1,25-Dihydroxyvitamin D3 receptors in rat kidney cytosol

Rat kidney cytosol contains a 3.3 S high affinity binding component for 1,25-dihydroxyvitamin D₃ as detected by DNA-cellulose chromatography and subsequent sucrose gradient analysis. The semipurified aporeceptor demonstrates specificity for 1,25-dihydroxyvitamin D₃ and an apparent dissociation constant for this sterol-hormone of 3.4 × 10^?10M at 25°C. The physicochemical properties of this binding component are in agreement with those observed for the chick intestinal 1,25-dihydroxyvitamin D₃ receptor, suggesting that this component may function as a specific receptor for the hormone in the kidney.     

Inactivation of porcine corpus luteum lutropin receptors by a microsomal enzyme: Activation in ageing and regressing corpora lutea

The binding of ¹²⁵I-labelled human choriogonadotropin (hCG) to homogenates of porcine corpora lutea showed a marked departure from ideal behaviour, due to a time- and temperature-dependent inactivation of both free hormone and unoccupied receptors. Occupied receptors were not affected. Lutropin (LH)-receptor-inactivating activity was detected after preincubation of homogenates, particulate fractions and microsomes, but little activity could be demonstrated in cytosol fractions. Inactivation was dependent on the temperature and pH of preincubation, and on tissue concentration: LH-receptor inactivation was first-order with respect to preincubationtime. Lutropin-receptor-inactivating activity was low in early-luteal and mid-luteal phase in pig corpora lutea, but was increased significantly in late-luteal and regressing corpora lutea.     

Evidence for a reactive sulfhydryl in the DNA binding domain of the 1,25-dihydroxyvitamin D3 receptor

Evidence is presented here that organomercurial binding to a reactive sulfhydryl group is capable of altering the DNA-binding characteristics of the 1,25-dihydroxyvitamin D receptor (D-receptor). Accordingly, hormone-free receptor (R_o) binding to DNA-cellulose is inhibited in a concentration-dependent fashion with both HgCl₂ and p-chloromercuribenzene sulfonate (pCMBS) with complete inhibition evident at 1.0 mM. Further, low concentrations (0.5 mM) of mercurials are also capable of dissociating preformed DNA-receptor complexes, a process reversible with excess thiol reagent such as monothioglycerol. These findings are in contrast to alkylating reagents such as iodoacetamide, which is capable of only partially inhibiting the formation of the receptor-DNA duplex (37% at 25 mM). Once created, however, the duplex is completely insensitive to dissociation (even at 25 mM). These results imply that in addition to the association of a cysteine(s) moiety in or near the sterol binding site, modification of a similarly reactive group(s) can also alter the D-receptor's DNA-binding domain.     

Effects of human choriogonadotropin on mitochondrial and microsomal cytochrome P-450 levels in mouse testes

The mitochondrial and microsomal cytochrome P-450 contents of $\text{C57B1}\text{6}$ mouse testis have been measured using difference spectroscopy on stable enzyme preparations containing the ferrous-carbon monoxide complex. Results were obtained on control animals (52 ± 3 days of age) and on animals injected subcutaneously with human choriogonadotropin (0.017 μg/g body weight 24 h prior to sacrifice). The high ratio of testicular mitochondrial cytochrome oxidase to P-450, which has previously precluded measurements of basal P-450 levels, was overcome by using N,N,N′,N′-tetramethyl-p-phenylene diamine to bypass site II, in combination with antimycin A to prevent reverse electron flow. The basal levels of mitochondrial and microsomal P-450 in mouse testis were 37.9 ± 3.5 and 28.9 ± 1.6 pmol/mg protein, respectively. Following administration of a desensitizing dose of gonadotropin, the respective values were lowered to 19.9 ± 1.4 and 19.6 ± 2.1 pmol/mg protein in 24 h. This is the first report of a gonadotropin-mediated decrease in mitochondrial P-450 and thus demonstrates that desensitization leads to alterations in both microsomal and mitochondrial P-450 in mouse testis.     

Lipoxygenbase-derived products of arachidonic acid mediate stimulation of hexose uptake in human polymorphonuclear leukocytes

The titration of metal-freed bovine α-lactalbumin with Mg²⁺ ions causes a two-stepped decrease in the tryptophan fluorescence quantum yield and a pronounced spectral shift towards shorter wavelengths, which seems to be a result of the binding of two magnesium ions to the protein molecule. The magnesium binding constants evaluated from the fluorimetric Mg²⁺-titration are 2·10³ and 2·10² M^?1. Mg²⁺ ions in millimolar concentrations almost do not influence the binding of Ca²⁺ ions to the protein.     

Androgen receptor complex binding in murine skeletal muscle nuclei

Examination of binding of androgen-receptor complexes from murine skeletal muscle cytosol was performed by modified nuclear retention assay and modified nuclear acceptor assay. The experiments showed the binding of androgen-receptor complexes to the nuclear acceptor sites to be a cooperative process. Hill analysis of the data obtained resulted in a Hill coefficient of 3,6. The apparent dissociation constant for binding of cytosolic [³H]-testosterone-receptor complexes to nuclei was found to be in the range of K_D = 6 ? 8 × 10^?11 M. The nuclear matrix was able to bind androgen-receptor complexes in a saturable way, too.     

Recombinant Gaussia luciferase with a reactive cysteine residue for chemical conjugation: Expression, purification and its application for bioluminescent immunoassays

The mutated recombinant Gaussia luciferase (hgGLase) having the hinge sequence with a reactive cysteine residue at the carboxyl terminal region was purified from Escherichia coli cells by nickel-chelate affinity chromatography and hydrophobic chromatography. The biotinylated hgGLase (Biotin-hgGLase) was prepared by chemical conjugation with a maleimide activated biotin and apply to bioluminescent immunoassay. In the streptavidin and biotin complex system using Biotin-hgGLase, the measurable range of α-fetoprotein as a model analyte was 0.02–100 ng/ml with the coefficient of variation between 2.5% and 5.2%. The sensitivity of Biotin-hgGLase was similar to that by using the detection system of aequorin, alkaline phosophatase and horseradish peroxidase as a label enzyme.     

The static head method for determining the charge stoichiometry of coupled transport systems Applications to the sodium-coupled d-glucose transporters of the renal proximal tubule

The static head method for determining the charge stoichiometry (the number of moles of charge translocated per mole of substrate) of a coupled transport system is presented. The method involves establishing experimental conditions under which a membrane potential exactly balances the thermodynamic driving force of a known substrate gradient. The charge stoichiometry can then be calculated from thermodynamic principles. In contrast to the usual steady-state method for determining charge stoichiometry in cell suspensions and vesicle preparations, the static head method is applicable to systems which are not capable of maintaining a constant membrane potential over time. The charge stoichiometries of two renal sodium coupled d-glucose transporters previously identified in brush-border membrane vesicle preparations from the outer cortex (early proximal tubule) and outer medulla (late proximal tubule) are determined. The charge stoichiometries of these transporters are in good agreement with their sodium/glucose coupling ratios arguing against the possibility that glucose transport is coupled to ions other than sodium in these membranes.     

Characterization of the carcinoma-associated Tk antigen in helminth parasites

Expression of Tk antigen, a truncated carbohydrate antigen, was examined in helmith parasites. Using the monoclonal antibody LM389, this antigen was detected in extracts from Taenia hydatigena, Mesocestoides vogae (syn corti), and Taenia crassiceps. No reactivity was observed in Thysanosoma spp., Dipylidium caninum, Fasciola hepatica, and Nyppostrongylus brasiliensis. On the basis of their electrophoretic mobility, different patterns of Tk-bearing glycoproteins were observed among T. hydatigena, M. corti and T. crassiceps by immunoblotting, with certain components resolved as broad bands typical of mucin-like glycoproteins. Most Tk-reactive material remained in the 0.6 N perchloric acid-soluble fraction, confirming that Tk epitopes are carried by mucin-type glycoproteins. Immunohistochemical analysis revealed that in T. hydatigena, Tk antigen is mainly expressed in the tegument, whereas in M. corti the reactivity was principally observed in the subtegumental parenchyma. The presence of a novel tumor-associated carbohydrate antigen in invertebrates, contributes to strengthen the notion that truncated mucin-type O-glycosylation is a normal phenomenon in parasitic worms and may help identify new biological characteristics of helminth parasites.     

DNA polymerase and thymidine kinase activities in MC-29 virus-induced transplantable hepatoma and the effect of cytostatic treatment on these activities

The activity of DNA polymerases and thymidine kinase was compared in the MC-29 leukosis virus-induced transplantable hepatoma and in the livers of rats treated with cyclophosphamide (CP), cytosine-arabinoside (ara-C) and 5-fluoro-uracil (5-FU). The specific activity of DNA polymerase was twenty times greater in the MC-29 leukosis virus-induced hepatoma, while thymidine kinase was only 3–5 times greater than in the liver.All three enzymes showed Michaelis-Menten kinetics in their substrate and template saturation curves. The template utilization of DNA polymerases from hepatoma and from liver was compared. Both had higher activities on a poly(dA) · poly(dT) template at pH 8.0, than on DNA at pH 7.5. After chromatography on a phosphocellulose column two polymerases were separated. The first peak eluted by 0.15 M KCl preferred DNA as template (polymerase α). The second eluted by 0.5 M KCl worked better on poly(dA) · poly(dT) (polymerase β). Thymidine kinase was eluted by 0.25 M KCl. Inhibition by N-ethylmaleimide (NEM) showed the polymerase α to be sensitive and the polymerase β to be resistant to the sulfhydryl blocking agent; similar to the respective enzymes of other eukaryotic cells. The specific acitivity of DNA polymerase decreased after CP treatment at 6 h and 72 h and after ara-C treatment at 72 h. The specific activities of thymidine kinase were not changed significantly in response to the drug administrations.     

Tektin interactions and a model for molecular functions

Tektins from echinoderm flagella were analyzed for microheterogeneity, self-associations and association with tubulin, resulting in a general model of tektin filament structure and function applicable to most eukaryotic cilia and flagella. Using a new antibody to tektin consensus peptide RPNVELCRD, well-characterized chain-specific antibodies and quantitative gel densitometry, tektins A, B and C were found to be present in equimolar amounts in Sarkosyl-urea-stable filaments. In addition, two isoforms of tektin A are present in half-molar ratios to tektins B and C. Cross-linking of AB filaments indicates in situ nearest neighbor associations of tektin A1B and A2B heterodimers, -trimers, -tetramers and higher oligomers. Soluble purified tektin C is cross-linked as homodimers, trimers and tetramers, but not higher oligomers. Tektin filaments associate with both loosely bound and tightly bound tubulin, and with the latter in a 1:1 molar ratio, implying a specific, periodic association of tightly bound tubulin along the tektin axis. Similarly, in tektin-containing Sarkosyl-stable protofilament ribbons, two polypeptides ( approximately 67/73 kDa, homologues of rib72, efhc1 and efhc2) are present in equimolar ratios to each other and to individual tektins, co-fractionating with loosely bound tubulin. These results suggest a super-coiled arrangement of tektin filaments, the organization of which has important implications for the evolution, assembly and functions of cilia and flagella.     

Molecular heterogeneity in McArdle's disease

Biopsies were taken from a group of eleven patients with McArdle's disease, a congenital deficiency in muscle glycogen phosphorylase. The biopsies were screened by Western and Northern blotting for phosphorylase protein, phosphotrylase-bound pyridoxal-5′-phosphate (the cofactor of the enzyme) and for phosphorylase mRNA. Of the eleven patients, three expressed phosphorylase mRNA at near normal levels and at the expected size. One of these patients also expressed low levels of phosphorylase protein that correlated with a small amount of measurable phosphorylase activity. These data support the contention of molecular heterogeneity in the presentation of this phenotype.     

Thermodynamics of RTA3 peptide binding to membranes and consequences for antimicrobial activity

RTA3 is an α-helical, amphipathic peptide with broad-spectrum activity against Gram-negative bacteria and low mammalian cell toxicity. RTA3 contains a cysteine residue, replacement of which with an alanine or serine (RTA3-C15S) virtually abolishes antimicrobial activity. Much of the activity of RTA3 can be recovered in RTA3-C15L, indicating that the C15 residue functions largely as a bulky hydrophobic side chain promoting target cell membrane interactions. The poorly active RTA3-C15S is a useful variant for assessing the mechanistic aspects of RTA3 activity. Binding and membrane perturbation in vesicles containing different proportions of negative surface charge are analyzed in terms of amino acid-specific free energy contributions to interfacial binding, which likely underlie variations in antimicrobial activity amongst RTA3 variants. Comparison with published free energy scales indicates that the reduced electrostatic contribution to binding to membranes having reduced negative surface charge can be compensated in RTA3 (but not RTA3-C15S) by a slightly deeper insertion of the C-terminus of the peptide to maximize hydrophobic contributions to binding. Analysis of inner membrane (IM)- and outer membrane (OM)-selective permeabilization of Escherichiacoli demonstrates a broad similarity between peptide effects on vesicles with low negative surface charge (20% negatively charged lipids), E.coli membrane perturbation, and antimicrobial activity, supporting a role for membrane perturbation in the killing mechanism of RTA3. The results demonstrate that large variations in antimicrobial activity on subtle changes in amino acid sequence in helical amphipathic peptides can be rationalized in terms of the thermodynamics of peptide binding to membranes, allowing a more systematic understanding of antimicrobial activity in these peptides.     

A radioimmunoassay of plasma estradiol-17beta with the use of polyethylene glycol to separate free and antibody-bound hormone

A radioimmunoassay for plasma estradiol-17β was developed using polyethylene glycol to separate free from antibody-bound hormone. Specificity for estradiol-17β was achieved by a modified celite microcolunm procedure in which estradiol was.separated from interfering estrogens, including estrone. Using trace ³H-estradiol to monitor procedural losses, the method was shown to be sensitive and accurate. Intra- and inter-assay coefficient of variation of the method was 8.7 and 10.6%, respectively. Polyethylene glycol used for antibody precipitation appears to be a generally applicable method for steroid hormone radioimmunoassays. The simplicity, precision and rapid analysis, coupled with its lack of time dependence and ease in automation, makes this a convenient and practical method.     

A population of serum deprivation-induced bone marrow stem cells (SD-BMSC) expresses marker typical for embryonic and neural stem cells

The bone marrow represents an easy accessible source of adult stem cells suitable for various cell based therapies. Several studies in recent years suggested the existence of pluripotent stem cells within bone marrow stem cells (BMSC) expressing marker proteins of both embryonic and tissue committed stem cells. These subpopulations were referred to as MAPC, MIAMI and VSEL-cells. Here we describe SD-BMSC (serumdeprivation-induced BMSC) which are induced as a distinct subpopulation after complete serumdeprivation. SD-BMSC are generated from small-sized nestin-positive BMSC (S-BMSC) organized as round-shaped cells in the top layer of BMSC-cultures. The generation of SD-BMSC is caused by a selective proliferation of S-BMSC and accompanied by changes in both morphology and gene expression. SD-BMSC up-regulate not only markers typical for neural stem cells like nestin and GFAP, but also proteins characteristic for embryonic cells like Oct4 and SOX2. We hypothesize, that SD-BMSC like MAPC, MIAMI and VSEL-cells represent derivatives from a single pluripotent stem cell fraction within BMSC exhibiting characteristics of embryonic and tissue committed stem cells. The complete removal of serum might offer a simple way to specifically enrich this fraction of pluripotent embryonic like stem cells in BMSC cultures.     

Conformational conversion of PDE5 by incubation with sildenafil or metal ion is accompanied by stimulation of allosteric cGMP binding

Phosphodiesterase-5 (PDE5) is a dimer containing a cGMP-specific catalytic domain and an allosteric cGMP-binding subdomain (GAF A) on each subunit. PDE5 exhibits three conformational forms that can be separated by Native PAGE and are denoted as Bands 1, 2, and 3 in decreasing order of mobility. A preparation comprised mainly of Band 2 PDE5 was partially converted to Band 3 PDE5 by 1 h incubation with cGMP or the PDE5-specific inhibitors sildenafil, vardenafil, or tadalafil, but not with cAMP, milrinone (PDE3-specific), or rolipram (PDE4-specific). Band 2 PDE5 was converted almost entirely to Band 3 PDE5 by overnight incubation with sildenafil at 30 °C. This time-dependent conversion was accompanied by a 7-fold increase in allosteric cGMP-binding activity, suggesting that Band 3 PDE5 is a much more active form than Band 2 PDE5 for allosteric cGMP binding. Conversion of Band 2 PDE5 to Band 3 PDE5 occurred faster by pre-incubation with cGMP, which binds to both the allosteric and catalytic sites of PDE5, than with catalytic site-specific sildenafil. Overnight incubation of a Band 2/Band 3 PDE5 mixture with EDTA caused time-dependent conversion to Band 1 PDE5 (apoenzyme), and this conversion was accompanied by a 50% loss in cGMP-binding activity. After incubation with EDTA, addition of Mn⁺⁺ or Mg⁺⁺ caused reversion of Band 1 to a Band 2/Band 3 PDE5 mixture in which Band 3 PDE5 predominated. This reversion was accompanied by a 3-fold increase in allosteric cGMP-binding activity. The combination of results implied that physiological conversion of Band 2 to Band 3 PDE5 by cGMP and/or divalent metal ion occupancy of the catalytic domain would increase allosteric cGMP binding to the enzyme. This conversion would produce a greater negative feedback effect on cGMP action by increasing sequestration of cGMP at the allosteric cGMP-binding site of PDE5 and by increasing cGMP degradation at the catalytic site of the enzyme. This conversion would also increase PDE5 inhibitor binding to the enzyme.