Industry-relevant magnetron sputtering and cathodic arc ultra-high vacuum deposition system for in situ x-ray diffraction studies of thin film growth using high energy synchrotron radiation.

We present an industry-relevant, large-scale, ultra-high vacuum (UHV) magnetron sputtering and cathodic arc deposition system purposefully designed for time-resolved in situ thin film deposition/annealing studies using high-energy (>50 keV), high photon flux (>10(12) ph/s) synchrotron radiation. The high photon flux, combined with a fast-acquisition-time (<1 s) two-dimensional (2D) detector, permits time-resolved in situ structural analysis of thin film formation processes. The high-energy synchrotron-radiation based x-rays result in small scattering angles (<11°), allowing large areas of reciprocal space to be imaged with a 2D detector. The system has been designed for use on the 1-tonne, ultra-high load, high-resolution hexapod at the P07 High Energy Materials Science beamline at PETRA III at the Deutsches Elektronen-Synchrotron in Hamburg, Germany. The deposition system includes standard features of a typical UHV deposition system plus a range of special features suited for synchrotron radiation studies and industry-relevant processes. We openly encourage the materials research community to contact us for collaborative opportunities using this unique and versatile scientific instrument.

The roles of RgpB and Kgp in late onset gingipain activity in the vimA-defective mutant of Porphyromonas gingivalis W83.

Previous studies have shown that VimA, an acetyltransferase, can modulate gingipain biogenesis in Porphyromonas gingivalis. Inactivation of the vimA gene resulted in isogenic mutants that showed a late onset of gingipain activity that only occurred during the stationary growth phase. To further elucidate the role and contribution of the gingipains in this VimA-dependent process, isogenic mutants defective in the gingipain genes in the vimA-deficient genetic background were evaluated. In contrast with the wild-type strain, RgpB and Kgp gingipain activities were absent in exponential phase in the ∆rgpA::tetQ-vimA::ermF mutant. However, these activities increased to 31 and 53%, respectively, of that of the wild-type during stationary phase. In the ∆rgpA::cat-∆kgp::tetQ-vimA::ermF mutant, the RgpB protein was observed in the extracellular fraction but no activity was present even at the stationary growth phase. There was no gingipain activity observed in the ∆rgpB::cat-∆kgp::tetQ-vimA::ermF mutant whereas Kgp activity in ∆rgpA::cat-∆rgpB::tetQ-vimA::ermF mutant was 24% of the wild-type at late stationary phase. In contrast to RgpA, the glycosylation profile of the RgpB catalytic domain from both W83 and P. gingivalis FLL92 (vimA::ermF) showed similarity. Taken together, the results suggest multiple gingipain activation pathways in P. gingivalis. Whereas the maturation pathways for RgpA and RgpB are different, the late-onset gingipain activity in the vimA-defective mutant was due to activation/maturation of RgpB and Kgp. Moreover, unlike RgpA, which is VimA-dependent, the maturation/activation pathways for RgpB and Kgp are interdependent in the absence VimA.

A lily stylar pectin is necessary for pollen tube adhesion to an in vitro stylar matrix.

Pollen tube cells adhere to the wall surface of the stylar transmitting tract epidermis in lily. This adhesion has been proposed as essential for the proper delivery of the sperm cells to the ovule. An in vitro adhesion bioassay has been used to isolate two stylar molecules required for lily pollen tube adhesion. The first molecule was determined to be a small, cysteine-rich protein with some sequence similarity to lipid transfer proteins and now called stigma/stylar cysteine-rich adhesin (SCA). The second, larger, molecule has now been purified from style fragments and characterized. Chemical composition, specific enzyme degradations, and immunolabeling data support the idea that this molecule required for pollen tube adhesion is a pectic polysaccharide. In vitro binding assays revealed that this lily stylar adhesive pectin and SCA are able to bind to each other in a pH-dependent manner.

Proteoglycans and related components in plant cells.

After the context is set by a brief description of the plant cell surface, emphasis is placed on one class of cell surface components, the arabinogalactan proteins. An expansion of knowledge regarding the structure, expression, and function of these proteoglycans has been initiated and is being sustained through new experimental approaches, including the development of monoclonal antibody probes and the cloning of cDNAs corresponding to core polypeptides. An examination of the structure of both the polypeptide and carbohydrate components of arabinogalactan proteins is presented with emphasis placed on recently deduced core polypeptide sequences. Information about the biosynthesis and turnover of arabinogalactan proteins is incomplete, especially with regard to the carbohydrate component. Although functions of arabinogalactan proteins have not been clearly identified, regulated expression and several other lines of evidence point to involvement in plant reproductive development, pattern formation, and somatic embryogenesis, as well as in the underlying processes of cell division, cell expansion, and cell death. Arabinogalactan proteins are compared with animal proteoglycans and mucins, and the results of searches for plant analogues of other animal extracellular matrix components are examined.

Hydrodynamic models of viscous coupling between motile myosin and endoplasm in characean algae.

Cytoplasmic streaming in characean algae is thought to be driven by interaction between stationary subcortical actin bundles and motile endoplasmic myosin. Implicit in this mechanism is a requirement for some form of coupling to transfer motive force from the moving myosin to the endoplasm. Three models of viscous coupling between myosin and endoplasm are presented here, and the hydrodynamic feasibility of each model is analyzed. The results show that individual myosinlike molecules moving along the actin bundles at reasonable velocities cannot exert enough viscous pull on the endoplasm to account for the observed streaming. Attachment of myosin to small spherical organelles improves viscous coupling to the endoplasm, but results for this model show that streaming can be generated only if the myosin-spheres move along the actin bundles in a virtual solid line at about twice the streaming velocity. In the third model, myosin is incorporated into a fibrous or membranous network or gel extending into the endoplasm. This network is pulled forward as the attached myosin slides along the actin bundles. Using network dimensions estimated from published micrographs of characean endoplasm, the results show that this system can easily generate the observed cytoplasmic streaming.

Effects of exogenous proteins on cytoplasmic streaming in perfused Chara cells.

Cytoplasmic streaming in characean algae is thought to be generated by interaction between subcortical actin bundles and endoplasmic myosin. Most of the existing evidence supporting this hypothesis is of a structural rather than functional nature. To obtain evidence bearing on the possible function of actin and myosin in streaming, we used perfusion techniques to introduce a number of contractile and related proteins into the cytoplasm of streaming Chara cells. Exogenous actin added at concentrations as low as 0.1 mg/ml is a potent inhibitor of streaming. Deoxyribonuclease I (DNase I), an inhibitor of amoeboid movement and fast axonal transport, does not inhibit streaming in Chara. Fluorescein-DNase I stains stress cables and microfilaments in mammalian cells but does not bind to Chara actin bundles, thus suggesting that the lack of effect on streaming is due to a surprising lack of DNase I affinity for Chara actin bundles. Heavy meromyosin (HMM) does not inhibit streaming, but fluorescein-HMM (FL-HMM), having a partially disabled EDTA ATPase, does. Quantitative fluorescence micrography provides evidence that inhibition of streaming by FL-HMM may be due to a tendency for FL-HMM to remain bound to Chara actin bundles even in the presence of MgATP. Perfusion with various control proteins, including tubulin, ovalbumin, bovine serum albumin, and irrelevant antibodies, does not inhibit streaming. These results support the hypothesis that actin and myosin function to generate cytoplasmic streaming in Chara.