Biomedical subjects

C P Cartwright

Publications and source records attributed to C P Cartwright.

25 records · Page 2Linked to original sources

PubMed1994

Improved enrichment broth for cultivation of fastidious organisms.

An enrichment broth developed in our laboratory, fastidious broth (FB), was compared with two commercially available broth media, supplemented thioglycolate broth and enriched eugonic broth. FB supported the growth of a number of organisms that were not cultivatable in either of the other two media, including Corynebacterium jeikeium, Haemophilus influenzae, Neisseria gonorrhoeae, and Streptococcus pneumoniae. In addition, for several organisms that were able to grow in all three broths, including Neisseria meningitidis, Nocardia asteroides, and Actinomyces spp., both the time of incubation and the starting inoculum necessary to enable detection of growth were decreased significantly by using FB.

Bacteria↗

PubMed1993

Rapid method for presumptive identification of Corynebacterium jeikeium.

Corynebacterium jeikeium causes systemic infections, particularly in immunocompromised hosts. A minitube assay has been developed for the presumptive identification of C. jeikeium. With our rapid sucrose-urea test and conventional biochemical tests, sixty isolates of gram-positive, catalase-positive bacilli were identified in our laboratory. Results indicated that our assay has a sensitivity of 100% and a specificity of 90%.

Algorithms↗

PubMed1993

Reconstitution of a plasma-membrane H(+)-ATPase into bilayer lipid membrane.

The plasma membrane H(+)-ATPase of Neurospora has been reconstituted into planar lipid bilayer membranes by means of the vesicle-fusion technique described by Finkelstein and his collaborators (Zimmerberg et al., 1980; Cohen et al., 1980, 1984; Akabas et al., 1984). Enzyme was first transferred from isolated plasma membrane fragments into asolectin vesicles by a detergent-dialysis procedure (Perlin et al., 1984). After H(+)-pumping activity had been checked by quenching of acridine orange fluorescence, the vesicles were fused into performed bilayers. Critical features of the fusion process include (i) attachment of the vesicles to the bilayer in the presence of divalent cations (Mg++), and (ii) rapid osmotic swelling, which was enhanced by prior sonication or freeze-thawing of the vesicles, and/or by inclusions of physiologic channels. Enough proton pumps could be thus incorporated into bilayers to achieve ATP-driven, vanadate-sensitive currents of 0.04-0.4 pA. Aqueous solutions of low ionic strength were used to suppress conductance fluctuations due to the channels, and when that precaution was taken, we could demonstrate the proton pump the work against membrane potentials of at least 50 mV.

Acridine Orange↗

PubMed1992

Efficient secretion in yeast based on fragments from K1 killer preprotoxin.

The alpha and beta components of the secreted K1 killer toxin of Saccharomyces cerevisiae are derived from residues 45-147 and 234-316, respectively, of the 316 residue preprotoxin (ppTox). The beta N-terminus is produced by Kex2 cleavage after Lys Arg233; when beta la (the mature sequence of beta-lactamase) is fused at this site and the fusion is expressed from the PGK promoter in pDT17, a multicopy plasmid, unexpectedly modest levels of beta la secretion resulted. Over-expression of Kex2 failed to increase beta la secretion while a kex2-null mutation reduced secretion by 98%. beta la secretion in a Kex+ strain was not enhanced by inactivation of the alpha toxin component or by deletion of most of its central hydrophobic segments. However SP-beta la, produced by deletion of ppTox residues 35-176, expressed 10-fold higher beta la activity and the precursor was now secreted with similar efficiency in a kex2-null strain. Fusions of beta la to ppTox at Ala34 or Ala46 also led to efficient secretion in both KEX2 and kex2-null strains. Since these beta la fusions differ only in segments well downstream of the signal peptide and all had similar transcript levels, the efficiency of beta la secretion is apparently determined by the efficiency with which these fusions are translocated to the Golgi compartment where Kex2 is active. Efficiency is high for the shorter fusions, but is 10% or less for the longer fusions; even this fraction is apparently diverted to the vacuole if not cleaved by Kex2. SP-beta la was the most efficient construct tested; secreted beta la reached 4% of total cell protein, modestly exceeding levels produced by fusion to the MF alpha 1-encoded prepro alpha-factor, suggesting potential for the production of foreign proteins in yeast.

Amino Acid Sequence↗

PubMed1992

Kex2-dependent processing of yeast K1 killer preprotoxin includes cleavage at ProArg-44.

The K1 killer toxin of Saccharomyces cerevisiae consists of 103- and 83-residue alpha and beta components whose derivation, from a 316-residue precursor preprotoxin, requires processing at the alpha N-terminus (after ProArg-44), the alpha C-terminus (after ArgArg-149) and at the beta N-terminus (after LysArg-233). These processing events occur after translocation to the Golgi and have been investigated using beta-lactamase fusions. Signal peptidase cleavage of the precursor, predicted to occur after Ala-26, was confirmed by N-terminal sequence analysis of Ala-34 and Ile-52 fusions. Cleavage at all of the other predicted processing sites, including ProArg-44, is dependent on activity of the Kex2 protease. A fourth Kex2-dependent cleavage occurs at LysArg-188. Implications for the specificity of Kex2 cleavage and preprotoxin processing are discussed.

Amino Acid Sequence↗

PubMed1991

In vivo topological analysis of Ste2, a yeast plasma membrane protein, by using beta-lactamase gene fusions.

Gene fusions were constructed between Ste2, the receptor for the Saccharomyces cerevisiae alpha-factor, and beta la, the secreted form of beta-lactamase encoded by the bla gene of pBR322. The Ste2 and beta la components were linked by a processing fragment (P) from the yeast killer preprotoxin containing a C-terminal lysine-arginine site for cleavage by the Golgi-associated Kex2 protease. Ste2 is predicted to have a rhodopsinlike topology, with an external N terminus and seven transmembrane segments. Fusions to three of the four Ste2 domains predicted to be external resulted in beta la secretion from yeast cells. A fusion at a site just preceding the first transmembrane segment was an exception; the product was cell associated, indicating that the first 44 residues of Ste2 are insufficient to direct secretion of beta la; translocation of this domain presumably requires the downstream transmembrane segment. Expression of fusions located in two domains predicted to be cytoplasmic failed to result in beta la secretion. Following insertion of the preprotoxin signal peptide (S) between the Ste2 and P components of these cytoplasmic fusions, secretion of beta la activity occurred, which is consistent with inversion of the orientation of the beta la reporter. Conversely, insertion of S between Ste2 and P in an external fusion sharply reduced beta la secretion. Complementary information about both cytoplasmic and external domains of Ste2 was therefore provided, and most aspects of the predicted topology were confirmed. The steady-state levels of beta la detected were low, presumably because of efficient degradation of the fusions in the secretory pathway; levels, however, were easily detectable. This method should be valuable in the analysis of in vivo topologies of both homologous and foreign plasma membrane proteins expressed in yeast cells.

Amino Acid Sequence↗

PubMed1987

Effect of ethanol on activity of the plasma-membrane ATPase in, and accumulation of glycine by, Saccharomyces cerevisiae.

The pH optimum of the ATPase activity in plasma membranes from Saccharomyces cerevisiae NCYC 431 from 8 h cultures was around 6.5 and that in membranes from organisms from 16 h cultures near 6.0. The Km[ATP] of the enzyme was virtually unaffected by the age of the culture from which organisms were harvested, although the Vmax of the enzyme in membranes from organisms from 8 h cultures was higher than that for organisms from 16 h cultures. Ethanol non-competitively inhibited ATPase activity in membranes, although the inhibition constant for the enzyme from organisms from 8 h cultures was lower than that from organisms from 16 h cultures. Glycine accumulation by the general amino acid permease was non-competitively inhibited by ethanol. Inhibition constants were virtually the same for glycine uptake by deenergized organisms from 8 h and 16 h cultures, but under energized conditions the value was greater for organisms from 16 h rather than 8 h cultures. The data indicate that inhibition of plasma-membrane ATPase activity by ethanol could account, at least in part, for inhibition of glycine accumulation by ethanol.

Adenosine Triphosphatases↗