In vitro acetylation of the liver HMG non-histone proteins and its modulation by spermine and dexamethasone during aging of rats

The in vitro acetylation of HMG proteins was studied using liver slices of young (18-week) and old (138-week) male rats. Acetylation of total HMG proteins is lower in old age. The incorporation of (¹⁴C) acetate into individual HMG proteins varies remarkably with advancing age. Whereas acetylation of high mol. wt. proteins (HMG 1 and 2) is higher, that of low mol. wt. proteins (HMG 14 and 17) is lower in the liver of young rats as compared to the old ones. Spermine stimulates the acetylation of HMG 1 and 14 in young and HMG 1, 2 and 14 in old age. It inhibits the acetylation of HMG 17 in both ages. Dexamethasone decreases the level of incorporation of (¹⁴C) into HMG 1 and 17 in young and HMG 14 and 17 in old rats. On the other hand, it stimulates the acetylation of HMG 14 by two-fold in young and that of HMG 1 and 2 by more than three-fold in old rats. Such alteration in the acetylation of HMG proteins may account for age-related changes in the structure and function of chromatin.     

Protein Kinase C of Sympathetic Neuronal Membrane Is Activated by Phorbol Ester-Correlation Between Transmitter Release, 45Ca2+ Uptake, and the Enzyme Activity

The effects of phorbol esters [phorbol 12,13-dibutyrate (PDB), 12-O-tetradecanoylphorbol 13-acetate (TPA), and phorbol 13-acetate] were investigated on the release of [3H]norepinephrine, 45Ca2+ accumulation, and protein kinase C activity in cultured sympathetic neurons of the chick embryo. Sympathetic neurons derived from 10-day-old chick embryo were cultured in serum-free medium supplemented with insulin, transferrin, and nerve growth factor. After 3 days, neurons were loaded with [3H]-norepinephrine and the release of [3H]norepinephrine was determined before and after electrical stimulation. Stimulation at 1 Hz for 15 s increased the release of [3H]-norepinephrine over the nonstimulation period. Stimulation-evoked release gradually declined with time during subsequent stimulation periods. Incubation of neurons in Ca2+-free Krebs solution containing 1 mM EGTA completely blocked stimulation-evoked release of [3H]-norepinephrine. Stimulation-evoked release of [3H]-norepinephrine was markedly facilitated by 3 and 10 nM PDB or TPA. The spontaneous release was also enhanced by PDB and TPA. The net accumulation of 45Ca2+ during stimulation of sympathetic neurons was increased by two- to fourfold in the presence of PDB or TPA. PDB at 1-100 nM produced a concentration-dependent increase in the activation of protein kinase C. PDB at 30 nM increased the activity of protein kinase C of the particulate fraction from 0.09 to 0.58 pmol/min/mg protein. There was no significant change in protein kinase C activity of the cytosolic fraction (0.14 pmol/min/mg versus 0.13 pmol/min/mg protein). The ratio of the particulate to cytosolic protein kinase C increased from a control value of 0.62 to 4.39 after treatment with 30 nM PDB. TPA (10 and 30 nM) also increased protein kinase C activity of the particulate fraction by six- to eightfold. Phorbol 13-acetate had no effect on protein kinase C activity, [3H]norepinephrine release, and 45Ca2+ accumulation. These results provide direct evidence that activation of protein kinase C enhances Ca2+ accumulation, which in turn leads to the facilitation of transmitter release in sympathetic neurons.     

Structure determination of a monoclonal Fab fragment specific for histidine-containing protein of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli

Jel 42 is a monoclonal antibody specific for histidine-containing protein, a small phosphocarrier protein required for sugar transport in Escherichia coli. Fab fragments prepared from this antibody by papain digestion consisted of three major isoelectric forms which were separated on a chromatofocusing column. Two of these forms produced large crystals in space group P21 and unit cell dimensions a = 117.48 A, b = 66.56 A, c = 67.31 A, and beta = 118.7 degrees, with two Fab fragments per asymmetric unit. Data were collected to 3.5-A resolution. The structure of Fab Jel 42 was solved by the Molecular Replacement method (least-squares refined to R = 0.282) using the known structure of Fab HED 10 (12) as the search model; the amino acid residues of the hypervariable and elbow regions of Fab HED 10 were omitted from the starting model. A Fourier map calculated at this stage revealed electron density which corresponded to the hypervariable loops forming the antigen-binding crevice and the elbow region of Fab Jel 42. The elbow angles for the two independent Fab molecules are 159 and 167 degrees, similar to that of the Fab HED 10 search model which has an elbow angle of 162 degrees. There is no local noncrystallographic axis of symmetry relating the two molecules in the asymmetric unit.     

Altered amount and activity of superoxide dismutase in sickle cell anemia

The amount and activity of superoxide dismutase (SOD) (EC 1.15.1.1) were measured in red cells collected from 50 white controls, 101 black controls, 50 patients with sickle hemoglobin (SS Hb), 12 with sickle trait, and 11 with other sickling hemoglobinopathies. Red cells from normal black subjects had more SOD amount and activity than normal whites (1.77 U/mg Hb and 2.96 micrograms/mg Hb vs. 1.47 U/mg Hb and 2.64 micrograms/mg Hb, respectively) or blacks with SS Hb or other sickling hemoglobinopathies. Patients with more severe manifestations of SS Hb had lower levels of SOD activity than those with milder symptoms but had the same amount of enzyme protein. Individuals with sickle trait had amounts and activities of SOD comparable to black controls. An alteration in defense to free radical oxygen may play a role in the severity of symptoms experienced by patients with homozygous sickle cell disease.     

Efficacy of medicinal plant (Andrographis peniculata) extract on aflatoxin production and growth of Aspergillus flavus

The efficacies of four different concentrations (3, 5, 8 and 10 mg/ml) of an aqueous extract of the Andrographis peniculata were tested on growth and aflatoxin production by Aspergillus flavus in liquid SMKY medium. The maximum inhibition of aflatoxin production and growth of A. flavus were marked at 10 mg/ml (i.e. 78.6% aft. B₁ and 75.1% growth). Growth and aflatoxin production were co-related processes.     

Serum antioxidant enzyme activity in Parkinson's disease

Summary The activities of superoxide dismutase (SOD; EC 1.15.1.1) and glutathione peroxidase (GSHP_x; EC 1.11.1.9.), the enzymes that metabolize the superoxide anion and hydrogen peroxide, respectively, were measured in serum from healthy subjects and patients with Parkinson's disease (PD). The activities of SOD and GSHP_x in patients with PD were higher than those in normal healthy individuals. These results suggest that the increased activities of these enzymes could be due to oxidative stress in the initial stages of this disease.     

Oxygen free radicals in volume overload heart failure

It has been suggested that oxygen free radicals (OFR) depress the excitation-contraction coupling in cardiac muscle. It is possible that a decrease in the cardiac contractility in the failing heart may be due to an increased OFR producing activity of polymorphonuclear (PMN) leukocytes. We studied the OFR producing activity (chemiluminescence) of PMN leukocytes from blood in dogs with heart failure due to chronic volume overload. The animals were divided into two groups: I) normal, (n = 10): II) dogs with mitral insufficiency (MI) of 6 to 9 months duration, (n = 10). Hemodynamic studies were done to establish the presence of heart failure. Blood samples were collected to measure PMN leukocyte chemiluminescence. There was a decrease in the cardiac index and index of myocardial contractility (dp/dt/IIP) and an increase in the left ventricular end-diastolic pressure in dogs with MI indicating left ventricular failure. The peak chemiluminescent activity of the PMN leukocytes in blood of dogs with failure was about four folds greater than that in the blood from normal dogs. These results suggest that there may be an increased OFR generation in dogs with volume overload heart failure. The decrease in the myocardial contractility in the failing heart might be due to an increase in the OFR produced by the PMN leukocytes.     

Oxygen-derived free radicals and hemolysis during open heart surgery

Reperfusion injury occurs during open-heart surgery after prolonged cardioplegic arrest. Cardiopulmonary bypass also is known to cause hemolysis. Since reperfusion of ischemic myocardium is associated with the generation of oxygen free radicals, and since free radicals can attack a protein molecule, it seems reasonable to assume that hemolysis might be the consequence of free radical attack on hemoglobin protein. The results of this study demonstrated that reperfusion following ischemic arrest caused an increase in free hemoglobin and free heme concentrations, simultaneously releasing free iron and generating hydroxyl radicals. In vitro studies using pure hemoglobin indicated that superoxide anion generated by the action of xanthine oxidase on xanthine could release iron from the heme ring and cause deoxygenation of oxyhemoglobin into ferrihemoglobin. This study further demonstrated that before the release of iron from the heme nucleus, oxyhemoglobin underwent deoxygenation to ferrihemoglobin. The released iron can catalyze the Fenton reaction, leading to the formation of cytotoxic hydroxyl radical (OH·). In fact, the formation of OH. in conjunction with hemolysis occurs during cardiac surgery, and when viewed in the light of the in vitro results, it seems likely that oxygen-derived free radicals may cause hemolysis during cardiopulmonary bypass and simultaneously release iron from the heme ring, which can catalyze the formation of OH·.     

Effect of atrazine on some aspects of lipid metabolism in fresh water fish

The impact of atrazine, a triazine herbicide, on the lipid metabolism of fish, Sarotherodon mossambica was studied. Significant changes were reported in the lipid profiles of liver and muscle as a function of exposure period.