Pre-mRNA from erythroid enriched bone marrow cells of the rabbit

Different fractions of cellular RNA from erythroid enriched bone marrow cells of the rabbit, extracted by the temperature fractionation method, were investigated by hybridization to globin cDNA. 97.4% of all globin sequences were found in the 4 degrees C franction (cytoplasmic RNA) 0.11% are in the 40 degrees / 50 degrees C fraction and 2.47% in the 65 degrees C and 85 degrees C franctions (pre-mRNA). This shows a substantial purification of the pre-mRNA fractions from cytoplasmic mRNA. 33% of the globin sequences in the 65 degrees C and 85 degrees C fractions are polyadenylated. The poly(A)+-RNA from the 65 degrees C and 85 degrees C fractions separated in a formamide sucrose gradient showed a clear hybridization to globin cDNA in the region between 9S and 28S and around 4S. In a control experiment in which RNA from baby hamster kidney cells (BHK) was mixed with globin mRNA and separated in the same manner hybridization was observed at the 9S position of the gradient only.     

Photoreactivating enzyme from Escherichia coli: isolated enzyme lacks absorption in its actinic wavelength region and its ribonucleic acid cofactor is partially double stranded when associated with apoprotein

Isolated photoreactivating enzyme (PRE) from Escherichia coli exhibits some optical density at wavelengths greater than 300 nm. After correcting for the effects of light scattering, however, we find no true absorption in the spectral region that is required for enzymatic activity (320-450 nm). At shorter wavelengths, there is an absorption maximum near 260 nm that is due primarily to an RNA cofactor. Heating to 60 degrees C and subsequently cooling to 4 degrees C release the RNA cofactor from association with apoprotein and result in hyperchromicity. Circular dichroism indicates that the RNA associated with native enzyme is partially double stranded. At low ionic strength (mu = 0.01), heating to 15 degrees C or protease treatment at 4 degrees C results in irreversible loss of part of the double strandedness. We show that the difference spectrum at 4 degrees C between the absorption spectra of native enzyme and heat-treated enzyme can be fit by a superposition of reference spectra for denaturation of A-U and G-C base pairs derived from model polynucleotides. The coefficients of the linear combination of reference spectra were used to calculate the fraction of A-U and G-C base pairs. We find that both A-U and G-C base pairs are present in equal concentrations and that about 20% are in a double-stranded conformation in the native enzyme.     

X-ray diffraction studies of cross-bridges weakly bound to actin in relaxed skinned fibers of rabbit psoas muscle.

X-ray diffraction patterns were obtained from skinned rabbit psoas muscle under relaxing and rigor conditions over a wide range of ionic strengths (50-170 mM) and temperatures (1 degree C-30 degrees C). For the first time, an intensification of the first actin-based layer line is observed in the relaxed muscle. The intensification, which increases with decreasing ionic strength at various temperatures, including 30 degrees C, parallels the formation of weakly attached cross-bridges in the relaxed muscle. However, the overall intensities of the actin-based layer lines are low. Furthermore, the level of diffuse scattering, presumably a measure of disorder among the cross-bridges, is little affected by changing ionic strength at a given temperature. The results suggest that the intensification of the first actin layer line is most likely due to the cross-bridges weakly bound to actin, and that the orientations of the weakly attached cross-bridges are hardly distinguishable from the detached cross-bridges. This suggests that the orientations of the weakly attached cross-bridges are not precisely defined with respect to the actin helix, i.e., nonstereospecific. Intensities of the myosin-based layer lines are only marginally affected by changing ionic strength, but markedly by temperature. The results could be explained if in a relaxed muscle the cross-bridges are distributed between a helically ordered and a disordered population with respect to myosin filament structure. Within the disordered population, some are weakly attached to actin and others are detached. The fraction of cross-bridges in the helically ordered assembly is primarily a function of temperature, while the distribution between the weakly attached and the detached within the disordered population is mainly affected by ionic strength. Some other notable features in the diffraction patterns include a approximately 1% decrease in the pitch of the myosin helix as the temperature is raised from 4 degrees C to 20 degrees C.     

The transformation and toxicity of anthraquinone dyes during thermophilic (55 degrees C) and mesophilic (30 degrees C) anaerobic treatments

We studied in batch assays the transformation and toxicity of anthraquinone dyes during incubations with anaerobic granular sludge under mesophilic (30 degrees C) and thermophilic (55 degrees C) conditions. Additionally, the electron shuttling capacity of the redox mediator anthraquinone-2-sulfonic acid (AQS) and subsequent increase on decolourisation rates was investigated on anthraquinone dyes. Compared with incubations at 30 degrees C, serum bottles at 55 degrees C presented distinctly higher decolourisation rates not only with an industrial wastewater containing anthraquinone dyes, but also with model compounds. Compared with batch assays at 30 degrees C, the first-order rate constant "k" of the Reactive Blue 5 (RB5) was enhanced 11-fold and 6-fold for bottles at 55 degrees C supplemented and free of AQS, respectively. However, the anthraquinone dye Reactive Blue 19 (RB19) demonstrated a very strong toxic effect on volatile fatty acids (VFA) degradation and methanogenesis at both 30 degrees C and 55 degrees C. The apparent inhibitory concentrations of RB19 exerting 50% reduction in methanogenic activity (IC50-value) were 55 mg l(-1) at 30 degrees C and 45 mg l(-1) at 55 degrees C. Further experiments at both temperatures revealed that RB19 was mainly toxic to methanogens, because the glucose oxidizers including acetogens, propionate-forming, butyrate-forming and ethanol-forming microorganisms were not affected by the dye toxicity.     

The effect of medium on functional and structural properties of serum albumins. II. The effect of temperature on the N-form of human serum albumin

In order to investigate effects of temperature in the physiological range (from 10 to 50 degrees C) on structural, physical and functional properties of the N-form of human serum albumin (HSA), the temperature dependences of fluorescence parameters of Trp-214 residue of HSA and of the specifically bound dye ANS, as well as of association constants of ANS binding in the primary and secondary binding sites on HSA molecule were measured. The temperature-induced changes of these properties of HSA are essentially dependent on pH (7.0 or 5,6) and ionic strength (0.001-0.008 or 0.2 M NaCl). At pH 7.0 and 0.2 M NaCl the environment of Trp-214 remained invariant at temperature changes between 10 and 50 degrees C. On the other hand, the affinity to ANS of a primary binding site doubled and that of secondary ones halved. These affinity changes seem to be due, are least partly, to the heating-induced dissociation of Cl-ions, which are inhibitors of the primary dye binding. By lowering pH (to 5.6) and ionic strength the temperature-induced changes in the Trp-214 environment were observed. The changes are interpreted as indole group transition into the buried region, inaccesible to water (the "closing" of a structural slit). The affinity of secondary binding sites of ANS was halved.     

Kinetic and spectrophotometric studies on the renaturation of deoxyribonucleic acid

The kinetics of the renaturation of Escherichia coli DNA in 0.4-1.0m-sodium chloride at temperatures from 60 degrees to 90 degrees have been studied. The extent of renaturation was a maximum at 65 degrees to 75 degrees and increased with ionic strength, and the rate constant increased with both ionic strength and temperature. The energy and entropy of activation of renaturation were calculated to be 6-7kcal.mole(-1) and -40cal.deg.(-1)mole(-1) respectively. It has been shown that renaturation is a second-order process for 5hr. under most conditions. The results are consistent with a reaction in which the rate-controlling step is the diffusion together of two separated complementary DNA strands and the formation of a nucleus of base pairs between them. The kinetics of the renaturation of T7-phage DNA and Bordetella pertussis DNA have also been studied, and their rates of renaturation related quantitatively to the relative heterogeneity of the DNA samples. By analysis of the spectra of DNA at different stages during renaturation it was shown that initially the renatured DNA was rich in guanine-cytosine base pairs and non-random in base sequence, but that, as equilibrium was approached, the renatured DNA gradually resembled native DNA more closely. The rate constant for the renaturation of guanine-cytosine base pairs was slightly higher than for adenine-thymine base pairs.     

Conformational studies on the inactivation of glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile Bacillus coagulans KU

Among the various proposals that have been made in attempting to explain the ability of thermophiles to reproduce at high temperatures, there is no doubt that obligate and extreme thermophiles synthesize proteins (and other molecules) that have sufficient intrinsic molecular stability to withstand increased thermal stress. In contrast, the glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile Bacillus coagulans KU has been shown to be quite thermolabile in vitro. Thermal inactivation is not due to loss of bound NAD+. It has also been shown that the enzymatic activity can be thermostabilized in vitro by increased ionic strength. As previously reported [J. W. Crabb, A. L. Murdock, and R. E. Amelunxen (1975) Biochem. Biophys. Res. Commun. 62, 627; (1977) Biochemistry 16, 4840], the enzyme loses 94-97% of enzymatic activity after heat treatment at 55 degrees C for 5 min in 0.05 M sodium phosphate buffer (pH 7.1); however, by increasing the ionic strength to 1.8, complete protection was conferred at this temperature. Gel-filtration chromatography has been used to study the initial dissociation and subsequent aggregation of the glyceraldehyde-3-phosphate dehydrogenase after thermal inactivation. Aggregation occurs when the enzyme is heated at 50 degrees or 55 degrees C. Loss of enzymatic activity is correlated with changes in the tertiary structure as measured by the near-uv CD spectrum of the enzyme following heat inactivation, with essential disappearance of the peaks at 263 and 296 nm, and a blue shift of the far-uv spectrum, which is a measure of secondary structure. Estimation of secondary structure of the unheated protein from the far-uv CD data showed the enzyme contains approximately 26% alpha-helix, approximately 21% beta-structure, and approximately 53% disordered structure. Heat treatment at various temperatures resulted in only slight changes of the estimated secondary structure. Increased ionic strength prevents thermal alteration of the CD spectrum in both near- and far-uv regions. The data support the previous proposal that thermolabile enzymes such as the glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile B. coagulans are thermostabilized in vivo mainly by the intracellular charged macromolecular environment.     

Effect of head-group structure and counterion condensation on phase equilibria in anionic phospholipid-water systems studied by 2H, 23Na, and 31P NMR and X-ray diffraction

The phase equilibria, hydration, and sodium counterion association for the systems DOPA-2H2O, DOPS-2H2O, DOPG-2H2O, and DPG-2H2O were investigated with 2H, 23Na, and 31P NMR and X-ray diffraction. The following one-phase regions were found in the DOPA-water system: a reversed hexagonal liquid-crystalline (HII) phase up to about 35 wt % water and a lamellar liquid-crystalline (L alpha) phase between about 55 and 98 wt % water. The area per DOPA molecule was 36-65 A2 in the HII phase (10-40 wt % water) and 69 A2 in the L alpha phase (60 wt % water). DOPS and DOPG with 10-98 wt % water, and DPG with 20-95 wt % water formed an L alpha phase at temperatures between 25 and 55 degrees C. At temperatures above 55 degrees C, DPG with 20 and 30 wt % water formed a mixture of L alpha, HII, and cubic liquid-crystalline phases, the mole percent of lipid forming nonlamellar phases being smaller at 30 wt % water than at 20 wt % water. DPG with 10 wt % water probably formed a mixture of an L alpha phase and at least one nonlamellar liquid-crystalline phase at 25 and 35 degrees C, and a pure HII phase at 45 degrees C and higher temperatures. At water concentrations above about 50 wt % the 23Na quadrupole splitting was constant for all four lipid-water systems studied, implying that the counterion association to the charged lipid aggregates did not change upon dilution. These experimental observations can be described with an ion condensation model but not with a simple equilibrium model. The fraction of counterions located close to the lipid-water interface was calculated to be greater than 95%. The 2H and 23Na NMR quadrupole splittings of 2H2O and sodium counterions, respectively, indicate that the molecular order in the polar head-group region decreases for the L alpha phase in the order DOPA approximately DPG greater than DOPS greater than DOPG.     

The effect of pyrophosphate on the rigor fibers of rabbit m. psoas fibers

The effect of MgPPi on the rigor force of glycerinated fibres in the range of ionic strength 75-250 mM at two temperatures 18 degrees and 5 degrees C was studied. At 18 degrees C the maximum of this effect was above the range of average ionic strength. At 5 degrees C the greatest effect of MgPPi was observed at low ionic strength.     

Thermophilic (55-65 degrees C) and extreme thermophilic (70-80 degrees C) sulfate reduction in methanol and formate-fed UASB reactors

The feasibility of thermophilic (55-65 degrees C) and extreme thermophilic (70-80 degrees C) sulfate-reducing processes was investigated in three lab-scale upflow anaerobic sludge bed (UASB) reactors fed with either methanol or formate as the sole substrates and inoculated with mesophilic granular sludge previously not exposed to high temperatures. Full methanol and formate degradation at temperatures up to, respectively, 70 and 75 degrees C, were achieved when operating UASB reactors fed with sulfate rich (COD/SO4(2-)=0.5) synthetic wastewater. Methane-producing archaea (MPA) outcompeted sulfate-reducing bacteria (SRB) in the formate-fed UASB reactor at all temperatures tested (65-75 degrees C). In contrast, SRB outcompeted MPA in methanol-fed UASB reactors at temperatures equal to or exceeding 65 degrees C, whereas strong competition between SRB and MPA was observed in these reactors at 55 degrees C. A short-term (5 days) temperature increase from 55 to 65 degrees C was an effective strategy to suppress methanogenesis in methanol-fed sulfidogenic UASB reactors operated at 55 degrees C. Methanol was found to be a suitable electron donor for sulfate-reducing processes at a maximal temperature of 70 degrees C, with sulfide as the sole mineralization product of methanol degradation at that temperature.     

Comparative performance and microbial diversity of hyperthermophilic and thermophilic co-digestion of kitchen garbage and excess sludge

The objective of this study was to evaluate the performance characteristics of a hyperthermophilic digester system that consists of an acidogenic reactor operated at hyperthermophilic (70 degrees C) conditions in series with a methane reactor operated at mesophilic (35 degrees C), thermophilic (55 degrees C), and hyperthermophilic (65 degrees C) conditions. Lab-scale reactors were operated continuously, and were fed with co-substrates composed of artificial kitchen garbage (TS 9.8%) and excess sludge (TS 0.5%) at the volumetric ratio of 20:80. In the acidification step, COD solubilization was in the range of 22-46% at 70 degrees C, while it was 21-29% at 55 degrees C. The average protein solubilization was 44% at 70 degrees C. The double bond fatty acid removal ratio at 70 degrees C was much higher than at 55 degrees C. These results suggested that the optimal operation conditions for the acidogenic fermenter were about 3.1 days of HRT and 4 days of SRT at 70 degrees C. Methane conversion efficiency and the VS removal percentage in the methanogenic step following acidification was around 65% and 64% on average at 55 degrees C, respectively. The optimal operational conditions for this system are acidogenesis performed at 70 degrees C and methanogenesis at 55 degrees C. The key microbes determined in the hyperthermophilic acidification step were Anaerobic thermophile IC-BH at 6.4 days of HRT and Thermoanaerobacter thermohydrosulfuricus DSM 567 at 2.4 days of HRT. These results indicated that the hyperthermophilic system provides considerable advantages in treating co-substrates containing high concentrations of proteins, lipids, and nonbiodegradable solid matter.     

Effects of oxygen on aerobic solid-state biodegradation kinetics

Oxygen is a critical control variable for composting and other solid-state biodegradation processes. In this study we examined the effect of varying oxygen concentrations (1%, 4%, and 21% O2 (v/v)) on biodegradation kinetics under different substrate (sewage sludge and synthetic food waste), temperature (35, 45, 55, and 65 degrees C), and moisture (36-60% H2O) conditions. Three forms of a saturation or Monod-type model and one form of an exponential model were evaluated against data from extensive experiments under carefully controlled environmental conditions. The exponential model performed well at temperatures from 35 to 55 degrees C but had problems at higher temperatures. The Monod-type models yielded the best fit based on R2 values. Multiple linear regression was used to express the oxygen half-saturation coefficient as a function of temperature and moisture. For a modified one-parameter saturation model the half-saturation coefficient varied from -0.67% to 1.74% v/v O2 under the range of conditions typical of composting systems. While the positive correlation of biodegradation rate with oxygen concentration reported by previous researchers held true for temperatures below 55 degrees C, an inverse relationship was found at 65 degrees C. Although this study did not directly examine anaerobic conditions, the results under microaerophilic conditions suggest oxygen may not offer kinetic advantages for extreme thermophilic biodegradation processes.     

Acetate Degradation at 70 degrees C in Upflow Anaerobic Sludge Blanket Reactors and Temperature Response of Granules Grown at 70 degrees C

Anaerobic acetate degradation at 70 degrees C and at 55 degrees C (as a reference) was studied by running laboratory upflow anaerobic sludge blanket (UASB) reactors inoculated with mesophilic granular sludge. In UASB reactors fed with acetate-containing media (3 g of chemical oxygen demand [COD] per liter, corresponding to 47 mM acetate) approximately 50 days was needed at 70 degrees C and less than 15 days was needed at 55 degrees C to achieve an effluent COD of 500 to 700 mg/liter. In the UASB reactors at both 70 and 55 degrees C up to 90% of the COD was removed. Batch assays showed that sludges from two 70 degrees C UASB reactors, one run at a low effluent acetate concentration and the other run at a high effluent acetate concentration, exhibited slightly different responses to temperatures in the range from 37 to 70 degrees C. Both 70 degrees C sludges, as well as the 55 degrees C sludge, produced methane at temperatures of 37 to 73 degrees C. The 55 degrees C sludge exhibited shorter lag phases than the 70 degrees C sludges and higher specific methane production rates between 37 and 65 degrees C.     

Enthalpy relaxation of freeze concentrated sucrose-water glass

The enthalpy relaxation of freeze concentrated sucrose-water glass was investigated using 40% sucrose, differential scanning calorimetry (DSC) with isothermal ageing for 1-6 days at various temperatures (-70, -65, -60, and -55 degrees C). The enthalpy relaxation was observed as an endothermic peak superimposed on the endothermic step-wise change due to the glass transition around -47 degrees C. The enthalpy relaxation was found to increase with ageing time and temperature. An 80% sucrose glass was also investigated at ageing temperatures of -60 and -65 degrees C, and this material exhibited a similar glass transition and enthalpy relaxation to that observed with the frozen 40% sucrose solution. The calculated activation energy of the enthalpy relaxation of the sucrose-water glass was smaller than that reported for pure sucrose. These results suggest that the freeze concentrated sucrose-water glass could have a higher molecular mobility and less stability than pure sucrose glass.     

Fluorescence of proflavine--DNA complexes: heterogeneity of binding sites

Measurements of the relative quantum yield of fluorescence of proflavine bound to DNA as a function of the number of bound dyes per nucleotide and the ionic strength allow the determination of the binding constants and respective number of the two types of sites previously postulated. It is demonstrated that 2–3% of the base pairs form sites where the dye is strongly bound and fluoresces normally while in the other set of sites the binding constant is 3–4 times weaker and the fluorescence completely quenched. Comparison with complexes of Pro with double stranded polynucleotides poly (A + U), poly (I + C), poly(G + C), confirm that the strong binding sites correspond to A-T-rich regions of the DNA while the quenched sites seem to require the presence of a neighboring guanine. The role of charge transfer in quenching of fluorescence and mutagnic action is considered. An original method for the determination of free dye and bound dye, based upon the use of an external quencher is described in the Appendix.     

Magnesium ion-dependent contraction of skinned frog muscle fibers in calcium-free solution

Skinned frog fibers were reversibly activated in Ca-free solutions containing 0 mM KCl, 23 microM free Mg, and having an ionic strength of approximately 50 mM. Contractile force was nearly maximal at 22 degrees - 25 degrees C and decreased at lower temperatures. Maximal force in Ca-free solution at 50 mM ionic strength was close to twice the calcium-activated force with pCa 5 and 190 mM ionic strength. The force in Ca-free solution could be reduced to zero by raising the concentration of free Mg from 23 microM to 1.0 mM at the same ionic strength (50 mM). On stretching the fiber from 2.0 to 3.2 micron the force decreased; this effect was similar to that seen with Ca-activated fiber and the data support the idea that Ca-free tension is made at the cross-bridge level. Isotonic contraction during Ca-free activation showed a velocity transient as in Ca-activated fiber at 190 mM ionic strength, but the transient in the present case was very much prolonged. This finding suggests that contraction mechanisms for force generation and for shortening are essentially the same in the two conditions, but that certain rate constants of cross-bridge turnover are slower for the Ca-free contraction. Also, the results indicate that, in low ionic strength, Ca binding to thin filaments is not essential for unmasking the cross-bridge attachment sites, which suggests that the steric blocking mechanism is modified under these conditions.     

Affinity-purified tetanus neurotoxin interaction with synaptic membranes: properties of a protease-sensitive receptor component

The pharmacokinetic interaction of an affinity-purified 125I-labeled tetanotoxin fraction with guinea pig brain synaptosomal preparations was investigated. Binding of tetanotoxin was time- and temperature-dependent, was proportional to protein concentration, and was saturable at about 8 X 10(-9) M as estimated by a solid-surface binding assay. Binding was optimal at pH 6.5 under low ionic strength buffer and was almost entirely blocked by gangliosides or antitoxin. In analogy to intact nerve cells, binding of toxin to membranes resulted in a tight association operationally defined as sequestration. Binding and sequestration were abolished after membrane pretreatment with sialidase. The enzyme could not dissociate the membrane-bound toxin formed at 4 or 37 degrees C under low ionic strength conditions, which is in part compatible with internalization as defined in nerve cell cultures. In the latter system the toxin could be removed at 4 degrees C but not at 37 degrees C. Binding was significantly reduced upon pretreatment of guinea pig brain membranes by a variety of hydrolytic enzymes. Trypsin and chymotrypsin inhibited binding between 55% and 68% while bacterial protease abolished it by 91-95%. The effect was species-specific as it was not seen in rat or bovine synaptosomes. Collagenase and hyaluronidase had little or no inhibitory effect when applied to synaptosomes (27% and 9%) but inhibited binding to synaptic vesicles by 56% and 49%, respectively. Phospholipases A2 and C caused 42-43% inhibition of binding in vesicles and less than 22% in synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Duplex formation of a nonionic oligo(deoxythymidylate) analogue (heptadeoxythymidylyl-(3'-5')-deoxythymidine heptaethyl ester (d-(Tp(Et))7T)) with poly(deoxyadenylate). Evaluation of the electrostatic interaction.

The heptaethyl ester of heptadeoxythymidylyl-(3'-5')-deoxythymidine (d-[Tp(Et)]7T or d-T8-Et) has been prepared by chemical methods. The material, consisting of a mixture of diastereoisomers, forms a 1:1 complex with (dA)n in neutral aqueous buffer; this interaction is virtually independent of ionic strength. The octamer triester does not bind to (dA)n-(dT)n, and it interacts with (rA)n only at low temperatures. By cochromatography with (dA)n on Sephadex G-50, d-T8-Et fractions with different binding affinities for the polyadenylates were obtained. This heterogeneity in binding affinity is ascribed to the diastereoisomerism of d-T8-Et. Enthalpies of dupoex formation were determined by the concentration variation method. At 0.1 M sodium ion concentration, the enthalpy of binding of the various d-T8-Et fractions to (dA)n is essentially invariant (-8.1 kcal/mol of base pairs at 0 degrees C to -8.6 kcal at 25 degrees C) and 1.6 kcal/mol of base pairs more negative than the enthalpy of binding of the phosphodiester analogue, d-(Tp)7T, to (dA)n (-6.8 kcal/mol of base pairs at 11 degrees C). This difference is the electrostatic contribution to the enthalpy of duplex formation, arising from the interstrand electrostatic repulsion and the intrastrand repulsion in d-(Tp)7T. The entropy of binding to (dA)n is more negative for the octamer triesters than for the diester analogue, and is different for the various d-T8-Et fractions. This is interpreted in terms of varying degrees of restriction of rotational freedom for the ethyl substituents upon double helix formation.     

Temperature-induced chromatin changes in ethanol-fixed cells

We studied the chromatin structure of rat thymocytes fixed in 70% ethanol at 0-44 degrees C by flow cytometry and gel electrophoresis. The fluorescence of the DNA-specific dye mithramycin increased by 93% when thymocytes were exposed at 44 degrees C in the fixative compared to cells kept at 0 degrees C. Antibody labeling (X-ANA) of the core histones was 65% lower for the 44 degrees C-treated cells compared to the control cells (0 degree C). The emission anisotropies of the DNA-specific dye Hoechst 33258 bound to chromatin were 0.341 and 0.318 for thymocytes fixed at 0 degree C and 44 degrees C, respectively. Increased mobility of DNA in chromatin of 44 degrees C-treated cells, as revealed by the emission anisotropy of Hoechst 33258, was not due to denaturation of DNA but was probably caused by removal of constraints situated at short intervals (less than or equal to 50 BP) along the DNA helix. The short intervals between these constraints in chromatin fixed at 0 degree C suggests that they were histones. PAGE of 0.5 N H2SO4-extracted histones showed that the 44 degrees C treatment reduced total core histone content by 65% and that the different histones were lost in unequal amounts. The loss was about 75% and 54% for the histone pairs H3/H4 and H2A/H2B, respectively. The amount of H1 was reduced by about 25% on temperature treatment. The temperature-induced change in the chromatin structure of the cells in 70% ethanol was biphasic. A change in the three-dimensional structure of chromatin occurred for temperatures up to 20 degrees C (no histones were released but binding of mithramycin increased by approximately 15%, whereas the binding of X-ANA decreased by the same amount). Sixty-five percent of core histones were released in the second phase (20-44 degrees C), which may explain the further increase and decrease in the binding of mithramycin and X-ANA, respectively.     

Aerobic and two-stage anaerobic-aerobic sludge digestion with pure oxygen and air aeration

The degradability of excess activated sludge from a wastewater treatment plant was studied. The objective was establishing the degree of degradation using either air or pure oxygen at different temperatures. Sludge treated with pure oxygen was degraded at temperatures from 22 degrees C to 50 degrees C while samples treated with air were degraded between 32 degrees C and 65 degrees C. Using air, sludge is efficiently degraded at 37 degrees C and at 50-55 degrees C. With oxygen, sludge was most effectively degraded at 38 degrees C or at 25-30 degrees C. Two-stage anaerobic-aerobic processes were studied. The first anaerobic stage was always operated for 5 days HRT, and the second stage involved aeration with pure oxygen and an HRT between 5 and 10 days. Under these conditions, there is 53.5% VSS removal and 55.4% COD degradation at 15 days HRT - 5 days anaerobic, 10 days aerobic. Sludge digested with pure oxygen at 25 degrees C in a batch reactor converted 48% of sludge total Kjeldahl nitrogen to nitrate. Addition of an aerobic stage with pure oxygen aeration to the anaerobic digestion enhances ammonium nitrogen removal. In a two-stage anaerobic-aerobic sludge digestion process within 8 days HRT of the aerobic stage, the removal of ammonium nitrogen was 85%.