Acylation of 1-alkenyl-glycerophosphocholine and 1-acyl-glycerophosphocholine in guinea pig heart.

The deacylation-reacylation process has been shown to be an important pathway for phospholipids to attain the desired acyl groups at the C-2 position. The acylation of 1-acyl-glycerophosphocholine (-GPC) in mammalian hearts has been well documented, but the acylation of 1-alkenyl-GPC has not been described. In this paper, we demonstrate the presence of acyl-CoA: 1-alkenyl-GPC acyltransferase for the acylation of 1-alkenyl-GPC in mammalian hearts; the highest activity is found in guinea pig heart. The guinea pig heart 1-alkenyl-GPC acyltransferase has only 10-40% of the 1-acyl-GPC acyltransferase activity, and both activities are located in the microsomal fraction. However, these two enzymes respond differently to cations, detergents and heat treatment, and the two enzymes also display different acyl specificity. Kinetic studies indicate that both reactions could not be accommodated by the same catalytic site. The results provide strong evidence that the two activities are from separate and distinct proteins. The specificity of 1-alkenyl-GPC acyltransferase for unsaturated species of acyl-CoA may play an important role in the maintenance of the high degree of unsaturated acyl groups found in guinea pig heart plasmalogens.     

Comparison of anti-inflammatory compounds in the carrageenan induced paw edema model and the reversed passive Arthus model utilizing the same animal

In order to better define antiinflammatory activity in new agents, a test was devised utilizing both carrageenan induced paw edema and the reversed passive Arthus reaction in the same animal. The model of carrageenan induced rat paw edema is a standard laboratory assay used to predict classical "aspirin-like" antiinflammatory molecules. The reversed passive cutaneous Arthus reaction involves precipitating antigen-antibody complexes, complement and infiltrating polymorphonuclear leukocytes (PMN's) and can be used to identify agents that affect one or more of these factors specifically. Antiinflammatory compounds were given orally one hour prior to the administration of carrageenan and goat anti-rat serum. Comparisons were made between several non-steroidal compounds and the steroid hydrocortisone. All of the compounds tested gave good carrageenan activity, but only hydrocortisone produced significant Arthus lesion inhibition in this assay.     

The acylation of lysophosphoradylglycerocholines in guinea-pig heart mitochondria.

The importance of the deacylation-reacylation pathway for attaining the desired fatty acid composition in microsomal phospholipids has been well established. It is not clear, however, whether this mechanism is of equal importance in mitochondria. The absence of acyltransferase activity in mammalian heart mitochondria has been reported in a number of studies. In the present study we report the presence of acyltransferase activities for lysophosphoradylglycerocholines in guinea-pig heart mitochondria. This enzyme showed properties that were considerably different from those of the microsomal enzymes. Of all the acyl-CoAs tested (C18:0, C18:1, C18:2 and C20:4) the mitochondrial enzyme utilized only linoleoyl-CoA as fatty acyl donor and utilized both 1-acyl-sn-glycero-3-phosphocholine and 1-alkenyl-sn-glycero-3-phosphocholine as fatty acyl acceptors. The presence of significant quantities of fatty acids other than linoleate at the C-2 position of mitochondrial acylglycerophosphocholines, coupled with the specificity of the enzyme for linoleoyl-CoA, suggest that, in addition to reacylation, other mechanisms play a significant role in producing the molecular composition of these phospholipids found in the mitochondria.     

Choline glycerophospholipid biosynthesis in the guinea pig heart

The aims of this study were to (i) elucidate the biosynthetic pathways for the formation of plasmenylcholine in the mammalian heart and (ii) investigate whether the control of choline glycerophospholipid production is different in hearts with high plasmenylcholine content. Guinea pig hearts were used throughout this study, since 34% of the cardiac choline glycerophospholipids in this species is present in the plasmenylcholine form. By perfusion of the guinea pig heart in the Langendorff mode with labeled choline, we demonstrated that the majority of plasmenylcholine in the heart was synthesized via the CDP-choline pathway. The ability of the heart to form plasmenylcholine from CDP-choline and 1-alkenyl-2-acylglycerol was also shown. We postulate that 1-alkenyl-2-acylglycerol in the guinea pig heart might originate from the hydrolysis of plasmenylethanolamine. In mammalian liver and other tissues, the CDP-choline pathway is the major pathway for phosphatidylcholine biosynthesis and the rate-limiting step is catalyzed by CTP:phosphocholine cytidylyltransferase. The results obtained from the present study support this supposition. In addition, evidence was obtained indicating that phosphorylation of choline by choline kinase in the CDP-choline pathway may also be rate limiting. Although the involvement of choline kinase as a rate-limiting enzyme in the CDP-choline pathway has been shown in a number of cell cultures, the rate-limiting role of this enzyme in intact mammalian organs has not been previously reported. The rationale for the presence of more than one rate-limiting step in the CDP-choline pathway in the guinea pig heart remains undefined.     

Occurrence of 3-O-( -D-glucopyranosyluronic acid)-D-galactose in the capsular polysaccharide of Klebsiella K-type 20

Partial hydrolysis and methylation studies have been used to show that the capsular polysaccharide of Klebsiella K-type 20 is composed of a main chain of d-mannose and d-galactose units with the aldobiouronic acid attached as a side chain to d-mannose     

Inhibition of protein synthesis in Krebs 2 ascites cells and cell-free systems by phenylalanine and its effect on leucine and lysine in the amino acid pool

1. The inhibition of incorporation of ¹⁴C-labelled amino acids into protein of whole cells by phenylalanine has been reproduced in a cell-free system. In both cases only the l-isomer was inhibitory. 2. The effect of phenylalanine on incorporation of [¹⁴C]leucine and [¹⁴C]lysine into protein was different in both whole cells and cell-free systems. 3. In whole cells inhibition of incorporation of leucine at 2·5μg./ml. was very rapid, but when the concentration was increased to 100μg./ml. the inhibition was not apparent for about 1hr. The kinetics of inhibition of lysine was the same at both these concentrations and was similar to that found with leucine at 100μg./ml. 4. Neither a lower specific radioactivity of the two amino acids in the pool nor a decrease in their pool size could be consistently related with inhibition of protein synthesis. 5. In the cell-free system l-phenylalanine inhibited the incorporation of leucine but not of lysine. 6. Charging of transfer RNA by leucine was markedly decreased in the presence of phenylalanine, whereas charging of transfer RNA by lysine was not.     

Maturation of the hypothalamo-neurohypophysial system

Summary Sections of the hypothalamus, median eminence and pituitary from fetal and neonatal rats were examined with the immunoperoxidase staining technique and light microscopy. Purified antisera raised against vasopressin and oxytocin, and antisera cross-reactive with rat neurophysin were used to localize these antigens in the hypothalamo-neurohypophysial system (HNS). Neurophysin was detected throughout the HNS of the 18-day fetal rat. Vasopressin was present in the hypothalamus and pituitary of the 19-day fetus, and in the median eminence of the 4-day neonate. Oxytocin was not detected in the pituitary until 1–2 days after birth, in the hypothalamus after 4 days, and in the median eminence after 8 days. During the first days after birth the supraoptic nucleus was more mature than the paraventricular nucleus. The HNS did not approach maturity until at least 7 days after birth. The relative maturity of the supraoptic nucleus compared with the paraventricular nucleus, and the detection of vasopressin before oxytocin are evidence for the one-neuron-one-hormone theory. The data do not exclude the possibility that the fetal hypothalamo-neurohypophysial system, and perhaps the fetal hormone, vasotocin, affect the initiation and course of parturition.This work was financed by the Medical Research Council of New Zealand     

Concanavalin A-induced apoptosis in murine macrophages through a Ca(2+)- independent pathway

Concanavalin A (ConA), normally a mitogen of T lymphocytes, was found to induce apoptosis or programmed cell death in murine peritoneal macrophages. The following observations support this assertion: 1) incubation of peritoneal macrophages or cultured PU5-1.8 macrophage cells with ConA caused a dose- and time-dependent reduction of mitochondrial dehy-drogenase activity as measured by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, 2) treatment of cells with ConA induced formation of apoptotic bodies as seen under the confocal laser scanning microscope, 3) challenge of cells with ConA produced a considerable amount of cell debris with DNA content next to G0 phase as revealed by flow cytometry and 4) ConA was able to elicit DNA fragmentation in these cells. The involvement of Ca(2+) in mediating the apoptosis was studied in single cells by confocal laser scanning microscope using the Ca(2+) fluorescence dye, fluo-3. Our results show that ConA induced an immediate rise of intracellular free Ca(2+) concentration as well as opening of Ca(2+) channels on cell surface. But when the cells were treated with 1,2-bis(o-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid/AM (BAPTA/AM), a Ca(2+) chelator, to buffer the rise of internal Ca(2+), ConA still caused DNA fragmentation. Furthermore, injection of Ca(2+) into the cell with ionomycin had no stimulatory effect on DNA fragmentation. These results suggest that Ca(2+) changes induced by ConA are not a prerequisite for apoptosis in macrophages.     

Adipsin and an endogenous pathway of complement from adipose cells.

The alternative complement pathway is best known for its role in humoral suppression of infectious agents. We have previously shown that adipose cells synthesize adipsin, the mouse homolog of human complement factor D, and that the synthesis of this protein is reduced in several rodent models of obesity. We show here that adipose cells and adipose tissue also synthesize two other essential components of the alternative pathway of complement, factors C3 and B, and activate the proximal portion of this pathway. This activation occurs in the absence of infectious agents and without triggering the terminal, lytic part of this pathway. We demonstrate the production in vitro of several polypeptides characteristic of complement activation that are known to have potent biological activities, including the anaphylatoxin C3a. Cultured adipocytes require stimulation with cytokines to activate complement, while explanted adipose tissue has no such requirement. The adipose tissue from obese mice is deficient in this localized activation of the alternative pathway. These results indicate that complement activation occurs in a localized site, adipose tissue, in normal mice and is impaired in a state of metabolic dysfunction. This suggests a novel function for the proximal portion of this complement pathway related to adipose cell biology or energy balance.