A Note on the Recognition of Pollution Stress in Populations of Estuarine Bacteria

Water samples were collected from the Humber estuary at a site subject to pollution by metal refinery effluent. V _max/bacterium for glucose mineralization, the percentage of glucose uptake by bacteria which was mineralized (% R ) and dissolved copper concentrations were measured. V _max/bacterium was negatively correlated with copper concentration and might be of use as an indicator of pollution stress. % R was not related to copper concentration and was not suitable as an indicator of pollution stress at this site.     

Relationships between suspended solids and standing crops and activities of bacteria in an estuary during a neap-spring-neap tidal cycle

Summary During a neap-spring-neap tidal cycle in the Humber estuary concentrations of suspended solids and oxygen depletion were greatest at spring tides. Most bacteria were attached to particles and attached bacteria (counted using epifluorescence microscopy) increased as solids increased. Concentrations of free bacteria however were unrelated to suspended solids. Heterotrophic activities of bacteria per unit volume of water (measured using ¹⁴C glucose uptake and Michaelis-Menton kinetics) varied irregularly and were not related to bacterial crops, suspended solids or oxygen depletion. Activity per bacterium however decreased as suspended solids increased.     

Metabolism of nitric oxide in soil and denitrifying bacteria

Abstract Production and consumption of NO was measured under anaerobic conditions in a slightly alkaline and an acidic soil as well as in pure cultures of denitrifying Pseudomonas aeruginosa, P. stutzeri, P. fluorescens, Paracoccus denitrificans, Azospirillum brasilense , and A. lipoferum . Growing bacterial cultures reduced nitrate and intermediately accumulated nitrite, NO, N₂O, but not NO₂. Addition of formaldehyde inhibited NO production and NO consumption. In the presence of acetylene NO was reduced to N₂O. Net NO release rates in denitrifying bacterial suspensions and in soil samples decreased hyperbolically with increasing NO up to mixing ratios of about 5 ppmv NO. This behaviour could be modelled by assuming a constant rate of NO production simultaneously with a NO consumption activity that increased with NO until V _max was reached. The data allowed calculation of the gross rates ( P ) of NO production, of the rate constants ( k ), V _max and K _m of NO consumption, and of the NO compensation mixing ratio ( m _c). In soil, P was larger than V _max resulting in net NO release even at high NO mixing ratios unless P was selectively inhibited by chlorate + chlorite or by aerobic incubation conditions. In bacteria, V _max was somewhat larger than P resulting in net NO uptake at high NO mixing ratios. Both P and V _max were dependent on the supply of electron donor (e.g. glucose). Both in soil (aerobic or anaerobic) and in pure culture, the K _m values of NO consumption were in a similar low range of about 0.5–6.0 nM. Anaerobic soil and denitrifying bacteria exhibited m _c values of 1.6–2.1 ppmv NO and 0.2–4.0 ppmv NO, respectively.     

Use of perfused cores for evaluating extracellular enzyme activity in stream-bed sediments

Abstract β-Glucosidase activity was investigated in stream-bed sediments using 4-methylumbelliferyl-β- d -glucopyranoside (MUF-β-Glc) as a model substrate. In a perfused core technique, water containing MUF-β-Glc was perfused up through sediment cores. β-glucosidase activity quantified from the release of fluorescent MUF in water discharge from the cores. At low rates of perfusion, maximum β-glucosidase activity ( V _max) in perfused sediments was similar to that in suspended (unperfused) sediments. Substrate affinity( K m)was higher in the suspended sediments. V _maxand K _m both increased when the perfusion rate was raised, although naturally-low substrate concentrations could mean that variability in perfusion rates has little effect on enzyme activity in the field. V _max was uninfluenced by whether ground or stream water was perfused through the sediments, but K _m was higher in cores perfused with groundwater. Increasing concentrations of glucose in the perfusion water resulted in a progressive inhibition of β-glucosidase activity. Although natural concentrations of glucose were low, the high turnover of enzymatically-released glucose probably means that β-glucosidase activity could be regulated by product concentration.     

The populations, characterization and activity of suspended bacteria in the Welsh River Dee

In a 29 month study of bacterial populations at three sites on the Welsh River Dee, the aerobic heterotrophic bacteria increased from an average basal level of about 1.2 times 10⁴ colony-forming units (cfu)/ml near the source of the river, to 2 times 10⁵ cfu/ml in the lower reaches. The ratio, total bacterial cell count: viable count, decreased from 70 in the upland reaches to 10 in the lower parts of the river. There was no apparent seasonal variation in bacterial numbers but on occasions the bacterial populations in both upland and lowland reaches of the river increased simultaneously. Fluctuations in bacterial numbers over a 50-fold range were observed in this study. Bacterial isolates from both upland and lowland sites were predominated by two groupings of bacteria, the Pseudomonas—Agrobacterium—Alcaligenes group and the Flavobacterium—Cytophaga—Flexibacter group. Results suggested that the latter group may have been part of the autochthonous population. Seasonal variation in heterotrophic potential ( V _max) for acetate uptake was shown to occur over a 30-fold range in the lowland reaches of the River Dee. Peaks in activity at the lowland site occurred during the summer months, the range of V _max values for acetate ranged from 0.2 to 30 μg/1/h. Fluctuations in V _max values from the upland site were not seasonal but were instead linked to faecal pollution, V _max values from this site range from 0.04 to 3 μg/1/h.     

Ferric iron-reducing Shewanella putrefaciens and N2-fixing Bradyrhizobium japonicum with uptake hydrogenase are unable to oxidize atmospheric H2

Abstract Bradyrhizobium japonicum and Shewanella putrefaciens were unable to oxidize hydrogen at atmospheric concentrations (0.55 ppmv), neither in suspension nor when added to sterile soil. The K _m-value of S. putrefaciens for H₂ (39 ppmv in gas phase, 0.22 μM in aqueous phase), using Fe(III) as electron acceptor, showed a 4–5-fold higher affinity for H₂ than that of B. japonicum (1200 ppmv; 0.84 μM) or other hydrogen-oxidizing bacteria. However, the V _max (4.54 fmol H₂ h⁻¹ cell ⁻¹) and threshold (> 0.5 ppmv; 0.35 nM) of S. putrefaciens and the V _max (7.19 fmol H₂ h⁻¹ cell⁻¹) and threshold (> 0.5 ppmv; 0.35 nM) of B. japonicum were in the same order of magnitude as data for Knallgas bacteria from relevant literature. To enable hydrogen oxidation in soil the soil-samples with S. putrefaciens even had to be supplemented with Fe(III). Fresh soil, on the other hand, oxidized hydrogen very efficiently below atmospheric mixing ratios, demonstrating that there must be other oxidation activities in soil.     

KINETICS OF BLOOD-BRAIN BARRIER TRANSPORT OF PYRUVATE, LACTATE AND GLUCOSE IN SUCKLING, WEANLING AND ADULT RATS

Abstract— The kinetics of the uptake from blood to brain of pyruvate, lactate and glucose have been determined in rats of different ages. The carotid artery single injection technique was used in animals anaesthetized with pentobarbital. The rates of influx for each substrate were determined over a range of concentrations for the different age-groups. Data were analysed in terms of the Michaelis-Menten equation with a component to allow for non-saturable diffusion. Values are given for K _m, V _max and K _d. In suckling rats (15-21 days) the V _max values for both pyruvate and lactate were 2.0 μmol g⁻¹ min⁻¹. In 28-day-old rats the V _max values had fallen to one-half and in adults they were less than one-tenth. K _m, values were higher in the younger animals. The rate of glucose transport in suckling rats was half that of 28-day-old and adults although there was no difference with age in the K _m values.
The results are discussed in relation to the net flux of these substrates in and out of brain during different stages of post-natal development.     

THE EFFECT OF IRRADIANCE ON THE GROWTH OF DIFFERENT FORMS OF FRESHWATER DIATOMS: IMPLICATIONS FOR SUCCESSION IN ATTACHED DIATOM COMMUNITIES

The growth rates of 12 strains of attached freshwater diatoms were examined as a function of irradiance. The results were used to determine physiological parameters in the growth–irradiance relationships. Values of G _max (maximum growth rate) and I  _k (irradiance at half of G _max) correlated with successional phase. The development of attached diatom communities appears to proceed from high irradiance species to low irradiance species that appear later in the successional sequence. Thus, efficiency of light use is linked to successional strategy, as has been documented for terrestrial plants.     

Microcalorimetric assessment of microbial activity in long-term fertilization experimental soils of Southern China

Microcalorimetry, plate count and PCR–denaturing gradient gel electrophoresis (DGGE) were employed to investigate microbial diversity and activity in soils from the Red Soil Experimental Station of the Chinese Academy of Agricultural Sciences, Hunan Province, China, where a wheat–corn rotation with 12 fertilization treatments was established in 1990. Fertilization greatly increased microbial biomass carbon (C) and nitrogen (N) (C_mic and N_mic) as well as the activities of phosphatase, urease, invertase, protease, catalase and dehydrogenase. Manure alone (M) enhanced the number of denitrifying and aerobic bacteria by 54.4% and 20.5%, respectively, whereas fallow (H) increased the number of aerobic cellulose decomposing bacteria by 31.4%. Fallow and soils amended with mineral fertilizers plus pig manure or straw increased both the DGGE band patterns and the Shannon index compared with mineral fertilizers or the control. Mineral treatments with lower bacterial numbers enhanced the values of the peak time ( t _max) more than did organic treatments. The peak height ( P _max) was positively correlated ( P <0.01), with soil enzymes, C_mic and N_mic, and the number of microorganisms, whereas the peak time ( t _max) was negatively connected ( P <0.01) with these parameters. The microbial growth rate constant ( k ) was linked to bacteria ( P <0.01), actinomycetes ( P <0.05) and catalase ( P <0.05). The total heat evolution ( Q ) had no relationships with any soil microbial properties (except for catalase). We propose that P _max and t _max could be used as indices of soil microbial activity, while the values of k and Q are poor indicators.     

N-SUBSTITUTED CHOLINE ANALOGUES AS SUBSTRATES FOR CHOLINE ACETYLTRANSFERASE

Abstract— Alkyl, phenyl, phenylalkyl and pyridiniumalkyl derivatives of choline were studied as substrates for choline acetyltransferase from bovine brain. When one methyl group of choline was replaced by an ethyl group, pK _m (negative logarithm of apparent K _m) decreased, whereas V _max was not significantly changed in comparison with choline. The n-propyl derivative showed an even lower pK _m with unchanged V _max Further elongation of the n-alkyl chain had little effect on the substrate parameters until the n-decyl derivative was reached, when a pronounced decrease of V _max occurred. The highest n-alkyl homologue studied, n-pentadecyl choline, was a very poor substrate. Phenylcholine was also a poor substrate, but introduction of an alkyl chain between the phenyl group and the quaternary nitrogen resulted in compounds with better substrate properties, although they were still inferior to choline. The lowest homologue of the pyridiniumalkyl cholines studied, pyridiniumpropylcholine, had a very low pK _m and a lower V _m in comparison with choline. Increasing the chain length of the alkyl residue resulted in an increase of pK _m, whereas V _max was little affected. The results demonstrate that replacement of one methyl group of choline with a more bulky substituent resulted in impaired substrate properties, presumably due to steric effects. No evidence was obtained for hydrophobic interactions between the enzyme and non-polar substituents in the choline analogues studied.     

Diaminopimelic acid uptake by Bacillus megaterium: influence of growth conditions and other amino acids

Diaminopimelic acid (DAP) uptake was studied in Bacillus megaterium. The K _m and V _max were determined for bacteria grown with or without DAP. The uptake of DAP was shown to be constitutive and unaffected by the presence of other amino acids (including cystine). The concentration of DAP and lysine in the amino acid pool was examined and a procedure for pulse-labelling cell walls developed.     

Comparisons of swimming performance in rainbow trout using constant acceleration and critical swimming speed tests

Maximum swimming performance of seasonally acclimated rainbow trout Oncorhynchus mykiss was compared among short-duration constant acceleration tests ( U _max) and with the well established, but longer duration critical swimming speed ( U _crit) test. The present results show that U _max was insensitive to a range of acceleration rates that differed by more than three-fold. Thus, test duration could be reduced from 58 to 18 min without affecting the estimate of U _max. The value of U _max, however, was up to 57% higher than U _crit. Only the slowest acceleration rate tested (an increase of 1 cm s⁻¹ every min) had a significantly lower U _max, and this was up to 19% higher than U _crit. Even so, the potential saving in the test duration was small (70 v. 90 min) when compared with a ramp- U _crit test (a standard U _crit test but with the water velocity initially ramped to c . 50% of the estimated U _crit). Therefore, swim tests that are appreciably shorter in duration than a ramp- U _crit test result in U _max being appreciably greater than U _crit. An additional discovery was that the ramp- U _crit performance of cold-acclimated rainbow trout was independent of the recovery period between tests. These results may prove useful in making comparisons among different swim test protocols and in designing swim tests that assess fish health and toxicological impacts.     

Deamination of 5-Hydroxytryptamine by Both Forms of Monoamine Oxidase in the Rat Brain

Abstract: K _m and V _max values of monoamine oxidase (MAO) A and B towards 5-hydroxytryptamine were determined for rat brain homogenates after the in vitro inhibition of one of the two forms by the selective inhibitors clorgyline and l -deprenyl. K _m values of 178 and 1170μ m , and V _max values of 0.73 and 0.09 nmol·mg protein⁻¹·min⁻¹ towards 5-hydroxytryptamine were found for MAO-A and -B, respectively. The K ₁ for 5-hydroxytryptamine as a competitive inhibitor of β-phenethylamine oxidation by MAO-B was found to be 1400 μm. The significance of these findings is discussed.     

Uptake and turnover of acetate in hypersaline environments

Abstract: Acetate uptake and turnover rates were determined for the heterotrophic community in hypersaline environments (saltern crystallizer ponds, the Dead Sea) dominated by halpphilic Archaea. Acetate was formed from glycerol, which is potentially the major available carbon source for natural communities of halophilic Archaea. Values of [ K _t+ S _n] (the sum of the substrate affinity and the substrate concentration present in situ) for acetate measured in saltern crystallizer ponds were around 4.5–11.5 μM, while in the Dead Sea during a Dunaliella bloom values up to 12.8 μM were found. Maximal theoretical rates ( V _max) of acetate uptake in saltern crystallizer ponds were 12–56 nmol l⁻¹ h⁻¹, with estimated turnover times for acetate ( T _t) between 127–730 h at 35°C. V _max values measured in the Dead Sea were between 0.8 and 12.8 nmol l⁻¹ h⁻¹, with turnover times in the range of 320–2190 h. V _max values for acetate were much lower than those for glycerol. Comparisons with pure cultures of halophilic Archaea grown under different conditions showed that the natural communities were not adapted for preferential use of acetate. Both in natural brines and in pure cultures of halophilic Archaea, acetate incorporation rates rapidly decreased above the optimum pH value, probably since acetate enters the cell only in its unionized form. The low affinity for acetate, together with low potential utilization rates result in the long acetate turnover times, which explains the accumulation of acetate observed when low concentrations of glycerol are supplied as a nutrient to natural communities of halophilic Archaea.     

Tyrosine Hydroxylase from Bovine Striatum: Catalytic Properties of the Phosphorylated and Nonphosphorylated Forms of the Purified Enzyme

Abstract: The properties of purified tyrosine hydroxylase (TH) from bovine corpus striatum, both native and phosphorylated forms of the enzyme, were studied. TH had a tendency toward greater affinity for tetrahydrobiopterin (BH₄) than for the synthetic cofactor 6-methyltetrahydropterin (6-MPH₄), although the maximal velocity of the TH-catalyzed reaction was greater with 6-MPH4. Phosphorylation increased the affinity of TH for cofactor at pH 6.0, with little change in V _max. At pH 7.0, phosphorylation caused increased activation of TH by increasing V _max as well as reducing the K _m for cofactor. The K _m for dopamine was increased twofold by phosphorylation at pH 6.0, but eightfold at pH 7.0. Phosphorylation was not associated with a change in K _m for tyrosine at any pH or with any cofactor studied, although the K _m for tyrosine of TH was cofactor-dependent and seven to eight times greater with 6-MPH₄ than with BH₄ as cofactor. Heparin and NaCl activated native TH at pH 6.0, but not at pH 7.0. Phosphorylated TH was unaffected by heparin or salt at pH 6.0, but was relatively inhibited at pH 7.0. The data are presented in the context of the physiological environment of TH.     

l-threonine transport in the obligate methylotroph Methylobacillus flagellatum KT

Abstract The accumulation of l -threonine by the methylotrophic bacterium Methylobacillus flagellatum KT occurs via a specific system that is capable of transporting l -threonine against a 100-fold concentration gradient. This transport system demonstrates the following kinetic parameters: K _m= 0.2 mM and V _max= 2.5 nmol/min/mg of cells (dry weight). The activity of the system is inhibited by oxidative phosphorylation uncouplers and valinomycin. Cytoplasmic l -threonine does not leak from the cell, but bacteria are capable of exchanging exogenous l -threonine for its intracellular counterpart.     

Measuring and modelling environmental influences on photosynthetic gas exchange in Sphagnum and Pleurozium

A mechanistic model has been used to examine the environmental regulation of photosynthetic gas exchange in moss. The effects of water content on conductance to CO₂ and on photosynthetic capacity during desiccation were calculated from the carbon isotope discrimination data of Williams & Flanagan (1996 , Oecologia 108, pp. 38–46) and combined with the biochemical model of Farquhar et al. (1980 , Planta 149, pp. 78–90). The model includes a simple light attenuation function that imparts curvature to the light response curve for net assimilation, enabling the use of physiologically realistic values for the biochemical parameters. Measurements of gas exchange for Sphagnum and Pleurozium were made in an old black spruce ecosystem over a growing season in order to assign values to parameters in the model. The calculated maximum rates of carboxylation by Rubisco ( V _max) were 5, 14 and 6 μ mol m^–2 s^–1 for Sphagnum during the spring, summer and autumn seasons of 1996, respectively. The increase in V _max during the summer was consistent with an increased allocation of resources to the photosynthetic apparatus. In contrast, no seasonal variation in V _max was observed in Pleurozium with average values of 7, 5 and 7 μ mol m^–2 s^–1 during the spring, summer and autumn, respectively.     

Uptake of Amino Acids by Brain Microvessels Isolated from Rats after Portacaval Anastomosis

Abstract: The uptake of amino acids by microvessels isolated from brains of rats was studied. Previous studies have demonstrated alterations in blood-brain amino acid transport after portacaval shunt in rats. In order to elucidate whether such changes in the blood-brain barrier were located in the microvessels, brain microvessels were isolated from both rats with portacaval shunt and controls. Brain microvessels from rats 2 weeks after shunt operations took up significantly greater amounts of ¹⁴C-labeled neutral amino acids, but not of glutamic acid. lysine, or α-methylaminoisobutyric acid than microvessels from sham-operated controls. Measurement of uptake kinetics showed a higher V _max for phenylalanine and leucine uptake and a lower V _max for lysine uptake in microvessels from shunted rats compared with control, whereas the respective K _m's of uptake were similar in both preparations. The results suggest that changes in brain microvessel transport activity account for altered brain neutral amino acid concentrations after portacaval shunt and that such changes can be studied in vitro in isolated microvessels.     

Phorbol Esters Increase Dopamine Transporter Phosphorylation and Decrease Transport Vmax

Abstract: Sodium- and chloride-coupled transport of dopamine from synapses into presynaptic terminals plays a key role in terminating dopaminergic neurotransmission. Regulation of the function of the dopamine transporter, the molecule responsible for this translocation, is thus of interest. The primary sequence of the dopamine transporter contains multiple potential phosphorylation sites, suggesting that the function of the transporter could be regulated by phosphorylation. Previous work from this laboratory has documented that phorbol ester activation of protein kinase C (PKC) decreases dopamine transport V _max in transiently expressing COS cells. In the present report, we document in vivo phosphorylation of the rat dopamine transporter stably expressed in LLC-PK₁ cells and show that phosphorylation is increased threefold by phorbol esters. Dopamine uptake is also regulated by phorbol esters in these cells; phorbol 12-myristate 13-acetate (PMA) reduces transport V _max by 35%. Parallels between the time course, concentration dependency, and staurosporine sensitivity of alterations in transporter phosphorylation and transporter V _max suggest that dopamine transporter phosphorylation involving PKC could contribute to this decreased transporter function. Phosphorylation of the dopamine transporter by PKC or by a PKC-activated kinase could be involved in rapid neuroadaptive processes in dopaminergic neurons.     

Photosynthetic rates and partitioning of absorbed light energy in photoinhibited leaves

Parameters for the evaluation of the effects of photoinhibition on photosynthetic carbon gain were studied in Chenopodium album leaves. The light-response curve of photosynthetic rate was determined at 36 Pa CO₂ partial pressure and fitted by a non-rectangular hyperbola. Both the initial slope of the curve and the light-saturated rate decreased in photoinhibited leaves, although the decrease in the latter was small. The convexity of the curve was also smaller in photoinhibited leaves. The capacities of ribulose-1,5-bisphosphate carboxylation ( V _cmax) and electron transport ( J _max) were estimated from the CO₂-response curves. V _cmax and J _max decreased similarly with increasing photoinhibition. Energy partitioning in photosystem II (PSII) was estimated using chlorophyll fluorescence parameters. The fraction of energy that was consumed by photochemistry decreased with increasing photoinhibition. However, an increase in inactive PSII, decreasing energy partitioning to active PSII, relaxed the excitation pressure in PSII, and led to a reduction in the fraction of excess energy that was neither consumed by photochemistry nor dissipated as heat.