Structures of manganese(II) complexes with ATP, ADP, and phosphocreatine in the reactive central complexes with creatine kinase: electron paramagnetic resonance studies with oxygen-17-labeled ligands

Coordination of Mn(II) to the phosphate groups of the substrates and products in the central complexes of the creatine kinase reaction mixture has been investigated by electron paramagnetic resonance (EPR) spectroscopy with regiospecifically 17O-labeled substrates. The EPR pattern for the equilibrium mixture is a superposition of spectra for the two central complexes, and this pattern differs from those observed for the ternary enzyme-Mn(II)-nucleotide complexes and from that for the dead-end complex enzyme-Mn(II)ADP-creatine. In order to identify those signals that are associated with each of the central complexes of the equilibrium mixture, spectra were obtained for a complex of enzyme, Mn(II)ATP, and a nonreactive analogue of creatine, 1-(carboxymethyl)-2-iminoimidazolidin-4-one, which is a newly synthesized competitive inhibitor. This inhibitor permits an unobstructed view of the EPR spectrum for Mn(II)ATP in the closed conformation of the active site. The EPR spectrum for this nonreactive complex with Mn(II)ATP matches one subset of signals in the spectrum for the equilibrium mixture, i.e., those due to the enzyme-Mn(II)-ATP-creatine complex. Chemical quenching of the samples followed by chromatographic assays for both ATP and ADP indicates that the enzyme-Mn(II)ADP-phosphocreatine and the enzyme-Mn(II)ATP-creatine complexes are present in a ratio of approximately 0.7 to 1. A similar value for the equilibrium constant for enzyme-bound substrates is obtained directly from the EPR spectrum for the equilibrium mixture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oligonucleotides covalently linked to intercalating agents. Influence of positively charged substituents on binding to complementary sequences

A pentamethylene chain was used to covalently link the 3'-phosphate of oligothymidylates to the 9-amino group of an acridine derivative. Positively charged substituents were further attached to the 3'-phosphate group to form 3'-phosphotriesters. These molecules form specific complexes with poly(rA) which involve the formation of a number of A X T base pairs equal to that of thymines in the oligonucleotide. Absorption changes induced in the acridine absorption bands are similar to those expected upon intercalation of the acridine dye between A X T base pairs. The acridine covalently linked to the 3'-phosphate strongly stabilizes the complexes formed with poly(rA) as compared with the corresponding unsubstituted oligodeoxynucleotide. The presence of a positively charged substituent on the 3'-phosphate together with the acridine dye further enhances the interaction. The effect of salt concentration on complex stability depends on the number of negatively charged phosphate groups of the oligodeoxynucleotide and on the nature of the substituents borne by the 3'-phosphate group. When the oligothymidylate is substituted by an acridine dye, the stability of the poly(rA) complexes increases when salt concentration increases. If an additional positively charged substituent is present on the 3'-phosphate group, stability decreases when salt concentration increases for the shortest oligonucleotide (trimer) and increases with longer oligonucleotides. Thermodynamic parameters have been calculated from the concentration dependence of melting temperatures.     

SERGE, the subcellular site of initial hepatic glycogen deposition in the rat: a radioautographic and cytochemical study

Hormonal control of hepatic glycogen and blood glucose levels is one of the major homeostatic mechanisms in mammals: glycogen is synthesized when portal glucose concentration is sufficiently elevated and degraded when glucose levels are low. We have studied initial events of hepatic glycogen synthesis by injecting the synthetic glucocorticoid dexamethasone (DEX) into adrenalectomized rats fasted overnight. Hepatic glycogen levels are very low in adrenalectomized rats, and DEX causes rapid deposition of the complex carbohydrate. Investigation of the process of glycogen deposition was performed by light and electron microscopic (EM) radioautography using [3H]galactose as a glycogen precursor. Rats injected with DEX for 2-3 h and [3H]galactose one hour before being killed displayed an increasing number of intensely labeled hepatocytes. EM radioautography revealed silver grains over small (+/- 1 micron) ovoid or round areas of the cytosome that were rich in smooth endoplasmic reticulum (SER) and contained a high concentration of small dense particles. These distinct areas or foci of SER and presumptive glycogen (SERGE) were most numerous during initial periods of glycogen synthesis. After longer exposure to DEX (4-5 h) more typical deposits of cytoplasmic glycogen were evident in the SERGE regions. Several criteria indicated that the SERGE foci contained glycogen or presumptive glycogen: resemblance of the largest dense particles to beta-glycogen particles in EM; association of 3H-carbohydrate with the foci; removal of particles and label with alpha-amylase; and positive reaction with periodic acid-chromic acid-silver methenamine. The concentration of SER in the small foci and the association of newly formed glycogen particles with elements of SER suggest a role for this organelle in the initial synthesis of glycogen.     

Dolichodorus miradvulvus n. sp. (Nematoda: Tylenchida) with a Key to Species

Dolichodorus miradvulvus n. sp. from Anubias nana Engler in Florida is described and illustrated. The female is characterized by deep grooves in the cuticle on the ventral surface just anterior and posterior to the vulva, and by transversely elongate pouches anterior and posterior to the vulva. Both sexes have a constricted area of the stylet shaft just anterior to the knobs, and on the male the intersection of the lateral field and bursa appear sclerotized.     

A stochastic model of mutant growth due to mutation in tumors,based on stem cell considerations

A stochastic model of the evolution of mutant subpopulations from stem cells in a human tumor system is derived. From the model, the growth of mutants (both stem cell mutants and overall mutants) due to mutation of tumor stem cells during growth is explored in detail. This allows one to relate the mutant stem cell and overall tumor mutant cell population sizes. The relation of these average sizes is derived for large tumor size and confirms the result of the model due to MacKillop et al. [4], which is based on three tumor cell subpopulations: stem, transitional, and end cells. Furthermore, the results of the stem cell statistics obtained are the same as those obtained from the filtered Poisson process approach [2].     

3α, 11α-Dihydroxy-23-oxo-lup-20(29)-en-28-oic acid from Acanthopanax trifoliatus

The new triterpene 3α,11α-dihydroxy-23-oxo-lup-20(29)-en-28-oic acid was isolated from Acanthopanax trifoliatus. Its structure has been determined on the basis of spectroscopic data and chemical transformations.     

Dopamine Sulfoconjugation in the Rat Brain: Regulation by Monoamine Oxidase

An increase of free 3,4-dihydroxyphenylethylamine (DA, dopamine) in the rat brain such as is found following 3,4-dihydroxyphenylalanine (L-DOPA) administration or an intraventricular injection of free dopamine did not result in DA sulfate formation, despite the presence of phenolsulfotransferase activity in various regions of the brain and the high affinity of DA for this enzyme. However, when rats were pretreated with pargyline, a monoamine oxidase inhibitor, the same treatment with L-DOPA or free DA led to active synthesis of DA sulfate. The increase in DA sulfate was significantly correlated with the degree of monoamine oxidase inhibition and directly proportional to free DA concentrations in the hypothalamus (r = 0.86), striatum (r = 0.54), and brainstem (r = 0.89). The highest ratio of DA sulfate to free DA was found in the hypothalamus, suggesting that sulfoconjugation is most active in this region. Prior treatment of rats with 6-hydroxydopamine did not decrease DA sulfate concentrations, indicating that sulfoconjugation occurs most likely in extraneuronal tissues not destroyed by the neurotoxin. The results are compatible with the notion that phenolsulfotransferase may be highly compartmentalized and that inhibition of monoamine oxidase allows the newly generated free DA to become accessible to the sulfoconjugating enzyme, resulting in increase in DA sulfation.     

Disturbances in toxicological experiments caused by Microsporum canis

Cutaneous lesions which can lead to false positive results have been observed in several rabbits used for the determination of the cutaneous irritation capacity of a product (ITA, PII). The responsible agent was Microsporum canis. A preventive treatment by an antifungal agent did not modify toxicological experimental results.     

Evidence for a fucose-binding protein in boar spermatozoa

A fucose binding protein was detected in boar spermatozoa by means of a specifically developed modified enzyme-linked-lectin-assay using glycosylated peroxidase derivatives. The distribution of the fucose binding protein was assessed by means of fluorescence microscopy with fluoresceinyl-glycosylated peroxidase. Fucose binding was particularly prominent at the apical region of the sperm head. In order to gain more insight into the precise localization of the carbohydrate binding protein electron microscopical studies were performed using fucosyl peroxidase coupled to colloidal gold. In ultrathin sections as well as in specimens prepared in toto for TEM an intensive binding of fucosylperoxidase-colloidal gold was predominantly found at the apical part of the acrosome appearing as a crescent-like area. In some cases this binding pattern was replaced by a triangle-like intensive labelling at the equatorial segment as revealed clearly by specimens prepared in toto. By SDS-PAGE of the SDS-extractable sperm-proteins, followed by transblotting to nitrocellulose and visualization with the fucosylperoxidase by enzymatic amplification with 4-chloro-1-naphthol mainly one protein with the reduced molecular weight of approximately 53 kdal and some small proteins with apparent molecular weights less than 20 kdal was found to be responsible for the fucose-binding ability of porcine spermatozoa.     

Correlation between limitation of growth ofPachysolen tannophilus on D-xylose with the formation of ethanol and other products

The formation of ethanol, xylitol, ribitol, arabitol and acetic acid from D-xylose byPachysolen tannophilus correlated with the limitation of growth. The correlation was consistent with these products being secondary metabolites.Issued as NRCC Publication Number 24259.