The rate of cleavage of GTP on the binding of Phe-tRNA.elongation factor Tu.GTP to poly(U)-programmed ribosomes of Escherichia coli

Interaction of Phe-tRNA.elongation factor Tu.GTP with poly(U)-programmed ribosomes containing an occupied P site can be described by a three-step kinetic mechanism. Initial binding is followed by the cleavage of GTP, and then a new peptide bond is formed. Rate constants controlling the first and third of these reactions are known, but only a lower limit for the rate constant of the cleavage step has been reported. We have determined this rate constant to be 20 s-1 at 5 degrees C, 30 s-1 at 15 degrees C, and 50 s-1 at 25 degrees C. This is much faster than the reverse step of the initial binding process and implies that the intrinsic accuracy of the ribosome in the initial selection step is sacrificed in favor of speed. The similarity of the kinetic and chemical mechanism of this GTP cleavage step with other nucleoside 5'-triphosphatases is discussed.     

Immunoassay of the Neuronal and Neuroendocrine Marker PGP 9.5 in Human Tissues

An inhibitory, coated-well immunoassay for the neurone-specific protein PGP 9.5 has been devised and used to measure the concentrations of the protein in human tissues. Concentrations of PGP 9.5 between 40 ng/ml and 10 micrograms/ml could be measured using this assay. In brain PGP 9.5 was present at 100.58 +/- 16.18 micrograms/mg protein. Of the other organs examined only kidney and testis showed significant concentrations of PGP 9.5 (3.97 +/- 0.87 microgram/mg protein and 3.25 +/- 0.36 microgram/mg protein, respectively). All other organs contained less than 2% of the brain level. The tissue levels determined by coated-well immunoassay confirmed the tissue specificity of PGP 9.5 originally determined by high-resolution two-dimensional gel electrophoresis.     

Rescue of a herpes simplex virus type 1 neurovirulence function with a cloned DNA fragment.

A herpes simplex virus type 1 (HSV-1) genetic function that is required for viral replication in the murine central nervous system was unambiguously localized. Thus, cosmid clones of either HSV-1 HindIII fragment C (0.64 to 0.87 map units) or fragment B (0.64 to 0.83 plus 0.91 to 1.0 map units) were employed to restore neurovirulence to an intertypic recombinant (RE6) that is specifically deficient in this property. The neurovirulent recombinants were generated in cell culture by cotransfecting the clone fragments and unit-length RE6 DNA and then selected in mouse brains. Either fragment efficiently conferred neurovirulence to RE6, demonstrating that no short region unique sequences are required. Analyses of the genomic structures of the neurovirulent recombinants showed that, in every case, HSV-1 information from 0.71 to 0.83 map units was incorporated into the RE6 genome. Cleavage of HindIII fragment C with EcoRI eliminated its capacity to rescue RE6. Virulence could be restored by the addition of HSV-1 BamHI fragment L (0.71 to 0.74 map units) that spans an EcoRI site at 0.72 map units. The precise location of this HSV-1 neurovirulence function is discussed.     

Lipoxygenase-generated hydroperoxides account for the nonphysiological features of ethylene formation from 1-aminocyclopropane-1-carboxylic acid by microsomal membranes of carnations

Several lines of evidence indicate that the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by microsomal membranes from carnation flowers is attributable to hydroperoxides generated by membrane-associated lipoxygenase (EC 1.13.11.12). As the flowers senesce, the capability of isolated microsomal membranes to convert ACC to ethylene changes. This pattern of change, which is distinguishable from that for senescing intact flowers, shows a close temporal correlation with levels of lipid hydroperoxides formed by lipoxygenase in the same membranes. Specific inhibitors of lipoxygenase curtail the formation of lipid hydroperoxides and the production of ethylene from ACC to much the same extent, whereas treatment of microsomes with phospholipase A₂, which generates fatty-acid substrates for lipoxygenase, enhances the production of hydroperoxides as well as the conversion of ACC to ethylene. Lipoxygenase-generated lipid hydroperoxides mediate the conversion of ACC to ethylene in a strictly chemical system and also enhance ethylene production by microsomal membranes. The data collectively indicate that the in-vitro conversion ACC to ethylene by microsomal membranes of carnation flowers is not reflective of the reaction mediated by the native in-situ ethylene-forming enzyme.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EDTA ethylenediaminetetraacetic acid     

Equol: a contributor to enigmatic immunoassay measurements of estrogen

The efficacy of radioimmunoassay (RIA) for the measurement of estradiol-17 beta (E2) in murine plasma was investigated. When Sephadex LH-20 or celite column chromatography was used to separate E2 from estrone (E1) and other cross-reacting compounds, the results were erratic if small volumes of mouse plasma were resolved. Assay of a diethyl ether extract of plasma (500 microL) was the most practical method for estimating the concentration of estradiol-17 beta in mice. This method was used to determine the pattern of estrogen secretion during the estrous cycle, on the day of implantation and during pregnancy. No convincing change in estrogen secretion was observed in the diestrous/proestrous mouse. By comparison, estrogen levels were elevated during pregnancy. Taken together, these results implied that cross-reactive components in plasma masked low levels of endogenous estrogen. Further evaluation of mouse plasma and urine using a co-chromatography technique to examine estrogen elution from a reverse-phase HPLC system followed by GC/MS analysis indicated the presence of equol [7-hydroxy-3-(4-hydroxyphenyl)chroman], a phytoestrogen metabolite with a ring structure similar to estradiol-17 beta. Equol and possibly other cross-reactive components of plasma may account for the apparent lack of increased estrogen secretion during the mouse estrous cycle and on the day of implantation as determined by the radioimmunoassay of ether extracts of plasma.     

Effect of calf removal on serum luteinizing hormone and cortisol concentrations in postpartum beef cows

Serum luteinizing hormone (LH) and cortisol concentrations were measured in ten fall calving, Angus cows averaging 38 +/- 8 days postpartum. Calves from five cows were weaned at the beginning of the study. Blood samples were collected at 20 min. intervals for 48 h after weaning and for 8 h on day 4 and day 6 postweaning. Mean serum LH concentrations increased (P<0.01) in weaned cows (W) from 0.55 +/- 0.01 ng/ml at time of calf removal to 1.3 +/- 0.04 ng/ml 48 h afterwards. Comparable LH concentrations for suckled cows (S) were 0.65 +/- 0.08 ng/ml and 0.62 +/- 0.03 ng/ml respectively. Average serum LH concentrations at 48 h after weaning were greater (P<0.01) for W cows than S cows and a treatment by time interaction occurred (P<0.01) with serum LH concentrations increasing (P<0.01) from time of calf removal to 48 h after calf removal in W cows. Frequency of LH peaks increased (P<0.01) in W cows and by 48 h after weaning was greater (P<0.01) in W cows than in S cows. Magnitude of LH peaks did not differ between the two groups. Serum cortisol concentrations were not different between W and S cows except for a transient elevation (P<0.01) in W cows from 7.6 +/- 0.9 ng/ml to 11.9 +/- 1.0 ng/ml 9 to 12 h after calf removal. Since serum LH concentrations were increased in W cows but not in S cows at 48 h and serum cortisol concentrations increased transiently in W cows we suggest that circulating cortisol levels may not be a physiological inhibitor of LH secretion in the suckled postpartum beef cow.     

Ultrastructural localization of an extracellular protease in Pseudomonas fragi by using the peroxidase-antiperoxidase reaction

An extracellular protease, which previously has been found to correlate with the appearance of bleblike evaginations on the cell wall of Pseudomonas fragi ATCC 4973, was purified 38-fold by ammonium sulfate precipitation and Sephadex chromatography to yield a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyclonal rabbit antiserum raised against the purified enzyme had an enzyme-linked immunosorbent assay titer of 4 X 10(7). The peroxidase antiperoxidase method was used to localize the neutral protease in P. fragi at the ultrastructural level. Electron microscopy of cell sections of this organism revealed that high concentrations of positive immunoperoxidase reaction product were located near the cell wall, whereas control sections stained with preimmune or heterologous serum did not show similar deposits to be present. These results are consistent with the hypothesis that blebs appearing on P. fragi contain high concentrations of neutral protease.     

Epidemiological characteristics of platelet aggregability.

The epidemiological characteristics of platelet aggregability were established in 958 participants in the Northwick Park Heart Study. The main analyses were based on the dose of adenosine diphosphate at which primary aggregation occurred at half its maximum velocity. Aggregability increased with age in both sexes, was greater in whites than blacks (particularly among men), and tended to decrease with the level of habitual alcohol consumption. Aggregability was, however, greater in women than men and in nonsmokers than smokers. There was no relation between aggregability on the one hand and obesity, current or past oral contraceptive use, menopausal state, or blood cholesterol and triglyceride concentrations on the other. Aggregability was somewhat, though not significantly, higher in men with a history of ischaemic heart disease and in those with electrocardiographic evidence of ischaemia than in those without. There was a strong association between the plasma fibrinogen concentration and aggregability. The widely held concept of platelet aggregability and its implications is probably an oversimplification. In the prevention of thrombosis it may be as useful to consider modifying external influences on platelet behaviour, such as plasma fibrinogen concentration or thrombin production, as it is to rely solely on platelet active agents.     

Epidemic of prosthetic valve endocarditis caused by Staphylococcus epidermidis.

In an epidemic of prosthetic valve endocarditis caused by Staphylococcus epidermidis the surgeon was found to be the source of contamination. The probable route was accidental puncture of gloves during operation. During the epidemiological investigation a second cluster of patients contaminated with Staph epidermidis during open heart surgery was found also related to one surgeon. This strain caused no detectable signs or symptoms of infection. Carriage of virulent staph epidermidis has rarely been recognised as a hazard but may have serious consequences.