Characterization of a female-specific hepatic mitochondrial cytochrome P-450 whose steady-state level is modulated by testosterone

Polyclonal antibody to mitochondrial P-450c27/25 reacted with two proteins of apparent molecular masses of 52 kilodaltons (kDa) and 50 kDa from the female rat liver mitochondrial proteins bound to an omega-octylaminoagarose column. The two proteins were purified to greater than 85% homogeneity by DEAE-Sephacel and hydroxylapatite column chromatography, and both were found to be P-450 as judged by dithionite-reduced CO difference spectra. Both of the P-450 forms required mitochondrial-specific ferredoxin and ferredoxin reductase for in vitro reconstitution of enzyme activities, suggesting that they are mitochondrial forms. The 52-kDa P-450 exhibited the properties of mitochondrial 27/25-hydroxylase with respect to high vitamin D3 25-hydroxylase activity [1.4 nmol (nmol of P-450)-1 min-1] and N-terminal amino acid sequence. The 50-kDa P-450, on the other hand, lacked significant vitamin D3 25-hydroxylase activity, but showed 17 beta-reductase [0.380-0.400 nmol (nmol of P-450)-1 min-1] and 17 beta-oxidase [0.1-0.16 nmol (nmol of P-450)-1 min-1] activities with both androgens and estrogens as substrates. Immunoblot analysis of proteins using a monoclonal antibody specific for P-450c27/25 showed a 2-3-fold higher level of this enzyme in the female liver mitochondria than in the males. Similarly, use of a polyclonal antibody in the immunoblot analysis showed that the 50-kDa P-450 is female-specific. The relative level of P-450c27/25 was reduced significantly in castrated females, while the level of the female-specific 50-kDa P-450 was increased. However, the levels of both enzymes were increased in castrated males.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence complementarity between the 5'-terminal regions of mRNAs for rat mitochondrial cytochrome P-450c27/25 and a growth hormone-inducible serine protease inhibitor. A possible gene overlap.

Recently we reported that a P-450c27/25 cDNA probe hybridizes to two RNA species of about 1.9 and 2.3-2.4 kilobase pairs (kb) in some rat tissues. To understand the molecular relationship between the two mRNAs, we have isolated and characterized a cDNA for the larger, previously uncharacterized 2.3-kb mRNA species. The 2.3-kb cDNA is identical to the previously reported 1.9-kb P-450c27/25 cDNA excepting a 400-nucleotide-long 5' extension. The terminal 291 nucleotides of this extension exhibit 100% complementarity with the 5'-translated region of the mRNA belonging to a family of growth hormone-inducible serine protease inhibitors (SPI). Northern blot analysis, using strand-specific probes, and S1 nuclease protection revealed the presence of the 2.3-kb mRNA exhibiting the sequence characteristics of the larger cDNA. These results were further confirmed by polymerase chain reaction amplification of reverse transcribed RNA. Expression of the 2.3-kb cDNA in COS cells resulted in the correct mitochondrial targeting of a 52-kDa protein exhibiting the properties of P-450c27/25. Furthermore, both the 1.9- and 2.3-kb mRNAs appear to direct the synthesis of a similarly sized 55-kDa precursor protein in a reticulocyte lysate system. Restriction mapping, polymerase chain reaction amplification and partial sequencing of a 25-kb genomic DNA clone suggest the proximal location of the SPI and the P-450c27/25 protein coding regions in the rat genome on either side of a common overlap region. The results also show that the P-450c27/25 mRNAs are regulated by growth hormone in parallel to the SPI mRNAs. These results collectively suggest that a growth hormone-inducible SPI family mRNA and the P-450c27/25 mRNA are encoded by two closely linked, possibly overlapping genes.     

Alleviation of water and osmotic stress-induced changes in nitrogen metabolizing enzymes in <Emphasis Type="Italic">Triticum aestivum</Emphasis> L. cultivars by potassium

Present communication reports laboratory and pot experiments conducted to study the influence of water and osmotic stress on nitrogen uptake and metabolism in two wheat (Triticum aestivum L) cultivars with and without potassium supplementation. Polyethylene glycol 6000-induced osmotic stress/restricted irrigation caused a considerable decline in the activity of nitrate reductase, glutamate synthase, alanine and aspartate aminotransferases, and glutamate dehydrogenase. Potassium considerably improved nitrogen metabolism under normal water supply conditions and also resulted in amelioration of the negative impact of water and osmotic stresses indicating that potassium supplementation can be used as a potential tool for enhancing the nitrogen use efficiency in wheat for exploiting its genetic potential.     

Large scale production of phage antibody libraries using a bioreactor

One of the limitations of the use of phage antibody libraries in high throughput selections is the production of sufficient phage antibody library at the appropriate quality. Here, we successfully adapt a bioreactor-based protocol for the production of phage peptide libraries to the production of phage antibody libraries. The titers obtained in the stirred-tank bioreactor are 4 to 5 times higher than in a standard shake flask procedure, and the quality of the phage antibody library produced is indistinguishable to that produced using standard procedures as assessed by Western blotting and functional selections. Availability of this protocol will facilitate the use of phage antibody libraries in high-throughput scale selections.     

The Yo-Yo intermittent recovery test level 1 is reliable in young high-level soccer players

The aim of the study was to investigate test reliability of the Yo-Yo intermittent recovery test level 1 (YYIR1) in 36 high-level youth soccer players, aged between 13 and 18 years. Players were divided into three age groups (U15, U17 and U19) and completed three YYIR1 in three consecutive weeks. Pairwise comparisons were used to investigate test reliability (for distances and heart rate responses) using technical error (TE), coefficient of variation (CV), intra-class correlation (ICC) and limits of agreement (LOA) with Bland-Altman plots. The mean YYIR1 distances for the U15, U17 and U19 groups were 2024 ± 470 m, 2404 ± 347 m and 2547 ± 337 m, respectively. The results revealed that the TEs varied between 74 and 172 m, CVs between 3.0 and 7.5%, and ICCs between 0.87 and 0.95 across all age groups for the YYIR1 distance. For heart rate responses, the TEs varied between 1 and 6 bpm, CVs between 0.7 and 4.8%, and ICCs between 0.73 and 0.97. The small ratio LOA revealed that any two YYIR1 performances in one week will not differ by more than 9 to 28% due to measurement error. In summary, the YYIR1 performance and the physiological responses have proven to be highly reliable in a sample of Belgian high-level youth soccer players, aged between 13 and 18 years. The demonstrated high level of intermittent endurance capacity in all age groups may be used for comparison of other prospective young soccer players.     

Comparing ambient, air-convection, and fluid-convection heating techniques in treating hypothermic burn patients, a clinical RCT

Background

Hypothermia in burns is common and increases morbidity and mortality. Several methods are available to reach and maintain normal core body temperature, but have not yet been evaluated in critical care for burned patients. Our unit's ordinary technique for controlling body temperature (Bair Hugger^®+ radiator ceiling + bed warmer + Hotline^®) has many drawbacks e.g.; slow and the working environment is hampered. The aim of this study was to compare our ordinary heating technique with newly-developed methods: the Allon?2001 Thermowrap (a temperature regulating water-mattress), and Warmcloud (a temperature regulating air-mattress).

Methods

Ten consecutive burned patients (> 20% total burned surface area and a core temperature < 36.0°C) were included in this prospective, randomised, comparative study. Patients were randomly exposed to 3 heating methods. Each treatment/measuring-cycle lasted for 6 hours. Each heating method was assessed for 2 hours according to a randomised timetable. Core temperature was measured using an indwelling (bladder) thermistor. Paired t-tests were used to assess the significance of differences between the treatments within the patients. ANOVA was used to assess the differences in temperature from the first to the last measurement among all treatments. Three-way ANOVA with the Tukey HSD post hoc test and a repeated measures ANOVA was used in the same manner, but included information about patients and treatment/measuring-cycles to control for potential confounding. Data are presented as mean (SD) and (range). Probabilities of less than 0.05 were accepted as significant.

Results

The mean increase, 1.4 (SD 0.6°C; range 0.6-2.6°C) in core temperature/treatment/measuring-cycle highly significantly favoured the Allon?2001 Thermowrap in contrast to the conventional method 0.2 (0.6)°C (range -1.2 to 1.5°C) and the Warmcloud 0.3 (0.4)°C (range -0.4 to 0.9°C). The procedures for using the Allon?2001 Thermowrap were experienced to be more comfortable and straightforward than the conventional method or the Warmcloud.

Conclusions

The Allon?2001 Thermowrap was more effective than the Warmcloud or the conventional method in controlling patients' temperatures.     

Entomopathogenic Nematodes Are Not an Alternative to Fenamiphos for Management of Plant-Parasitic Nematodes on Golf Courses in Florida

With the cancellation of fenamiphos in the near future, alternative nematode management tactics for plant-parasitic nematodes (PPN) on golf courses need to be identified. The use of entomopathogenic nematodes (EPN) has been suggested as one possible alternative. This paper presents the results of 10 experiments evaluating the efficacy of EPN at managing PPN on turfgrasses and improving turf performance. These experiments were conducted at various locations throughout Florida over the course of a decade. In different experiments, different EPN species were tested against different species of PPN. Separate experiments evaluated multiple rates and applications of EPN, compared different EPN species, and compared single EPN species against multiple species of PPN. In a few trials, EPN were associated with reductions in certain plant-parasite species, but in other trials were associated with increases. In most trials, EPN had no effect on plant parasites. Because EPN were so inconsistent in their results, we conclude that EPN are not acceptable alternatives to fenamiphos by most turf managers in Florida at this time.     

Current versus historical population sizes in vertebrate species with high gene flow: a comparison based on mitochondrial DNA lineages and inbreeding theory for neutral mutations

Using inbreeding theory as applied to neutral alleles inherited maternally, we generate expected probability distributions of times to identity by descent for random pairs of mitochondrial genotypes within a population or within an entire species characterized by high gene flow. For comparisons with these expectations, empirical distributions of times to most recent common ancestry were calculated (by conventional mtDNA clock calibrations) from mtDNA haplotype distances observed within each of three vertebrate species--American eels, hardhead catfish, and redwinged blackbirds. These species were chosen for analysis because census population size in each is currently large and because both genetic and life-history data are consistent with the postulate that historical gene flow within these species has been high. The observed molecular distances among mtDNA lineages were two to three orders of magnitude lower than predicted from census sizes of breeding females, suggesting that rate of mtDNA evolution is decelerated in these species and/or that long-term effective population size is vastly smaller than present-day population size. Several considerations point to the latter possibility as most likely. The genetic structure of any species is greatly influenced by historical demography; even for species that are currently abundant, mtDNA gene lineages appear to have been channeled through fairly small numbers of ancestors.