Fluoride-induced filaments of Erwinia carotovora

Sodium fluoride induces filamentous growth of Erwinia carotovora when it is grown in liquid media containing aspartic acid only as the sole source of nitrogen. It is proposed that a stable complex of F(-)-Mg2+-enzyme and PO2(4) resulting in Mg2+ deficiency and consequent inability of E. carotovora cells to oxidize aspartic acid normally, is responsible for the formation of filaments in the presence of fluoride ions.     

Toxic components on the larval surface of the corn earworm (Heliothis zea) and their effects on germination and growth of Beauveria bassiana

Caprylic acid is present on the surface of corn earworm, Heliothis zea, and fall armyworm, Spodoptera fragiterda, larvae. Because caprylic acid inhibits germination of Beauveria bassiana, presence of this compound will be determined to the establishment of an infection of larvae by this fungus. Other free fatty acids present on the surface of the H. zea and S. frugiterda are tentatively identified as valeric and nonanoic acids; these also possess mycostatic activity toward B. bassiana. Depending on concentration, caprylic acid inhibits germination of conidia for different amounts of time (R. J. Smith and E. A. Grula, 1981, J. Invertebr. Pathol., 37, 222–230). We now further report that inhibition and/or growth is also related to the source of carbon, nitrogen, and energy present in the growth medium. This observation of selective toxicity in the presence of different nutrients was also observed using nonanoic acid. Our data therefore make it necessary to interpret the effects of certain fatty acids on germination and growth of B. bassiana (and probably other fungi as well) in terms of nutrients for the germination process.     

Andrenocorticotropin and β-lipotropin in the hypothalamus

Adrenocorticotropin and β-lipotropin (β-LPH) have been localized by immunoperoxidase methods in nerve cells and fibers of the hypothalamus and brain stem of the ewe. 6-μm sections were immunostained first for either ACTH or β-LPH. The reaction products and the antibody complexes were then eluted completely from the tissue, and the same section was immunostained for the second peptide. Absorption of the primary antisera with a variety of peptide fragments of ACTH and β-LPH demonstrated, immunocytochemically as well as by radioimmunoassay, that the ACTH and β-LPH antisera were directed to the COOH- and NH(2)-termini of the peptides, respectively. Neither antiserum recognized any portion of the heterologous peptide. In the sequential staining procedure on the same tissue section, preincubation of the antisera with the homologous peptide abolished the staining, whereas preincubation with the heterologous peptide did not affect it, regardless of the order followed. Every nerve cell in the arcuate nucleus that contained ACTH also contained β-LPH, but β-LPH cells appeared, probably falsely, to be twice as numerous as ACTH cells. β-LPH-positive fibers in and beyond the hypothalamus were also more numerous and stained more intensively than ACTH fibers. The salient exception was fibers in the infundibular zona externa, where the opposite was true. Our observations establish that ACTH and β-LPH are contained in the same nerve cells They stongly favor biosynthesis in brain, probably from a common precursor molecule, as has been demonstrated in the pituitary gland. The complexity of the cytologic distribution pattern described suggests that the two peptides are not processed in the same manner by the nerve cell.     

Structural origins of fibrin clot rheology

The origins of clot rheological behavior associated with network morphology and factor XIIIa-induced cross-linking were studied in fibrin clots. Network morphology was manipulated by varying the concentrations of fibrinogen, thrombin, and calcium ion, and cross-linking was controlled by a synthetic, active-center inhibitor of FXIIIa. Quantitative measurements of network features (fiber lengths, fiber diameters, and fiber and branching densities) were made by analyzing computerized three-dimensional models constructed from stereo pairs of scanning electron micrographs. Large fiber diameters and lengths were established only when branching was minimal, and increases in fiber length were generally associated with increases in fiber diameter. Junctions at which three fibers joined were the dominant branchpoint type. Viscoelastic properties of the clots were measured with a rheometer and were correlated with structural features of the networks. At constant fibrinogen but varying thrombin and calcium concentrations, maximal rigidities were established in samples (both cross-linked and noncross-linked) which displayed a balance between large fiber sizes and great branching. Clot rigidity was also enhanced by increasing fiber and branchpoint densities at greater fibrinogen concentrations. Network morphology is only minimally altered by the FXIIIa-catalyzed cross-linking reaction, which seems to augment clot rigidity most likely by the stiffening of existing fibers.     

Identification and characterization of a prevacuolar compartment in stigmas of nicotiana alata

The stigmas of the ornamental tobacco plant Nicotiana alata accumulate large quantities of a series of 6-kD proteinase inhibitors (PIs) in the central vacuole that are derived from a 40-kD precursor protein, Na-PI. The sorting information that directs Na-PI to the vacuole is likely to reside in a C-terminal propeptide domain of 25 amino acids that forms an amphipathic alpha helix. Using cell fractionation techniques, we have examined transit of Na-PI through the endomembrane system and have identified a prevacuolar compartment that contains Na-PI with an intact targeting signal. In contrast, the targeting signal is not present on the predominant form of Na-PI in the vacuole. The prevacuolar compartment is marked by the presence of homologs of both the t-SNARE, PEP12p, and the putative vacuolar sorting receptor BP-80. Cross-linking and affinity precipitation studies revealed that Na-PI associates with BP-80 within this compartment, providing in vivo evidence for the function of BP-80 as a sorting receptor for a protein with a C-terminal vacuolar targeting signal.     

Requirement for sequential enzymatic activities for penetration of the integument of the corn earworm (Heliothis zea)

When larvae of the corn earworm, Heliothis zea, are boiled in 1% sodium dodecyl sulfate for 2 to 4 hr, larval contents are solubilized leaving a thin and transparent but intact skeletal structure that we are naming ghosts. The thinnest and most transparent ghosts are produced by boiling first-instar larvae. As larvae mature, later instar forms possess substantial quantities of melanized areas that remain largely undissolved in the detergent. Chemical analyses reveal that ghosts consist largely, and perhaps exclusively, of chitin and protein. Scanning electron microscopy shows that the chitin-protein complex is a continuous layer that does not possess holes sufficiently large for fungal hyphae to pass through without some type of accompanying enzymatic activity. Digestion of ghosts is possible using the combination of a proteolytic enzyme followed by chitinase. Because this sequence is necessary for dissolution of the structure, the protein material is most likely structural and wrapped about the chitin. Such an arrangement would allow protection of the structure from direct chitinase attack. The number of proteins involved is not known; however, because they survive the detergent-heat treatment this strongly suggests that the protein(s) is covalently bonded to chitin. Several different proteolytic enzymes allow attack of the underlying chitin by chitinase. This indicates that the accompanying protein(s) is exposed and therefore accessible to several types of proteolytic enzymes. Such a conclusion may not be warranted, however, if the accessibility and conformational state of the protein(s), and therefore digestibility, is affected by the relatively vigorous preparative procedure.     

Requirement for pantothenate for filament formation by Erwinia carotovora.

Pantothenate is required for the formation of filaments by Erwinia carotovora. This has been demonstrated for the following division-inhibiting agents: D-serine, D-cycloserine, penicillin, vancomycin, fluoride ion, and ultraviolet light. D-Serine inhibits pantothenate synthesis in an ammonia-glucose or an ammonia-pyruvate medium; therefore, it is necessary to add pantothenate to obtain filament formation in these media, using D-serine as the division-inhibiting agent. Under conditions in which pantothenate synthesis is not inhibited by the agent producing filaments, the need for it for filamentation was shown by the use of salicylate, an inhibitor of endogenous pantothenate synthesis. Evidence is presented that the production of filaments is a specific response to pantothenate, rather than a nonspecific growth stimulation.     

Evolution of sea urchin non-repetitive DNA

Summary New methods have been applied to the determination of single copy DNA sequence differences between the sea urchin speciesStrongylocentrotus purpuratus, S. franciscanus, S. drobachiensis, andLytechinus pictus. The thermal stability of interspecies DNA duplexes was measured in a solvent (2.4 M tetraethylammonium chloride) that suppresses the effect of base composition on melting temperature. The lengths of duplexes were measured after digestion with S1 nuclease and correction made for the effect of length on thermal stability. The degree of base substitution that has occurred in the single copy DNA during sea urchin evolution is significantly larger than indicated by earlier measurements. We estimate that 19% of the nucleotides of the single copy DNA are different in the genomes of the two sea urchin congeners,S. purpuratus, andS. franciscanus, which apparently diverged only 15 to 20 million years ago.