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Vint proteins have been identified in unicellular metazoans as a novel hedgehog‐related gene family, merging the von Willebrand factor type A domain and the Hedgehog/INTein (HINT) domains. We present the first three‐dimensional structure of the Vint domain from Tetrahymena thermophila corresponding to the auto‐processing domain of hedgehog proteins, shedding light on the unique features, including an adduct recognition region (ARR). Our results suggest a potential binding between the ARR and sulfated glycosaminoglycans like heparin sulfate. Moreover, we uncover a possible regulatory role of the ARR in the auto‐processing by Vint domains, expanding our understanding of the HINT domain evolution and their use in biotechnological applications. Vint domains might have played a crucial role in the transition from unicellular to multicellular organisms. [ABSTRACT FROM AUTHOR]

The continuous evolution of molecular biology and gene synthesis methods paired with an ever‐increasing potential of synthetic biology approaches and genome engineering toolkits enables the rapid design of genetic bioparts and genetically modified organisms. Although various software solutions assist with specific design tasks and challenges, lab internal documentation and ensuring compliance with governmental regulations on biosafety assessment of the generated organisms remain the responsibility of individual academic researchers. This results in inconsistent and redundant documentation regimes and a significant time and labor burden. GMOCU (GMO documentation) is a standardized semi‐automatic user‐oriented software approach —written in Python and freely available— that unifies lab internal data documentation on genetic parts and genetically modified organisms (GMOs). It automatizes biological risk evaluations and maintains a shared up‐to‐date inventory of bioparts for team‐wide data navigation and sharing. GMOCU further enables data export into customizable formats suitable for scientific publications, official biosafety documents, and the research community. [ABSTRACT FROM AUTHOR]

Preclinical investigations have shown that N‐ethyl‐N‐isopropyllysergamide (EIPLA) exhibits lysergic acid diethylamide (LSD)‐like properties, which suggests that it might show psychoactive effects in humans. EIPLA is also an isomer of N6‐ethylnorlysergic acid N,N‐diethylamide (ETH‐LAD), a lysergamide known to produce psychedelic effects in humans that emerged as a research chemical. EIPLA was subjected to analysis by various forms of mass spectrometry, chromatography (GC, LC), nuclear magnetic resonance (NMR) spectroscopy, and GC condensed‐phase infrared spectroscopy. The most straightforward differentiation between EIPLA and ETH‐LAD included the evaluation of mass spectral features that reflected the structural differences (EIPLA: N6‐methyl and N‐ethyl‐N‐isopropylamide group; ETH‐LAD: N6‐ethyl and N,N‐diethylamide group). Proton NMR analysis of blotter extracts suggested that EIPLA was detected as the base instead of a salt, and two blotter extracts suspected to contain EIPLA revealed the detection of 96.9 ± 0.5 μg (RSD: 0.6%) and 85.8 ± 2.8 μg base equivalents based on LC–MS analysis. The in vivo activity of EIPLA was evaluated using the mouse head‐twitch response (HTR) assay. Similar to LSD and other serotonergic psychedelics, EIPLA induced the HTR (ED50 = 234.6 nmol/kg), which was about half the potency of LSD (ED50 = 132.8 nmol/kg). These findings are consistent with the results of previous studies demonstrating that EIPLA can mimic the effects of known psychedelic drugs in rodent behavioral models. The dissemination of analytical data for EIPLA was deemed justifiable to aid future forensic and clinical investigations. [ABSTRACT FROM AUTHOR]

The development of novel lysergamides continues to occur, based on both the needs of psychedelic medicine and commercial interest in new recreational substances. The present study continues the authors' research on novel lysergamides and describes the analytical profile of 1‐cyclopropanoyl‐AL‐LAD (IUPAC name: 1‐(cyclopropanecarbonyl)‐N,N‐diethyl‐6‐(prop‐2‐en‐1‐yl)‐9,10‐didehydroergoline‐8β‐carboxamide; 1cP‐AL‐LAD), using various chromatographic, mass spectrometric, and spectroscopic methods. Analysis of a powdered sample of 1cP‐AL‐LAD, obtained from an online vendor, by high performance liquid chromatography‐electrospray ionization‐quadrupole time‐of‐flight mass spectrometry in full scan/AutoMS/MS mode revealed the detection of 17 impurities based on high‐resolution tandem mass spectral data; tentative determination of their identity was based on mass spectral grounds alone, though detection of AL‐LAD and 1P‐AL‐LAD was confirmed using available reference standards. Other tentative compound identifications included 1‐acetyl‐AL‐LAD and several other substances potentially reflecting oxidation of the N6‐allyl group as well as other positions on the ergoline ring system. These data may assist those interested in the chemistry of lysergamides. Finally, 1cP‐AL‐LAD was also detected in samples of "blotters" sold online for recreational use. [ABSTRACT FROM AUTHOR]

Lysergic acid diethylamide (LSD) is known to induce powerful psychoactive effects in humans, which cemented its status as an important tool for clinical research. A range of analogues and derivatives has been investigated over the years, including those classified as new psychoactive substances. This study presents the characterization of the novel lysergamide N,N‐diethyl‐1‐propanoyl‐6‐(prop‐2‐en‐1‐yl)‐9,10‐didehydroergoline‐8β‐carboxamide (1P‐AL‐LAD) using various mass spectrometric, gas‐ and liquid chromatographic and spectroscopic methods. In vitro metabolism studies using pooled human liver microsomes (pHLM) confirmed that 1P‐AL‐LAD converted to AL‐LAD as the most abundant metabolite consistent with the hypothesis that 1P‐AL‐LAD may act as a prodrug. Fourteen metabolites were detected in total; metabolic reactions included hydroxylation of the core lysergamide ring structure or the N6‐allyl group, formation of dihydrodiol metabolites, N‐dealkylation, N1‐deacylation, dehydrogenation, and combinations thereof. The in vivo behavioral activity of 1P‐AL‐LAD was evaluated using the mouse head twitch response (HTR), a 5‐HT2A‐mediated head movement that serves as a behavioral proxy in rodents for human hallucinogenic effects. 1P‐AL‐LAD induced a dose‐dependent increase in HTR counts with an inverted U‐shaped dose–response function, similar to lysergic acid diethylamide (LSD), psilocybin, and other psychedelics. Following intraperitoneal injection, the median effective dose (ED50) for 1P‐AL‐LAD was 491 nmol/kg, making it almost three times less potent than AL‐LAD (174.9 nmol/kg). Previous studies have shown that N1‐substitution disrupts the ability of lysergamides to activate the 5‐HT2A receptor; based on the in vitro metabolism data, 1P‐AL‐LAD may induce the HTR because it acts as a prodrug and is metabolized to AL‐LAD after administration to mice. [ABSTRACT FROM AUTHOR]

Lysergic acid diethylamide (LSD) is a potent psychoactive substance that has attracted great interest in clinical research. As the pharmacological exploration of LSD analogs continues to grow, some of those analogs have appeared on the street market. Given that LSD analogs are uncontrolled in many jurisdictions, it is important that these analogs be differentiated from LSD. This report presents the analysis of blotters found to contain the N‐methyl‐N‐isopropyl isomer of LSD (MIPLA), and techniques to differentiate it from LSD and the N‐methyl‐N‐propyl isomer (LAMPA) under routine conditions. Gas chromatography (GC)‐solid phase infrared spectroscopy was particularly helpful. GC‐electron ionization‐tandem mass spectrometry of the m/z 72 iminium ion also provided sufficient information to distinguish the three isomers on mass spectral grounds alone, where chromatographic separation proved challenging. Derivatization with 2,2,2‐trifluoro‐N,N‐bis (trimethylsilyl)acetamide (BSTFA) also led to improved GC separation. Liquid chromatography single quadrupole mass spectrometry (LC‐Q‐MS) and in‐source collision‐induced dissociation allowed for the differentiation between MIPLA and LAMPA based on distinct m/z 239 ion ratios when co‐eluting. An alternative LC‐MS/MS method improved the separation between all three lysergamides, but LSD was found to co‐elute with iso‐LSD. However, a comparison of ion ratios recorded for transitions at m/z 324.2 > 223.2 and m/z 324.2 > 208.2 facilitated their differentiation. The analysis of two blotters by LC‐Q‐MS revealed the presence of 180 and 186 μg MIPLA per blotter. These procedures may be used to avoid inadvertent misidentification of MIPLA or LAMPA as LSD. [ABSTRACT FROM AUTHOR]

Purpose: The assessment of cellular energy metabolism is crucial for understanding myocardial physiopathology. Here, we conducted a pilot study to develop an alternative imaging approach for the assessment of myocardial energy metabolism. Methods: We developed a deuterium MRSI method to noninvasively monitor the accumulation of deuterated downstream metabolites and deuterated water in rat hearts infused with deuterated glucose or acetate substrate on a 16.4 Tesla animal scanner. Results: We found that the deuterated water accumulation rate and isotopic turnover rate of deuterated glutamate/glutamine via the tricarboxylic acid cycle and exchange in rat hearts were much higher when infused with acetate compared to that with glucose, demonstrating the myocardium substrate preference for acetate over glucose. Conclusion: We demonstrated the feasibility of deuterium MRSI for noninvasive imaging and assessment of myocardial energy metabolism in vivo. Although the strong signal and large dynamics of myocardial deuterated water may provide a sensitive imaging biomarker, quantifying the metabolic rates still poses a challenge due to the confounding effects of blood recirculation, perfusion, and multiple deuterated water production pathways. In contrast, the deuterated glutamate/glutamine signal and change should directly reflect the metabolic activity of the myocardial tricarboxylic acid cycle, which can be used to study the metabolic shift in substance preference between acetate and glucose in the diseased state. Deuterium MRSI is noninvasive and robust and may have the potential to assess myocardial energy metabolism in human patients. [ABSTRACT FROM AUTHOR]

Purpose: Oxygen‐17 (17O) MRS imaging, successfully used in the brain, is extended by imaging the oxygen metabolic rate in the resting skeletal muscle and used to determine the total whole‐body oxygen metabolic rate in the rat. Methods: During and after inhalations of 17O2 gas, dynamic 17O MRSI was performed in rats (n = 8) ventilated with N2O or N2 at 16.4 T. Time courses of the H217O concentration from regions of interest located in brain and muscle tissue were examined and used to fit an animal‐adapted 3‐phase metabolic model of oxygen consumption. CBF was determined with an independent washout method. Finally, body oxygen metabolic rate was calculated using a global steady‐state approach. Results: Cerebral metabolic rate of oxygen consumption was 1.97 ± 0.19 μmol/g/min on average. The resting metabolic rate of oxygen consumption in skeletal muscle was 0.32 ± 0.12 μmol/g/min and >6 times lower than cerebral metabolic rate of oxygen consumption. Global oxygen consumed by the body was 24.2 ± 3.6 mL O2/kg body weight/min. CBF was estimated to be 0.28 ± 0.02 mL/g/min and 0.34 ± 0.06 mL/g/min for the N2 and N2O ventilation condition, respectively. Conclusion: We have evaluated the feasibility of 17O MRSI for imaging and quantifying the oxygen consumption rate in low metabolizing organs such as the skeletal muscle at rest. Additionally, we have shown that CBF is slightly increased in the case of ventilation with N2O. We expect this study to be beneficial to the application of 17O MRSI to a wider range of organs, although further validation is advised. [ABSTRACT FROM AUTHOR]

Inteins catalyze self‐excision from host precursor proteins while concomitantly ligating the flanking substrates (exteins) with a peptide bond. Noncatalytic extein residues near the splice junctions, such as the residues at the −1 and +2 positions, often strongly influence the protein‐splicing efficiency. The substrate specificities of inteins have not been studied for many inteins. We developed a convenient mutagenesis platform termed "QuickDrop"‐cassette mutagenesis for investigating the influences of 20 amino acid types at the −1 and +2 positions of different inteins. We elucidated 17 different profiles of the 20 amino acid dependencies across different inteins. The substrate specificities will accelerate our understanding of the structure–function relationship at the splicing junctions for broader applications of inteins in biotechnology and molecular biosciences. [ABSTRACT FROM AUTHOR]

Interpretation of amphetamine‐type stimulant (ATS) findings in urine samples can be challenging without chiral information. We present a sensitive enantioselective high‐performance liquid chromatography–tandem mass spectrometry method for the quantification of (R)‐amphetamine, (S)‐amphetamine, (R)‐methamphetamine, (S)‐methamphetamine, (1R,2R)‐pseudoephedrine, (1S,2S)‐pseudoephedrine, (1R,2S)‐ephedrine, (1S,2R)‐ephedrine, (1R,2S)‐norephedrine, (1S,2R)‐norephedrine, (R)‐cathinone, (S)‐cathinone, and (1S,2S)‐norpseudoephedrine (cathine) in urine. The method was successfully applied to more than 100 authentic urine samples from forensic casework. In addition, samples from a controlled self‐administration of (1S,2S)‐pseudoephedrine (Rinoral, 1200 mg within 6 days) were analyzed. The results strengthen the hypothesis that (1R,2S)‐norephedrine is a minor metabolite of amphetamine and methamphetamine. We suggest cathine and (1S,2R)‐norephedrine as minor metabolites of amphetamine racemate in humans. Small methamphetamine concentrations detected in samples with high concentrations of amphetamine could result from a metabolic formation by methylation of amphetamine although in samples with an (R)/(S) ratio for methamphetamine < 1 an additional (previous) (S)‐methamphetamine consumption seems likely. Our data suggest that even amphetamine concentrations exceeding methamphetamine concentrations in urine can be caused by the biotransformation of methamphetamine to amphetamine as long as no (R)‐amphetamine is detected. However, without chiral information, such findings might be (falsely) assumed as a co‐consumption of both substances. Cathinone enantiomers detected in urine samples with high amphetamine concentrations can be interpreted as metabolites of amphetamine. In addition, the results of the self‐administration study revealed that both cathinone enantiomers are minor metabolites of (1S,2S)‐pseudoephedrine, which is the active ingredient of various medicines used for cold. The enantioselective analysis is a powerful tool to avoid the misinterpretation of ATS findings in urine samples. [ABSTRACT FROM AUTHOR]

Among the increasing number of new psychoactive substances, 3′,4′‐methylenedioxy‐α‐pyrrolidinohexanophenone (MDPHP) belongs to the group of synthetic cathinones, which are the derivatives of the naturally occurring compound cathinone, the main psychoactive ingredient in the khat plant. Currently, only limited data are available for MDPHP, and no information is available on its human metabolism. We describe the toxicological investigation of nine cases associated with the use of MDPHP during the period February–June 2019. Serum MDPHP concentrations showed a high variability ranging from 3.3 to 140 ng/mL (mean 30.3 ng/mL and median 16 ng/mL). Intoxication symptoms of the described cases could not be explained by the abuse of MDPHP alone because in all cases the co‐consumption of other psychotropic drugs with frequent occurrence of opiates and benzodiazepines could be verified. Therefore, the patients showed different clinical symptoms, including aggressive behaviour, delayed physical response, loss of consciousness and coma. Liquid chromatography–high‐resolution mass spectrometry was successfully used to investigate the human in vivo metabolism of MDPHP using authentic human urine samples. The metabolism data for MDPHP were further substantiated by the analysis of human urine using gas chromatography–mass spectrometry (GC–MS, a widely used systematic toxicological analysis method appropriate for the toxicological detection of MDPHP intake), which revealed the presence of seven phase I metabolites and three phase II metabolites as glucuronides. GC‐MS spectral data for MDPHP and metabolites are provided. The identified metabolite pattern corroborates the principal metabolic pathways of α‐pyrrolidinophenones in humans. [ABSTRACT FROM AUTHOR]

Protein trans‐splicing catalyzed by split inteins has increasingly become useful as a protein engineering tool. We solved the 1.0 Å‐resolution crystal structure of a fused variant from the naturally split gp41‐1 intein, previously identified from environmental metagenomic sequence data. The structure of the 125‐residue gp41‐1 intein revealed a compact pseudo‐C2‐symmetry commonly found in the Hedgehog/Intein superfamily with extensive charge–charge interactions between the split N‐ and C‐terminal intein fragments that are common among naturally occurring split inteins. We successfully created orthogonal split inteins by engineering a similar charge network into the same region of a cis‐splicing intein. This strategy could be applicable for creating novel natural‐like split inteins from other, more prevalent cis‐splicing inteins. Database: Structural data are available in the RCSB Protein Data Bank under the accession number 6QAZ. [ABSTRACT FROM AUTHOR]

Telomerase reverse transcriptase (TERT) maintains telomeres and is rate limiting for replicative life span. While most somatic tissues silence TERT transcription resulting in telomere shortening, cells derived from cancer or cardiovascular diseases express TERT and activate telomerase. In the present study, we demonstrate that histone deacetylase (HDAC) inhibition induces TERT transcription and promoter activation. At the protein level in contrast, HDAC inhibition decreases TERT protein abundance through enhanced degradation, which decreases telomerase activity and induces senescence. Finally, we demonstrate that HDAC inhibition decreases TERT expression during vascular remodeling in vivo. These data illustrate a differential regulation of TERT transcription and protein stability by HDAC inhibition and suggest that TERT may constitute an important target for the anti-proliferative efficacy of HDAC inhibitors. J. Cell. Physiol. 231: 1276-1282, 2016. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Hysteretic processes have been recognized for decades as an important characteristic of soil hydraulic behavior. Several studies confirmed that wetting and drying periods cannot be described by a simple functional relationship, and that some nonequilibrium of the water retention characteristics has to be taken into account. A large number of models describing the hysteresis of the soil water retention characteristic were successfully tested on soil cores under controlled laboratory conditions. However, its relevance under field conditions under natural forcings has rarely been investigated. In practice, the modeling of field soils usually neglects the hysteretic nature of soil hydraulic properties. In this study, long-term observations of water content and matric potential in lysimeters of the lysimeter network TERENO-SoilCan are presented, clearly demonstrating the hysteretic behavior of field soils. We propose a classification into three categories related to different time scales. Based on synthetic and long-term monitoring data, three different models of hysteresis were applied to data sets showing different degrees of hysteresis. We found no single model to be superior to the others. The model ranking depended on the degree of hysteresis. All models were able to reflect the general structure of hysteresis in most cases but failed to reproduce the detailed trajectories of state variables especially under highly transient conditions. As an important result we found that the temporal dynamics of wetting and drying significantly affects these trajectories which should be accounted for in future model concepts. [ABSTRACT FROM AUTHOR]

Purpose Measurement of the cerebral metabolic rate of oxygen (CMRO2) by means of direct imaging of the 17O signal can be a valuable tool in neuroscientific research. However, knowledge of the longitudinal and transverse relaxation times of different brain tissue types is required, which is difficult to obtain because of the low sensitivity of natural abundance H217O measurements. Methods Using the improved sensitivity at a field strength of 16.4 Tesla, relaxation time measurements in the rat brain were performed in vivo and postmortem with relatively high spatial resolutions, using a chemical shift imaging sequence. Results In vivo relaxation times of rat brain were found to be T1 = 6.84 ± 0.67 ms and T2* = 1.77 ± 0.04 ms. Postmortem H217O relaxometry at enriched concentrations after inhalation of 17O2 showed similar T2* values for gray matter (1.87 ± 0.04 ms) and white matter, significantly longer than muscle (1.27 ± 0.05 ms) and shorter than cerebrospinal fluid (2.30 ± 0.16 ms). Conclusion Relaxation times of brain H217O were measured for the first time in vivo in different types of tissues with high spatial resolution. Because the relaxation times of H217O are expected to be independent of field strength, our results should help in optimizing the acquisition parameters for experiments also at other MRI field strengths. Magn Reson Med 75:1886-1893, 2016. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The nuclear receptor NOR1 is an immediate‐early response gene implicated in the transcriptional control of proliferation. Since the expression level of NOR1 is rapidly induced through cAMP response element binding (CREB) protein‐dependent promoter activation, we investigated the contribution of histone acetylation to this transient induction. We demonstrate that NOR1 transcription is induced by histone deacetylase (HDAC) inhibition and by depletion of HDAC1 and HDAC3. HDAC inhibition activated the NOR1 promoter, increased histone acetylation and augmented the recruitment of phosphorylated CREB to the promoter. Furthermore, HDAC inhibition increased Ser133 phosphorylation of CREB and augmented NOR1 protein stability. These data outline previously unrecognized mechanisms of NOR1 regulation and illustrate a key role for histone acetylation in the rapid induction of NOR1.NOR1 transcription is induced by histone deacetylase inhibition through chromatin modification. HDAC inhibition increases CREB phosphorylation and its recruitment to CRE consensus motifs within the NOR1 promoter. HDAC inhibition augments NOR1 protein stability. [ABSTRACT FROM AUTHOR]

The cerebral metabolic rate of oxygen of small animals can be reliably imaged using the in vivo 17O magnetic resonance approach at high field. However, a separate measurement is required for imaging the cerebral blood flow in the same animal. In this study, we demonstrate that the 17O NMR signal of metabolically produced H217O in the rat brain following an 17O2 inhalation can serve as a perfusion tracer and its decay rate can be used to determine the absolute values of cerebral blood flow across a wide range of animal conditions. This finding suggests that the in vivo 17O magnetic resonance approach is capable of imaging both cerebral metabolic rate of oxygen and cerebral blood flow simultaneously and noninvasively; and it provides new utilities for studying the cerebral oxygen metabolism and perfusion commonly associated with brain function and diseases. Magn Reson Med 70:309-314, 2013. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Excessive accumulation of reactive oxygen species (ROS) in adipose tissue has been implicated in the development of insulin resistance and type 2 diabetes. However, emerging evidence suggests a physiologic role of ROS in cellular signaling and insulin sensitivity. In this study, we demonstrate that pharmacologic depletion of the antioxidant glutathione in mice prevents diet-induced obesity, increases energy expenditure and locomotor activity, and enhances insulin sensitivity. These observations support a beneficial role of ROS in glucose homeostasis and warrant further research to define the regulation of metabolism and energy balance by ROS. [ABSTRACT FROM AUTHOR]

Changes in the status of DNA methylation are among the most common molecular alterations in human neoplasia, Recent demonstrations of tumor-derived methylated DNA in the blood stream of cancer patients allow the use of these epigenetic markers for risk assessment in cancer patients. We were interested in evaluating the prognostic value of several methylated genes in the serum of cancer patients. Using MethyLight, a high-throughput DNA methylation assay, we analyzed 215 serum samples from patients with cervical (a = 93) or breast cancer (a 122) for DNA methylation changes. In cervical cancer, hypermethylation of three genes (MYOD1, CDH1, and CDH13) in pre-treatment sera was statistically significantly associated with a poorer disease outcome. Additionally, for the first time we used a so-called gene evaluation set to identify the most important DNA methylation changes in the serum of breast cancer patients from a long list of candidate genes. In the gene evaluation set, we detected five genes (ESR1, APC, HSD1 7B4, HId, and RASSF1A) using our criteria for further analysis. Finally, two of the evaluated genes (APC and RASSF1A) proved to be independent prognostic parameters in breast cancer patients. In summary, we detected several prognostic DNA methylation markers in the serum of cervical and breast cancer patients. This finding indicates great potential for the use of these epigenetic markers in clinical, routine risk assessment in patients with various malignancies. [ABSTRACT FROM AUTHOR]

Cervical cancer is the principal cause of death due to cancer in women. Five-year survival rate ranges from 15–80%, depending on the extent of the disease. New predictive markers for relapse may increase survival rates by improving treatment of patients at high risk for relapse. The gene products of CDH1 and CDH13, namely E-cadherin and H-cadherin, play a key role in cell–cell adhesion. Inactivation of the cadherin-mediated cell adhesion system, caused by aberrant methylation, is a common finding in human cancers. To test the hypothesis that CDH1/CDH13 methylation is a prognostic marker in cervical cancer we determined the methylation status of CDH1/CDH13 in serum samples from 93 cervical cancer patients. Methylation analysis was carried out using MethyLight. Aberrant methylation of the 5'-region of CDH1 or CDH13 was observed in 43% (40 of 93) of the patients. Cervical cancer patients with unmethylated CDH1/CDH13 in serum samples showed significantly better disease-free survival in univariate and multivariate analysis. Median disease-free survival for CDH1/CDH13 methylation negative and positive patients was 4.3 years and 1.2 years, respectively. Our results suggest that detection of aberrant methylation of CDH1/CDH13 may be of potential use as a marker for selecting cervical cancer patients at high risk for relapse who could benefit from additional systemic therapy. © 2003 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Photoactivation of the α,β-unsaturated ketones of natural and synthetic steroid molecules by light of λ ≥ 330 nm allows their covalent attachment to steroid-binding proteins. The general validity of this method is demonstrated with two steroid hormone receptors and the steroid-binding protein uteroglobin. Progesterone can be covalently attached to the partially purified progesterone receptor and to uteroglobin, and comigrates with the binding proteins upon elcetrophoresis in polyacrylamide gels containing sodium doclecyl sulfate. Similarly the synthetic glucocorticoid triamcinolone acetonide can be covalently bound to the partially purified glucocorticoid of rat liver. This method allows the identification of steroid hormone receptors after electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Labeling with radioactive steroids is specific since it can be prevented by the addition of an excess of non-radioactive ligand. Digestion of the labeled binding proteins with trypsin or chymotrypsin yields a defined pattern of radioactive peptides, demonstrating that covalent attachment takes place at specific binding sites. [ABSTRACT FROM AUTHOR]

The activated form of the hepatic glucocorticoid receptor has been purified 60000-fold taking advantage of the differential affinity for phosphocellulose of the activated and the nonactivated forms of the receptor. Rat liver cytosol was incubated with [3H]triamcinolone acetonide at low temperature and low ionic strength and adsorbed to a large excess of phosphocellulose. The unbound fraction was heat-activated, passed through a column of DEAE-cellulose and precipitated with ammonium sulfate. The activated receptor was then bound to a small phosphocellulose column and eluted with a gradient of NaC1. Additional steps included chromatography on DNA-cellulose, precipitation with ammonium sulfate and centrifugation through a sucrose density gradient. The final preparation was identified as the steroid-receptor complex by electrophoresis on poly-acrylamide gels in the presence of heparin; it exhibits a single band of molecular weight 40000 ± 4000 in gels containing sodium dodecyl sulfate. An independent calculation of the molecular weight under nondenaturing conditions based on the Stokes' radius (2.7 nm) and the sedimentation coefficient (S20,w, = 3.0 S) yields similar results, indicating that the activated form of the receptor we have isolated is composed of a single polypeptide chain and contains a single steroid-binding site per molecule. Since this homogenous preparation of receptor has retained its affinity for DNA it may be useful for cell-free studies. [ABSTRACT FROM AUTHOR]

A response by Hannes M. Findeisen et al to a letter to the editor about the article "Lack of Evidence to Support a Beneficial Role for Glutathione Depletion on Body Weight or Glucose Intolerance" that was published in a previous issue is presented.