Helical tubes of FtsZ from Methanococcus jannaschii

Bacterial cell division depends on the formation of a cytokinetic ring structure, the Z-ring. The bacterial tubulin homologue FtsZ is required for Z-ring formation. FtsZ assembles into various polymeric forms in vitro, indicating a structural role in the septum of bacteria. We have used recombinant FtsZ1 protein from M. jannaschii to produce helical tubes and sheets with high yield using the GTP analogue GMPCPP [guanylyl-(alpha,beta)-methylene-diphosphate]. The sheets appear identical to the previously reported Ca++-induced sheets of FtsZ from M. jannaschii that were shown to consist of 'thick'-filaments in which two protofilaments run in parallel. Tubes assembled either in Ca++ or in GMPCPP contain filaments whose dimensions indicate that they could be equivalent to the 'thick'-filaments in sheets. Some tubes are hollow but others are filled by additional protein density. Helical FtsZ tubes differ from eukaryotic microtubules in that the filaments curve around the filament axis with a pitch of approximately 430 A for Ca++-induced tubes or 590 - 620 A for GMPCPP. However, their assembly in vitro as well-ordered polymers over distances comparable to the inner circumference of a bacterium may indicate a role in vivo. Their size and stability make them suitable for use in motility assays.     

HURP wraps microtubule ends with an additional tubulin sheet that has a novel conformation of tubulin

HURP is a newly discovered microtubule-associated protein (MAP) required for correct spindle formation both in vitro and in vivo. HURP protein is highly charged with few predicted secondary and tertiary folding domains. Here we explore the effect of HURP on pure tubulin, and describe its ability to induce a new conformation of tubulin sheets that wrap around the ends of intact microtubules, thereby forming two concentric tubes. The inner tube is a normal microtubule, while the outer one is a sheet composed of tubulin protofilaments that wind around the inner tube with a 42.5° inclination. We used cryo-electron microscopy and unidirectional surface shadowing to elucidate the structure and conformation of HURP-induced tubulin sheets and their interaction with the inner microtubule. These studies clarified that HURP-induced sheets are composed of anti-parallel protofilaments exhibiting P2 symmetry. HURP is a unique MAP that not only stabilizes and bundles microtubules, but also polymerizes free tubulin into a new configuration.     

Myocardial tissue engineering: toward a bioartificial pump

Regenerative therapies, including cell injection and bioengineered tissue transplantation, have the potential to treat severe heart failure. Direct implantation of isolated skeletal myoblasts and bone-marrow-derived cells has already been clinically performed and research on fabricating three-dimensional (3-D) cardiac grafts using tissue engineering technologies has also now been initiated. In contrast to conventional scaffold-based methods, we have proposed cell sheet-based tissue engineering, which involves stacking confluently cultured cell sheets to construct 3-D cell-dense tissues. Upon layering, individual cardiac cell sheets integrate to form a single, continuous, cell-dense tissue that resembles native cardiac tissue. The transplantation of layered cardiac cell sheets is able to repair damaged hearts. As the next step, we have attempted to promote neovascularization within bioengineered myocardial tissues to overcome the longstanding limitations of engineered tissue thickness. Finally, as a possible advanced therapy, we are now trying to fabricate functional myocardial tubes that may have a potential for circulatory support. Cell sheet-based tissue engineering technologies therefore show an enormous promise as a novel approach in the field of myocardial tissue engineering.     

Stable L-forms of Clostridium perfringens and their growth on glass surfaces.

L-forms of Clostridium perfringens were induced in brain heart infusion broth containing 10% sucrose and 2 units of penicillin. After a few hours of growth, spheroplasts, granules, and elongated bacilli were apparent. At 24-h intervals, serial subcultures were made in the above medium which resulted in a culture composed entirely of spheroplasts (or protoplasts) and granules. Upon the withdrawal of penicillin these L-form cultures grew well and, after 100 passages, there was no reversion to the bacillary form. Sucrose could also be withdrawn from the medium. The effects of centrifugation, osmotic stabilizer, ultraviolet light, temperature, pH, and lyophilization upon stable L-forms were examined. L-forms were found to attach to the walls of culture tubes during trowth and sheets of L-form growth were obtained on cover slips in Leighton tubes and on the sides of medicine bottles.     

Occurrence,Formation and Function of Organic Sheets in the Mineral Tube Structures of Serpulidae (Polychaeta,Annelida)

A scanning electron microscopy study of organic sheets in serpulid tube mineral structures was carried out to discern their function, formation and evolution. The organic sheets may have some taxonomic value in distinguishing the two major clades of serpulids previously identified. The organic sheets in the mineral tube structure occur only in certain taxa belonging to clade A, but not all species in clade A have them. Organic sheets are best developed in genus Spirobranchus. One could speculate that organic sheets have evolved as an adaption to further strengthen the mechanical properties of the tubes in clade A, which contains serpulids with the most advanced mineral tube microstructures. The organic sheets are presumably secreted with at least some mineral phase.     

Morphopathology of mixed sweat-gland tumors

Mixed sweat-gland tumors were studied on 11 biopsies, of which two showed clear-cut morphological features of malignancy. Stress was laid upon the sudoriferous-duct histogenesis of such tumors. Several peculiarities were carefully investigated, especially epithelial proliferation in sheets, cords, tubes, isolated cells and certain epithelial metaplasias correlated with various mesenchymal stroma changes, such as fibrosis, myxoid, chondroid or even osseous changes. Special emphasis was also laid upon the comparative aspects between benign and malignant mixed sweat-gland tumors and other similar body tumors.     

Degradation of poly (3-hydroxybutyrate) and its copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by a marine Streptomyces sp. SNG9

A marine Streptomyces sp. SNG9 was characterized by its ability to utilize poly(3-hydroxybutyrate) (PHB) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate P (3HB-co-HV). The bacterium grew efficiently in a simple mineral liquid medium enriched with 0.1% poly(3-hydroxybutyrate) powder as the sole carbon source. Cells excreted PHB depolymerase and degraded the polymer particles to complete clarity in 4 days. The degradation activity was detectable by the formation of a clear zone around the colony (petri plates) or a clear depth under the colony (test tubes). The expression of PHB depolymerase was repressed by the presence of simple soluble carbon sources. Bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). Morphological alterations of the polymers sheets were evidence for bacterial hydrolysis.     

Degradation of poly (3-hydroxybutyrate) and its copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by a marine Streptomyces sp. SNG9.

A marine Streptomyces sp. SNG9 was characterized by its ability to utilize poly(3-hydroxybutyrate) (PHB) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate P (3HB-co-HV). The bacterium grew efficiently in a simple mineral liquid medium enriched with 0.1% poly(3-hydroxybutyrate) powder as the sole carbon source. Cells excreted PHB depolymerase and degraded the polymer particles to complete clarity in 4 days. The degradation activity was detectable by the formation of a clear zone around the colony (petri plates) or a clear depth under the colony (test tubes). The expression of PHB depolymerase was repressed by the presence of simple soluble carbon sources. Bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). Morphological alterations of the polymers sheets were evidence for bacterial hydrolysis.     

Novel morphology in Enteromorpha (Ulvophyceae) forming green tides

"Green tides" are vast accumulations of unattached green macroalgae associated with eutrophicated marine environments. They have major ecological and economic impacts globally, so an understanding of their origin and persistence is required in order to make management decisions. Blooms predominantly consist of two common fouling genera of the Ulvales, Ulva (distromatic sheets) and Enteromorpha (monostromatic tubes). In the Baltic Sea and elsewhere green tides have increased over the last few decades. On the west coast of Finland, summer blooms consist of monostromatic sheets resembling Monostroma (Codiolales). We identified these as Enteromorpha intestinalis by comparative analyses of rDNA internal transcribed spacer 1 (ITS1), 5.8S, and ITS2 sequences, the first time monostromatic sheets have been found in the genus Enteromorpha. Ordinary attached E. intestinalis sporulated freely in culture, but the sheets reproduced only by cell regeneration into typical tubular thalli. The ITS sequences were identical to those of attached E. intestinalis populations in southwestern Finland, but differed by two substitutions from other Baltic sequences. We infer that this bloom originated from local attached populations and now reproduces clonally by fragmentation. This study provides further evidence of radical changes in gross morphology of green algae under eutrophicated conditions and the need for molecular identification.     

Straight and curved conformations of FtsZ are regulated by GTP hydrolysis

FtsZ assembles in vitro into protofilaments that can adopt two conformations-the straight conformation, which can assemble further into two-dimensional protofilament sheets, and the curved conformation, which forms minirings about 23 nm in diameter. Here, we describe the structure of FtsZ tubes, which are a variation of the curved conformation. In the tube the curved protofilament forms a shallow helix with a diameter of 23 nm and a pitch of 18 or 24 degrees. We suggest that this shallow helix is the relaxed structure of the curved protofilament in solution. We provide evidence that GTP favors the straight conformation while GDP favors the curved conformation. In particular, exclusively straight protofilaments and protofilament sheets are assembled in GMPCPP, a nonhydrolyzable GTP analog, or in GTP following chelation of Mg, which blocks GTP hydrolysis. Assembly in GDP produces exclusively tubes. The transition from straight protofilaments to the curved conformation may provide a mechanism whereby the energy of GTP hydrolysis is used to generate force for the constriction of the FtsZ ring in cell division.     

Assembly of archaeal cell division protein FtsZ and a GTPase-inactive mutant into double-stranded filaments

We have studied the assembly and GTPase of purified FtsZ from the hyperthermophilic archaeon Methanococcus jannaschii, a structural homolog of eukaryotic tubulin, employing wild-type FtsZ, FtsZ-His6 (histidine-tagged FtsZ), and the new mutants FtsZ-W319Y and FtsZ-W319Y-His6, with light scattering, nucleotide analyses, electron microscopy, and image processing methods. This has revealed novel properties of FtsZ. The GTPase of archaeal FtsZ polymers is suppressed in Na+-containing buffer, generating stabilized structures that require GDP addition for disassembly. FtsZ assembly is polymorphic. Archaeal FtsZ(wt) assembles into associated and isolated filaments made of two parallel protofilaments with a 43 A longitudinal spacing between monomers, and this structure is also observed in bacterial FtsZ from Escherichia coli. The His6 extension facilitates the artificial formation of helical tubes and sheets. FtsZ-W319Y-His6 is an inactivated GTPase whose assembly remains regulated by GTP and Mg2+. It forms two-dimensional crystals made of symmetrical pairs of tubulin-like protofilaments, which associate in an antiparallel array (similarly to the known Ca2+-induced sheets of FtsZ-His6). In contrast to the lateral interactions of microtubule protofilaments, we propose that the primary assembly product of FtsZ is the double-stranded filament, one or several of which might form the dynamic Z ring during prokaryotic cell division.     

The endoplasmic reticulum network in the ascidian egg: localization and calcium content

In contrast with most other eggs, where the endoplasmic reticulum is mixed with many other organelles, in ascidians, continuous sheets and tubes of endoplasmic reticulum constitute the only prominent organelle in the immediate layer (0.5-1μm) beneath the plasma membrane, and occupies 16–20% of the cortical volume. We took advantage of this unusual stratification of the organelles in the ascidian egg, to carry X-ray microanalysis. Our measurements provide the first estimate of the calcium content of the endoplasmic reticulum network in an egg, and show it is the main calcium store.     

Ultrastructure of the internal medullary substance of the kidney in rodents of an arid zone

Peculiar ultrastructural features of the kidney internal medullary substance have been studied in some typical representatives of the Middle Asia desert and semi-desert fauna (Spermophilopsis leptodactylus and Meriones erythrourus). Papillary organization in the species studied has a specific high differentiation of the fine nephron loop (Henle's) parts; flattening (especially in Meriones erythrourus) of epithelium in the collecting tubes while its cytoplasm is rather poor in organelle; high contents of the interstitial cells and their tight junction with canaliculi, capillaries, floccular material of the interstitial tissue; thinning of the interstitial sheets, especially between the collecting tubes and vessels, presence of collagene fibres in the interstitium, that is especially, specific for all the species of the Spermophilopsis leptodactylus studied. It is possible to suppose that development of similar features in the structure of the kidney internal medullary substance, where the main water resorption takes place in the species investigated, is connected with their life in similar environmental conditions with low hydratation level and is aimed to increase efficiency of the concentrating apparatus work.     

Evaluation of sampling methods for the detection of Salmonella in broiler flocks

The present study compares four different sampling methods potentially applicable to detection of Salmonella in broiler flocks, based on collection of faecal samples (i) by hand, 300 fresh faecal samples (ii) absorbed on five sheets of paper (iii) absorbed on five pairs of socks (elastic cotton tubes pulled over the boots and termed 'socks') and (iv) by using only one pair of socks. Twenty-three broiler flocks were included in the investigation and 18 of these were found to be positive by at least one method. Seven serotypes of Salmonella with different patterns of transmission (mainly horizontal or vertical) were found in the investigation. The results showed that the sock method (five pairs of socks) had a sensitivity comparable with the hand collection method (60 pools of five faecal samples); the paper collection method was inferior, as was the use of only one pair of socks. Estimation of the effective pool sizes for the different methods was also carried out.     

Two-dimensional structure of the membrane domain of human band 3, the anion transport protein of the erythrocyte membrane.

The membrane domain of human erythrocyte Band 3 protein (M(r) 52,000) was reconstituted with lipids into two-dimensional crystals in the form of sheets or tubes. Crystalline sheets were monolayers with six-fold symmetry (layer group p6, a = b = 170 A, gamma = 60 degrees), whereas the symmetry of the tubular crystals was p2 (a = 104 A, b = 63 A, gamma = 104 degrees). Electron image analysis of negatively stained specimens yielded projection maps of the protein at 20 A resolution. Maps derived from both crystal forms show that the membrane domain is a dimer of two monomers related by two-fold symmetry, with each monomer consisting of three subdomains. In the dimer, two subdomains of each monomer form a roughly rectangular core (40 x 50 A in projection), surrounding a central depression. The third subdomain of the monomer measures approximately 15 x 25 A in projection and appears to be connected to the other two by a flexible link. We propose that the central depression may represent the channel for anion transport while the third subdomain appears not to be directly involved in channel formation.     

ESCRT-III polymers in membrane neck constriction

The endosomal sorting complex required for transport (ESCRT)-III machinery contributes to membrane deformation and scission in cytokinesis, intraluminal vesicle formation, autophagy and virus budding. Recombinant ESCRT-III subunits polymerize in vitro into filaments, tubes, sheets or rings, and ESCRT-III-dependent filaments have been observed in cells at virus bud necks and at the cytokinetic abscission site. These observations have inspired speculation about how ESCRT-III could mediate constriction and fission of membrane necks. Based on the polymer structures observed in vitro and in vivo, we discuss models for ESCRT-III function and outline how emerging technologies could be used to test these models.     

The straight and curved conformation of FtsZ protofilaments-evidence for rapid exchange of GTP into the curved protofilament

Bacterial cell division protein FtsZ assembles into protofilaments, which can adopt a straight or curved conformation, similar to its eukaryotic homolog, tubulin. The straight protofilaments can assemble into sheets with a lattice similar to the microtubule wall. The curved protofilaments can form rings when adsorbed to a lipid monolayer, but in solution they form helices. 4 helices assemble together to make a tube, the characteristic polymer of the curved protofilament. GTP favors the straight conformation, while GDP favors the curved. We show here that addition of EDTA and GTP to tubes causes a rapid transformation to straight protofilament sheets. Apparently when the magnesium is chelated the GDP in the curved protofilaments dissociates rapidly and is replaced with GTP, and this GTP induces the transition to straight protofilaments.     

The aerolysin membrane channel is formed by heptamerization of the monomer.

The cytolytic toxin aerolysin has been found to form heptameric oligomers by SDS-PAGE electrophoresis, STEM mass measurements of single oligomers and image analysis of two-dimensional membrane crystals. Two types of crystal, flat sheets and long regular tubes, have been obtained by reconstitution of purified protein and Escherichia coli phospholipids. A noise-filtered image of the best crystalline sheets reveals a structure with 7-fold symmetry containing a central strongly stain-excluding ring that encircles a dark stain-filled channel 17 A in diameter. The ring is surrounded by seven arms each made up of two unequal sized domains. By combining projected views and side-views, a simplified model of the aerolysin channel complex has been constructed. The relevance of this structure to the mode of action of aerolysin is discussed.     

Evidence of non-vascular land plants from the early Silurian (Llandoverian) of Virginia,U.S.A.

Evidence is presented for the earliest known occurrences of non-vascular land plants and of higher, septate fungi. Macerates of carbonaceous silstone lenses from the lower Massanutten Sandstone, early Silurian (Llandoverian) of Virginia, have yielded a diverse assemblage of microfossil elements. Parallel aligned, banded tubes with annular to spiral ribbing and rounded to papilliform ends, membranous cellular sheets, cuticles, trilete spores, small spore tetrads, and septate higher filamentous fungi were recovered from the macerates. The banded tubes are probably a significant analogue with supportive or conductive cell types, but are not considered tracheidal. The heterogeneous plant assemblage may represent a thalloid, non-vascular land plant, in part, with a tubular-filamentous (nematophytic) organization associated with a membranous cellular layer and cuticular covering. While no spores were established as nematophytic, the presence of trilete spores adds to the indirect evidence of multiple evolutionary convergence toward land-plant characters. This assemblage is interpreted as of land-plant origin, based on the inferred fluvial depositional model of the fossiliferous rocks. A glacio-eustatic sea-level drop in the late Ordovician is suggested as a stimulus to the advent of land plants in the early Silurian.     

Enzyme immobilization on nylon-optimization and the steps used to prevent enzyme leakage from the support

Because of its low cost, chemical and mechanical properties and ready availability in a number of different forms (e.g. powders, beads, nets, tubes, film, sheets, etc.) Nylon is an attractive matrix for enzyme immobilization. We report here a thorough evaluation of a protocol for enzyme immobilization on nylon film with relatively inexpensive and non-toxic reagents, involving acid hydrolysis, glutaraldehyde coupling and spacer molecules and employing beta-glucosidase and trypsin as model enzymes. We also describe steps for virtually eliminating enzyme leakage and non-specific binding. Individual steps in the procedure are simple and conditions flexible so, whilst evaluated in terms of binding proteins to nylon film, they should be applicable to other forms of nylon and suitable for binding most enzymes and proteins, including antibodies, providing a method having potential in both affinity chromatography/adsorption and in bioreactor applications.