Effect of free fatty acids on the bioavailability of plasma testosterone and dihydrotestosterone

Free fatty acids (FFA) are known to interfere with the binding of thyroid hormone and estrogens to circulating proteins, but their effect on androgen binding is unknown. The effect of linoleic, oleic and palmitic acids at physiological concentrations on the binding of testosterone (T) and dihydrotestosterone (DHT) to circulating proteins was evaluated in vitro, using equilibrium dialysis and ammonium sulfate precipitation techniques. The results indicate that FFA can inhibit T binding to albumin and SHBG. They also can inhibit DHT binding to albumin, whereas DHT binding to SHBG is not altered, suggesting that FFA at physiological concentrations may be important regulators of bioavailability of T to tissues.     

Isolation and characterization of luciferase from Indian firefly,Luciola praeusta

Luciferase from Indian firefly Luciola praeusta (Coleoptera: Lampyridae: Luciolinae) was isolated and the properties compared with that of the North American firefly, Photinus pyralis. Luciola praeusta luciferase was purified using acetone extraction, gel‐filtration column chromatography, ammonium sulfate precipitation and anion exchange chromatography. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis indicates a homogeneous preparation and the molecular mass was slightly higher than that of Photinus pyralis. The effect of pH, buffer composition and metal ions on the spectral characteristics was studied. The maximum bioluminescence activity of luciferase was observed in ACES buffer at pH 6.5. The emission maximum of 562 nm (in crude extract) was red shifted to 570 nm in Tricine buffer at pH 7.8. In addition, the effect of bovine serum albumin on the storage stability of the protein was investigated. Based on the unique spectral characteristics observed, we propose that Luciola praeusta luciferase in the native form is suitable for the assay of biochemical metabolites in acidic pH. Copyright © 2013 John Wiley & Sons, Ltd.     

“Linearity assessment methods for sex steroid hormones and carrier proteins among men in the National Health and Nutrition Examination Survey (NHANES III)”

IntroductionIt has been hypothesized that racial disparities among several diseases are explained by differences in testosterone (T), 17-β estradiol (E), sex hormone binding globulin (SHBG) and albumin (A) levels, yet epidemiologic results have been mixed. Statistical advice regarding appropriate adjustment methods for carrier proteins of sex steroid hormones in the literature is scant. Therefore, we investigated different adjustment methods for carrier proteins.MethodsData for 1496 men, >17 years from the Third National Health and Nutrition Examination Survey (NHANES III) 1988–91 were used to analyze linearity between sex hormones and carrier proteins by examining correlation, plots, and regression models. The statistical importance of age, body mass index (BMI), and race/ethnicity were examined for changes in results by the adjustment method.ResultsT was weakly correlated with SHBG and A (r-squared, 0.25, 0.13, respectively) and E was weakly negatively correlated with A (p < 0.0001), but not SHBG (p < 0.1799). Based on the model residual plots and r-squared, the categorical model performed better than linear models. Regression coefficients for age, BMI, and race/ethnicity groups using quotient (e.g., T/A and E/A) models differed from continuous and categorical models.ConclusionChoosing an appropriate adjustment for carrier proteins is important to prevent bias in analyses and inconsistency in findings across studies. Linearity between variables should not be assumed when adjusting models, and should be conducted and reported. An independent categorical carrier protein variable is recommended in analysis exploring factors predicting sex hormone levels, although statistical testing should always be employed.     

Screening,Purification and Properties of a Specific Antiphage Substance,AFS, against Filamentous Phage of Escherichia coli

We have made screening for antiphage antibiotics using an assay system of a male-specific filamentous phage, fl, and Escherichia coli as its host, and found a proteinous active substance named AFS (anti-filamentous phage substance) which was produced by an Actinomyces belonging to the group of Streptomyces lavendulae. It was purified from the filtered broth of the strain by ammonium sulfate precipitation and chromatographies of SP-Sephadex, CM-Sephadex and Sephadex G–50. The purified preparation was judged to be homogeneous by several column chromatographies and electrophoresis. Its molcular weight was about 5100 and isoelectric point was pH 9.9. Fourty two residues of amino acids, rich in the hydrophobic ones, were contained in one mole of AFS, but no carbohydrate was detected.     

Specific cytosol binding of diethylstilbestrol in rat skeletal muscle

Rat skeletal muscle cytosol proteins bound ³H-diethylstilbestrol (³H-DES). More than 90% of this binding was high capacity and low affinity. Serum albumin accounted for roughly 50–60% of the binding, as evidenced by its precipitation with anti-rat albumin IgG. About half of the binding was distinguishable from albumin and other serum proteins by its precipitation in 40% saturated ammonium sulfate. This material sedimented at 4–5S in high-salt sucrose gradients, and resolved into two components (8S and 4–5S) in low-salt. Following incubation at 23–27°C for one hour, 2% of the bound ³H-DES in whole cytosol (approximately 2 fmole/mg cytosol protein) was retained by DNA-cellulose, and was eluted with 0.6 M KCl. This small fraction of the total binding was inhibited by estrogens and DES analogues: estradiol-17β, DES, dienestrol, and hexestrol were strong inhibitors; isodienestrol, dimethylstilbestrol, estradiol-17α, estrone, tamoxifen, MER-25, CI-628, and nafoxidine were weak inhibitors; dihydrotestosterone, testosterone, and prednisone did not compete. These observations indicate that specific estrogen-binding sites exist in rat skeletal muscle.     

荔枝果皮过氧化酶的纯化与性质研究(英)

荔枝果皮采后褐变是影响这一重要热带水果经济价值的主要问题,酚类物质的酶促氧化一直被认为是造成植物组织褐变的关键因素,其中多酚氧化酶被研究得最多.过氧化物酶在植物体中分布很广,能够氧化多种底物,在荔枝果皮中的含量也很高.非结合性过氧化物酶已经被证明在果实的采后成熟与老化过程中参与多种过程.在这项研究中,用磷酸缓冲液提取荔枝果皮的非结合性过氧化物酶,并通过硫酸铵沉淀,DEAE Sephadex A-50离子交换柱层析以及Sephadex G-100凝胶过滤进行纯化.对得到的酶溶液进行了酶学性质的研究,发现荔枝果皮过氧化物酶具有较高的热稳定性和高的最适反应pH值（6.8）,能够氧化许多底物尤其是单酚和各种多酚类物质,反应抑制剂专一性与其他植物来源的过氧化物酶略有不同.显示了过氧化物酶参与荔枝果皮褐变过程的可能性,并为提高荔枝采后贮藏性提供了新的思路.     

Are There Variances Of Calculated Free Testosterone Attributed To Variations In Albumin And Sex Hormone-Binding Globulin Concentrations In Men?

ObjectiveCalculated free testosterone (cFT) is determined from total testosterone (TT), sex hormone binding globulin (SHBG), and albumin (Alb) levels using mathematical formulae. Variations in cFT due to changes in SHBG or Alb have not been investigated. We evaluated potential cFT variances determined with fixed Alb (4.3 g/dL) and measured Alb, and the point at which low SHBG and Alb combinations produced significant cFT variance.MethodWe analyzed 11,176 data points from 5,797 men. cFT values with fixed versus actual Alb values were evaluated and compared. cFT levels were theoretically determined for all possible combinations of TT, SHBG, and Alb (8,343,552 combinations). Agreement between the 2 measures was assessed with Lin’s concordance coefficient.ResultsMean Alb was 4.06 ± 0.32 g/dL. Mean SHBG was 39.0 ± 23.6 nmol/L. A fixed Alb of 4.3 g/dL did not produce significant variance for most cFT evaluations. Accuracy decreased when Alb was ≤3.5 g/dL in combination with SHBG ≤30 nmol/L, and this occurred in 1.2% of all data points.ConclusionA fixed Alb of 4.3 g/dL is acceptable for most clinical evaluations. If Alb is <3.5 g/dL and SHBG is <30 nmol/L, the variance increases, and a free testosterone (FT) measurement by equilibrium dialysis is warranted for better accuracy. (Endocr Pract. 2013;19:236-242)     

What is pea legumin — Is it glycosylated?

Since there is some question as to whether or not legumin is glycosylated, this storage protein was isolated by various procedures from developing cotyledons of Pisum sativum L. supplied with [¹⁴C]-labeled glucosamine and analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Legumin isolated by the classical method of Danielsson [(1949) Biochem. J. 44, 387–400] a procedure in which globulins extracted with a buffered salt solution are precipitated with ammonium sulfate (70% saturation) and legumin separated from vicilin by isoelectric precipitation, was labeled. The glucosamine incorporated into legumin was associated with low-molecular-weight polypeptides. In contrast, legumin isolated by the method of Casey [(1979) Biochem. J. 177, 509–520], a procedure where legumin is prepared by zonal isoelectric precipitation from globulins precipitated with 40–70% ammonium sulfate, was not labeled. However, the globulin fraction precipitated with 40% ammonium sulfate was labeled and the radioactive glucosamine was associated with low-molecular-weight polypeptides. Legumin isolated from protein bodies [Thomson et al. (1978) Aust. J. Plant Physiol. 5, 263–279] was not extensively labeled. However, the saltinsoluble fraction of protein body extracts was labeled and the radioactivity was associated with low-molecular-weight polypeptides. These results indicate that protein bodies contain a glycoprotein of low-molecular-weight that co-purifies with legumin isolated by the method of Danielsson but that is discarded when isolation methods developed more recently are used.     

Relationship between androgenic hormones and arterial stiffness, based on longitudinal hormone measurements

Circulating testosterone levels (T) decrease with age in men. Low T has been associated with coronary disease and with risk factors for atherosclerosis. This study examines the relationship in men between androgenic hormones and arterial stiffness, a major risk factor for cardiovascular events. T, sex hormone-binding globulin (SHBG), and dehydroepiandrosterone sulfate (DHEAS) were measured longitudinally over 33 yr (follow-up 11.8 +/- 8.3 yr) in 901 men from the Baltimore Longitudinal Study of Aging, of whom 206 (68.1 +/- 13.7 yr) underwent carotid duplex ultrasonography. The 901 men were used to characterize age-associated hormone levels by means of mixed-effects models. Hormone values were estimated for the 206 men at the time of ultrasonography. Free T index (FTI) was calculated by dividing T by SHBG. The arterial stiffness index was calculated from peak systolic and end diastolic diameters of the common carotid artery and simultaneous brachial artery blood pressure. T, FTI, and DHEAS were correlated negatively with age, pulse pressure (PP), and stiffness index (each P < 0.01), whereas SHBG was correlated positively with age and stiffness index (P < 0.01). However, T was the only hormone that predicted the stiffness index after adjustment for age, PP, fasting plasma glucose, body mass index, and total cholesterol. T values 5-10 yr before the carotid study also predicted the stiffness index (P < 0.05). Thus the adverse influence of low T on the cardiovascular system in men may be mediated in part via the effects of T on vascular structure and function.     

Solubilization and partial purification of n,n'-dicyclohexylcarbodiimide-sensitive ATPase from pea cotyledon mitochondria

The N,N′-dicyclohexylcarbodiimide (DCCD)-sensitive ATPase of pea (Pisum sativum L.) cotyledon mitochondria was solubilized from submitochondrial particle membranes with sodium cholate and ammonium sulfate. Ammonium sulfate precipitation of the enzyme resulted in an increase in specific activity. At between 38% and 45% saturated ammonium sulfate, 20% of the ATPase activity was precipitated, with a specific activity 4 to 5 times higher than that of the crude enzyme. The precipitate was highly sensitive to DCCD.

The properties of the ammonium sulfate preparation were investigated. It contained levels of cytochrome and NADH dehydrogenase contamination comparable to those of the highly purified F₀F₁ preparations from animal tissue. The high degree of purification was corroborated by sodium dodecyl sulfate electrophoresis.

     

Studies on a gram-positive hydrogen bacterium,Nocardia opaca 1 b

Nocardia opaca strain 1 b has a NAD-dependent hydrogenase (hydrogen dehydrogenase). The enzyme has been purified from autotrophically grown cells and tested for optimal assay conditions and stability. The purification procedure involved protamine sulfate treatment, ammonium sulfate precipitation, and separation by DEAE-cellulose and Sephadex G-200 chromatography and resulted in a 63-fold increase of specific activity at a 11.7% enzyme recovery. The final specific activity was 103 μmoles H₂/min·mg protein. The purified enzyme was dependent on nickel and magnesium ions at 0.5 and 5.0 mM concentrations, respectively, as well as flavin mononucleotide at a 5–10 μM concentration. Straight enzyme kinetics were achieved by preincubating the enzyme in the presence of NADH₂. A high stability of the enzyme was observed in 0.1 M potassium phosphate buffer, pH 6.5, in the presence of 0.5 mM nickel and 5 mM magnesium ions under hydrogen atmosphere. Even under air the enzyme was remarkably stable, although less than under hydrogen. From double reciprocal plots of substrate saturation curves the Michaelis-Menten constants were calculated: For saturating NAD-concentration the K _m was 0.063 mM H₂ and for saturating hydrogen concentration the K _m was 0.123 mM NAD.     

Isolation of alpha-lactalbumin,beta-lactoglobulin,and bovine serum albumin from cow's milk using gel filtration and anion-exchange chromatography including evaluation of their antigenicity

The aim of this study was to introduce a simple, reproducible, and less expensive method for isolation of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin from cow's milk while retaining their antigenicity. Whey (lactoserum) was obtained by isolating casein from defatted milk using hydrochloric acid. Globulins were then precipitated from whey by half-saturated ammonium sulfate and beta-lactoglobulin was purified further using Sephadex G-50 gel filtration. The proteins in the supernatant were also fractionated using diethylaminoethyl cellulose chromatography in which beta-lactoglobulin was separated from alpha-lactalbumin and bovine serum albumin. The latter two proteins that co-eluted in anion-exchange chromatography were then gently isolated from each other by Sephadex G-50 gel filtration. Pure beta-lactoglobulin was also obtained by anion-exchange chromatography of the ammonium sulfate-precipitated globulins. Using enzyme-linked immunosorbent assay (ELISA), Western blotting, and ELISA inhibition assay, antigenicity of the purified proteins was evaluated. Our results showed high purity and well-preserved antigenicity of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin thus purified.     

Valoración de niveles de testosterona mediante diferentes métodos analíticos en varones sanos

Plasma testosterone concentrations are essential for the diagnosis of several causes of hypogonadism, including late-onset hypogonadism. Defining the normal range for testosterone concentrations poses certain difficulties due to the changes that occur with age and the variability of the different analytical methods used.ObjectivesTo study normal ranges of testosterone in healthy young men and to compare the results of distinct analytical methods.Material and methodsWe recruited 20 healthy men with a mean age of 24.5 years (standard deviation (SD): 5.04) and a mean body mass index (BMI) of 23.8% (SD: 3.3). Total testosterone (TT) was measured by immunochemiluminescence (ICLA) and free testosterone (FT) by radioimmunoassay (RIA). Calculated free testosterone (FTc) and bioavailable testosterone (BT) were calculated using Vermeulen's formula. Serum lutropin (LH), follitropin (FSH) and sex hormone binding globulin (SHBG) were measured by immunoradiometric assays (IRMA).ResultsThe mean concentrations were 20 nmol/l (SD: 4.96) for TT, 0.054 nmol/L (SD: 0.01) for FT, 0.3834 nmol/L (SD: 0.09) for FTc and 9.9 nmol/L (SD: 2.8) for BT. There was no correlation between testosterone measured by different methods other than an association between FT and FTc (r=0.662, p<0.003) and between FTc and BT (r=0.979, p<0.0001). An inverse correlation was found between BMI and TT concentrations (r: ?0.52, p<0.017).ConclusionsThe normal range for testosterone in healthy young men should be established in each laboratory based on the analytical method used.     

Purification and Characterization of Extracellular Pectinolytic Enzymes Produced by Sclerotinia sclerotiorum

An exopolygalacturonase (exoPG) and an exopolymethylgalacturonase (exoPMG) produced by Sclerotinia sclerotiorum have been purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. The exoPG and the exoPMG were purified 66- and 50-fold, respectively, by using a series of separation procedures that included ammonium sulfate precipitation and gel chromatography. Molecular masses of the native proteins were 68 kDa for exoPG and 140 kDa for exoPMG. The pH optima of the enzymes were about pH 5, and their optimum temperature was 45°C. Activities of both enzymes were inhibited by Hg²⁺, Zn²⁺, Cu²⁺, and p-chloromercuribenzoate. ExoPMG activity, in contrast to exoPG activity, was stimulated by Mn²⁺ and Co²⁺. ExoPMG hydrolyzed only citrus pectin, while exoPG degraded sodium polygalacturonate and, to a lesser extent, citrus pectin. The exo mode of action of the enzymes was revealed by thin-layer chromatography of substrate hydrolysates. Antibodies raised against each purified protein exhibited no cross-reaction, thus confirming the biochemical specificities of the enzymes.     

Dextransucrase as an Enzyme Associating with Alkaline Earth Metal Ions

Dextransucrase (EC. 2.4.1.5) of Leuconostoc mesenteroides was purified from the culture filtrate by precipitation with solid ammonium sulfate in the presence of egg white albumin followed by successively treating with columns of DEAE-cellulose and Bio Gel P-150.

The purified enzyme lost the activity upon dialysis against EDTA, and was reactivated by the addition of alkaline arth metal ions. The best reactivation was brought about by calcium ion. The enzyme inactivated by EDTA was unstable and readily denatured irreversibly. Several other properties of the purified enzyme were also investigated and discussed.     

The Synthesis of Gibberethiones,Gibberellin-related Diterpenoids

The factors affecting desalting by gel chromatography were investigated both experimentally and theoretically. In most cases, the desalting of bovine serum albumin dissolved in an ammonium sulfate solution on a Sephadex G-25 Coarse column was chosen as a model experiment, and the effects of sample volume and concentration on the desalting were examined. The elution curves observed were in fairly good agreement with those calculated on the basis of a mass balance model, when the concentration of ammonium sulfate was lower than 0.4 m. Otherwise, the concentrations of solutes in the eluate were considerably diluted in comparison with the calculated results. Finally, useful charts were illustrated, from which appropriate operational conditions for desalting could be predicted.     

Dosimetric comparison of MRI-based HDR brachytherapy and stereotactic radiotherapy in patients with advanced cervical cancer: A virtual brachytherapy study

AimTo evaluate the treatment plans of 3D image-guided brachytherapy (BT) and stereotactic robotic radiotherapy with online image guidance – CyberKnife (CK) in patients with locally advanced cervix cancer.Methods and materialsTen pairs of plans for patients with locally advanced inoperable cervical cancer were created using MR based 3D brachytherapy and stereotaxis CK. The dose that covers 98% of the target volume (HR CTV D98) was taken as a reference and other parameters were compared.ResultsOf the ten studied cases, the dose from D100 GTV was comparable for both devices, on average, the BT GTV D90 was 10–20% higher than for CK. The HR CTV D90 was higher for CK with an average difference of 10–20%, but only fifteen percent of HR CTV (the peripheral part) received a higher dose from CK, while 85% of the target volume received higher doses from BT. We found a significant organ-sparing effect of CK compared to brachytherapy (20–30% lower doses in 0.1 cm³, 1 cm³, and 2 cm³).ConclusionBT remains to be the best method for dose escalation. Due to the significant organ-sparing effect of CK, patients that are not candidates for BT could benefit from stereotaxis more than from classical external beam radiotherapy.     

Purification and Some Properties of a Neutral α-Glucosidase from Pig Serum

A neutral α-glucosidase was purified from pig serum by precipitation with ammonium sulfate, chromatographies on DEAE-cellulose and -Sephadex A–50, and gel filtration on Bio-Gel P–300 and Sephadex G–200. The purified enzyme was homogeneous in ultracentrifugal and disc electrophoretic analysis. The sedimentation coefficient (s_20,w) was calculated to be 10.7 S, and the isoelectric point, 4.0. The molecular weight was estimated to be approximately 2.7 × 10⁵ by thin-layer gel filtration and SDS-disc electrophoresis.

The enzyme exhibited also glucoamylase activity. The optimal pH was found to be in the pH range of 6.0 to 7.0 for maltose and soluble starch. The ratio of velocity of hydrolysis for maltose (Km, 0.72 mg/ml), soluble starch (Km, 9.8 mg/ml) and shellfish glycogen (Km, 55.6 mg/ml) was calculated to be 100: 110: 5.15 in this order.     

Comparative dosimetrical analysis of intensity-modulated arc therapy,CyberKnife therapy and image-guided interstitial HDR and LDR brachytherapy of low risk prostate cancer

BackgroundThe objective of the study was to dosimetrically compare the intensity-modulated-arc-therapy (IMAT), Cyber-Knife therapy (CK), single fraction interstitial high-dose-rate (HDR) and low-dose-rate (LDR) brachytherapy (BT) in low-risk prostate cancer.Materials and methodsTreatment plans of ten patients treated with CK were selected and additional plans using IMAT, HDR and LDR BT were created on the same CT images. The prescribed dose was 2.5/70 Gy in IMAT, 8/40 Gy in CK, 21 Gy in HDR and 145 Gy in LDR BT to the prostate gland. EQD2 dose-volume parameters were calculated for each technique and compared.ResultsEQD2 total dose of the prostate was significantly lower with IMAT and CK than with HDR and LDR BT, D90 was 79.5 Gy, 116.4 Gy, 169.2 Gy and 157.9 Gy (p < 0.001). However, teletherapy plans were more conformal than BT, COIN was 0.84, 0.82, 0.76 and 0.76 (p < 0.001), respectively. The D₂ to the rectum and bladder were lower with HDR BT than with IMAT, CK and LDR BT, it was 66.7 Gy, 68.1 Gy, 36.0 Gy and 68.0 Gy (p = 0.0427), and 68.4 Gy, 78.9 Gy, 51.4 Gy and 70.3 Gy (p = 0.0091) in IMAT, CK, HDR and LDR BT plans, while D_0.1 to the urethra was lower with both IMAT and CK than with BTs: 79.9 Gy, 88.0 Gy, 132.7 Gy and 170.6 Gy (p < 0.001). D₂ to the hips was higher with IMAT and CK, than with BTs: 13.4 Gy, 20.7 Gy, 0.4 Gy and 1.5 Gy (p < 0.001), while D₂ to the sigmoid, bowel bag, testicles and penile bulb was higher with CK than with the other techniques.ConclusionsHDR monotherapy yields the most advantageous dosimetrical plans, except for the dose to the urethra, where IMAT seems to be the optimal modality in the radiotherapy of low-risk prostate cancer.