Intracellular proteolytic activity during sporulation ofBacillus megaterium

Intracellular proteolytic activity increased during incubation of the sporogenic strain ofBacillus megaterium KM in a sporulation medium together with excretion of an extracellular metalloprotease. The exocellular protease activity in a constant volume of the medium reached a 100-fold value with respeot to the intracellular activity. Maximal values of the activity of both the extracellular and intracellular enzyme were reached after 3 – 5 h of incubation. After 7 h 20 – 50% cells formed refractile spores. The intracellular proteolytic system hydrolyzed denatured proteinsin vitro at a rate up to 150 μg mg_-1 h_-1 and native proteins at a rate up to 70 μg mg^-1 h^-1. Degradation of proteinsin vivo proceeded from the beginning of transfer to the sporulation medium at a constant rate of 40 μg mg^-1 h^-1 and the inactivation of beta-galactosidase at a rate of 70 μg mg^-1 h^-1. The intracellular proteolytic activity was inhibited to 65 – 88% by EDTA, to 23 – 76% by PMSF. Proteolysis of denatured proteins was inhibited both by EDTA and PMSF more pronouncedly than proteolysis of native proteins; 50 – 65% of the activity were localized in protoplasts. Another strain ofBacillus megaterium (J) characterized by a high (up to 90%) and synchronous sporulation activity was found to behave in a similar way, but the rate of protein turnover in this strain was almost twice as high. The asporogenic strain ofBacillus megaterium KM synthesized the exocellular protease in the sporulation medium, but its protein turnover was found to decrease substantially after 3 – 4 h. The intraeellular proteolytic system of the sporogenic strain J and the asporogenic strain KM were also inhibited by EDTA and PMSF.     

NITRATE REDUCTASE ACTIVITY OF AMPHIDINIUM CARTERI AND CACHONINA NIEI (DINOPHYCEAE) IN BATCH CULTURE: DIEL PERIODICITY AND EFFECTS OF LIGHT INTENSITY AND AMMONIA1,2

The activity of extracted NADH-NO₃^? reductase was measured in the marine dinoflagellates Amphidinium carteri Hulburt and Cachonina niei Loeblich. Its activity showed a diel periodicity and was ca. twice as great at midday as at midnight. The enzyme activity was unstable, with an in vitro half-life of 2–3 h. Values of enzyme activity were low or undetectable during lag phase but paralleled the instantaneous growth rate value during log phase. Nitrate reductase activity was not found in the stationary phase of growth, but additions of NO₃^? resulted in enzyme activity after 24h. When A. carteri was exposed to a series of light intensities for several weeks, the division rate and enzyme activity increased with increasing light intensity up to saturating intensities. In 6 h exposures, enzyme activity decreased with decreasing light intensities below light intensities saturating division rate. Additions of NH₄⁺ (0.5–50 μm) to A. carteri cultures decreased the amount of extractable enzyme. The in vitro activity was not inhibited by similar NH⁺₄ concentrations.     

Cell density dependent regulation of phenylalanine hydroxylase activity in hepatoma cells : Evidence for both an active and inactive enzyme form

The cell density dependent regulation of phenylalanine hydroxylase activity in Reuber hepatoma (H4) cells growing in monolayer culture has been examined in detail. We found that 48 h or more after subculture phenylalanine hydroxylase activity in the cells is an exponential function of cell density (cells/cm²). No discontinuity in the relationship is seen with the formation of a confluent monolayer.A rapid loss or a rapid gain in enzyme activity in the cells is observed after diluting or concentrating the cell cultures. The two processes appear qualitatively different. The loss in activity is a first order process which starts at the time of subculture with the rate of loss dependent on the density of subculture. The gain in activity begins 6–8 h after subculture to a higher density; it can be blocked by cycloheximide and has a maximum rate of increase that is about 10% of the maximum rate of loss of activity.Using immunochemical procedures, we found the same amount of phenylalanine hydroxylase associated antigen in Reuber cells from low density as from high density cultures, over a range of phenylalanine hydroxylase specific activities from 0.2 to 4.2. After concentrating cells to a higher density, no increase in enzyme antigen was observed, despite a several-fold increase in enzyme activity and a requirement for protein synthesis during the process. These observations imply the presence of an active and inactive phenylalanine hydroxylase with the relative amounts of each determined by the cell density. The effects of db-cAMP are discussed. Evidence is presented here that the hydrocortisone stimulation of phenylalanine hydroxylase activity works through a different mechanism than the cell density dependent process.     

Phase-Dependent Effects of Stimuli Locked to Oscillatory Activity in Cultured Cortical Networks

The archetypal activity pattern in cultures of dissociated neurons is spontaneous network-wide bursting. Bursts may interfere with controlled activation of synaptic plasticity, but can be suppressed by the application of stimuli at a sufficient rate. We sinusoidally modulated (4 Hz) the pulse rate of random background stimulation (RBS) and found that cultures were more active, burst less frequently, and expressed oscillatory activity. Next, we studied the effect of phase-locked tetani (four pulses, 200 s⁻¹) on network activity. Tetani were applied to one electrode at the peak or trough of mRBS stimulation. We found that when tetani were applied at the peak of modulated RBS (mRBS), a significant potentiation of poststimulus histograms (PSTHs) occurred. Conversely, tetani applied at the trough resulted in a small but insignificant depression of PSTHs. In addition to PSTHs, electrode-specific firing rate profiles within spontaneous bursts before and after mRBS were analyzed. Here, significant changes in firing rate profiles were found only for stimulation at the peak of mRBS. Our study shows that rhythmic activity in culture is possible, and that the network responds differentially to strong stimuli depending on the phase at which they are delivered. This suggests that plasticity mechanisms may be differentially accessible in an oscillatory state.     

Cyclic Adenosine Monophosphate in the Nervous System of Aplysia californica : I. Increased synthesis in response to synaptic stimulation

In the isolated abdominal ganglion of Aplysia, previously incubated in adenine-³H, the amount of ³H-labeled adenosine-3',5' monophosphate (cAMP) doubled after electrical stimulation of nerves at a physiological rate (1/sec). No change was detected after 4 min of stimulation. An increase in cAMP was first seen after 15 min; lengthening the period of stimulation to 1 hr did not increase the extent of the effect. ATP contained 50% of the total radioactivity taken up from adenine-³H, cAMP about 0.1%. During stimulation both the total amount and the specific radioactivity of adenosine triphosphate (ATP) did not change. Thus, the increased amount of radioactivity found in cAMP after stimulation represented an increase in its rate of synthesis. During stimulation formation of cAMP-³H was not altered in nerves or in the cell body of an identified neuron (R2). In addition, no changes were detected in the total amounts of cAMP in the ganglion and in the cell body of R2. It seems likely that the increase was initiated by synaptic activity rather than by action potentials. It was blocked by elevating the concentration of Mg, which also blocks synaptic activity without impairing conduction of impulses. Moreover, impulse activity induced by ouabain and glutamate did not result in increased formation of cAMP.     

The effect of low-intensity laser irradiation in the blue, green, and red spectral ranges on healing of experimental skin wounds in rats

The effects of low-intensity laser irradiation in the red (632.8 nm), green (532 nm), and blue (441.2 nm) spectral ranges on wound healing has been studied in rats. The effect of the traditionally used red laser irradiation has been compared with the effect caused by laser irradiation in other spectral ranges, aiming to support the provisional hypothesis that a similar healing effect could be achieved at lower doses of wound irradiation by lasers emitting in the blue and green spectral ranges. The following parameters have been used to characterize healing of the experimental wounds: the functional activity of phagocytes in the wound exudate, which was determined from luminol-dependent chemiluminescence, the phagocyte number; the wound exudates’ antioxidant activity; and the rate of healing, which was determined as the change of the wound surface area. It was found that in all cases the laser irradiation accelerated the healing of wounds. Exposure to red laser irradiation at the dose of 1.5 J/cm²), and to blue or green laser irradiation at a dose of 0.75 J/cm² shortened the time of the wound healing from 22 to 17 and 19 days, respectively. The functional activity of leukocytes in irradiated groups increased by day 5 after surgery, whereas in the control group it decreased. The superoxide dismutase activity increased in all experimental groups by day 5 after surgery. Laser irradiation in the red spectral range at a dose of 1.5 J/cm² resulted in a larger increase in superoxide dismutase activity, as compared to that found after exposure to laser irradiation in the blue and green spectral ranges at a dose of 0.75 J/cm².     

Optimization of extracellular xylanase production by Dictyoglomus sp. B1 in continuous culture

The thermophilic, xylanolytic, anaerobic organism, Dictyoglomus sp. B1, was cultivated in batch and continuous cultures in media containing insoluble beech-wood xylan. The extracellular xylanase activity levels obtained for the two cultivation methods were compared. Experiments were performed separately to determine the optimum substrate concentration, dilution rate, pH and temperature for xylanase production. Maximum xylanase activity was found at a substrate concentration of 1.5 g xylan/l, a dilution rate of 0.112 h^–1, pH 8.0 and at 7°C. Different combinations of these optimum values were used in a 2³ factorial experiment to investigate whether an increase in the xylanase production/activity could be achieved. A maximum xylanase activity of 2312 U/l was found when fermentors were operated at 73°C with a substrate concentration of 1.5 g xylan/l, pH 8.0, and a dilution rate of 0.112 h^–1. Thus, the optimum xylanase activity in the factorial experiment was obtained when the conditions that gave the maximum xylanase activities in the individual experiments were combined. Optimum xylanase activity obtained in the 2³ factorial experiment was 6.2 times higher than the activity found in the initial batch culture (373 U/l) and 3.0 times higher than the activity of a batch culture (783 U/l) grown at the same optimum conditions as the factorial experiment. The higher specific xylanase activity (217 U/mg protein) found in the 2³ factorial experiment was 4.1 times higher than the specific activity in the initial batch culture (53 U/mg protein).     

Properties of anionic peroxidases purified from Chinese cabbage roots

Two anionic peroxidases were isolated from Chinese cabbage roots and purified using gel filtration followed by ion-exchange chromatography. Following purification a specific activity of peroxidases was estimated as 50 units.mg^-1 (A1) and 30 units.mg^-1 (A2) compared with that of a crude extract of the peroxidases which was 2.31 units.mg^-1. The pH for its optimum activity was 5.0 and the addition of Ca²⁺ produced a 15 % increase in peroxidase activity. Isoelectric focusing techniques were carried out in order to classify the peroxidases based on their isoelectric point (pI). Two anionic peroxidases, A1 and A2, were found to have pI values of 4.83 and 4.78, respectively. The peroxidases were found to be heat-stable, with 20 % (A1) and 16 % (A2) of the enzymatic activity remaining after heat treatment at 70 °C for 20 min. The heat inactivation rate followed first-order kinetics with the activation energy; Ea, estimated as 38.2 kcal.mol^-1 and 36.4 kcal.mol^-1 for A1 and A2, respectively.     

Production of a membrane-bound proteins,the human gamma-glutamyl transferase,by CHO cells cultivated on microcarriers,in aggregates and in suspension

Recombinant Chinese Hamster Ovary (CHO) cells, engineered for the production of human gamma-glutamyl transferase (GGT), have been grown on Cytodex 1 microcarriers, as aggregates, or as single cells in suspension after adaptation. GGT is a membrane bound enzyme which was not secreted during the culture period. The maximal enzyme activity was found to be directly related to the achieved maximal cell density. Culture of CHO on microcarriers yielded the fastest growth, with a specific growth rate of 0.04 h^–1, the highest cell density (near 1.3×10⁶ cells ml^–1), and the highest enzyme activity around 300 mU ml^–1, which corresponded to a specific cellular level of 20 mU 10^–5 cells. GGT could also be produced by growing CHO cells in suspension as single cells or as aggregates. Under these conditions, however, the specific CHO growth rate was significantly slower and the GGT level per cell was divided by a factor 6. Growing CHO cells without microcarriers also resulted in differences in cell metabolism, with a higher conversion yield of glutamine into ammonia, and a higher cell lysis. The catalytic kinetic constants of the enzyme were found identical for the three culture systems.     

Degradation of ribulose-1,5-bisphosphate carboxylase by proteolytic enzymes from crude extracts of wheat leaves

In crude extracts from the primary leaf of wheat seedlings, Triticum aestivum L., cv. Olympic, maximum proteinase activity, as determined by measuring the rate of release of amino nitrogen from ribulose-bisphosphate carboxylase (RuBPCase), was found to be obtained only when EDTA and L-cysteine were included in the extraction buffer. Highest proteinase activity was obtained by grinding at pH 6.8, although the level of activity was similar in the pH range 5.6 to 8.0; this range also coincided with maximum extractability of protein. The lower amount of RuBPCase degrading proteinase extracted at low pH was not due to an effect of pH on enzyme stability. The optimum temperature of reaction was 50° C and reaction rates were linear for at least 120 min at this temperature. In the absence of substrate the proteinase was found to be very sensitive to temperatures above 30° C, with even short exposures causing rapid loss of activity. The relation between assay pH and RuBPCase degradation indicated that degradation was restricted to the acid proteinase group of enzymes, with a pH optimum of 4.8, and no detectable activity at a pH greater than 6.4. The levels of extractable RuBPCase proteinase exhibited a distinct diurnal variation, with activity increasing during the latter part of the light period and then declining once the lights were turned off. The effect of leaf age on the level of RuBPCase, RuBPCase proteinase and total soluble protein was investigated. Maximum RuBPCase activity occurred 9 days after sowing as did soluble protein. After the maximum level was obtained, the pattern of total soluble protein was shown to be characterised by three distinct periods of protein loss: I (day 9–13) 125 ng leaf^-1 day^-1; II (day 15–27) 11 ng leaf^-1 day^-1; III (day 29–49) 22 ng leaf^-1 day^-1. Comparison of the pattern of RuBPCase activity and total protein suggest that the loss of RuBPCase may be largely responsible for the high rate of protein loss during period I. Proteinase activity increased sharply during the period of most rapid loss of RuBPCase activity, and because the specific activity of RuBPCase also declined, we concluded that RuBPCase was being degraded more rapidly than the other proteins. Once the majority of the RuBPCase was lost, there did not appear to be a direct relation between RuBPCase proteinase activity and rate of total soluble protein loss, since the proteinase exhibited maximum activity during the slowest period of protein loss (II), and was declining in activity while the rate of protein loss remained stable during the third and final period of total protein loss.Abbreviations RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - TCA trichloroacetic acid Supported by the Wheat Industry Research Council of Australia and the Australian Research Grants Committee D2 74/15052     

Influence of boron deficiency on 3H indole-3yl-acetic acid uptake and efflux in cell cultures of Daucus carota L.

³H-IAA net uptake and efflux was measured in carrot cell suspensions after two and five days in either boron (B) sufficient or B deficient media. Contrary to earlier observations with substances such as phosphate, glucose and potassium (or Rb⁺), ³H-label uptake from IAA over a 3 h period did not differ significantly after two days of treatment. After five days of B deficiency, cells accumulated higher ³H-activity than those receiving B. Total ³H-efflux from deficient and sufficient cells did not differ significantly but B deficient cells released more water soluble (pH 3.0) ³H, and less methanol soluble ³H activity, which may be due to a higher IAA oxidase activity. Correspondingly, we found an elevated peroxidase (POD) activity in B deficient cell cultures which was 54% higher after two days of deficieny and which increased further threefold after five days. Similar changes have been found in the amount of POD activity released to the nutrient solution with even more pronounced differences after two days. Thus, B deficient cells released less authentic ³H-IAA (1.7% vs. 3.1% related to total ³H efflux), as determined by HPLC separation.     

Pentobarbital anaesthesia in the chronobiological studies

Administration of anaesthesia may influence specific aspects of in vivo animal experiments and is an especially important consideration for experiments conducted during the daytime. Although chronobiological studies investigating interactions between general anaesthesia and circadian rhythms are sparse, all suggest that general anaesthetic agents have a significant effect on circadian rhythms. To assess the suitability of pentobarbital anaesthesia in chronobiological studies, this study was performed using pentobarbital-anaesthetized (40 mg/kg, intraperitoneal) female Wistar rats after adaptation to a light–dark (LD) cycle (12 h:12 h). Heart rate, rectal temperature (RT), electrocardiographic parameters, autonomic nervous system activity, acid–base balance and plasma concentrations of Na⁺, K⁺, Ca²⁺ and Cl^- were evaluated for their dependence on the LD cycle. LD differences were found in heart rate and RT, measured before the administration of the anaesthetic agent. Pentobarbital anaesthesia eliminated LD differences in all electrophysiological parameters, parameters of heart rate variability (except RR intervals) and parameters of acid–base balance and ion concentrations. LD differences with borderline statistical significance were found only for Na⁺ levels, with a higher level in the light period (i.e. nonactive) of the rat regimen day. During pentobarbital anaesthesia, parasympathetic tone predominates and sympathetic activity is depressed. Spontaneously breathing rats under pentobarbital anaesthesia are in an asphyxic state independent of the LD cycle in in vivo experiments. Results of this study suggest that pentobarbital anaesthesia is not suitable for chronobiological studies. However, it is suitable for cardiovascular research that is done regardless of the circadian rhythmicity, because it does not cause significant changes in heart activity.     

EFFECT OF VILLUS LENGTH ON CELL PROLIFERATION AND MIGRATION IN SMALL INTESTINAL EPITHELIUM

For the interpretation of data supporting the hypothesis of a feedback regulation of proliferative activity in intestinal crypts by the functional villus cell compartment the life span and migration rate of epithelial cells on villi of experimentally reduced length should be known. Autoradiographic studies and scintillation counting of isolated villi at different time intervals after ³H-thymidine labelling were carried out 36, 48 and 60 hr intervals after X-irradiation. The results showed that the life span of epithelial cells in rat small intestine (36–48 hr) is independent of the villus length. In villi of reduced length the migration rate of the epithelial cells was found to be decreased compared with controls. Changes in the migration rate in turn seem to be dependent on the production of epithelial cells in the crypt. Comparative studies on the recovery of crypt and villus epithelium after various doses (300 and 700 R) of X-radiation support the hypothesis that increased proliferative activity in the crypt cell compartment is related to a reduction of the number of functional villus cells below a critical villus length. The importance of these findings in the interpretation of data on (micro) biochemical analyses of certain cell differentiation characteristics during increased proliferative activity is discussed.     

Non-linearity of human renal (Na+ + K+)-ATPase; elimination by purification and correlation with cyclic AMP-dependent protein kinase activity.

Human renal (Na⁺ + K⁺)-ATPase exhibits a non-linear reaction rate which can be eliminated by purification of the enzyme. The activity in the microsomes and the first several stages of purification is nearly zero after 8 minutes of assay. The reaction of the purified enzyme, however, is linear with assay time. This change to a linear reaction rate correlates with the loss of cyclic AMP-dependent protein kinase activity from the (Na⁺ + K⁺)-ATPase preparation.     

Subcellular distribution of 35S-sulfur in spinach leaves after application of 35SO 4 2- , 35SO 3 2- , and 35SO2

³⁵SO₂, ³⁵SO ₃ ^2- , and ³⁵SO ₄ ^2- , respectively, were applied to leaves of Spinacia oleracea L. for 60 min in the light. Thereafter, the specific activity was determined in the organelles separated by means of sucrose density gradient centrifugation. In mitochondria and peroxisomes, the specific activity was equally distributed in their protein moieties. After application of ³⁵SO₂ or ³⁵SO ₃ ^2- , the chloroplast lamellae are characterized by elevated specific activity, which is not found after application of ³⁵SO ₄ ^2- . Chloroplast stroma shows a low specific incorporation rate after application of either compound, which may be due to the low turnover rate of Fraction I protein.     

Complete anaerobic oxidation of hydroquinone by Desulfococcus sp. strain Hy5: indications of hydroquinone carboxylation to gentisate

The sulfate-reducing strain Hy5 was able to grow with hydroquinone as sole source of carbon and energy. In experiments with dense cell suspensions, several indications were found that gentisate was the first intermediate in anaerobic degradation of hydroquinone: (1) degradation of hydroquinone was accelerated by addition of bicarbonate; (2) cell suspensions grown with hydroquinone oxidized gentisate at a rate similar to that of suspensions grown with gentisate, whereas the latter were not able to degrade hydroquinone in the presence of chloramphenicol; (3) in SDS-PAGE analysis of cell-free extracts of strain Hy5, two additional protein bands were found after growth with hydroquinone that were not detected in cells grown with gentisate, probably representing a hydroquinone carboxylating enzyme. A corresponding enzyme activity could not be detected. In cell-free extracts of hydroquinone-grown strain Hy5, the specific acyl-CoA ligase activity with gentisate as substrate was detected at 70 nmol x mg^-1 x min^-1. Gentisyl-CoA was enzymatically reduced to several unidentified nonaromatic products in the presence of dithionite-reduced methyl viologen.     

Oxidation of flavonoids by hypochlorous acid: reaction kinetics and antioxidant activity studies

Flavonoids, plant polyphenols, ubiquitous components of human diet, are excellent antioxidants. Hypochlorous acid (HOCl), produced by activated neutrophils, is highly reactive chlorinating and oxidizing species. It has been reported earlier that flavonoids are chlorinated by HOCl. Here we show that flavonoids from flavonol subclass are also oxidized by HOCl, but only if the latter is in a large molar excess (≥?10). The kinetics of this reaction was studied by stopped-flow spectrophotometry, at different pH. We found that flavonols were oxidized by HOCl with the rate constants of the order of 10⁴–10⁵ M^?1 s^?1 at pH 7.5. Antioxidant activity of HOCl-modified flavonoids was measured by 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) method. Slightly higher antioxidant activity, compared to parent compounds, was observed for flavonols after their reaction with equimolar or moderate excess of HOCl whereas flavonols treated with high molar excess of HOCl exhibited decrease in antioxidant activity. The mechanism of flavonoid reaction with HOCl at physiological pH is proposed, and biological consequences of this reaction are discussed.     

Studies on the methods of inorganic nutrient application in coconut

Summary Using carrier free³²P, tagged single superphosphate and⁸⁶Rb, the efficiency of different methods of plant injection and soil placement techniques for fertilizer application was examined. In the plant injection techniques the radioactivity was fed to the palms through growing roots tips, cut ends of roots, stem injection and leaf axils. The application of radioactivity through the cut ends of roots was most efficient since³²P was detected in 10 m tall palms, four hours after application. In stem, leaf axil and growing roots tips injection the³²P was detected after 8, 12 and 18 h. Out of four methods of soil application, the quickest recovery of³²P in the palms was detected after 7 days of placement when applied by the hole method. The³²P activity in the palms through circular trenches, strips and basin methods was recorded after 8, 8 and 11 days of application respectively. The accumulation of⁸⁶Rb was significantly higher than³²P. With plant injection technique the accumulation of activity was found to be significantly higher than with soil placement methods. The rate of radioactivity absorption was 10 to 60 time faster in the former technique as compared to that of the latter. The application of radioactivity through cut ends of roots and circular trench methods, were found to be better and may be recommended for nutrient application in coconut.     

Detection and properties of l-histidinol dehydrogenase in wheat germ

The presence and partial characterization of the properties of l-histidinol dehydrogenase (EC 1.1.1.23), the enzyme catalysing the last step in the pathway of histidine biosynthesis, has been described in higher plants for the first time. The activity has been found in cell-free extracts from wheat germ, turnip root, radish root and squash fruit. The enzyme has been partially purified and characterized from extracts of acetone powders of wheat germ. DEAE-cellulose chromatography revealed two peaks of histidinol dehydrogenase activity. In one there was a rapid reduction of NAD⁺ in the absence of histidinol; however, the rate was stimulated by the addition of histidinol. The rate in the absence of substrate became quite low after several min and the histidinol-dependent rate was then easily observed. The second peak of activity did not reduce NAD⁺ unless l-histidinol was present in the assay mixture. The K^ms for l-histidinol and NAD⁺ were determined for this latter enzyme. The values obtained at saturating concentrations of the other substrate were l-histidinol, 8.8 μM and NAD⁺, 0.14 mM. The product of the dehydrogenase reaction was histidine as determined by paper chromatography.