Evidence that de novo protein synthesis participates in a time-dependent augmentation of the chemotactic peptide-induced respiratory burst in neutrophils: Effects of recombinant human colony stimulating factors and dihydrocytochalasin B

We examined the effects of the recombinant human colony stimulating factors GM-CSF and G-CSF, cycloheximide (a protein synthesis inhibitor) and dihydrocytochalasin B (a microfilament disrupting agent) upon FMLP (N-formyl-methionyl-leucylphenylalanine)-stimulated O₂ ⁻ production by neutrophils. We confirmed a time dependent augmentation of O₂ ⁻ production following preincubation of neutrophils either alone or with colony stimulating factors. Furthermore, we found that GM-CSF, but not G-CSF, increased O₂ ⁻ production at some concentrations of the stimulus. Preincubation of neutrophils with cycloheximide in the absence of CSF caused a marked fall in O₂⁻-production that was first evident at 2 hours. The fall in O₂⁻-forming capacity caused by cycloheximide was much less pronounced if dihydrocytochalasin B was also included in the preincubation buffer. These findings suggest a previously unrecognized role for de novo protein synthesis in maintaining the ability of neutrophils to manufacture O₂⁻, and support earlier studies indicating that the cycling of FMLP receptors between the cell membrane and an intracellular compartment is important in determining the magnitude of the respiratory burst in FMLP-stimulated neutrophils.     

噬菌体T7基因6.5和6.7的克隆及其缺失变种的构建

 用适当的限制性内切酶,将噬菌体T7基因6.5和6.7从整个噬菌体T7基因组中分离出来,插入到质粒pBR322中去,转化E.Coli HMS174,筛选出这两个基因的成功克隆。运用同样手段,从整个噬菌体T7基因组中分离出含有部分基因6编码序列,而不含基因6.5和6.7编码序列的T7DNA片段,插入到pBR322的衍生质粒中去,转化Ecoli C1757,再用含有基因6和基因7的双突变噬菌体T7去感染这一转化菌,通过同源交叉而得到缺失基因6.5和6.7的噬菌体T7缺失变种。这种噬菌体只能在载有噬菌体T7基因6.5和6.7,或者只载有基因6.7质粒的寄主中增殖。通过噬菌体结构蛋白电泳分析证明,这种噬菌体丢失了野生型菌体T7所具有的两条结构蛋白带。     

Purification of the Lewis blood-group gene associated α-3/4-fucosyltransferase from human milk: an enzyme transferring fucose primarily to Type 1 and lactose-based oligosaccharide chains

A soluble Lewis blood-group gene associated -3/4-L-fucosyltransferase has been purified from human milk by a series of steps involving hydrophobic chromatography on Phenyl Sepharose 4B, ion exchange chromatography on CM-Sephadex C-50, affinity chromatography on GDP-hexanolamine Sepharose 4B and gel filtration on Sephacryl S-200. The first step separated -3-L-fucosyltransferase activity directed towardsN-acetylglucosamine in Type 2 (Gal1-4GlcNAc-R) acceptors from an -3/4-fucosyltransferase fraction acting on both Type 1 (Gal1-3GlcNAc-R) and Type 2 acceptors. Further purification of this latter fraction on CM-Sephadex and GDP-hexanolamine Sepharose gave a single peak of fucosyltransferase activity that catalysed the addition of fucose toN-acetylglucosamine in both Type 1 and Type 2 acceptors and to theO-3 position of glucose in lactose-based oligosaccharides. The enzyme preparation at this stage resembled previously described -3/4-fucosyltransferase preparations purified from human milk. However, gel filtration of this preparation on Sephacryl S-200 or Sephadex G-150 separated further amounts of -3-fucosyltransferase activity acting solely on Type 2 acceptors and left a residual -3/4-fucosyltransferase that retained strong -4 activity with the Type 1 acceptor, lacto-N-biose 1, and -3 activity with 2-fucosyllactose, but had relatively little -3 activity withN-acetyllactosamine and virtually no capacity to transfer fucose to glycoproteins withN-linked oligosaccharide chains having unsubstituted terminal Type 2 structures.     

Effect of adrenergic agonists and antagonists on alanine amino transferase,fructose-1:6-bisphosphatase and glucose production in hepatocytes

Using rat hepatocytes we confirmed our previous results that glucagon and -adrenergic agonists increased the enzyme activity of alanine aminotransferase (AAT) and propranolol abolished their effects. Only the enzyme activity was measured and other parameters like quantity of the enzyme or activation due to modification were not looked for. As in perfusion experiment phenylephrine and phenoxybenzamine (-agonist and -antagonist respectively) also increased the AAT activity in isolated rat hepatocytes and propranolol reversed these effects. The additive effect of glucagon and phenoxybenzamine on AAT was also persistant in hepatocyte system.Fructose- 1:6-bisphosphatase (Fru-P₂ase), another key enzyme in gluconeogenic pathway, was elevated by glucagon and other -adrenergic agonists both in liver perfusion and isolated hepatocyte experiments and was brought back to the normal level by propranolol. In this case also only the enzyme activity was measured and no other parameters were looked for. Unlike AAT this enzyme was not stimulated by phenylephrine or phenoxybenzamine. But AAT and Fru-P₂-ase activities were increased significantly by adenylate cyclase activators like fluoride or forskolin. Thus, it appears that the regulation of fru-P₂-ase by glucagon is purely a -receptor mediated process whereas AAT activation shows a mixed type of regulation where some well known -agonist and antagonists are behaving as -agonists.Results further indicate the presence of phosphodiesterase in hepatocyte membrane which was stimulated by glucagon and brought back to the normal level by propranolol.The different adrenergic compounds stated above, not only modified the activity of the above two enzymes but also stimulated glucose production by hepatocytes from alanine which was in turn abolished by propranolol as well as amino oxyacetate (AOA), a highly specified inhibitor of AAT. This confirm the participation of AAT in gluconeogenesis from alanine in liver. Forskolin and fluoride also increased the glucose production from alanine and showed additive effects with glucagon, phenylephrine and phenoxybenzamine.     

The “ON”-bipolar agonist,L-2-amino-4-phosphonobutyrate,blocks light-evoked cone contraction in xenopus eye cups

Rhythmic photoreceptor metabolism in relationship to light-dark cycles is now thought be regulated through a retinal feed-back mechanism with dopamine serving as a principal signal initiating light-evoked events. In order to test the hypothesis that depolarizing ON-bipolar neurons participate in the retinal signalling pathway, we determined the effects of L-2-amino-4-phosphonobutyrate (L-APB) on light-evoked cone contraction in eye cups fromXenopus laevis. L-APB blocked the response stereospecifically when applied over a broad concentration range. The high specificity of L-APB in retina suggests that sign-inverting bipolar neurons which depolarize in light are in the signalling pathway. One possibility is that this pathway conveys signals that regulate dopamine release.Special issue dedicated to Dr. Frederick E. Samson.     

Gene variation and gene flow inOrchis morio (Orchidaceae) from Italy

Data are presented on genetic variation at 27 enzyme loci of the Green-Winged orchid,Orchis morio, in 18 population samples from Italy. The existence in Italy of two subspecies, i.e. subspp.morio andpicta, is not supported by allozyme data. No genetic heterogeneity was found betweenmorio-like andpicta-like samples and specimens. Moreover, morphological transition between the two forms was observed in different Italian populations. The parameters of genetic variability estimated forO. morio populations are consistent with those found among monocotyledon plants, and among those outcrossing, animal-pollinated and with wind-dispersed seeds. Genetic diversity of ItalianO. morio is mostly within populations. Correspondingly, low values of interpopulational genetic distance were found. This appears to be due to high levels of gene flow, which were estimated with different methods. The lack ofO. longicornu from Italian samples, as well as of any hybrid withO. morio (F₁, backcrossed or recombinant individuals) is demonstrated on the basis of genetic data. It is concluded that recurrent reports ofO. longicornu from Italy are due to confusion withO. morio or with otherOrchis species.     

Phenetic,biogeographical, and evolutionary study ofOrnithogalum subg.Heliocharmos (Hyacinthaceae) in the western Mediterranean basin

A macromorphological study is made on taxa of the genusOrnithogalum subg.Heliocharmos in North Africa, Spain, and France. The results obtained are consistent with data from cytogenetics, reproductive biology and strategies of reproduction. They allow the retention of two species:O. algeriense andO. umbellatum. A biogeographical and phylogenetic interpretation of the subgenus is proposed for the western Mediterranean. Theoretical views on phenetics are discussed.     

Infradian cycles of oxygen consumption in diapausing pupae of the flesh fly, Sarcophaga crassipalpis, monitored by a scanning microrespirographic method.

Fine details of the infradian O2 consumption cycles that characterize pupal diapause in flesh flies have been monitored by a newly designed microrespirographic method coupled with an electronically regulated O2 generator. During the 4-5 days between the peaks of elevated O2 consumption, the diapausing pupae maintained a very low and fairly constant respiratory rate (13 microl O2 x g-1.h-1). During the intercalated peaks of increased respiratory metabolism, which lasted an average of 33.6 h to 24-27 degrees C, the average maximum rate of O2 consumption was 86.9 microl.g-1.h-1, a value of 6.7 times higher than the interpeak values. The respiratory peaks started abruptly in some cases while the decline was consistently gradual. During the periods between the peaks there were no discontinuous bursts of CO2 release, a feature common to diapause in many other insects. Diapause was characteristically terminated during a peak of the O2 consumption cycle. At diapause termination O2 consumption remained at the maximum values of the peak for many hours and then gradually increased to levels characteristic of nondiapause development.     

ATP-dependent Ca2+ transport and Ca2+-ATPase activities in an enriched plasma membrane fraction from gypsy moth larval midgut tissue

A subcellular fraction enriched in plasma membranes was obtained from gypsy moth (Lymantria dispar) larval midgut tissue. Using [⁴⁵Ca]²⁺ as a tracer, Ca²⁺ transport activity by membrane vesicles in the enriched fraction was measured and shown to be ATP-dependent, with a very high affinity for Ca²⁺ (apparent K_m for [Ca²⁺ _free]

1 Abbreviations used: [Ca²⁺free] = concentration of free (unbound) calcium ion;CaM = calmodulin; F = fraction; IOV = inside-out membrane vesicles; W-5 = N-(6-aminohexyl)-1-naphthalenesulfonamide; W-7 = N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide.

= 22 nM). Ca²⁺ transport was abolished upon addition of the calcium ionophore, A23187. Ca²⁺-stimulated, Mg²⁺-dependent ATPase activity peaked between 100 and 200 nM Ca²⁺_free. Ca²⁺-Mg²⁺-ATPase activity was inhibited by vanadate, 2 phenothiazine drugs (trifluoperazine and chlorpromazine), and the naphthalene sulfonamide, W-7; the related compound, W-5, and ouabain had a negligible effect. These results suggest the presence of a high affinity plasma membrane Ca²⁺ pump in gypsy moth larval midgut cells and are discussed in light of earlier work involving calcium transport in isolated midguts of larval Hyalophora cecropia. Ionic and other conditions that characterize the midgut physiology of larval Lepidoptera (e.g., luminal pH; electrochemical gradient for Ca²⁺; effect of certain ions and inhibitors on Ca²⁺ transport) contrast significantly with those found in adult Diptera. The implications that these differences may have for calcium regulation are discussed. © 1992 Wiley-Liss, Inc.     

Studies on the morphology and anatomy of the Antarctic nudibranch genera Pseudotritonia Thiele, 1912 and Telarma Odhner, 1934 with a discussion of the family Gharcotiidae Odhner, 1926 (Nudibranchia: Opisthobranchia)

The external morphology and anatomy of the Antarctic nudibranchs Pseudotritonia quadrangularis Thiele, 1912, P. gracilidens Odhner, 1944 and Telarma antarctica Odhner, 1934 are redescribed. Both genera, which were only known by two or one specimen, do not possess a cnidosac, but there is a terminal swelling at the termination of the digestive glandular ramifications in P. gracilidens. This sac is composed of cells with a large vacuole. A glandular stripe on the right side of the body above the genital openings, nephroproct and anal papilla is considered to be a synapomorphy for both genera. The validity of the family Charcotiidae and the affinities to the aeolid genus Notaeolidia Eliot, 1905 and taxa of the Arminacea are discussed.