Scale Growth and Squamation Chronology for the Laboratory-Reared Hermaphroditic Fish <Emphasis Type="Italic">Rivulus marmoratus</Emphasis> (Cyprinodontidae)

Scale morphology, growth and the squarnation chronology are described for the hermaphroditic fish Rivulus marmoratus reared in the laboratory. The scales are round or oval shaped cycloid type, and their sizes are about 0.3–1.0 mm in diameter. The number of ridges increases more rapidly relative to the body growth of the fish in early stages, but this increase is proportionate to growth subsequently. Three loci of scale development have been identified. The scales first appeared on the center of the parietal region at 8 days after hatching. The second locus of scale formation was on the lateral line of the posterior end of the caudal peduncle. A third locus was later observed on the lower right corner of the operculum: The final squamation was completed at 6 weeks after hatching.     

Cultural characteristics and zearalenone production by strains of<Emphasis Type="Underline">Fusarium</Emphasis> from wheat

Conidia of the speciesFusarium culmorum /W.G.Sm./ Sacc. andFusarium graminearum Schwabe are characterized by variability in zearalenone production and dimensions depending on the substrate. The sporulation of isolates from some wheat eultivars have been deprived in vivo and in vitro in the first passage, but not their pathogenicity and toxic metabolites production. Nonsporulating strains produced lower quantités of zearalenone than sporulating ones. Liquid filtrates of such nonsporulating strains had a high phytotoxic effect on wheat caryopses. The crystalline toxin DAS /0,25 ug/ml/ had low phytotoxic effect on wheat caryopses.     

Cumulation of mycotoxins in maize cobs infected with<Emphasis Type="Italic">Fusarium gramihearum</Emphasis>

Maize cobs withFusarium ear rot were collected at 1986 season and five infected byFusarium graminearum were analyzed for presence of triohothecenes and zearalenone. Collected material was subsampled forFusarium damaged kernels and corresponding axial stems and healthy looking kernels. All investigated cobs contained deoxynivalenol (DON) (range 18.0–131.5 mg/kg) and zearalenone (ZEA) (range 0.38–2.17 mg/kg), in four cobs 15-acetyl-deoxynivalenol (15-AcDON) (range 5.2–6.2 mg/kg) was present and two cobs besides three all metabolites contained 3-acetyl-deoxynivalenol (3-AcD0N) (range 0.5–0.8 mg/kg).The average of individual toxins amount in axial stems: in mg/kg was equal to: DON — 110.36, ZEA — 4.57, 15-AcD0N — 16.66, and 3-AcD0N — 1.32.Fusarium damaged kernels contained in average the following amount (mg/kg) of: DON 77.00, ZEA 0.98, 15-AcD0N 3.78 and 3-AcD0N 0.06. Healthy looking kernels contained DON 1.96 mg/kg and ZEA 0.07 mg/kg only. Cooccurrence of 3-AcDON and 15-AcDON in two samples was an interesting finding. The amount of DON in total cob was highly correlated (r = 0.94) with percentage ofFusarium damaged kernels in given ear.     

Reduced bovine pancreatic trypsin inhibitor has a compact structure

The conformation of reduced bovine pancreatic trypsin inhibitor (R-BPTI) under reducing conditions was monitored by measurements of nonradiative excitation energy-transfer efficiencies (E) between a donor probe attached to the N-terminal Arg1 residue and an acceptor attached to one of the lysine residues (15, 26, 41, or 46) [Amir, D., & Haas, E. (1987) Biochemistry 26, 2162-2175]. High-excitation energy-transfer efficiencies that approach those found in the native state were obtained for the reduced labeled BPTI derivatives in 0.5 M guanidine hydrochloride (Gdn.HCl) and 4 mM DTT. Unlike the dependence expected for a random coil chain, E does not decrease as a function of the number of residues between the labeled sites. The efficiency of energy transfer between probes attached to residues 1 and 15 in the reduced state is higher than that found for the same pair of sites in the native state or reduced unfolded (in 6 M Gdn.HCl) state. This segment also shows high dynamic flexibility. These results indicate that the overall structure of reduced BPTI under folding (but still reducing) conditions shows a high population of conformers with interprobe distances similar to those of the native state. Reduced BPTI seems to be in a molten globule state characterized by a flexible, compact structure, which probably reorganizes into the native structure when the folding is allowed to proceed under oxidizing conditions.     

Pertussis toxin as a probe of neutrophil activation

In reviewing our own and other work, it is clear that pertussis toxin treatment of neutrophils causes a time- and concentration-dependent inhibition of granule enzyme secretion induced by formylmethionylleucylphenylalanine (fMet-Leu-Phe), C5a, leukotriene (LT) B4 and platelet-activating factor (PAF). Chemotaxis, O2- generation, aggregation, and arachidonic acid production induced by fMet-Leu-Phe are also inhibited by pertussis toxin. Granule enzyme release caused by A23187 or phorbol 12-myristate 13-acetate is not inhibited. The inhibition of neutrophil function correlates closely with the NAD-ribosylation of a 41,000-dalton protein in the neutrophil plasma membrane, presumably the GTP-binding regulatory protein Ni. Pertussis toxin treatment prevents or obtunds the increased influx of Ca2+ induced by fMet-Leu-phe and LTB4, but not that caused by stimulation of neutrophils with PAF. Pertussis toxin prevents the receptor-induced breakdown of polyphosphoinositides in intact neutrophils and isolated membrane and prevents or decreases the production of inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol. The hypothesis advanced by us and others is that pertussis toxin interacts with a GTP-binding regulatory protein identical or similar to Ni, which couples receptor-chemotactic factor interaction to phospholipase C activation. Inhibition of the activation prevents the production of IP3 and the resulting release of Ca2+ from intracellular stores and of 1,2-diacylglycerol and thus, the activation of protein kinase C. The lack of these two mediators is the immediate cause of the depression of neutrophil activation resulting from pertussis toxin. Some of the limitations and uncertainties of our present knowledge with respect to this hypothesis are discussed.     

Is a rise in intracellular concentration of free calcium necessary or sufficient for stimulated cytoskeletal-associated actin?

The addition of the calcium ionophore A23187 to rabbit neutrophils increases the amount of actin associated with the cytoskeleton regardless of the presence or absence of calcium in the incubation medium. In the presence of extracellular calcium, the effect of A23187 is biphasic with respect to concentration. The action of the ionophore is rapid, transient, and is inhibited by pertussis toxin, hyperosmolarity, and quinacrine. On the other hand, the addition of pertussis toxin or hyperosmolarity has small if any, effect on the rise in intracellular calcium produced by A23187. While quinacrine does not affect the fMet-Leu-Phe-induced increase in cytoskeletal actin and the polyphosphoinositide turnover, its addition inhibits completely the stimulated increase in Ca-influx produced by the same stimulus. The results presented here suggest that a rise in the intracellular concentration of free calcium is neither necessary nor sufficient for the stimulated increase in cytoskeletal-associated actin. A possible relationship between the lipid remodeling stimulated by chemoattractants and the increased cytoskeletal actin is discussed.     

The inhibition of neutrophil granule enzyme secretion and chemotaxis by pertussis toxin

Pertussis toxin treatment of rabbit peritoneal neutrophils causes a concentration-dependent inhibition of granule enzyme secretion induced by formylmethionyl-leucyl-phenylalanine, C5a, and leukotriene B4. It also inhibits chemotaxis induced by formylmethionyl-leucyl-phenylalanine. The same toxin treatment, however, has no effect on granule enzyme secretion induced by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate. Moreover, pertussis toxin treatment does not affect either the number or affinity of the formylpeptide receptors on the neutrophil nor does it have any effect on the unstimulated levels of cyclic AMP (cAMP) or the transient rise in cAMP induced by chemotactic factor stimulation in these cells. We hypothesize that pertussis toxin, as in other cells, interacts with a GTP binding regulatory protein identical with or analogous to either Ni or transducin which mediates the receptor-induced inhibition or activation of a target protein or proteins required in neutrophil activation. The nature of the target protein is unknown, but it is not the catalytic unit of adenylate cyclase. The target protein acts after binding of chemotactic factor to its receptor in the sequence that leads to the receptor-induced rise in intracellular Ca2+. It does not affect the responses elicited by the direct introduction of calcium into the cells or the activity of protein kinase C.     

Control of K+ influx in 3T3 cells transformed by a conditional mutant of Rous sarcoma virus

Mouse 3T3 cells transformed by a conditional mutant of Rous sarcoma virus (LA90) can assume either a normal or a transformed phenotype, depending on the temperature of cultivation. These cells (LA90) were arrested at the G0/G1 phase of the cell cycle by starvation for serum growth factors at the nonpermissive temperature (39 degrees C). Release from the G0/G1 phase by serum growth factors resulted in a rapid stimulation of Rb+ influx. To investigate whether the stimulation of Rb+ influx is obligatory for cell proliferation, the cultures were released from the G0/G1 phase by a temperature decrease in the absence of serum. A temperature decrease from 39 to 32 degrees C activated the viral pp60src gene mitogenic activity. Under these conditions, no rapid stimulation of Rb+ influx was observed. These results suggest that the rapid stimulation of Rb+ influx induced by serum growth factors is not an essential signal for cell release from the G0/G1 phase. However, a delayed increase in Rb+ influx concomitant with an increase in the cell content of K+ was observed in the cultures released from the G0/G1 phase by temperature decrease in the absence of serum growth factors. We found that the LA90 cells incubated at the permissive temperature (32 degrees C) secreted a mitogenic activity into the medium. Moreover, the conditioned medium from cultures incubated at 32 degrees C, but not at 39 degrees C, stimulate Rb+ influx in G0/G1 cells. These results indicate that Rous sarcoma virus pp60src induces a slow autocrine secretion of a mitogenic activity. This mitogenic activity slowly modulates the K+ content. Therefore, the slow elevation in cellular content of K+ is proposed to be an obligatory event for proliferation in normal and transformed cells.     

Chemotactic factor causes rapid decreases in phosphatidylinositol,4,5-bisphosphate and phosphatidylinositol 4-monophosphate in rabbit neutrophils

Stimulation of rabbit neutrophils prelabeled with 32P by the synthetic chemotactic peptide f-Met-Leu-Phe induces a rapid decrease in the radioactivity in both phosphatidylinositol, 4,5 bis phosphate and phosphatidylinositol 4-monophosphate. The mean +/- standard error of the mean values of the maximum decrease in phosphatidylinositol, 4,5 bis phosphate occurred at 10 seconds following stimulation and is equal to 19 +/- 3% of the control value. The corresponding value for phosphatidylinositol 4-monophosphate occurred at 60 seconds following stimulation and is equal to 37 +/- 7% of the control value. On the other hand, the radioactivity in phosphatidic acid and lysophospholipids increased continuously with time following stimulation. The relationship of these changes to calcium release and neutrophil activation is discussed.     

Fluorescence polarization as a tool to study lectin-sugar interaction

The binding ofRicinus communis agglutinin andAbrus agglutinin to 4-methylumbelliferyl β-D-galactopyranoside was studied by equilibrium dialysis, fluo-rescence quenching and fluorescence polarization. The number of binding sites and the association constant value obtained by fluorescence polarization for bothRicinus communis agglutinin andAbrus agglutinin are in close agreement with those obtained by the other methods. This indicates the potential of ligand-fluorescence polarization measurements in the investigation of lectin-sugar interactions.