A histological and immunohistochemical study of the effects of N-acetyl cysteine on retinopathy of prematurity by modifying insulin-like growth factor-1

Retinopathy of prematurity (ROP) is a vasoproliferative disorder that occurs in premature infants and may lead to permanent visual impairment. We investigated both the possible protective role of N-acetyl cysteine (NAC) for preventing ROP and the role of IGF-1 in the disorder. Forty-five newborn rats were divided into three groups. Group 1 was raised in room air as controls. Group 2 was exposed to 60% oxygen for 14 days after birth, then transferred to room air. Group 3 was exposed to the same conditions as group 2, but received intraperitoneal injections of NAC on postnatal days 7–17. After 35 days, both eyes of all rats were processed for histology. Some sections were stained with hematoxylin and eosin to assess structural changes and other sections were immunostained to determine the location of IGF-1. Frozen sections also were prepared and stained for adenosine triphosphatase to detect retinal blood vessels. Compared to the controls, more blood vessels, many of which were abnormal, and increased IGF-1 expression were observed in group 2. In group 3, abnormal blood vessels and IGF-1 expression were less evident. NAC appeared to be an effective vascular-protective agent for ROP by decreasing IGF-1 expression.     

Metabolism of some possible ecdysone precursors in Calliphora stygia

A study has been made, using Calliphora stygia at the time of puparium formation, of the incorporation of a number of labelled sterols into β-ecdysone. [1-³H]-Cholesterol and [4-¹⁴C]-cholesterol are incorporated to a similar extent (0·01-0·02%). [1-³H]-7-Dehydrocholesterol is better incorporated (0·025%) than cholesterol while [1-³H]-cholesterol sulphate, (22R)-22-hydroxy-[22-³H]-cholesterol, and 25-hydroxy-[26-¹⁴C]-cholesterol are not incorporated to a significant extent.     

Polymeric inhibitors of platelet aggregation. Synergistic effects and proposals for a new mechanism

We have studied the inhibition of ADP-induced platelet aggregation in sheep platelet-rich plasma by water-soluble polymers bound to the prostaglandin analogue 5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl)hydantoin ('BW 245' C, (I). The use of unambiguous modes of binding this antiplatelet drug to polymers has enabled us to study some structural features which influence inhibitory activity. Evidence is adduced which indicates that the chemical mechanisms responsible for inhibition by free and coupled BW 245 are similar. The most important observation is a remarkable synergism demonstrated by the greatly enhanced activity of a mixture of a polymer coupled to BW 245 with the uncoupled parent polymer. In some cases (e.g., with high-molecular-weight dextran) the effect may reach (and possibly exceed) two orders of magnitude. The influence of polymer molecular weights and 'cross-polymer' effects have both been examined. A mechanism has been proposed to account for these phenomena, involving adsorption of the added (inactive) polymer on to the platelet membranes, facilitating interaction of the polymer-bound drug with receptors, made more accessible by alteration to the surface geometry. This mechanism is based on physical processes, unlike other explanations of synergistic behaviour, e.g., that of prostaglandins used in conjunction with non-polymeric drugs. The observed dependences of synergistic effects upon polymer molecular weight and type and distribution of drug molecules along chains are typical 'polymer' phenomena which are all consistent with the proposed mechanism.     

Regulation of dolichol and of cholesterol biosynthesis in cholesterol-fed rabbits

The feeding of rabbits with a diet supplemented with 2% cholesterol caused a significant increase in the concentration of serum and hepatic microsomal cholesterol while not affecting serum high-density lipoprotein cholesterol concentration. The concentration of cytochrome b₅ was also increased in the cholesterol-fed rabbits but no change in the concentration of cytochrome P-450 was apparent. The increase in microsomal cholesterol was accompanied by an inhibition of hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase and a marked stimulation of acyl-coenzyme A:cholesterol acyltransferase activity. The incorporation of [1-¹⁴C]acetate into cholesterol and dolichol was strongly inhibited in liver slices of cholesterol-fed animals. In contrast, while incorporation of [2-¹⁴C]mevalonate into cholesterol was also inhibited by approximately 90%, incorporation of this precursor into dolichol was stimulated fourfold. The increased incorporation of mevalonate into dolichol was consistent with a threefold increase in the activity of the dolichol phosphate-dependent mannosyl transferase. The possible significance of these differences is discussed.     

Absolute rates of adenosine formation during ischaemia in rat and pigeon hearts.

1. The activities of ecto- and cytosolic 5'-nucleotidase (EC 3.1.3.5), adenosine kinase (EC 2.7.1.20), adenosine deaminase (EC 3.5.4.4) and AMP deaminase (EC 3.5.4.6) were compared in ventricular myocardium from man, rats, rabbits, guinea pigs, pigeons and turtles. The most striking variation was in the activity of the ecto-5'-nucleotidase, which was 20 times less active in rabbit heart and 300 times less active in pigeon heart than in rat heart. The cytochemical distribution of ecto-5'-nucleotidase was also highly variable between species. 2. Adenosine formation was quantified in pigeon and rat ventricular myocardium in the presence of inhibitors of adenosine kinase and adenosine deaminase. 3. Both adenosine formation rates and the proportion of ATP catabolized to adenosine were greatest during the first 2 min of total ischaemia at 37 degrees C. Adenosine formation rates were 410 +/- 40 nmol/min per g wet wt. in pigeon hearts and 470 +/- 60 nmol/min per g wet wt. in rat hearts. Formation of adenosine accounted for 46% of ATP plus ADP broken down in pigeon hearts and 88% in rat hearts. 4. The data show that, in both pigeon and rat hearts, adenosine is the major catabolite of ATP in the early stages of normothermic myocardial ischaemia. The activity of ecto-5'-nucleotidase in pigeon ventricle (16 +/- 4 nmol/min per g wet wt.) was insufficient to account for adenosine formation, indicating the existence of an alternative catabolic pathway.     

Biochemical correlates of selection for weight-for-age in chickens: twenty-fold higher muscle ornithine decarboxylase levels in modern broilers

Summary Little is known about the biochemical correlates of selection for growth in farm or laboratory animals, or the identity of the gene products affected or produced by trait-genes. Modern broiler chickens have about 8-fold greater breast muscle mass than layer chickens at 7 weeks of age and over 2-fold greater breast muscle mass than their 1972 counterparts. This increase in muscle mass is associated with over 20-fold higher levels of ornithine decarboxylase (ODC) in broiler chickens at 1 week of age as compared with layer strain chickens; there is a comparable increase in a relaxed-selection strain of broilers. The increase in ODC levels is larger than the differences in muscle or body weight between broilers and layers at 7 weeks of age, occurs at an age when there is no difference in weights between the strains and precedes the major growth spurt. Increases in ODC levels and hence polyamine synthesis have been associated with, and usually precede, rapid growth and cell proliferation in a wide range of cell types and organisms in response to many different stimuli. Therefore, the correlation of ODC levels with genetic differences in muscle growth make it worth investigating the control of ODC gene expression in these strains.     

Function of arginine-166 in the active site of Escherichia coli alkaline phosphatase

The function of arginine residue 166 in the active site of Escherichia coli alkaline phosphatase was investigated by site-directed mutagenesis. Two mutant versions of alkaline phosphatase, with either serine or alanine in the place of arginine at position 166, were generated by using a specially constructed M13 phage carrying the wild-type phoA gene. The mutant enzymes with serine and alanine at position 166 have very similar kinetic properties. Under conditions of no external phosphate acceptor, the kcat for the mutant enzymes decreases by approximately 30-fold while the Km increases by less than 2-fold. When kinetic measurements are carried out in the presence of a phosphate acceptor, 1.0 M Tris, the kcat for the mutant enzymes is reduced by less than 3-fold, while the Km increases by more than 50-fold. For both mutant enzymes, in either the absence or the presence of a phosphate acceptor, the catalytic efficiency as measured by the kcat/Km ratio decreases by approximately 50-fold as compared to the wild type. Measurements of the Ki for inorganic phosphate show an increase of approximately 50-fold for both mutants. Phenylglyoxal, which inactivates the wild-type enzyme, does not inactivate the Arg-166----Ala enzyme. This result indicates that Arg-166 is the same arginine residue that when chemically modified causes loss of activity [Daemen, F.J.M., & Riordan, J.F. (1974) Biochemistry 13, 2865-2871]. The data reported here suggest that although Arg-166 is important for activity is not essential. The analysis of the kinetic data also suggests that the loss of arginine-166 at the active site of alkaline phosphatase has two different effects on the enzyme. First, the binding of the substrate, and phosphate as a competitive inhibitor, is reduced; second, the rate of hydrolysis of the covalent phosphoenzyme may be diminished.     

Analysis of T cell subsets in Graves' disease: alterations associated with carbimazole.

Conflicting data on subpopulations of peripheral blood lymphocytes in patients with autoimmune disease largely reflect variations in methods of study. An investigation was therefore conducted aimed at avoiding this difficulty. Serial samples of peripheral blood mononuclear cells from 42 patients with hyperthyroid Graves'' disease were collected at monthly intervals before, during, and for 12 months after a six month course of carbimazole. Samples were stored in liquid nitrogen until completion of the study, when they were thawed and all samples from each patient analysed within the same assay using mouse monoclonal antibodies to human cell subsets and a fluorescence activated cell sorter. Proportions of cytotoxic/suppressor (OKT8) positive cells before treatment (mean 17.4 (SEM 0.8)%) were significantly lower (p less than 0.001) than those in normal controls (29.8 (1.9)%; n = 10) and returned to normal by the end of treatment. In contrast, the proportions of activated T cells (OKIa-OKM1) were significantly raised before treatment as compared with normal (14.4 (0.6)% versus 4.6 (0.8)%; p less than 0.001) and fell to normal by the end of treatment. Proportions of OKT3 and OKT4 positive T cells remained unchanged throughout treatment and in the succeeding 12 months. In patients who relapsed after treatment there was a rise in the proportion of activated T cells and a fall in OKT8 positive T cells, which returned towards normal with retreatment. The explanation for the alterations in numbers of circulating T cells remains to be determined but they may provide a means for predicting more accurately the outcome of Graves'' disease after treatment with carbimazole.