODC) declined in parallel from day 7 to 12. The administration in ovo of hydrocortisone reduced significantly, particularly at 8-10 days of incubation, both DNA synthesis and the four enzyme activities tested. The effect was dose dependent, reaching the maximum with 50-100 nmol of hydrocortisone, 8-16 h after treatment. The highest inhibition was found for ODC activity (70%), followed by thymidine kinase activity (62%) and DNA synthesis (45%), whereas activities of DNA polymerase and thymidylate synthetase were reduced only by 30%. The inhibitory effect was exerted by all the glucocorticoids tested, with dexamethasone and hydrocortisone being the most efficacious. The results support the view that glucocorticoids reduce the proliferative events in chick embryo retina, particularly at 8-10 days of embryonic life.     

The karyology of the cyprinid genera <Emphasis Type="Italic">Scardinius</Emphasis> and <Emphasis Type="Italic">Rutilus</Emphasis> in southern Europe

The chromosomes of eight species of Rutilus and Scardinius, mostly endemic to the Italian and the Balkan peninsula, were analyzed by conventional and other banding techniques. Parallel analyses were conducted also on two leuciscine species, Alburnus albidus, for which we provide the first karyological analysis, and Leuciscus cephalus. All species examined displayed the same karyotype (2n=50 chromosomes, 8 metacentric+13 submetacentric+4 subtelo/acrocentric pairs) with nucleolus organizer regions (NORs) on the ends of the shorter arms of a medium-sized submetacentric pair. In contrast, interspecific variation was observed in the distribution of constitutive heterochromatin. The variation observed in this genomic material proved to be systematically and phylogenetically informative. Indeed, a peritelomeric C-band on the first telocentric pair characterizes species of Rutilus and Scardinius. In both genera heterochromatin differentiation appears to be directed to a centromere–telomere direction, particularly evident along the metacentric elements of their karyotypes.An erratum to this article can be found at     

Proteomic identification of a two-component regulatory system in Pseudoalteromonas haloplanktis TAC125

The capability of microorganisms to utilize different carbohydrates as energy source reflects the availability of these substrates in their habitat. Investigation of the proteins involved in carbohydrate usage, in parallel with analysis of their expression, is then likely to provide information on the interaction between microorganisms and their ecosystem. We analysed the growth behaviour of the marine Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 in the presence and in the absence of different carbon source. A marked increase in the optical density was detected when l-malate was added to the growth medium. Bacterial proteins differently expressed in the presence of l-malate were identified by proteomic profiling experiments. On the basis of their relative increase, six proteins were selected for further analyses. Among these, the expression of a putative outer membrane porin was demonstrated to be heavily induced by l-malate. The presence of a functionally active two-component regulatory system very likely controlled by l-malate was found in the upstream region of the porin gene. A non functional genomic porin mutant was then constructed showing a direct involvement of the protein in the uptake of l-malate. To the best of our knowledge, the occurrence of such a regulatory system has never been reported in Pseudoalteromonads so far and might constitute a key step in the development of an effective inducible cold expression system.     

Maitotoxin-Induced Intracellular Calcium Rise in PC 12 Cells: Involvement of Dihydropyridine-Sensitive and ω-Conotoxin-Sensitive Calcium Channels and Phosphoinositide Breakdown

The biological activities of maitotoxin are strictly dependent on the extracellular calcium concentration and are always associated with an increase of the free cytosolic calcium level. We tested the effects of voltage-sensitive calcium channel blockers (nicardipine and omega-conotoxin) on maitotoxin-induced intracellular calcium increase, membrane depolarization, and inositol phosphate production in PC12 cells. Maitotoxin dose dependently increased the cytosolic calcium level, as measured by the fluorescent probe fura 2. This effect disappeared in a calcium-free medium; it was still observed in the absence of extracellular sodium and was enhanced by the dihydropyridine calcium agonist Bay K 8644. Nicardipine inhibited the effect of maitotoxin on intracellular calcium concentration in a dose-dependent manner. The maitotoxin-induced calcium rise was also reduced by pretreating cells with omega-conotoxin. Pretreatment of cells with maitotoxin did not modify 125I-omega-conotoxin and [3H]PN 200-110 binding to PC12 membranes. Nicardipine and omega-conotoxin inhibition of maitotoxin-evoked calcium increase was reduced by pertussis toxin pretreatment. Maitotoxin caused a substantial membrane depolarization of PC12 cells as assessed by the fluorescent dye bisoxonol. This effect was reduced by pretreating the cells with either nicardipine or omega-conotoxin and was almost completely abolished by the simultaneous pretreatment with both calcium antagonists. Maitotoxin stimulated inositol phosphate production in a dose-dependent manner. This effect was reduced by pretreating the cells with 1 microM nicardipine and was completely abolished in a calcium-free EGTA-containing medium. The findings on maitotoxin-induced cytosolic calcium rise and membrane depolarization suggest that maitotoxin exerts its action primarily through the activation of voltage-sensitive calcium channels, the increase of inositol phosphate production likely being an effect dependent on calcium influx. The ability of nicardipine and omega-conotoxin to inhibit the effect of maitotoxin on both calcium homeostasis and membrane potential suggests that L- and N-type calcium channel activation is responsible for the influx of calcium following exposure to maitotoxin, and not that a depolarization of unknown nature causes the opening of calcium channels.