An amino acid substitution in biobreeding rat corticosteroid binding globulin results in reduced steroid binding affinity

BioBreeding (BB) rats are derived from an outbred colony of Wistar rats and are used as a model of autoimmune diabetes mellitus. A corticosteroid binding globulin (CBG) variant with reduced affinity for glucocorticoids has now been found in the blood of these animals. The dissociation rate constants of BB CBG for cortisol (4.42 nM) and corticosterone (1.43 nM) are both about 50% higher than those associated with Wistar CBG, but no obvious difference in the steroid binding specificity of BB and Wistar CBGs was detected. Purified BB and Wistar CBGs exhibit the same size heterogeneity when examined by polyacrylamide gel electrophoresis under denaturing conditions, and the sizes of their respective hepatic mRNAs are identical. The genetic basis for this abnormality was therefore determined by comparing the cDNA sequences for BB and Wistar CBG, and this revealed a point mutation that results in a single amino acid substitution at residue 276 (Ile in BB CBG and Met in Wistar CBG). To confirm that this mutation is responsible for the reduced steroid binding affinity associated with BB CBG, the cDNAs for rat CBG-Ile276 and CBG-Met276 were expressed in Chinese hamster ovary cells. The steroid binding affinities of the CBGs secreted by these cells were essentially identical with those observed in the corresponding serum samples from these two rat strains. The amino acid substitution identified in BB rat CBG therefore clearly accounts for the reduction in its steroid binding affinity, and further analysis of this and other natural CBG variants may reveal important information about the CBG steroid binding site. It is also possible that this mutation may contribute to the etiology of pathological abnormalities that are characteristic of the BB rat.     

Network-based Phenome-Genome Association Prediction by Bi-Random Walk

MotivationThe availability of ontologies and systematic documentations of phenotypes and their genetic associations has enabled large-scale network-based global analyses of the association between the complete collection of phenotypes (phenome) and genes. To provide a fundamental understanding of how the network information is relevant to phenotype-gene associations, we analyze the circular bigraphs (CBGs) in OMIM human disease phenotype-gene association network and MGI mouse phentoype-gene association network, and introduce a bi-random walk (BiRW) algorithm to capture the CBG patterns in the networks for unveiling human and mouse phenome-genome association. BiRW performs separate random walk simultaneously on gene interaction network and phenotype similarity network to explore gene paths and phenotype paths in CBGs of different sizes to summarize their associations as predictions.ResultsThe analysis of both OMIM and MGI associations revealed that majority of the phenotype-gene associations are covered by CBG patterns of small path lengths, and there is a clear correlation between the CBG coverage and the predictability of the phenotype-gene associations. In the experiments on recovering known associations in cross-validations on human disease phenotypes and mouse phenotypes, BiRW effectively improved prediction performance over the compared methods. The constructed global human disease phenome-genome association map also revealed interesting new predictions and phenotype-gene modules by disease classes.     

Use of captive bolt as a method of euthanasia in larger laboratory animal species

The penetrative captive bolt pistol was tested on dogs (Canis familiaris) and rabbits (Oryctolagus cunniculi) to assess if it is a humane method of euthanasia to use when a physical method is required. The bolt was placed directly on the skull at the intersection of lines drawn from the lateral canthus of each eye to the opposite ear, and fired. In all cases, consciousness appeared to be lost immediately as evidenced by motoric collapse of the animal and loss of the corneal reflex. The only movement noted appeared to be reflex in nature. In order to determine the time of onset of brain death, the auditory evoked potential (AEP) and electroencephalogram (EEG) were measured in dogs. Within 15 seconds after firing the pistol, organized AEP activity could not be detected above the medulla and EEG activity became isoelectric. These findings suggest that cerebral death occurred almost immediately and, therefore, the captive bolt pistol is a humane method of euthanasia.     

Sex-specific variation in brown-headed cowbird immunity following acute stress: a mechanistic approach

There is some discrepancy in the literature regarding whether acute stress is immunostimulatory or immunosuppressive. Studies of domesticated (laboratory and food) animals and humans typically indicate that acute stress is immunostimulatory, whereas studies of non-domesticated species document both immunostimulatory and immunosuppressive results. Few studies have examined the mechanisms responsible for changes in immune activity in species other than those classically used in laboratory research. We examined the effect of both acute stress and exogenous corticosterone (CORT) on the bactericidal capacity (BC) of blood plasma from captive, wild-caught brown-headed cowbirds (Molothrus ater) to determine if CORT is responsible for changes in levels of immune activity. We conducted "stress tests" in which we handled birds to elicit a stress response and then measured the birds' total CORT and BC at 30 or 90 min post-stressor. We also conducted non-invasive tests in which we administered exogenous CORT by injecting it into mealworms that were fed to the cowbirds remotely. Total, free, and bound CORT levels, corticosteroid binding globulins (CBGs), and BC at 7 or 90 min post-mealworm ingestion were measured. Both males and females exhibited significant increases in total CORT following handling stress and the administration of exogenous CORT. Experimental males and females also exhibited a significant increase in CBG capacity at 7 min post-mealworm ingestion compared to controls. Male cowbirds exhibited a significant decline in their BC following both handling stress and the administration of exogenous CORT whereas female cowbirds exhibited no decline under either condition. Female CBG levels were not different than those of males, suggesting that differences in BC could be due to differences between the sexes in the number of corticosteroid receptors which, along with CBGs, regulate the stress response. Female cowbirds may modulate their stress response as an adaptive life-history strategy for maximizing current reproduction.     

Killing of experimental rabbits with captive bolt guns according to animal welfare regulations]

Captive bolt guns for small animals could be used for killing rabbits quickly with little alterations of tissue caused by agony. The application is acceptable for animal welfare if the captive bolt gun is placed on the head between the base of the ears and the rabbit is bled dry by cutting a large artery immediately after the shooting.     

Rat corticosteroid binding globulin: primary structure and messenger ribonucleic acid levels in the liver under different physiological conditions

Rat corticosteroid binding globulin (CBG) cDNAs were isolated from a lambda gt11 liver cDNA library. When rat hepatic mRNA was hybrid selected and translated in vitro, a major product reacted with antibodies against rat CBG and its Mr (approximately 43,000) was consistent with a nonglycosylated, CBG precursor polypeptide. Two overlapping cDNAs produced a 1,432 nucleotide sequence with an open reading frame comprising 396 amino acids. This includes a potential signal peptide of 22 residues followed by the amino terminus of purified rat CBG. Rat CBG therefore contains 374 amino acids (Mr = 42,196), and has six consensus sites for N-glycosylation. There is 60% identity in the primary structures of rat and human CBG over 383 residues that comprise the human sequence. Furthermore, the single cysteine in rat CBG corresponds to one of two cysteines in human CBG, and this may be significant because a cysteine is located in the human CBG steroid binding site. Northern analysis of RNA from various rat tissues revealed an approximate 1.8 kilobase CBG mRNA only in the liver. Its relative abundance in a pregnant rat was only 30% higher than in an adult female; approximately 3-fold higher than in an adult male, and 25-fold higher than in the fetuses from the same animal. Southern analysis of rat genomic DNA suggests the presence of a single gene for CBG.     

Identification and Confirmation of Differentially Expressed Fucosylated Glycoproteins in the Serum of Ovarian Cancer Patients Using a Lectin Array and LC-MS/MS

In order to discover potential glycoprotein biomarkers in ovarian cancer, we applied a lectin array and Exactag labeling based quantitative glycoproteomics approach. A lectin array strategy was used to detect overall lectin-specific glycosylation changes in serum proteins from patients with ovarian cancer and those with benign conditions. Lectins, which showed significant differential response for fucosylation, were used to extract glycoproteins that had been labeled using isobaric chemical tags. The glycoproteins were then identified and quantified by LC-MS/MS, and five glycoproteins were found to be differentially expressed in the serum of ovarian cancer patients compared to benign diseases. The differentially expressed glycoproteins were further confirmed by lectin-ELISA and ELISA assay. Corticosteroid-binding globulin (CBG), serum amyloid p component (SAP), complement factor B (CFAB), and histidine-rich glycoprotein (HRG) were identified as potential markers for differentiating ovarian cancer from benign diseases or healthy controls. A combination of CBG and HRG (AUC = 0.825) showed comparable performance to CA125 (AUC = 0.829) in differentiating early stage ovarian cancer from healthy controls. The combination of CBG, SAP, and CA125 showed improved performance for distinguishing stage III ovarian cancer from benign diseases compared to CA125 alone. The ability of CBG, SAP, HRG, and CFAB to differentiate the serum of ovarian cancer patients from that of controls was tested using an independent set of samples. Our findings suggest that glycoprotein modifications may be a means to identify novel diagnostic markers for detection of ovarian cancer.     

Interaction of human CBG with cell membranes

Specific binding sites for corticosteroid-binding globulin (CBG) and its pregnancy-associated variant (pCBG), having a modified carbohydrate moiety, were found in the plasma membranes of human liver, decidual endometrium and placental syncytiotrophoblast. The membrane binding was influenced by the conformation of the glycoprotein molecules and structure of their carbohydrate chains. CBG receptor was solubilized from the endometrium membrane and partially characterized. It was found to have a subunit structure, with a homooligomeric sialoglycoprotein consisting of four 20 kDa protomeric species being involved in the recognition of the CBG molecules complexed with progesterone or cortisol. A kinetic study using membrane microvesicles derived from the syncytiotrophoblast brush border revealed that neither CBG nor pCBG restricted cortisol accumulation in the intravesicular space, whereas only normal CBG could penetrate the syncytiotrophoblast membrane. Action of the CBG-cortisol complex on trophoblast cells resulted in the activation of membrane adenylate cyclase and growth of the cAMP accumulation within these cells. Collectively, these findings suggest that both normal CBG and pCBG are involved in the guided transport of steroid hormones to the target cells and transmembrane transfer of hormones and/or hormonal signals.     

Genetic studies of low-abundance human plasma proteins. XII. A new variant of corticosteroid-binding globulin detected by isoelectric focusing/immunoblotting

Thin-layer polyacrylamide gel isoelectric focusing over the pH range 3.5-5 followed by immunoblotting was used to investigate the occurrence and frequency of genetic variation in corticosteroid-binding globulin (CBG). Plasma samples from US Caucasians (n = 105) and US Blacks (n = 106) from Pittsburgh, Pa., Canadian Indians from Vancouver Island (n = 91) and Nigerian Blacks (n = 116) were analyzed. A complex isoprotein pattern was observed in all individuals tested. Reduction of this pattern to a single primary band following neuraminidase treatment indicates that the observed intraindividual variation is due to variation in the number of sialic acid residues associated with CBG. The CBG variant pattern consisted of a series of isoprotein bands having the same mobility as the common pattern, and a second series of bands at a more acidic isoelectric point. This pattern is consistent with heterozygosity for a rare CBG allele.     

Disparity in the response of sex steroid-binding protein and corticosteroid-binding globulin to thyroxine in the primate

There is uncertainty regarding the differential effects of thyroid hormone on the circulating levels of sex steroid-binding protein (SBP) and corticosteroid-binding globulin (CBG). Therefore, we studied the effects of elevated thyroxine (T4) on SBP and CBG concentrations in serum in 4 baboon (Papio anubis) infants between 6 and 7 mo of age. The infants were given levothyroxine in increasing doses (25 to 600 micrograms) over a 1-mo period until the peripheral T4 levels were 3-4 times higher than baseline values. Each animal served as its own control. Blood samples were obtained at 2- to 3-day intervals prior to and during treatment. Serum T4 was measured by RIA. SBP and CBG were measured by diethylaminoethyl cellulose filter paper assays. Elevations in serum T4 resulted in a dose-response increase in circulating SBP concentrations with a maximal increase at the 600-micrograms dose of T4 (p less than 0.001). In contrast, significant elevations in peripheral CBG levels occurred with 50 and 100 micrograms of T4 (p less than 0.025 and p less than 0.01, respectively), but decreased thereafter with higher doses of T4. At the 600-micrograms dose of T4, CBG concentrations returned to baseline values. We conclude that in the baboon, T4 increases the circulating levels of SBP in a graded fashion, while the effect on CBG is biphasic.     

An ultrastructural study of sporidium formation during infection of a rhabditid nematode by large gun cells of Haptoglossa heteromorpha

Recently fired gun cells of Haptoglossa heteromorpha, an aplanosporic nematode parasite, were examined ultrastructurally. The everted tubes of the fired cells had penetrated the cuticle of a nematode, and infective sporidia were developing inside the host body. The nematode cuticle was penetrated by the narrow, walled part of the tube below the needle chamber. The lower unwalled part of the tube tail formed the sporidium. The developing sporidium had a multilayered fibrous outer coating and the plasma membrane was separated from the wall in places. Sporidia contained biphasic membrane-bound vesicles that had been generated by the Golgi dictyosome during gun cell development. Immediately following gun cell firing, the nuclear envelope of the sporidium nucleus was not apparent, and the sporidium nucleus contained clusters of electron-dense particles concentrated in the nucleolar region. We compare the structures and organelles found in the mature gun cell with those in the fired cell and attempt to identify the membranous layers around the sporidium.     

Mechanical energy oscillations of two brachiation gaits: measurement and simulation

How do arm‐swinging apes locomote effectively over a variety of speeds? One way to reduce the metabolic energy cost of locomotion is to transfer energy between reversible mechanical modes. In terrestrial animals, at least two transfer mechanisms have been identified: 1) a pendulum‐like mechanism for walking, with exchange between gravitational potential energy and translational kinetic energy, and 2) a spring‐like mechanism for running, where the elastic strain energy of stretched muscle and tendon is largely returned to reaccelerate the animal. At slower speeds, a brachiator will always have at least one limb in contact with the support, similar to the overlap of foot contact in bipedal walking. At faster speeds, brachiators exhibit an aerial phase, similar to that seen in bipedal running. Are there two distinct brachiation gaits even though the animal appears to simply swing beneath its overhead support? If so, are different exchange mechanisms employed? Our kinetic analysis of brachiation in a white‐handed gibbon (Hylobates lar) indicates that brachiation is indeed comprised of two mechanically distinct gaits. At slower speeds in “continuous contact” brachiation, the gibbon utilizes a simple pendulum‐like transfer of mechanical energy within each stride. At faster speeds in “ricochetal” brachiation, translational and rotational kinetic energy are exchanged in a novel “whip‐like” transfer. We propose that brachiators utilize the transfer between translational and rotational kinetic energy to control the dynamics of their swing. This maneuver may allow muscle action at the shoulder to control the transfer and adjust the ballistic portion of the step to meet the requirements for the next hand contact. Am J Phys Anthropol 115:319–326, 2001. © 2001 Wiley‐Liss, Inc.     

Evaluation of the kinetic energy of the torso by magneto-inertial measurement unit during the sit-to-stand movement

Sit-to-stand tests are used in geriatrics as a qualitative issue in order to evaluate motor control and stability. In terms of measured indicators, it is traditionally the duration of the task that is reported, however it appears that the use of the kinetic energy as a new quantitative criterion allows getting a better understanding of musculoskeletal deficits of elderly subjects. The aim of this study was to determine the feasibility to obtain the measure of kinetic energy using magneto-inertial measurement units (MIMU) during sit-to-stand movements at various paces. 26 healthy subjects contributed to this investigation. Measured results were compared to a marker-based motion capture using the correlation coefficient and the normalized root mean square error (nRMSE). nRMSE were below 10% and correlation coefficients were over 0.97. In addition, errors on the mean kinetic energy were also investigated using Bland-Altman 95% limits of agreement (0.63 J–0.77 J), RMSE (0.29 J–0.38 J) and correlation coefficient (0.96–0.98). The results obtained highlighted that the method based on MIMU data could be an alternative to optoelectronic data acquisition to assess the kinetic energy of the torso during the sit-to-stand test, suggesting this method as being a promising alternative to determine kinetic energy during the sit-to-stand movement.     

Hormonal effects on the secretion and glycoform profile of corticosteroid-binding globulin

Corticosteroid-binding globulin (CBG) is a plasma glycoprotein that is primarily synthesized in the liver and binds cortisol and progesterone with high affinity. In this study, a CBG secreting hepatocellular carcinoma derived cell line (HepG2) was used to investigate the hormonal regulation of hepatic CBG synthesis. HepG2 cells were grown for 72 h in 30, 300 and 3000 nM concentrations of estradiol (E₂), testosterone (T), insulin, thyroxin (T₄) and dexamethasone (DMZ) and the secreted CBG quantified by a novel enzyme-linked immunosorbent assay (ELISA). Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was carried out to determine the effects of these hormones on the relative distribution of CBG glycoforms.

Insulin, T₄ and high concentrations of E₂ decreased the secretion of CBG by HepG2 cells (p < 0.05). Ethanol, the solvent used for E₂, T and DMZ, also significantly attenuated CBG secretion. 2D-PAGE resolved 13–14 glycoforms of CBG produced by HepG2 cells. Insulin caused a reduction in the synthesis of more acidic, while T₄ and DMZ decreased the production of more basic CBG glycoforms. Stimulation with E₂ resulted in the synthesis of additional isoforms of increased acidity, which may represent a type of CBG only seen during pregnancy in vivo. Possible physiological implications of these findings are discussed.     

Penetrating Captive Bolt Stunning and Exsanguination of Cattle in Abattoirs

Abattoirs commonly use penetrating captive bolt pistols to stun cattle. Humane slaughter requires that an animal immediately becomes unconscious and does not regain consciousness. In this review we consider the concepts of concussion, unconsciousness, and return to consciousness. We conclude that an animal effectively stunned with a penetrating captive bolt pistol, as indicated by the presence of certain signs and the absence of others, has little possibility of brain function returning. Appropriate monitoring at all stages between stunning and sticking would ensure immediate detection and restunning on those few occasions when brain function was returning. When a captive bolt irreversibly stuns animals, sticking has no role in terms of animal welfare; its only function is to relieve the carcass of blood. With effective initial stunning and subsequent monitoring, specifying a stun-to-stick interval appears unnecessary. Abattoir surveys have indicated that it is possible to approximate, or even actually obtain, 100% successful stunning from a single shot. Furthermore, low prevalence of recovery follows. Thus, penetrating captive bolt stunning can form part of a process for the humane slaughter of cattle. This article outlines an approach to implementing standard operating procedures for checking the effectiveness of captive bolt stunning.     

Penetrating Captive Bolt Stunning and Exsanguination of Cattle in Abattoirs

Abattoirs commonly use penetrating captive bolt pistols to stun cattle. Humane slaughter requires that an animal immediately becomes unconscious and does not regain consciousness. In this review we consider the concepts of concussion, unconsciousness, and return to consciousness. We conclude that an animal effectively stunned with a penetrating captive bolt pistol, as indicated by the presence of certain signs and the absence of others, has little possibility of brain function returning. Appropriate monitoring at all stages between stunning and sticking would ensure immediate detection and restunning on those few occasions when brain function was returning. When a captive bolt irreversibly stuns animals, sticking has no role in terms of animal welfare; its only function is to relieve the carcass of blood. With effective initial stunning and subsequent monitoring, specifying a stun-to-stick interval appears unnecessary. Abattoir surveys have indicated that it is possible to approximate, or even actually obtain, 100% successful stunning from a single shot. Furthermore, low prevalence of recovery follows. Thus, penetrating captive bolt stunning can form part of a process for the humane slaughter of cattle. This article outlines an approach to implementing standard operating procedures for checking the effectiveness of captive bolt stunning.     

降雨能量对东北典型黑土区土壤溅蚀的影响

溅蚀特征研究可揭示溅蚀发生机理,而现有研究大多用溅蚀量来表征溅蚀特征,不能全面准确地反应溅蚀作用过程。为此,基于改进的试验土槽进行室内模拟降雨试验,研究降雨能量对坡面不同方向溅蚀量及溅蚀过程的影响。试验设计包括2种降雨强度(50 mm/h和100 mm/h)和10个降雨能量,其中10个降雨能量是通过2种降雨强度(50 mm/h和100 mm/h)和5个雨滴降落高度(3.5,5.5,7.5,9.5、11.5 m)来实现的。结果表明:在相同降雨强度下,坡面总溅蚀分量均随降雨能量的增加而增大。次降雨坡面溅蚀量均为向下坡最大,其次为侧坡溅蚀量,而向上坡溅蚀量最小。当降雨强度由50mm/h增加至100mm/h时,坡面向上坡溅蚀量增加2.3—5.0倍,向下坡溅蚀量增加1.7—5.1倍,侧坡溅蚀量增加1.9—4.3倍,总溅蚀量增加1.9—4.5倍,净溅蚀量增加1.2—6.4倍。对于不同降雨能量处理,坡面溅蚀率均表现为坡面产流前随降雨历时的增加而递增,产流后迅速达到峰值,之后逐渐减小并趋于稳定。定量分析了各溅蚀分量、总溅蚀量、净溅蚀量与降雨能量的关系,提出了溅蚀发生的降雨能量阈值,发现雨滴溅蚀发生的临界能量为3—6 J m~(-2)mm~(-1),且向上坡溅蚀量,向下坡溅蚀量,净溅蚀量和总溅蚀量皆与降雨能量呈幂函数关系,而侧坡溅蚀量与降雨能量呈二次多项式关系。     

The cortisol response to ACTH in pigs,heritability and influence of corticosteroid-binding globulin

In the search for biological basis of robustness, this study aimed (i) at the determination of the heritability of the cortisol response to ACTH in juvenile pigs, using restricted maximum likelihood methodology applied to a multiple trait animal model, and (ii) at the study of the relationships between basal and stimulated cortisol levels with corticosteroid-binding globulin (CBG), IGF-I and haptoglobin, all important players in glucose metabolism and production traits. At 6 weeks of age, 298 intact male and female piglets from 30 litters (30 dams and 30 boars) were injected with 250 µg ACTH(1–24) (Synacthen). Blood was taken before ACTH injection to measure basal levels of cortisol, glucose, CBG, IGF-I and haptoglobin, and 60 min later to measure stimulated cortisol levels and glucose. Cortisol increased 2.8-fold after ACTH injection, with a high correlation between basal and stimulated levels (phenotypic correlation, r_p=0.539; genetic correlation, r_g=0.938). Post-ACTH cortisol levels were highly heritable (h²=0.684) and could therefore be used for genetic selection of animals with a more reactive hypothalamic–pituitary–adrenocortical axis. CBG binding capacity correlated with cortisol levels measured in basal conditions in males only. No correlation was found between CBG binding capacity and post-ACTH cortisol levels. Basal IGF-I concentration was positively correlated with BW at birth and weaning, and showed a high correlation with CBG binding capacity with a strong sexual dimorphism, the correlation being much higher in males than in females. Basal haptoglobin concentrations were negatively correlated with CBG binding capacity and IGF-I concentrations. Complex relationships were also found between circulating glucose levels and these different variables that have been shown to be related to glucose resistance in humans. These data are therefore valuable for the genetic selection of animals to explore the consequences on production and robustness traits, but also point at pigs as a relevant model to explore the underlying mechanisms of the metabolic syndrome including the contribution of genetic factors.     

Clinical use of unbound plasma cortisol as calculated from total cortisol and corticosteroid-binding globulin

A method to calculate unbound cortisol from total cortisol (measured by competitive protein binding) and CBG (measured by radial immunodiffusion) based on the binding equilibrium has been evaluated. The calculated results (y) correlate well with those (x) obtained by centrifugal ultrafiltration at 37 degrees C (y = 1.04 x - 2.11 ng/ml; r = 0.975; n = 150). The concentration of CBG is similar in normal men (37.7 +/- 3.5 (SD) micrograms/ml; n = 12) and women (39.5 +/- 3.7 (SD) micrograms/ml; n = 7) and shows no diurnal variation, but marked diurnal variation is observed for total cortisol (193.7 +/- 35.0 (SD) ng/ml at 08.00 h vs 43.2 +/- 23.3 (SD) ng/ml at 22.00 h; n = 19) and particularly for unbound cortisol (16.5 +/- 5.6 (SD) ng/ml at 08.00 h vs 2.3 +/- 1.8 (SD) ng/ml at 22.00 h; n = 19). The concentration of CBG (89.1 +/- 11.2 (SD) micrograms/ml) and of total cortisol (395.6 +/- 103.3 (SD) ng/ml at 08.00 h; 110.3 +/- 16.6 (SD) ng/ml at 22.00 h) are clearly elevated in estrogen treated women (n = 11) but unbound cortisol levels (17.2 +/- 7.7 (SD) ng/ml at 08.00 h; 2.5 +/- 0.5 (SD) ng/ml at 22.00 h) are similar to the control group. The concentration of CBG is significantly decreased in patients with Cushing's syndrome (33.2 +/- 5.6 micrograms/ml; n = 17) and unbound cortisol is relatively more elevated than total cortisol in these patients. In adrenal insufficiently CBG is normal, but total and unbound cortisol are markedly decreased. There is a significant decrease of CBG in hyperthyroidism (35.7 +/- 5.5 micrograms/ml; n = 22), in cirrhosis (32.0 +/- 8.0 micrograms/ml; n = 14) and in renal disease and a significant increase in patients treated with antiepileptic drugs (47.5 +/- 6.3 micrograms/ml; n = 14), but total and unbound cortisol are normal in all these conditions. We conclude that unbound cortisol can be calculated in a simple and reliable way from total cortisol and CBG and permits a better evaluation of adrenal function, particularly in patients with altered CBG concentrations.     

A simple method for the purification of murine corticosteroid binding globulin: characterization of a monomer

A fast and reproducible two-step method with high resolution was developed for purification of murine corticosteroid-binding globulin (CBG). The first step was liquid chromatography on a Sephacryl-S-200 column, and the CBG-containing residual was subsequently chromatographed by fast protein liquid chromatography (FPLC). This enabled us to quickly obtain a highly purified protein and the apparently isolated CBG was tested for its purity by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) and sedimentation equilibrium centrifugation. The CBG concentration in pregnant mouse serum was estimated to 0.78 g/l (1.5% of the total protein). The monomeric organization of the protein was demonstrated by mercaptoethanol treatment. No NH2-terminal amino acid could be detected, probably owing to a blocked amino acid. The mol. wt (Mr) of murine CBG was determined to be 52,000 and the sedimentation constant S20 degrees, w to 3.9 S by analytical ultracentrifugation. The protein showed 5 bands when subjected to isoelectric focusing: 3 bands with apparent isoelectric points (pI) between pH 3.15-3.25 and two between pH 3.40-3.50.