Fine structure of DNA melting curves.

Theoretical calculations predict that the differential melting curves for random polynucleotide sequences having lengths up to several tens of thousands of base pairs have a clear-cut fine structure. This structure appears in the form of multiple narrow peaks 0.3–0.4°C wide on the bell shaped main curve. The differential melting curves have different shapes for different specific sequences. The theory also predicts the disappearance of the fine structure when the length of the sequence increases and when circular, covalently closed DNA is considered instead of the open structure. The predictions of the theory were confirmed by the measurements of differential melting curves for open and covalently closed circular forms of DNA for PM2 phage (N = 10⁴ base pairs) and also for other phage DNA's of different length: T7 (N = 3.8 × 10⁴); S_D (N = 9.2 × 10⁴); T2 (N = 17 × 10⁴). It was shown that the effect of fine structure results mainly from the cooperative melting out of DNA regions 300–500 base pairs long.     

Preparation and antithrombogenic activities of heparinoid from 6-O-(carboxymethyl)chitin

The highest antithrombogenic activity was achieved by the sulphation of partially N-deacetylated O-(carboxymethyl)chitin among variously modified chitin derivatives. It was also suggested that the distribution of N-sulphate and N-acetyl groups on the C-2 position might be essential to the selective binding by antithrombin-III to inhibit thrombin activity. Kinetic evaluations demonstrated the non-competitive inhibition on direct interaction with thrombin (K_i = 9.26 × 10⁻⁸m) and the competitive inhibition with antithrombin-III (K_i = 3.33 × 10⁻⁸) m as well as with heparin. 6-O-Carboxymethyl groups were found, from the data of intravenous injection in mice, to suppress the toxicity of chitin heparinoids.     

Analysis of the binding of phenylalanine to phenylalanyl-tRNA synthetase

Using the complete rate equation for the PP_i-ATP exchange reaction at equilibrium, the dissociation constants of phenylalanine (10^?5m), phenylalanine butyl ester (8 × 10^?5m), benzyl alcohol (6 × 10^?4m), phenylalaninol (2 × 10^?4m), hydrocinnamic acid (3 × 10^?3m) and glycine (>1 m) with the phenylalanyl-tRNA synthetase (Escherichia coli K12) were determined. Taking the model of Koshland (1962) for the estimation of the configurational free energy change due to proximity and orientation, and decomposing the process of binding into several thermodynamic steps, the contribution to binding of the benzyl group, glycine unit, protonated amino group, carboxylate group and joint interactions were estimated. The results are: (1) the standard free energy contributions for binding phenylalanine are benzyl group (?8.2 kcal/mol), glycine unit (?2.5 kcal/mol), protonated amino group (?0.8 kcal/mol) and carboxylate group (1 kcal/mol). (2) The standard free energy change due to the change in the interaction between the protonated amino group and carboxylate group when they are transferred from the aqueous environment to the enzyme environment is ?2.7 kcal/mol. (3) A dissociation constant for glycine of 7.5 m is calculated without the hypothesis that a conformational change occurs in the enzyme when the benzyl unit of phenylalanine binds, permitting an interaction of the enzyme with the protonated amino and/or carboxylate groups.The detection of E·AA² and E·ATP shows that a sequential addition of substrates is not necessary for binding. A comparison of the dissociation constants of E·AA (10^?5m), E·ATP (1.5 × 10^?3m), E·PP (5.5 × 10^?4m), E·I (8 × 10^?5m) and the mixed complexes E·I·ATP (6 × 10^?8m²), E·I·PP (5 × 10^?8m²) and E·AA·PP (7 × 10^?9m²), with phenylalanine butyl ester as the inhibitor, indicates no strong interaction between the binding of ATP or PP_i with the binding of phenylalanine.     

Relationship between the wavelength maximum of a protein and the temperature dependence of its intrinsic tryptophan fluorescence intensity

Proper determination of the temperature dependence of intrinsic tryptophan fluorescence intensity in native and denatured states is an essential prerequisite for extracting the free energy of protein unfolding from the thermal denaturation profile. The most common method employed in determining the temperature dependence of these conformations is through the determination of slopes of pre- and post-transition baselines. However, simulations of protein unfolding profiles suggest that this method does not work for marginally stable proteins. We show herein that the temperature dependence of the fluorescence intensity of N-acetyl tryptophanamide (NATA) in organic solvents and water may be used to represent the temperature dependence of the fluorescence intensity of tryptophan in native and denatured conformations of a protein, respectively. The wavelength of the emission maximum, λ _max, of N-acetyl tryptophanamide (NATA) in a particular solvent or tryptophan in proteins is related to the temperature dependence (m) of its fluorescence intensity by the equation: m (K⁻¹) = (−0.000299 ± 2.2 × 10⁻⁵ K⁻¹ nm⁻¹) × λ _max (nm) + (0.0919 ± 0.0025 K⁻¹).     

Direct chemiluminescence determination of penicillin G potassium and a chemometrical optimization approach

A highly selective and simple chemiluminescence (CL) method for determination of penicillin G potassium (PGK) was developed. In the proposed method, CL was elicited from PGK upon its oxidation with H₂O₂. The light emission was enhanced in the presence of N‐cetyl‐N,N,N‐trimethylammonium bromide (CTMAB). An experimental design, central composite design (CCD), was used to realize the optimized variables, including pH, surfactant (CTMAB) and H₂O₂ concentrations. Under optimum condition, the calibration graph was linear in the range 3.3 × 10^?3–3.3 × 10^?1 mmol/L, with a detection limit of 8.8 × 10^?4 mmol/L for PGK. The precision was calculated by analysing samples containing 1.6 × 10^?1 mmol/L PGK (n = 5) and the relative standard deviation (RSD) was 1.40%. The utility of this method was demonstrated by determining PGK in pharmaceutical formulations for injection. The proposed method was validated by a reference method. Copyright © 2011 John Wiley & Sons, Ltd.     

Complexes of antiparallel telomeric G-quadruplex d(TTAGGG)4 with carboxymethyl tetracationic porphyrins

Porphyrins are a chemical class that is widely used in drug design. Cationic porphyrins may bind to DNA guanine quadruplexes. We report the parameters of the binding of 5,10,15,20-tetrakis(N-carboxymethyl-4-pyridinium) porphyrin (P1) and 5,10,15,20-tetrakis(N-etoxycarbonylmethyl-4-pyridinium) porphyrin (P2) to antiparallel telomeric G-quadruplex formed by d(TTAGGG)₄ sequence (TelQ). The binding constants (K _i ) and the number of binding sites (N _j ) were determined from absorption isotherms generated from the absorption spectra of complexes of P1 and P2 with TelQ. Compound P1 demonstrated a high affinity to TelQ (K _i = (40 ± 6) × 10⁶ M^?1, N ₁ = 1; K ₂ = (5.4 ± 0.4) × 10⁶ M^?1, N ₂ = 2). In contrast, the binding constants of P2-TelQ complexes (K ₁ = (3.1 ± 0.2) × 10⁶ M^?1, N ₁ = 1; K ₂ = (1.2 ± 0.2) × 10⁶ M^?1, N ₂ = 2) were one order of magnitude smaller than the corresponding values for P2-TelQ complexes. Measurements of the quantum yield and fluorescence lifetime of the drug’s TelQ complexes revealed two types of binding sites for P1 and P2 on the quadruplex oligonucleotide. We concluded that strong complexes can result from the interaction of the porphyrins with TTA loops whereas the weaker complexes are formed with G-quartets. The altered TelQ conformation detected by the circular dichroism spectra of P1-TelQ complexes can be explained by the disruption of the G-quartet. We conclude that peripheral carboxy groups contribute to the high affinity of P1 for the antiparallel telomeric G-quadruplex.     

The role of water in the crystal structure of N-acetyl-L-4-hydroxyproline

The crystal structure of N-acetyl-L -4-hydroxyproline (Hyp) was determined by direct methods. (The crystal is orthorhombic with the space group P2₁2₁2₁.) The acetyl group is in the trans conformation and the pyrrolidine ring puckers at C^γ (C^sC^γ envelope), as in most Hyp residues. According to the rotation angle ψ = ?30°, the N-acetyl-L -4Hyp has the same conformation as an α-helix of prolyl residues. The crystal packing is stabilized by hydrogen bonds between three different molecules and the same molecule of water. One of the water bridges involves the carbonyl of the N-acetyl group of one molecule and the hydrogen atom of the 4-OH group of another. Such an arrangement has been proposed to explain the high stability of (Gly-L -Pro-L -4Hyp)_n. A second bridge involves the two hydrogens of the water molecule and the carbonyl groups of two neighbouring molecules, as already proposed in a dihydrated model of collagen. These experimental features, which are discussed in relation to the different models of collagen, allow us to propose an hypothetical arrangement for the water molecule which is strongly retained in the triple helix of (Gly-L -Pro-L -4Hyp)_n.     

Conformational Preferences of Modified Nucleoside N(4)-Acetylcytidine, ac4C Occur at “Wobble” 34th Position in the Anticodon Loop of tRNA

Conformational preferences of modified nucleoside, N(4)-acetylcytidine, ac⁴C have been investigated using quantum chemical semi-empirical RM1 method. Automated geometry optimization using PM3 method along with ab initio methods HF SCF (6-31G**), and density functional theory (DFT; B3LYP/6-31G**) have also been made to compare the salient features. The most stable conformation of N(4)-acetyl group of ac⁴C prefers “proximal” orientation. This conformation is stabilized by intramolecular hydrogen bonding between O(7)···HC(5), O(2)···HC2′, and O4′···HC(6). The “proximal” conformation of N(4)-acetyl group has also been observed in another conformational study of anticodon loop of E. coli elongator tRNA^Met. The solvent accessible surface area (SASA) calculations revealed the role of ac⁴C in anticodon loop. The explicit molecular dynamics simulation study also shows the “proximal” orientation of N(4)-acetyl group. The predicted “proximal” conformation would allow ac⁴C to interact with third base of codon AUG/AUA whereas the ‘distal’ orientation of N(4)-acetyl cytidine side-chain prevents such interactions. Single point energy calculation studies of various models of anticodon–codon bases revealed that the models ac⁴C₍₃₄₎(Proximal):G₃, and ac⁴C₍₃₄₎(Proximal):A₃ are energetically more stable as compared to models ac⁴C₍₃₄₎(Distal):G₃, and ac⁴C₍₃₄₎(Distal):A₃, respectively. MEPs calculations showed the unique potential tunnels between the hydrogen bond donor–acceptor atoms of ac⁴C₍₃₄₎(Proximal):G₃/A₃ base pairs suggesting role of ac⁴C in recognition of third letter of codons AUG/AUA. The “distal” conformation of ac⁴C might prevent misreading of AUA codon. Hence, this study could be useful to understand the role of ac⁴C in the tertiary structure folding of tRNA as well as in the proper recognition of codons during protein biosynthesis process.     

Effect of sodium selenite and methyl methanesulfonate or N-hydroxy-2-acetylaminofluorene co-exposure on sister-chromatid exchange production in human whole blood cultures

Sodium selenite (Na₂Se0₃) was tested for its sister-chromatid exchange (SCE)-inducing ability in human whole blood cultures and for the effect of its co-exposure with methyl methanesulfonate (MMS) or N-hydroxy-2-acetyl aminofluorene (N-OH-AAF) on SCE frequency. Long exposure times (77 h and 96 h) to 3.95 × 10^-6 M Na₂SeO₃ resulted in cell death as measured by mitotic indices, but mitotic figures were present after exposure to higher concentrations for a shorter time (19 h). High Na₂SeO₃ concentrations (7.90 × 10^?6 and 1.19 × 10^?5 M) resulted in a three-fold increase in the SCE frequency above background level (6–7 SCEs/cell). Exposure of lymphocytes to 1 × 10^?4 M MMS for the last 19 h of culture yielded an average SCE frequency of 30.17 ± 0.75 while a similar exposure to 2.7 × 10^?5 M N-OH-AAF resulted in 13.61 ± 0.43 SCEs/cell. Simultaneous addition of the high Na₂Se0₃ concentrations and MMS or N-OH-AAF to the cultures resulted in SCE frequencies that were 25–30% and 11–17%, respectively, below the sum of the SCE frequencies produced by the individual compounds.     

Determination of DNA Damage in Experimental Liver Intoxication and Role of N-Acetyl Cysteine

The present study aimed at detecting DNA damage and fragmentation as well as histone acetylation depending on oxidative stress caused by CCl₄ intoxication. Also, the protective role of N-acetyl cysteine, a precursor for GSH, in DNA damage is investigated. Sixty rats were used in this study. In order to induce liver toxicity, CCl₄ in was dissolved in olive oil (1/1) and injected intraperitoneally as a single dose (2 ml/kg). N-acetyl cysteine application (intraperitoneal, 50 mg/kg/day) was started 3 days prior to CCl₄ injection and continued during the experimental period. Control groups were given olive oil and N-acetyl cysteine. After 6 and 72 h of CCl₄ injection, blood and liver tissue were taken under ether anesthesia. Nuclear extracts were prepared from liver. Changes in serum AST and ALT activities as well as MDA, TAS, and TOS levels showed that CCl₄ caused lipid peroxidation and liver damage. However, lipid peroxidation and liver damage were reduced in the N-acetyl cysteine group. Increased levels in 8-hydroxy-2-deoxy guanosine and histone acetyltransferase activities, decreased histone deacetylase activities, and DNA breakage detected in nuclear extracts showed that CCl₄ intoxication induces oxidative stress and apoptosis in rat liver. The results of the present study indicate that N-acetyl cysteine has a protective effect on CCl₄-induced DNA damage.     

Determination on the binding of chlortetracycline to bovine serum albumin using spectroscopic methods

In this work, the interaction of chlortetracycline with bovine serum albumin (BSA) was investigated by fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular docking. Results indicated that chlortetracycline quenches BSA fluorescence mainly by a static quenching mechanism. The quenching constants (K_SV) were obtained as 5.64 × 10⁴, 4.49 × 10^4/, and 3.44 × 10^4/ M_?1 at 283, 295, and 307 K, respectively. The thermodynamic parameters of enthalpy change Δ H°, entropy change Δ S°, and free energy change Δ G° were ?5.12 × 10^4/ J mol?1, ?97.6 J mol?1 K?1, and ?2.24 × 10^4/ J mol?1 (295 K), respectively. The association constant (K_A) and the number of binding sites (n) were 9.41 × 10^3/ M?1 and 0.86, respectively. The analysis results suggested that the interaction was spontaneous, and van der Waals force and hydrogen‐bonding interactions played key roles in the reaction process. In addition, CD spectra proved secondary structure alteration of BSA in the presence of chlortetracycline. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:331–336, 2012; View this article online at wileyonlinelibrary.com . DOI 10:1002/jbt.21424     

Determination of pioglitazone hydrochloride by flow injection chemiluminescence tris(2,2′-bipyridyl)ruthenium(II)–silver(III) complex system

A novel flow injection-chemiluminescence (FI–CL) approach is proposed for the assay of pioglitazone hydrochloride (PG-HCl) based on its enhancing influence on the tris(2,2′-bipyridyl)ruthenium(II)–silver(III) complex (Ru(bipy)₃²⁺-DPA) CL system in sulfuric acid medium. The possible CL reaction mechanism is discussed with CL and ultraviolet (UV) spectra. The optimum experimental conditions were found as: Ru(bipy)₃²⁺, 5.0 × 10⁻⁵ M; sulfuric acid, 1.0 × 10⁻³ M; diperiodatoargentate(III) (DPA), 1.0 × 10⁻⁴ M; potassium hydroxide, 1.0 × 10⁻³ M; flow rate 4.0 ml min⁻¹ for each flow stream and sample loop volume, 180 μl. The CL intensity of PG-HCl was linear in the range of 1.0 × 10⁻³ to 5.0 mg L⁻¹ (R² = 0.9998, n = 10) with limit of detection [LOD, signal-to-noise ratio (S/N) = 3] of 2.2 × 10⁻⁴ mg L⁻¹, limit of quantification (LOQ, S/N = 10) of 6.7 × 10⁻⁴ mg L⁻¹, relative standard deviation (RSD) of 1.0 to 3.3% and sampling rate of 106 h⁻¹. The methodology was satisfactorily used to quantify PG-HCl in pharmaceutical tablets with recoveries ranging from 93.17 to 102.77 and RSD from 1.9 to 2.8%.     

Effects of negative air ions,noise, sex and age on maze learning in rats

An equal number of male and female rats of the King-Holtzman hybrid breed, divided into two age groups (group A₁ : 21–30 days old and group A₂ : 90–100 days old), were exposed to two levels of noise: N₀ = 30 db and N₁ = 90 db, (Ref. 0.0002μ bar), and three levels of negative air ions: I_O = no measurable ion concentration, I₁ = 7 × 10⁶ ions/cm³ and I₂ = 7 × 10⁷ ions/cm³. Time and error scores of 240 rats running a modified Lashley left-right maze with an escape-from-water motive served as criteria. A randomized complete blocks design with replications (2×3×2×2×10) was selected for treatment by analysis of variance. The results indicate that (a) the males show significantly lower error score in negatively ionized air; and (b) the females swim significantly faster than males under all investigated conditions with no apparent effect of noise or ions on their performance.

---

Zusammenfassung Eine gleiche Anzahl m?nnlicher und weiblicher Ratten (n = 120) des King-Holtzman Stammes, unterteilt in 2 Altersgruppen (21–30 und 90–100 Tage) wurden zwei Ger?uschintensit?ten (30 db und 90 db) und 3 Konzentrationen negativer Luftionen I₀: nicht messbar niedrig, I₁:7×10⁶, I₂:7×10⁷ Ionen/cm³ ausgesetzt. Die Wirkung wurde geprüft an der Schwimmzeit und der Anzahl Fehler der Tiere im Lasley-Links-Rechts-Labyrinth. Das Motiv zum Schwimmen war, aus dem Wasser zu entkommen. Die Auswertung durch Varianzanalyse erfolgte unter Nachbildung randomisierter kompletter Blockentwürfe. Die Ergebnisse zeigen, dass (a) die M?nnchen bei negativ ionisierter Luft signifikant weniger Fehler machten und (b) die Weibchen unter allen Bedingungen schneller schwammen als die M?nnchen, ohne dass das L?rmniveau oder die Luftionisation darauf einen Einfluss hatten.

---

Resume Un nombre égal de rats males et femelles (120 de chaque) de la race King-Holtzman futdivisé en deux groupes d'age (21 à 30 jours et 90 à 100 jours). Ils furent exposés à 2 intensités auditives (30 et 90 db) et à 3 concentrations d'ions négatifs: I₀ non mesurables (faible), I₁ : 7 × 10⁶ et I₂: 7 × 10⁷ ions/cm³. Les effets d'un tel traitement furent examinés au moyen de deux critères: le temps pris pour sortir de l'eau et le nombre de fautes commises dans un labyrinthe droitegauche de Laslay. Pour l'analyse des variances, on a adopté une répartition due au hasard avec répétitions (2×3×2×2×10). On peut en conclure que: (a) les males font moins d'erreurs dans l'air ionisé négativement et cela de fa?on significative et (b) les femelles ont nagé plus vite que les males dans toutes les conditions de l'essai. Ni le bruit ni le degré d'ionisation ne semblent avoir d'influence sur leur comportement.

---

---

This study was supported in part by a grant from the Faculty Research Fund of the University of Oklahoma.     

Characterisation of acidic sites of Pseudomonas biomass capable of binding protons and cadmium and removal of cadmium via biosorption

Summary The ability of Pseudomonas aeruginosa to accumulate Cd(II) ions from wastewater industries was experimentally investigated and mathematically modelled. From the potentiometric titration and non-ideal competitive analysis (NICA) model, it was found that the biomass contains three acidic sites. The values of proton binding (pK _i =1.66±3.26×10⁻³, 1.92±1.63×10⁻⁴ and 2.16±3.79×10⁻⁴) and binding constant of cadmium metal ions (pK _M1=1.99±2.45×10⁻³ and pK _M2=1.67±4.08×10⁻³) on the whole surface of biomass showed that protonated functional groups and biosorption of Cd(II) ions could be attributed to a monodentate binding to one acidic site, mainly the carboxylic group. From the isothermal sorption experimental data and Langmuir model, it was also found that the value of Langmuir equilibrium (pK _f) constant is 2.04±2.1×10⁻⁵ suggesting that the carboxyl group is the main active binding site. In addition, results showed that the maximum cadmium capacity (q _max) and affinity of biomass towards cadmium metal ions (b) at pH 5.1 and 20 min were 96.5±0.06 mg/g and 3.40×10⁻³± 2.10×10⁻³, respectively. Finally, interfering metal ions such as Pb(II), Cu(II), Cr(III), Zn(II), Fe(II), Mn(II), Ca(II) and Mg(II) inhibited Cd(II) uptake. Comparing the biosorption of Cd(II) by various Pseudomonas isolates from contaminated environment samples (soil and sewage treatment plant) showed that maximum capacities and equilibrium times were different, indicating that there was a discrepancy in the chemical composition between biomasses of different strains.     

Amino acid conjugates as metabolites of the plant growth regulator dihydrojasmonic acid in barley (Hordeum vulgare)

The biotransformation of [2-¹⁴C](±)9, 10-dihydrojasmonic acid (DJA) was studied in excised shoots of 6-day-old barley seedlings after 72 h. From the ethyl acetate extract, some minor metabolites were isolated and purified by DEAE-Sephadex A-25 chromatography, thin-layer chromatography (TLC), C₁₈-cartridges, and high-performance liquid chromatography (HPLC). The structural identification of these metabolites was performed by gas chromatography-mass spectrometry (GC-MS), circular dichroism (CD), and amino acid analysis, and the following amino acid conjugates were found:N-[(–)9,10-dihydrojasmonoyl]valine,N-[(–)9,10-dihydrojasmonoyl]isoleucine,N-[9,10-dihydrojasmonoyl]leucine,N-[11-hydroxy-9,10-dihydrojasmonoyl]valine,N-[11-hydroxy-9,10-dihydrojasmonoyl]isoleucine,N-[12-hydroxy-9,10-dihydrojasmonoyl]isoleucine; and the cucurbic acid-related compoundsN-{[3-hydroxy-2(4-hydroxypentyl)-cyclopent-1-yl]-acetyl}isoleucine andN-{[3-hydroxy-2(5-hydroxypentyl)-cyclopent-1-yl]-acetyl}isoleucine. The results suggest conjugation with isoleucine and valine, as well as preferential hydroxylation at position C-11 or hydrogenation at position C-6, as being important steps in the metabolism of (±)DJA in barley shoots.     

Geochemical and Microbiological Studies of Nitrous Oxide Variations within the New NEEM Greenland Ice Core during the Last Glacial Period

Deep polar ice cores provide atmospheric records of nitrous oxide (N₂O) and other trace gases reflecting climate history along with a parallel archive of microbial cells transported with mineral dust, marine and volcanic aerosols from around the globe. Our interdisciplinary study of 32 samples from different depths of the recently drilled NEEM Greenland ice core addressed the question whether the identified microorganisms were capable of post-depositional biological production of N₂O in situ. We used high-resolution geochemical and microbiological approaches to examine the N₂O concentrations, the quantitative distributions of dust, Ca⁺², NH₄⁺ and NO₃^? ions related to N cycle pathways, the microbial abundance and diversity at specific NEEM core depths from 1758 m to 1867.8 m. Results showed varying concentrations of N₂O (220–271.5 ppb). Microbial abundance fluctuated between 3.3 × 10⁴ and 3.3 × 10⁶ cells mL^?1 in direct correlation with dust and Ca²⁺ concentrations with higher cell numbers deposited during colder periods. The average values of NH₄⁺ and NO₃^? indicated that substrates were available for the microorganisms capable of utilizing them. PCR amplification of selected functional genes involved in bacterial and archaeal nitrification and denitrification was not successful. Sanger and Illumina MiSeq sequence analyses of SSU rRNA genes showed variable representation of Alpha-, Beta- and Gammaproteobacteria, Firmicutes, Actinobacteria, chloroplasts and fungi. The metabolic potential of the dominant genera of Proteobacteria and Firmicutes as possible N₂O producers suggested that denitrification activity may have led to in-situ production and accumulation of N₂O.     

Oxygen transfer to slurries treated in a rotating drum operated at atmospheric pressure

The objective of this work was to determine (1) the effect of rotational speed (N) and lifters on the oxygen transfer coefficient (k _L) of a mineral solution and (2) the effect of solids concentration of a slurry soil-mineral solution on k _L, at a fixed value N (0.25 s⁻¹); in both cases the treatment was carried out in an aerated rotating drum reactor (RDR) operated at atmospheric pressure. First, the k _L for the mineral solution was in the range 6.38 × 10⁻⁴–7.69 × 10⁻⁴ m s⁻¹, which was of the same order of magnitude as those calculated for closed rotating drums supplied with air flow. In general, k _L of RDR implemented with lifters was superior or equal to that of RDR without lifters. For RDR implemented with lifters, k _L increased with N in the range 6.65 × 10⁻⁴–10.51 × 10⁻⁴ m s⁻¹, whereas k _L of RDR without lifters first increased with N up to N = 0.102 s⁻¹, and decreased beyond this point. Second, regarding soil slurry experiments, an abrupt fall of k _L (ca. 50%) at low values of the solid concentration (C _v) and an asymptotic pattern at high C _v were observed at N = 0.25 s⁻¹. These results suggest that mass transfer phenomena were commanded by the slurry properties and a semi-empirical equation of the form Sh = f(Re, Sc) seems to corroborate this finding.     

Forssman hapten N-acetyl-alpha-D-galactosaminidase in rat brain and kidney

—Forssman hapten (N-acetyl-α-galactosaminosyl-N-acetyl-β-galactosaminosyl-α-galactosyl-β-galactosyl-glucosylceramide), prepared from sheep erythrocytes was specifically labelled with tritium at the terminal N-acetyl-α-galactosamine moiety by the galactose oxidase-sodium [³H]borohydride method. Activities to cleave the terminal N-acetyl-α-galactosamine from Forssman hapten were detected in the high-speed supernatant of the frozen-thawed and sonicated crude mitochondrial fraction from adult rat brain and kidney. The optimal pH of the reaction was approximately 4·4. The reaction was linear for at least 1 h for the kidney enzyme and up to 3 h for the brain enzyme. Taurocholate was required for the activity. The optimal concentration was 1·5-2 mg/ml. Several other detergents and bile salts tested could not replace taurocholate. The apparent K_m of the brain and kidney enzymes were 1·0×10^?4M and 3·5×10^?4m , respectively. During development, Forssman hapten-cleaving activities of both brain and kidney gradually declined in specific activity as the animal matured. These changes were similar to those of nonspecific p-nitrophenyl N-acetyl-α-galactosaminidase. Several rat organs examined all showed detectable activities to cleave Forssman hapten.     

Purification and properties of a β-N-acetylaminoglucohydrolase from malted barley

β-N-Acetylaminoglucohydrolase (β-2-acetylamino-2-deoxy-D-glucoside acetylaminodeoxyglucohydrolase, EC 3.2.1.30) was extracted from malted barley and purified. The partially purified preparation was free from α-and β-glucosidase, α- and β-galactosidase, α-mannosidase and β-mannosidase. This preparation was free from α-mannosidase only after affinity chromatography with p-amino-N-acetyl-β-D-glucosaminidine coupled to Sepharose. The enzyme was active between pH 3 and 6.5 and had a pH optimum at pH 5. A MW of 92000 was obtained by sodium dodecyl sulfate-acrylamide gel electrophoresis and a sedimentation coefficient of 4.65 was obtained from sedimentation velocity experiments. β-N-Acetylaminoglucohydrolase had a K_m of 2.5 × 10^?4 M using the p-nitrophenyl N-acetyl β-D-glucosaminidine as the substrate.     

Production of Pseudomonas Cells Having a High Fumarate Hydratase Activity

An extracellular 45 kDa endochitosanase was purified and characterized from the culture supernatant of Bacillus sp. P16. The purified enzyme showed an optimum pH of 5.5 and optimum temperature of 60°C, and was stable between pH 4.5-10.0 and under 50°C. The K _m and V _max were measured with a chitosan of a D.A. of 20.2% as 0.52 mg/ml and 7.71×10^?6 mol/sec/mg protein, respectively. The enzyme did not degrade chitin, cellulose, or starch. The chitosanase digested partially N-acetylated chitosans, with maximum activity for 15-30% and lesser activity for 0-15% acetylated chitosan. The chitosanase rapidly reduced the viscosity of chitosan solutions at a very early stage of reaction, suggesting the endotype of cleavage in polymeric chitosan chains. The chitosanase hydrolyzed (GlcN)₇ in an endo-splitting manner producing a mixture of (GlcN)_2-5. Time course studies showed a decrease in the rate of substrate degradation from (GlcN)₇ to (GlcN)₆ to (GlcN)₅, as indicated by the apparent first order rate constants, k ₁ values, of 4.98×10^?4, 2.3×10^?4, and 9.3×10^?6 sec^?1, respectively. The enzyme hardly catalyzed degradation of chitooligomers smaller than the pentamer.