Isotope-exchange enhancement studies of Escherichia coli glutamine synthetase

Isotope-exchange enhancement studies, a variation on positional isotope-exchange enhancement as described by Raushel and Garrard [Raushel, F. M., & Garrard, L. J. (1984) Biochemistry 23, 1791-1795], are used to establish the point in the biosynthetic reaction of Escherichia coli glutamine synthetase at which gamma-glutamyl phosphate is formed. In these experiments, the behavior of the reverse biosynthetic reaction, i.e., the reaction of ADP, L-glutamine, and phosphate to form NH4+, L-glutamate, and ATP, is examined as a function of the concentration of ammonium ion. By varying the concentration of NH4+, the ratio of the velocity of isotope exchange to the velocity of net reaction, as measured by the rate of 18O depletion from labeled phosphate and the rate of production of L-glutamate, respectively, can be modulated in a mechanism-dependent manner. Evidence is presented demonstrating the presence of a branch point in the mechanism. The enzyme-ATP-glutamate complex may partition in two ways, one involving binding of ammonium ion and the other involving the chemical transformation to form the enzyme-ADP-gamma-glutamyl phosphate complex. The alternate pathways then rejoin upon formation of the enzyme-ADP-NH4+-gamma-glutamyl phosphate complex. Because of the branch point, there is no absolute requirement that ammonium ion be absent or present in order for the formation of gamma-glutamyl phosphate to occur. At high concentrations of ammonia, one pathway through the branch can be eliminated, effectively making that portion of the pathway ordered, with ATP, L-glutamate, and NH4+ binding consistent with our previously reported steady-state kinetic mechanism [Meek, T. D., & Villafranca, J. J. (1980) Biochemistry 19, 5513-5519].     

Aluminium salts accelerate peroxidation of membrane lipids stimulated by iron salts

Aluminium salts do not themselves stimulate peroxidation of ox-brain phospholipid liposomes, but they greatly accelerate the peroxidation induced by iron(II) salts at acidic pH values. This effect of Al(III) is not seen at pH 7.4, perhaps because Al(III) salts form insoluble complexes at this pH in aqueous solution. Peroxidation of liposomes in the presence of Al(III) and Fe(II) salts is inhibited by the chelating agent desferrioxamine, and by EDTA and diethylenetriaminepentaacetic acid at concentrations greater than those of Fe(II) salt. Aluminium salts slightly stimulate the peroxidation of peroxide-depleted linolenic acid micelles, but they do not accelerate the peroxidation induced by addition of iron(II) salts to the micelles at acidic pH. Aluminium salts accelerate the peroxidation observed when human erythrocytes are treated with hydrogen peroxide at pH 7.4. Desferrioxamine decreases the peroxidation. We suggest that Al(III) ions produce an alteration in membrane structure that facilitates lipid peroxidation, and that the increased formation of fluorescent age pigments in the nervous system of patients exposed to toxic amounts of Al(III) may be related to this phenomenon. The ability of desferal to bind both iron (III) and aluminium(III) salts and to inhibit lipid peroxidation makes it an especially useful chelating agent in the treatment of 'aluminium overload'.     

The survival of anaerobes during sewage treatment employing biological filter beds

During the passage of sewage through a typical treatment plant employing biological filter beds and operating under dry weather flow conditions, about 7% of the input of anaerobic organisms survive to be released in the effluent. The greatest fall in numbers occurs in the first of two primary settling tanks operating in series and during passage through the filter beds. The predominant organisms were Bacteroides species, gram-positive cocci and clostridial species. There is no significant difference in the rate of survival of any of the genera through the different stages of treatment.     

Therapeutic potential of rat monoclonal antibodies: isotype specificity of antibody-dependent cell-mediated cytotoxicity with human lymphocytes

The ability of rat monoclonal antibodies to promote antibody-dependent cell-mediated cytotoxicity with human effector cells was tested by using a variety of antibodies against different human and mouse leukocyte antigens. It was found that only IgG2b antibodies were effective. This isotype has already been shown to be efficient in fixing human complement, which suggests that among rat monoclonal antibodies, the IgG2b subclass might be a good choice for attempts at serotherapy. Further studies with other antibody-mediated effector mechanisms as well as suitable clinical trials are merited.     

14C labelling of octulose bisphosphates by L-type pentose pathway reactions in liver in situ and in vitro

The complete reaction sequence of the pentose pathway in vitro was studied by incubating [1-14C] ribose 5-phosphate with rat liver enzyme preparation and assessed by both the rate and extent of formation of the glucose 6-P product. The reactions formed, as intermediates, the 1,8-bisphosphates of D-glycero D-ido octulose (D-g D-i Oct) and D-glycero D-altro octulose, both heavily labelled at C-4 with 14C isotope during the 12h incubation. The formation of the octulose phosphates and the specificity of their isotopic labelling confirms an important prediction of, and contribution by reactions of the L-type pentose phosphate pathway (L-PP) in liver in vitro. Infusion in situ of [6-14C] glucose into the liver of the anaesthetized rabbit resulted in the formation of high specific activity [8-14C] D-g D-i Oct 1,8-P2. The specificity of labelling indicates that the octulose intermediate is formed according to the options of the L-PP mechanism of glucose metabolism in intact liver.     

Sociality in a nocturnal “solitary” prosimian:Galago crassicaudatus

A 16-month field study of the nocturnal prosimianGalago crassicaudatus umbrosus in the Northern Transvaal, South Africa, revealed extensive homerange overlaps between adults of both sexes, age-graded male tolerance, and a high rate of direct affiliative contact between individuals of most age-sex classes. These results contrast with the general characterization of nocturnal lorisid sociality given by Charles-Dominique (1978). Nocturnal prosimians’ dispersion while foraging supports the predation model of group cohesion in diurnal primates. The problem of group size and cohesion should be divorced from that of social networks and social complexity. There may be benefits to social interactions which transcend group living. The more subtle differences in nocturnal prosimian sociality need to be reconsidered in the light of current sociobiological and socioecological theory.     

Pharmacological investigation of the mechanisms of platelet-activating factor induced mortality in the mouse

Although the platelets of the mouse are refractory to the direct effects of platelet-activating-factor (PAF), tail vein injection of 10-150 micrograms/kg PAF produces lethal anaphylactic shock. Sensitivity varies with strain and source: Swiss Webster mice show a range of sensitivity and DBA/2 (complement C5-deficient) mice are very resistant. At lethal doses of PAF, animals show labored respiration and general depression; death occurs within 15-45 min. Dexamethasone administered at least 1.5 hr prior consistently protects, whereas the cyclooxygenase inhibitors do not. Antihistamines, adrenergic antagonists, and methysergide have no effect, but cyproheptadine is partially protective at near lethal doses. Calcium entry blockers and calcium chelators, tetracycline and chlortetracycline are partially protective at very high doses consistent with non-specific effects on calcium dependent processes. The arachidonic acid lipoxygenase inhibitors BW755c, phenidone, nordihydroguaiaretic acid and diphenyldisulfide provide nearly complete protection after oral administration of 50-200 mg/kg. Phosphodiesterase inhibitors and dapsone are also effective orally. The leukotriene antagonist FPL55712 administered intraperitoneally (10 mg/kg) 5 min. prior to PAF challenge provides almost complete protection. PAF-induced mortality in the mouse represents a small animal model of systemic anaphylaxis particularly useful for the systemic testing of arachidonic acid lipoxygenase inhibitors and leukotriene antagonists.     

Experimentally induced defects of mitochondrial metabolism in rat skeletal muscle. Biological effects of the NADH: coenzyme Q reductase inhibitor diphenyleneiodonium.

An animal model for the human condition of mitochondrial myopathy has been established and characterized physiologically and biochemically. The NADH: coenzyme Q reductase inhibitor diphenyleneiodonium [Bloxham (1979) Biochem. Soc. Trans. 7, 103-106] was either infused acutely in vivo into rat hind limb or injected chronically into rats. Both modes of delivery resulted in a reduced muscle oxidative capacity and increased fatigue. Analysis of muscle metabolites by h.p.l.c. and 31P-n.m.r. indicated that ATP concentrations were similar to control values during periods of stimulation and these were maintained by the phosphocreatine pool. During the recovery period after muscle stimulation in the experimental animals the muscle pH remained depressed and the rate of phosphocreatine synthesis was markedly delayed as compared with controls. Factors thought to be involved in the fatigue response are discussed in relation to this model.     

Selectivity of the insulin-like actions of vanadate on glucose and protein metabolism in skeletal muscle.

The 1H-n.m.r. spectrum of casein micelles consists of a small number of moderately sharp (linewidth approx. 60 Hz) resonances superimposed on the envelope of very broad lines expected for particles of this size. These sharp lines resemble, in chemical shift and relative intensity, the spectrum of the isolated 'macropeptide' released from the micelles by treatment with chymosin. The sharp lines in the casein micelle spectrum are further sharpened by addition of chymosin and broadened markedly by addition of ethanol. These observations are consistent with the proposal that the 'macropeptide' (the C-terminal 64 residues of K-casein) forms flexible 'hairs' on the surface of the micelles.