Ramifications of kinetic partitioning on usher-mediated pilus biogenesis

The biogenesis of diverse adhesive structures in a variety of Gram-negative bacterial species is dependent on the chaperone/usher pathway. Very little is known about how the usher protein translocates protein subunits across the outer membrane or how assembly of these adhesive structures occurs. We have discovered several mechanisms by which the usher protein acts to regulate the ordered assembly of type 1 pili, specifically through critical interactions of the chaperone-adhesin complex with the usher. A study of association and dissociation events of chaperone-subunit complexes with the usher in real time using surface plasmon resonance revealed that the chaperone-adhesin complex has the tightest and fastest association with the usher. This suggests that kinetic partitioning of chaperone-adhesin complexes to the usher is a defining factor in tip localization of the adhesin in the pilus. Furthermore, we identified and purified a chaperone-adhesin-usher assembly intermediate that was formed in vivo. Trypsin digestion assays showed that the usher in this complex was in an altered conformation, which was maintained during pilus assembly. The data support a model in which binding of the chaperone-adhesin complex to the usher stabilizes the usher in an assembly-competent conformation and allows initiation of pilus assembly.     

The pilH gene encodes an ABC transporter homologue required for type IV pilus biogenesis and social gliding motility in Myxococcus xanthus

Type IV pilus genes have been shown to be required for social gliding motility in Myxococcus xanthus. We report the discovery of four additional pil genes: pilD, a homologue of type IV prepilin leader peptidases; and pilG, pilH and pilI, which have no known homologues in other type IV pilus systems. pilH encodes an ATP-binding cassette (ABC) transporter homologue, the first such homologue to be required for the biogenesis of any bacterial pilus type. pilG and pilI are co-transcribed with pilH and appear to be functionally related to pilH. Null mutants of pilG, pilH and pilI all lack social motility, are deficient in pilus production, have elevated sporulation efficiencies and display similar developmental abnormalities. In addition, all three mutations reduced the amount of PilA found in the supernatant after cells were sedimented from liquid culture. We suggest that the products of these three genes form a single ABC exporter complex, in which pilI is an integral membrane protein with membrane-spanning domains, and pilG is an accessory factor. The complex may participate in pilus assembly and/or the export of PilA pilin.     

Cloning of an Aeromonas hydrophila type IV pilus biogenesis gene cluster: complementation of pilus assembly functions and characterization of a type IV leader peptidase/N-methyltransferase required for extracellular protein secretion

Aeromonas hydrophila secretes several extracellular proteins that are associated with virulence including an enterotoxin, a protease, and the hole-forming toxin, aerolysin. These degradative enzymes and toxins are exported by a conserved pathway found in many Gram-negative bacteria. In Pseudomonas aeruginosa this export pathway and type IV pilus biogenesis are dependent on the product of the pilD gene. PilD is a bifunctional enzyme that processes components of the extracellular secretory pathway as well as a type IV prepilin. An A. hydrophila genomic library was transferred into a P. aeruginosa pilD mutant that is defective for type IV pilus biogenesis. The A. hydrophila pilD homologue, tapD, was identified by its ability to complement the pilD mutation in P. aeruginosa. Transconjugants containing tapD were sensitive to the type IV pilus-specific phage, PO4. Sequence data revealed that tapD is part of a cluster of genes (tapABCD) that are homologous to P. aeruginosa type IV pilus biogenesis genes (pilABCD). We showed that TapB and TapC are functionally homologous to P. aeruginosa PilB and PilC, the first such functional complementation of pilus assembly demonstrated between bacteria that express type IV pili. In vitro studies revealed that TapD has both endopeptidase and N-methyltransferase activities using P. aeruginosa prepilin as substrate. Furthermore, we show that tapD is required for extracellular secretion of aerolysin and protease, indicating that tapD may play an important role in the virulence of A. hydrophila.     

Suppression of an absolute defect in type IV pilus biogenesis by loss-of-function mutations in pilT, a twitching motility gene in Neisseria gonorrhoeae

Type IV pili of Neisseria gonorrhoeae, the Gram-negative etiologic agent of gonorrhea, facilitate colonization of the human host. Gonococcal PilT, a protein belonging to a large family of molecules sharing a highly conserved nucleotide binding domain motif, has been shown to be dispensable for organelle biogenesis but essential for twitching motility and competence for genetic transformation. Here, we show that the defect in pilus biogenesis resulting from mutations in the pilC gene, encoding a putative pilus-associated adhesin for human tissue, can be suppressed by the absence of functional PilT. These data conclusively demonstrate that PilT influences the Type IV pilus biogenesis pathway and strongly suggest that organelle expression is a dynamic process. In addition, these findings imply that PilT antagonizes the process of organelle biogenesis and provide the basis for a model for how the counteractive roles of PilT and PilC might relate mechanistically to the phenomenon of twitching motility.     

Increased eotaxin-mRNA expression in non-atopic and atopic nasal polyps: comparison to RANTES and MCP-3 expression

The Escherichia coli low molecular mass penicillin-binding proteins (PBP4, PBP5 and PBP6) are a group of penicillin-sensitive enzymes involved in the final stages of cell wall assembly. It has been suggested that these proteins may interact with the periplasmic face of the inner membrane via C-terminal amphiphilic alpha-helices. Theoretical analysis has predicted that these C-terminal helical regions may be membrane interactive. We have tested this hypothesis by assaying PBP C-terminal homologues (P4, P5 and P6) for haemolytic activity. Our results show that the PBP5 and PBP6 C-terminal homologues readily lyse sheep erythrocytes in a pH-dependent manner with LD50's of 3.5 x 10(-6) M and 6.8 x 10(-7) M respectively at pH 7. These results appear to support the present model for the membrane anchoring of PBP5 and PBP6. The PBP4 C-terminal homologue shows no evidence of haemolytic activity which could imply a different means of membrane association for PBP4.     

Influence of duodenal slaframine infusion on site of nutrient disappearance from the digestive tract of steers fed a high-concentrate diet

The effect of duodenal slaframine (SF) infusion on site and extent of digestion was determined using four steers equipped with ruminal, duodenal, and ileal cannulas in a 4 x 4 Latin square. A 77% dry-rolled corn diet was provided in 12 equal portions daily at a DMI of 2.26% BW. Slaframine in a .9% saline excipient was infused into the duodenum every 12 h with total daily dose of 0, 30, 60, or 90 micrograms /kg of BW. Slaframine infusion had no effect on ruminal pH, ruminal NH3 N, or solids and liquids passage rate. Slaframine increased (linear, P < .10) total tract OM and starch disappearance and digestibility and tended to increase (linear, P = .14) total tract N digestibility. Ruminal starch disappearance tended to be decreased (quadratic, P = .16) by SF. Small intestinal OM digestibility was increased (linear, P < .10) but starch digestibility in the small intestine was not affected by SF. Increased total tract starch digestibility was caused by increased (quadratic, P < .10) starch fermentation in the large intestine. Ruminal feed N digestibility decreased at the intermediate doses of SF (quadratic, P < .10). Total N digestibility in the small intestine tended to be increased (cubic, P = .13) with 30 and 90 micrograms of SF/kg of BW. Decreased ruminal feed N digestion was compensated for by increased (quadratic, P < .10) small intestinal feed N disappearance for steers treated with intermediate doses of SF. The potential of SF to increase starch digestion in the rumen and small intestine seems to be limited.     

Expression of collagens and decorin during aortic arch artery development: implications for matrix pattern formation

The elastic matrix of the large arteries shows a high level of spatial order. However, the mechanisms by which such order is established and maintained are largely unknown. The embryonic development of the avian heart and great vessels provides an appropriate model to investigate these mechanisms. In control embryos, an elastic matrix with a high level of spatial order develops in the nascent great vessels. But after the normal vascular smooth muscle (VSM) progenitor cells in the great vessels are experimentally replaced by other VSM progenitor cells, the elastic extracellular matrix is congenitally disordered. The present study used this model to test the hypothesis that the proteoglycan decorin was involved in the establishment and maintenance of the normal three-dimensional spatial order of the vascular elastic matrix. The temporospatial expression of decorin was analysed during development of normal vessels and in experimental vessels with surrogate VSM. The results showed the following: (1) the expression of decorin was related in time and space to the establishment of large helical collagen type III fibers that are characteristic of the normal elastic extracellular matrix; (2) in the experimental extracellular matrix there were few helical fibers of collagen type III, but those that were present remained positive for decorin; and (3) in both control and experimental vessels, decorin associated with neither fibers of collagen type I nor fibers of collagen type III in any conformation other than the large helical fibers. These data indicate a previously unrecognized relationship between decorin and the spatial order of the physiologically significant helical fibers of collagen type III.     

Characterization of type IV pilus genes in Pseudomonas syringae pv. tomato DC3000

Many strains of Pseudomonas syringae produce retractile pili that act as receptors for lytic bacteriophage phi 6. As these are also characteristics of type IV pili, it was postulated that P. syringae may possess genes for type IV pilus biogenesis. A cosmid clone bank of P. syringae pv. tomato DC3000 genomic DNA was used to complement a mutant of Pseudomonas aeruginosa defective in the PilD (XcpA) prepilin peptidase gene by selection for restoration of extracellular protein secretion, a function also known to require PilD. A cosmid able to complement this mutant was also able to complement mutations in the pilB and pilC genes, suggesting that, if the organization of these genes is similar to that of P. aeruginosa, the cosmid may contain the P. syringae pilA. This was confirmed by sequencing a region from this plasmid that was shown to hybridize at low stringency to the P. aeruginosa pilA gene. The deduced P. syringae PilA polypeptide possesses the characteristic properties of the type IV pilins. Heterologous expression of the P. syringae pilA in P. aeruginosa was also shown, conferring not only phi 6 phage sensitivity to P. aeruginosa pilA mutants but also sensitivity to PO4, a lytic bacteriophage specific for the pilus of P. aeruginosa. This suggests that additional components might be present in the mature pilus of P. aeruginosa that are the true receptors for this phage. Chromosomal mutations in P. syringae pv. tomato DC3000 pilA and pilD genes were shown to abolish its sensitivity to bacteriophage phi 6. To determine the importance of P. syringae pilus in plant leaf interactions, these mutations were tested under laboratory and field conditions. Although little effect was seen on pathogenicity, culturable leaf-associated population sizes of the pilA mutant were significantly different from those of the wild-type parent. In addition, the expression of the DC3000 pilA gene appears to contribute to the UV tolerance of P. syringae and may play a role in survival on the plant leaf surface.     

Crystal structure of the DpnM DNA adenine methyltransferase from the DpnII restriction system of streptococcus pneumoniae bound to S-adenosylmethionine

BACKGROUND:. Methyltransferases (Mtases) catalyze the transfer of methyl groups from S-adenosylmethionine (AdoMet) to a variety of small molecular and macromolecular substrates. These enzymes contain a characteristic alpha/beta structural fold. Four groups of DNA Mtases have been defined and representative structures have been determined for three groups. DpnM is a DNA Mtase that acts on adenine N6 in the sequence GATC; the enzyme represents group alpha DNA Mtases, for which no structures are known. RESULTS:. The structure of DpnM in complex with AdoMet was determined at 1.80 A resolution. The protein comprises a consensus Mtase fold with a helical cluster insert. DpnM binds AdoMet in a similar manner to most other Mtases and the enzyme contains a hollow that can accommodate DNA. The helical cluster supports a shelf within the hollow that may recognize the target sequence. Modeling studies indicate a potential site for binding the target adenine, everted from the DNA helix. Comparison of the DpnM structure and sequences of group alpha DNA Mtases indicates that the group is a genetically related family. Structural comparisons show DpnM to be most similar to a small-molecule Mtase and then to macromolecular Mtases, although several dehydrogenases show greater similarity than one DNA Mtase. CONCLUSIONS:. DpnM, and by extension the DpnM family or group alpha Mtases, contains the consensus fold and AdoMet-binding motifs found in most Mtases. Structural considerations suggest that macromolecular Mtases evolved from small-molecule Mtases, with different groups of DNA Mtases evolving independently. Mtases may have evolved from dehydrogenases. Comparison of these enzymes indicates that in protein evolution, the structural fold is most highly conserved, then function and lastly sequence.     

Specific detection and quantification of palmitoyl-stearoyl-phosphatidylserine in human blood using normal-phase liquid chromatography coupled with electrospray mass spectrometry

We have made an immunohistochemical study of the vomeronasal (VN) complex of 12-day-old rats to characterize the innervation of its blood vessels. The VN complex can be subdivided into rostral, middle and caudal segments, each one with a particular vascularization pattern. Several small vessels were associated with the rostral segment, whereas a large venous sinus ran along the middle and caudal segments. Immunostaining for alpha-smooth muscle actin demonstrated that the muscular sheath was asymmetric, with more cells layers in its lateral than in its medial walls. Nerves were demonstrated with antisera against protein gene product 9.5 (PGP), and against several molecules associated with specific classes of nerve fibers: the C-terminal peptide of neuropeptide Y (CPON), calcitonin gene-related peptide (CGRP), substance P (SP), galanin (GAL), vasoactive intestinal peptide (VIP) and neuronal nitric oxide synthase (NOS). The latter, was also studied with NADPH-diaphorase. Vascular associated fibers exhibited NOS-, CPON-, GAL-, CGRP-, SP- and VIP-immunoreactivity. Only the vessels of the rostral segment showed VIP-immunoreactive fibers. Each wall of the venous sinus exhibited different types of nerve fibers. CPON-, GAL-, CGRP- and SP-immunoreactive fibers concentrated in the medial wall, whereas NOS-immunoreactive ones concentrated in the lateral wall. This distribution of vascular fibers, plus the presence of sensory fibers exhibiting CGRP-, SP- and GAL-immunoreactivity within the pseudostratified epithelium of the VN tube, would be relevant to understand the operation of the pumping mechanism regulating influx and efflux from the VN tube.     

Benefits of helical final gas-cooling systems for coke-oven gas

The benefits of cooling coke-oven gas by means of helical final gas-cooling systems are considered. By data analysis, the effectiveness of helical final gas-cooling systems is assessed, for the example of Nexson Group equipment. A general conclusion is formulated regarding the economic benefits of using helical final gas-cooling systems for coke-oven gas, by the reconstruction of existing equipment or by the construction of new closed systems. The results may be useful to steelmakers in formulating corporate development plans and to regional governments in environmental and investment planning.     

Influence of dietary magnesium level on growth-performance and metabolic responses of Holstein steers to laidlomycin propionate

We used 216 Holstein steers (151 kg) in a 262-d trial to evaluate the influence of dietary magnesium level (.19, .25, and .32%) and laidlomycin propionate (LP; 0 vs 11 ppm, air-dry basis) on growth performance and NE value of the diet. During the initial 112 d of the trial, LP increased (P < .01) ADG (6.3%) and feed efficiency (4.2%). From d 112 until slaughter, LP increased (P < .05) ADG (9.7%) and feed efficiency (4.5%). Across the 262-d feeding period, LP supplementation enhanced (P < .01) ADG (8.9%) and feed efficiency (6.3%). There was an interaction (P < .05) between dietary Mg and LP on NE value of the diet. The enhancement in NE value of the diets owing to LP with .19, .25, and .32% dietary Mg were .5, 3.0, and 5.9%, respectively. Six Holstein steers (302 kg) were used in a 6 x 6 Latin square experiment to evaluate treatment effects on characteristics of ruminal and total tract digestion. There were no treatment interactions (P > .10) on site and extent of digestion of OM, starch, and N. Supplemental Mg increased (quadratic effect, P < .10) ruminal OM digestion. Neither LP nor dietary Mg level affected (P > .10) ruminal digestion of starch and feed N. Supplemental LP decreased (15%, P < .05) ruminal microbial efficiency. Total tract digestion of OM and N increased (linear effect, P < .01) with increasing dietary Mg level. There were interactions between LP and dietary Mg level on ruminal soluble-Mg concentration (linear effect, P < .01) and Mg absorption (quadratic effect, P < .05). Apparent total tract Mg digestion increased owing to LP (P < .01) and dietary Mg level (linear effect, P < .01). There were no treatment effects (P > .10) on ruminal pH. Dietary Mg level did not influence (P > .10) ruminal VFA concentrations or molar proportions. Supplemental LP increased (14%; P < .10) total ruminal VFA concentration but did not affect (P > .10) VFA molar proportions. We conclude that LP will increase daily weight gain and feed efficiency of calf-fed Holstein steers and that this response may be enhanced by increasing dietary Mg level.     

Numerical simulation of die compaction and sintering

Abstract

A method to simulate die compaction and sintering is presented. By implementing user defined routines for both processes into the general purpose finite element program ABAQUS quantitative predictions of density distributions and shape distortions can be obtained as well as the stresses in the tool components. By computational optimisation of the individual production steps suggestions can be made to improve the final properties. As an example for both die pressing and sintering a complex three-dimensional part is simulated and suggestions to improve dimensional accuracy are made. Finally, stresses in the tools are calculated showing that the deflections are large enough to cause punch to punch contact so that tool wear must be expected.     

Periplasmic chaperone recognition motif of subunits mediates quaternary interactions in the pilus

The class of proteins collectively known as periplasmic immunoglobulin-like chaperones play an essential role in the assembly of a diverse set of adhesive organelles used by pathogenic strains of Gram-negative bacteria. Herein, we present a combination of genetic and structural data that sheds new light on chaperone-subunit and subunit-subunit interactions in the prototypical P pilus system, and provides new insights into how PapD controls pilus biogenesis. New crystallographic data of PapD with the C-terminal fragment of a subunit suggest a mechanism for how periplasmic chaperones mediate the extraction of pilus subunits from the inner membrane, a prerequisite step for subunit folding. In addition, the conserved N- and C-terminal regions of pilus subunits are shown to participate in the quaternary interactions of the mature pilus following their uncapping by the chaperone. By coupling the folding of subunit proteins to the capping of their nascent assembly surfaces, periplasmic chaperones are thereby able to protect pilus subunits from premature oligomerization until their delivery to the outer membrane assembly site.     

Modulation of neuronal activity by target uncertainty

Visual scenes are composed of many elements and although we can appreciate a scene as a whole, we can only move our eyes to one element of the scene at a time. As visual scenes become more complex, the number of potential targets in the scene increases, and the uncertainty that any particular one will be selected for an eye movement also increases. How motor systems accommodate this target uncertainty remains unknown. The activities of neurons in both the cerebral cortex and superior colliculus are modulated by this selection process. We reasoned that activity associated with target uncertainty should be evident in the saccadic motor system at the final stages of neural processing, in the superior colliculus. By systematically changing the number of stimuli from which a selection must be made and recording from superior colliculus neurons, we found that as the target uncertainty increased, the neural activity preceding target selection decreased. These results indicate that neurons within the final common pathway for movement generation are active well in advance of the selection of a particular movement. This early activity varies with the probability that a particular movement will be selected.     

Correlation between secretagogue-induced Ca2+ influx, intracellular Ca2+ levels and secretion of catecholamines in cultured adrenal chromaffin cells

BACKGROUND: Complex branched muscle fibers are frequently observed in the muscles of mdx mutant mice and/or in damaged muscles. To investigate whether the complex branched fibers were present in the compensatory hypertrophied muscles of rats, we examined the morphological changes in these muscles. METHODS: We examined the hypertrophied plantaris (PLA) muscle of the Wistar male rats, prepared by surgical ablation of synergistic muscles. The muscle was examined using three-dimensional analysis with scanning electron microscopy, immunohistochemical detection of proliferating cells using 5-bromo-2'-deoxyuridine (BrdU) and histological and histochemical characterization. Studies were performed at 48 hours, 2, 4, 6, 10, and 15 weeks after surgical preparation. RESULTS: The muscle hypertrophy ratio (muscle weight relative to the contralateral intact control side), gradually increased from 2 to 10 weeks, and the peak value (48.6%) occurred at the 10th week. The total number of fibers did not change significantly at any time interval. However, the number of branched muscle fibers increased significantly (P < 0.05) after 6 weeks, and accounted for about 2.5% of the total fibers at the 15th week. Most branched fibers showed complex features resembling the "anastomosing syncytial reticulum" described in myopathic animals. The fibers were observed mainly in the middle and distal portions of the PLA muscle. The proportion and distribution of proliferating cells in the entire PLA muscle corresponded with the distribution of the complex branched fibers. These results were also observed in muscle tissues prepared for histological and histochemical examination. CONCLUSIONS: The presence of a large proportion of complex branched fibers in a limited segment of the compensatory hypertrophied muscle suggests that this hypertrophy model represents a pathological and/or pathophysiological hypertrophy model rather than a normal physiological process.     

A nucleated assembly mechanism of Alzheimer paired helical filaments

Alzheimer's disease is characterized by two types of fibrous aggregates in the affected brains, the amyloid fibers (consisting of the Abeta-peptide, generating the amyloid plaques), and paired helical filaments (PHFs; made up of tau protein, forming the neurofibrillary tangles). Hence, tau protein, a highly soluble protein that normally stabilizes microtubules, becomes aggregated into insoluble fibers that obstruct the cytoplasm of neurons and cause a loss of microtubule stability. We have developed recently a rapid assay for monitoring PHF assembly and show here that PHFs arise from a nucleated assembly mechanism. The PHF nucleus comprises about 8-14 tau monomers. A prerequisite for nucleation is the dimerization of tau because tau dimers act as effective building blocks. PHF assembly can be seeded by preformed filaments (made either in vitro or isolated from Alzheimer brain tissue). These results suggest that dimerization and nucleation are the rate-limiting steps for PHF formation in vivo.     

Morphological stability in the presence of fluid flow in the melt

Recent experiments have shown that the presence of a vertical buoyancy-driven flow adjacent to an initially cylindrical crystal-melt interface may produce a time-dependent helical deformation of the interface, with a rotation period ranging from several minutes to many hours, depending upon the width of the melt. The temperature distribution is such that the interface is expected to be morphologically stable in the absence of fluid flow. A linear stability analysis reveals that the instability is due to a coupling between a basic hydrodynamic instability in the buoyant flow and the deformable boundary separating the two phases. The crystal-melt interface lowers the critical Grashof number of an analogous rigid-walled system by an order of magnitude for succinonitrile with a Prandtl number P = 22.8; furthermore, the hydrodynamic mode that is actually destabilized by the interface is not the least stable mode in the rigid-walled system for P = 22.8. The results show that the instability may be regarded either as a rather large alteration of a basic hydrodynamic instability by the crystal-melt interface, or as a significant modification of the morphological stability of the interface by the presence of the buoyant flow. This paper is based on a presentation made at the symposium “Fluid Flow at Solid-Liquid Interfaces” held at the fall meeting of the TMS-AIME in Philadelphia, PA on October 5, 1983 under the TMS-AIME Solidification Committee.     

Interactions of the components of the general secretion pathway: role of Pseudomonas aeruginosa type IV pilin subunits in complex formation and extracellular protein secretion

The general secretion pathway (GSP), found in a wide range of bacteria, is responsible for extracellular targeting of a subset of proteins from the periplasm. In Pseudomonas aeruginosa, the GSP requires the participation of 12 proteins, of which XcpT, XcpU, XcpV, XcpW are homologues of PilA, the major subunit of type IV pili. The interaction between the pilin-like Xcp proteins was investigated using bifunctional crosslinking reagents. Cross-linking analysis of whole cells of wild-type P. aeruginosa, followed by immunoblot analysis, revealed a 34-kDa XcpT-containing complex. This complex was shown to consist of XcpT/PilA heterodimers. The role of PilA in the GSP was examined, using P. aeruginosa mutants in the pilA gene, or in rpoN, a gene regulating pilA expression. Each mutant showed a significant reduction in the efficiency of extracellular protein secretion, and this defect could be restored by expression of the cloned pilA gene in the mutant cells. The formation of the PilA/XcpT complex did not require XcpR or XcpQ, two other components of the secretion machinery, nor did it require the pilus biogenesis factors PilB and PIlC. The dimeric XcpT/PilA complex was also formed in a pilD mutant, which lacks the leader peptidase enzyme, demonstrating that the leader peptide at the N-terminus or PilA or XcpT did not have to be removed for the dimerization to occur. XcpW and XcpU can also be crosslinked to form dimeric complexes with PilA. When expression of XcpT is increased, its homodimers, as well as XcpT/XcpW heterodimers, can be detected. Finally, an oligohistidine-tagged XcpT was shown to form stoichiometric complexes with PilA, and with XcpT, U, V and W. These dimers were co-purified by nickel-affinity chromatography. The results of this study suggest that XcpT can form heterodimers with PilA, and Xcp U, V and W, which may be assembly intermediates of the secretion apparatus. Alternatively, these may represent dynamic intermediates that facilitate protein secretion by continuous association and dissociation. The requirement for PilA for efficient protein secretion argues for a critical role played by PilA in two related processes during P. aeruginosa infections: formation of an adhesive pilus organelle and secretion of exoenzymes.