Alpha 2 giardin is an assemblage A-specific protein of human infective Giardia duodenalis.

Of the 7 genetic assemblages of the parasite Giardia duodenalis only 2 (A and B) are known to cause infections in humans. These assemblages have been characterized in detail at the genomic level but few studies have examined differences in the proteins expressed. Employing one and two-dimensional PAGE we have identified an assemblage A-specific protein of human infective G. duodenalis; alpha 2 giardin. The protein difference was evident using both electrophoretic techniques. Alpha 2 giardin is known to be a structural protein and associates with the caudal flagella and the plasma membrane; however, its exact function is unknown. Although several proteins unique to assemblage B were also observed, we were unable to identify these proteins due to a lack of genomic data available for assemblage B isolates. Together, these proteins represent distinct phenotypic differences between the human infective assemblages of G. duodenalis and support the need to revise the taxonomy of this parasite.

A short-term in vitro gill culture system to study the effects of toxic (copper) and non-toxic (cortisol) stressors on the rainbow trout, Oncorhynchus mykiss (Walbaum).

A short-term (24 h) method of gill filament culture system was developed to predict the effects of environmental contamination and stress in fish. Gill culture system containing two or three rainbow trout gill filaments in sterile glutamine supplemented Leibovitz 15 (L-15) media was submitted for 24 h to six different treatments: (i) CONT (control, medium only); (ii) CORT (cortisol, 0.28 microM cortisol); (iii) BLOCK (glucocorticoid receptor blocker, 14 microM RU 486); (iv) CORT+BLOCK (cortisol and blocker, 0.28 microM cortisol+14 microM RU 486); (v) CORT+CU (cortisol and copper, 100 microM CuSO4+0.28 microM cortisol); (vi) CU (copper, 100 microM CuSO4). After 24 h, the overall gill structure and cellular components resembled those of salmonids in vivo. Lactate dehydrogenase (LDH) activity in the culture media increased in the CORT+CU and CU groups but was significantly lower in the CORT+CU compared to CU group. Apoptotic cells increased in the CORT and CORT+BLOCK. The numbers of glucocorticoid (GR) receptor-positive cells were lower in the CU group. This short-term culture system seems to be suitable for studying the effects of both external and internal stress effectors (toxicants and hormones respectively), as it contains all cell types found in the gills and the cells give similar biological response as in vivo.

Effects of mining activities on heavy metal concentrations in water, sediment, and macroinvertebrates in different reaches of the Pilcomayo River, South America.

From 1997 until 1999 the extent and the ecological effects of zinc, copper, lead, and cadmium pollution were studied in different reaches of the South American Pilcomayo River. A comparison of metal concentrations in water, sediment, and chironomid larvae, as well as the diversity of macroinvertebrate species, was made between sites near the origin of the Pilcomayo River, with hardly any mining activities, sites in the Potosí region, with intensive mining, and sites located 500 km or further downstream of Potosí, in the Chaco plain. Samples were also collected in an unpolluted river (Cachi Mayu River) and in the Tarapaya River, which is strongly contaminated by mine tailings (1000 tons a day). The upper parts of the Pilcomayo River are strongly affected by the release of mine tailings from the Potosí mines where mean concentrations of lead, cadmium, copper, and zinc in water, filtered water, sediment, and chironomid larvae were up to a thousand times higher than the local background levels. The diversity of the benthic macroinvertebrate community was strongly reduced in the contaminated parts; 97% of the benthic macroinvertebrates consisted of chironomid larvae. The degree of contamination in the lower reaches of the river, however, was fairly low because of sedimentation processes and the strong dilution of mine tailings with enormous amounts of clean sediment from erosion processes. Analysis of sediment cores from the Ibibobo floodplain, however, reveal an increase of the heavy metal concentrations in the lower reaches since the introduction of the contaminating flotation process in the mine industry in 1985.

Proteome analysis of Helicobacter pylori: major proteins of type strain NCTC 11637.

Proteome analysis involves the simultaneous resolution and display of proteins produced by an organism, followed by the quantitation, characterisation and identification of these proteins. As part of an ongoing study mapping and comparing the proteins expressed by various strains of the pathogenic bacterium Helicobacter pylori, we have resolved and identified 93 of the most abundant proteins expressed by type reference strain NCTC 11637. Proteins were separated by two-dimensional gel electrophoresis and stained with Coomassie G250. Intensely-stained spots were excised and digested with trypsin, and the resulting peptides were characterised by mass spectrometry. Proteins were then identified by correlating actual peptide profiles with theoretical profiles generated from published nucleotide sequences. Ninety-three of the most intensely-stained protein spots were identified as the products of 35 genes, giving a ratio of 2.7 protein gene-products per gene. The products of the tsaA, pfr, ureA and ureB genes were amongst several proteins present in multiple isoforms. Peptide mass fingerprinting data were used to identify probable post-translational protein modifications. These results suggest that H. pylori proteins are subject to a high degree of post-translational modification. Comparative proteomics of H. pylori strains should greatly assist in investigating the pathogenic properties of this bacterium.

Morphological and metabolic changes in common carp, Cyprinus carpio, during short-term copper exposure: interactions between Cu2+ and plasma cortisol elevation.

The effects of increased endogenous cortisol levels were compared with those of sublethal copper exposure in the freshwater common carp, Cyprinus carpio. Fish were exposed to either increased levels of endogenous cortisol (200 ng/ml) or sublethal copper (1.9 microM) alone or were pretreated by elevating plasma cortisol levels prior to copper exposure to assess whether interactions between both treatments occurred. Effects induced by increased cortisol levels included increased Na+/K(+)-adenosine triphosphate (ATPase) activity and increased plasma Na+ and plasma osmolarity, while copper exposure induced anaerobic metabolism, gill damage, decreasing Na+/K(+)-ATPase activity, decreasing plasma ion levels, and blood thickening. Pretreatment of copper-exposed fish with cortisol partially protected these fish by reducing the copper-induced decrease in Na+/K(+)-ATPase activity. Overall, the results obtained in this study argue against a major role for cortisol as an intermediate for the toxic effects of copper.

Metallothionein and cortisol receptor expression in gills of Atlantic salmon, Salmo salar, exposed to dietary cadmium.

Commercial fish feeds may contain significant levels of cadmium (Cd). However, little is known about the effects of dietary cadmium on fish organs, especially gills, the key osmoregulatory organ. We therefore studied the effects of dietary cadmium on metallothionein (MT) and cortisol receptor (GR) immunoreactivity in the branchial epithelium of the Atlantic salmon (Salmo salar). Cadmium was daily administered via food at 0.2mg (control), 5mg (low dose) and 125 mg (high dose) Cd per kilogram dry pellet weight. Fish were sampled after four and eight weeks. After both four and eight weeks, plasma cadmium concentration had increased significantly only in fish fed the high cadmium dose. Plasma calcium, sodium, chloride and cortisol levels were not affected. In the controls, most MT was colocated with the chloride cell marker, Na(+)/K(+)-ATPase, but some MT was present in pavement and respiratory cells. GR expression was found in chloride, pavement, respiratory and undifferentiated cells in all fish groups, but cadmium accumulation and a marked stimulation of MT expression were seen only in the chloride cells in the gills of fish fed the high cadmium dose. Cadmium treatment did not alter GR expression. When the double staining technique for MT and GR was applied, a marked heterogeneity became apparent in the chloride, pavement and respiratory cells of both groups of cadmium-treated fish and in the control fish. Some fish showed double staining, others stained only for one of the antibodies, whereas other cells were negative for both. We conclude that cadmium entering the gut also enters the gills, where it accumulates in chloride cells and stimulates MT expression.

Evidence for an osmoregulatory role of thyroid hormones in the freshwater mozambique tilapia Oreochromis mossambicus.

The existing equivocal reports on the osmoregulatory role of triiodothyronine (T(3)) and thyroxine (T(4)) in teleosts prompted a reinvestigation of their osmoregulatory function in the euryhaline teleost Oreochromis mossambicus. Evidence is presented for thyroidal involvement in hydromineral balance in freshwater tilapia. Dose- and tissue-related responses to various T(3) and T(4) concentrations were observed in the branchial and renal tissues. The branchial Na(+),K(+)-ATPase activity, known to reflect sodium pump dynamics, increased significantly after the administration of low doses of T(3) (20 and 40 ng. g(-1)) or T(4) (40 and 80 ng. g(-1)). Higher doses of T(3) and T(4) (>160 ng. g(-1)) did not change the enzyme activity, compared to sham-injected fish. Conversely, the specific activity of renal Na(+),K(+)-ATPase decreased significantly at all doses of T(3) or T(4). Further, immunoreactive Na(+),K(+)-ATPase in T(4)-treated fish increased in branchial chloride cells and this was coupled with a significant increase in the size of chloride cells. T(4) treatment, however, did not change branchial chloride cell density. Plasma osmolality, [Na(+)], and [Cl(-)] increased, whereas [K(+)] decreased following low doses of T(3) or T(4). As expected, plasma levels of T(3) and T(4) increased significantly in a dose-dependent manner after a single injection of either T(3) or T(4). The basal levels of T(3) and T(4) were 4.45 +/- 0.49 and 1.25 +/- 0.26 nmol. L(-1), respectively. This study shows that physiological concentrations of T(3) (<10.57 nmol. L(-1)) and T(4) (<6.64 nmol. L(-1)) enhance branchial Na(+) pump activity and chloride cell morphometric dynamics, favoring hyperosmoregulatory capacity in freshwater tilapia. These data are consistent with the hypothesis that thyroid hormones perform a role in hydromineral regulation in freshwater teleosts.

Cortisol increases Na(+)/K(+)-ATPase density in plasma membranes of gill chloride cells in the freshwater tilapia Oreochromis mossambicus.

The effect of cortisol on Na(+)/K(+)-ATPase expression in the gill chloride cells of tilapia Oreochromis mossambicus was studied by immunocytochemistry at the light and electron microscope levels. One of three doses of cortisol (low, 125 mg kg(-1 )food; middle, 375 mg kg(-1 )food; high, 750 mg kg(-1) food) was administered via the food (at a ration of 1.5 % of body mass) and the fish were sampled after 5 days. Plasma osmolality and Na(+) levels were elevated in the middle- and high-dose groups, and plasma cortisol levels in the high-dose groups. Hematocrit values were not affected by the treatments. Opercular membrane chloride cell density increased by 94 % and 286 % in the middle- and high-dose fish, respectively, whereas the gill chloride cell frequency increased by up to 28 % maximally in the high-dose fish. Lamellar gill chloride cells were absent in the control and low-dose groups, but were observed in the middle- and high-dose groups. Cortisol increased the volume of the tubular membrane system in mature gill chloride cells. Quantification of immunogold-labelled Na(+)/K(+)-ATPase antigen (a 104 kDa protein species, as demonstrated by western blot) revealed that the high dose of cortisol increases the Na(+)/K(+)-ATPase density in the tubular system of chloride cells. This is the first direct evidence that cortisol not only increases chloride cell numbers but also Na(+)/K(+)-ATPase density in these cells.

Na(+)/K(+)-ATPase immunoreactivity in branchial chloride cells of Oreochromis mossambicus exposed to copper.

Chloride cells were identified by Na(+)/K(+)-ATPase immunocytochemistry at the light and electron microscope levels in gills of freshwater tilapia Oreochromis mossambicus. Turnover of chloride cells was enhanced by exposing the fish to waterborne copper (3.2 micromol l(-)(1)) for 14 days, as indicated by a 38 % increase in cells expressing proliferating cell nuclear antigen (PCNA) relative to controls. The expression of PCNA was most marked in the central area of the filamental epithelium, from where the chloride cells are thought to originate and migrate. In control fish, chloride cells were associated exclusively with the filamental epithelium. In both controls and copper-exposed fish, two chloride cell populations were seen after Na(+)/K(+)-ATPase immunostaining. These probably represent subpopulations of newly emerged chloride cells: (1) strongly stained cells (mature chloride cells) in the filamental and lamellar epithelium and (2) weakly stained cells, identified by electron microscopy as apoptotic and necrotic chloride cells, mainly in the filamental epithelium. Absolute numbers of mature chloride cells fell, while necrotic and apoptotic chloride cell numbers increased, in copper-exposed fish. A strong correlation could be established for gill Na(+)/K(+)-ATPase specific activity and the number of strongly stained chloride cells in controls and copper-exposed fish and for Na(+)/K(+)-ATPase specific activity and total numbers of immunoreactive cells in copper-exposed fish owing to an increased incidence of weakly staining cells.

Metallothionein response in gills of Oreochromis mossambicus exposed to copper in fresh water.

Freshwater Oreochromis mossambicus (tilapia) were exposed to 3.2 micromol/l Cu(NO(3))(2) in the water for up to 80 days, and copper (Cu) and immunoreactive metallothionein (irMT) were localized in the branchial epithelium. Cu was demonstrated in mucous cells (MC), chloride cells (CC), pavement cells (PC), respiratory cells (RC), and basal layer cells (BLC) via autometallography combined with alcian blue staining for MC and Na(+)-K(+)-ATPase immunostaining for CC and, on the basis of their location in the epithelium of PC, RC, and BLC. In control fish (water with Cu concentration

Stress responsiveness of the pituitary-interrenal axis during early life stages of common carp (Cyprinus carpio).

Whole-body levels of ACTH, alpha-MSH and cortisol in eggs and larvae of the common carp (Cyprinus carpio) were determined periodically up until 168 h after fertilisation. ACTH, alpha-MSH and cortisol immunoreactivity was detected in unfertilised eggs, and endogenous production of ACTH and alpha-MSH was observed 24 h after fertilisation and that of cortisol 36 h after fertilisation. ACTH immunoreactivity reached peak levels before hatching (56-72 h after fertilisation) and remained relatively stable thereafter, while alpha-MSH immunoreactivity started to increase after hatching. At 36 h after fertilisation, whole-body cortisol levels increased rapidly reaching peak levels at the end of hatching (72 h after fertilisation), remaining stable until the end of the experiment. From 50 h after fertilisation onwards, embryos and larvae increased their whole-body cortisol levels when subjected to handling (mechanical pressure during egg stage or netting during the larval stage). It is concluded that the pituitary-interrenal axis in carp is fully functional at the time of hatching. No indications of a stress non-responsive period after hatching were observed. To characterise ACTH and alpha-MSH immunoreactivities in carp larvae, whole-body homogenates were analysed by HPLC, with pituitary homogenates of adult carp serving as a reference. ACTH and alpha-MSH immunoreactivity in carp larvae homogenates consisted of three and two products respectively. HPLC of adult carp pituitaries revealed the presence of two ACTH immunoreactive products, which may represent a phosphorylated and a non-phosphorylated ACTH variant, while the three alpha-MSH peaks most likely represent des-acetylated, mono-acetylated and di-acetylated alpha-MSH, the latter being the predominant form. In carp larvae, however, one of the ACTH immunoreactive products co-eluted with the non-phosphorylated ACTH, while the two alpha-MSH products identified co-eluted with des-acetylated and mono-acetylated alpha-MSH, indicating that POMC processing at this stage of development is different from prohormone processing in adult fish.

Expression, purification and preliminary X-ray crystallographic analysis of PsaA, a putative metal-transporter protein of Streptococcus pneumoniae.

The putative metal-transporter protein PsaA of Streptococcus pneumoniae is of potential interest both as a vaccine and also as a drug target. The overexpression of the protein in E. coli, and its subsequent purification and crystallization are described. The crystals are rectangular rods and diffract to beyond 2.7 A resolution. The crystal space group is P212121 with unit-cell dimensions a = 59.9, b = 66.5 and c = 69.9 A.

Molecular analysis of putative pneumococcal virulence proteins.

Although the polysaccharide capsule has been recognized as a sine qua non of virulence, recent attention has focused on the role of pneumococcal proteins in pathogenesis, particularly in view of their potential as vaccine antigens. The contribution of pneumolysin, two distinct neuraminidases, autolysin, hyaluronidase, and the 37 kDa pneumococcal surface adhesin A has been examined by specifically mutagenizing the respective genes in the pneumococcal chromosome and examining the impact on virulence in animal models. The vaccine potential of these proteins has also been assessed by immunization of mice with purified antigens, followed by challenge with virulent pneumococci.

Kinetics of Cu2+ inhibition of Na+/K(+)-ATPase.

The interaction of Cu2+ with enzymatic activity of rabbit kidney Na+/K(+)-ATPase was studied in media with buffered, defined free Cu2+ levels. The IC50-values are 0.1 mumol/l for Na+/K(+)-ATPase and 1 mumol/l for K(+)-pNPPase. Dithiothreitol (DTT) reverses the inhibitory effect of Cu2+ in vitro. Cu2+ exerts non-competitive effects on the enzyme with respect to Na+, K+, ATP or pNPP, but has a mixed-type inhibitory effect with respect to Mg2+. It is concluded that the appreciation of the inhibitory effect of Cu2+ on this enzyme requires carefully composed assay media that include a buffer for Cu2+, and that the IC50-values calculated according to this model indicate that Cu2+ may be more toxic than previously anticipated.

Cloning and characterization of nanB, a second Streptococcus pneumoniae neuraminidase gene, and purification of the NanB enzyme from recombinant Escherichia coli.

Streptococcus pneumoniae is believed to produce more than one form of neuraminidase, but there has been uncertainty as to whether this is due to posttranslational modification of a single gene product or the existence of more than one neuraminidase-encoding gene. Only one stable pneumococcal neuraminidase gene (designated nanA) has been described. In the present study, we isolated and characterized a second neuraminidase gene (designated nanB), which is located close to nanA on the pneumococcal chromosome (approximately 4.5kb downstream). nanB was located on an operon separate from that of nanA, which includes at least five other open reading frames. NanB has a predicted size of 74.5 kDa after cleavage of a 29-amino-acid signal peptide. There was negligible amino acid homology between NanA and NanB, but NanB did exhibit limited homology with the sialidase of Clostridium septicum. NanB was purified from recombinant Escherichia coli and found to have a pH optimum of 4.5, compared with 6.5 to 7.0 for NanA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis suggested that NanB has a molecular size of approximately 65 kDa. The discrepancy between this estimate and the size predicted from the nucleotide sequence is most likely a consequence of C-terminal processing or anomalous electrophoretic behavior.

Sequence variation in the Streptococcus pneumoniae pneumolysin gene affecting haemolytic activity and electrophoretic mobility of the toxin.

When 30 clinical isolates of Streptococcus pneumoniae, representing 16 capsular serotypes, were analysed by Western blot for production of the haemolytic toxin pneumolysin (Ply), all strains produced an immunoreactive band of similar intensity. However, six isolates of serotype 8 and two of type 7F expressed Ply whose mobility on SDS-PAGE was anomalously slow. Culture lysates from these strains also had low haemolytic activities compared with those for clinical isolates of other serotypes, suggesting the possibility of mutations affecting specific activity. Genes encoding Ply from one type 8 isolate and one type 7F isolate were cloned into Escherichia coli and sequenced. Compared with the published sequence for Ply, the deduced amino acid sequence for the type 8 Ply variant contained three amino acid substitutions, and the type 7F variant four amino acid substitutions. Both variants also had Val270 and Lys271 deleted. The variant Ply proteins were purified from recombinant E. coli expressing the cloned genes, and shown to have substantially reduced specific haemolytic activities [6.8 x 10(4) haemolytic units (HU)/mg and 2.3 x 10(4) HU/mg for type 8 Ply and type 7F Ply respectively] compared with Ply itself (1.2 x 10(6) HU/mg). Studies with chimeric toxin gene constructs indicated that both the reduced haemolytic specific activity and the anomalous electrophoretic mobility of the variant Plys were attributable to a single amino acid substitution (Thr172-->Ile).

Interactions between copper and cadmium modify metal organ distribution in mature tilapia, Oreochromis mossambicus.

Sexually mature female tilapia were exposed to sublethal concentrations of waterborne Cu and/or Cd over 6 days, and subsequent body concentrations of these metals were determined in several organs. The results show that the distribution of Cu and Cd was metal and organ specific. This is demonstrated, for example, by the observation that in tilapia, Cu exposure did not result in Cu accumulation in the liver, whereas in the intestinal wall, notably high concentrations of Cu and Cd were measured in metal exposed fish. In addition to single metal exposed fish, we also determined Cu and Cd body distribution in Cu?Cd co-exposed fish. The observed interactions in metal accumulation were most pronounced in the organs of fish exposed to low, environmentally realistic, metal concentrations.

Immunization of mice with pneumolysin toxoid confers a significant degree of protection against at least nine serotypes of Streptococcus pneumoniae.

Pneumolysin is the thiol-activated cytolysin produced by Streptococcus pneumoniae. Mice were immunized with a genetically engineered toxoid version of pneumolysin, which was derived from a serotype 2 pneumococcus. The toxoid carried the mutation Trp-433-->Phe. Alum was used as the adjuvant. Immunized mice had significantly increased levels of anti-pneumolysin antibodies, principally immunoglobulin G1. Mice were challenged intraperitoneally or intranasally with 12 strains covering capsular serotypes 1 to 6, 7F, 8, and 18C. Following challenge, the survival rate and/or the time of death of nonsurvivors (survival time) was significantly greater than that of sham-immunized mice for all nine serotypes. However, differences in the degree of protection were noted between different strains. The route of challenge also appeared to influence the degree of protection. Nevertheless, the significant, albeit in some cases partial, protection provided against all nine pneumococcal serotypes supports the conclusion that pneumolysin toxoids warrant consideration for inclusion in a human vaccine.

Copper toxicity in cultured human skeletal muscle cells: the involvement of Na+/K(+)-ATPase and the Na+/Ca(2+)-exchanger.

Copper (Cu2+) intoxication has been shown to induce pathological changes in various tissues. The mechanism underlying Cu2+ toxicity is still unclear. It has been suggested that the Na+/K(+)-ATPase and/or a change of the membrane permeability may be involved. In this study we examined the effects of Cu2+ on the Na+ and Ca2+ homeostasis of cultured human skeletal muscle cells using the ion-selective fluorescent probes Na(+)-binding benzofuran isophatalate (SBFI) and Fura-2, respectively. In addition, we measured the effect of Cu2+ on the Na+/K(+)-ATPase activity. Cu2+ and ouabain increase the cytoplasmic free Na+ concentration ([Na+]i). Subsequent addition of Cu2+ after ouabain does not affect the rate of [Na+]i increase. Cu2+ inhibits the Na+/K(+)-ATPase activity with an IC50 of 51 microM. The cytoplasmic free Ca2+ concentration ([Ca2+]i) remains unaffected for more than 10 min after the administration of Cu2+. Thereafter, [Ca2+]i increases as a result of the Na+/Ca(2+)-exchanger operating in the reversed mode. The effects of Cu2+ on the Na+ homeostasis are reversed by the reducing and chelating agent dithiothreitol and the heavy metal chelator N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN). In conclusion, SBFI is a good tool to examine Na+ homeostasis in cultured human skeletal muscle cells. Under the experimental conditions used, Cu2+ does not modify the general membrane permeability, but inhibits the Na+/K(+)-pump leading to an increase of [Na+]i. As a consequence the operation mode of the Na+/Ca(2+)-exchanger reverses and [Ca2+]i rises.

Protection of infant mice from challenge with Streptococcus pneumoniae type 19F by immunization with a type 19F polysaccharide--pneumolysoid conjugate.

The immunogenicity and protective efficacy of a conjugate of Streptococcus pneumoniae type 19F polysaccharide and a genetically toxoided derivative of the pneumococcal toxin pneumolysin was investigated in an infant mouse model. The conjugate was administered to Balb/c mice during pregnancy and/or lactation, and to their offspring during early infancy. The anti-polysaccharide and anti-pneumolysin titres of the immunized infant mice were significantly higher than those of non-immunized controls. When the infant mice were challenged with type 19F pneumococci, the bacteria were cleared more effectively from the blood of immunized mice than from that of control mice. The survival rate for the immunized mice was also significantly higher than that for the control group. These results indicate that highly protective anti-pneumococcal responses can be induced in infant mice by immunization with the conjugate during gestation or early infancy, and suggest a possible role for pneumolysoid-polysaccharide conjugates as human vaccine components.