Effects of o-cresol co-disposal and pH on the methanogenic degradation of refuse

SULISTI, I.A. WATSON-CRAIK AND E. SENIOR. 1996. Both maximum o -cresol degradation and activity of sulphate-reducing bacteria (SRB) were observed at refuse pH values between 7.0 and 8.0. Optimum pH values for methane release were between 6.5 and 7.5. Partial inhibition of methane production was recorded at pH 5.7, 6.0 and 8.0, whilst sulphate reduction was inhibited partially at pH values 5.7–6.5. Both sulphate reduction and methanogenesis were completely inhibited in refuse with initial pH 4.0. The catabolism of acetate occurred under similar conditions to methane production, and was promoted at pH 6.5–7.5. It appeared that propionate oxidation depended upon the activities of SRB. Optimum conditions for the metabolism of propionate and other volatile fatty acids were between pH 7.0 and 8.0.     

Alkaline phosphatases of the mosquito, Culex tarsalis Coquillett

Spectrophotometric and isoelectric focusing (IEF) electrophoretic characterization of the alkaline phosphatase (ALKP) of the mosquito, Culex tarsalis, are presented. With p-nitrophenylphosphate (Pnp) as substrate, ALKP was optimally active at 37 degrees C, pH 8.0, 30 mM MgCl2, Vmax was 35.8 mumoles/10 min and the Km was 5.7 mM, with no demonstrable requirement for Zn2+. The spectrophotometric enzyme(s) was stimulated by dithiothreitol, 2-mercaptoethanol, and poly-vinylpyrollidone (PVP); inhibited by NaF, several alternative cations (Ca2+, Ba2+, Fe2+, Cu2+), and EDTA. ALKP activity was cyclic during the 15 day post-adult emergence period of the study. No significant differences were noted between the specific activities of males and females. IEF electrophoresis revealed 6 ALKP isozymes detected with alpha-naphthylphosphate within the pH range 4.0-5.5, with a second group of 3 rather indistinct species in the pH 6.0-7.0 range. IEF ALKP isozymes were stimulated by Mg2+ and PVP and inhibited by EDTA (except ALKP5.0) and cysteine; partial inhibition with phenylalanine. IEF detection of ALKP activity with Pnp indicated that the majority of the activity was localized in the pH 4.0-5.5 range, in close agreement with the alpha-naphthylphosphate results.     

Glycosphingolipid glycosyl hydrolases and glycosidases of synchronized human KB cells.

KB cells were synchronized by a double thymidine block procedure. An investigation was made of the activities of alpha-L-fucosidase (EC 3.2.1.51), alpha-D-galactosidase (EC 3.2.1.22), beta-D-galactosidase (ec 3.2.1.23), alpha-D-glucosidase (EC 3.2.1.20), beta-D-glucosidase (EC 3.2.1.21), alpha-D-mannosidase (EC 3.2.1.24), beta-D-N-acetylgalactosaminidase (EC 3.2.1.53), and beta-D-N-acetylglucosaminidase (EC 3.2.1.52) from synchronized cultures, using appropriate artificial substrates. Ceramide glucosidase (EC 3.2.1.45) and ceramide trihexosidase levels (EC 3.2.1.47) were also investigated at various stages in the cell cycle, using appropriate glycosphingolipid substrates. Whereas each of these enzymes exhibited some activity throughout the cell cycle, peak activity (2- to 6-fold increase) occurred late in the S phase. Two molecular forms of ceramide glucosidase (optimal activity at pH 4.0 and pH 6.0) and two forms of ceramide trihexosidase (pH 4.0 and pH 7.5) were identified. Peak levels of the forms that preferred the relatively acid pH occurred earlier in the S phase of the cell cycle than those of the forms that were more active at the higher pH. The possibility that the forms with optimal activity at pH 4 are precursors of those with optimal activity at pH 6 to 7.5 is discussed. Precipitation of beta-galactosidase of synchronized KB cells with specific antibody revealed that changes in the activity of this enzyme during the cell cycle were the result of fluctuations in the amount of the enzyme.     

Degradation of DNA during the autolysis of<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The autolysis of yeast cells has practical implications in the production of fermented foods and beverages and flavourants for food processing. Protein and RNA degradation during yeast autolysis are well described but the fate of DNA is unclear. Yeast cells (Saccharomyces cerevisiae) were autolysed by incubating suspensions at 30–60°C (pH 7.0), and at pH 4.0–7.0 (40°C) for 10–14 days. Up to 55% of total DNA was degraded, with consequent leakage into the extracellular environment of mainly 3′- and 5′-deoxyribonucleotides, and lesser amounts of polynucleotides. The rate and extent of DNA degradation, composition of the DNA degradation products and DNase activity were affected by temperature and pH. The highest amount of DNA degradation occurred at 40°C and pH 7.0, where the highest DNase activity was recorded. DNase activity was lowest at 60°C and pH 4.0, where the proportion of polynucleotides in the degradation products was higher. Electronic Publication     

The isolation and properties of a non-pepsin proteinase from human gastric mucosa.

1. A non-pepsin proteinase, proteinase 2, was successfully isolated free from pepsinogen (by repetitive chromatography on DEAE- and CM-celluloses) from the gastric mucosa of a patient with a duodenal ulcer and the uninvaded mucosa of a patient with a gastric adenocarcinoma. 2. Proteinases 1a and 1b, found in gastric adenocarcinoma, were not found in the gastic mucosa of these patients. 3. Proteinase 2 was shown to have an asymmetrical broad pH-activity curve with a maximum over the pH range 3.0-3.7. 4. Proteolytic activity of proteinase 2 was inhibited by pepstatin; the concentration of pepstatin giving 50% inhibition is of the order of 3nm. 5. Inhibition of proteolytic activity by carbenoxolone and related triterpenoids indicated that at pH 4.0 proteinase 2 possesses structural characteristics relating it to the pepsins and at pH 7.4 to the pepsinogens. 6. The sites of cleavage of the B-chain of oxidized insulin for proteinase 2 at pH 1.7 and pH 3.5 were shown to be similar to those previously established for human pepsin 3 and for the cathepsin E of rabbit bone marrow. 7. The non-pepsin proteinase 2 (cathepsin) of human gastric mucosa has properties more similar to cathepsin E than to the cathepsins D.     

Redox potential of plastoquinone A in spinach chloroplasts.

The redox potential of plastoquinone A in spinach chloroplasts was determined. The midpoint potential of the quinone is about +80 mV at pH 7.0 with an n value of 2. The pH-dependence of the potential is -30 mV per pH between pH 4.0 and 5.7, and -60 mV per pH between pH 5.7 and 8.0. The change of the slope at pH 5.7 is interpreted as the protonation of the oxidized plastoquinone A.     

Properties of intracellular ribonuclease utilized for RNA reduction in disintegrated cells of Saccharomyces cerevisiae.

The properties of intracellular RNase in disintegrated cell suspensions of Saccharomyces cerevisiae have been studied. The influence of salt addition and/or incubation of the suspension on the activity of RNase and on the degradation of endogenous RNA was determined. No significant change in the RNase activity in the disintegrated suspensions was obtained by addition of 3% NaCl or by incubation at 50 degrees C with 3% NaCl. During the incubation with NaCl the active RNase was able to degrade endogenous RNA. By incubation without salt the RNase was inactivated. Inactivation also occurred after extraction at alkaline pH. The RNase had an optima at pH 5-6 and temperatures between 50-60 degrees C. The main part of the RNase in the unincubated suspension was soluble also at pH 4.0. No serious protein degradation occurred during the short time incubation needed for RNA reduction. 70% of the protein in the suspensions was recovered in the precipitate at pH 4.0 after 20 min of incubation. The corresponding protein recovery from unincubated suspensions was 77%.     

Redox potential of plastoquinone A in spinach chloroplasts

The redox potential of plastoquinone A in spinach chloroplasts was determined. The midpoint potential of the quinone is about +80 mV at pH 7.0 with an n value of 2. The pH-dependence of the potential is ?30 mV per pH between pH 4.0 and 5.7, and ?60 mV per pH between pH 5.7 and 8.0. The change of the slope at pH 5.7 is interpreted as the protonation of the oxidized plastoquinone A.     

Activity and isoenzyme composition of vacuolar peroxidase in the roots of red beet at different stages of development and upon changes in storage conditions

Activity and isoenzyme composition of phenol-dependent vacuolar peroxidase (PO) were examined at different stages of development of red beet (Beta vulgaris L.) roots. The enzyme activity was found to increase during growth and decrease during the period of root dormancy. The isoenzyme composition of PO was also altered; additional cationic and anionic isoforms were revealed at certain stages of growth and dormancy. The dormant roots are often subjected to stresses, such as water deficiency and pathogenesis. For this reason, the enzyme activity in beet roots showing obvious signs of shrivel and infection with pathogenic microorganisms was investigated. The activation of PO was observed in infected roots, whereas the increase in the number of cationic PO isoforms was noted in water-stressed roots. The pH optima of the enzyme were found to shift depending on the developmental stage and the nature of stress factors: during dormancy and under water stress the pH optima shifted towards the acidic values. The shift in pH optima occurred concurrently with the appearance of cationic inducible isoforms. The pH dependences of cationic PO isoforms (these POs were mainly associated with the tonoplast) showed that their activity was optimal at pH 4.0 and 5.0. The observed changes in activity and composition of isoenzymes suggest the active involvement of the vacuolar peroxidase in metabolic processes and cell defense responses in beet roots.     

Succinyl CoA Synthetase of Tobacco

The isouition and partial purification of succinyl CoA synthetase from Nicotiana tabacum is described. Enzynio activity had a pH optimum of pH 7.0 and apparent Km values for succinic acid. ATF and MgCl(2) . are 3.5 × 10(-2) 5.7 × 10(-3) and 4.0 × 10(-3) M, respectively. The euzyme is specific in requirement for ATP. Enzyme activity was low in seed, decreased during germination and seedling development, then increased to maximum in physiologically mature leaf tissue. Plauts with the apical apical meristem removed had less euzyme activity in leaves but more enzyme activity in the roots than plants with apical meristem intact. Most enzyme activity observed was associated with the soluble fractions of the cell.     

Alteration of the anterior acrosome of motile bovine spermatozoa by fructose and hydrogen ion concentration

Storing cauda epididymal spermatozoa in seminal plasma or in defined media at 1 x 10(9) spermatozoa/ml for 24 h at 4 degrees C caused swelling of the apical ridge on motile spermatozoa (SAR) provided concentrations of fructose in the range normally found in seminal plasma or comparable levels of glucose were present. Evaluation of these conditions indicated that, with glycolysable sugars in the media, pH dropped from 6.6-6.7 to 5.7-6.0. Most of the pH decrease occurred during the first 2 h of slow cooling from 37 to 4 degrees C. pH decrease was undoubtedly due to sperm organic acid production which overwhelmed the relatively weak buffering capacity of the defined media and/or seminal plasma. Inducing pH decreases with HCl in fructose-free conditions, and using NaOH to prevent a pH decrease when fructose was included in media, demonstrated that exposing spermatozoa to pH values of 5.7-6.0 and not a specific response to fructose was the major cause of SAR.     

Increased cervical electrical activity during oestrus in progestagen treated ewes: Possible role in sperm transport

The aim of this investigation was to characterize the pattern of cervical myoelectrical activity (EMG) in the sheep, during the periovulatory period, after synchronization of oestrus with progestagen and eCG. EMG was measured with a computerised modular system in five ewes previously fitted with a pair of monopolar Teflon needle electrodes in the muscle layers of the cervix. Each ewe was submitted to oestrus synchronization treatment with intravaginal progestagen sponge during 12 days, and the administration of 500 IU of eCG at the withdrawal of sponge. EMG was recorded in each animal during 19 h, starting 44 h after withdrawal of sponge. The number and duration of events were determined every hour during the experiment. Two distinct event durations were identified: one lasting less than 200 s and another lasting between 300 and 500 s. The two types of events analysed (less than 200 s and lasting between 300 and 500 s) had a similar pattern during the period of observation although they were not in synchrony. For events lasting less than 200 s, activity increased between 48 and 50 h after sponge withdrawal, with the peak of activity being observed between 51 and 53 h. For events of 300-500 s duration, the peak of activity was observed between 48 and 50h after sponge withdrawal and activity was maintained until 51-53 h. The increase in cervical motility observed in progestagen-eCG treated ewes is in keeping with the increase in cervical activity observed by others in natural cycling animals, and suggests that exogenous hormones used in synchronization protocols had no deleterious action on cervical motility during periovulatory period. The enhanced activity of cervical muscle layer found around the time of mating and/or AI suggests it may play an important role as a regulatory mechanism of sperm transport. Taking advantage of the cervical responsiveness to various drugs, experimental modulation of cervical activity could be used to facilitate cervical sperm transport and consequent improve of fertility after cervical AI.     

Effect of dicarboxylic acids on the peroxidase-IAA oxidase isozymes of soybean callus

Multiple forms of peroxidase with indole-3-acetic acid (IAA) oxidase activity were detected in callus cultures from soybean seeds [ Glycinc max (L.) Merrill, cv. Acme] using ion-exchange chromatography and polyacrylamide gel electrophoresis. The properties of the IAA oxidase were studied with a partially purified fraction eluted from a DEAE cellulose column. At pH 5.7. p-coumaric acid and MnCl₂ were required as cofactors and H₂O₂ was not able to replace them, but H₂O₂ eliminated the usual lag period of the reaction. Activation effects obtained with some dicarboxylic acids acting only on IAA oxidase are shown. These effects were studied at different pH values and oxalic acid was found to be the most efficient activator, particularly at pH 4.6. Activation by oxalic acid occurred even in the absence of MnCl₂, but the presence of this salt produced a synergistic effect. IAA oxidase showed a sigmoidal kinetic behaviour at pH 5.7 changing to hyperbolic at pH 4.6     

Studies on the meta and para O-sulphation of the catechol compound 3,4-dihydroxybenzoic acid by rat liver sulphotransferase in vitro.

The enzymic meta and para O-sulphation of 3,4-dihydroxybenzoic acid was investigated in vitro with a dialysed high-speed supernatant from rat liver. The O-sulphated products were identified by comparison with the reference compounds. The chemical synthesis and identification of the reference O-sulphate esters is described in detail. The sulphotransferase activity of the dialysed supernatant from rat liver towards 3,4-dihydroxybenzoic acid was 580 pmol of 3-O-sulphate and 120 pmol of 4-O-sulphate formed/min per mg of protein at the optimal pH of 7.4. The meta/para ratio of O-sulphation was independent of pH, time of incubation, concentration of enzyme and presence of dithiothreitol. The O-sulphate esters of 3,4-dihydroxybenzoic acid were found to be good substrates for the arylsulphatase reaction at pH 5.6. The arylsulphatase activity of a dialysed preparation from rat liver was 4.0 nmol of 3-O- and 5.7 nmol of 4-O-sulphate ester hydrolysed/min per mg of protein, respectively. Arylsulphatase from Helix pomatia had an activity of 620 pmol of 3-O-sulphate and of 16.6 nmol of 4-O-sulphate ester hydrolysed/min per unit (mumol/h) of sulphatase.     

Further evaluation of an EMG technique for assessment of the deep cervical flexor muscles

A novel surface electromyographic (EMG) technique was recently described for the detection of deep cervical flexor muscle activity. Further investigation of this technique is warranted to ensure EMG activity from neighbouring muscles is not markedly influencing the signals recorded. This study compared deep cervical flexor (DCF) muscle activity with the activity of surrounding neck and jaw muscles during various anatomical movements of the neck and jaw in 10 volunteer subjects. DCF EMG activity was recorded with custom electrodes inserted via the nose and fixed by suction to the posterior mucosa of the oropharynx. Surface electrodes were placed over the sternocleidomastoid, anterior scalene, masseter and suprahyoid muscles. Positioned in supine, subjects performed isometric cranio-cervical flexion, cervical flexion, right and left cervical rotation, jaw clench and resisted jaw opening. Across all movements examined, EMG amplitude of the DCF muscles was greatest during neck movements that would require activity of the DCF muscles, particularly during cranio-cervical flexion, their primary anatomical action. The actions of jaw clench and resisted jaw opening demonstrated significantly less DCF EMG activity than the cranio-cervical flexion action (p < 0.05). Across all other movements, the neighbouring neck and jaw muscles demonstrated greatest EMG amplitude during their respective primary anatomical actions, which occurred in the absence of increased EMG amplitude recorded from the DCF muscles. The finding of substantial EMG activity of the DCF muscles only during neck actions that would require their activity, particularly cranio-cervical flexion, and not during actions involving the jaw, provide further assurance that the majority of myoelectric signals detected from the nasopharyngeal electrode are from the DCF muscles.     

Morphogenic change in the cervix of the ewe after prolonged exposure to oestradiol-17 beta

This study examined the effects of prolonged exposure to oestradiol-17 beta on the morphology of the cervix of the ewe. Merino ewe lambs were implanted subcutaneously with 3 Silastic capsules which released a total of approximately 300 micrograms oestradiol-17 beta per day. After exposure for 200 days the uterus was more markedly bicornuate, and the cervix was broader and softer, than in controls. The cervical folds were shorter and contained many stromal cells. The amount of lamina propria under the folds was increased and altered so that it contained tubular glands and more stromal cells. The endocervix thus came to resemble endometrium. This appearance developed within 80 days of exposure, and remained for at least 170 days after implant removal. In a second experiment, mature multiparous Merino ewes were ovariectomized and implanted with 1, 2 or 4 similar oestradiol capsules for 140 days. Similar features developed in these ewes, and the degree of change was almost as great with 1 implant as with 4. Changes of a similar nature can be produced in other species by oestrogen given during organogenesis but not during adult life. The changes indicate that the ewe has an ability to display a degree of morphogenic response to oestradiol during adult life.     

Kinetic control of ethanol production by Zymomonas mobilis

Summary Zymomonas mobilis UQM 2716 was grown anaerobically in continuous culture (D = 0.1/h; 30° C) 3nder glucose or nitrogen limitation at pH 6.5 or 4.0. The rates of glucose consumption and ethanol production were lowest during glucose-limited growth at pH 6.5, but increased during growth at pH 4.0 or under nitrogen limitation, and were highest during nitrogen-limited growth at pH 4.0. The uncoupling agent CCCP substantially increased the rate of glucose consumption by glucose-limited cultures at pH 6.5, but had much less effect at pH 4.0. Washed cells also metabolised glucose rapidly, irrespective of the conditions under which the original cultures were grown, and the rates were variably increased by low pH and CCCP. Broken cells exhibited substantial ATPase activity, which was increased by growth at low pH. It was concluded that the fermentation rates of cultures growing under glucose or nitrogen limitation at pH 6.5, or under glucose limitation at pH 4.0, are determined by the rate at which energy is dissipated by various cellular activities (including growth, ATP-dependent proton extrusion for maintenance of the protonmotive force and the intracellular pH, and an essentially constitutive ATP-wasting reaction that only operates in the presence of excess glucose). During growth under nitrogen limitation at pH 4.0 the rate of energy dissipation is sufficiently high for the fermentation rate to be determined by the inherent catalytic activity of the catabolic pathway.Abbreviations CCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - qG rate of glucose consumption (g glucose/g dry wt cells/h) - qE rate of ethanol production (g ethanol/g dry wt cells/h) - Y growth yield (g dry wt cells/g glucose) - D dilution rate Offprint requests to: C. W. Jones     

Fly abundance and climate as determinants of sheep blowfly strike incidence in southwest England

The relationships between abundance of the blowfly Lucilia sericata (Meigen) (Diptera: Calliphoridae), climate, animal management procedures and the incidence of cutaneous myiasis (blowfly strike) in sheep were examined in three sheep pasture systems in southwest England during the summers of 2002 and 2003. In each year, flies were collected using liver-baited sticky targets, daily weather and routine husbandry practices were noted and the age-class of each animal infested and body position of each strike were recorded. On sites where no strike control was used, 5.8-12.1% of ewes and 5.7-15.8% of lambs were struck. Ewe strikes predominated at the beginning of the season. The incidence of ewe strikes was significantly associated with higher mean temperature, rainfall and shearing; shearing was associated with a 95% reduction in the risk of ewe strike. In lambs, the incidence of strike was significantly related to higher fly abundance, ewe shearing, treatment and mean ambient temperature. Lambs were 4.6 times more likely to be struck after the ewes had been shorn than before; however, the strongest relationship was with mean L. sericata abundance. Average minimum threshold temperatures of 9.5 degrees C for lamb strikes and 8.5 degrees C for all strikes were extrapolated, below which oviposition did not occur. Over 80% of ewe strikes occurred in the breech region in 2002, as did 100% in 2003. However, in lambs both body and breech strikes occurred in both years. The distribution of lamb strikes appeared to change over time, with breech strikes predominating in May, June and July and body strikes occurring increasingly later in the season. The incidence of lamb breech strikes was significantly associated with higher L. sericata abundance and ewe shearing but there was no relationship with weather conditions. By contrast, the risk of body strike in lambs was significantly associated with higher blowfly abundance, higher rainfall and higher maximum temperatures. The relationship between strike incidence and L. sericata abundance is important because it enhances our understanding of strike incidence patterns and management of this disease. Clearly, any factors that facilitate larger L. sericata populations, such as inappropriate carcass burial or increased average ambient temperatures, are likely to increase the incidence of strike.     

The adaptive acid response in Mesorhizobium sp.

Mesorhizobium huakuii strain LL56 and Mesorhizobium sp. strain LL22, which nodulate Lotus glaber, developed an adaptive acid response during exponential growth upon exposure to sublethal acid conditions. The adaptive acid response was found to be dependent on the sublethal pH and the strain intrinsic acid tolerance: the lowest adaptation pH was 4.0 for strain LL56 and 5.7 for strain LL22, and the lowest pH values tolerated after adaptation were 3.0 and 4.0, respectively. Both complex and minimal medium allowed the development of the adaptive acid response, although in complex medium this response was more effective. Three low molecular weight polypeptides (LMWPs) showed increased expression in strain LL56 during the adaptation to pH 4.0. However, the adaptive acid tolerance was only partially dependent on de novo protein synthesis, and constitutive systems may play a significant role on the acid tolerance of Mesorhizobium huakuii strain LL56.     

Oxytocin response to conditioned and nonconditioned stimuli in lactating ewes

We have measured oxytocin release during lactation in the ewe in response to normal tactile sucking stimuli as well as exteroceptive stimuli emanating from the lamb. Four puerperal ewes that had indwelling catheter inserted in the femoral artery while still pregnant were used. Each nursed a single lamb. Each was studied 2 or 3 times between Days 1 and 15 of lactation during a 2.5-h experimental period that was preceded by a 2-h separation from the lamb in the early morning. Samples were taken before and after the lamb was brought within sight and sound of the ewe but without contact, and then 0.5, 1, 5, 10, 15, 30, and 60 min after suckling began. When another suckling episode intervened, the same sampling schedule was immediately restarted. Suckling occurred in an intermittent fashion; 3 to 4 episodes of 2.9 +/- 2.0 (SD)-min duration took place with variable intervals during the 2.5-h experimental period. Exteroceptive stimuli emanating from the lamb caused plasma oxytocin to rise significantly from basal levels of 10.0 +/- 4.5 to 21.8 +/- 5.7 pg/ml (mean +/- SE, n 10, p less than 0.05). This rise was not seen on Day 1 and in only half of the ewes on Day 2, but thereafter the rise occurred in every instance. A further rise in plasma oxytocin was observed in almost all instances (86%) at suckling. Peak levels were usually observed within 1 min. They were quite variable, ranging from 15 pg/ml to 287 pg/ml, and not related to the milk yield, but were significantly greater than spontaneous pulses observed in nonlactating puerperal sheep.(ABSTRACT TRUNCATED AT 250 WORDS)