Relationships between growth, morphology and wall stress in the stalk of Acetabularia acetabulum.

With carbon particles we analyzed patterns of growth in Acetabularia acetabulum (Lam.) P.C. Silva, a giant unicell famous for classic development studies. We focused on the stalk apex, which generates the stalk, whorls of hairs, and whorls of gametophores. To gain visual and physical accessibility, we amputated the youngest whorls of hair and the original apex and performed experiments on the apex that regenerated. Video analysis indicated that most growth occurred near the tip of the new apex. Less growth occured throughout the cut-interwhorl. We also analyzed cell wall thickness along stalks cleared of cytoplasm. Correlating growth data to wall morphology suggests growth near the apex may be proportional to stress on the cell wall. We propose that turgor-pressure wall stress modulates local apical cell wall growth rates. A supplementary model, relating cell wall curvature and growth rate in the cut-interwhorl, characterizes how the stalk's final dimensions and nearly cylindrical shap may arise. See http://faculty.washington.edu/mandoli/vondassow for supplementary data, analysis, and mathematical appendices. We believe this is the first quantiative description of apex morphogenesis of A. acetabulum.

The segment polarity network is a robust developmental module.

All insects possess homologous segments, but segment specification differs radically among insect orders. In Drosophila, maternal morphogens control the patterned activation of gap genes, which encode transcriptional regulators that shape the patterned expression of pair-rule genes. This patterning cascade takes place before cellularization. Pair-rule gene products subsequently 'imprint' segment polarity genes with reiterated patterns, thus defining the primordial segments. This mechanism must be greatly modified in insect groups in which many segments emerge only after cellularization. In beetles and parasitic wasps, for instance, pair-rule homologues are expressed in patterns consistent with roles during segmentation, but these patterns emerge within cellular fields. In contrast, although in locusts pair-rule homologues may not control segmentation, some segment polarity genes and their interactions are conserved. Perhaps segmentation is modular, with each module autonomously expressing a characteristic intrinsic behaviour in response to transient stimuli. If so, evolution could rearrange inputs to modules without changing their intrinsic behaviours. Here we suggest, using computer simulations, that the Drosophila segment polarity genes constitute such a module, and that this module is resistant to variations in the kinetic constants that govern its behaviour.

Microtubules and mitotic cycle phase modulate spatiotemporal distributions of F-actin and myosin II in Drosophila syncytial blastoderm embryos.

We studied cyclic reorganizations of filamentous actin, myosin II and microtubules in syncytial Drosophila blastoderms using drug treatments, time-lapse movies and laser scanning confocal microscopy of fixed stained embryos (including multiprobe three-dimensional reconstructions). Our observations imply interactions between microtubules and the actomyosin cytoskeleton. They provide evidence that filamentous actin and cytoplasmic myosin II are transported along microtubules towards microtubule plus ends, with actin and myosin exhibiting different affinities for the cell's cortex. Our studies further reveal that cell cycle phase modulates the amounts of both polymerized actin and myosin II associated with the cortex. We analogize pseudocleavage furrow formation in the Drosophila blastoderm with how the mitotic apparatus positions the cleavage furrow for standard cytokinesis, and relate our findings to polar relaxation/global contraction mechanisms for furrow formation.

The venomous hair structure, venom and life cycle of Lagoa crispata, a puss caterpillar of Oklahoma.

The presence of a unique population of Lagoa crispata, puss caterpillar, in western Oklahoma is reported. A detailed microscopic examination shows the structure of the L. crispata spines resemble the type 4 spines described by [Kawamoto, F., Kumada, N., 1984. Biology and venoms of lepidoptera. In: Tu, A.T. (Ed.), Handbook of Natural Toxins, Insect Poisons, Allergens and other invertebrate venoms, vol. 2, pp. 291-332 (ch. 9)]. The major food source of L. crispata are the leaves of oak (shin oak). The high tannin content of this food source results in spine extracts high in oak tannins. These extracts have activity but enzyme and toxin activity is lost with time. The gel filtration protein fractions are colored from brown to yellow and are inactive as enzymes or toxins. No hyaluronidase, protease or phosphohydrolase activity is detected in these protein fractions. The life cycle shows these caterpillars have 6 instars. Characterizations and annual emerging times of each instar are included.

Citrate inhibition of snake venom proteases.

Thirty snake venoms had a citrate content of 2.3 to 12.9%, dry basis, by an aconitase isocitric dehydrogenase coupled enzyme assay. This is a venom concentration range of approximately 30 to 150 mM citrate assuming 25% venom solids content. Inhibition of snake venom protease activity by the addition of exogenous citrate was obtained using azure blue hide powder and azocasein as substrates. Protease inhibitions of 7.5% for Crotalus atrox venom to 78% for Bothrops picadoi venom were observed with citrate. Complete inhibition of snake venom protease activity by citrate was not observed. Bothrops asper (Pacifico) venom showed a 41% protease inhibition by citrate with azocasein as the substrate and 46% inhibition of Bothrops asper (Alantico) venom protease with azure blue hide power as a substrate. Trypsin was not inhibited in this system. Citrate may inhibit some venom protease activity by forming a complex with the zinc of zinc-dependent enzymes. reserved.

Chemical kinetic theory: understanding cell-cycle regulation.

Progress of a cell through its reproductive cycle of DNA synthesis and division is governed by a complex network of biochemical reactions controlling the activities of both M-phase- and S-phase-promoting factors. Standard chemical kinetic theory provides a disciplined method for expressing the molecular biologists' diagrams and intuition in precise mathematical form, so that qualitative and quantitative implications of our 'working models' can be derived and compared with experiment.

Arthropod venom citrate inhibits phospholipase A2.

Citrate has been identified as a major component of honey bee (Apis mellifera) venom by gas liquid chromatography-mass spectrometry. A citrate concentration of 9% was found for dried bee venom by a coupled enzyme assay, aconitase-isocitric dehydrogenase. A liquid honey bee venom would contain 140 mM citrate concentration (if the solids content were 30%). Bee venom phospholipase was inhibited at a 43% level with a citrate concentration of 20 mM and calcium ion at 3 mM with the enzyme assay. Citrate was also found in the venoms of bumble bee, Bombus fervidus, 7%; yellow jacket, Vespula maculifrons, 4%; scorpion, Centruroides sculpturatus, 8%; tarantula, Grammastola cala, 8% and brown recluse spider venom gland extract, Loxoceles reclusa, 1.5% based on dried venom solids. Citrate may serve as an endogenous inhibitor of divalent metal ion-dependent enzymes in arthropod venoms as described by Francis et al. (1992, Toxicon 30, 1239-1246). Many arthropod venoms contain calcium-dependent phospholipases. A direct effect of citrate as a venom component may be possible. The presence of citrate in venoms must be considered in research on receptors, ion channels and divalent ion-dependent toxins.

Identification of an N-acetylated microsomal glutathione S-transferase by mass spectrometry.

Microsomal glutathione S-transferase (mGST) was purified to homogeneity from male Sprague-Dawley rat liver, as determined by SDS-PAGE. Removal of Triton X-100 and further separation by reversed phase HPLC revealed two proteins, mGST 1 and mGST 2, in a 1:3 ratio. Analysis of mGST 1 and mGST 2 by electrospray ionization mass spectrometry determined their molecular weights to be 17,354.2 +/- 6.6 and 17,397.9 +/- 6.6, respectively. mGST 1 was in close agreement with the calculated molecular weight of 17,348, as predicted by the previously reported cDNA sequence. Cyanogen bromide digestion and peptide mapping by fast atom bombardment mass spectrometry (FAB-MS) localized the mass increase to the N-terminal peptide, 1-7. FAB-tandem mass spectrometry of this peptide in conjunction with Edman reactions on the intact protein demonstrated the N-terminal alanine to be acetylated.

Primary structures of two proteins from the venom of the Mexican red knee tarantula (Brachypelma smithii).

Venom of the Mexican red knee tarantula (Brachypelma smithii) was fractionated by gel filtration over Sephadex G-50 Fine. Small polypeptides present in the second and third peaks were subfractionated by cation exchange and reversed-phase FPLC. One major, basic protein was isolated and sequenced from each G-50 fraction using a gas-phase protein sequencer. Primary structures were completed and confirmed using tandem mass spectrometry and carboxypeptidase digestions. Protein 1 contains 39 residues, including six cysteine residues in three disulfide bonds. It is identical to one of the isoforms of ESTX from the venom of the tarantula Eurypelma californicum. Brachypelma smithii Protein 5 contains 34 residues, including six cysteine residues in three disulfide bonds. Disulfide bond assignments for both proteins are provided. Protein 5 shows most similarity with toxin Tx2-9 from the Brazilian 'armed' spider, but only displays 41% sequence identity. Similarities with other proteins are lower. Proteins 1 and 5 appear unrelated to each other.

Bilirubin levels in subarachnoid clot and effects on canine arterial smooth muscle cells.

BACKGROUND AND PURPOSE: Previous studies have suggested that bilirubin is a potential contributor to cerebral vasospasm. The purpose of this investigation was to determine whether bilirubin accrues in subarachnoid clot, whether its vasoconstrictive effect could involve a direct action on arterial smooth muscle cells, and, if so, whether bilirubin affects their Ca2+ uptake. METHODS: Subarachnoid clots were analyzed for bilirubin using high-performance liquid chromatography. The length and 45Ca2+ uptake of vascular smooth muscle cells enzymatically dissociated from canine carotid arteries were measured before and after exposure to bilirubin solution. Additional experiments were conducted on cultured smooth muscle cells from canine basilar artery and on ATP-depleted cardiac myocytes. RESULTS: Mean +/- SE bilirubin concentration in experimental clot was 263 +/- 35.7 mumol/L. Vascular smooth muscle cells exposed to bilirubin showed progressive shortening (P < .01) and an increased uptake of 45Ca2+ (P < .001). Contraction was prevented by Ca(2+)-free media but not by verapamil. Experiments with heart myocytes showed that bilirubin caused an increased uptake of 45Ca2+ but not of [14C]sucrose. CONCLUSIONS: The results indicate that bilirubin accrues in subarachnoid clot, that it exerts a direct constrictive effect on arterial smooth muscle cells, and that this effect is associated with an increased uptake of Ca2+. Studies on heart myocytes suggest that the Ca2+ uptake induced by bilirubin could be due to a selective increase in membrane permeability to Ca2+.

Hemin: levels in experimental subarachnoid hematoma and effects on dissociated vascular smooth-muscle cells.

Although hemin is known to exert toxic effects on a variety of cell types, its possible participation in the genesis of cerebral vasospasm has received little attention. The authors measured the concentration of hemin in experimental subarachnoid clot and studied its effects on the morphology and 45Ca++ uptake of vascular smooth-muscle cells dissociated from canine carotid artery. Craniectomies were performed in five dogs under general anesthesia, and 3 to 5 ml of autologous whole blood was deposited in the supraclinoid subarachnoid compartment. The concentration of hemin recovered by Folch extraction from clotted material removed 7 days after surgery was 390 +/- 247 microM (mean +/- standard error of the mean). Mean vascular smooth-muscle cell length after 40 minutes of exposure to 50 microM hemin was 37.3 +/- 1.2 microns (control 51.6 +/- 1.6 microns) (p < 0.01). The mean percent permeation of 45Ca++, measured by a dual label technique, of cells exposed to hemin was 200.9% +/- 23% (control 102.9% +/- 4.3%) (p < 0.01). These findings indicate that hemin accrues in subarachnoid hematoma, that it exerts a constrictive effect on vascular smooth-muscle cells, and that this effect is associated with an increased uptake of Ca++. This study demonstrates that hemin should be included in the list of potential agents that participate in the development of cerebral vasospasm.

Cellular motions and thermal fluctuations: the Brownian ratchet.

We present here a model for how chemical reactions generate protrusive forces by rectifying Brownian motion. This sort of energy transduction drives a number of intracellular processes, including filopodial protrusion, propulsion of the bacterium Listeria, and protein translocation.

Citrate is a major component of snake venoms.

Citrate has been identified as a major component of snake venoms by gas liquid chromatography and mass spectrometry. The venoms of Bothrops asper, Crotalus atrox, Crotalus viridis viridis, Crotalus adamanteus, Sistrurus miliarius barbouri, Crotalus horridus horridus, Agkistrodon contortrix mokasen, Agkistrodon contortrix contortrix and Agkistrodon piscivorus piscivorus contain citrate at concentration levels which can serve as effective buffers. Calcium, magnesium, zinc, iron, sodium and potassium salts of citrate would be constituents of venom.

Identification of glutathione conjugates of the dimethyl ester of bilirubin in the bile of Gunn rats.

The Gunn rat, which is deficient in the UDP-glucuronosyltransferase for bilirubin, promptly excreted polar conjugates of the dimethyl ester of bilirubin in bile after intravenous infusion of this ester. The conjugates proved to be monoglutathione thioether adducts of the vinyl groups of the parent tetrapyrrole. High performance liquid chromatographic analysis of the conjugates as their dipyrrolic azosulfanilates demonstrated that only one of the dipyrroles of each tetrapyrrole was conjugated. The nonconjugated dipyrrole eluted as either the methyl endo- or exovinyl azodipyrrole. The amino acid composition of the pigments was consistent with that of a monoglutathione conjugate. NMR spectroscopy of the two major pigments demonstrated the loss of the proton signals of the C-18 vinyl group, indicating it to be the site of conjugation. Cation fast atomic bombardment tandem mass spectrometry demonstrated a molecular ion, [M + H]+, of m/z 937, which fragmented with a loss of 307 atomic mass units, consistent with glutathione. A molecular ion of m/z 807 was observed for the conjugate treated with gamma-glutamyltranspeptidase, consistent with the loss of glutamate. The mass spectrometry data indicated that the conjugates also contained a functional group whose mass was equivalent to hydroxyl, suggesting initial formation of an epoxide, which then reacts with glutathione. Pretreatment of the rat with 2,3,7,8-tetrachlorodibenzo-p-dioxin to induce cytochrome P-450 resulted in a 6-fold increase of the biliary excretion of the glutathione conjugates. Such induction also resulted in the excretion of a glutathione conjugate of bilirubin itself.

In vitro formation of glutathione conjugates of the dimethylester of bilirubin.

Rat hepatic microsomes catalyzed the formation of two distinct glutathione conjugates of bilirubin dimethylester (DMB). The two conjugates were identical to those isolated from the bile of Gunn rats infused with DMB. The microsomal reaction was dependent on NADPH, oxygen and glutathione and was inhibited by nitrogen and the cytochrome P450 inhibitors metyrapone, 1-benzyl-imidazole, and alpha-naphthoflavone. Conjugate formation was inducible with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) but not phenobarbital pretreatment. The rate of formation of conjugates was not affected by washings of the microsomal pellet or by the presence of superoxide dismutase and/or catalase. Cation fast atom bombardment mass spectrometry (FAB/MS) of the conjugates indicated a molecular ion of 937 atomic mass units (amu). Fragmentation revealed a loss of 307 amu, consistent with glutathione, and a residual mass of 629 amu suggesting a hydroxylated derivative of DMB (612 amu). Cation FAB/MS/MS of conjugates formed in vitro under an atmosphere of oxygen-16 and oxygen-18 demonstrated the incorporation of molecular oxygen by a difference of 2 amu in the respective molecular ions. Our results suggest that DMB is oxidized by the cytochrome P450 IA gene family to an epoxide intermediate which is then subsequently conjugated with glutathione.

Hormonal control of gene expression: interactions between two trans-acting regulators in Drosophila.

The steroid hormone 20-hydroxyecdysone (20HE) and the Broad-Complex locus (BRC) are involved in regulating developmental changes in gene expression around the time of metamorphosis in Drosophila. We have investigated the regulatory interactions between 20HE, BRC, and a set of genes expressed in the fat body of third-instar Drosophila larvae. RNA levels for two hormone-inducible genes, Larval Serum Protein-2 and P1, accumulate to normal levels in BRC-mutant larvae. In contrast, RNA levels for the P6 gene were affected by mutations at BRC. On the basis of the results of experiments in which hormone concentrations were varied in BRC-mutant or wild-type larvae, we conclude that 20HE can both increase and decrease P6 RNA levels in the absence of BRC product(s). BRC appears to be a trans-acting modulator of the response of P6 to the hormone. We propose that BRC attenuates the repressive effect of the hormone, expanding the range of hormone concentrations that induce the gene, thus allowing P6 RNA to reach high levels during the third larval instar. The results are discussed in relation to other genes that are regulated by the same two trans-acting factors. A model is presented that refines the model of Ashburner et al. (1974, Cold Spring Harbor Symp. Quant. Biol. 38: 655-662) for the hormonal regulation of gene activity.

High-performance liquid chromatographic analysis of bile pigments as their native tetrapyrroles and as their dipyrrolic azosulfanilate derivatives.

A reversed-phase high-performance liquid chromatographic (HPLC) analysis of bile pigments is described that provides baseline separation of the major bilirubin conjugates found in bile. The advantage of the technique is that the bile pigments can be analyzed directly as their native tetrapyrroles without prior solvent extractions or derivatization. The use of ammonium acetate in place of sodium salts permits preparative isolation and lyophilization of the pigments for mass spectroscopy. The derivatization of the pigments as their dipyrrolic azosulfanilates with subsequent HPLC analysis demonstrates baseline separation of the endo- and exovinyl azodipyrroles and allows identification of that half of the tetrapyrrole which contains the conjugate in the instances of monoglycosides.