Subcellular distribution of carbonic anhydrase in Solanum tuberosum L. leaves

The intracellular compartmentation of carbonic anhydrase (CA; EC 4.2.1.1), an enzyme that catalyses the reversible hydration of CO₂ to bicarbonate, has been investigated in potato (Solanum tuberosum L.) leaves. Although enzyme activity was mainly located in chloroplasts (87% of total cellular activity), significant activity (13%) was also found in the cytosol. The corresponding CA isoforms were purified either from chloroplasts or crude leaf extracts, respectively. The cytosolic isoenzyme has a molecular mass of 255 000 and is composed of eight identical subunits with an estimated M _r of 30000. The chloroplastic isoenzyme (M _r 220000) is also an octamer composed of two different subunits with M _r estimated at 27 000 and 27 500, respectively. The N-terminal amino acid sequences of both chloroplastic CA subunits demonstrated that they were identical except that the M _r-27 000 subunit was three amino acids shorter than that of the M _r-27 500 subunit. Cytosolic and chloroplastic CA isoenzymes were found to be similarly inhibited by monovalent anions (Cl^–, I^–, N ₃ ^- and NO ₃ ^- ) and by sulfonamides (ethoxyzolamide and acetozolamide). Both CA isoforms were found to be dependent on a reducing agent such as cysteine or dithiothreitol in order to retain the catalytic activity, but 2-mercaptoethanol was found to be a potent inhibitor. A polyclonal antibody directed against a synthetic peptide corresponding to the N-terminal amino acid sequence of the chloroplastic CA monomers also recognized the cytosolic CA isoform. This antibody was used for immunocytolocalization experiments which confirmed the intracellular compartmentation of CA: within chloroplasts, CA is restricted to the stroma and appears randomly distributed in the cytosol.Abbreviations BSA bovine serum albumin - CA carbonic anhydrase - PMSF phenylmethylsulphonyl fluoride - BAM benzamidine - DTT dithiothreitol - 2-ME 2-mercaptoethanol - PVDF polyvinylidene difluoride The authors thanks P. Carrier and Dr. B. Dimon for technical assistance with the mass-spectrometry measurements.     

Prevalence of fungi in carpeted floor environment: analysis of dust samples from living-rooms,bedrooms, offices and school classrooms

The results of 100 carpet dust analyses from atopic individuals' environment were compared according to the sampling period or the location. Dust samples were collected with a standard domestic vacuum cleaner, in locations with carpeted floor: in residences (living-room and/or bedroom), in school classrooms and in offices. The quantities of fungi vary from 5000 CFU/g to 66 000 000 CFU/g of dust. More than 100 species were isolated by dilution plating. The main species found in carpet dust wereEurotium repens, Penicillium chrysogenum, Alternaria alternata, Aureobasidium pullulans andPhoma herbarum. Strict xerophilic species were rather rare and detected in small quantities. Differences in the distribution of the CFU concentrations were examined for the four different sampling locations and were statistically significant (P=0.0174). In this study, schools were open spaces, and offices, mostly with air conditioning systems, were locations in which air is not confined. This, added to frequent professional carpet cleaning, probably explains the lowest levels of fungal concentration found in these locations. The majority of the homes had the largest fungal concentration in the living-room (median: 2×10⁵ CFU/g) while some bedrooms (median: 7×10⁴ CFU/g) had the highest concentrations. It is suggested that, when fungi are suspected to be the origin of respiratory allergy or irritating symptoms, the mycoflora of the bedroom, principally, should be investigated first.     

R-(+)-alpha-lipoic acid inhibits endothelial cell apoptosis and proliferation: involvement of Akt and retinoblastoma protein/E2F-1

Lipoic acid was recently demonstrated to improve endothelial dysfunction or retinopathy not only in rats but also in diabetic patients. We tested the hypothesis that R-(+)-alpha-lipoic acid (LA) directly affects human endothelial cell (EC) function (e.g., apoptosis, proliferation, and protein expression), independent of the cells' vascular origin. Macrovascular EC (macEC), isolated from umbilical (HUVEC) and adult saphenous veins and from aortae, as well as microvascular EC (micEC) from retinae, skin, and uterus, were exposed to LA (1 mumol/l-1 mmol/l) with/without different stimuli (high glucose, TNF-alpha, VEGF, wortmannin, LY-294002). Apoptosis, proliferation, cell cycle distribution, and protein expression were determined by DNA fragmentation assays, [(3)H]thymidine incorporation, FACS, and Western blot analyses, respectively. In macro- and microvascular EC, LA (1 mmol/l) reduced (P < 0.05) basal (macEC, -36 +/- 4%; micEC, -46 +/- 6%) and stimulus-induced (TNF-alpha: macEC, -75 +/- 11%; micEC, -68 +/- 13%) apoptosis. In HUVEC, inhibition of apoptosis by LA (500 mumol/l) was paralleled by reduction of NF-kappaB. LA's antiapoptotic activity was reduced by PI 3-kinase inhibitors (wortmannin, LY-294002), being in line with LA-induced Akt phosphorylation (Ser(437), +159 +/- 43%; Thr(308), +98 +/- 25%; P < 0.01). LA (500 mumol/l) inhibited (P < 0.001) proliferation of macEC (-29 +/- 3%) and micEC (-29 +/- 3%) by arresting the cells at the G(1)/S transition due to an increased ratio of cyclin E/p27(Kip) (4.2-fold), upregulation of p21(WAF-1/Cip1) (+104 +/- 21%), and reduction of cyclin A (-32 +/- 11%), of hyperphosphorylated retinoblastoma protein (macEC: -51 +/- 7%; micEC: -50 +/- 15%), and of E2F-1 (macEC: -48 +/- 3%; micEC: -31 +/- 10%). LA's ability to inhibit apoptosis and proliferation of ECs could beneficially affect endothelial dysfunction, which precedes manifestation of late diabetic vascular complications.     

Elimination of a bacterial pore-forming toxin by sequential endocytosis and exocytosis

Staphylococcus aureus α-toxin is the archetype of bacterial pore forming toxins and a key virulence factor secreted by the majority of clinical isolates of S. aureus. Toxin monomers bind to target cells and oligomerize to form small β-barrel pores in the plasma membrane. Many nucleated cells are able to repair a limited number of lesions by unknown, calcium-independent mechanisms. Here we show that cells can internalize α-toxin, that uptake is essential for cellular survival, and that pore-complexes are not proteolytically degraded, but returned to the extracellular milieu in the context of exosome-like structures, which we term toxosomes.     

The Mechanism of Amino Acid Activation: the Work of Mahlon Hoagland

Enzymatic Carboxyl Activation of Amino Acids(Hoagland, M. B., Keller, E. B., and Zamecnik, P. C. (1956) J. Biol. Chem. 218, 345–358)Mahlon Bush Hoagland was born in Boston, Massachusetts in 1921. He attended Harvard University and graduated in 1943. Knowing that he wanted to be a surgeon, Hoagland then enrolled at Harvard Medical School. However, he was diagnosed with tuberculosis, and his poor health prevented him from becoming a surgeon when he received his M.D. in 1948. Instead, he accepted a research position at Massachusetts General Hospital. In 1953, he became a postdoctoral fellow with Journal of Biological Chemistry (JBC) Classic author Fritz Lipmann (1) at Huntington Laboratories (also at Massachusetts General Hospital), and a year later, he moved to an adjoining laboratory to work on protein synthesis with JBC Classic author Paul Zamecnik (2).Open in a separate windowMahlon HoaglandInspired by Lipmann''s insights into acyl activation mechanisms, Hoagland used a cell-free system created by Zamecnik that carried out net peptide bond formation using ¹⁴C-amino acids (3) to uncover the mechanism of amino acid activation. As reported in the JBC Classic reprinted here, he isolated an enzyme fraction that, in the presence of ATP and amino acids, catalyzed the first step in protein synthesis: the formation of aminoacyl adenylates or activated amino acids. Using data from analysis of this fraction, Hoagland presented a scheme for amino acid activation in his Classic paper.A few years later, Zamecnik and Hoagland discovered a molecule that is essential for protein synthesis: tRNA. This discovery is the subject of the Zamecnik Classic (2).After the discovery of tRNA, Hoagland spent the next year (1957–1958) at Cambridge University''s Cavendish laboratories working with Francis Crick. During that year he traveled to France to visit the Institute Pasteur in Paris. Experiments begun at the Institute would, by 1960, lead to the discovery of messenger RNA (mRNA).When he returned to the United States, Hoagland was appointed associate professor of microbiology at Harvard Medical School. He remained there until 1967 when he accepted a position as professor at Dartmouth Medical School. In 1970, he became the director of the Worcester Foundation for Experimental Biology, a Massachusetts research institute founded by his father. He retired in 1985 and currently lives in Thetford, Vermont.Hoagland has received several awards and honors in recognition of his contributions to science. These include the 1976 Franklin Medal, the 1982 and 1996 Book Awards from the American Medical Writers Association, and membership in the American Academy of Arts and Sciences and the National Academy of Sciences.     

Detection of SHV β-lactamases in Gram-negative bacilli using fluorescein-labeled antibodies

Background  

β-lactam resistance in Gram-negative bacteria is a significant clinical problem in the community, long-term care facilities, and hospitals. In these organisms, β-lactam resistance most commonly results from the production of β-lactamases. In Gram-negative bacilli, TEM-, SHV-, and CTX-M-type β-lactamases predominate. Therefore, new and accurate detection methods for these β-lactamase producing isolates are needed.     

Mutations in TMEM76* cause mucopolysaccharidosis IIIC (Sanfilippo C syndrome)

Mucopolysaccharidosis IIIC (MPS IIIC, or Sanfilippo C syndrome) is a lysosomal storage disorder caused by the inherited deficiency of the lysosomal membrane enzyme acetyl-coenzyme A: alpha -glucosaminide N-acetyltransferase (N-acetyltransferase), which leads to impaired degradation of heparan sulfate. We report the narrowing of the candidate region to a 2.6-cM interval between D8S1051 and D8S1831 and the identification of the transmembrane protein 76 gene (TMEM76), which encodes a 73-kDa protein with predicted multiple transmembrane domains and glycosylation sites, as the gene that causes MPS IIIC when it is mutated. Four nonsense mutations, 3 frameshift mutations due to deletions or a duplication, 6 splice-site mutations, and 14 missense mutations were identified among 30 probands with MPS IIIC. Functional expression of human TMEM76 and the mouse ortholog demonstrates that it is the gene that encodes the lysosomal N-acetyltransferase and suggests that this enzyme belongs to a new structural class of proteins that transport the activated acetyl residues across the cell membrane.     

How shrub encroachment under climate change could threaten pollination services for alpine wildflowers: A case study using the alpine skypilot,Polemonium viscosum

Under climate change, shrubs encroaching into high altitude plant communities disrupt ecosystem processes. Yet effects of encroachment on pollination mutualisms are poorly understood. Here, we probe potential fitness impacts of interference from encroaching Salix (willows) on pollination quality of the alpine skypilot, Polemonium viscosum. Overlap in flowering time of Salix and Polemonium is a precondition for interference and was surveyed in four extant and 25 historic contact zones. Pollinator sharing was ascertained from observations of willow pollen on bumble bees visiting Polemonium flowers and on Polemonium pistils. We probed fitness effects of pollinator sharing by measuring the correlation between Salix pollen contamination and seed set in naturally pollinated Polemonium. To ascertain whether Salix interference occurred during or after pollination, we compared seed set under natural pollination, conspecific pollen addition, and Salix pollen addition. In current and past contact zones Polemonium and Salix overlapped in flowering time. After accounting for variance in flowering date due to latitude, Salix and Polemonium showed similar advances in flowering under warmer summers. This trend supports the idea that sensitivity to temperature promotes reproductive synchrony in both species. Salix pollen is carried by bumble bees when visiting Polemonium flowers and accounts for up to 25% of the grains on Polemonium pistils. Salix contamination correlates with reduced seed set in nature and when applied experimentally. Postpollination processes likely mediate these deleterious effects as seed set in nature was not limited by pollen delivery. Synthesis: As willows move higher with climate change, we predict that they will drive postpollination interference, reducing the fitness benefits of pollinator visitation for Polemonium and selecting for traits that reduce pollinator sharing.