Isolation and screening of potential actinobacteria for rapid composting of rice straw

Rice straw is produced as a by-product from rice cultivation, which is composed largely of lignocellulosic materials amenable to general biodegradation. Lignocellulolytic actinobacteria can be used as a potential agent for rapid composting of bulky rice straw. Twenty-five actinobacteria isolates were isolated from various in situ and in vitro rice straw compost sources. Isolates A2, A4, A7, A9 and A24 were selected through enzymatic degradation of starch, cellulose and lignin followed by the screening for their adaptability on rice straw powder amended media. The best adapted isolate (A7) was identified as Micromonospora carbonacea. It was able to degrade cellulose, hemicelluloses and carbon significantly (P ≤ 0.05) over the control. C/N ratio was reduced to 18.1 from an initial value of 29.3 in 6 weeks of composting thus having the potential to be used in large scale composting of rice straw.     

Rat ovarian angiotensin II receptors. Characterization and coupling to estrogen secretion

Angiotensin II receptor agonist (125I-angiotensin II) and antagonist (125I-[Sar1,Ile8]angiotensin II) bind in a specific and saturable manner to rat ovarian membranes. Agonist and antagonist binding affinity (KD approximately 0.5 nM) and the number of sites estimated (Bmax approximately 60 fmol/mg of protein) were similar. Dissociation of receptor-bound agonist was more rapid than the dissociation of receptor-bound antagonist, and agonist, but not antagonist, dissociation from the receptor was accelerated by GTP gamma S. A 0-150 mM increase in Na+ produced a 27% increase in the KD of agonist binding. Antagonist binding was not modified by Na+. These studies suggest that both agonist and antagonist identify putative angiotensin II receptors in the ovary but that the properties of agonist and antagonist binding are distinct. Angiotensin II antagonist binding sites are present on the granulosa cell layer of rat ovarian follicles (Speth, R. C., Bumpus, F. M., and Husain, A. (1986) Eur. J. Pharmacol. 130, 351-352). To determine the role of angiotensin II in ovarian function, we examined angiotensin II receptors and function during the onset of puberty. High affinity and low capacity angiotensin II receptors were present in ovaries from immature rats. After pregnant mare's serum gonadotropin induced ovulation in immature rats, antagonist binding to total ovarian membranes increased over 3-fold. In vitro incubation of peripubertal ovaries with 1 microM angiotensin II produced a stimulation of estrogen, but not progesterone, secretion. This steroidogenic effect of angiotensin II was most pronounced in the luteal phase of the estrus cycle. These studies point toward the involvement of angiotensin II in the regulation of ovarian function, possibly through modulation of follicular estrogen levels.     

Quantitative DNA analysis of low grade cervical intraepithelial neoplasia and human papillomavirus infection by static and flow cytometry.

Quantitative deoxyribonucleic acid (DNA) analysis of cervical biopsy specimens from 26 women with cytological, colposcopic, and histological evidence of mild cervical atypia consistent with cervical intraepithelial neoplasia grade I, reactive atypia, or human papillomavirus infection alone or in combination was performed in a comparative evaluation of Feulgen microspectrophotometry, the fast interval processor image analysis system, and flow cytometry. The fast interval processor image analysis system showed a distinct advantage over the other methods, being faster and allowing the operator to see the cells that were selected for measurement. The three methods of measurement together showed that the DNA content of at least 2% of the cells measured exceeded 5C (C being the haploid amount of DNA in a normal cell and 2C representing the diploid complement of a normal cell) in all cases of cervical intraepithelial neoplasia grade I and reactive atypia and in 87% of those reported as showing human papillomavirus infection alone. In contrast, the DNA content of cervical biopsy specimens from the transformation zone of 11 normal controls did not exceed 4C. This study shows the value of using a DNA threshold--that is, the "5C exceeding rate"--to distinguish between normal and neoplastic appearances of the cervix. These results support the view that cervical infection by human papillomavirus is a true precursor of neoplasia.     

Polygalacturonase activity and variation in ripening of papaya fruit with tissue depth and heat treatment

Effects of tissue position (viz. outer vs inner mesocarp) and heat treatment (48°C, 20 min) on variations in polygalacturonase (EC 3.2.1.15 and EC 3.2.1.67) activity and ripening of fruits of Carica papaya L. cv. Backcross Solo were investigated. Polygalacturonase activity increased during ripening concomitantly with an increase in tissue softness and soluble polyuronide level. Throughout ripening, inner mesocarp tissue was softer and contained higher polygalacturonase activity than outer mesocarp tissue. Titratable acidity as well as ß-galactosidase (EC 3.2.1.23) activity also increased during ripening; however, unlike polygalacturonase, their level or activity was lower in inner than in outer mesocarp. Ascorbic acid could partially account for the increase in titratable acidity during ripening but contributed very little to the differences in titratable acid levels between outer and inner mesocarp. Heat treatment had no effect on either fruit softness or titratable acidity, but it markedly reduced the increase in ascorbic acid and polygalacturonase activity during ripening. Ripening, as reflected by changes in tissue softness and polygalacturonase activity, progressed outwardly from the interior towards the exterior of the fruit. The effect of heat treatment in suppressing polygalacturonase activity was relatively greater in inner than in outer mesocarp, suggesting that sensitivity of the enzyme to heat treatment may vary with stage of ripeness of the tissue.     

N-succinimidyl methoxyphenylacetic acid ester, an amine-directed chiral derivatizing reagent suitable for enzymatic scale resolutions

A chiral derivatizing reagent, N-succinimidyl-2-(S)-methoxy-2-phenylacetic acid ester (SMPA), directed toward reaction with primary amine-containing compounds has been synthesized and characterized. This reagent is suitable for HPLC resolution from enzymatic-scale reactions where only microgram quantities of chiral products may be obtainable. SMPA derivatization was shown to be effective in the resolution of the enantiomers of a number of different racemic compounds. SMPA was used to resolve the diastereoisomeric derivatives of a previously unknown enzymatically oxygenated product, allowing determination of the stereochemical course of the enzymatic reaction. SMPA is easily prepared from an inexpensive, commercially available, and enantiomerically pure precursor with the formation of a shelf-stable crystalline product which is utilizable in water-containing solutions. In addition to its usefulness for micro-determinations, SMPA is useful for preparative-scale resolutions of enantiomers since the reagent is cleaved from the diastereoisomeric derivative by acid hydrolysis.     

Unusual redox properties of electron-transfer flavoprotein from Methylophilus methylotrophus

The most positive redox potential ever recorded for a flavin adenine dinucleotide (FAD) containing protein has been measured for an electron-transfer flavoprotein (ETF) synthesized by Methylophilus methylotrophus. This potential value, 0.196 V versus the standard hydrogen electrode (vs SHE), was measured at pH 7.0 for the one-electron reduction of fully oxidized ETF (ETFox) to the red anionic semiquinone form of ETF (ETF.-). Quantitative formation of ETF.- was observed. The first successful reduction of ETF from M. methylotrophus to its two-electron fully reduced form was also achieved. Although addition of the second electron to ETF.- was extremely slow, the potential value measured for this reduction was -0.197 V vs SHE, suggesting a kinetic rather than thermodynamic barrier to two-electron reduction. These data are believed to be consistent with the postulated catalytic function of ETF to accept one electron from the iron-sulfur cluster of trimethylamine dehydrogenase (TMADH). The second electron reduction appears to have no catalytic function. The very positive potential measured for this ETF and the wide separation of potentials for the two electron reduction steps show that this ETF is a unique and interesting flavoprotein. In addition, this work highlights that while ETFs exhibit similar structural and spectral properties, they display wide variations in redox properties.     

Resonance Raman spectroscopy of amicyanin, a blue copper protein from Paracoccus denitrificans

The copper binding site of amicyanin from Paracoccus denitrificans has been examined by resonance Raman spectroscopy. The pattern of vibrational modes is clearly similar to those of the blue copper proteins azurin and plastocyanin. Intense resonance-enhanced peaks are observed at 377, 392, and 430 cm-1 as well as weaker overtones and combination bands in the high frequency region. Most of the peaks below 500 cm-1 shift 0.5-1.5 cm-1 to lower energy when the protein is exposed to D2O. Based on the pattern of conserved amino acids, the axial type EPR spectrum, and the resonance Raman spectrum, it is proposed that the copper binding site in amicyanin contains a Cu(II) ion in a distorted trigonal planar geometry with one cysteine and two histidine ligands and an axial methionine ligand at a considerably longer distance. Furthermore, the presence of multiple intense Raman peaks in the 400 cm-1 region which are sensitive to deuterium substitution leads to the conclusion that the Cu-S stretch is coupled with internal ligand vibrational modes and that the sulfur of the cysteine ligand is likely to be hydrogen-bonded to the polypeptide backbone.     

Properties of Paracoccus denitrificans amicyanin

Paracoccus denitrificans synthesizes an inducible, periplasmic, blue copper protein [Husain, M., & Davidson, V.L. (1985) J. Biol. Chem. 260, 14626-14629] that can be classified as an amicyanin on the basis of its ability to accept electrons from methylamine dehydrogenase. The amino acid composition and sequence of the 10 N-terminal residues of this protein have been determined. From these data, it is evident that amicyanin is structurally distinct from azurins as it contains no disulfide bond and an N-terminal sequence that is completely different from the highly conserved N-terminal azurin sequences. Dialysis of reduced amicyanin against potassium cyanide resulted in a nearly quantitative yield of apoamicyanin. Amicyanin and apoamicyanin exhibit fluorescence emission maxima at 314 nm when excited at 280 nm. Addition of 6 M guanidine hydrochloride shifts these emission maxima to 350 nm. The fluorescence intensity of apoamicyanin is 10-fold greater than that of amicyanin. Addition of copper to the apoprotein caused a stoichiometric quenching of fluorescence and restoration of visible absorbance with no concomitant change in absorbance at 280 nm. At least one cysteine residue, which reacts with 5,5'-dithiobis(2-nitrobenzoic acid) in apoamicyanin, does not react in the holoprotein, even in the presence of 6 M guanidine hydrochloride. Reductive and oxidative titrations of amicyanin indicate that it is a one-electron carrier. This amicyanin is also able to accept electrons from the methylamine dehydrogenase isolated from bacterium W3A1, which is taxonomically very different from P. denitrificans.     

Preliminary X-ray crystallographic study of amicyanin from Paracoccus denitrificans

Single crystals have been prepared of Paracoccus denitrificans amicyanin, a blue copper protein that serves as an electron acceptor for methylamine dehydrogenase. The crystals belong to the monoclinic space group P2(1), and have unit cell parameters a = 20.90 A, b = 56.61 A, c = 27.55 A and beta = 96.41. There is one molecule in the asymmetric unit. The crystals diffract to beyond 1.5 A resolution.