Mechanism of neutrophil activation by NAF, a novel monocyte-derived peptide agonist

The rise in cytosolic free Ca2+, shape change, superoxide formation, and granule exocytosis induced in human neutrophils by N-formyl-Met-Leu-Phe (fMLP) and by a newly discovered activating peptide, neutrophil-activating factor, termed NAF, were compared. NAF was effective in the concentration range of 0.1-10 nM and was 10- to 100-fold more potent than fMLP. In qualitative terms, the single responses to either stimulus were remarkably similar: they showed virtually identical onset and initial kinetics, and were all inhibited by pretreatment of the neutrophils with Bordetella pertussis toxin. In addition, the respiratory burst elicited by either stimulus was inhibited by 17-hydroxywortmannin and staurosporine. Two conclusions are drawn from these results: 1) neutrophil activation by NAF (as by fMLP) is dependent on a GTP-binding protein and on protein kinase C; 2) a similar, or even identical, mechanism of signal transduction must be assumed on stimulation of human neutrophils with NAF, fMLP, and other chemotactic agonists. Human monocytes, lymphocytes, and platelets did not show cytosolic free Ca2+ changes when exposed to NAF, which suggests that NAF is selective for the neutrophils.     

The localization of calcium release by inositol trisphosphate in Limulus photoreceptors and its control by negative feedback

Microvillar photoreceptors of invertebrates exhibit a light-induced rise in the intracellular concentration of free calcium (Cai) that results in part from release of calcium from an intracellular compartment. This light-induced release of calcium appears to result from a cascade of reactions that involve rhodopsin, a GTP-binding protein and a phospholipase-C which releases inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) from the plasma membrane; the Ins(1,4,5)P3 acts to release calcium from smooth endoplasmic reticulum. In the ventral photoreceptor of the horseshoe crab Limulus polyphemus not all of the endoplasmic reticulum is subject to calcium release by Ins(1,4,5)P3. Only endoplasmic reticulum in the light-sensitive region of the cell is competent to release calcium in response to Ins(1,4,5)P3. The release of calcium by Ins(1,4,5)P3 in ventral photoreceptors appears to be subject to feedback inhibition through elevated Cai. We suggest that this feedback inhibition contributes to sensory adaptation in the photoreceptor and may account for oscillatory membrane responses sometimes observed with large injections of Ins(1,4,5)P3.     

Inactivation of recombinant plasmid DNA from a human erythropoietin-producing mouse cell line grown on a large scale

Summary Experiments were carried out to assess the survival of recombinant plasmid DNA during large-scale production of recombinant human erythropoietin (rhuEPO) in a fermentation pilot plant. The analyses revealed DNA-degrading activities in the fermentation broth and in the waste-water, leading to rapid destruction of plasmid DNA added to medium or waste-water. The capability of the plasmid-DNA-spiked samples to transform competent bacteria was drastically reduced. The DNA-degrading activity in the waste-waters could be blocked by addition of EDTA or by boiling, indicating the presence of DNA-degrading enzymes (DNases). No plasmid-specific DNA sequences were detected in waste-water samples by in-vitro amplification with Taqpolymerase. Genomic DNA preparations of cell debris collected from waste-water samples only contained degraded plasmid DNA. Furthermore, it was shown that intact plasmid DNA could be degraded to fragments of less than 1000 bp by incubation at 121°C for 20 min, leading to a decrease in the plasmid-specific transforming capacity by a factor of 10³ per minute. Thus, DNA from the rhuEPO production pilot plant was efficiently inactivated at three different levels: (i) in the fermentation medium (DNase), (ii) in the waste-water container (DNase), and (iii) by heat inactivation for 20 min at 120°C. These results indicate that the probability of delivery of recombinant DNA into the environment is extremely low in such biotechnological production processes. Offprint requests to: M. R. Fibi     

Modulation of the beta-adrenergic response in cultured rat heart cells

Incubation of rocker-cultured neonatal rat heart cells with 3 mM L(+)-lactate led to a sharp increase in the sensitivity of cardiomyocytes to the beta-adrenergic agonist isoprenaline, as measured by their chronotropic response. This effect was accompanied by a reduction in the arachidonic acid content of the total phospholipids. The phospholipase A₂-activator melittin as well as free arachidonic acid induced this supersensitivity to the same degree. On the other hand, the L(+)-lactate-evoked supersensitivity could be blocked by the phospholipase A₂ inhibitors mepacrine and n-bromophenacyl-bromide, suggesting an involvement of phospholipase A₂ in the process of beta-adrenergic sensitization. The sensitizing action of arachidonic acid was blocked by the lipoxygenase inhibitors esculetin and nordihydroguaiaretic acid, but not by the cyclooxygenase inhibitor indomethacin. Supersensitivity was likewise evoked by 15-S-hydroxyeicosatetraenoic acid (15-S-HETE), but not by 5-S-HPETE or 5-S-HETE. These findings suggest that the phospholipase A₂-15-lipoxygenase pathway plays a role in the induction of beta-adrenergic supersensitivity in the cultured cardiomyocytes and point to a new physiological role of the lipoxygenase product 15-S-HETE.Abbreviations NDGA nordihydroguaiaretic acid - HETE hydroeicosatetraenoic acid - HPETE hydroperoxyeicosatetraenoic acid     

Circular dichroism of platelet factor 4

The circular dichroism of platelet factor 4 was investigated and it was found to contain 15% alpha-helix, 25% beta-structure, and the rest of the molecule in unordered conformation. In the presence of heparin, no change in the circular dichroism was observed, suggesting no significant changes in the secondary structure of platelet factor 4 when heparin binds. The CD spectrum of platelet factor 4 was also investigated in the presence of increasing concentrations of guanidine hydrochloride. A two-state transition was observed with midpoints at 0.125 and 2.0 M guanidine hydrochloride. Based on gel filtration studies, the first unfolding transition was correlated with the dissociation of the tetrameric structure. This first unfolding domain was not observed in the presence of heparin, suggesting that heparin stabilizes the tetrameric structure. The second unfolding transition corresponds to the disruption of the overall secondary structure which is generally observed with most proteins. It is concluded that a relatively weak force of attraction holds the tetrameric structure of platelet factor 4 and the dissociation of the subunits is accompanied by loss of some helical secondary structure.     

The Nutrimentary Egg Development of the Mite,Varroa jacobsoni (Acari,Arachnida), an Ectoparasite of Honey Bees

The fine structure of the female gonad of Varroa jacobsoni is described. There are two components: the ovary proper and the so-called lyrate organ. The ovary is the place where oocytes mature, embedded in a supporting tissue composed of two cell types: somacells 1 and somacells 2. The lyrate organ has a nutrimentary function and is comprised of two components: supporting cells and nutritive tissue. The supporting cells are similar to the somacells 2 in that they contain abundant microtubules. The nutritive tissue is an extensive syncytium. It is connected with the oocytes via intercellular bridges, the nutritive cords. Oocytes and nutritive tissue are thought to have derived from common stem cells. From fine structural evidence it is concluded that ribosomes are one of the most important components to be transported via the nutritive cords into the oocytes. However, an increase in number of mitochondria in the middle-stage oocytes may also be a consequence of transport of these organelles from the nutritive tissue into the oocytes. Further characteristics make plausible that the interdependences of oocytes and nutritive tissue are comparable to those found in meroistic ovarioles of insects. The somatic components do not seem to be as important as the follicle cells of insects, however. It is assumed that the evolution of a nutrimentary oogenesis speeds up embryogenesis. Thus, the differentiation of the female gonad of Varroa jacobsoni may have facilitated the species' adaptation to a development completed in a short and limited time within the shelter of the covered brood cell of the bee.     

Phosphate-Activated Glutaminase in the Crude Mitochondrial Fraction (P2 Fraction) from Human Brain Cortex

The kinetics and other properties of phosphate-activated glutaminase have for the first time been studied in the crude mitochondrial fraction (P2 fraction) from human brain. The enzyme is for unexplained reasons inactivated postmortem. The enzyme activity decreases by storing the tissue or homogenate at 37 degrees C. The inactivation is not caused by formation of a dialysable inhibiting compound. No large proteolytic degradation has occurred, since the phosphate-activated glutaminase-like immunoreactive band did not disappear during the storage. The molecular weight of the subunit of the enzyme as determined by immunoblots of sodium dodecyl sulfate-treated homogenates from human brain is estimated to be approximately 64 K. The enzyme has been shown to have a pH optimum of 8.6; it is activated by phosphate, inhibited by glutamate, and partially inhibited by ammonia. Double-inverse plots of enzyme activity against phosphate are concave-upward, and more so in the presence of an inhibitor. The inhibition by glutamate appears to be noncompetitive with the substrate glutamine, and competitive with the activator phosphate. These kinetic properties are not significantly different from our earlier observations concerning phosphate-activated glutaminase from pig brain and pig kidney.     

AFA-I, a cloned afimbrial X-type adhesin from a human pyelonephritic Escherichia coli strain. Purification and chemical, functional and serologic characterization

AFA-I, a mannose-resistant, P-independent, X-binding afimbrial Escherichia coli adhesin was purified from a recombinant strain and chemically, functionally and serologically characterized. AFA-I exists on the bacterial surface and free as a macromolecular aggregate in the supernatant of spent culture medium. It is composed of a single, repeating 16-kDa polypeptide subunit. The AFA-I protein amino acid composition is remarkable for the presence of 22% non-polar hydrophobic residues and 2.5-3.0 cysteines per subunit. Since AFA-I travels as a monomer in sodium dodecyl sulfate/polyacrylamide gel electrophoresis under non-reducing conditions, no disulfide bonds exist between subunits and at least one free sulfhydryl per subunit is available. The AFA-I N-terminal amino acid sequence residues 1-24 was unrelated to E. coli fimbrial sequences; however, the N-terminus of AFA-I and GV-12, another E. coli afimbrial protein, was asparagine. HB101 (pIL 14), the AFA-I recombinant strain, agglutinated only human and gorilla erythrocytes, indicating a preference for receptor molecules on the red cells of man and the anthropoid apes. AFA-I did not bind glycophorin A or sialyl glycosides and is therefore distinct from the E. coli X-binding adhesins with M and S specificity. The AFA-I receptor was found to be abundant and diffusely distributed on HeLa tissue culture monolayer cell surfaces by indirect fluorescent microscopy. Anti-AFA-I sera bound AFA-I in Western blots of 4 out of 16 X-binding E. coli urine isolates. They did not bind MS or P pili. AFA-I may be exemplary of an adhesin class significant for the pathogenesis of human urinary tract infections.     

Phenobarbital induction of rat liver cytochromes P-450b and P-450e. Quantitation of specific RNAs by hybridization to synthetic oligodeoxyribonucleotide probes

Cytochromes P-450b and P-450e are extremely homologous and immunochemically indistinguishable proteins that are coordinately induced by phenobarbital in rat liver. To assess the effect of phenobarbital on mRNA levels for each of these hemoproteins we performed solution hybridization and Northern blot experiments with synthetic oligodeoxynucleotide probes of defined sequence. Our data demonstrate that phenobarbital administration to rats resulted in marked increases in levels of hepatic mRNA for both cytochrome P-450b and cytochrome P-450e, with a 4- to 5-fold greater accumulation of P-450b mRNA vis à vis P-450e mRNA. The level of hepatic mRNA increased from less than 3 molecules/cell of each mRNA in untreated rats, to 630 and 130 molecules/cell for P-450b and P-450e, respectively, in phenobarbital-treated rats. Data obtained in Northern blot hybridization experiments demonstrated that the size of the mRNAs for each protein were identical, being approximately 1800 bases in length.     

Action of neurotensin on size, composition, and growth of pancreas and stomach in the rat

Since the gastrointestinal peptide neurotensin has a stimulatory effect on the secretion of the exocrine pancreas and an inhibitory effect on secretion and motility of the stomach, we investigated whether chronic parenteral administration of neurotensin would affect pancreatic and gastric growth. We therefore infused synthetic neurotensin subcutaneously (dose, 43 and 282 pmol X kg-1 X min-1) in 20 Wistar rats for 2 weeks using Alzet osmotic minipumps and compared pancreatic weight, DNA, RNA, protein, lipase, amylase, pancreatic polypeptide and insulin with these parameters in 10 control rats from the same litter with subcutaneously implanted plastic cylinders approximately the size of the minipumps. In another experiment, synthetic neurotensin (836 pmol X kg-1) was injected intraperitoneally three times a day for 3 days in 12 rats. Thereafter, we measured pancreatic DNA and in vitro incorporation of [3H]thymidine into pancreatic DNA. These effects were compared with the actions of caerulein and normal saline. Long term infusion of the high neurotensin dose induced an increase of pancreatic weight (control: 0.87 g, neurotensin: 1.02 g) and of DNA (control: 2.5 micrograms; neurotensin: 3.5 micrograms) and pancreatic polypeptide (control: 2.4 ng; neurotensin: 7.4 ng) contents, whereas pancreatic protein, RNA, amylase and lipase contents were not stimulated. In relation to DNA, these parameters even were significantly depressed. Insulin remained unchanged. Intraperitoneal injection of neurotensin induced an increase of pancreatic DNA content and stimulated [3H]thymidine incorporation into DNA (control: 11 000 dpm/g; neurotensin: 15 800 dpm/g pancreas). Moreover, long-term neurotensin infusion with the high dose led to a rise in protein concentration and an increase in the thickness of the gastric antrum; antral DNA concentration was insignificantly stimulated. Parenteral neurotensin in the doses and at the times administered, led therefore, to hyperplasia of the pancreas and induced growth of the gastric antrum. It is concluded that neurotensin can act as a trophic factor on pancreas and gastric antrum of the rat. It remains to be determined whether this represents a physiological effect of neurotensin.