The new Eurotransplant Kidney Allocation System: report one year after implementation. Eurotransplant International Foundation

BACKGROUND: Upon the availability of a cadaveric donor kidney, a delicate allocation process precedes every transplantation. A remodeled Eurotransplant Kidney Allocation System (ETKAS)-derived from simulation studies-was installed in March 1996. The purpose was to adjust long waiting times and international exchange balances, while aiming at an optimal HLA-mismatch distribution. The new ETKAS consisted of a point-score system that was 100% patient oriented. METHODS: The impact of the new ETKAS on the composition of the waiting list, and the outcome of the allocation procedures during its first year, were evaluated and compared with the results obtained in 1995. RESULTS: The percentage of long-waiting patients and of patients with poorly matchable HLA phenotype increased significantly, from 9% to 19% and from 19% to 29%, respectively. Zero HLA-A-, HLA-B-, HLA-DR-mismatched patients still comprised 23% of the kidney transplant activity. The kidney exchange of the different Eurotransplant countries became balanced within 4 months; this persisted during the rest of the year. Pediatric patients had a high transplantation rate due to an assignment of extra points. The composition of the waiting list showed, after 1 year, fewer long-waiting patients and fewer patients with rare HLA phenotypes. CONCLUSIONS: The new ETKAS was able in its first year to meet the goals set at its introduction. In comparison with the old ETKAS, there was a better trade-off between HLA matching and waiting time. The value of computer simulation studies has been demonstrated impressively in the context of organ allocation.     

Application of polymerase chain reaction for the correlation of Salmonella serovars recovered from greyhound feces with their diet

The polymerase chain reaction was employed to correlate Salmonella serovars isolated from fecal material of greyhounds suffering from gastroenteritis with those isolated from the diet fed to the greyhounds prior to onset of diarrhea. Kennels around the Abilene, Kansas, area were contacted and supplied with materials needed to collect a portion of the diet each day. With the onset of diarrhea, the kennels were instructed to ship the fecal material and diet from the previous 10 days to the laboratory for testing. Forty-one fecal samples and corresponding diets were screened for Salmonella, Clostridium perfringens, Campylobacter jejuni, Staphylococcus aureus, Staphylococcus intermedius, and pathogenic (piliated) Escherichia coli by direct culture using standard procedures. The fecal material was also screened for coronavirus and parvovirus using electron microscopy. Thirty-five "normal" fecal samples were screened for all of the above mentioned microorganisms as a control. In addition, the fecal material was screened for E. coli verotoxins I and II and clostridial enterotoxins. A total of 61 Salmonella isolates were recovered from the 41 samples of feces and diet submitted for testing; 31 were recovered from the feces and 30 from the diet. Four Salmonella isolates were recovered from the normal fecal samples. Results obtained by PCR, plasmid profiles, antigenic analysis, and antibiogram profiles indicated that 16 of the 31 isolates recovered from the fecal material were the same strain as that recovered from the diet.     

Effect of estrous expression on timing and failure of ovulation of Holstein dairy cows using automated activity monitors

The aim of this study was to determine if estrous expression, as measured by an automated activity monitor (AAM), affects timing and failure of ovulation of lactating Holstein dairy cows. Cows were equipped with 2 AAM, 1 neck-mounted (AAM_C) and 1 leg-mounted (AAM_L), by 10 d postpartum and enrolled into the trial when their activity crossed the alert threshold on the AAM_C. A total of 850 episodes of estrus from 293 different cows were used for this study. When cows were enrolled, their ovaries were scanned by transrectal ultrasonography and gait and body condition scored. Ovaries of cows detected in estrus were scanned twice daily for a maximum of 3 d to determine the disappearance of the preovulatory follicle (ovulation) and the interval from estrus to ovulation was calculated. Physical activity data recorded from the AAM were used to determine estrus behavior using 2 traits: (1) peak activity and (2) duration. Peak activity was only available for the AAM_L. Peak activity was defined as the maximum activity during an estrus episode. Duration of estrus was defined as the time the activity of the cow exceeded threshold values set by the AAM software. The AAM_C correctly identified 87.8% of the estrus alerts, with 12.2% false positives. The average (±standard deviation) intervals from activity alert to ovulation were 25.8 ± 10.2 and 24.7 ± 9.3 h for the AAM_C and AAM_L, respectively. Changes in estrous expression were associated with differences in the interval from alert to ovulation. Cows with short intervals to ovulation were found to have less intense estrous expression than cows with medium and long length intervals to ovulation using the AAM_C, whereas using the AAM_L, cows with short intervals to ovulation exhibited less intense estrous expression than cows with medium but the same as those with long intervals to ovulation. Furthermore, irrespective of the AAM, estrus events with less estrous expression had increased odds of having a short interval to ovulation (below the median of 20 h) when compared with those having greater estrous expression (2.6 and 1.9 increased odds for the AAM_C and AAM_L, respectively). Ovulation failure was affected by estrous expression because estrus events with greater peak activity or longer duration had reduced ovulation failure compared with those with less estrous expression (AAM_C peak activity: 1.9 ± 1.4 vs. 9.5 ± 1.7%; AAM_L peak activity: 2.3 ± 1.4 vs. 6.2 ± 1.5%; AAM_C duration: 2.1 ± 1.4 vs. 8.9 ± 1.7%). In addition, cows with more estrous expression had greater pregnancy per artificial insemination than those with less estrous expression with both the AAM_C (42.3 ± 0.4 vs. 31.7 ± 0.4%) and the AAM_L (43.1 ± 0.4 vs. 36.3 ± 0.4%). Pregnancy per artificial insemination results were consistent even when removing cows that failed to ovulate. In conclusion, expression of estrus was highly associated with ovulation timing, ovulation failure, and fertility when using 2 different AAM. Cows with greater estrous expression have longer intervals from activity alert to ovulation, experience less ovulation failure, and have greater pregnancy per artificial insemination.     

High resolution footprinting of a type I methyltransferase reveals a large structural distortion within the DNA recognition site

The type I DNA methyltransferase M.EcoR124I is a multi-subunit enzyme that binds to the sequence GAAN6RTCG, transferring a methyl group from S-adenosyl methionine to a specific adenine on each DNA strand. We have investigated the protein-DNA interactions in the complex by DNase I and hydroxyl radical footprinting. The DNase I footprint is unusually large: the protein protects the DNA on both strands for at least two complete turns of the helix, indicating that the enzyme completely encloses the DNA in the complex. The higher resolution hydroxyl radical probe shows a smaller, but still extensive, 18 bp footprint encompassing the recognition site. Within this region, however, there is a remarkably hyper-reactive site on each strand. The two sites of enhanced cleavage are co-incident with the two adenines that are the target bases for methylation, showing that the DNA is both accessible and highly distorted at these sites. The hydroxyl radical footprint is unaffected by the presence of the cofactor S-adenosyl methionine, showing that the distorted DNA structure induced by M.EcoR124I is formed during the initial DNA binding reaction and not as a transient intermediate in the reaction pathway.     

Effects of hypertonia on voltage-gated ion currents in freshly isolated hippocampal neurons, and on synaptic currents in neurons in hippocampal slices

A NAD-dependent mannitol dehydrogenase (MtlD) was purified to homogeneity from P. fluorescens DSM50106 and the N-terminal amino acid sequence was determined. An oligonucleotide deduced from this peptide sequence was used as a probe to isolate the mannitol dehydrogenase gene (mtlD) from a genomic library of P. fluorescens. Nucleotide sequence analysis of a 1.8 kb NruI fragment containing the entire mtlD gene revealed an open reading frame of 1482 bp encoding a protein with a calculated molecular weight of 54.49 kDa. The enzyme shared a high similarity with a mannitol dehydrogenase from Rhodobacter sphaeroides and a putative mannitol dehydrogenase of Saccharomyces cerevisae with an overall identity in amino acid sequence of 44% and 42%, respectively, whereas the similarity to mannitol-1-phosphate dehydrogenases of Escherichia coli or Enterococcus faecalis was only about 23% of identical amino acids. By construction of inducible expression plasmids the specific activity of the mannitol dehydrogenase synthesized in E. coli was increased from 0.02 U (mg protein)(-1) to 10 U (mg protein)(-1). After fusion of six histidine codons to the 3' end of mtlD gene and expression in E. coli active mannitol dehydrogenase could be purified in a two-step procedure by affinity chromatography using a Ni2+ matrix column. The purified enzyme exhibited a specific activity of 46 U (mg protein)(-1) and was shown to be a polyol dehydrogenase with a broad substrate spectrum oxidizing efficiently mannitol, sorbitol and arabitol.     

Intensity of estrus following an estradiol-progesterone-based ovulation synchronization protocol influences fertility outcomes

The objective of this study was to examine the association between increased physical activity at the moment of timed artificial insemination (AI), detected by an automated activity monitor (AAM), and fertility outcomes. This paper also investigated factors affecting estrous expression in general. A total of 1,411 AI events from 1,040 lactating Holstein cows were recorded, averaging 1.3 ± 0.6 (±standard deviation) events per cow. Activity (measured as steps/h) was monitored continuously by a leg-mounted AAM located on the rear leg of the cow. Ovulation was synchronized by a timed AI protocol based on estradiol and progesterone. Ovarian ultrasonography was performed in all cows on d ?11 (AI = d 0) and in a subset of cows on d 0 (n = 588) and d 7 (n = 819) to determine the presence of a corpus luteum and follicles. The body condition score (1 to 5 scale) was assessed on d 0 and a blood sample was collected for progesterone measurement on d 7. Using the AAM, an estrus event was determined when the relative increase (RI) in physical activity of the cow exceeded 100% of the baseline activity. The physical activity was classified as strong RI (≥300% RI), moderate RI (100–300% RI), or no estrus (<100% RI). Milk production was measured daily and averaged between d ?11 and 0. Pregnancy was diagnosed at 32 and 60 d post-AI and pregnancy losses were calculated. The mean RI at estrus was 328.3 ± 132.1%. Cows with strong RI had greater pregnancy per AI than those with moderate RI and those that did not express estrus (35.1 vs. 27.3 vs. 6.2%). When including only cows that successfully ovulated after timed AI, those that displayed strong intensity RI still had greater pregnancy per AI than those with moderate intensity RI or those that did not express estrus (45.1 vs. 34.8 vs. 6.2%). Cows expressing strong RI at timed AI had greater ovulation rates compared with moderate RI and cows that did not express estrus (94.9 vs. 88.2 vs. 49.5%). Furthermore, pregnancy losses were reduced in cows with strong RI compared with cows expressing moderate RI (13.9 vs. 21.7%). Cows with a strong RI at estrus were more likely to have a corpus luteum at the beginning of the protocol and had greater concentration of progesterone 7 d post-AI. Multiparous cows expressed lower RI compared with primiparous cows. Cows with lower body condition score tended to have decreased RI at estrus. No correlation between estrous expression and pre-ovulatory follicle diameter was observed. Also, no correlation was observed between milk production at AI and RI. In conclusion, strong intensity RI of estrus events at timed AI was associated with improved ovulation rates and pregnancy per AI, and reduced pregnancy losses. These results provide further evidence that measurements of estrous expression can be used to predict fertility at the time of AI and possibly be used as a tool to assist decision making strategies of reproduction programs.     

Zeolites in biomedical application: Zn-exchanged clinoptilolite-rich rock as active carrier for antibiotics in anti-acne topical therapy

A clinoptilolite-rich rock was evaluated as inorganic Zn²⁺ releasing carrier for antibiotic erythromycin. The perspective is its use in the topical treatment of acne, a diffused skin pathology, given the efficacy of zinc-erythromycin combination against resistant Propionibacterium strains. The tested rock is an ash-rich epiclastite collected in Northern–Central part of Sardinia island (Italy). ICP chemical analyses of the bulk rock evidenced a composition compatible with topical applications. A 66 wt.% of clinoptilolite content was determined by means of XRD analysis (reference intensity ratio [RIR] technique). EDS chemical analyses of zeolite crystals were performed on polished thin section. The CEC of the rock is 1.45±0.08 meq/g. Using a specific exchange method, the material was previously Na-conditioned then Zn-conditioned. A substantially complete Zn-form was obtained, as demonstrated by AAS analyses. A back-exchange reaction toward Na-form was performed in the same conditions (65 °C in 1 M NaCl solution): zinc release was fast and almost complete (94%). Zn-conditioned powder was then micronized to achieve a volume/surface ratio suitable for a topical therapy. After micronization, the specific surface area, determined by BET gas adsorption, was 30.2 m²/g, and 92% of the powdered rock was lower than 30 μm in size (measured by a Coulter Counter apparatus); the so-called “volume-surface diameter” was 6.48 μm, compatible with the intended topical application. Zn²⁺ release was measured on micronized rock at 37 °C both in physiologic solution as in 0.05 M KH₂PO₄/Na₂HPO₄ buffer. Also in these conditions, a prompt and significant zinc release was recorded: after 30 min, 68% and 60%, respectively. Erythromycin was charged onto the micronized material using a solvent evaporation method. HPLC determinations showed that 85% of the drug contacted with the carrier was loaded. The simultaneous release of zinc and erythromycin were evaluated in phosphate buffer. Eighty-two percent of the loaded antibiotic was released after 30 min. Zinc exchange is substantially unaffected by the contemporary drug release. The request to file an international patent for this pharmaceutical application has been accepted by the European International Preliminary Examination Office.