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.

Field fertility of sex-sorted and non-sorted frozen-thawed stallion spermatozoa.

In the 2004/2005 breeding season, the fertility of sex-sorted (SS) and non-sorted (NS) frozen stallion spermatozoa from two Hannovarian stallions was compared. A hysteroscopic insemination technique [Morris, L.H., Tiplady, C., Allen, W.R., 2003a. Pregnancy rates in mares after a single fixed time hysteroscopic insemination of low numbers of frozen-thawed spermatozoa onto the uterotubal junction. Equine Vet. J. 35, 197-201] was used to deposit low doses (6, 13 or 25 x 10(6) frozen-thawed SS or NS spermatozoa) onto the utero-tubal junction at 32 or 38 h after the administration of Chorulon (2500 IU, Intervet). Fertility was low, with one pregnancy (13 x 10(6) spermatozoa, 500 microL) obtained after artificial insemination with frozen SS spermatozoa (n=29 cycles) which resulted in the birth of a filly. Two pregnancies were obtained in mares inseminated with 6 x 10(6) NS spermatozoa in 250 microL (n=31 cycles). Mares failing to conceive on two experimental cycles were allocated to the conventional insemination group. Insemination with >500 x 10(6) motile NS frozen-thawed spermatozoa, yielded satisfactory per cycle conception rates (35.5%, 22/62) for both stallions combined and was within the values of their normal fertility as quoted by the stud's records. This suggests that the quality of the frozen semen was acceptable and that the freezing processes yielded viable spermatozoa capable of fertilisation. The poor fertility after hysteroscopic insemination with low doses of sex-sorted or non-sorted spermatozoa from the same stallions may be directly attributable to the low dose insemination conditions with frozen-thawed rather than sex-sorted spermatozoa.

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.

DNA sequences corresponding to the ariel gene family of Entamoeba histolytica are not present in E. dispar.

For the identification of quantitative genetic differences between pathogenic Entamoeba histolytica and the closely related but nonpathogenic species E. dispar, a set of 68 independent probes that had previously been isolated from an E. histolytica cDNA library were hybridized to total genomic DNA of both amoeba species. Besides ehcp5, the sequence that codes for cysteine proteinase 5 and has recently been shown to be missing in E. dispar, only one of the probes exclusively reacted with E. histolytica DNA, whereas the remainder hybridized to DNA of both species. Sequence analysis revealed that the specific probe represents a copy of the multicopy ariel gene family, which has 80% sequence identity to srehp, the gene encoding a serine-rich E. histolytica membrane protein. In contrast to ariel, srehp is present in both amoeba species, suggesting that the ariel gene product might have a particular function in E. histolytica.

Small bowel ischaemia-reperfusion increases plasma concentrations of oxidised proteins in rats.

OBJECTIVES: To find out whether plasma concentrations of protein carbonyl (a specific marker of oxidative damage of proteins) are increased during intestinal ischaemia-reperfusion and whether they are correlated with von Willebrand's factor (vWF, a marker of endothelial injury) or myeloperoxidase (a marker of neutrophil activation). DESIGN: Randomised experimental study. SETTING: University department of surgery, New Zealand. ANIMALS: Thirty anaesthetised adult Wistar rats. INTERVENTIONS: The sham operated group (n = 10) had laparotomy and isolation of the superior mesenteric artery without clamping. The ischaemia-reperfusion group (IR, n = 10) had the superior mesenteric artery clamped for 1 hour and reperfusion for 15 minutes. The control group (n = 10) had direct puncture of the heart to sample blood. MAIN OUTCOME MEASURES: Plasma concentrations of protein carbonyl, vWF, and myeloperoxidase. RESULTS: Plasma protein carbonyl concentrations were significantly higher in the IR group than in the sham group (p < 0.02, Mann-Whitney test, median (range) 0.187 (0.141-0.242) compared with 0.144 (0.121-0.185) nmol/mg) and in the control group (p < 0.01, Mann-Whitney test, median (range) 0.187 (0.141-0.242) compared with 0.136 (0.108-0.175) nmol/mg). There was a significant correlation between protein carbonyl and vWF concentrations (r = 0.54, F = 10.9, p < 0.003, linear regression) but not with those of myeloperoxidase. CONCLUSION: Intestinal ischaemia-reperfusion caused an increase in the plasma protein carbonyl concentration, which is possibly produced by endothelial cells.

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.

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.

[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.

Continuous quality improvement: adaptation of the 10-step model with postanesthesia care unit application.

In the 1986 Agenda for Change the Joint Commission on Accreditation of Health Care Organizations (JCAHO) introduced a systematic conversion changing the blueprint for quality from quality assurance to continuous quality improvement (CQI). CQI is an evolutionary philosophy that incorporates leadership concepts from total quality management. It includes substantial changes regarding quality to be initiated by leaders of health care organizations. Ultimately, a commitment to quality and continuous improvement by all personnel is essential. The JCAHO is facilitating this transition through annual revisions in accreditation standards. CQI standards and indicators focusing on patient processes and outcomes are to be finalized in the 1994 Accreditation Manual for Hospitals. Monitoring and evaluation to assess quality and improvement remains intact as an integral part of CQI. Standards furnish the basis for measuring and assuring quality. The Marker Model provides clear, concise descriptions of all types of standards and their use in assuring quality. The 10-step model is an organized method developed by JCAHO to monitor compliance with standards and to evaluate improvement. The framework for this monitoring model uses a multidisciplinary approach achieved as a result of the effort and expertise of caregivers. The 10-step model, now in transition at Tri-City Medical Center, is described with application to the PACU.

Influence of the lectins and polycation on the configuration of renal podocytes: a scanning electron microscopic study of renal podocytes after micropuncture of the glomerulus in vivo.

The lectins concanavalin A (Con A), wheat germ agglutinin (WGA), Ricinus communis agglutinin (RCA) I+II and the polycation protamine sulfate were applied directly to renal glomerular podocytes by micropuncture techniques in vivo; others received a control solution. To make visible the distribution of lectins, some rats were given fluorescein isothiocyanate-conjugated Con A. The glomeruli undergoing the micropuncture experiments were labeled and then prepared for SEM and TEM observation, in some cases also for histochemical analysis. Comparatively, the effect of application of the Con A and protamine sulfate solution by intraarterial infusion was studied. The glomeruli of a total of 100 Munich-Wistar rats were studied. Con A and WGA cause varying degrees of 'retraction' of the foot processes of the podocytes when applied using the techniques of micropuncture. Intraarterial infusion of a Con A solution, on the other hand, causes no changes in the podocytes. RCA II, applied for 10 min using micropuncture techniques, causes thickening and swelling of the foot processes as well as the formation of intercellular junctions ('agglutination'). RCA I, on the other hand, causes no changes in the podocytes of the rat glomerulus. Glomeruli treated with the micropuncture application of the polycation protamine sulfate demonstrate largely 'agglutination' and only sometimes localized minimal retraction of the foot processes of the podocytes. The intraarterial infusion of protamine sulfate causes almost exclusively 'agglutination' of the podocyte foot processes. Retraction of the podocyte foot processes is probably a result of the active movement of the podocytes, which in turn induced by attachment of lectines to the lectin receptors in glycocalyx of the podocyte cell membrane. Simple reduction of the polyanions in the podocyte cell membrane by protamine sulfate appears to cause only simple electrostatic interaction which then results in 'agglutination' of the podocyte foot processes.

Application of scanning electron microscopy to diagnostic pathology. A critical review.

A survey with critical comment of the present state of SEM knowledge is given for those internal organs that are most important for the surgical and anatomical pathologist. With the exception of microanalysis, today SEM does not aid diagnosis by the pathologist because the SEM results are, at present, not certain enough to justify their application to a diagnosis with clinical consequences where the same results can be obtained by the accustomed LM and TEM with their respective adjuvant techniques like histochemistry or immune histology. In the future, SEM will only be established as an essential diagnostic tool when SEM reveals hitherto unknown morphological patterns which can be related to clinical syndromes; some suggestions are made where this can be achieved.

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.