Borrelia burgdorferi periplasmic flagella have both skeletal and motility functions

Bacterial shape usually is dictated by the peptidoglycan layer of the cell wall. In this paper, we show that the morphology of the Lyme disease spirochete Borrelia burgdorferi is the result of a complex interaction between the cell cylinder and the internal periplasmic flagella. B. burgdorferi has a bundle of 7–11 helically shaped periplasmic flagella attached at each end of the cell cylinder and has a flat-wave cell morphology. Backward moving, propagating waves enable these bacteria to swim in both low viscosity media and highly viscous gel-like media. Using targeted mutagenesis, we inactivated the gene encoding the major periplasmic flagellar filament protein FlaB. The resulting flaB mutants not only were nonmotile, but were rod-shaped. Western blot analysis indicated that FlaB was no longer synthesized, and electron microscopy revealed that the mutants were completely deficient in periplasmic flagella. Wild-type cells poisoned with the protonophore carbonyl cyanide-m-chlorophenylhydrazone retained their flat-wave morphology, indicating that the periplasmic flagella do not need to be energized for the cell to maintain this shape. Our results indicate that the periplasmic flagella of B. burgdorferi have a skeletal function. These organelles dynamically interact with the rod-shaped cell cylinder to enable the cell to swim, and to confer in part its flat-wave morphology.

Relationship between the oxidation potential and electron spin density of the primary electron donor in reaction centers from Rhodobacter sphaeroides

The primary electron donor in bacterial reaction centers is a dimer of bacteriochlorophyll a molecules, labeled L or M based on their proximity to the symmetry-related protein subunits. The electronic structure of the bacteriochlorophyll dimer was probed by introducing small systematic variations in the bacteriochlorophyll–protein interactions by a series of site-directed mutations that replaced residue Leu M160 with histidine, tyrosine, glutamic acid, glutamine, aspartic acid, asparagine, lysine, and serine. The midpoint potentials for oxidation of the dimer in the mutants showed an almost continuous increase up to ≈60 mV compared with wild type. The spin density distribution of the unpaired electron in the cation radical state of the dimer was determined by electron–nuclear–nuclear triple resonance spectroscopy in solution. The ratio of the spin density on the L side of the dimer to the M side varied from ≈2:1 to ≈5:1 in the mutants compared with ≈2:1 for wild type. The correlation between the midpoint potential and spin density distribution was described using a simple molecular orbital model, in which the major effect of the mutations is assumed to be a change in the energy of the M half of the dimer, providing estimates for the coupling and energy levels of the orbitals in the dimer. These results demonstrate that the midpoint potential can be fine-tuned by electrostatic interactions with amino acids near the dimer and show that the properties of the electronic structure of a donor or acceptor in a protein complex can be directly related to functional properties such as the oxidation–reduction midpoint potential.

Effects of elevated atmospheric CO2 concentration on leaf dark respiration of Xanthium strumarium in light and in darkness

Leaf dark respiration (R) is an important component of plant carbon balance, but the effects of rising atmospheric CO2 on leaf R during illumination are largely unknown. We studied the effects of elevated CO2 on leaf R in light (RL) and in darkness (RD) in Xanthium strumarium at different developmental stages. Leaf RL was estimated by using the Kok method, whereas leaf RD was measured as the rate of CO2 efflux at zero light. Leaf RL and RD were significantly higher at elevated than at ambient CO2 throughout the growing period. Elevated CO2 increased the ratio of leaf RL to net photosynthesis at saturated light (Amax) when plants were young and also after flowering, but the ratio of leaf RD to Amax was unaffected by CO2 levels. Leaf RN was significantly higher at the beginning but significantly lower at the end of the growing period in elevated CO2-grown plants. The ratio of leaf RL to RD was used to estimate the effect of light on leaf R during the day. We found that light inhibited leaf R at both CO2 concentrations but to a lesser degree for elevated (17–24%) than for ambient (29–35%) CO2-grown plants, presumably because elevated CO2-grown plants had a higher demand for energy and carbon skeletons than ambient CO2-grown plants in light. Our results suggest that using the CO2 efflux rate, determined by shading leaves during the day, as a measure for leaf R is likely to underestimate carbon loss from elevated CO2-grown plants.

Blue Light and Abscisic Acid Independently Induce Heterophyllous Switch in Marsilea quadrifolia1

In natural habitats Marsilea quadrifolia L. produces different types of leaves above and below the water level. In aseptic cultures growth conditions can be manipulated so that leaves of the submerged type are produced continuously. Under such conditions the application of either blue light or an optimal concentration of abscisic acid (ABA) induced the development of aerial-type leaves. When fluridone, an inhibitor of ABA biosynthesis, was added to the culture medium it did not prevent blue light induction of aerial leaf development. During blue light treatment the endogenous ABA level in M. quadrifolia leaves remained unchanged. However, after the plants were transferred to an enriched medium, the ABA level gradually increased, corresponding to a transition in development from the submerged type of leaves to aerial leaves. These results indicate that the blue light signal is not mediated by ABA. Therefore, in the regulation of heterophyllous determination, discrete pathways exist in response to environmental signals.

Changes in Organization of the Endoplasmic Reticulum during Xenopus Oocyte Maturation and ActivationV⃞

The organization of the endoplasmic reticulum (ER) in the cortex of Xenopus oocytes was investigated during maturation and activation using a green fluorescent protein chimera, immunofluorescence, and electron microscopy. Dense clusters of ER developed on the vegetal side (the side opposite the meiotic spindle) during maturation. Small clusters appeared transiently at the time of nuclear envelope breakdown, disappeared at the time of first polar body formation, and then reappeared as larger clusters in mature eggs. The appearance of the large ER clusters was correlated with an increase in releaseability of Ca2+ by IP3. The clusters dispersed during the Ca2+ wave at activation. Possible relationships of ER structure and Ca2+ regulation are discussed.

Light and Excess Manganese1 : Implications for Oxidative Stress in Common Bean

The effect of light intensity on antioxidants, antioxidant enzymes, and chlorophyll content was studied in common bean (Phaseolus vulgaris L.) exposed to excess Mn. Leaves of bean genotypes contrasting in Mn tolerance were exposed to two different light intensities and to excess Mn; light was controlled by shading a leaflet with filter paper. After 5 d of Mn treatment ascorbate was depleted by 45% in leaves of the Mn-sensitive genotype ZPV-292 and by 20% in the Mn-tolerant genotype CALIMA. Nonprotein sulfhydryl groups and glutathione reductase were not affected by Mn or light treatment. Ten days of Mn-toxicity stress increased leaf ascorbate peroxidase activity of cv ZPV-292 by 78% in low light and by 235% in high light, and superoxide dismutase activity followed a similar trend. Increases of ascorbate peroxidase and superoxide dismutase activity observed in cv CALIMA were lower than those observed in the susceptible cv ZPV-292. The cv CALIMA had less ascorbate oxidation under excess Mn-toxicity stress. Depletion of ascorbate occurred before the onset of chlorosis in Mn-stressed plants, especially in cv ZPV-292. Lipid peroxidation was not detected in floating leaf discs of mature leaves exposed to excess Mn. Our results suggest that Mn toxicity may be mediated by oxidative stress, and that the tolerant genotype may maintain higher ascorbate levels under stress than the sensitive genotype.

ADP-Glucose Pyrophosphorylase Is Localized to Both the Cytoplasm and Plastids in Developing Pericarp of Tomato Fruit1

The intracellular location of ADP-glucose pyrophosphorylase (AGP) in developing pericarp of tomato (Lycopersicon esculentum Mill) has been investigated by immunolocalization. With the use of a highly specific anti-tomato fruit AGP antibody, the enzyme was localized in cytoplasm as well as plastids at both the light and electron microscope levels. The immunogold particles in plastids were localized in the stroma and at the surface of the starch granule, whereas those in the cytoplasm occurred in cluster-like patterns. Contrary to the fruit, the labeling in tomato leaf cells occurred exclusively in the chloroplasts. These data demonstrate that AGP is localized to both the cytoplasm and plastids in developing pericarp cells of tomato.

Regulation of herbivore growth by the balance of light and nutrients.

Experiments using planktonic organisms revealed that the balance of radiant energy and available nutrients regulated herbivore growth rates through their effects on abundance and chemical composition of primary producers. Both algae and herbivores were energy limited at low light/nutrient ratios, but both were nutrient limited at high light/nutrient ratios. Herbivore growth increased with increasing light intensity at low values of the light/nutrient ratio due to increases in algal biomass, but growth decreased with increasing light at a high light/nutrient ratio due to decreases in algal quality. Herbivore production therefore was maximal at intermediate levels of the light/nutrient ratio. The results contribute to an understanding of mass transfer mechanisms in ecosystems and illustrate the importance of integration of energy-based and material-based currencies in ecology.

Protein-protein interactions in the rigor actomyosin complex.

Since it has not been possible to crystallize the actomyosin complex, the x-ray structures of the individual proteins together with data obtained by fiber diffraction and electron microscopy have been used to build detailed models of filamentous actin (f-actin) and the actomyosin rigor complex. In the f-actin model, a single monomer uses 10 surface loops and two alpha-helices to make sometimes complicated interactions with its four neighbors. In the myosin molecule, both the essential and regulatory light chains show considerable structural homology to calmodulin. General principles are evident in their mode of attachment to the target alpha-helix of the myosin heavy chain. The essential light chain also makes contacts with other parts of the heavy chain and with the regulatory light chain. The actomyosin rigor interface is extensive, involving interaction of a single myosin head with regions on two adjacent actin monomers. A number of hydrophobic residues on the apposing faces of actin and myosin contribute to the main binding site. This site is flanked on three sides by charged myosin surface loops that form predominantly ionic interactions with adjacent regions of actin. Hydrogen bonding is likely to play a significant role in actin-actin and actin-myosin interactions since many of the contacts involve loops. The model building approach used with actomyosin is applicable to other multicomponent assemblies of biological interest and is a powerful method for revealing molecular interactions and providing insights into the mode of action of the assemblies.

Recruitment of hepatocyte nuclear factor 4 into specific intranuclear compartments depends on tyrosine phosphorylation that affects its DNA-binding and transactivation potential.

Hepatocyte nuclear factor 4 (HNF-4) is a prominent member of the family of liver-enriched transcription factors, playing a role in the expression of a large number of liver-specific genes. We report here that HNF-4 is a phosphoprotein and that phosphorylation at tyrosine residue(s) is important for its DNA-binding activity and, consequently, for its transactivation potential both in cell-free systems and in cultured cells. Tyrosine phosphorylation did not affect the transport of HNF-4 from the cytoplasm to the nucleus but had a dramatic effect on its subnuclear localization. HNF-4 was concentrated in distinct nuclear compartments, as evidenced by in situ immunofluorescence and electron microscopy. This compartmentalization disappeared when tyrosine phosphorylation was inhibited by genistein. The correlation between the intranuclear distribution of HNF-4 and its ability to activate endogenous target genes demonstrates a phosphorylation signal-dependent pathway in the regulation of transcription factor activity.

A 20-nucleotide element in the intestinal fatty acid binding protein gene modulates its cell lineage-specific, differentiation-dependent, and cephalocaudal patterns of expression in transgenic mice.

A sequence of epithelial cell proliferation, allocation to four principal lineages, migration-associated differentiation, and cell loss occurs along the crypt-villus axis of the mouse intestine. The sequence is completed in a few days and is recapitulated throughout the life-span of the animal. We have used an intestine-specific fatty acid binding protein gene, Fabpi, as a model for studying regulation of gene expression in this unique developmental system. Promoter mapping studies in transgenic mice identified a 20-bp cis-acting element (5'-AGGTGGAAGCCATCACACTT-3') that binds small intestinal nuclear proteins and participates in the control of Fabpi's cephalocaudal, differentiation-dependent, and cell lineage-specific patterns of expression. Immunocytochemical studies using confocal and electron microscopy indicate that it does so by acting as a suppressor of gene expression in the distal small intestine/colon, as a suppressor of gene activation in proliferating and nonproliferating cells located in the crypts of Lieberkühn, and as a suppressor of expression in the growth factor and defensin-producing Paneth cell lineage. The 20-bp domain has no obvious sequence similarities to known transcription factor binding sites. The three functions modulated by this compact element represent the types of functions required to establish and maintain the intestine's remarkably complex spatial patterns of gene expression. The transgenes described in this report also appear to be useful in characterizing the crypt's stem cell hierarchy.

The alpha and gamma 1 isoforms of protein phosphatase 1 are highly and specifically concentrated in dendritic spines.

Protein phosphatase 1 (PP1) is a highly conserved enzyme that has been implicated in diverse biological processes in the brain as well as in nonneuronal tissues. The present study used light and electron microscopic immunocytochemistry to characterize the distribution of two PP1 isoforms, PP1 alpha and PP1 gamma 1, in the rat neostriatum. Both isoforms are heterogeneously distributed in brain with the highest immunoreactivity being found in the neostriatum and hippocampal formation. Further, both isoforms are highly and specifically concentrated in dendritic spines. Weak immunoreactivity is present in dendrites, axons, and some axon terminals. Immunoreactivity for PP1 alpha is also present in the perikaryal cytoplasm and nuclei of most medium- and large-sized neostriatal neurons. The specific localization of PP1 in dendritic spines is consistent with a central role for this enzyme in signal transduction. The data support the concept that, in the course of evolution, spines developed as specialized signal transduction organelles enabling neurons to integrate diverse inputs from multiple afferent nerve terminals.

Cell cycle-related shifts in subcellular localization of BCR: association with mitotic chromosomes and with heterochromatin.

The disruption of the BCR gene and its juxtaposition to and consequent activation of the ABL gene has been implicated as the critical molecular defect in Philadelphia chromosome-positive leukemias. The normal BCR protein is a multifunctional molecule with domains that suggest its participation in phosphokinase and GTP-binding pathways. Taken together with its localization to the cytoplasm of uncycled cells, it is therefore presumed to be involved in cytoplasmic signaling. By performing a double aphidicolin block for cell cycle synchronization, we currently demonstrate that the subcellular localization of BCR shifts from being largely cytoplasmic in interphase cells to being predominantly perichromosomal in mitosis. Furthermore, with the use of immunogold labeling and electron microscopy, association of BCR with DNA, in particular heterochromatin, can be demonstrated even in quiescent cells. Results were similar in cell lines of lymphoid or myeloid origin. These observations suggest a role for BCR in the phosphokinase interactions linked to condensed chromatin, a network previously implicated in cell cycle regulation.

Localization of neuropeptide Y Y1 receptors in cerebral blood vessels

The localization of neuropeptide Y (NPY) Y1 receptor (R) -like immunoreactivity (LI) has been studied in cerebral arteries and arterioles of the rat by immunohistochemistry using fluorescence, confocal, and electron microscopy. High levels of Y1-R-LI were observed in smooth muscle cells (SMCs) in the small arterioles of the pial arterial network, especially on the basal surface of the brain, and low levels in the major basal cerebral arteries. The levels of Y1-R-LI varied strongly between adjacent SMCs. Y1-R-LI was associated with small endocytosis vesicles, mainly on the outer surface of the SMCs, but also on their endothelial side and often laterally at the interface between two SMCs. NPY-immunoreactive (Ir) nerve fibers could not be detected in association with the Y1-R-rich small arterioles but only around arteries with low Y1-R levels. A dense network of central NPY-Ir nerve fibers in the superficial layers of the brain was lying close to the strongly Y1-R-Ir small arterioles. The results indicate that NPY has a profound effect on small arterioles of the brain acting on Y1-Rs, both on the peripheral and luminal side of the SMCs. However, the source of the endogenous ligand, NPY, remains unclear. NPY released from central neurons may play a role, in addition to blood-borne NPY.

Fluid-Phase Markers in the Basolateral Endocytic Pathway Accumulate in Response to the Actin Assembly-promoting Drug Jasplakinolide

To investigate the role of filamentous actin in the endocytic pathway, we used the cell-permeant drug Jasplakinolide (JAS) to polymerize actin in intact polarized Madin–Darby canine kidney (MDCK) cells. The uptake and accumulation of the fluid-phase markers fluorescein isothiocyanate (FITC)-dextran and horseradish peroxidase (HRP) were followed in JAS-treated or untreated cells with confocal fluorescence microscopy, biochemical assays, and electron microscopy. Pretreatment with JAS increased the uptake and accumulation of fluid-phase markers in MDCK cells. JAS increased endocytosis in a polarized manner, with a marked effect on fluid-phase uptake from the basolateral surface but not from the apical surface of polarized MDCK cells. The early uptake of FITC-dextran and HRP was increased more than twofold in JAS-treated cells. At later times, FITC-dextran and HRP accumulated in clustered endosomes in the basal and middle regions of JAS-treated cells. The large accumulated endosomes were similar to late endosomes but they were not colabeled for other late endosome markers, such as rab7 or mannose-6-phosphate receptor. JAS altered transport in the endocytic pathway at a later stage than the microtubule-dependent step affected by nocodazole. JAS also had a notable effect on cell morphology, inducing membrane bunching at the apical pole of MDCK cells. Although other studies have implicated actin in endocytosis at the apical cell surface, our results provide novel evidence that filamentous actin is also involved in the endocytosis of fluid-phase markers from the basolateral membrane of polarized cells.