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1.  Release of spectrin-free vesicles from human erythrocytes during ATP depletion: 1. characterization of spectrin-free vesicles 
The Journal of Cell Biology  1977;73(3):548-560.
Human erythrocytes incubated without glucose at 37 degrees C (in vitro aging) release spectrin-free vesicles after 12 or more hours. The release of vesicles is dependent upon ATP depletion. If the endogenous level of ATP is maintained, vesicle release is completely inhibited up to 54 h. Vesicle release is independent of hemolysis because in vitro aged cells and cells that maintain their ATP levels lose identical amounts of hemoglobin up to 45 h. 93 percent of all membrane particles released constitute a uniform population of spheres with a diameter of 185 +/- 23nm. These vesicles are of slightly varying densities due to varying contents of hemoglobin. Vesicles contain half the amount of membrane protein that is found in intact membranes when referred to the content of phospholipids phosphorus. This is primarily due to the absence of spectrin. However, their content of protein component III, glycophorin, and cholesterol remains the same as in intact membranes. Thus, the major integral membrane proteins are present in vesicles in similar quantities were surface area as in cells except for the enzyme acetylcholinesterase that is enriched up to twofold. The phospholipids composition of these vesicles is representative of the intact membrane except that the amount of phosphatidic acid is 10-fold higher and the amount of phosphatidylethanolamine is slightly lower than in erythrocytes. These results suggest a selective release of membrane domains that lack peripheral membrane proteins and are enriched in acetylcholinesterase. This release of spectrin-free vesicles from cells aged in vitro could represent an acceleration of the physiological aging process.
PMCID: PMC2111411  PMID: 873988
2.  Two modes of metabolic regulation of lysyl-transfer ribonucleic acid synthetase in Escherichia coli K-12. 
Journal of Bacteriology  1977;131(2):589-597.
Lysyl-transfer ribonucleic acid (tRNA) synthetase activity was compared in three independently isolated Escherichia coli K-12 mutants of the enzyme S-adenosyl-L-methionine synthetase (metK mutants) and their isogenic parents. In all three cases the activity of the lysyl-tRNA synthetase was elevated two- to fourfold in the mutant strains. Glycyl-L-leucine (3 mM) usually enhanced lysyl-tRNA synthetase activity two- to threefold in wild-type cells but did not further stimulate the synthetase activity in metK mutants. By two other criteria, the lysyl-tRNA synthetase from wild-type cells grown with the peptide and from the metK mutant RG62, grown in minimal medium, were similar. These criteria are enhanced resistance to thermal inactivation and altered susceptibility to endogenous proteases when compared with the synthetase from wild-type cells grown in minimal medium. In a separate set of experiments, the activities of the lysyl-, arginyl-, seryl-, and valyl-tRNA synthetases were measured in an isogenic pair of relt and rel strains of E. coli grown in a relatively poor growth medium (acetate) and in enriched medium. In the rel+ strain the level of all four synthetases was higher (two- to fourfold) in the enriched medium as expected. In the rel strain the difference in the activities of the synthetases between the two media were diminished. In all four cases the activities of the synthetases were higher in acetate medium in the rel strain. Evidence is presented that these two modes of metabolic regulation act independently.
PMCID: PMC235468  PMID: 328487
3.  Potentiation of the function of Hageman factor fragments by high molecular weight kininogen. 
Patients lacking high molecular weight (HMW) kininogen have profound abnormalities of the Hageman factor-dependent pathways of coagulation, kinin formation, and fibrinolysis. The ability of HMW kininogen to potentiate the Hageman factor fragments (HFf) activation of prekallikrein and Factor XI in plasma was studied. HFf only partially converted Factor XI to XIa and prekallikrein to kallikrein in plasma deficient in HMW kininogen (Williams trait), while enhanced activation of Factor XI and prekallikrein by HFf resulted after reconstitution with HMW kininogen. In a system using highly purified components, HMW kininogen increased the initial rate of prekallikrein activation whether the kallikrein formed was assayed by arginine esterase activity or kininforming ability. The potentiation of prekallikrein activation occurred over a 12-fold range of enzyme (HFf) concentration and was nonhyperbolic with respect to substrate (prekallikrein). HMW kininogen exerted its effect even in the absence of prekallikrein since the hydrolysis of acetylglycyl-lysine methyl ester by HFf was increased by HMW kininogen. These results suggest that one of the functions of HMW kininogen is to augment the catalytic action of HFf.
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PMCID: PMC372338  PMID: 874091
4.  Genetic complementation analysis of Escherichia coli type 1 somatic pilus mutants. 
Journal of Bacteriology  1977;130(1):506-511.
A genetic complementation analysis of 75 stable nonpiliated mutants of a type 1 piliated strain of Escherichia coli K-12, AW405, was performed. Strains containing pairs of pil mutations were constructed by the infectious transfer of an F101 plasmid containing one pil mutation into E. coli K-12 AW 405 containing another pil mutation. The presence or absence of type 1 pili on the merodiploid strains was determined by agglutination with type 1 pilus antiserum. All 75 mutants fell into one of four complementation groups. The pattern of complementation defined three cistrons involved in pilus formation, pilA, pilB, and pilC. The fourth complementation group was composed of a large number of mutants defective in both pilA and pilB functions.
PMCID: PMC235229  PMID: 323241
5.  Isolation and characterization of Escherichia coli phase variants and mutants deficient in type 1 pilus production. 
Journal of Bacteriology  1977;130(1):495-505.
Type 1 pili of Escherichia coli are the prototype of the somatic class of pili found on many strains of bacteria. As a first step in the genetic analysis of type 1 piliation, an extensive series of nonpiliated derivatives of E. coli K-12 strain AW405, was characterized to produce attached or free pili when examined in the antiserum or appeared to produce attached or free pili when examined in the electron microscope. The derivatives fell into two classes; phase variants and mutants. Phase variants that formed colonies of two distinctive types, one associated with a predominantly piliated (P+), and the other associated with a nonpiliated (P-) phase, were obtained. Each phase could give rise to the other at a relatively high rate, which was greater in the P- to P+ direction during culture in unshaken liquid medium. In addition, 77 Pil- mutants were selected on the basis of a subtle difference in colonial morphology. The mutants reverted, if at all, at a much lower rate than that of the P- to P+ change. The stability of Pil- derivatives grown in unshaken liquid medium was used as a criterion for distinguishing between phase variants and mutants, Phase variation also effected colonial morphology and chemotactic swarming. These properties did not directly depend upon piliation since Pil- mutants were only slightly altered in colonial form and unaltered in chemotactic swarming. Piliation of Pil+ bacteria was quantitatively affected by growth conditions.
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PMCID: PMC235228  PMID: 323240
6.  Improving the performance of anaerobic bacteriology in a hospital laboratory. 
Journal of Clinical Pathology  1977;30(2):157-159.
A comparison was made of the performance of a newly established anaerobic section of a clinical laboratory and the routine diagnostic section in terms of isolation and identification of anaerobic bacteria. Both sections attempted to isolate obligate anaerobes from the same clinical specimens which were not transported in anaerobic containers. Anaerobic and diagnostic sections isolated anaerobes from 35% and 6% respectively of clinical specimens. The use of antibiograms greatly improved the identification of anaerobic organisms.
PMCID: PMC476341  PMID: 321477
7.  "Active" one-carbon generation in Saccharomyces cerevisiae. 
Journal of Bacteriology  1977;129(2):926-933.
A new mutation introducing a one-carbon requirement (e.g., formate) for the glycine-supplemented growth of a serine-glycine auxotroph (ser1) was correlated with a lack of glycine decarboxylase activity. The presence of oxalate decarboxylase activity or glyoxylate decarboxylase activity did not overcome the one-carbon requirement. Another mutation characterized by the absence of oxalate decarboxylase activity did not introduce a one-carbon requirement. The presence and physiological significance of glycine decarboxylase activity in Saccharomyces are thus inferred.
PMCID: PMC235031  PMID: 320197
8.  Mechanism of action of Pseudomonas aeruginosa exotoxin Aiadenosine diphosphate-ribosylation of mammalian elongation factor 2 in vitro and in vivo. 
Infection and Immunity  1977;15(1):138-144.
Previous studies showed that Pseudomonas aeruginosa exotoxin A (PA toxin) catalyzes nicotinamide adenine dinucleotide (NAD)-dependent inhibition of protein synthesis in a rabbit reticulocyte lysate and transfer of radioactivity from [14C]adenine-labeled NAD to a protein having the same molecular weight as elongation factor 2 (EF-2) (B.H.Iglewski and D. Kabat, 1975). Such an inhibited protein-synthesizing lysate was restored to activity by addition of a protein from normal mouse liver which co-purifies with EF-2. In addition, EF-2 activity was almost totally absent in livers of mice which had been injected 24 h earlier with PA toxin. On the contrary, EF-2 concentrations were only partially reduced in other organs and were normal in brains of intoxicated mice. Studies using NAD labeled in various positions show that PA toxin, like fragment A of diphtheria toxin, catalyzes transfer of the adenosine 5'-diphosphate-ribosyl moiety of NAD. Furthermore, reversal occurred when the modified protein was incubated with excess concentrations of PA toxin and nicotinamide, and NAD was identified as a product of the reverse reaction. The protein modification catalyzed either by PA toxin or by fragment A of diphtheria toxin could be reversed by incubation with other toxin. These results support the proposal that these two toxins adenosine 5'-diphosphate-ribosylate and same amino acid of EF-2 in a stereochemically identical fashion. Furthermore, PA toxin inactivates EF-2 in intoxicated mice to an extent which would ultimately result in death.
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PMCID: PMC421339  PMID: 188760

Results 1-8 (8)