The impact of feeding supplemental minerals to sheep on the return of micronutrients to pasture via urine and faeces.

The form (organic versus inorganic) of minerals (Se, Zn, Cu and Mn), supplemented to sheep (Charolais × Suffolk-Mule (mean weight = 57 ± 2.9 kg) at two European industrial doses, on the return of micronutrients to pasture via nutrient partitioning and composition in sheep urine and faeces was investigated. This gave four treatments in total with 6 animals per treatment (n = 24). The form of the supplemented minerals did not influence the excretory partitioning of micronutrients (Se, Zn, Cu and Mn) between urine and faeces, nor on their concentrations in the excreta. The two doses trialed however, may influence the Se flux in the environment through altering the ratios of Se:P and Se:S ratios in the faeces and Se:S ratio in the urine. Administration of the mineral supplements also improved the retention of P in sheep reducing its excretion via urine. Although the concentrations of readily bioavailable micronutrients in the faeces were not affected by the mineral forms, there were differences in the more recalcitrant fractions of Se, Zn and Cu (as inferred via a sequential extraction) in faeces when different forms of supplemental minerals were offered. The potential impact of these differences on micronutrient flux in pasture requires further investigation.

Effect of staining and freezing media on sortability of stallion spermatozoa and their post-thaw viability after sex-sorting and cryopreservation.

Sex-sorted, frozen-thawed stallion spermatozoa remain out of reach of commercial horse breeders because of the low efficiency of the sex-sorting process and unacceptable fertility rates after insemination. Two experiments were designed to test the effects of alternative staining and freezing media to improve the viability of sex-sorted frozen-thawed stallion spermatozoa. Experiment 1 compared two freezing media, INRA 82(®) and a modified lactose-ethylenediaminetetraacetic acid (EDTA), for the cryopreservation of sex-sorted stallion spermatozoa. No significant differences between the two freezing media could be identified, suggesting that both cryodiluents would be suitable for incorporation into a sex-preselection protocol for stallion spermatozoa. Experiment 2 compared Kenney's modified Tyrode's (KMT) and Sperm TALP (Sp-TALP) as the staining and incubation medium for stallion spermatozoa prior to sex-sorting. A significant increase in the percentage of acrosome-reacted spermatozoa occurred after staining and incubation in the clarified Sp-TALP compared with KMT. As no improvements in sorting rates were achieved using Sp-TALP, it was concluded that stallion sorting protocols could include KMT as the staining and incubation medium while either INRA 82(®) or lactose-EDTA could be employed as a cryodiluents.

Vitamin C supplementation in very preterm infants: a randomised controlled trial.

OBJECTIVE: To determine whether regulating vitamin C (ascorbic acid: AA) intake to achieve higher or lower plasma concentrations was associated with improved clinical outcome. DESIGN: A double blind, randomised controlled trial. SETTING: Neonatal intensive care unit at Christchurch Women's Hospital. PATIENTS: Infants with birth weight <1500 g or gestation <32 weeks, admitted to the unit within 48 hours of birth. INTERVENTION: Infants were randomised to one of three protocols with regard to AA supplementation for the first 28 days of life: group LL received low supplementation throughout; group LH received low until day 10 and then high: group HH received high throughout. MAIN OUTCOME MEASURES: Primary outcome measures were oxygen requirement at 28 days and 36 weeks postmenstrual age, total days supplemental oxygen, and retinopathy of prematurity. AA concentrations were measured at study entry (day 2), and days 10, 21, and 28. RESULTS: A total of 119 infants were enrolled over 24 months (mean gestation 28.4 weeks; birth weight 1161 g). Six infants died, and these had significantly higher AA concentrations before randomisation than surviving infants (116 micromol/l (95% confidence interval 90 to 142) v 51 micromol/l (45 to 58), p<0.0001). There were no significant differences in primary outcomes between the groups. However, the proportion of surviving infants with an oxygen requirement at 36 weeks postmenstrual age in group HH (19%) was half that in group LL (41%) (p=0.06). CONCLUSIONS: In a randomised controlled trial, no significant benefits or harmful effects were associated with treatment allocation to higher or lower AA supplementation throughout the first 28 days of life.

Protein carbonyl measurement by a sensitive ELISA method.

We describe a new immunoassay for measuring protein carbonyls as an index of oxidative injury. Protein samples were reacted with dinitrophenylhydrazine then adsorbed to wells of an ELISA plate before probing with a commercial antibody raised against protein-conjugated dinitrophenylhydrazine. The biotin-conjugated primary antibody was then reacted with streptavidin-biotinylated horseradish peroxidase for quantification. The method was calibrated using oxidized albumin and results correlated well with the colorimetric carbonyl assay. The method required only 60 microg protein and was used to analyze the amount of protein carbonyls in plasma and lung aspirate samples. It was sensitive in the 0-2.5 nmol/mg protein range within which clinical samples fell and was linear up to 10 nmol/mg protein. The ELISA method for protein carbonyls is more sensitive and discriminatory than the colorimetric assay and should have wide application for analysing experimental and clinical samples, especially where concentrations are low and where only small amounts of sample are available.

Tetracycline-controlled gene expression in Entamoeba histolytica.

To provide further tools for functional genetics of the protozoan parasite Entamoeba histolytica, we have tested the suitability of the bacterial TN10-encoded tet-repressor/tet-operator system for gene regulation in ameba trophozoites. Expression of the tet-repressor within the ameba was driven by the wild-type endogenous lectin gene promoter from episomal transfected plasmids. Tetracycline-inducible expression of a reporter gene driven by a modified tet-operator-bearing lectin gene promotor was monitored by transient and episomal transfection. Promotor activity was dependent on the position of the tet-operator insertion. Under appropriate conditions, expression of the reporter gene in tet-repressor expressing cells revealed only background levels but was inducible up to 240-fold by the addition of non-toxic amounts of tetracycline reaching full activity within 36 to 48 h. Because of the tight and rapid control by tetracycline, the tet-repressor controlled lectin gene promotor should be a usefull tool for reverse genetic approaches in E. histolytica as well as for recombinant protein expression within this anaerobic organism.

Analysis of the 170-kDa lectin gene promoter of Entamoeba histolytica.

The promoter region driving the gene for the 170-kDa heavy subunit of the Entamoeba histolytica galactose-inhibitable lectin was analysed by transient transfection using the chloramphenicol acetyltransferase gene as reporter. S1 mapping confirmed our previous notion that the promoter is located within a 1.35-kb intergenic sequence preceding the structural lectin gene. Transcripts derived from the chloramphenicol acetyltransferase gene of transfected trophozoites were found to be polyadenylated and the transcriptional start mapped to a position similar to that of the wild-type lectin gene. By deletion analysis the entire promoter was restricted to a fragment covering about 550 bp upstream from the start of transcription. On the other hand, residual promoter activity required a sequence of about 140 bp only, encompassing a newly identified CCAAT-box like element around position -100, as well as the amebic specific TATA-box. This 140-bp fragment as well as a stretch of 15 bp, which is located some 100 nt further upstream, were found to be conserved within the 5' noncoding region of a second E. histolytica lectin gene. Point-mutation analyses indicated that the 15-bp fragment, the likely CCAAT-box, as well as the TATA-box are required for full promoter activity.

Analysis of cDNA expressed sequence tags from Entamoeba histolytica: identification of two highly abundant polyadenylated transcripts with no overt open reading frames.

Upon analysis of 304 expressed sequence tags derived from the protozoan parasite Entamoeba histolytica, a number of novel protein encoding amoeba sequences were isolated. In addition, two unrelated, abundantly expressed transcripts were identified, and designated, ehapt1 and ehapt2. Although these transcripts do not contain any overt open reading frame, both are polyadenylated and together represent about 19% of total polyA+-RNA(11.6% for ehapt1 and 7.5% for ehapt2), thus being the most highly expressed polyA-containing transcripts so far identified in E. histolytica trophozoites. Northern blot and primer extension analyses revealed single-sized transcripts of 0.5 and 0.6 kb for ehapt1 and ehapt2, respectively, and Southern blot analysis suggests that both are encoded by multiple genes, which are distributed throughout the amoeba genome. Comparison between various ehapt1- and ehapt2-derived cDNAs indicated that both transcripts are highly polymorphic. Whereas nucleotide substitutions in ehapt2 are distributed throughout the sequence, variations in ehapt1 are mainly restricted to two regions, one of which comprises a deletion of variable length within an 8 nt tandem repeat unit. At present there is no convincing explanation for the possible role of ehapt1 and ehapt2 in E. histolytica, and analogous sequences have not been described so far for any other organism. Most likely they might represent regulatory RNAs or transcribed transposable elements.

The endothelial surface of large veins of rabbit: scanning electron microscopic observations.

The following veins of the rabbit were fixed by perfusion and studied systematically by scanning electron microscopy: sagittal sinus, confluence of sinuses, external jugular vein, superior vena cava, inferior vena cava, greater saphenous, and femoral veins. One result is that the shape and arrangement of endothelial cells of the veins are obviously influenced by hemodynamic shear forces. Two types of subendothelial fibres were demonstrated: "cross-fibers" which correspond to the circular inner muscle cells of the media, and "longitudinal fibers" which correspond to the intimal meshwork of connective tissue fibers. Regional differences are demonstrated in the occurrence of these fibres. Moreover, five morphologically different venous valve types are observed. The functional significance of these different valve types is not yet known.

[Simultaneous occurrence of tracheobronchopathia osteochondropastica and mucoepidermoid carcinoma]. / Gleichzeitiges Auftreten von Tracheobronchopathia osteochondroplastica und Mucoepidermoidcarcinom.

In a 51 year old male patient with tracheobronchopathia osteochondroplastica a mucodermoid carcinoma of the middle lobe of the lung was diagnosed. Tracheobronchopathia osteochondroplastica is a rare condition which is occasionally diagnosed by fiberoptic bronchoscopy. More frequently, however, it is only found at autopsy. Mucoepidermoid carcinomas derive from the glands of the bronchial tree and are divided in low-grade and high-grade tumors. The tumor should be locally resected, adjuvant radio- or chemotherapy is ineffective.