recO and recR mutations delay induction of the SOS response in Escherichia coli

RecF, RecO and RecR, three of the important proteins of the RecF pathway of recombination, are also needed for repair of DNA damage due to UV irradiation. recF mutants are not proficient in cleaving LexA repressor in vivo following DNA damage; therefore they show a delay of induction of the SOS response. In this communication, by measuring the in vivo levels of LexA repressor using anti-LexA antibodies, we show that recO and recR mutant strains are also not proficient in LexA cleavage reactions. In addition, we show that recO and recR mutations delay induction of -galactosidase activity expressed from a lexA-regulated promoter following exposure of cells to UV, thus further supporting the idea that recF, recO and recR gene products are needed for induction of the SOS response.     

Hypothesis: Iron chelation plays a vital role in neutrophilic inflammation

Neutrophil influx into tissues occurs in many diverse diseases and can be associated with both beneficial and injurious effects. We hypothesize that the stimulus for certain neutrophilic inflammatory responses can be reduced to a series of competing reactions for iron, with either a labile or reactive coordination site available, between host chelators and chelators not indigenous to that specific living system. The iron focuses the transport of host phagocytic cells through a metal catalyzed generation of oxidant sensitive mediators including cytokines and eicosanoids. Many of these products are chemotactic for neutrophils. We also postulate that the iron increases the activity of the phagocyte associated NADPH oxidoreductase in the neutrophil. The function of this enzyme is likely to be the generation of superoxide in the hostÕs attempt to chemically reduce and dislodge the iron from its chelate complex. After the reoxidation of Fe in an aerobic environment, Fe will be coordinated by host lactoferrin released by the neutrophil. When complexed by this glycoprotein, the metal does not readily undergo oxidation/reduction and is safely transported to the macrophages of the reticuloendothelial system where it is stored in ferritin. Finally, we propose that the neutrophil will attempt to destroy the chelator not indigenous to the host by releasing granular contents other than lactoferrin. Inability to eliminate the chelator allows this sequence to repeat itself, which can lead to tissue injury. Such persistence of a metal chelate in the host may be associated with biomineralization, fibrosis, and cancer.     

Comparison of the pacemaker properties of chick embryonic atrial and ventricular heart cells

Summary We have investigated the pacemaker properties of aggregates of cells dissociated from the atria and ventricles of 10 to 14-day-old chick embryonic hearts using a two-microelectrode current and voltage-clamp technique. These preparations usually beat spontaneously and rhythmically in tissue culture medium containing 1.3mm potassium with a beat rate typically in the range of 15–60 beats per minute. The beat rate results show considerable variability, which precludes any statistically significant comparison between the spontaneous activity of atrial and ventricular cell preparations at 10–14 days of development. However, the shapes of pacemaker voltage changes do exhibit differences characteristic of cell type. Spontaneous atrial preparations rapidly depolarize from maximum diastolic potential (–90 mV) to a plateau range of pacemaker potentials (–80 to –75 mV). The membrane subsequently depolarizes more gradually until threshold (–65 mV) is reached. In contrast, spontaneously beating ventricular cell preparations slowly hyperpolarize after maximum diastolic potential to the –100 to –95 mV range before gradually depolarizing toward threshold. Voltage-clamp analysis reveals a virtual lack of any time-dependent pacemaker current in atrial preparations. These preparations are characterized by an approximately linear background current (I _bg) having a slope resistance of 100 K cm². Ventricular preparations have a potassium ion pacemaker current with slow kinetics (I _{K 2}), and a second time-dependent component (I _x) which is activated at potentials positive to –65 mV. The background current of these preparations displays inward rectification. Computer simulations of pacemaking reveal that the initial rapid phase of pacemaker depolarization in atrial cells is determined by the membrane time constant, which is the product of membrane capacitance and the slope resistance ofI _bg. The hyperpolarization after maximum diastolic potential of ventricular cells is caused byI _{K 2}. The final slow phase of depolarization in both cell types is caused in part by the steady-state amplitude of the fast inward sodium current (I _Na). This component has negative slope conductance which effectively increases the slope resistance in the vicinity of threshold compared to TTX-treated preparations. This mechanism is sufficient to produce interbeat intervals several seconds in duration, even in the absence of time-dependent pacemaker current, provided that the background current is at the appropriate level.     

Subcellular distribution of enzymes determined by rapid digitonin fractionation of isolated hepatocytes

Conditions were determined for rapid separation of cytosolic and mitochondrial compartments by digitonin fractionation of rat hepatocytes. The minimum time required for separation of mitochondrial and cytosolic enzyme markers decreased rapidly with increasing temperature. Kyro EOB, a non-ionic detergent, increases the release of cytosolic enzymes, particularly at lower temperatures. Experimental procedures are described for greater than 90% release of cytosolic enzymes and less than 2% release of mitochondrial enzymes in 3s. By using appropriate concentrations of digitonin and Kyro EOB in a fractionation medium maintained at 1°C and a minimum time of exposure to the medium, nearly separate patterns of release were obtained for enzyme markers for the cytosol, mitochondrial matrix and mitochondrial intermembrane space. The distribution of enzymes that exist in more than one of these compartments was quantified by comparing their rates of release with those of marker enzymes. The cytosol/mitochondrial-matrix distributions for such enzymes in hepatocytes from starved rats were 16%/84% for aspartate aminotransferase, 34%/66% for fumarase and 77%/23% for ATP citrate lyase. In hepatocytes from rats that were induced to synthesize ATP citrate lyase by starvation and re-feeding, the ratio had increased to 95%/5%. The maximum cytosol/intermembrane-space ratio for adenylate kinase was 8%/92%. A procedure is also described for treating commercial digitonin that increases its solubility in water from about 1mg/ml to more than 800mg/ml.     

Outer membrane protein a and other polypeptides regulate capsular polysaccharide synthesis in E. coli K-12.

Summary capR (lon) mutants of Escherichia coli K-12 are mucoid on minimal agar because they produce large quantities of capsular polysaccharide. When such mutants are transformed to tetracycline resistance by plasmid pMC44, a hybrid plasmid that contains a 2 megadalton (Mdal) endonuclease EcoR1 fragment of E. coli K-12 DNA joined to the cloning vehicle-pSC101, capsular polysaccharide synthesis is inhibited and the transformed colonies exhibit a nonmucoid phenotype. Re-cloning of the 2 Mdal EcoR1 fragment onto plasmid pHA105, a min-colE1 plasmid, yielded plasmid pFM100 which also inhibited capsular polysaccharide synthesis in the capR mutants. A comparison of the polypeptides specified by both plasmids pFM100 and pMC44 in minicells demonstrated that seven polypeptide bands were specified by the 2 Mdal DNA, one of which was previously demonstrated to be outer membrane protein a; also known as 3b or M2 (40 kilodaltons, Kdal). Plasmid mutants no longer repressing capsular polysaccharide synthesis were either unable to specify the 40 K dal outer membrane protein a or were deficient in synthesis of 25 K dal and 14.5 K dal polypeptides specified by the 2 Mdal DNA fragment. Studies with a minicell-producing strain that also contained a capR mutation indicated that the capR gene product regulated processing of at least one normal protein, the precursor of outer membrane protein a.     

Time course of active Na transport and oxidative metabolism following transepithelial potential perturbation in toad urinary bladder

Summary The use of an Ussing chamber with well-defined mixing characteristics coupled to a mass spectrometer permits the concurrent evaluation of transepithelial current and oxidative metabolism with improved temporal resolution. The time-course of the amiloride-sensitive currentI ^a and the rate of suprabasal CO₂ productionJ _CO2 ^sb were observed in 10 toad urinary bladders at short-circuit and after clamping at 100 mV, serosa positive. Following perturbation of (0100mV),I ^a declined sharply within 1/2 min, remained near constant 15 min, and then increased slightly.J _CO2 ^sb declined more gradually, remained near constant at 4–7 min, and then declined further. Detailed analysis revealed an early quasi-steady state with near constancy ofJ _CO2 ^sb starting at 2.9±1.1 (sd) min and lasting 4.7 ±1.8 (sd) min, followed by relaxation to a later steady state at about 15 min. During the early quasi-steady state,I ^a was also nearly constant. Considering that in steady statesI ^a/FJ _Na ^a , the rate of transepithelial active Na transport, during the early quasi-steady state mean values ±se ofJ _Na ^a ,J _CO2 ^sb and (J _Na ^a /J _CO2 ^sb ) were, respectively, 29.9±1.7%, 59.4 ±3.2%, and 56.4±5.7% of values at short-circuit. Corresponding values during the late steady state were 41.4±6.0%, 38.2±6.1%, and 111.3±8.6%. Thus the flow ratioJ _Na ^a /J _CO2 ^sb was depressed significantly during the early quasi-steady state, but returned later to the original value. The results of measurements ofI ^a andJ _CO2 ^sb in three hemibladders were qualitatively similar. In terms of a phenomenological black-box treatment the findings are consistent with earlier studies indicating incomplete coupling between transport and metabolism. Further studies will be required to clarify the molecular basis for these observations.     

Insulin sensitivity of liver glycogen synthase b into a conversion

Summary Liver glycogen synthase b phosphatase, chromatographically separable from phosphorylase a phosphatase, is decreased in 48-hour alloxan diabetic rats. The phosphatase activities are measured in an in vitro system using exogenous isolated phospho-enzyme as substrates with added phosphatases. Synthase and phosphorylase phosphatases were shown to have differential catalytic properties by their reactivity in the presence of Pi, the heat-stable inhibitor of phosphorylase phosphatase and after incubation with added cAMP-dependent protein kinase.Supported by NIH Grants HD-07788, AM-21149 and, in part, by grants from the Greater St. Louis Diabetic Childrens Welfare Association and the American Diabetes Association, N.Y.     

Separation Of Fuchsin Analogs Using Thin Layer Chromatography

The four analogs comprising basic fuchsin have been separated using thin layer chromatography (TLC). Mixtures spotted on reverse phase TLC plates were developed with a solution of 25% methanol, 10% ammonium hydroxide, and 65% distilled water. The R_f values of the analogs were for pararosaniline, 0.54; rosaniline, 0.41; magenta II, 0.31; new fuchsin, 0.19.     

Increased phosphorylation of a specific protein in mitogen-stimulated lymphocytes

The phosphorylation of proteins from murine splenic lymphocytes was studied. When the phosphorylation of proteins in extracts from Concanavalin A (ConA)-treated lymphocytes was compared with that of resting lymphocytes, there was only one detectable difference between them. A protein of 135,000 mol. wt was highly phosphorylatable in extracts from ConA-treated cells while phosphate incorporation into this protein was slight in extracts from untreated cells. This effect could be observed 12 h after ConA treatment and was maximal during S phase. This soluble protein was also phosphorylated in intact lymphocytes.