Lipoprotein lipase activity in cultured macrophage cell line J7742 and its increase in variants deficient in adenylate cyclase and cyclic AMP-dependent protein kinase

Three macrophage cell lines, J774₂, CT₂ and J7H₁ were compared with respect to synthesis and secretion of lipoprotein lipase. The enzyme activity measured was characterized as lipoprotein lipase on the basis of serum dependence and inhibition by 1 M NaCl. Enzyme activity in all three lines increased with time in culture and the highest activity was found in the medium of the CT₂ line which is adenylate cyclase deficient while that in the J7H₁ line, cyclic AMP-dependent protein kinase deficient, was intermediate. The half life of the enzyme activity in conditioned medium from all three lines was 30–40 min, suggesting that the different levels of activity observed do represent different levels of enzyme production by the cells. About 80% of the lipoprotein lipase activity from all three lines was present in the medium and 50–70% of cellular activity could be released into the medium by a 3-min exposure to heparin. In addition, 24 h incubation with heparin enhanced enzyme secretion in all three lines. To determine the role of cyclic AMP in the regulation of lipoprotein lipase activity use was made of dibutyryl cAMP, methyl isobutylxanthine (IBMX) and cholera toxin. These agents strikingly depressed lipoprotein lipase activity in the J774₂ line but only dibutyryl cAMP was active in the CT₂ line (adenylate cyclase deficient). In the J7H₁ (protein kinase deficient) line there was no response to dibutyryl cAMP or IBMX over the first 4 h of incubation. Addition of these agents did not affect total cell protein synthesis. The present findings indicate that in the intact cells changes in cyclic AMP levels are associated with a change in the activity of lipoprotein lipase.     

Role of caveolin-1 and cholesterol in transmembrane fatty acid movement

We have created by transfection a series of HEK 293 cell lines that express varying amounts of caveolin-1 to test the possible effect of this protein on the transport and metabolism of long chain fatty acids (FA) in cells with this gain of function. We used an extracellular fluorescent probe (ADIFAB) to monitor binding of exogenous FA to the plasma membrane and an intracellular pH probe to monitor FA equilibration across the plasma membrane. Real-time fluorescence measurements showed rapid binding of oleic acid to the extracellular side of the plasma membrane and a rapid translocation across the lipid bilayer by the flip-flop mechanism (<5 s). Two cell lines expressing levels of caveolin-1 roughly comparable to that of adipocytes, which have a very high level of endogenous expression of caveolin-1, showed a relatively slow change in intracellular pH (t(1/2) < 100 s) in addition to the fast changes in fluorescence. We interpret this additional second phase to represent translocation of additional FA from the outer to inner leaflet of the plasma membrane. The slower kinetics could represent either slower flip-flop of FA across highly organized, rigid regions of the plasma membrane or binding of FA to caveolin-1 in the intracellular leaflet of the plasma membrane. The kinetics of palmitate and elaidate (a trans FA) transmembrane movement were identical to that for oleate. These results were observed in the absence of the putative FA transport protein, CD36, and in the absence of any changes in expression of fatty acid transport proteins (FATP) 2 and 4, and are in direct correlation with increased cellular free cholesterol content. FA metabolism was slow in all cell lines and was not enhanced by caveolin-1 expression. We conclude that transport of FA across the plasma membrane is modulated by caveolin-1 and cholesterol and is not dependent on the putative FA transport proteins CD36 and FATP.     

Analysis of calcium responses mediated by the A3 adenosine receptor in cultured newborn rat cardiac myocytes

Intracellular calcium signaling cascade induced by adenosine A(3) receptor activation was studied in this work. It was found that adenosine A(3) receptor activation (and not A(1) or A(2A) adenosine receptors activation) leads to an increase in cytosolic calcium and its further extrusion. A selective A(3) agonist Cl-IB-MECA (2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide) induced an increase in cytoplasmic calcium in a dose-dependent manner, and was independent on extracellular calcium. The Ca(2+) signal in newborn cardiomyocytes, induced by A(3) receptor activation, is dependent on a pertussis toxin-sensitive G-protein. The action of Cl-IB-MECA was not inhibited by an inhibitor of phospholipase C (PLC), and by antagonists to inositol 1,4,5-trisphosphate (IP(3)) receptor. In contrast, inhibition of ryanodine receptor prevented calcium elevation induced by this agonist. It was shown that extrusion of the elevated cytosolic Ca(2+) was achieved via activation of sarcoplasmic reticulum (SR) Ca(2+)-reuptake and of sarcolemmal Na(+)/Ca(2+) exchanger (NCX). The increase in the SR Ca(2+)-uptake and NCX Ca(2+) efflux were sufficient not only for compensation of Ca(2+) release from SR after A(3) receptor activation, but also for an effective prevention of extensive increase in intracellular Ca(2+) and may provide mechanism against cellular Ca(2+) overload. In cells with elevated [Ca(2+)](i) (due to increase of [Ca(2+)](o)), adenosine or Cl-IB-MECA decreased the [Ca(2+)](i) toward diastolic control level, whereas agonist of A(1) receptor was ineffective. The protective effect of A(3) receptor agonist was abolished in the presence of selective A(3) receptor antagonist MRS1523.     

VASCULARIZATION OF ARTICULATED CHENOPODIACEAE AND THE NATURE OF THEIR FLESHY CORTEX

Fahn , A. (Hebrew U., Jerusalem.), and Tova Arzee . Vascularization of articulated Chenopodiaceae and the nature of their fleshy cortex. Amer. Jour. Bot. 46(5): 330–338. Illus. 1959.—The primary vascular system and venation pattern of the following chenopodiacean species with articulated forms were examined: Arthrocnemum glaucum, Salicornia fruticosa, S. herbacea, Halocnemum strobilaceum, Anabasis articulata, A. haussknechtii, A. setifera, taloxylon persicum, H. salicornicum. On the basis of the primary vascular system, classification into 2 types was possible: (a) Salicornia-Arthrocnemum type, in which the number of the stelar strands alternates between 6 and 8; and (b) Anabasis type, in which the number alternates between 4, 6 and 8. The latter type is assumed to be derived from the former. Similarly, on the basis of the venation pattern of the cortex and reduced leaves, 2 types were recognized: (a) Salicornia-Arthrocnemum type, in which the cortical network is connected with the lateral branches of the leaf strands only; and (b) Anabasis type, in which the network is connected with both foliar and stelar strands. The view of the foliar origin of the cortex, as postulated by previous authors, is refuted and the present authors consider the fleshy tissue of the internode to be true cortex.     

The Involvement of Immune Semaphorins in the Pathogenesis of Inflammatory Bowel Diseases (IBDs)

ResultsThe percentage (%) of T regulatory cells (Tregs) expressing sema3A in patients with active CD (64.5% ±14.49%) and active UC (49.8% ±16.45%) was significantly lower when compared to that of healthy controls (88.7% ±3.6%, p< 0.001 and p< 0.0001, respectively). This expression was seen to be in negative correlation with CD activity. Serum levels of Sema4A were significantly lower in patients with CD and UC when compared to that of controls (5.69±1.48ng\ml for CD, 5.26±1.23 ng/ml for UC patients vs 9.74±2.73ng/ml for normal controls, P<0.001). Sema4A was highly expressed in lymphocytes of the lamina propria of CD and UC patients but absent in patients with diverticulitis or in normal individuals.ConclusionsAltered % of Tregs expressing sema3A in patients with inflammatory bowel diseases (IBD) is partially responsible for their failure in preventing CD4+ effector T cell induced inflammation in IBD in peripheral blood. The increased expression of sema4A in bowel biopsies from CD and UC patients is suggestive of its central role in regulating local tissue inflammation in the bowel.     

Relationship of glutamate dehydrogenase levels to free amino acids, amides and ammonia in excised oat leaves

Levels of glutamate dehydrogenase (GDH) increase 12 fold indetached oat leaves during 96 hr incubation with 15 mM ammonia.The slight elevation of GDH detected within the first 24 hrwas followed by increasing rates of enzyme production in subsequentperiods. Rapid increases in free ammonia and amino acids witha marked synthesis of glutamine and decreased of glutamate andaspartate were observed during the initial stages of ammoniumassimilation. (Received December 26, 1973; )     

The Efficacy of an Immunoisolating Membrane System for Islet Xenotransplantation in Minipigs

Developing a device that protects xenogeneic islets to allow treatment and potentially cure of diabetes in large mammals has been a major challenge in the past decade. Using xenogeneic islets for transplantation is required in light of donor shortage and the large number of diabetic patients that qualify for islet transplantation. Until now, however, host immunoreactivity against the xenogeneic graft has been a major drawback for the use of porcine islets. Our study demonstrates the applicability of a novel immunoprotective membrane that allows successful xenotransplantation of rat islets in diabetic minipigs without immunosuppressive therapy. Rat pancreatic islets were encapsulated in highly purified alginate and integrated into a plastic macrochamber covered by a poly-membrane for subcutaneous transplantation. Diabetic Sinclair pigs were transplanted and followed for up to 90 days. We demonstrated a persistent graft function and restoration of normoglycemia without the need for immunosuppressive therapy. This concept could potentially offer an attractive strategy for a more widespread islet replacement therapy that would restore endogenous insulin secretion in diabetic patients without the need for immunosuppressive drugs and may even open up an avenue for safe utilization of xenogeneic islet donors.     

High myopia caused by a mutation in LEPREL1, encoding prolyl 3-hydroxylase 2

Autosomal-recessive high-grade axial myopia was diagnosed in Bedouin Israeli consanguineous kindred. Some affected individuals also had variable expressivity of early-onset cataracts, peripheral vitreo-retinal degeneration, and secondary sight loss due to severe retinal detachments. Through genome-wide linkage analysis, the disease-associated gene was mapped to ～1.7 Mb on chromosome 3q28 (the maximum LOD score was 11.5 at θ = 0 for marker D3S1314). Sequencing of the entire coding regions and intron-exon boundaries of the six genes within the defined locus identified a single mutation (c.1523G>T) in exon 10 of LEPREL1, encoding prolyl 3-hydroxylase 2 (P3H2), a 2-oxoglutarate-dependent dioxygenase that hydroxylates collagens. The mutation affects a glycine that is conserved within P3H isozymes. Analysis of wild-type and p.Gly508Val (c.1523G>T) mutant recombinant P3H2 polypeptides expressed in insect cells showed that the mutation led to complete inactivation of P3H2.