Cryptosporidium spp., Giardia intestinalis and microsporidia are unicellular opportunistic pathogens that can cause gastrointestinal infections in both animals and humans. Since companion animals may serve as a source of infection, the aim of the present screening study was to analyse the prevalence of these intestinal protists in fecal samples collected from dogs living in 10 animal shelters in central Europe (101 dogs from Poland and 86 from the Czech Republic), combined with molecular subtyping of the detected organisms in order to assess their genetic diversity. Genus-specific polymerase chain reactions were performed to detect DNA of the tested species and to conduct molecular subtyping in collected samples, followed by statistical evaluation of the data obtained (using χ2 or Fisher's tests). The observed prevalence was 15.5, 10.2, 1 and 1% for G. intestinalis, Enterocytozoon bieneusi, Cryptosporidium spp. and Encephalitozoon cuniculi, respectively. Molecular evaluation has revealed the predominance of dog-specific genotypes (Cryptosporidium canis XXe1 subtype; G. intestinalis assemblages C and D; E. cuniculi genotype II; E. bieneusi genotypes D and PtEbIX), suggesting that shelter dogs do not pose a high risk of human transmission. Interestingly, the percentage distribution of the detected pathogens differed between both countries and individual shelters, suggesting that the risk of infection may be associated with conditions typical of a given location.

Barramundi (Lates calcarifer), a catadromous teleost of significant and growing commercial importance, are reported to have limited fatty acid bioconversion capability and therefore require preformed long-chain PUFA (LC-PUFA) as dietary essential fatty acid (EFA). In this study, the response of juvenile barramundi (47·0 g/fish initial weight) fed isolipidic and isoenergetic diets with 8·2 % added oil was tested. The experimental test diets were either devoid of fish oil (FO), and thus with no n-3 LC-PUFA (FO FREE diet), or with a low inclusion of FO (FO LOW diet). These were compared against a control diet containing only FO (FO CTRL diet) as the added lipid source, over an 8-week period. Interim samples and measurements were taken fortnightly during the trial in order to define the aetiology of the onset and progression of EFA deficiency. After 2 weeks, the fish fed the FO FREE and FO LOW diets had significantly lower live-weights, and after 8 weeks significant differences were detected for all performance parameters. The fish fed the FO FREE diet also had a significantly higher incidence of external abnormalities. The transcription of several genes involved in fatty acid metabolism was affected after 2 weeks of feeding, showing a rapid nutritional regulation. This experiment documents the aetiology of the onset and the progression of EFA deficiency in juvenile barramundi and demonstrates that such deficiencies can be detected within 2 weeks in juvenile fish.

The isoenzymatic pattern of glutamate dehydrogenase (GDH) has been described for Ascaris suum, a parasite of Sus scrofa domestica. Only one band of activity has been revealed, suggesting a monomorphic condition for this enzyme. Also, the structure of GDH has been assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and silver staining. Only one subunit was present with a molecular weight of about 55 000. A hexameric structure for GDH of A. suum is suggested.

The effect of accelerated aging (AA) and seed priming on specific seed enzymes was studied in soybeans. Aging seeds for 48 and 96 h at 41°C and 100% humidity reduced germination from 92% to 68% and 0%, respectively. Malate dehydrogenase (MDH) glutamate dehydrogenase (GDH), and esterase were assayed in extracts from dry and imbibing seeds. Malate dehydrogenase activity was the least affected by AA treatments. Certain GDH isozyme activity was reduced by 17% after 48 h of AA and almost completely lost after 96 h. Imbibing for 24 h increased GDH activity in 48 h-accelerated-aged seeds but not the 96 h-aged seeds. Two esterase isozymes in dry seeds showed a 77% loss of activity after 48 h of AA. This treatment increased the total esterase activity, with the greatest activity observed after 96 h of AA. This was due to the appearance of a diffuse high molecular weight (HMW) region of activity observed on native polyacrylamide gels and specifically induced in the embryonic axes. Imbibing 48-h-accelerated-aged seeds for 48 h caused a reappearance of the two esterase enzymes lost during aging. The HMW esterase staining region also appeared after imbibing unaged seeds for 24 h. Seed priming, caused a response similar to imbibition for each enzyme except the HMW esterase activity. Priming 48-h-accelerated-aged seeds reduced the HMW esterase activity. This was not observed during imbibition of aged seeds and indicates the first identification of a specific enzymatic response unique to a seed priming treatment.

In this paper, elevated pressures up to 750 atm (1 atm = 101 kPa) were found to have a strong stabilizing effect on two extremely thermophilic glutamate dehydrogenases (GDHs): the native enzyme from the hyperthermophile Pyrococcus furiosus (Pf), and a recombinant GDH mutant containing an extra tetrapeptide at the C-terminus (rGDHt). The presence of the tetrapeptide greatly destabilized the recombinant mutant at ambient pressure; however, the destabilizing effect was largely reversed by the application of pressure. Electron spin resonance (ESR) spectroscopy of a spin-label attached to the terminal cysteine of rGDHt revealed a high degree of mobility, suggesting that destabilization is due to weakened intersubunit ion-pair interactions induced by thermal fluctuations of the tetrapeptide. For both enzymes, the stabilizing effect of pressure increased with temperature as well as pressure, reaching 36-fold for rGDHt at 105 °C and 750 atm, the largest pressure-induced thermostabilization of an enzyme reported to date. Stabilization of both native GDH and rGDHt was also achieved by adding glycerol. Based on the kinetics of thermal inactivation and the known effects of glycerol on protein structure, a mechanism of pressure-induced thermostabilization is proposed.

NAD+ facilitates high-yield reactivation of clostridial glutamate dehydrogenase (GDH) after unfolding in urea. The specificity of this effect has been explored by using analogues and fragments of NAD+. The adenine portion, unlike the nicotinamide portion, is important for reactivation. Alteration in the nicotinamide portion, in acetylpyridine adenine dinucleotide, has little effect, whereas loss of the 6-NH2 substitution on the adenine ring, in 6-deamino NAD, diminishes the effectiveness of the nucleotide in promoting refolding. Also ADP-ribose, lacking nicotinamide, promotes reactivation whereas NMN-phosphoribose, lacking the adenine, does not.

Of the smaller fragments, those containing an adenosine moiety, and especially those with one or more phosphate groups, impede the refolding ability of NAD+, and are able to bind to the folding intermediate though unable to facilitate refolding.

These results are interpreted in terms of the known 3D structure for clostridial glutamate dehydrogenase. It is assumed that the refolding intermediate has a more or less fully formed NAD+-binding domain but a partially disordered substrate-binding domain and linking region. Binding of NAD+ or ADP-ribose appears to impose new structural constraints that result in completion of the correct folding of the second domain, allowing association of enzyme molecules to form the native hexamer.

Vaccination of lambs with the membrane-bound (S3) thiol-Sepharose binding protein (TSBP) fraction derived from the gut of Haemonchus contortus confers significant protection against homologous challenge. The S3 TSBP peptide profile is dominated by a major protein of ca. 60 kDa which is strongly recognized by antisera from sheep demonstrably protected following immunization with S3 TSBP. In an attempt to identify this protein, sera from protected lambs were employed to screen a λgt11 cDNA library of the adult parasite and resulted in the isolation of numerous clones encoding a homologue of the mitochondrial enzyme, glutamate dehydrogenase (GDH). GDH enzyme activity was readily demonstrable in S3 TSBP material and immunolocalization studies showed that the enzyme was localized to the cytoplasm of the parasite's gut. Furthermore, the enzyme appeared to be developmentally regulated, with both GDH mRNA and protein expressed almost exclusively during the blood-feeding parasitic stages.

Dry deposition of NH3 and NOx (NO and NO2) can affect plant metabolism at the cellular and whole-plant level. Gaseous pollutants enter the plant mainly through the stomata, and once in the apoplast NH3 dissolves to form NH4+, whereas NO2 dissolves to form NO3− and NO2−. The latter compound can also be formed after exposure to NO. There is evidence that NH3-N and NOx-N can be reversibly stored in the apoplast. Temporary storage might affect processes such as absorption rate, assimilation and re-emission. Once formed, NO3− and NO2− can be reduced, and NH4+ can be assimilated via the normal enzymatic pathways, nitrate reductase (NR), nitrite reductase and the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle. Fumigation with low concentrations of atmospheric NH3 increases in vitro glutamine synthetase activity, but whether this involves both or only one of the GS isoforms is still an open question. There seems to be no correlation between fumigation with low concentrations of NH3 and in vitro GDH activity. The contribution of atmospheric NH3 and NO2 deposition to the N budget of the whole plant has been calculated for various atmospheric pollutant concentrations and relative growth rates (RGRs). It is concluded that at current ambient atmospheric N concentrations the direct impact of gaseous N uptake by foliage on plant growth is generally small.