Metabolic Changes in Nicotinamide Adenine Dinucleotide in Response to Anthrax Toxin

Bacillus anthracis produces a toxin both in vitro and in vivo which, when injected intravenously into rats, brings about the death of the animals accompanied by gross pulmonary edema. Lung tissue removed prior to death showed, in vitro, a 30% reduction in overall oxidative metabolism (Q(o2)), whereas the nicotinamide adenine dinucleotide (NAD)-independent succinic dehydrogenase remained unaffected. The NAD concentration in the lungs of injected animals was reduced by 50%. Upon addition of NAD, the Q(o2) of lung tissue from injected animals rose to control values. At 45 min after toxin injection, the serum lactate concentration began to rise, showing about a 3.5-fold increase over controls after 75 min. No changes occurred in the pyruvate concentration. These changes may be explained by increased use of the pyruvate for glycolytic energy production with further loss of NAD. Additional experiments with liver, spleen, kidney, and brain tissues showed that the toxin-induced reduction of Q(o2) is an effect specific for lung tissue. Brain tissue showed a significant increase in oxidative metabolism upon the addition of the toxin, whereas the other tissues remained unaffected. It is suggested that a principal effect of the toxin is to inhibit, in lung tissue, the regeneration of NAD in the respiratory chain.     

Endothelial and leukocyte forms of IL-8. Conversion by thrombin and interactions with neutrophils

We have recently shown that endothelial cell-derived IL-8 inhibits neutrophil adhesion to IL1-beta-activated human umbilical vein endothelial cell monolayers. IL-8 secreted by T lymphocytes or monocytes has been characterized as a promoter of neutrophil degranulation and chemotaxis. The IL-8 isolated from each of these cell types is a mixture of two IL-8 polypeptides, one consisting of 72 amino acids (herein called [ser-IL-8]72) and the other 77 amino acids (an N-terminal extended form herein called [ala-IL-8]77). IL-8 derived from T lymphocytes and monocytes is predominantly [ser-IL-8]72, whereas endothelial-derived IL-8 is highly enriched (greater than 80%) in [ala-IL-8]77. We address the relationship and activities of these two forms of IL-8 using recombinant proteins expressed by both mammalian cells and Escherichia coli. Thrombin was found to efficiently convert [ala-IL-8]77 to [ser-IL-8]72. In contrast, urokinase and tissue-type plasminogen activator were unable to cleave [ala-IL-8]77, and trypsin generated multiple IL-8 cleavage fragments. In competitive binding assays using 125I[ala-IL-8]77 neutrophils exhibited a twofold preference for [ser-IL-8]72 over [ala-IL-8]77. Both forms of IL-8 inhibited neutrophil adhesion to IL-1-beta-activated HUVEC monolayers by up to 90%. However, [ser-IL-8]72 was approximately 10-fold more potent than [ala-IL-8]77 in these assays (ED50 approximately 0.3 nM for [ser-IL-8]72 vs approximately 3 nM for [ala-IL-8]77. Both forms of IL-8 promoted degranulation of cytochalasin B-treated neutrophils [[ser-IL-8]72 (ED50 greater than 10 nM) was two- to three-fold more potent than [ala-IL-8]77], although in this regard they were less active than FMLP. Our data suggest that [ala-IL-8]77 and [ser-IL-8]72 have qualitatively similar and potentially complex biological activities, and that full activation of IL-8 requires cleavage to the [ser-IL-8]72 form. In the case of inflamed endothelial cells this activation could be mediated by thrombin generated in the procoagulant environment associated with these cells.     

Histamine Stimulation of Cyclic AMP Accumulation in Astrocyte-Enriched and Neuronal Primary Cultures from Rat Brain

Histamine stimulates cyclic AMP accumulation in astrocyte-enriched and neuronal primary cultures from rat brain in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. The response in the astrocyte cultures (Emax = 304 +/- 44% over basal, EC50 = 43 +/- 5 microM) was much higher than in neuronal cultures (Emax = 24 +/- 2%, EC50 = 14 +/- 7 microM). The histamine effect in astrocytes was competitively inhibited by the H2 antagonists cimetidine (Ki = 1.1 +/- 0.2 microM) and ranitidine (Ki = 46 +/- 10 nM) but was insensitive to the H1 antagonist mepyramine (1 microM). The two selective H2 agonists impromidine and dimaprit behaved as partial agonists and showed relative potencies (139 and 0.5, respectively) consistent with an interaction with H2 receptors. The more selective H1 agonist 2-thiazolylethylamine (0.01-1 mM) did not potentiate the response to impromidine (10 microM). Thus, in contrast to what is generally observed in intact cell preparations from brain, the histamine-induced cyclic AMP accumulation in astroglial cells is mediated solely by H2 receptors. The small effect shown in neuronal cultures also appears to be mediated by H2 receptors.     

Serotypes and antibiotic resistance of Verotoxigenic (VTEC) and Necrotizing (NTEC) Escherichia coli strains isolated from calves with diarrhoea

Serotypes and antibiotic resistance of 51 Verotoxigenic (VTEC) and 33 Necrotizing (NTEC) bovine Escherichia coli strains were determined and compared with those shown by 205 non-VTEC non-NTEC strains isolated from the same batch of calves. E. coli untypable for O-antigen represented 47% of the VTEC, 12% of the NTEC and 8.8% of the non-VTEC non-NTEC. Typable VTEC belonged to serotypes 02:K?, 0103:K-, 0104:K?, 0128:K?, 0153:K- and O157:K-:H7, whereas typable NTEC were of serotypes 08:K87, 015:K14, 015:K-, 054:K?, 076:K-, 078:K(80), 088:K?, 0123:K-, 0139:K- and 0153:K-. Non-VTEC non-NTEC showed a wide variety of serotypes which were generally unrelated to those found in VTEC and NTEC. VTEC were resistant to antibiotics at higher rates than NTEC and non-VTEC non-NTEC, and showed also the highest multidrug-resistant pattern. Our results show that bovine VTEC strains belonged to O-groups usually found in human VTEC causing sporadic diarrhoea, haemorrhagic colitis and/or haemolytic uraemic syndrome, such as 02, 0103, 0104, 0153 and especially 0128 and O157. In contrast, bovine NTEC strains belonged to serotypes different from those previously found in necrotizing E. coli strains of human origin.     

Purification and properties of glutamine synthetase from the non-N2-fixing cyanobacterium Phormidium laminosum.

Soluble glutamine synthetase activity (L-glutamate:ammonia ligase, ADP forming, EC 6.3.1.2) was purified to electrophoretic homogeneity from the filamentous non-N2-fixing cyanobacterium Phormidium laminosum (OH-1-p.Cl1) by using conventional purification procedures in the absence of stabilizing ligands. The pure enzyme showed a specific activity of 152 mumol of gamma-glutamylhydroxamate formed.min-1 (transferase activity), which corresponded to 4.4 mumol of Pi released.min-1 (biosynthetic activity). The relative molecular mass of the native enzyme was 602 kilodaltons and was composed of 12 identically sized subunits of 52 kilodaltons. Biosynthetic activity required the presence of Mg2+ as an essential activator, although Co2+ and Zn2+ were partially effective. The kinetics of activation by Mg2+, Co2+, and Zn2+ were sigmoidal, and concentrations required for half-maximal activity were 18 mM (h = 2.2), 6.3 mM (h = 5.6), and 6.3 mM (h = 2.45), respectively. However, transferase activity required Mn2+ (Ka = 3.5 microM), Cu2+, Co2+, or Mg2+ being less effective. The substrate affinities calculated for L-Glu, ammonium, ATP, L-Gln, and hydroxylamine were 15, 0.4, 1.9 (h = 0.75), 14, and 4.1 mM, respectively. Optimal pH and temperature were 7.2 and 55 degrees C for biosynthetic activity and 7.5 and 45 degrees C for transferase activity. The biosynthetic reaction mechanism proceeded according to an ordered three-reactant system, the binding order being ammonium, L-Glu, and ATP. The presence of Mn2+ or Mg2+ drastically affected the thermostability of transferase and biosynthetic activities. Heat inactivation of biosynthetic activity in the presence of Mn2+ obeyed first-order kinetics, with an Ea of 76.8 kcal (ca. 321 kJ) mol-1. Gly, L-Asp, L-Ala, L-Ser and, with lower efficiency, L-Lys and L-Met, L-Lys, and L-Glu inhibited only transferase activity. No cumulative inhibition was observed when mixtures of amino acids were used. Biosynthetic activity was inhibited by AMP (Ki= 7 mM), ADP (Ki= 2.3 mM), p-hydroxymercuribenzoate (Ki= 25 microM), and L-methionine-D, L-sulfoximine (Ki= 2 microM). The enzyme was not activated in vitro by chemically reduced Anabaena thioredoxin. This is the first report of glutamine synthetase activity purified from a filamentous non-N2-fixing cyanobacterium.     

Surface and virulence properties of environmental Vibrio cholerae non-O1 from Albufera Lake (Valencia, Spain).

A total of 140 environmental Vibrio cholerae non-O1 isolates, together with several culture collection strains from both environmental and clinical sources, were studied in relation to hemagglutination, surface hydrophobicity, and the enzymatic, hemolytic, cytotoxic, and enterotoxic activities of their extracellular products. A total of 78 and 62% of the strains produced hemagglutinins and exohemagglutinins, respectively. Four different hemagglutinating and two exohemagglutinating activities were found by using eight sugars in the inhibition assays. Cell-bound mannose-sensitive hemagglutination was detected mainly in chicken blood, whereas fucose-sensitive hemagglutination was recorded only in human blood. Cell-bound hemagglutinin resistant to all sugars tested was the only one related to surface hydrophobicity. The surface properties varied along the growth curves. The non-O1 strains displayed strong enzymatic and hemolytic activities, except for esculin hydrolysis. Of 26 non-O1 isolates selected for cytotoxin and enterotoxin production, 23 showed a wide spectrum of cytotoxic effects on cell lines of poikilothermic and homoiothermic species, but they were weakly enterotoxigenic in the infant mouse test. All extracellular products of cytotoxic strains were proteolytic, lipolytic, and hemolytic, and a high percentage produced hemagglutination of chicken blood. The cytotoxic factors in the non-O1 strains analyzed were not R plasmid mediated.     

Effect of Quin-2 on 45Ca2+ uptake mediated by Na+i/Ca2+o exchange and 45Ca2+ efflux in rat brain synaptosomes: a requirement for [Ca2+]i

The Na+/Ca2+ exchanger of squid axons, barnacle muscle and sarcolemma requires micromolar intracellular calcium for activation in the Na+i/Ca2+o exchange mode ('reverse' Na+/Ca2+ exchange). The requirement for [Ca2+]i has been demonstrated with the use of intracellular calcium buffers, such as Quin-2, to inhibit Na+i/Ca2+o exchange. However, the inhibition of Na+i/Ca2+o exchange in mammalian nerve terminals loaded with Quin-2 has not been observed [7], suggesting a lower sensitivity to low [Ca2+]i for this system. In contrast, the results reported herein indicate that 45Ca2+ uptake in synaptosomes through Na+i/Ca2+o exchange is inhibited by Quin-2 much in the same way as it is in the squid, provided that synaptosomes are preincubated in low Ca2+ medium to avoid saturation of Quin-2. Under these conditions, 45Ca2+ efflux via Ca2+i/Ca2+o exchange is also inhibited. Our results indicate that the Na+i/Ca2+o and Ca2+i/Ca2+o modes of the Na+/Ca2+ exchanger from rat brain synaptosomes require intracellular calcium for activation. However, because no clear relationship between the observed [Ca2+]i values and the inhibition of Na+i/Ca2+o exchange has been found, it is suggested that localised submembrane calcium concentrations not detected by the [Ca2+]i probe might regulate the exchanger.     

Effect of hyperoxia (PaO2 50-90 mmHg) on fetal breathing movements in the unanaesthetized fetal sheep

Hypoxia inhibits fetal breathing movements but after birth it stimulates breathing. These differences have long been thought to involve central nervous inhibitory mechanisms. Such mechanisms might exert a tonic inhibition of fetal breathing movements at normal fetal PaO2 and the rise in PaO2 at birth might lift this inhibitory effect. To test this hypothesis 7 fetal sheep were chronically instrumented at 125-130 days for recording electrocortical activity (ECoG), and the electromyograph (EMG) activity of the diaphragm and neck muscles. Catheters were placed in a fetal carotid and a brachial artery and in the fetal trachea. For an extracorporeal membrane oxygenation system a 12 F gauge silastic catheter was placed in the right atrium for draining fetal blood and a 9.6 F gauge catheter was placed in a carotid artery to return oxygenated blood. Three days after operation the fetuses were connected to the extracorporeal membrane oxygenation system and fetal PaO2 was raised to 65.2 +/- 4.4 mmHg (SEM) for 6 to 19 h without changing pH or PaCO2. Neither the incidence of high voltage ECoG (48.5 +/- SEM 2.0% vs 52.8 +/- 3.3%) nor of fetal breathing movements (37.3 +/- 2.6% vs 23.8 +/- 5.9%) changed during the periods of hyperoxia. Since fetal breathing movements did not become continuous, we conclude that the lower PaO2 in the fetus compared to the neonate does not exert a tonic inhibitory influence on fetal breathing movements.     

A new species of Lernanthropus De Blainville, 1822 (Copepoda: Lernanthropidae) parasitic on Menticirrhus ophicephalus (Jenyns) (Teleostei: Sciaenidae) from the Peruvian coast

Lernanthropus huamani n. sp. (Copepoda: Lernanthropidae), a parasite of the Peruvian sciaenid fish Menticirrhus ophicephalus (Jenyns), is described and illustrated. The new species differs from all other species of Lernanthropus by a combination of characters, including the dorsal plate, legs and other appendages. L. guacoldae Villalba & Fernández, 1984 is considered a synonym of L. pacificus Oliva & Duran, 1982.