Oxygen-dependent regulation of in vivo replication of simian virus 40 DNA is modulated by glucose.

Simian virus 40 (SV40)-infected CV1 cells exposed to hypoxia show an inhibition of viral replication. Reoxygenation after several hours of hypoxia results in new initiations followed by a nearly synchronous round of SV40 replication. In this communication, we examined the effect of glucose on inhibition of viral DNA replication under hypoxia. We found that glucose stimulated SV40 DNA replication under hypoxia in two different ways. First, the rate of DNA synthesis, i.e. the fork propagation rate, increased. This effect seemed to be mediated by inhibition of mitochondrial respiration by glucose (Crabtree effect). Inhibition of mitochondrial respiration probably resulted in a higher intracellular oxygen concentration and an activation of oxygen-dependent ribonucleotide reductase, which provides the precursors for DNA synthesis. This glucose effect was consequently strongly dependent on the strength of hypoxia and the extent of intracellular respiration; hypoxic gassing with 10 ppm instead of 200-400 ppm O(2) or treatment of hypoxic cells with a mitochondrial uncoupler (carbonyl cyanide m-chlorophenylhydrazone) reduced the glucose effect on replication, whereas antimycin A, an inhibitor of respiration, increased it. The second effect of glucose concerned initiation, i.e. stimulation of unwinding of the viral origin. This effect was not influenced by the strength of hypoxia or the extent of cellular respiration and seemed, therefore, not to be mediated through a Crabtree effect. No evidence for a direct correlation between the cellular ATP concentration and the extent of SV40 replication under hypoxia was found. The effect of glucose on replication under hypoxia was not restricted to SV40-infected CV1 cells but was also detectable in HeLa cells. This suggests it to be a mechanism of more general validity.

A low-molecular-weight cytosolic inhibitor of the specific testosterone binding in bovine seminal vesicles.

Bovine seminal vesicle cytosol contains a low-molecular-weight and thermostable substance which specifically inhibits the binding of testosterone to its cognate receptor. The mass and the ether phospholipid structure of the inhibitor were elucidated by mass spectrometry. Saturation and binding experiments indicate that the inhibitor acts in a dose-dependent and competitive manner, altering the apparent dissociation constant (K(D)) while maintaining the number of androgen-binding sites (B(max)). Its possible role in the regulation of androgen binding activity is discussed.

Molecular symmetry of the dodecamer subunit of Lumbricus terrestris hemoglobin.

The principal functional subunit of the approximately 3500 kDa extracellular Lumbricus terrestris hemoglobin is a 213 kDa dodecamer of four chemically distinct globin chains, consisting of a non-covalent complex of three trimer submits (disulfide-bonded chains a, b and c) and three monomer subunits (chain d). X-ray diffraction of crystals of the dodecamer grown at neutral pH, were found to be monoclinic, with the unit cell dimensions: a = 112.3 A, b = 190.0 A, c = 69.6 A, beta = 102.0 degrees with h + k + l = 2n + 1 absent, characteristic of space group I121. In addition, these crystals exhibit a pseudo trigonal P321 symmetry with unit cell dimensions a = 190.5 A, b = 190.5 A, c = 69.5 A, gamma = 120.0 degrees. Assuming that the assymetric unit contains an entire dodecamer, a model of the latter was constructed that satisfies the symmetry of the trigonal pseudo cell and is consistent with the symmetry of the I121 crystallographic cell. The resulting model has strong implications concerning the hexagonal bilayer structure of the native hemoglobin.

A biotin-streptavidin amplified enzymeimmunoassay for 13,14-dihydro-15-keto-PGF2 alpha.

As an alternative for radioimmunoassays a new enzyme immunoassay (EIA) for the determination of 13,14-dihydro-15-keto PGF2 alpha (PGFM) has been developed. Biocytin was linked to PGFM by the N-hydroxysuccinimide method and the product (biocytinyl-PGFM) purified by reversed phase column chromatography. Biocytinyl-PGFM was used in the EIA as a bridge between the immobilized PGFM-antibody and streptavidin-peroxidase. The absolute sensitivity of the assay was about 160 amol (92% rel. binding) and required only 2 microliters plasma for PGFM estimation within the whole physiological range (0.08-20 pmol/ml). All variabilities were less than 14%. The described assay procedure may be of general applicability for other prostaglandins.

Reactivation of the androgen receptor from murine preputial gland by thioredoxin or GSH.

The androgen receptor from murine preputial gland was inactivated by density gradient centrifugation, affinity chromatography and isoelectric focusing. The hormone binding of the receptor could be partially restored (roughly 40%) by the thioredoxin-thioredoxinreductase system, by thioredoxin plus dithiothreitol or by glutathione. Dithiothreitol, by itself, was unable to reactivate the androgen receptor. These findings show that the receptor inactivation is, at least partially, due to thiol group oxidation and removal of SH-reducing and/or -stabilizing substances from the receptor during purification.

Effects of available P and N:P ratios on non-symbiotic dinitrogen fixation in tallgrass prairie soils.

Prescribed burning is a major control over element cycles in Tallgrass prairie (Eastern Kansas, USA). In this paper we report potential effects of fire on nonsymbiotic nitrogen fixation. Fire resulted in additions of available P in ash, which may stimulate nitrogen fixation by terrestrial cyanobacteria. Cyanobacterial nitrogenase activity and biomass responded positively to additions of ash or P in laboratory assays using soil. Further assays in soil showed that cyanobacteria responded to changes in available N:available P ratio (aN:P) across a range of concentrations. Nitrogen fixation rate could be related empirically to aN:P via a log-linear relationship. Extrapolation of laboratory results to the field yielded a maximal estimate of 21 kg N ha-1 y-1. Results support arguments from the marine and terrestrial literature that P availability is central to regulation of ecosystem N budgets.

cAMP binds with high affinity and specificity to the androgen receptor from murine skeletal muscle.

cAMP binding of the androgen receptor (AR) from murine skeletal muscle was studied. Testosterone affinity chromatography yielded androgen receptor with about 4000-fold purification. Determination of the cAMP binding in the affinity eluate, by adsorption of protein-cAMP complexes to cellulose ester filters or removal of unbound cAMP by dextran-coated charcoal, was not possible, as the observed binding was not stable during the assays. Displacement studies suggest that this is due to a very fast dissociation kinetics of the binding. The problem could be solved by assaying the components of affinity eluate immobilized to a testosterone affinity resin that stabilizes the cAMP-protein complexes. The cAMP binding found in the affinity eluate shows an upward concave Scatchard plot and is compatible with a model containing two independent binding sites with dissociation constants of 7 and 58 nM. However, a larger number of binding sites or negative cooperativity cannot be excluded. Sixteen cAMP binding sites were observed per testosterone binding site. The binding affinity of cAMP exceeds that of cGMP 200-fold, that of cCMP 2000-fold, and that of AMP and 2',3'-cAMP more than 10,000-fold. Results indicate that cAMP is bound by the AR, although it only represents about 1% of the total protein in the affinity eluate: (i) Specific testosterone and cAMP binding of affinity eluate was copurified by affinity chromatography, density gradient centrifugation, and gel filtration. The ratio of cAMP to testosterone binding in each peak was about 16:1, identical with that found in the total affinity eluate.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of testosterone and testosterone receptor complexes with nuclei of skeletal muscle from intact male mice and from mice bearing the testicular feminization (Tfm) mutant gene.

With the nuclear exchange assay and the nuclear retention assay it is shown that the androgen insensitivity of Tfm mice is probably due to a defect of the nuclear acceptor sites for the testosterone receptor complex. Further on the results obtained point strongly to the possibility that hormone free androgen receptor is localized in the nuclei and in the cytoplasma according to the "equilibrium model". A practicable method for separation of unbound steroids from nuclei is described.

Isolation and characterization of the androgen receptor of murine preputial gland.

The androgen receptor of murine preputial gland showed in binding experiments a biphasic saturation curve and a biphasic Scatchard plot. The receptor converted from an 8.5-9 S form to a 4.5-5 S form in high ionic strength buffer. The apparent dissociation constant was KD 0.5 +/- 0.2 nM for the 8.5-9 S receptor form. A 6.5-7 S receptor form could be detected in some experiments. The ligand specificity was evaluated by competition experiments: testosterone greater than androstenedione greater than dihydrotestosterone greater than androstanediol greater than estradiol greater than progesterone greater than dexamethasone. The receptor of murine preputial gland was less stable than the androgen receptor of skeletal muscle of the same mice.

Inhibition of cAMP-phosphodiesterase by molybdate.

Inhibition of 3':5'-cyclic-AMP-5'-nucleotidohydrolase (EC 3.1.4.17) type II (cAMP-phosphodiesterase) by sodium molybdate was studied: While determination of inorganic phosphate and 5'-ribonucleotide phosphohydrolase (5'-nucleotidase) (EC 3.1.3.5) activity was not disturbed by sodium molybdate in concentrations up to 10 mM, cAMP-phosphodiesterase was inhibited by millimolar concentrations of molybdate. The half maximal effect was observed at about 2 mM sodium molybdate (0.75 mM cAMP in the assay).

In vitro transformation of androgen receptor from murine skeletal muscle by cAMP.

Sedimentation constants and DNA-cellulose-binding of cytosolic androgen receptor from murine skeletal muscle were determined in presence of cyclic nucleotides. Without cAMP, two testosterone-binding fractions of similar amount at 4-5S and 8-9S were obtained. With 3 microM cAMP the receptor sedimented predominantly at 4-5S. Binding of testosterone-receptor-complexes to DNA-cellulose was enhanced by increasing cAMP-concentrations and reached a maximum at 20-90 nM cAMP depending on the DNA-concentrations in the test. A similar DNA-binding characteristic was obtained after partial purification of the receptor by affinity chromatography. cGMP had no effect. We conclude that the androgen receptor is transformed in vitro by cAMP.

Piracetam improves visuomotor and cognitive deficits in early Parkinsonism--a pilot study.

The influence of Piracetam on Parkinsonism was studied in 18 patients and 18 matched controls. Clinical, visuomotor and psychometric variables were measured. Piracetam improved visuomotor reaction time (RT) and accuracy in 6 mildly affected and tracing time in 6 moderately affected patients, the clinical condition and the organic brain syndrome in all patients investigated. The improvement of the prolonged RT seems to be correlated with bradyphrenia. No drug influence could be observed in the prolonged interhemispheric transfer time. As the mildly affected patients displayed the clearest effect of Piracetam, its administration in early and mild stage of parkinsonism is recommended.

Correlation of the 4-5 S form and the 8 S form of the cytosolic androgen receptor in murine skeletal muscle.

Binding experiments with the cytosolic androgen receptor from murine skeletal muscle yield with testosterone a biphasic saturation curve and a biphasic Scatchard plot. These binding characteristics result from the conversion of 8 S receptor (KD = 1,4 X 10(-10) M) into 4-5 S receptor (KD = 1,2 X 10(-9) M). This conversion is androgen dependent and is facilitated in vitro by either UV-irradiation or by methods known to activate steroid hormone receptor complexes to a nuclear binding form (e.g. high ionic strength or elevated temperature). The measured data show that both receptor forms are in a complex dissociation equilibrium. The reassociation of the 4-5 S receptor to form the 8 S complex is inhibited by RNase.

Structure and activity of insulin, XVI. Semisyntheses of desheptapeptide-(B24--30)- up to destripeptide-(B28--30)-insulin with lysine or alanine in place of arginine in position B22: influence on the three-step-increase of activity in positions B24--26 (Phe-Phe-Tyr).

The desonapeptide-(B22--30)-insulin pentamethyl ester, protected with Boc- at the two N-terminal amino groups, was prepared as described in the preceding XVth communication[6]. The free carboxyl group of the glutamic acid residue B21 of this compound was coupled to the following synthetic oligopeptide esters (X = Lys or Ala): X-Gly-OMe X-Gly-Phe-OMe X-Gly-Phe-Phe-OME X-Gly-Phe-Phe-Tyr-OMe X-Gly-Phe-Phe-Tyr-Ala-OMe After coupling, the semisynthetic products were deprotected and purified. Their biological activities were determined in the mouse fall test and by measurement of blood glucose levels. There were no statistical differences between the values obtained for the lysine B22 and alanine B22 products. The three-step increase in activity due to the amino acids Phe-Phe-Tyr (B24--26) was still recognizable, but compared with the analogues containing arginine B22, the activities were very stronly diminished. These results are in contrast with the assumption that activity of insulin is dependent on the formation of a strong ionic linkage between the asparagine-A21 carboxyl group and any positive charge in B22. The results, however, demonstrate the high specificity of the arginine guanidino group in position B22.

Further studies on the three-step-increase in activity due to the aromatic amino acids B24-26 (-Phe-Phe-Tyr-).

Using a reaction suite which was suggested by Ruttenberg [5] for the semisynthesis of insulin variants, insulin hexamethyl ester was digested by trypsin, then the N-terminal amino groups of the resulting desoctapeptide insulin pentamethyl ester were protected with the Boc residue. The free carboxyl group of the arginyl residue (B22) of this product was coupled to two different series of synthetic peptide methyl esters: I) Gly-OMe, Gly-Phe-OMe, Gly-Phe-Phe-OMe, Gly-Phe-Phe-Tyr-OMe and II) Gly-Ala-OMe, Gly-Phe-Ala-OMe, Gly-Phe-Phe-Ala-OMe, Gly-Phe-Phe-Tyr-Ala-OMe. Removal of all protecting groups yielded the corresponding insulin variants. The syntheses of these peptide methyl esters are described. Following the original prescription of Ruttenberg[5], we were not able to prepare the desired variants. That is why we were forced to change some important details of the Ruttenberg[5] recipe. The activity determinations by the mouse fall test showed the weak activity (ca. 4%) of the desoctapeptide insulin (C-terminus Arg B22). This activity increases drastically in three steps, when the amino acids Phe, Phe, Tyr (B24-26) are added successively to the insulin trunk. Coupling of Gly-Phe yields 14%, -Gly-Phe-Phe 36%, and -Gly-Phe-Phe-Tyr 61% of the biological activity (cryst. insulin=100%). The same peptides, elongated at their C-terminis with an alanyl residues (see above, series II) yield higher activities. Coupling these peptides to the arginyl residue B22 increases the activity as follows: -Gly-Phe-Ala, 36%, -Gly-Phe-Phe-Ala, 59%, and -Gly-Phe-Phe-Tyr-Ala, 91%. Comparing the activities of the variants with the C-termini-Gly-Phe-Phe (36%) and -Gly-Phe-Ala (36%) or -Gly-Phe-Phe-Tyr (61%) and -Gly-Phe-Phe-Ala (59%), it becomes clear that the aromatic amino acids Phe (B25) and Tyr (B26) can be substituted by Ala without loss of activity. In our preceding work (published 1969-1973 [3, 6-8]), we synthesized successively shortened insulin B-chains which yielded, after combination with natural A-chain, practically the same activity values as we have now obtained with the Ruttenberg semisynthesis. As we have already mentioned l.c.[1-4], it is obvious that the activity of insulin proceeds from the arginyl residue (B22) and is only intensified by the aromatic amino acids (B24-26). We[2,3] observed the same three-step increase in activity in the case of our synthetic oligopeptides Arg-Gly-Phe, Arg-Gly-Phe-Phe and Arg-Gly-Phe-Phe-Tyr (B22-26), which we assume to be the active region of insulin (1971[2]).

[Identification and biological activity of peptides containing a partially benzyloxycarbonylated L-arginine on their amino terminus (author's transl)]. / Identifizierung und biologische Aktivität von Peptiden mit aminoendständigem, partiell benzyloxycarbonyliertem L-Arginin

Nomega-Z-L-Arg-L-Phe-L-Phe was shown to be the antibiotically active compound in a peptide mixture which was obtained by treating Z3-L-Arg-L-Phe-L-Phe with hydrogen bromide/trifluoroacetic acid or 4N HBr/glacial acetic acid, respectively. Identification of this compound was achieved by thin-layer chromatography, enzymatic digestion and autobiograms with fungi. The pure Nomega-Z-L-Arg-L-Phe-L-Phe was not the only compound with antibiotic qualities; generally it could be said that all peptides with the sequence Nomega-Z-L-Arg-X-L-Phe (X might be any amino acid) are antibiotically active. All of them are antagonized by L-aspartic acid and asparagine in the crossstrip test (on fungi). The antibiotical activity of all these peptides must be due to the Nomega-Z-L-Arg-residue provided that it is coupled to a dipeptide X-L-Phe, or to an aromatic system (e.g. L-Phe or benzyl amine).

[Sequence variation on an antibiotically active tripeptide (author's transl)]. / Sequenzvariationen an einem antibiotisch wirksamen tripeptid

Seven tripeptides with the sequence L-Arg-D,L-X-L-Phe were synthesised. Three of these peptides showed antibiotical activity on fungi. The amino- and the carboxylic terminus of the peptide L-Arg-D,L-Phe-L-Phe, which showed antibiotical activity, were changed to give the sequences L-X-D,L-Phe-L-Phe or L-Arg-D,L-Phe-L-X respectively. The resulting tripeptides showed no antibiotical activity.

[Antibiotically active tripeptides of the sequence L-arg-D-X-L-phe] (author's transl)]. / Antibiotisch wirksame Tripeptide der Sequenz L-Arg-D-X-L-Phe

Five tripeptides with the sequence L-Arg-D-X-L-Phe showed antibiotic activity on fungi and on some pathogenic moulds. The action of the peptides could be neutralized in the cross-strip test by the L-amino acid corresponding to the D-amino acid in the middle position.