Homologous Recombination Involving a Large Heterology in Escherichia Coli

Recombination between two different deletion alleles of a gene (neo) for neomycin and kanamycin resistance was studied in an Escherichia coli sbcA- recB-C- strain. The two homologous regions were in an inverted orientation on the same plasmid molecule. Kanamycin-resistant plasmids were selected and analyzed. The rate of recombination to form kanamycin-resistant plasmids was decreased by mutations in the recE, recF and recJ genes, but was not decreased by a mutation in the recA gene. It was found that these plasmids often possessed one wild-type kanamycin-resistant allele (neo+) while the other neo allele was still in its original (deletion) form. Among kanamycin-resistant plasmids with one wild-type and one parental allele it was often found that the region between the inverted repeats had been flipped (turned around) with respect to sites outside the inverted repeats. These results were interpreted as follows. Gene conversion, analogous to gene conversion in eukaryotic meiosis, is responsible for a unidirectional transfer of information from one neo deletion allele to the other. The flipping of the region between the inverted repeats is interpreted as analogous to the crossing over associated with gene conversion in eukaryotic meiosis. In contrast with a rec+ strain, these products cannot be explained by two rounds of reciprocal crossing over involving a dimeric form as an intermediate. In the accompanying paper we present evidence that gene conversion by double-strand gap repair takes place in the same E. coli strain.     

Angiotensin-converting enzyme and anorexia nervosa

Angiotensin-converting enzyme (ACE) activity was measured in 10 patients with anorexia nervosa, 6 with hyperthyroid Graves' disease, and 7 with primary hypothyroidism. Patients with anorexia nervosa had a low serum ACE activity (9.8 +/- 2.2 IU/l), as compared to findings in normal subjects (13.4 +/- 3.5 IU/l) (P less than 0.05). Patients with hyperthyroid Graves' disease had high serum ACE activity (23.7 +/- 5.8 IU/l), as compared to levels in normal subjects (P less than 0.01), and patients with primary hypothyroidism tended to have low serum ACE activity (10.1 +/- 1.8 IU/l), compared to the normal subjects (P less than 0.1). Following weight gain (before; 71.3 +/- 10.2% of ideal body weight, after; 88.7 +/- 5.6% of ideal body weight), serum ACE activity in patients with anorexia nervosa reverted to within the normal range (13.8 +/- 3.5 IU/l), and serum T3 concentration was restored to the normal range (before; 0.7 +/- 0.2 ng/ml, after; 1.1 +/- 0.3 ng/ml). In these patients, ACE activity correlated with the per cent of ideal body weight (P less than 0.05). These data suggest that, in underweight subjects with anorexia nervosa, decreased serum ACE activities may relate to emaciation.     

Effects of monoenergetic X-rays with resonance energy of bromine K-absorption edge on bromouracil-labelled E. coli cells

In order to examine enhanced killing that might be induced by Auger cascades in the incorporated atoms in cells, bromouracil(BrU)-labelled E. coli cells were irradiated with monoenergetic X-rays at 13.49 and 12.40keV, just above and below the K-absorption edge of bromine. In both cases BrU-labelled cells were more sensitive for killing than were normal cells. However, when the degree of BrU-sensitization was compared between the two energies of X-rays, the enhanced killing at 13.49 keV was only small, 2 +/- 8 per cent based on the D0 value in saline. By the addition of DMSO, which is believed to suppress radical-mediated effects, killing of BrU-labelled cells was enhanced at 13.49 keV by 8 +/- 4 per cent as compared with 12.40 keV, based on D0. These results have been examined in terms of absorbed energy in BrU-labelled cells and in terms of the number of induced Auger events.     

Use of ryanodine for functional removal of the calcium store in smooth muscle cells of the guinea-pig

Calcium store of the skinned fibers of the guinea-pig portal vein, pulmonary artery and taenia caeci consisted of two classes: one with both Ca-induced Ca release (CICR) and inositol 1,4,5-trisphosphate (IP3)-induced Ca release (IICR) mechanisms (S alpha) and the other only with IICR mechanisms (S beta). Ryanodine, applied during the CICR was activated, locked the CICR channels open, but the drug had practically no effect on the IICR mechanism. Thus, after the ryanodine treatment the Ca store with the CICR (S alpha) lost its capacity to hold Ca. Changes in the agonist-evoked contraction of intact muscle due to the ryanodine treatment suggest that agonists release Ca from S alpha which produces the initial phase of contractures.     

Branching Photocycle of Sensory Rhodopsin in Halobacterium Halobium

Temperature effect of the photocyle of sensory rhodopsin (sR) was studied by nanosecond spectroscopy. Though the formation yield of sR_M (sR₃₇₀) was sharply decreased with temperature, those of sR_K (sR₆₈₀) and sR_L were insensitive to temperature changes. These results show the existence of the branching process back to sR from sR_L. The absorption maxima for sR_K and sR_L were 595 ± 5 and 555 ± 15 nm, respectively.     

Scattered light intensity fluctuation in the canine ventricular myocardium: correlation with inotropic drug effect

Scattered light intensity fluctuation (SLIF) of coherent light by a strip of ventricular muscle during diastole is believed to be due to asynchronous cellular motion within the myocyte as a result of spontaneous release of Ca from the sacoplamic reticulum. Previous studies have shown a correlation between inotropic agents, such as ouabain and elevated extracellular Ca or decreased extracellular Na, and SLIF. The purpose of this study was to see if this correlation could be extended to other inotropic agents. The digitalis genin, ouabagenin, produces inotropy by increasing intracellular free Ca. In toxic concentrations the drug produces abnormal aftercontractions by spontaneous Ca release from the sarcoplasmic reticulum. On the other hand, the Ca channel agonist BAY k 8644 is also positively inotropic, but its effect is associated with a decrease in Ca release from the sarcoplasmic reticulum, manifested by conversion of "rest potentiation" to "rest depression." The effects of these inotropic agents on the power spectra of SLIF were dissimilar. Both frequency and amplitude of SLIF were increased after ouabagenin (1 microM), but these changes were most marked after the onset of toxicity, at which time contractility was decreased, rather than during the positive inotropic response. In contrast, BAY k 8644 (1 microM) decreased SLIF at all levels of inotropic response. The beta-adrenoceptor stimulant drug, dobutamine, and the adenylate cyclase activator, forskolin, produced minimal increase in SLIF at inotropic concentrations but caused a large increase in SLIF only after the onset of toxicity. These results suggest that SLIF is a better indicator of intracellular Ca overload and toxic oscillatory contractions in the presence of an inotrope and not of increased inotropy, per se.     

Metabolism of galactosylceramide in the twitcher mouse, an animal model of human globoid cell leukodystrophy

The metabolism of galactosylceramide was investigated in normal and twitcher mice, an animal model for human globoid cell leukodystrophy. The findings were compared with data obtained on human tissues. In vitro studies demonstrated that there were two genetically distinct enzymes that hydrolyze galactosylceramide: galactosylceramidase I and II. The former was deficient in the twitcher, while the latter was intact. beta-Galactosidase preparations purified from normal mouse liver possessed the activity to hydrolyze galactosylceramide when the assay conditions for galactosylceramidase II was used. Therefore, galactosylceramidase II was considered to be identical to GM1 ganglioside beta-galactosidase. In contrast to the human enzyme, the murine beta-galactosidase had a relatively high Km value toward galactosylceramide. The galactosylceramide-loading test demonstrated that the twitcher fibroblasts hydrolyzed the lipid at lower rates than seen in cases of human globoid cell leukodystrophy fibroblasts. These differences in galactosylceramidase II between murine and human tissues suggest that galactosylceramide accumulates in twitcher mice but not in humans with globoid cell leukodystrophy, even though galactosylceramidase I is genetically deficient in both human and this mouse model.     

Sequence-specific modification of DNA by 6-hydroxybenzo[a]pyrene

6-Hydroxybenzo[a]pyrene cleaved phi X174 supercoiled DNA to open circular DNA in the presence of heavy metal ions. It induced an alkali-labile modification in DNA via an oxygen-radical-mediated reaction; the most frequent alkali-labile sites were on the 3' side of the pyrimidine residues of the pyrimidine cluster.     

Time-resolved x-ray study of effect of sinusoidal length change on tetanized frog muscle.

Time-resolved x-ray diffraction studies were done on frog skeletal muscles with synchrotron radiation by applying sinusoidal length changes of frequency 10 Hz and amplitude approximately 1% to isometrically contracting muscles at approximately 17 degrees C. Distinct periodic intensity changes were observed in the 14.3-nm myosin meridional reflection and the equatorial 1,0 and 1,1 reflections. Response of the 14.3-nm reflection to the sinusoidal length change was nonlinear, as evidenced by a large second harmonic in its oscillatory intensity change, whereas the response of the equatorial 1,1 reflection was closely linear, as evidenced by almost sinusoidal intensity change. Intensity change of the 1,0 reflection was nearly antiphase to that of the 1,1 reflection. Integral widths of the 14.3-nm meridional reflection measured along the meridian and of the equatorial 1,1 reflection remained almost constant during tension development, while that of the 1,0 reflection tended to decrease. The widths of the 14.3-nm meridional reflection perpendicular to the meridian and of the equatorial 1,0 reflection appeared to undergo oscillatory changes in response to the sinusoidal length changes.     

Eimeria tenella, E. necatrix, E. acervulina, E. maxima, and E. brunetti: potent anticoccidial activity of an uridine analog, 1-(beta-D-ribofuranosyl)-2(1H)-pyrazinone 4-oxide

The anticoccidial activity of an uridine analog, 1-(beta-D-ribofuranosyl)-2(1H)-pyrazinone 4-oxide (emimycin riboside), against five species of chicken Eimeria was tested individually in battery experiments. With 16 ppm of the compound in feed, marked anticoccidial activity was obtained against Eimeria tenella, E. necatrix, E. acervulina, E. maxima, and E. brunetti. The last named species was more drug-sensitive than the others--dietary levels of at least 8 ppm of the drug exhibited good protection and eliminated practically all clinical signs. The battery tests with delayed and restricted medications showed that emimycin riboside affected the development of parasites in first and second generation schizogony of the life cycle of E. tenella.