Ether phospholipids in control and 20∶4-depleted rat PMN: Additional evidence for a 1-0-alkyl-2-20∶4-sn-glycerol-3-phosphocholine specific phospholipase A2

Earlier studies from our laboratory have shown that PAF and LTB₄ biosynthesis are inhibited in rat PMN depleted of its 203 (JBC. '86,261, 7592). To test whether these cells contain sufficient 1-O-alkyl-2-acyl-GPC to support PAF synthesis, phosphotidyl choline was isolated from these 204-depleted cells, fractionated into different subclasses and their fatty acid composition determined. These results were compared with those obtained with control PMN. Both control and 204-depleted PMN contained significantly large amounts of alkylacyl-GPC and diacyl-GPC. Small amounts (4%) of alkenylacyl-GPC were also present. The amount of 203 in 204-depleted cells was more or less equal to the amount of 204 in control cells. About 62% of PC-bond 204 in control PMN and about 56% of PC-bound 203 in the 204-depleted PMN was found associated with the alkylacyl species. These results show that both control and 204 depleted PMN have ample precursor substrates to support PAF biosynthesis and these substrates are enriched with 204 in control cells and with 203 in 204-depleted cells. These findings are consistant with the existance of a highly specific phospholipase A₂ capable of distinguising 204 from 203 containing phospholipids.     

SpecialEscherichia coli serotypes among enterotoxigenic strains from diarrhoea in adults and children

106 enterotoxigenicEscherichia coli strains from children and adults from many parts of the world were serotyped for O and H antigens. Some OH types,i.e. O6H16, O8H9, O15H11, O25H42, O78H11 and O78H12, were found repeatedly from different geographical locations. Some of these OH serotypes were only found rarely among more than 20000E. coli strains collected over many years from different locations and sources. It is suggested that these special OH serotypes represent clones which have been selected to the special conditions in the small intestine and selected to carry the plasmids necessary to provoke diarrhoea.     

Increasing incidence of meningococcal disease in Spain associated with a new variant of serogroup C

Serogroup B has been the main cause of meningococcal disease in Spain since at least 1979, but in recent years an increase in the prevalence of infection due to serogroup C meningococci has been detected. In 1996, for the first time, most cases of meningococcal disease were caused by serogroup C strains. The sero/subtype of all serogroup C meningococci received from 1993 to June 1996 was determined, and the results showed that C2bP1.2,5, the most common phenotype in 1995 and 1996 (63% and 65%, respectively), represented only 4.8% of strains in 1993. The C2bP1.2,5 epidemic strains appear to be responsible for the high prevalence of serogroup C in Spain. One hundred fifty-one randomly selected serogroup C strains were analyzed by multilocus enzyme electrophoresis, ribotyping, and pulsedfield gel electrophoresis. Pulsed-field gel electrophoresis provided the most accurate information: more than 80% of the C2bP1.2,5 and C2bP1.2 isolates exhibited one of two very closely related profiles, while most of the C:2b:NST and C2bP1.5 strains had a pattern located at a genetic distance of 0.24 from those two profiles. The results show that C2bP1.2,5 strains represent a subclone or a genetic variant of the previously identified Spanish epidemic clone C2bnon-subtypable strains.     

Effects of sonication on physicochemical and biological properties of synthetic double-stranded polyribonucleotides as interferon inducer

Summary Two kinds of synthetic double-stranded polyribonucleotides, poly IGC¹ and poly I:C and their constituent single-stranded polymers were subjected to sonication. Sonication of both poly IGC and poly IC resulted in decreases in viscosity, molecular size and heterogeneity in the size distribution. In poly IGC, whose average sedimentation constant was larger than or around 11 S, these changes were accompanied with enhancements of interferon inducing activity in rabbits and mice and antiviral activity in mice, and moreover with a decrease in the systemic toxicity in mice. In poly IC, however, such an enhancement in the interferon inducing activity was observed only when its molecular size corresponded to that of poly IGC. Previous sonication of poly C of the relatively large molecular size (> 10 S) has also been shown effective, to a certain extent, in obtaining double-stranded RNA of smaller size distribution with increased interferon inducing activity and lowered toxicity. It has been shown that these changes induced by sonication were based on the breakage of phosphodiester bonds of both double and single-stranded polyribonucleotides. On the basis of the analyses of the correlations between molecular sizes and the biological activities, it has been suggested that, while toxicity decreases always when the molecular size becomes smaller, the optimal size of the double-stranded polyribonucleotide complexes for interferon production ranges roughly from 10 S (9.1×10⁵ daltons) to 5 S (1.2 ×10⁵ daltons).     

Biochemical characteristics,phage patterns,and O1 factor analysis ofEscherichia coli O1∶K1∶H7∶F11 and O1∶K1∶H−∶F9 strains isolated from patients with urinary tract infections

Biochemical characteristics, O1 antigen factors and phage patterns were examined in 35 urinary O1K1 isolates ofEscherichia coli different in H and F antigen. Fermentation of dulcitol, decarboxylation of ornithine, requirement for nicotinamide, and determination of O1 factor d allowed maximum differentiation. On the basis of these tests the strains could be divided into two major groups which are obviously of different clonal origin. Members of clone 1 represented by serotypes O1K1H7F11 (12 strains) and O1K1H^–F11 (5 strains) were characterized by positive biochemical reactions and absence of O1 antigen factor d. Negative biochemical tests and presence of O1 antigen factor d were shown by strains of clone 2 which were of serotypes O1K1H^–F9 (14 strains) and O1K1H^–F^– (3 strains). Phage patterns are less well correlated with clonal assignment. However, strains of clone 2 were not susceptible to K1-specific phage D and were non-typable with another set of 13 phages.     

Recognition by peptide mapping of three different structural groups of outer membrane protein YOP-1 of Yersinia enterocolitica and Yersinia pseudotuberculosis

The structural relation of YOP-1 of european and american Yersinia enterocolitica serotypes O3, O9, O5, 27, and O8 and O20, respectively, and Y. pseudotuberculosis serotypes I, II, and III was compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis and peptide mapping using Staphylococcus aureus protease V8. Apparent molecular weights of YOP-1 ranged from 206,000 (O3) to approx. 180,000 (O8). According to their respective peptide maps YOP-1 of the european and american Y. enterocolitica serotypes and Y. pseudotuberculosis serotypes could be assigned to three different groups. Evaluation of several isolates of Y. enterocolitica serotypes O3, O9, and O8 by peptide mapping indicated that YOP-1 is conserved within a serotype. However, one serotype O8 isolate differed from the consensus peptide pattern of the other serotype O8 and O20 isolates. The similarity of the peptide patterns of Yersinia serotypes which predominate in certain geographical locations, i. e., european and american Y. enterocolitica serotypes, suggest common evolution of YOP-1 of these serotypes independent of the evolution of the other serotypes.     

Influence of adoptively transferred thioglycollate-elicited peritoneal macrophages on metastasis formation in mice with depressed or stimulated NK activity

The effect of thioglycollate-elicited macrophages (TG-M) on natural killer (NK)-cell activity and metastases formation in mice was investigated. Intravenously (i.v.) inoculated TG-M inhibited spleen NK activity of normal mice and abrogated polyinosinic: polycytidylic (poly IC) induced augmentation of NK cell function. TG-M also inhibited the clearance of i.v.-injected radiolabeled B16 melanoma cells from the lungs of normal or poly IC stimulated mice. Formation of experimental B16 melanoma metastases was dramatically increased in mice pretreated with TG-M. Administration of TG-M increased metatasis formation to a greater extent than anti-asialo GM₁ serum, while anti-asGM₁ serum was more efficient than TG-M in depressing spleen NK cell activity. When mice with low NK reactivity (beige mice or mice treated with anti-asialo GM₁ serum) were inoculated with TG-M, there was a substantial additive augmenting effect on metastasis formation in the lungs. Treatment with poly IC elevated NK-cell activity and had profound antimetastatic effects in normal but not in TG-M pretreated mice. The metastasis augmenting effect of TG-M was fully expressed in poly IC-treated mice as well as in athymic nude mice. Inoculation of proteose peptone-elicited macrophages (PM), unlike TG-M, did not depress NK activity or augment metastasis formation in normal or poly IC-treated mice. However, since the inhibition of NK activity in TG-M-treated mice was relatively weak, and a substantial additional increase in metastases was observed in NK-depressed mice after transfusion of TG-M, it seems unlikely that the TG-M-induced inhibition of NK reactivity is entirely responsible for the augmented formation of metastases. Further studies revealed that i.v. inoculation of TG-M, but not PM, induced intravascular inflammatory reactions, and damage to endothelial cells and basement membrane of the lung vasculature. These reactions may contribute to increased tumor cell extravasation and metastasis formation in mice pretreated with TG-M.     

The length dependency of calcium activated contractions in the femoral artery smooth muscle studied with different methods of skinning

The relationship between the calcium concentration and the isometric tension obtained with different techniques of skinning provides information on the biochemical events of contraction in vascular smooth muscle. Muscle preparations of the rabbit femoral artery were skinned with triton X-100, saponin, -escin and -toxin and the relationship between the calcium concentration and isometric tension was determined at different preparation lengths. We determined the calcium sensitivity as a function of muscle length with different techniques of skinning. At a pCa of 6.0, triton X-100 skinned smooth muscle of the femoral artery generated 50% of the maximal tension. In -toxin skinned preparations, this calcium sensitivity was shifted to a pCa of 5.6. The sensitivity of the saponin and -escin skinned preparations were in between those of the triton X-100 and the -toxin skinned preparations. The cooperativity of the regulation of contraction varied among the differently skinned preparations between 3 (-toxin) and 6 (triton X-100). The relationships between the calcium concentration and the isometric tension of the differently skinned preparations up to the optimal length for tension generation did not exhibit any length dependency. The length tension relationship, obtained from the maximal response at the highest calcium concentration is in line with that from other studies. The presence of intracellular proteins and membranes affects the regulation of contraction in smooth muscle of the femoral artery.     

Identification and quantitation of PAF from psoriatic scales

Platelet activating factor was isolated from scales of psoriatic patients by the procedure of Bligh and Dyer and purified by silica gel thin layer chromatography. The purified PAF was digested with phospholipase C and the resulting diglyceride was derivatized into PFB ethers. The PAF-PFB ethers were analyzed using fused silica capillary chromatography-negative ion chemical ionization mass spectrometry. Different molecular species of PAF were identified by their negative ion mass spectra and by their elution time fromthe capillary column. All the molecular species had high abdundance (>90%) of the molecular anion. 1-0-Hexadecyl-2-acetyl-GPC (160) was the major PAF species representing 51% of the total PAF. 170 and 181 were the next abundant species representing 15 and 16%, respectively. Several minor PAF molecular species were also present. The amount of each PAF molecular species was quantitated from 1-0-hexadecyl-2-²H₃ acetyl-GPC used as the internal standard. Nanogram quantities of PAF were recovered from 100 mg of psoriatic scales. Significant amounts of lysoPAF were also present in these scales. The alkyl chain of the lysoPAF was compared with that of PAF.     

An autoradiographic identification of Purkinje axon terminals in the cat

Summary The Purkinje axon boutons terminating in nuclei fastigii and interpositus in the cat have been identified after injection of³H leucine into the cerebellar cortex overlying the nuclei. The animals survived from 4–48 h after injection of the isotope. Semithin and ultrathin sections were coated and exposed for 3 and 14 weeks, respectively. The electron micrographs showed labelling over myelinated axons down to a diameter of 0.8 m, and over boutons.Fifty labelled boutons were used for identification of the shape of their synaptic vesicles. The statistical analysis including the test for skewness showed that 39 boutons (78%) fall in one group. Most of the synaptic vesicles in this group are elliptical (ration from 11.1 – 11.7). Slightly ovoid vesicles (ration up to 11.3) are frequent, but flattened vesicles (ration above 11.7) are relatively few in this group of boutons. 8% of the boutons have a rather homogeneous vesicle population (prevalence of round vesicles, ratio 11).Synaptic specializations of Gray's type II or of an intermediate type were found in the boutons belonging to the first group (78% of the boutons). Specializations of Gray's type I were found in the other bouton groups (8% of the boutons).     

Induction of interferon and host resistance in vivo by double-stranded complexes of copolyribonucleotide of inosinic and guanylic acids with polyribocytidylic acid

Summary Several double-stranded complexes of copolyribonucleotide of inosinic and guanylic acids with polyribocytidylic acid (poly IGC) were found to possess interferon inducing activity stronger than poly ICin vivo, Their activity increased in parallel with increase in the ratio of guanine base to hypoxanthine base in these copolymers as far as double-strand formation was observed with polyribocytidylic acid. Many other combinations of copolyribonucleotide with homopolyribonucleotide were also investigated, and several of them were found to induce interferon. However, the interferon inducing effects of these combinations including complementary base-pairings of hypoxanthine and cytosine increased in parallel with the length of the base-pairings, thus approaching to that of poly IC. It is, therefore, supposed that the activity of poly IG C is somewhat different from poly IC and that those of other combinations owe to the essential structure of poly IC. Furthermore, kinetics of interferon induction, cross tolerance to reinduction, and antiviral effectsin vivo of poly IGC and poly IC were studied.     

Effects of priming exercise intensity on the dynamic linearity of the pulmonary V̇O2 response during heavy exercise

Prior heavy-intensity exercise facilitates the pulmonary oxygen uptake (O₂) response during subsequent exercise, such that its kinetics returns towards first-order. To better understand this priming phenomenon, we investigated the effect of priming exercise, over a range of intensities, on the O₂ response to heavy-intensity cycle ergometry at a work rate of 50% [halfway between lactate threshold (LT) and O_2max]. Eight subjects performed two consecutive 6-min bouts separated by 6 min at 20 W. The first bout was each of: no warm-up control (CON), sub-lactate threshold (LT) at 80% of LT, and three supra-LT conditions (20%, 40%, and 60%). The O₂ response during the subsequent bout was evaluated using the effective time constant (), and the O₂ difference between minutes 3 and 6 (O_2(6–3)). The goodness-of-fit, indicative of first-order kinetics, was determined by the residual profile, and the mean square of errors (MSEr). The heart rate and blood lactate concentration ([La]r) just prior to the second bout were also measured. Compared with CON, and O_2(6–3) were significantly reduced following all supra-LT priming bouts, while the goodness-of-fit was significantly improved following 40% exercise. O_2(6–3) and [La]r were negatively correlated (P<0.05), unlike HR. In conclusion, prior exercise just above, but not below, LT facilitated the O₂ response in a threshold-like manner. Supra-LT priming exercise influenced the O₂ response allowing it to return to within as little as 12% from first-order (compared to ~50% in CON). The associated increases in circulating lactate and/or related factors seem to be centrally involved in this phenomenon.     

Passive electrical properties of the atrio-ventricular node

Summary The passive electrical properties of the atrioventricular node and of the atrial muscle of the rabbit heart were determined. To polarize the cells, a small suction electrode was used. The space constant of the atrio-ventricular node was found to be small (430 ) compared to other cardiac fibers. The small value of in nodal cells is due to a high intracellular resistance (r_a, 40.9 ± 9 M/cm) which is higher than in atrial muscle (9.6 ± 2.2 M/cm). The input resistance of cells of the N layer was found equals to 880 K while in the right atrium was 320 K. The time constant of nodal cells in the AN layer was 3.4 ms, in the N layer 9 ms and in the atrium 5 ms. Assuming a specific membrane capacity (C _m) of nodal and atrial fibers of 1 F/cm²,R _m was found equals to 9.000 cm² in N layer, 3.400 cm² in AN layer and 3.800 cm² in the right atrium.Acetylcholine (5 g/ml) reduced the space constant of the atrio-ventricular node by 38% and the time constant of nodal cells by 33%.The delay of impulse conduction in the A-V node semms then related to a high intracellular resistance along the pathway of conduction.This work was supported by Grant # HL-10897 from the NHI, Bethesda, Maryland and in part by a Grant from the P. R. Heart Association     

Aerobactin production of serotyped Escherichia coli from urinary tract infections

Aerobactin production was examined by a bioassay in 467 Escherichia coli urinary strains from girls. All strains were of known OKH serotype. 139, 119 and 112 strains were isolates from pyelonephritis (Py), cystitis (Cy) and asymptomatic bacteriuria (ABU), respectively, and 97 were from fecal samples of healthy girls (FN). The incidence of aerobactin production was significantly higher among Py strains than among ABU and FN strains (P<0.001) and also significantly higher than among Cy strains (P<0.01). Aerobactin production was associated with serotype, e.g. the majority of 06K2H1 strains and of 016K1H6 were positive while e.g. the 06K13H1 strains were negative. There was no consistent pattern of coappearance of aerobactin and hemolysin.     

Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes: role of the α subunit in agonist sensitivity and desensitization

Neuronal nicotinic acetylcholine receptors (nAChRs) were expressed in Xenopus laevis oocytes after nuclear injection of complementary deoxyribonucleic acid (cDNA) expression vectors. The two receptor subtypes 4/n1 and 3/n1 were readily distinguishable from one another by ACh sensitivity and desensitization. 3/n1 receptors showed lower ACh sensitivity and stronger desensitization than 4/n1 receptors. Furthermore, although the current/voltage relationship was very similar in both receptor subtypes, the voltage dependence of desensitization was found to be strikingly different. As the n1 subunit was unchanged, the subunits must be responsible for these functional differences. Symmetric hybrid cDNAs, 43 and 34, were constructed and functional receptors were obtained by co-injection with n1. These hybrid receptors displayed an ACh sensitivity that was mainly defined by the extracellular sequence of the subunit. In contrast, no part of the subunit was found fully to determine desensitization.     

Effect of molsidomine on ex vivo platelet aggregation and plasma guanosine 3′∶5′-cyclic monophosphate levels in healthy volunteers

Summary To find out whether 3-morpholino-sydnonimine (SIN 1), the active metabolite of molsidomine, exerts its antiaggregatory effects not only in vitro but also in vivo, we tested ex vivo aggregation before and after intravenous application of molsidomine in healthy volunteers. We also measured plasma levels of guanosine 35-cyclic monophosphate (cyclic GMP) as SIN 1, the bioactive metabolite of molsidomine, becomes effective via activation of soluble guanylate cyclase. In eight out of ten subjects molsidomine had an inhibitory effect on platelet aggregation and a higher threshold concentration of platelet-activating factor was required after molsidomine application to induce irreversible aggregation. Despite the effect on platelets, plasma cyclic GMP levels did not increase. These results suggest that the nitric oxide-containing SIN 1 inhibits platelet aggregation not only in vitro but also in vivo and that this property can be a beneficial effect in antianginal therapy.Abbreviations Cyclic GMP guanosine 35-cyclic monophosphate - NO nitric oxide - PAF platelet-activating factor - PRP platelet-rich plasma - SIN 1 3-morpholino-sydnonimine     

Effect of increasing doses of amikacin with or without piperacillin in the serum bactericidal test

To determine whether high doses of amikacin would prevent the development of resistance in clinical isolates, the serum bactericidal activity and killing rate of conventional and high doses of amikacin and piperacillin alone and in combination were measured in volunteer sera against a series of ten strains each of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa.Amikacin serum levels were 24.9±6.0 mg/l 1 h after infusion of the 7.5 mg/kg dose and 44.8±5.0 mg/l after the two-fold dose. Median serum bactericidal titers for low dose piperacillin + amikacin were 18–164 and for high-dose piperacillin + amikacin 116–1128. Both were satisfactory, except against piperacillin-resistant Pseudomonas aeruginosa (median bactericidal titers 12), and both combinations had equivalent killing rates.     

Is potassium co-transported by the cardiac Na-Ca exchange?

It has been suggested that the stoichiometry of the electrogenic Na-Ca exchange is 3Na1Ca. Recently, however, it was reported in rod outer segments that the stoichiometry of Na-Ca exchange is not 3Na1Ca but 4Na1Ca+1K. In cardiac cells, the reversal potential has always been measured in the absence of K or at a very low K concentration. We have, therefore, re-examined the reversal potential of the Na-Ca exchange current by whole-cell voltage clamp in single guinea-pig ventricular cells in the presence of K on both sides of the membrane. The Na-Ca exchange current reversed at potentials close to the calculated values for 3Na1Ca stoichiometry even in the presence of K. The magnitude of the Na-Ca exchange current did not change in 1 and 10 mM [K]₀. We therefore conclude that K is not co-transported by cardiac Na-Ca exchange and its stoichiometry is 3Na1Ca.     

Recombinant interleukin-1 and tumor necrosis factor induce neutrophil migration “in vivo” by indirect mechanisms

The and forms of recombinant interleukin-1 (IL-1 and IL-1) and of recombinant Tumor Necrosis Factor (TNF and TNF) induced dose-dependent neutrophil migration into rat peritoneal cavities. Migration induced by both IL-1s showed a bell-shaped dose-response curve and IL-1 was 3-fold more potent than IL-1. Pretreatment of the animals with dexamethasone or depletion of the peritoneal macrophage population, abolished the neutrophil migration induced by the four cytokines. In vitro stimulation of macrophage monolayers with IL-1 and the TNFs released a factor into the supernatant which, unlike these cytokines, induced neutrophil migration in dexamethasone pretreated animals. These results suggest that the neutrophil migration induced by IL-1, IL-1 and TNF is not due to a direct effect on neutrophils, but occurs via the release of a chemotactic factors(s) from resident macrophages.     

The influence of Poly I∶C on the course of infection in mice inoculated with West Nile Virus

Summary In mice infected intraperitoneally with a hundred per cent lethal dose of West Nile virus a significant reduction in mortality was found if treatment with the complex of synthetic polyriboinosinic and polycytidylic acids (Poly IC) was given four hours prior to or twenty hours after virus challenge.Treatment induced large amounts of circulating interferon a few hours after inoculation. Only a slight difference in maximum viraemia in the various groups was found, but viraemia developed later in the mice given Poly IC a few hours before virus injection. Infection of the brain developed later in the groups treated with Poly IC.Using various doses of West Nile virus almost the same mortality was found in the group given a lethal virus dose but treated with Poly IC and the group receiving sublethal virus dose and no Poly IC treatment. Maximum of viraemia was high in the former group, while in the latter group it was significantly lower. Therefore it is supposed that Poly IC in these experiments did not protect through an interferon mediated suppression of the viraemia but rather through an effect of the interferon exerted directly upon the target organ. A reduction of circulating HI antibodies was found in the group on which Poly IC had the most pronounced effect.