Histologically validated scoring system for the assessment of hock burn in broilers.

The assessment of bird-based welfare indicators plays an important role in the evaluation of bird welfare. The aim of the study was to histologically validate a visual scoring system for hock burn in broilers and to detect threshold values of a visual score to define welfare-relevant alterations in terms of mild lesions or ulcers of the hock. We collected 200 hocks of 39- to 42-day-old Ross 308 broilers after the slaughter process. Each hock was scored visually ("macro scores" 0-4) and evaluated histologically ("micro scores" 0-3), with high scores representing more severe lesions. Although we found a tendency for higher micro scores with increasing macro scores, an exact allocation of macro to micro scores was not possible. For example, macro score 1 could represent micro scores 1, 2 and 3, whereas macro scores 3 and 4 always represented micro score 3 (ulcer). The conditional probability of certain micro scores for given macro scores was estimated using a multinomial logistic regression model. Ulcer showed the highest probability at macro score 1, whereas mild lesions were not found to have an estimated highest probability at any macro score. The depth of inflammation of hock burn lesions increased with increasing macro scores up to macro score 3 with an average depth of 1019 µm. Visually more severe and deeper lesions were also histologically rated with higher scores. Thus, considering limitations, the herein validated macroscopic assessment scheme for hock burn allows an estimation of histological alterations in hocks of broilers.RESEARCH HIGHLIGHTS Histological validation of a visual assessment scheme for hock burn in broilers.Tendency for higher micro scores with increasing macro scores.Estimation of histological score via macro score possible with limitations.Histological depth of inflammation increased with an increasing macro score.

Determination of body width in brown and white layer pullets by image analyses.

1. Specific legal requirements for keeping pullets are not available in the European Union. However, two of the most important rearing factors for pullets are sufficient perching and feeder space. Both factors represent horizontal space dimensions which derive from the body width of the birds. 2. The body width of two strains of layer pullets (brown (BL) and white (WL) layer pullets) based on the measurement of distances in digital images was conducted on front-view digital photographs of BL and WL pullets taken at 8, 12 and 19 weeks of life. 3. Depending on live weight, age and body position, BL pullets measured an average body width between 10.70 ± 1.10 and 13.96 ± 1.11 cm. The width of WL pullets ranged from 10.30 ± 0.86 to 13.00 ± 1.14 cm. 4. Compared with WL, BL pullets occupied more horizontal space during rearing. Age influenced the body width of BL and WL pullets at the end of rearing. The tested body positions of the pullets did not affect the measured body width. 5. The biometric data obtained in this study are a useful basis for developing legal requirements for pullets, especially for defining minimum perch width and feeder space allowances.

Spatial requirements of poultry assessed by using a colour-contrast method (KobaPlan).

Sufficient floor space is a fundamental precondition for poultry to perform normal behavioural patterns. To calculate and determine stocking densities, it is essential to know the absolute minimum surface area required by any given animal (body space). Additional space is required for characteristic behaviours (behavioural space) and for adequate inter-individual distances, group sizes and room to perform social interactions have to be taken into account. To calculate body space, planimetric measurements were carried out by the colour contrast method "KobaPlan" in various poultry species in standing and sitting positions and at a number of different ages. They included laying hens (Lohmann brown (LB), Lohmann selected Leghorn (LSL)), broiler breeders (Ross, both genders), broiler chickens (Ross 308, both genders), turkeys (BUT 6, males), Peking ducks (Cherry Valley, both genders) and Muscovy ducks (Canedins R51, males). Depending on live weight, age, plumage condition and body position, LB hens occupied an average area between 401 cm(2) and 542 cm(2), LSL hens between 353 cm(2) and 445 cm(2), broiler breeder females between 440 cm(2) and 537 cm(2), broiler breeder males 623 cm(2) up to 945 cm(2), broiler chickens up to 434 cm(2), male fattening turkeys up to 1808 cm(2), Muscovy drakes up to 873 cm(2) and Peking ducks up to 627 cm(2). The values can be regarded as necessary minimum spatial requirements for the measured poultry species and genotype. The current method offers the potential to record the area occupied by animals exhibiting species-specific behavioural patterns.

Use of an elevated platform with perforated surface and manure belt by fast-growing broilers on commercial farms.

Like other members of the species Gallus gallus, fast-growing broilers are motivated to perch. However, broilers in the European Union are kept in unstructured barns, with no opportunity to sit elevated and rest undisturbed. A possible solution to this problem is elevated platforms. The aim of this study was to evaluate the use of an elevated platform with perforated surface and manure belt by fast-growing broilers. On 2 commercial farms, an elevated platform was installed in 1 barn per farm. Approximately 35,000 Ross 308 broilers were housed in each barn for 3 fattening periods. On 1 d per wk, the number of broilers per m² on the platform and the ramp was determined every 30 min from video recordings. Besides, focal animals were observed to analyze their behavior on the platform at different ages and during light and dark periods. Broilers used the elevated platform and the ramp from the first week until the end of the fattening period (platform: 9.92 broilers per m², ramp: 6.47 broilers per m²), with a peak in the fourth week of life (platform: 13.00 broilers per m²). In wk 2, 4, and 5, platform use was higher during the light period than during the dark period. Broilers stayed longer on the platform in the dark period (dark: 01:54:23 [hh:mm:ss], light: 00:19:54 [hh:mm:ss]). In every phase of the fattening period, broilers on the platform were inactive to a high proportion (on average 80.60%). This indicates that broilers used the platform also for resting behavior. Thus, the elevated platform with perforated surface and manure belt is a suitable option to structure broiler barns. It allows the broilers to sit elevated and provides additional space. Factors such as a shallow ramp incline of 20°, wide ramps, and appropriate material used for the surface and ramps may have contributed to its high use by broilers of all ages. Further research is needed to evaluate the design of platforms that allow broilers to rest undisturbed.

Influence of environmental enrichment on circulating white blood cell counts and behavior of female turkeys.

Under commercial conditions turkeys are housed in large groups in poorly structured environments. This leads to stress and subsequently to pecking and cannibalism. Environmental enrichment is suggested to reduce stress and feather pecking, thus leading to an increase of the overall flock health. However, the effect of increasing age on the use of enrichment elements and on the behavior repertoire as well as its correlation with health parameters has scarcely been studied. Therefore, our objective was to investigate the influence of environmental enrichment on the behavioral repertoire and on health parameters of turkeys. In 3 consecutive trials, female turkeys were housed up to 12 wk either in an unstructured (control group) or enriched environment (EE group) featuring elevated plateaus at different levels ("turkey tree"). Behavior parameters, clinical health, and immune parameters were determined at selected time points. The percentage of birds using the turkey tree increased with age up to 55 to 77% at 22 to 30 d post hatch (dph). Thereafter, the number of birds located on the turkey tree decreased to 25 to 32% at 73 to 79 dph. Feather pecking and fighting was significantly lower in the EE group compared to the control group in 2 and 3 trials, respectively (P < 0.05). The integrity of feathers and integument, scored in the head/neck, wing, and tail regions was repeatedly better in the EE birds compared to control birds at most investigated time points (P < 0.05), suggesting a reduction in stress related aggression by the use of the turkey tree. Head pecking, running and flying activity, foraging, and preening were overall comparable between the EE and the control group (P > 0.05). Humoral immunity as determined by vaccination-induced anti-Newcastle disease virus antibody titers was not affected by the turkey tree use. The flow cytometric evaluation of blood monocyte and T-lymphocyte numbers showed no repeatable difference between control and EE groups. Interestingly, compared to the control groups, EE birds displayed significantly higher numbers of circulating MHC class II+ lymphocytes and lower numbers of thrombocytes at various time points compared to controls (P < 0.05). This study provides clear evidence that environmental enrichment with plateaus not only leads to an altered behavioral repertoire but also modifies some of the investigated immune parameters, implying that EE may have a modulatory effect on turkeys' immunity and overall fitness. Further studies are needed to understand the correlation between behavior and health parameters in birds more closely.

Corticosterone in feathers of laying hens: an assay validation for evidence-based assessment of animal welfare.

Studies indicate that the evaluation of animal welfare in birds may be carried out with the measurement of the stress-related hormone corticosterone in feathers. However a standardized procedure for corticosterone measurements in feathers is lacking, a validation needs to be carried out for each new species before implementation. The aim of the present study was to establish a valid method to measure corticosterone concentrations in feathers of laying hens in a precise and repeatable manner using an established and commercially available ELISA. Validation was performed with feather pools of tail and interscapular feathers of commercial Lohmann Brown laying hens. Assessment groups, consisting of 5 replicates, were created. All replicates of an assessment group were processed at the same time. Each replicate was run in 4 repetitions by ELISA. Intra-assay and interassay CV was 7.5 and 6.4%, respectively. The serial dilution showed linearity and parallelism. Examining the hormone extraction efficiency by using different methanol volumes resulted in no statistical differences (P > 0.05). Pulverized feathers showed higher corticosterone values than minced feathers (P > 0.05). Differences were shown between 2 feather types (tail vs. interscapular feathers; P < 0.05), as well as between vane and rachis (P < 0.05). Performance of a freeze-thaw cycle led to a decrease of corticosterone concentrations in the samples. A possible effect of UV-A radiation on the stability of corticosterone in the feathers was not found (P > 0.05). With the present study, a valid protocol, feasible for analyzing feather pools of laying hens, was developed. It may provide fundamentals for further investigations on corticosterone in feathers as a noninvasive indicator to evaluate aspects of animal welfare.

Effects of a partially perforated flooring system on animal-based welfare indicators in broiler housing.

Alternative flooring designs in broiler housing have been the subject of intensive research. Research comparing different floor types with a focus on animal-based welfare indicators might be of special interest to meet the animal's needs. This case-control study investigated the effect of a partially perforated vs. a littered flooring system on health- and behavior-based welfare indicators of fast-growing Ross 308 broilers. Furthermore, production performance was assessed. The experimental barn was partially (50%) equipped with a perforated floor directly underneath the feeders and water lines accessible by perforated ramps. Conventional wood shavings were used in the control barn, as usual in practice. There were 4 fattening periods (repetitions) of 31 to 32 D performed with 500 animals per barn (final density of 39 kg m-2). Beside the flooring system, management conditions were identical. Health- and behavior-based welfare indicators were assessed weekly. Production performance indicators were measured continuously during animal control. During the avoidance distance test, animals were less fearful on day 21 (P = 0.010) and tended to be less fearful on day 28 (P = 0.083) in the barn with the partially perforated flooring system compared to the littered control barn. More animals around the novel object were also assessed in the barn with the partially perforated flooring system during the novel object test on day 1 (P < 0.001) and a tendency was found on day 28 (P = 0.064). Results showed that the partially perforated flooring system had a positive influence on foot pad dermatitis from day 14 (all P ≤ 0.007) and hock burn on day 28 (P < 0.001). With regard to the production performance, animals showed no differences in final body weight for both floor types. In this study, the partially perforated flooring system had a positive effect on animal health and behavior as indicated by welfare indicators without a reduction in production performance.

Initial Bacterial Adhesion Properties of Anodically Oxidized Ti6Al4V.

This paper reports about the initial interaction of bacteria with anodically oxidized Ti6Al4V for the use as dental implant abutment surfaces. Ti6Al4V samples are anodically oxidized in hydrofluoric acid using different voltages. The resulting nanotopographies are characterized by atomic force microscopy, scanning electron microscopy and contact angle measurements. The topographies reach from micro-porous structures with small nanoporosities on top to fully hexagonally aligned nanotubes. For initial bacterial adhesion tests, Escherichia coli and Staphylococcus aureus are used. Samples are incubated for 2 h and afterwards non-adherent cells are washed off. The results of live/dead staining and cell counts are presented. Gram-negative and Gram-positive strains show different behavior in respect to total number of initially adherent cells on different micro/nanotopographies. The observed reduction of adhered microorganisms is mainly based on underlying microporous topographies.

Investigation of the prevalence and severity of foot pad dermatitis at the slaughterhouse in fattening turkeys reared in organic production systems in Germany.

The present study shows the prevalence and severity of foot pad dermatitis (FPD) in turkeys reared in organic production systems assessed at slaughterhouses in Germany. The investigations of altogether 1,860 turkeys of the strains Kelly Broad Breasted Bronze (Kelly BBB; 540 toms, 540 hens) and British United Turkeys (B.U.T.) 6 and the Test Product 7 (TP 7; 780 hens) showed that 97.7% of the examined turkeys were diagnosed with different degrees of FPD. Only 4.6% of the toms and 1.3% of the hens had feet without lesions. Most frequent were necrotic lesions measuring up to 2 cm in diameter (64.3% of all turkeys). Extensive necrotic lesions of the foot pads (toms: 29.8%; hens: 12.4%) and necrosis of superficial scales (toms: 11.3%; hens: 7.6%) were less frequent. Plantar abscesses were rare findings (1.9%). In general, the feet of the Kelly BBB hens were more affected by foot pad lesions than those of the Kelly BBB toms. There were significant differences between the investigated flocks concerning the occurrence of foot pad lesions. The aim in rearing turkeys must be the reduction of FPD.

Aldosterone receptors and the evaluation of plasma mineralocorticoid activity in normal and hypertensive states.

Aldosterone receptors from rat kidney slices were utilized in a competitive binding technique to analyze the contribution of various steroids to plasma "mineralocorticoid" activity and to assess their possible role in hypertension. To consider simultaneously the plasma binding, steroids were incubated with slices in undiluted plasma; competitor activities for [3H]aldosterone binding were aldosterone, 100%; deoxycorticosterone, 16.2%; cortisol, 0.4%; and 18-hydroxy-deoxy-corticosterone and d18-hydroxy-corticosterone, 0.1%. These steroids were more active in buffer than plasma, suggesting that they bind to plasma and that this reduces their receptor binding. Analysis of the competition data suggests that at normal plasma concentrations, aldosterone occupies the receptors to a major extent, cortisol occupies some of the receptors, and deoxycorticosterone and 8-hydroxydeoxycorticosterone contribute little to receptor occupancy. Two steroids implicated in low-renin essential hypertension, 16beta-hydroxy-dehydro-epiandrosterone and 16-oxoandrostenediol, did not have significant competitor activity. Competitor activity in plasmas from normal subjects taken at 12 noon (upright) was greater than that in those taken at 8 a.m. (supine). Since the 12 noon samples had higher aldosterone and lower cortisol levels than the 8 a.m. samples, the competitor activity under these physiological circumstances reflects aldosterone more than cortisol. The competitor activities of plasmas from patients relative to normal subjects (100+/-12.1%; mean+/-SEM) were: normal renin "essential" hypertension, 117+/-14%; low-renin essential hypertension, 101+/-6.6%; and primary aldosteronism, 176+/-14.3%. Thus a significant increase in activity of steroids that interact with mineralocorticoid receptors was detected in primary aldosteronism (P LESS THAN 0.01) BUT WAS NOT DETECTED IN LOW-RENIN OR NORMAL-RENIN ESSENTIAL HYPERTENSION.

Ras pathway activates epithelial Na+ channel and decreases its surface expression in Xenopus oocytes.

The small G protein K-Ras2A is rapidly induced by aldosterone in A6 epithelia. In these Xenopus sodium reabsorbing cells, aldosterone rapidly activates preexisting epithelial Na+ channels (XENaC) via a transcriptionally mediated mechanism. In the Xenopus oocytes expression system, we tested whether the K-Ras2A pathway impacts on XENaC activity by expressing XENaC alone or together with XK-Ras2A rendered constitutively active (XK-Ras2AG12V). As a second control, XENaC-expressing oocytes were treated with progesterone, a sex steroid that induces maturation of the oocytes similarly to activated Ras. Progesterone or XK-Ras2AG12V led to oocyte maturation characterized by a decrease in surface area and endogenous Na+ pump function. In both conditions, the surface expression of exogenous XENaC's was also decreased; however, in comparison with progesterone-treated oocytes, XK-ras2AG12V-coinjected oocytes expressed a fivefold higher XENaC-mediated macroscopic Na+ current that was as high as that of control oocytes. Thus, the Na+ current per surface-expressed XENaC was increased by XK-Ras2AG12V. The chemical driving force for Na+ influx was not changed, suggesting that XK-Ras2AG12V increased the mean activity of XENaCs at the oocyte surface. These observations raise the possibility that XK-Ras2A, which is the first regulatory protein known to be transcriptionally induced by aldosterone, could play a role in the control of XENaC function in aldosterone target cells.

[Pathological and histopathological findings in the joints of fattening turkeys]. / Pathologische und histo-pathologische Befunde an Gelenken von Mastputen.

This paper reports on pathological and histopathological findings in the joints of male turkeys (B.U.T. Big 6) reared in two different housing systems. Pathological investigations were conducted on all animals which died between the 6th week of life and the end of fattening (585 birds, 1170 hip, knee and intertarsal joints) from four fattening periods with 3000 birds each. This study included 297 birds kept in a conventional housing system (barn 2) with a stocking density of 2.8 birds/m2, and 288 birds from a barn (1) from which the animals had access to an outdoor scratching area (stocking density 2.5 birds/m2). In all, 157 (26.5%) of the 585 fallen turkeys (barn 1:93; barn 2:70) showed at least one pathological joint alteration. and there were macroscopically detectable changes in 274 (7.8%) of the 3510 investigated joints: 152 (7.8%) of the 1728 joints from barn 1, and 122 (8.8%) of the 1782 from barn 2. In both housing systems the most frequent pathological alterations were in the intertarsal joint affecting 16% (188) of these 1170 joints (barn 1:106; barn 2:82); next most frequent were changes in the knee joint with 4.5% (53) of the 1170 joints affected (barn 1:26; barn 2:27). In both barns, synovial alterations occurred predominantly in the intertarsal and the knee joints. Bacteriological investigation of altered synovial fluids (83) most frequently showed an infection with Staphylococcus spp. (44) and E. coli (15). Macroscopic investigation showed alterations in 31 (2.7%) of the 1170 hip joints (barn 1:19; barn 2:12); 2% (22) of these showed cartilage degenerations of the femoral head (barn 1:15; barn 2:7). Histological investigation of these cartilage lesions showed that they were predominantly (70%) due to osteochondrosis with complete epiphyseolysis affecting 16 femoral heads (barn 1:10; barn 2:6). Partial epiphysiolysis was found in 24% (5) of the femoral heads (barn 1:4; barn 2:1). Furthermore, 43% (9) of the 22 femoral heads with cartilage degeneration also had osteomyelitis and osteochondritis, as well (barn 1:6; barn 2:3). Histological investigation of the 104 femoral heads with no macroscopic findings (barn 1:56; barn 2:48), revealed osteochondrosis in 11 cases (10.6%) (barn 1:6; barn 2:5). Osteomyelitis and osteochondritis were found in 17 (16.3%) of these femoral heads (barn 1:9; barn 2:8). There were no significant (p < 0.05) differences between the two housing systems in the incidence and frequency of joint alterations. It is to be assumed that these joint alterations were the cause of acute, chronic pain.

Floor space covered by broiler chickens kept at stocking densities according to Council Directive 2007/43/EC.

It is controversially discussed whether the stocking densities set by the EU Directive 2007/43/EC allow a species-appropriate housing of broiler chickens. To calculate the exact area broilers occupy due to their physical size and shape, planimetric measurements using a colour-contrast method were carried out. In total, 1949 photographs of standing and 1482 of squatting chickens, taken from a top view, were analysed. A computer program counted the pixels representing the previously weighed animal in the photograph and calculated the animal area. The average area covered by chickens with 400 g live weight was 116.64±13.12 cm(2) in a standing and 138.61±12.92 cm(2) in a squatting position. These areas increased linearly as a function of live weight to 452.57±58.89 cm(2) (R(2)=0.90 standing) and 513.54±42.70 cm(2) (R(2)=0.82 squatting) at the end of the study (3200 g live weight). Squatting chickens occupied more space compared with a standing position in most of the tested weight classes (P<0.05). Depending on target weights, stocking densities and body positions, broilers occupied 48.5-77.7 per cent of 1 m(2) Thus, from a physical point of view, simultaneous resting is possible at any stocking density provided by the EU Directive and at common target weights.

Mineralocorticoid receptors in human kidney.

Cytosol binding of [3H]aldosterone was measured after incubation of the hormone at 37 C with slices of human kidney obtained after surgical removal. High affinity [3H]aldosterone binding with an apparent equilibrium dissociation constant (Kd) of approximately 0.5 nM was observed to probably "mineralo-corticoid receptors." [3H]aldosterone binding of lower affinity and higher capacity (probably to "glucocorticoid receptors") was also observed. Binding of other steroids by the mineralocorticoid receptors was determined by competitive analysis using a low concentration of [3H]aldosterone (so that [3H] binding is predominantly by the mineralocorticoid receptors). The binding activities relative to aldosterone (100%) were: deoxycorticosterone, 48%; cortisol, 1.5%; 18-hydroxy-deoxycorticosterone, 1.2%; and 18-hydroxy-corticosterone, 0.2%. The relative sodium-retaining potencies of these steroids in vivo correlate well with their binding activities. These data provide further support to the view that the major high affinity [3H]aldosterone binding is by mineralocorticoid receptors. Two steroids, 16beta-hydroxy-dehydroepiandrosterone and 16-oxo-androstenediol, recently shown to have sodium-retaining activity in the rat, and also implicated in low-renin "essential" hypertension in man, showed no competitive binding activity. The affinity of [3H]aldosterone for binding to these mineralocorticoid receptors and the relative steroid binding activities are similar to the values previously reported in the rat. Thus, human and rat mineralocorticoid receptors appear to be similar in their affinity for aldosterone and their specificity for binding a number of other steroids.

Functional heterodimeric amino acid transporters lacking cysteine residues involved in disulfide bond.

The protein mediating system L amino acid transport, AmAT-L, is a disulfide-linked heterodimer of a permease-related light chain (AmAT-L-lc) and the type II glycoprotein 4F2hc/ CD98. The Schistosoma mansoni protein SPRM1 also heterodimerizes with h4F2hc, inducing amino acid transport with different specificity. In this study, we show that the disulfide bond is formed by heavy chain C109 with a Cys residue located in the second putative extracellular loop of the multi-transmembrane domain light chain (C164 and C137 for XAmAT-L-lc and SPRM1, respectively). The non-covalent interaction of Cys-mutant subunits is not sufficient to allow coimmunoprecipitation, but cell surface expression of the light chains is maintained to a large extent. The non-covalently linked transporters display the same transport characteristics as disulfide bound heterodimers, but the maximal transport rates are reduced by 30-80%.

Value of the cytologic analysis of fine needle aspiration biopsy specimens in the diagnosis of pancreatic carcinomas.

Between 1982 and 1986, 410 preoperative percutaneous fine needle aspiration (FNA) biopsies of the pancreas were performed on 316 patients clinically suspected of having a malignant pancreatic tumor. Of 58 patients with pancreatic carcinomas subsequently confirmed by histologic investigation, the FNA biopsy yielded a cytologically positive diagnosis of carcinoma in 39 cases (67.2%) and suspicious findings in another 5 cases (8.6%). In 14 cases of malignancy (24.1%), the FNA puncture failed to sample material from the tumor; hence, the cytologic evaluation yielded false-negative results. Of 21 patients with inflammatory disorders of the pancreas, cytologically suspicious cells were observed in 5 cases (23.8%); in none of those 5 cases did the histologic examination show any evidence of carcinoma. This indicates that caution should be taken not to cytologically over-diagnose cases of pancreatitis. On the whole, cytology proved to be a valuable method for the diagnosis of pancreatic carcinoma; it provided the highest rate of positive results in comparison with other modern clinical diagnostic methods. Furthermore, cytology may improve the diagnostic results even in those cases with clinically negative or merely suspicious findings. FNA punctures of the pancreas produced no serious complications in this series.

Aldosterone action: induction of p21(ras) and fra-2 and transcription-independent decrease in myc, jun, and fos.

Adrenal steroids induce an increase in transcellular Na+ reabsorption across Xenopus laevis A6 cell epithelia that requires the action of transcriptionally regulated gene products. In a previous study we identified K-ras2 as an aldosterone-upregulated mRNA in A6 epithelia. Here, we show that in vivo injection of aldosterone in Xenopus (2.5 h) increases K-ras2 mRNA specifically in the kidney (2.5-fold) and that in A6 epithelia aldosterone (2.5 h) increases Ras protein synthesis ( approximately 6-fold). Xl-ras, another ras mRNA expressed at a low level in A6 cells, was also induced (2-fold). Aldosterone was shown to regulate the mRNA levels of several transcription factors as well. After 2 h of aldosterone treatment, fra-2 mRNA was upregulated by 130%, whereas c-myc, c-jun, c-fos, and glucocorticoid receptor mRNAs were downregulated by 23-43%. After 16 h, c-fos and GR mRNAs were further decreased, whereas levels of fra-2, c-jun, and c-myc began to return to control levels. Interestingly, the downregulation of the protooncogene mRNAs was independent of transcription. These results support the view that aldosterone exerts complex pleiotropic transcriptional and nontranscriptional actions that involve the regulation of signaling cascade elements (i.e., K-Ras2) as well as that of transcription factors.

Corticosteroid regulation of renal Na,K-ATPase.

The nature and the mechanisms of renal Na,K-ATPase (Na pump) regulation by corticosteroids remain a matter of debate. This is mainly due to the limitations of the experimental approaches, the inhomogeneity of the nephron and the complexity of homeostatic feedback mechanisms. However, 'classical' early and late cellular actions of corticosteroids, which are mineralocorticoid-specific in the collecting duct, can be recognized: The 'early action' is characterized by an increase in the number of active (versus inactive) pumps and/or by a change in their kinetic properties. The 'late action' is characterized by an increase in the number of pumps resulting from, at least in part, an increase in the rate of Na,K-ATPase gene transcription. Sodium delivery into the cells and other elements of the regulatory network play permissive or co-regulatory roles in both early and late actions.

Characterization of early aldosterone-induced RNAs identified in A6 kidney epithelia.

The early aldosterone-induced increase in Na reabsorption across tight epithelia is characterized by a transcription-dependent activation of epithelial Na channels (ENaC) and pumps (Na,K-ATPase). In order to contribute towards the identification of transcriptionally regulated mediators of this process, we first tested mRNAs of proteins previously suggested to be involved. Epithelia were treated for 1 h with 10(-6 )M aldosterone, a concentration which produces a maximal transport response and at which both mineralo- and glucocorticoid receptors are occupied. Northern blot analysis showed no change in mRNAs of trimeric G protein alpha subunits, calmodulin, and mitochondrial energy metabolism proteins, whereas Na,K-ATPase alpha1 and beta1 subunit mRNAs were slightly increased (1.2- to 1.4-fold). In a second approach, we visualized 5000 cDNA bands generated from A6 RNAs by differential display polymerase chain reaction (PCR). After 1 h of aldosterone treatment, approximately 0.5% of these appeared to be regulated. Four cDNA fragments corresponding to early adrenal-steroid-upregulated RNAs (ASURs) were cloned and for two of them cDNAs containing entire coding sequences were isolated by library screening. ASUR4 is the Xenopus laevis homologue of human E16 and rat TA1, a membrane protein structurally related to yeast and prokaryotic permeases, and ASUR5 is the A transcript of Xenopus K-ras2. The rapid inductions of the four ASURs correspond to direct transcriptional effects since they were not inhibited by cycloheximide but were blocked by actinomycin D. The K1/2 values were similar or slightly below those reported for stimulation of Na transport. These characteristics of RNA accumulation and their time courses suggest a possible role of one of these induced RNAs in the mediation of the early effect of aldosterone on Na transport.