Towards a blood-stage vaccine for malaria: Are we following all the leads?

Although the malaria parasite was discovered more than 120 years ago, it is only during the past 20 years, following the cloning of malaria genes, that we have been able to think rationally about vaccine design and development. Effective vaccines for malaria could interrupt the life cycle of the parasite at different stages in the human host or in the mosquito. The purpose of this review is to outline the challenges we face in developing a vaccine that will limit growth of the parasite during the stage within red blood cells - the stage responsible for all the symptoms and pathology of malaria. More than 15 vaccine trials have either been completed or are in progress, and many more are planned. Success in current trials could lead to a vaccine capable of saving more than 2 million lives per year.

Cytokine regulation of syndecan-1 and-2 gene expression in human periodontal fibroblasts and osteoblasts

Cell-surface proteoglycans participate in several biological functions including interactions with a variety of growth factors and cytokines. Regulation of syndecan-1 and -2 gene expression was investigated in human periodontal ligament fibroblasts (PDLF), osteoblasts (OB) and gingival fibroblasts (GF), in response to platelet-derived growth factor (PDGF-BB), transforming growth factor (TGF-beta(1)), and interleukin (IL-1beta) by Northern blot analyses. We also compared the effect of PDGF-BB and TGF-beta(1), separately and in combination, in the prolonged presence of IL-1beta on the expression of both syndecan genes. The results demonstrated that the three cell lines regulated the expression of syndecan-1 and -2 in response to growth factors and cytokines in different manners. These cell lines increased syndecan-1 mRNA levels in response to either PDGF-BB or TGF-beta(1) and decreased levels in response to IL-1beta. The effect of IL-1beta on syndecan-1 mRNA synthesis was partially reversed after adding PDGF-BB and TGF-beta(1), separately or in combination, in the presence of IL-1beta. In contrast, syndecan-2 mRNA level was markedly upregulated in response to either TGF-beta(1) or IL-1beta in OB when compared with the other two cell lines. However, the stimulatory effect of TGF-beta(1) on syndecan-2 mRNA production in OB was abolished in the prolonged presence of IL-1beta. These findings lend support to the notion that syndecan-1 and syndecan-2 have distinct functions which correlate with their source and functions within the periodontium.

Differential expression and distribution of syndecan-1 and-2 in periodontal wound healing of the rat

Cell-surface proteoglycans participate in several biological functions including interactions with adhesion molecules, growth factors and a variety of other effector molecules. Accordingly, these molecules play a central role in various aspects of cell-cell and cell-matrix interactions. To investigate the expression and distribution of the cell surface proteoglycans, syndecan-1 and -2, during periodontal wound healing, immunohistochemical analyses were carried out using monoclonal antibodies against syndecan-1, or -2 core proteins. Both syndecan-1 and -2 were expressed and distributed differentially at various stages of early inflammatory cell infiltration, granulation tissue formation, and tissue remodeling in periodontal wound healing. Expression of syndecan-1 was noted in inflammatory cells within and around the fibrin clots during the earliest stages of inflammatory cell infiltration. During granulation tissue formation it was noted in fibroblast-like cells and newly formed blood vessels. Syndecan-1 was not seen in newly formed bone or cementum matrix at any of the time periods studied. Syndecan-1 expression was generally less during the late stages of wound healing but was markedly expressed in cells that were close to the repairing junctional epithelium. In contrast, syndecan-2 expression and distribution was not evident at the early stages of inflammatory cell infiltration. During the formation of granulation tissue and subsequent tissue remodeling, syndecan-2 was expressed extracellularly in the newly formed fibrils which were oriented toward the root surface. Syndecan-2 was found to be significantly expressed on cells that were close to the root surface and within the matrix of repaired cementum covering root dentin as well as at the alveolar bone edge. These findings indicate that syndecan-1 and -2 may have distinctive functions during wound healing of the periodontium. The appearance of syndecan-1 may involve both cell-cell and cell-matrix interactions, while syndecan-2 showed a predilection to associate with cell-matrix interactions during hard tissue formation.

Growth of subtropical ECM fungi with different nitrogen sources using a new floating culture technique

Eight species of ectomycorrhizal (ECM) fungi in the genera Amanita. Gymnoboletus, Lactarius, and Russula were isolated from subtropical plant communities in eastem Australia. Two species were isolated from each of rainforest, Nothofagus forest, Eucalyptus forest, and Eucalyptus dominated wallum (heath) forest. These communities differ strongly in their soluble soil nitrogen (N) composition. The ability of the fungi to use inorganic (nitrate, ammonium) and organic (amide, peptide, protein) nitrogen sources was determined. As the fungi did not grow in liquid culture, a 'floating culture' technique was devised that allows hyphal growth on a screen floating on liquid medium. With some exceptions, fungal biomass production in floating culture closely reflected fungal growth on solid media assessed by total colony glucosamine content. Most isolates grown in floating culture had similar glucosamine concentrations on all N sources, with isolate specific concentrations ranging from 6 to 12 mug glucosamine g(-1) DW. However, Russula spp. had up to 1.7-fold higher glucosamine concentrations when growing with glutamine or ammonium compared to nitrate, glutathione or protein. Floating cultures supplied with 0.5, 1.5. 4.5, or 10 mm N mostly produced greatest biomass with 4.5 mM N. In vitro nitrate reductase activity (NRA) ranged from very low (0.03 mumol NO2- g(-1) fw h(-1)) in Russula sp. (wallum) to high (2.16 mumol NO2- g(-1) fw h(-1)) in Gymnoboletus sp. (rainforest) and mirrored the fungi's ability to use nitrate as a N source. All Russula spp. (wallum, Nothofagus and Eucalyptus forests), Lactarills sp, (rainforest) and.4manita sp. (wallum) utilized ammonium and glutamine but had little ability to use other N sources. In contrast,Amanita species (Nothofagus and Eucalyptus forests) grew on all N sources but produced most biomass with ammonium and glutamine. Only Gymnoboletus sp. (rainforest) showed similar growth with nitrate and ammonium as N sources. Fungal N source use was not associated with taxonomic groups, but is discussed in the context of soil N sources in the different habitats.

Nutritional strategies for the prevention of hypocalcaemia at calving for dairy cows in pasture-based systems

This review considers the current literature on the macro-mineral nutrition of the soon-to-calve, or transition, dairy cow. Calcium is the main focus, since milk fever (clinical hypocalcaemia) appears to be the most common mineral-related problem faced by the transition cow Australia-wide. The importance of minimising calcium intake and optimising the balance of the key dietary electrolytes, sodium, potassium, sulfate, and chloride, in the weeks before calving is highlighted. Excess dietary potassium can, in some situations, induce milk fever, perhaps even more effectively than excess calcium. Excess sodium remains under suspicion. In contrast, excess dietary chlorine and, to a lesser extent, sulfur can improve the ability of the cow to maintain calcium homeostasis. Diets that promote either a hypomagnesaemia or hyperphosphataemia have also the potential to precipitate milk fever at calving. Current prevention strategies focus on the use of forages with moderate to low levels of calcium, potassium, and sodium, and also rely on or utilise addition of chloride and sulfate in the form of 'anionic' feeds. Anionic salts are one example of an anionic feed. However, legitimate questions remain as to the effectiveness of anionic salts in pasture-feeding systems. The causes and prevention of milk fever are considered from the perspective of the variety of Australian feedbases. Impediments to the use of anionic feeds in Australia feeding systems are outlined. The potential for improving maternal reserves of calcium around calving to reduce the risk of milk fever is also discussed.

Cloud-point extraction and preconcentration of cyanobacterial toxins (microcystins) from natural waters using a cationic surfactant

A new cloud-point extraction and preconcentration method, using a cationic, surfactant, Aliquat-336 (tricaprylyl-methy;ammonium chloride), his-been developed for the determination of cyanobacterial toxins, microcystins, in natural waters. Sodium sulfate was used to induce phase separation at 25 degreesC. The phase behavior of Aliquat-336 with respect to concentration of Na2SO4 was studied. The cloud-point system revealed a very high phase volume ratio compared to other established systems of nonionic, anionic, and cationic surfactants: At pH 6-7, it showed an outstanding selectivity in ahalyte extraction for anionic species. Only MC-LR and MC-YR, which are known to be predominantly anionic, were extracted (with averaged recoveries of 113.9 +/- 9% and 87.1 +/- 7%, respectively). MC-RR, which is likely to be amphoteric at the above pH range, was. not cle tectable in.the extract. Coupled to HPLC/UV separation and detection, the cloud-point extraction method (with 2.5 mM Aliquat-336 and 75 mM Na2SO4 at 25 degreesC) offered detection limits of 150 +/- 7 and 470 +/- 72 pg/mL for MC-LR and MC-YR, respectively, in 25 mL of deionized water. Repeatability of the method was 7.6% for MC-LR and 7.3% for MC-YR: The cloud-point extraction process can be. completed within 10-15 min with no cleanup steps required. Applicability of the new method to the determination of microcystins in real samples was demonstrated using natural surface waters, collected from a local river and a local duck pond spiked with realistic. concentrations of microcystins. Effects of salinity and organic matter (TOC) content in the water sample on the extraction efficiency were also studied.

Current concepts in central nervous system regeneration

A dictum long-held has stated that the adult mammalian brain and spinal cord are not capable of regeneration after injury. Recent discoveries have, however, challenged this dogma. In particular, a more complete understanding of developmental neurobiology has provided an insight into possible ways in which neuronal regeneration in the central nervous system may be encouraged. Knowledge of the role of neurotrophic factors has provided one set of strategies which may be useful in enhancing CNS regeneration. These factors can now even be delivered to injury sites by transplantation of genetically modified cells. Another strategy showing great promise is the discovery and isolation of neural stem cells from adult CNS tissue. It may become possible to grow such cells in the laboratory and use these to replace injured or dead neurons. The biological and cellular basis of neural injury is of special importance to neurosurgery, particularly as therapeutic options to treat a variety of CNS diseases becomes greater. (C) 2002 Published by Elsevier Science Ltd.

Expression of Heparan Sulphate N-deacetylase/N-sulphotransferase by Vascular Smooth Muscle Cells

Heparan sulphate is an important mediator in determining vascular smooth muscle cell (SMC) phenotype. The sulphation pattern of the heparan sulphate chains is critical to their function. We have examined the initial step in the biosynthesis of the sulphated domains mediated by the enzyme heparan sulphate N-deacetylase/N-sulphotransferase (NDST). Rabbit aortic SMC in primary culture exhibited NDST enzyme activity and expressed NDST-1 in their Golgi apparatus, with maximal expression in SMC 2 days after dispersal in primary culture confirmed by Western blot analysis. Endothelial cells, macrophages and fibroblasts expressed NDST-1 but had generally less intense staining than SMC, although SMC expression decreased with culture. The uninjured rat aorta also showed widespread expression of NDST-1. After balloon de-endothelialisation, NDST-1 could not be detected in SMC of the neointima in the early stages of neointimal formation, but was re-expressed at later time points (after 12 weeks). In human coronary arteries, SMC of the media and the diffuse intimal thickening expressed NDST-1, while SMC in the atherosclerotic plaque were negative for NDST-1. We conclude that SMC may regulate their heparan sulphate sulphation at the level of expression of the enzyme heparan sulphate NDST in a manner related to their phenotypic state.

Regulation of the mouse Nas1 promoter by vitamin D and thyroid hormone

The renal sodium-sulfate cotransporter, NaSi-1, a protein implicated to control serum sulfate levels, has been shown to be regulated in vivo by 1,25-dihydroxyvitamin D-3 (1,25-(OH)(2)D-3) and tri-iodothyronine (T-3). Recently, we cloned the mouse NaSi-1 gene (Nas1) and in the present study identified a 1,25-(OH)(2)D-3- and T-3-responsive element located within the Nas1 promoter. Mutational analysis of the Nas1 promoter resulted in identification of a direct repeat 6-type vitamin-D-responsive element (DR6 VDRE) at -525 to -508 and an imperfect inverted repeat 0-type T-3-responsive element (IR0 T3RE) at -436 to -425 which conferred 1,25(OH)(2)D-3 and T3 responsiveness, respectively. In summary, we have identified responsive elements that mediate the enhanced transcription of Nas1 by 1,25-(OH)(2)D-3 and T-3, and these mechanisms may provide important clues to the physiological control of sulfate homeostasis.

Direct electrochemistry of a bacterial sulfite dehydrogenase

Sulfite dehydrogenase from Starkeya novella is an alphabeta heterodimer comprising a 40.6 kDa subunit (containing the Mo cofactor) and a smaller 8.8 kDa heme c subunit. The enzyme catalyses the oxidation of sulfite to sulfate with the natural electron acceptor being cytochrome c(550). Its catalytic mechanism is thought to resemble that found in eukaryotic sulfite oxiclases. Using protein film voltammetry and redox potentiometry, we have identified both Mo- and heme-centered redox responses from the enzyme immobilized on a pyrolytic graphite working electrode: E-m,E-8 (Fe-III/II) +177 mV; E-m,E-8 (Mo-VI/V) +211 mV and E(m,)8 (Mo-V/IV) -118 mV vs NHE; Upon addition of sulfite to the electrochemical cell a steady-state voltammogram is observed and an apparent Michaelis constant (K-m) of 26(l) muM was determined for the enzyme immobilized on the working electrode surface, which is comparable with the value obtained from solution assays.

Intramolecular electron transfer in a bacterial sulfite dehydrogenase

Sulfite dehydrogenase (SDH) from Starkeya novella, a sulfite-oxidizing molybdenum-containing enzyme, has a novel tightly bound αβ-heterodimeric structure in which the Mo cofactor and the c-type heme are located on different subunits. Flash photolysis studies of intramolecular electron transfer (IET) in SDH show that the process is first-order, independent of solution viscosity, and not inhibited by sulfate, which strongly indicates that IET in SDH proceeds directly through the protein medium and does not involve substantial movement of the two subunits relative to each other. The IET results for SDH contrast with those for chicken and human sulfite oxidase (SO) in which the molybdenum domain is linked to a b-type heme domain through a flexible loop, and IET shows a remarkable dependence on sulfate concentration and viscosity that has been ascribed to interdomain docking. The results for SDH provide additional support for the interdomain docking hypothesis in animal SO and clearly demonstrate that dependence of IET on viscosity and sulfate is not an inherent property of all sulfite-oxidizing molybdenum enzymes.

Characterization of Treponema phagedenis-like spirochetes isolated from papillomatous digital dermatitis lesions in dairy cattle

Four spirochete strains were isolated from papillomatous digital dermatitis (PDD) lesions in Iowa dairy cattle and compared with two previously described spirochete strains isolated from dairy cattle in California. These six strains shared an identical 16S ribosomal DNA sequence that was 98% similar to Treponema phagedenis and 99% similar to the uncultivated PDD spirochete sequence DDLK-4. The whole-cell protein profiles resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these six strains were similar. However, these strains showed differences in the antigenic diversity of lipopolysaccharide (LPS). Genetic diversity was also detected by pulsed-field gel electrophoresis of genomic DNA digests, revealing differences among five of the six strains. Serum immunoglobulin G antibodies from dairy cattle with active PDD lesions reacted with the LPS of all but one PDD spirochete strain. Likewise, peripheral blood mononuclear cells from cattle with active PDD lesions produced blastogenic responses to one of the two California isolates. Both antibody and lymphocyte blastogenic responses were reduced in convalescent dairy cattle, suggesting the immune response to these spirochetes has short duration. These results demonstrate genetic and antigenic diversity among T. phagedenis-like treponemes and provide further evidence for the involvement of these spirochetes in the pathogenesis of PDD.

'Iron lung': Distinctive bronchoscopic features of acute iron tablet aspiration

Three confirmed cases of acute iron tablet-induced necrosis due to a fulminant chemical burn injury to the tracheobronchial tree as a result of accidental inhalation and/or aspiration of iron tablets are described. Although histological confirmation has been relied upon for diagnosis, the distinctive bronchoscopic features may allow prompt recognition and treatment by bronchoscopists to prevent this potentially fatal condition.

The use of 16s rDNA restriction fragment length polymorphism analysis for the identification of non-fermenting gram negative bacilli in a cystic fibrosis microbiology referral service

The utility of 16s rDNA restriction fragment length polymorphism (RFLP) analysis for the partial genomovar differentiation of Burkholderia cepacia complex bacterium is well documented. We compared the 16s rDNA RFLP signatures for a number of non-fermenting gram negative bacilli (NF GNB) LMG control strains and clinical isolates pertaining to the genera Burkholderia, Pseudomonas, Achromobacter (Alcaligenes), Ralstonia, Stenotrophomonas and Pandoraea. A collection of 24 control strain (LMG) and 25 clinical isolates were included in the study. Using conventional PCR, a 1.2 kbp 16s rDNA fragment was generated for each organism. Following restriction digestion and electrophoresis, each clinical isolate RFLP signature was compared to those of the control strain panel. Nineteen different RFLP signatures were detected from the 28 control strains included in the study. TwentyoneyTwenty- five of the clinical isolates could be classified by RFLP analysis into a single genus and species when compared to the patterns produced by the control strain panel. Four clinical B. pseudomallei isolates produced RFLP signatures which were indistinguishable from B. cepacia genomovars I, III and VIII. The identity of these four isolates were confirmed using B. pseudomallei specific PCR. 16s rDNA RFLP analysis can be a useful identification strategy when applied to NF GNB, particularly for those which exhibit colistin sulfate resistance. The use of this molecular based methodology has proved very useful in the setting of a CF referral laboratory particularly when utilised in conjunction with B. cepacia complex and genomovar specific PCR techniques. Species specific PCR or sequence analysis should be considered for selected isolates; especially where discrepancies between epidemiology, phenotypic and genotypic characteristics occur.