Analysis of the DR β chains from two DRw6 cell lines (WT46 and WT52): Recombination in vivo may have generated new haplotypes

The HLA-DRw6 haplotype of the class II major histocompatibility complex (MHC) antigens exhibits unusual complexity and cannot be uniquely typed serologically. The DR chains expressed by consanguineous homozygous DRw6 typing cells WT46 and WT52 were biochemically analyzed using three monoclonal antibodies (mAb) that recognize denatured DR chains. The results of isoelectric focusing and N-terminal sequencing demonstrate that each DRw6 B-cell line expresses two DR chains. Evidence of an exchange of mAb epitopes involving the two DR chains of one of these cell lines was obtained and may be explained by a recombinational mechanism involving reciprocal exchange of genetic segments of the DR chains, one of which may encode the putative DRw6 chain and the other the chain carrying the MT2 allotypic determinant. Since a recombinational hot spot has been shown to occur uniquely in the mouse MHC within the E gene, the occurrence of a recombination within the human homolog, DR(MT2) , could reflect some specific feature of this MHC region. Comparison of the DR chains of the WT46 and WT52 cell lines with those of a third DRw6 cell line, LB, suggests that two alleles of MT2 occur.Abbreviations used in this paper IEF isoelectric focusing - mAb monoclonal antibody - MHC major histocompatibility complex - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Regional Cerebral Glucose Utilization During Insulin-Induced Hypoglycemia in Unanesthetized Rats

Regional cerebral glucose utilization (rCMRgl) was studied during insulin-induced hypoglycemia in unanesthetized rats. Rats were surgically prepared using halothane and nitrous oxide anesthesia and allowed 5 h to recover from the anesthesia before rCMRgl was measured. The rCMRgl was measured using [6-14C]glucose in a normoglycemic control group and two hypoglycemic groups, A (30 min after insulin injection) and B (2 h after insulin injection). The mean plasma glucose level was 7.03 mumol/ml in the normoglycemic group, 1.96 mumol/ml in hypoglycemic group A, and 1.40 mumol/ml in hypoglycemic group B. The rCMRgl in hypoglycemic group A decreased 8-18% in 17 brain regions measured; five changes were statistically significant. The rCMRgl in hypoglycemic group B decreased significantly in all but one of the brain regions measured; the decrease ranged from 15% in the pyramidal tract to 36% in the motor and auditory cortices. The rCMRgl in every brain region decreased when the plasma glucose level fell below 1.5-2.5 mumol/ml. No brain region could maintain rCMRgl at plasma glucose concentrations lower than predicted by regional glucose influx described in previous studies. Glucose utilization in all brain regions appears to be limited by the influx of glucose.     

Brain pyridoxal kinase. Purification and characterization

Pyridoxal kinase has been purified 9000-fold from sheep brain. The purification procedure involves ammonium sulphate fractionation, DEAE-cellulose chromatography, affinity chromatography and Sephadex G-100 gel filtration. The final chromatography step yields a homogeneous preparation of high specific activity with a pI of 5. The molecular mass of the native enzyme was estimated to be approximately 80 kDa by 10-25% gradient polyacrylamide gel electrophoresis and Sephadex G-200 gel filtration. The subunit molecular mass was determined by sodium dodecyl sulphate (SDS)/polyacrylamide gel electrophoresis to be 40 kDa compared with a series of molecular mass standards. This indicates that pyridoxal kinase is a dimeric enzyme. Further results obtained from electron microscopy, using a negative staining technique, provide evidence that pyridoxal kinase exists as a dispherical subunit structure.     

Echinostoma revolutum: resistance to secondary and superimposed infections in mice

A complete or almost complete resistance (94-100%) to a superimposed Echinostoma revolutum infection existed in mice harboring 20-, 30-, and 40-day-old infections in the range of 2-4 to 30-35 worms, but no resistance was found at challenge Day 10. A similar high level of resistance (85-100%) also existed in mice for at least 6 weeks after natural expulsion of a primary 6 metacercarial infection and for at least 5 weeks after anthelmintic termination of a 30-day-old 20 metacercarial infection. Thymus-deficient nude mice failed to develop resistance to a superimposed infection, and the resistance in normal mice was inhibited by corticosteroid treatment. These findings are all in favor of a host immune response being responsible for the resistance against both a secondary and a superimposed infection. Nearly all the worms of a superimposed infection were, in resistant mice, expelled prior to 24 hr following infection (rapid expulsion), and the few worms circumventing this early expulsion persisted for at least 8 days. Newly excysted juvenile worms implanted intraduodenally into resistant mice were rejected to the same degree as juvenile worms from an oral metacercarial infection indicating that the newly excysted juvenile worms are the target of the host immune response. However, 7-day-old worms implanted intraduodenally into resistant mice survived indicating that adaptation to the host immune response had occurred. In conclusion, this host-parasite model is an example of concomitant immunity because the immunological mechanism responsible for the expulsion of the superimposed infection had no effect on the number of primary worms present.     

Binding of plasminogen to extracellular matrix

We have previously demonstrated that plasminogen immobilized on various surfaces forms a substrate for efficient conversion to plasmin by tissue plasminogen activator (t-PA) (Silverstein, R. L., Nachman, R. L., Leung, L. L. K., and Harpel, R. C. (1985) J. Biol. Chem. 260, 10346-10352). We now report the binding of human plasminogen to the extracellular matrix synthesized in vitro by cultured endothelial cell monolayers. The binding was specific, saturable at plasma plasminogen concentrations, reversible, and lysine-binding site-dependent. Functional studies demonstrated that matrix immobilized plasminogen was a much better substrate for t-PA than was fluid phase plasminogen as shown by a 100-fold decrease in Km. Activation of plasminogen by t-PA and urokinase on the matrix was equally efficient. The plasmin generated on the matrix, in marked contrast to fluid phase, was protected from its fast-acting inhibitor, alpha 2-plasmin inhibitor. Matrix-associated plasmin converted bound Glu- into Lys-plasminogen, which in turn is more rapidly activated to plasmin by t-PA. The extracellular matrix not only binds and localizes plasminogen but also improves plasminogen activation kinetics and prolongs plasmin activity in the subendothelial microenvironment.     

Acyl-CoA-binding protein in the rat. Purification, binding characteristics, tissue concentrations and amino acid sequence.

Acyl-CoA-binding protein (ACBP) was purified from rat liver. The Mr was determined as 9932 +/- 10 by mass spectrometry and calculated as 9937.8 from the sequence. The protein binds acyl-CoA esters (C8-C16) with high affinity, but was unable to bind fatty acids. ACBP was found mainly (86%) in the soluble fraction, and the concentration was highest in liver, 5-6 micrograms/mg of soluble protein. The complete primary structure was determined by a combination of gas-phase Edman degradations and mass spectrometry. Extensive use of 252Cf plasma-desorption mass spectrometry facilitated the identification and verification of peptides. Comparison with the previously determined sequence of bovine acyl-CoA-binding protein revealed a very strong sequence similarity (83%), and all of the differences could be accounted for by single base changes.     

15-Hydroxy-eicosatetraenoic acid (15-Hete) inhibits carragheenan-induced experimental arthritis and reduces synovial fluid leukotriene B4 (LTB4)

15-Hydroxy-eicosatetraenoic acid (15-HETE), a product of arachidonic acid, has no proinflammatory capacity, but can inhibit the formation and the chemotactic response of neutrophils to leukotriene B₄ (LTB₄), a potent mediator of inflammation. The purpose of the present study was to determine whether intraarticular administration of 15-HETE in carragheenan-induced acute arthritis might decrease the levels of LTB₄ in synovial fluid and modify the arthritis. A bilateral acute knee joint arthritis was established in 7 dogs by intraarticular injections of carragheenan every third day. To the right joints, 15-HETE was administered both concomitantly with the carragheenan injections and continously via an osmotic pump. In samples of synovial fluid obtained on day 0, 3 and 10 PGE₂ and LTB₄ were determined using reversed phase high performance liquid chromatography combined with radioimmunoassays and neutrophil chemokinesis. In the presence of 15-HETE the clinical severity of arthritis was significantly reduced and the volume synovial effusate was decreased on an average by 42%. Furthermore, the relative number of neutrophils in histological sections of synovial tissue was decreased by 58%. Intaarticular carragheenan injection induced LTB₄ formation, and maximum levels were obtained on day 3 (279.2 ± 148.2 pg/joint). PGE₂ was also present on a day 3, but maximum levels were detected on day 10 (9.5 ± 4.8 ng/joint). In joints injected with both carragheenan and 15-HETE the levels of LTB₄ on days 3 and 10 were inhibited by 90% and 83%, respectively. For PGE₂ a small but significant decrease was found on both day 3 and on day 10. These results show that LTB₄ may be an important mediator of acute arthritis induced by carragheenan in dogs, and that intraarticular administration of 15- HETE can modify this arthritis by inhibiting LTB₄ formation.     

Effects of temperature on growth rates of fungi from subantarctic Macquarie Island and Casey,Antarctica

Summary The linear growth rates of fungal isolates were measured on agar plates at temperatures ranging from 4° to 35°C. Fungi tested included the major fungal colonizers of leaves and litter of the three dominant plant species on subantarctic Macquarie Island, and major fungal species associated with plant and soil communities near Australia's Casey Station on the Antarctic Continent. All fungi grew at 4°C and were classified as psychrotrophs. Maximum growth rates were recorded at temperatures of 10° to 20°C for 13 of the 15 isolates from Macquarie Island and for all six isolates from Casey. Most of the leaf colonizing fungi from Macquarie Island had optimum growth temperatures of 15°C whereas all litter fungi from Macquarie Island and Casey fungi except Thelebolus microsporus had optimum growth temperatures of 20°C or above. Maximum growth of all species was at temperatures above those normally prevailing in their natural environments, with most species growing at 4°C at between 10% and 30% of their maximum rates. However, microclimatic effects may have resulted at times in temperatures near their growth optima. The highest growth rates at 4°C were recorded for Phoma spp. 1 and 2, Phoma exigua and Mortierella gamsii from Macquarie Island and Mortierella sp. 1 from Casey. Thelebolus microsporus and sterile sp. G from Casey also grew relatively fast at 4°C, and these species, and Phoma sp. 3 and Phoma exigua from Macquarie Island had the lowest Q-₁₀ values for the temperature range 4° to 15°C.     

Periportal zonation of the cytosolic acetyl-CoA synthetase of male rat liver.

Several important metabolic functions of the mammalian liver have been shown to be located in zones with respect to the complex microcirculation of the organ. The zonal distribution of the cytosolic component of the acetyl-CoA synthetase activity has been investigated using the dual-digitonin-pulse-perfusion technique, which allows highly zone-selective sampling of cytosol from the periportal and perivenous zone of rat liver. Approximately 80% of the cytosolic enzymes are eluted from the hepatocytes in the periportal and perivenous sub-zones affected by digitonin, while less than 1% of the glutamate dehydrogenase activity (a marker enzyme of the mitochondrial compartment) is eluted. A twofold higher activity of the cytosolic form of acetyl-CoA synthetase is found in the periportal zone compared to the perivenous zone in fed male rats. Following a fasting/refeeding transition, this activity gradient is abolished in a manner similar to that observed for the enzyme acetyl-CoA carboxylase. Since the latter enzyme is utilizing the product of acetyl-CoA synthetase, acetyl-CoA, the similarity in the observed regulation suggests a functional coupling between cytosolic acetate activation and fatty-acid synthesis.