Effect of Cu Toxicity on Growth of Cowpea (Vigna unguiculata)

Accurate determination of the rhizotoxicity of Cu in dilute nutrient solutions is hindered by the difficulty of maintaining constant, pre-determined concentrations of Cu (micromolar) in solution. The critical Cu2+ activity associated with a reduction in the growth of solution-grown cowpea (Vigna unguiculata (L.) Walp. cv Caloona) was determined in a system in which Cu was maintained constant through the use of a cation exchange resin. The growth of roots and shoots was found to be reduced at solution Cu2+ activities ≥ 1.7 µM (corresponding to 90 % maximum growth). Although root growth was most likely reduced due to a direct Cu2+ toxicity, it is considered that the shoot growth reduction is attributable to a decrease in tissue concentrations of K, Ca, Mg, and Fe and the formation of interveinal chlorosis. At high Cu2+ activities, roots were brown in color, short and thick, had bent root tips with cracking of the epidermis and outer cortex, and had local swellings behind the roots tips due to a reduction in cell elongation. Root hair growth was reduced at concentrations lower than that which caused a significant reduction in overall root fresh weight.

Antibody responses and protective immunity to recombinant vaccinia virus-expressed bluetongue virus antigens

The role of individual viral proteins in the immune response to bluetongue virus (BTV) is not clearly understood. To investigate the contributions of the outer capsid proteins, VP2 and VP5, and possible interactions between them, these proteins were expressed from recombinant vaccinia viruses either as individual proteins or together in double recombinants, or with the core protein VP7 in a triple recombinant. Comparison of the immunogenicity of the vaccinia expressed proteins with BTV expressed proteins was carried out by inoculation of rabbits and sheep. Each of the recombinants was capable of stimulating an anti-BTV antibody response, although there was a wide range in the level of response between animals and species. Vaccinia-expressed VP2 was poorly immunogenic, particularly in rabbits. VP5, on the whole, stimulated higher ELISA titers in rabbits and sheep and in some animals in both species was able to stimulate virus neutralizing antibodies. When the protective efficacy of VP2 and VP5 was tested in sheep, vaccinia-expressed VP2, VP5 and VP2 + VP5 were protective, with the most consistent protection being in groups immunized with both proteins. (C) 1997 Elsevier Science B.V.

Characterization of serum antibodies to Porphyromonas gingivalis in individuals with and without periodontitis

Although Porphyromonas gingivalis is a defined pathogen in periodontal disease, many subjects control the infection without experiencing loss of attachment. Differences in host susceptibility to the disease may be reflected in the pattern of humoral antibodies against specific P. gingivalis antigens. The aim of this study was to determine the presence of antibodies against immunodominant P. gingivalis antigens as well as the isotype and subclass of anti-P. gingivalis antibodies against outer membrane antigens in four groups of patients: P. gingivalis-positive, 1) with and 2) without periodontitis, and P. gingivalis-negative, 3) with and 4) without periodontitis. Antigens of molecular weight 92, 63, and 32 kDa and lipopolysaccharide were found to be immunodominant. Group 1 subjects showed a significantly higher response to the 92 and 63 kDa antigens compared with other groups. The response to lipopolysaccharide was significantly higher in group 1, and lower in group 4 than in groups 2, 3. Immunoglobulin G(1) (IgG(1)), IgG(2) and IgM antibodies against P. gingivalis outer membrane were present in all subjects, while only some subjects were seropositive for IgG(3), IgG(4) and IgA. There were no differences in concentrations for IgG(1), IgG(3) and IgM. The IgG(2) concentration in group 4 was significantly higher than in groups 1 and 2, while the IgG(4) concentration in group 4 was significantly lower than in other groups. The frequency of seropositivity for IgG(4) and IgA was lowest in group 4, while IgG; seropositivity was almost exclusively seen in healthy patients iii groups 2, 4. These findings suggest that the presence of IgG(3) may reflect non-susceptibility to the disease, while lack of IgG(4) may be indicative of periodontal health and lack of infection.

On the mechanism of microbial cell disruption in high-pressure homogenisation

The tensions produced in the wall of a rigid, thin-walled, liquid-filled sphere as it moves with an axisymmetric straining flow are examined. This problem has not been previously addressed. A generalised correlation for the maximum wall tension, expressed in dimensionless form as a Weber number (We), is developed in terms of the acceleration number (Ac) and Reynolds number (Re) of the straining flow. At low Reynolds number We is dominated by viscous forces, while inertial forces due to internal pressure gradients caused by sphere acceleration dominate at higher Re. The generalised correlation has been used to examine the case of a typical yeast cell (a thin-walled, liquid-filled sphere) passing through a typical high-pressure homogeniser (a straining-flow device). At 56 MPa homogenising pressure, a 6 mu m yeast cell experiences tensions in the inertially dominated regime (Re = 100). The correlation gives We = 0.206, corresponding to a maximum wall tension of 8 Nm(-1). This is equivalent to an applied compressive force of 150 mu N and compares favourably with the force required to break yeast cells under compressive micromanipulation (40-90 mu N). Inertial forces may therefore be an important and previously unrecognised. mechanism of microbial cell disruption during high-pressure homogenisation. Further work is required to examine the likelihood of cell deformation in the high-strain-rate short-residence-time environment of the homogeniser, and the effect that such deformation may have on the contribution of inertial forces to disruption. (C) 1998 Published by Elsevier Science Ltd. All rights reserved.

The eyes of deep-sea fish II. Functional morphology of the retina

Three different aspects of the morphological organisation of deep-sea fish retinae are reviewed: First, questions of general cell biological relevance are addressed with respect to the development and proliferation patterns of photoreceptors, and problems associated with the growth of multibank retinae, and with outer segment renewal are discussed in situations where there is no direct contact between the retinal pigment epithelium and the tips of rod outer segments. The second part deals with the neural portion of the deep-sea fish retina. Cell densities are greatly reduced, yet neurohistochemistry demonstrates that all major neurotransmitters and neuropeptides found in other vertebrate retinae are also present in deep-sea fish. Quantitatively, convergence rates in unspecialised parts of the retina are similar to those in nocturnal mammals. The differentiation of horizontal cells makes it unlikely that species with more than a single visual pigment are capable of colour vision. In the third part. the diversity of deep-sea fish retinae is highlighted. Based on the topography of ganglion cells, species are identified with areae or foveae located in various parts of the retina, giving them a greatly improved spatial resolving power in specific parts of their visual fields. The highest degree of specialisation is found in tubular eyes. This is demonstrated in a case study of the scopelarchid retina, where as many as seven regions with different degrees of differentiation can be distinguished, ranging from an area giganto cellularis, regions with grouped rods to retinal diverticulum. (C) 1998 Elsevier Science Ltd. All rights reserved.

Retinal and lenticular ultrastructure in the aestivating salamanderfish, Lepidogalaxias salamandroides (Galaxiidae, Teleostei) with special reference to a new type of photoreceptor mosaic

The salamanderfish, Lepidogalaxias salamandroides (Galaxiidae, Teleostei) is endemic to southwestern Australia and inhabits shallow, freshwater pools which evaporate during the hot summer months. Burrowing into the substrate in response to falling water levels allows these fish to aestivate for extended periods of time while encapsulated in a mucous cocoon even when the pools contain no water. Only a few minutes after a major rainfall, these fish emerge into relatively clear water which subsequently becomes laden with tannin, turning the water black and reducing the pH to approximately 4.3. As part of a large study of the visual adaptations of this unique species, the retinal and lenticular morphology of the aestivating salamanderfish is examined at the level of the light and electron microscopes. The inner retina is highly vascularised by a complex system of vitreal blood vessels, while the outer retina receives a blood supply by diffusion from a choriocapillaris. This increased retinal blood supply may be an adaptation for reducing the oxygen tension during critical periods of aestivation. Large numbers of Muller cells traverse the thickness of the retina from the inner to the outer limiting membranes. The ganglion cells are arranged in two ill-defined layers, separated from a thick inner nuclear layer containing two layers of horizontal cells by a soma-free inner plexiform layer. The photoreceptors can be divided into three types typical of many early actinopterygian representatives; equal double cones, small single cones and large rods (2:1:1). These photoreceptors are arranged into a unique regular square mosaic comprising a large rod bordered by four equal double cones with a small single cone located at the corner of each repeating unit. The double cones may optimise perception of mobile prey which it tracks by flexion of its head and neck and the large rods may increase sensitivity in the dark tannin-rich waters in which it lives. Each single cone also possesses a dense collection of polysomes and glycogen (a paraboloid) beneath its ellipsoid, the first such finding in teleosts. The retinal pigment epithelium possesses melanosomes, pha,oocytes and a large number of mitochondria. The anatomy of the retina and the photoreceptor mosaic is discussed in relation to the primitive phylogeny of this species and its unique life history.

Spectroscopic identification of a dinuclear metal centre in manganese(II)-activated aminopeptidase P from Escherichia coli: implications for human prolidase

Electron paramagnetic resonance (EPR) spectra and X-ray absorption (EXAFS and XANES) data have been recorded for the manganese enzyme aminopeptidase P (AMPP, PepP protein) from Escherichia coli. The biological function of the protein, a tetramer of 50-kDa subunits, is the hydrolysis of N-terminal Xaa-Pro peptide bonds. Activity assays confirm that the enzyme is activated by treatment with Mn2+. The EPR spectrum of Mn2+-activated AMPP at liquid-He temperature is characteristic of an exchange-coupled dinuclear Mn(II) site, the Mn-Mn separation calculated from the zero-field splitting D of the quintet state being 3.5 (+/- 0.1) Angstrom. In the X-ray absorption spectrum of Mn2+-activated AMPP at the Mn K edge, the near-edge features are consistent with octahedrally coordinated Mn atoms in oxidation state +2. EXAFS data, limited to k less than or equal to 12 Angstrom(-1) by traces of Fe in the protein, are consistent with a single coordination shell occupied predominantly by O donor atoms at an average Mn-ligand distance of 2.15 Angstrom, but the possibility of a mixture of O and N donor atoms is not excluded. The Mn-Mn interaction at 3.5 Angstrom, is not detected in the EXAFS, probably due to destructive interference from light outer-shell atoms. The biological function, amino acid sequence and metal-ion dependence of E. coli AMPP are closely related to those of human prolidase, an enzyme that specifically cleaves Xaa-Pro dipeptides. Mutations that lead to human prolidase deficiency and clinical symptoms have been identified. Several known inhibitors of prolidase also inhibit AMPP. When these inhibitors are added to Mn2+-activated AMPP, the EPR spectrum and EXAFS remain unchanged. It can be inferred that the inhibitors either do not bind directly to the Mn centres, or substitute for existing Mn ligands without a significant change in donor atoms or coordination geometry. The conclusions from the spectroscopic measurements on AMPP have been verified by, and complement, a recent crystal structure analysis.

Novel nano-organisms from Australian sandstones

We report the detection of living colonies of nano-organisms (nanobes) on Triassic and Jurassic sandstones and other substrates. Nanobes have cellular structures that are strikingly similar in morphology to Actinomycetes and fungi (spores, filaments, and fruiting bodies) with the exception that they are up to 10 times smaller in diameter (20 nm to 1.0 mu m). Nanobes are noncrystalline structures that are composed of C, O, and N. Ultra thin sections of nanobes show the existence of an outer layer or membrane that may represent a cell wall. This outer layer surrounds an electron dense region interpreted to be the cytoplasm and a less electron dense central region that may represent a nuclear area. Nanobes show a positive reaction to three DNA stains, [4',6-diamidino-2 phenylindole (DAPI), Acridine Orange, and Feulgen], which strongly suggests that nanobes contain DNA. Nanobes are communicable and grow in aerobic conditions at atmospheric pressure and ambient temperatures. While morphologically distinct, nanobes are in the same size range as the controversial fossil nannobacteria described by others in various rock types and in the Martian meteorite ALH84001.

On uniquely determining cell-wall material properties with the compression experiment

A finite element model (FEM) of the cell-compression experiment has been developed in dimensionless form to extract the fundamental cell-wall-material properties (i.e. the constitutive equation and its parameters) from experiment force-displacement data. The FEM simulates the compression of a thin-walled, liquid-filled sphere between two flat surfaces. The cell-wall was taken to be permeable and the FEM therefore accounts for volume loss during compression. Previous models assume an impermeable wall and hence a conserved cell volume during compression. A parametric study was conducted for structural parameters representative of yeast. It was shown that the common approach of assuming reasonable values for unmeasured parameters (e.g. cell-wall thickness, initial radial stretch) can give rise to nonunique solutions for both the form and constants in the cell-wall constitutive relationship. Similarly, measurement errors can also lead to an incorrectly defined cell-wall constitutive relationship. Unique determination of the fundamental wall properties by cell compression requires accurate and precise measurement of a minimum set of parameters (initial cell radius, initial cell-wall thickness, and the volume loss during compression). In the absence of such measurements the derived constitutive relationship may be in considerable error, and should be evaluated against its ability to predict the outcome of other mechanical experiments. (C) 1998 Elsevier Science Ltd. All rights reserved.

Ca2+ sensitivity of phospholipid scrambling in human red cell ghosts

The phospholipids in plasma membranes of erythrocytes, as well as platelets, lymphocytes and other cells are asymmetrically distributed, with sphingomyelin and phosphatidylcholine residing predominantly in the outer leaflet of the bilayer, and phosphatidylserine and phosphatidylethanolamine in the inner leaflet. It is known that Ca2+ can disrupt the phospholipid asymmetry by activation of a protein known as phospholipid scramblase, which affects bidirectional phospholipid movement in a largely non-selective manner. As Ca2+ also inhibits aminophospholipid translocase, whose Mg2+-ATPase activity is responsible for active translocation of aminophospholipids from the outer to the inner leaflet, it is important to accurately determine the sensitivity of scramblase to intracellular free Ca2+. In the present study we have utilized the favourable K-d, of Mag-fura-2 for calcium in the high micromolar range to determine free Ca2+ levels associated with lipid scrambling in resealed human red cell ghosts. The Ca2+ sensitivity was measured in parallel to the translocation of a fluorescent-labelled lipid incorporated into the ghost bilayer. The phospholipid scrambling was found to be half-maximally activated at 63-88 mu M free intracellular Ca2+. The wider applicability of the method and the physiological implications of the calcium sensitivity determined is discussed.

Novel vascular graft grown within recipient's own peritoneal cavity

A method by which to overcome the clinical symptoms of atherosclerosis is the insertion of a graft to bypass an artery blocked or impeded by plaque. However, there may be insufficient autologous mammary artery for multiple or repeat bypass, saphenous vein may have varicose degenerative alterations that can lead to aneurysm in high-pressure sites, and small-caliber synthetic grafts are prone to thrombus induction and occlusion. Therefore, the aim of the present study was to develop an artificial blood conduit of any required length and diameter from the cells of the host for autologous transplantation. Silastic tubing, of variable length and diameter, was inserted into the peritoneal cavity of rats or rabbits. By 2 weeks, it had become covered by several layers of myofibroblasts, collagen matrix, and a single layer of mesothelium. The Silastic tubing was removed from the harvested implants, and the tube of living tissue was everted such that it now resembled a blood vessel with an inner lining of nonthrombotic mesothelial cells (the intima), with a media of smooth muscle-like cells (myofibroblasts), collagen, and elastin, and with an outer collagenous adventitia. The tube of tissue (10 to 20 mm long) was successfully grafted by end-to-end anastomoses into the severed carotid artery or abdominal aorta of the same animal in which they were grown. The transplant remained patent for at least 4 months and developed structures resembling elastic lamellae. The myofibroblasts gained a higher volume fraction of myofilaments and became responsive to contractile agonists, similar to the vessel into which they had been grafted. It is suggested that these nonthrombogenic tubes of living tissue, grown in the peritoneal cavity of the host, may be developed as autologous coronary artery bypass grafts or as arteriovenous access fistulae for hemodialysis patients.

On the Jager-Kaul theorem concerning harmonic maps

In 1983, Jager and Kaul proved that the equator map u*(x) = (x/\x\,0) : B-n --> S-n is unstable for 3 less than or equal to n less than or equal to 6 and a minimizer for the energy functional E(u, B-n) = integral B-n \del u\(2) dx in the class H-1,H-2(B-n, S-n) with u = u* on partial derivative B-n when n greater than or equal to 7. In this paper, we give a new and elementary proof of this Jager-Kaul result. We also generalize the Jager-Kaul result to the case of p-harmonic maps.

Synthesis of silica films at the air/water interface: Effect of template chain length and ionic strength

We have grown surfactant-templated silicate films at the air-water interface using n-alkyltrimethylammonium bromide and chloride in an acid synthesis with tetraethyl orthosilicate as the silicate source. The films have been grown with and without added salt (sodium chloride, sodium bromide) and with n-alkyl chain lengths from 12 to 18, the growth process being monitored by X-ray reflectometry. Glassy, hexagonal, and lamellar structures have been produced in ways that are predictable from the pure surfactant-water phase diagrams. The synthesis appears to proceed initially through an induction period characterized by the accumulation of silica-coated spherical micelles near the surface. All syntheses, except those involving C(12)TACl, show a sudden transformation of the spherical micellar phase to a hexagonal phase. This occurs when the gradually increasing ionic strength and/or changing ethanol concentration is sufficient to change the position of boundaries within the phase diagram. A possible mechanism for this to occur may be to induce a sphere to rod transition in the micellar structure. This transformation, as predicted from the surfactant-water phase diagram, can be induced by addition of salts and is slower for chloride than bromide counteranions. The hexagonal materials change in cell dimension as the chain length is changed in a way consistent with theoretical model predictions. All the materials have sufficiently flexible silica frameworks that phase interconversion is observed both from glassy to hexagonal and from hexagonal, to lamellar and vice versa in those surfactant systems where multiple phases are found to exist.