THE PROLIFERATION OF LYMPHOID CELLS IN GUINEA-PIG BONE MARROW

A double isotope DNA labelling method has been used to determine the duration of DNA synthesis (S) in bone marrow lymphoid cells classified by their nuclear diameters in smears. Incorporation of ³H-thymidine was confined almost entirely to marrow lymphoid cells of 8·0-15·0 μm nuclear diameter (large lymphoid cells). After exposure to ³H-thymidine in vivo and ¹⁴C-thymidine 40-104 min later in vitro , the proportion of cells labelled with ³H alone to those labelled with ¹⁴C(±³H) in radioautographic smears, plotted against time indicated the efflux from S per hour. Collectively, 28·3 ± 1·1% of all large lymphoid cells were in S and the efflux from S was 15·1% per hour. With decreasing cell size (nuclear diameter) the efflux fell progressively from 28·3% per hour (11·0 μm) to 9·2% per hour (8·0-8·9 μm) and the proportion of cells in S declined from 54·9 ± 2·3% to 14·8 ± 1·6%. Influx into S, measured in vitro by reversing the sequence of isotopes, closely resembled the corresponding efflux values in vivo relative to cell size. Most DNA synthesizing marrow large lymphoid cells belonged to a subgroup with deeply basophilic cytoplasm. The results demonstrate that basophilic large lymphoid cells in the marrow are actively proliferating and have a mean S phase duration of 6·6 hr. The largest marrow lymphoid cells (11·0 μm) proliferate most rapidly (S phase, 3·5 hr; maximum cell cycle time, 6·4 hr) while S duration is prolonged progressively to 10·9 hr for the smaller cells (8·0-8·9 μm).     

45Ca2+ Uptake into Rat Whole Brain Synaptosomes Unaltered by Dihydropyridine Calcium Antagonists

Abstract: Voltage-dependent ⁴⁵Ca²⁺ uptake into rat whole brain synaptosomes was measured after 3-s KCl-induced depolarization to investigate possible inhibitory effects of calcium antagonists, nitrendipine, nimodipine, and nisoldipine. At a Ca²⁺ concentration of 1.2 m M , nitrendipine, in concentrations ranging from 0.1 n M to 10 μ M , had no effect on ⁴⁵Ca²⁺ uptake. When the Ca²⁺ concentration was lowered to 0.06 and 0.12 m M , nitrendipine, 10 μ M , inhibited ⁴⁵Ca²⁺ uptake in response to 109 m M KCl depolarization. However, in a separate concentration response study, nitrendipine, nimodipine, and nisoldipine, 0.1 n M to 10 μ M , failed to alter the uptake of ⁴⁵Ca²⁺ (0.06 m M Ca²⁺) into 30 m M KCl-depolarized synaptosomes. The high concentrations of these agents required to depress ⁴⁵Ca²⁺ uptake indicate that the dihydropyridine calcium antagonists are considerably less potent in brain tissue than in peripheral tissue.     

The occurrence and ecological importance of dissolved ATP in fresh water

SUMMARY. Dissolved ATP, defined as ATP which passes through 0.2 μm filters, was found in fresh water. During the spring diatom bloom in two eutrophic Danish lakes, concentrations of dissolved ATP varied between 0.1 and 3.8 μgl⁻¹, constituting 14–76% of the total ATP (particulate plus dissolved ATP). The kinetics of the light emission obtained from mixing firefly enzyme with dissolved ATP demonstrated that the major proportion of the dissolved ATP was in fact ATP. Despite some variations, the seasonal changes in dissolved ATP paralleled the changes in the increasing phytoplankton population during the rise of the diatom blooms. The dissolved ATP increased after the diatom peak, indicating that release of ATP from the phytoplankton due to mortality may be a major source of dissolved ATP.
Consumption of dissolved ATP was evaluated in uptake experiments using ³H-ATP. Rates of uptake of ³H-ATP by micro-organisms (diameter 0.2–0.6 μm) proved to be close to the rates for ³H-D-glucose uptake. The variations in ³H-ATP uptake during the diatom blooms showed non-systematic changes and ranged between 1.0 and 15.8% h⁻¹ (mean = 4.9% h⁻¹) of the quantity added. Turnover rates for dissolved ATP varied between 12 and 730 ng l⁻¹ h⁻¹ (mean = 175 ng l⁻¹). These rather high rates of turnover suggest that dissolved ATP is an important compound in the metabolism of freshwater bacteria.     

Karyotype variability in successive generations after hybridization between the great sturgeon, Huso huso (L.), and the sterlet, Acipenser ruthenus L.

Effects of particle size, fish size and temperature on the filtration rate of silver carp were determined. When feeding at 20°C on zooplankton and spherical particles (yeast, micronic beads and pollen), 32-g silver carp filter particles larger than 70 urn at a maximum rate of 18.251 h⁻¹. For particles smaller than 70 μm, filtration rates decrease with decreasing particle size until there is no measured filtration for particles smaller than 10 μm. Filtering rates ( FR ) for particles between 10 and 50 μm are described by the equation, FR =−20.8 + 21.7 × log particle diameter. Filtration rates rise as fish size, particle size and temperature increase. Filtration rates per unit biomass, however, fall as fish size increases: FR = 1.54 W^0.713, where FR is the maximum filtration rate in ¹ h ¹ fish ¹ and W is weight of fish in grammes. The results of these trials are consistent with the hypothesis that particle selection by silver carp is a mechanical, passive function of gill raker morphology.     

Electrotransfection of Propionibacterium freudenreichii TL 110

The first highly efficient protocol is described for the electrotransfection of Propionibacterium freudenreichii with DNA phage. The transfection efficiency is 7 times 10⁵ transfectants per μg of DNA under optimal conditions. Optimized parameters included the field strength (12.5 kV, 200 Ohms, 25 μF), phage DNA concentration (1 μg ml^-1) and cell density (1.5 times 10¹⁰ cells ml^-1). Growth in the presence of glycine and harvesting of cells during the early exponential growth phase increased the transfection efficiency. This electrotransfection protocol is of importance for the genetic improvement of dairy propionibacteria.     

A note on the control of sulphate-reducing bacteria in seawater by u.v. irradiation

Continuous and batch cultures of marine sulphate-reducing bacteria (SRB) in North Sea water were irradiated with 110000 to 329500 μWs/cm² of ultraviolet radiation (wavelength 253.7 nm) with a commercial u.v. sterilizing unit. A 100% kill was obtained with logarithmic cultures of Desulfovibrio desulfuricans NCIMB 8400 at population densities of 10–10⁴/ml. A >99.99% kill was obtained with a mixture (ca 10⁵/ml) of batch grown Desulfovibrio spp. and oilfield SRB enrichments. Ultraviolet irradiation was less effective against the indigenous heterotrophic bacteria in the seawater ( ca 90% kill).     

Quantitative observations on the pulmonary anatomy of the domestic Muscovy duck (Cairina moschata)

The lungs of five female domestic Muscovy ducks, mean body weight 1.627 kg, total lung volume 48.07 cm³, were analysed by standard morphometric methods. Principal results obtained are: lung volume per unit body weight, 30.17 cm³/g; volume densities of exchange tissue relative to lung volume, 49.24%, blood capillaries relative to exchange tissue, 29.63%, tissue of the blood gas (tissue) barrier relative to exchange tissue, 5.88%; surface area of the blood-gas (tissue) barrier per unit body weight, 30.04 cm²/g; ratios of the surface area of the blood-gas (tissue) barrier per unit volume of the lung and per unit volume of exchange area, 979 cm²/cm³ and 200.06 mm²/mm³, respectively; harmonic and arithmetic mean thicknesses of the tissue barrier, 0.199 μm and 0.303 μm, respectively. The anatomical diffusing capacity of the tissue barrier for oxygen ( DtO₂ ) and the total pulmonary diffusing capacity ( DLO₂ ), 49.58 ml O₂/min/mmHg/kg and 4.55 ml O₂/min/mm Hg/kg, respectively. The lungs of the domestic Muscovy duck appear to be about as well adapted anatomically for gas exchange as the lungs of wild anatid species, and there is no clear evidence that domestication has been associated with any deterioration in the anatomical capacity for oxygen uptake. The weight-specific anatomical diffusing capacity of the lung for oxygen ( DLO₂/W ) was about 3.6 times greater than the weight-specific physiological value, a factor which falls within the expected range.     

ON THE MECHANISM OF OUABAIN INHIBITION OF SYNAPTOSOME PROTEIN SYNTHESIS

Abstract— Ouabain (200μ m ) inhibited incorporation of radiolabelled leucine or glycine into the protein of neonatal synaptosome fractions but had minimal effect on preparations from adult rats. Leucine uptake into synaptosomes was rapid but not influenced by 200μ m -ouabain in contrast to ouabain inhibition of [¹⁴C]glycine and [¹⁴C]γ-aminobutyric acid uptake. Ouabain blocked the Na⁺ -dependent (stimulated) component of synaptosome fraction protein synthesis in the presence of 25m m -K⁺. Ouabain inhibition was not alleviated by addition of ADP or ATP. 100μ m -atractylate failed to influence [³H]leucine uptake or incorporation. Synergistic inhibition by ouabain was observed with the cycloheximide-sensitive component of protein synthesis and the chloramphenicol sensitive phase. Increasing the medium Ca²⁺ concentration stimulated protein synthesis and this stimulated component was inhibited by ouabain. Ouabain inhibition was associated with decreasing intraterminal K⁺ concentration and [K]_i was linearly related to the protein synthesis rate in control and ouabain treated preparations.     

Metabolic utilization of body stores during the early life of whitefish, Coregonus lavaretus L.

Patterns of oxygen consumption, ammonia and urea excretion were monitored during late embryogenesis, i.e. 5 days before mass hatching and 12 days during the free-swimming stage of whitefish larvae, Coregonus lavaretus. Oxygen consumption increased from 1.31 to 2.53 mgO₂ h⁻¹× 10³ eggs⁻¹ at hatching. Fasted, free-swimming larvae showed increasing oxygen consumption to the tenth day after hatching when it reached 5.52 mgO₂h⁻¹× 10³ larvae⁻¹. Ammonia and urea excretion increased during pre-hatching period from 52.1 to 163.2 and 26.8 to 51.4 μgh⁻¹× 10³ eggs⁻¹, respectively. The nitrogen excretion rate increased between the sixth and tenth day of fasting, i.e. for ammonia from 117.7 to 160.9 and for urea from 35.8 to 52.5 μg h⁻¹× 10³ larvae⁻¹. Cumulative data on nitrogen and energy metabolism indicated that during late embryogenesis, and up to the fifth day after hatching, protein dominated in the energy expenditure. During the free swimming stage, the ratio of fat to protein in energy expenditure rose from 0.86 to 1.99. Combined data for several fish species indicated high dependance of oxygen uptake during the hatching period on egg size and temperature.     

Transformation of Methanococcus maripaludis and identification of a Pst I-like restriction system

Abstract An optimized polyethylene glycol (PEG) method of transformation was developed for Methanococcus maripaludis using the pKAS102 integration vector. The frequency of transformation with 0.8 μg of plasmid and 3×10⁹ cells was 4.8×10⁻⁵ transformants cfu⁻¹, or 1.8×10⁵ transformants μg⁻¹, which was four orders of magnitude greater than with the natural transformation method. A Pst I restriction activity in M. maripaludis was also identified. Methylation of the plasmid with Pst I methylase increased the methanococcal transformation frequency at least four-fold. Also, chromosomal DNA from M. maripaludis was resistant to digestion by the Pst I endonuclease.     

Calcium-dependent transferrin receptor recycling in bovine chromaffin cells

The release of regulated secretory granules is known to be calcium dependent. To examine the Ca²⁺-dependence of other exocytic fusion events, transferrin recycling in bovine chromaffin cells was examined. Internalised ¹²⁵I-transferrin was released constitutively from cells with a half-time of about 7 min. Secretagogues that triggered catecholamine secretion doubled the rate of ¹²⁵I-transferrin release, the time courses of the two triggered secretory responses being similar. The triggered ¹²⁵I-transferrin release came from recycling endosomes rather than from sorting endosomes or a triggered secretory vesicle pool. Triggered ¹²⁵I-transferrin release, like catecholamine secretion from the same cells, was calcium dependent but the affinities for calcium were very different. The extracellular calcium concentrations that gave rise to half-maximal evoked secretion were 0.1 m m for ¹²⁵I-transferrin and 1.0 m m for catecholamine, and the intracellular concentrations were 0.1 μ m and 1 μ m , respectively. There was significant ¹²⁵I-transferrin recycling in the virtual absence of intracellular Ca²⁺, but the rate increased when Ca²⁺ was raised above 1 n m , and peaked at 1 μ m when the rate had doubled. Botulinum toxin type D blocked both transferrin recycling and catecholamine secretion. These results indicate that a major component of the vesicular transport required for the constitutive recycling of transferrin in quiescent cells is calcium dependent and thus under physiological control, and also that some of the molecular machinery involved in transferrin recycling/fusion processes is shared with that for triggered neurosecretion.     

Transformation of Streptococcus bovis protoplasts by plasmid DNA

M. MAREKOVÁ, V. KMET' AND P. JAVORSKÝ. 1996. The transformation and subsequent regeneration of ruminal strain Streptococcus bovis AO24/85 protoplasts by plasmid DNA was studied. The best stabilizer for regeneration of protoplasted cells was 5% sucrose in the regeneration medium and in the agar plates. Optimal concentration of polyethylene glycol 6000 in the transformation medium was 25% for both plasmids tested. Addition of Ca²⁺ and Mg²⁺ ions (2.5 mmol l^-1) to the transformation medium increased the proportion of regenerated cells. Transformation frequencies were 3 times 10³ transformants per μg of pNZ12 and 2.4 times 10² per μg of pJK108, respectively.     

Key Residues Defining the μ-Opioid Receptor Binding Pocket: A Site-Directed Mutagenesis Study

Abstract: Structural elements of the rat μ-opioid receptor important in ligand receptor binding and selectivity were examined using a site-directed mutagenesis approach. Five single amino acid mutations were made, three that altered conserved residues in the μ, δ, and κ receptors (Asn¹⁵⁰ to Ala, His²⁹⁷ to Ala, and Tyr³²⁶ to Phe) and two designed to test for μ/δ selectivity (Ile¹⁹⁸ to Val and Val²⁰² to Ile). Mutation of His²⁹⁷ in transmembrane domain 6 (TM6) resulted in no detectable binding with [³H]DAMGO (³H-labeled d -Ala², N -Me-Phe⁴,Gly-ol⁵-enkephalin), [³H]bremazocine, or [³H]ethylketocyclazocine. Mutation of Asn¹⁵⁰ in TM3 produces a three- to 20-fold increase in affinity for the opioid agonists morphine, DAMGO, fentanyl, β-endorphin_1–31, JOM-13, deltorphin II, dynorphin_1–13, and U50,488, with no change in the binding of antagonists such as naloxone, naltrexone, naltrindole, and nor-binaltorphamine. In contrast, the Tyr³²⁶ mutation in TM7 resulted in a decreased affinity for a wide spectrum of μ, δ, and κ agonists and antagonists. Altering Val²⁰² to Ile in TM4 produced no change on ligand affinity, but Ile¹⁹⁸ to Val resulted in a four- to fivefold decreased affinity for the μ agonists morphine and DAMGO, with no change in the binding affinities of κ and δ ligands.     

THE SELECTIVE NEURONAL UPTAKE AND RELEASE OF [3H]DL-2,4-DIAMINOBUTYRIC ACID BY RAT CEREBRAL CORTEX

Abstract— At 25°C the accumulation of [³H] dl -2,4-diaminobutyric acid (DABA) into small rat cortical slices was linear with time and a tissue: medium ratio of 35:1 was attained after 60 min. At 37°C the uptake was no longer linear and the tissue: medium ratio at 60 min was 66:1. Uptake was unaffected by the addition of 10 μ m -AOAA and dependent on the presence of Na⁺ in the incubation media. The uptake was shown to have a high affinity component with a K _m of 20.7 μ m and a V _max of 28.6 nmol/g/min. IC50's for the inhibition of [³H]DABA uptake by dl -DABA, l -DABA and GABA were 80, 40 and 17 μ m respectively. Two m m β -alanine, however, caused less than 13% inhibition of [³H]DABA uptake. Electron microscopic autoradiographs showed the [³H]DABA to be accumulated by 22% of the identifiable nerve terminals and, after 14 days exposure, the density of silver grains over nerve terminals was 36–38 times higher than that over the rest of the electron micrograph. On the other hand, [³H]DABA was not taken up into rat sensory ganglia and light level autoradiography showed the small amount of [³H]DABA accumulated by the ganglia to be evenly distributed throughout the tissue. Both electrical stimulation for 30 s and exposure of the tissue to a medium containing 47 m m -K⁺ for 2 min caused a marked increase in the efflux of [³H]DABA from the tissue. Both these effects were abolished by a reduction in Ca²⁺ concentration and an increase in the Mg²⁺ concentration of the superfusing medium. These results suggest that l -DABA acts as a 'false transmitter' for the neuronal uptake, storage and release of GABA.     

SYNTHESIS AND RELEASE OF [14C]ACETYLCH0LINE IN SYNAPTOSOMES

Abstract— Synaptosomes took up [¹⁴C]choline, about half or more of which was converted to [^I4C]acetylcholine when incubated in an appropriate medium containing 1 to 5 μ M-[¹⁴C] choline and neostigmine. The amount of [¹⁴C]acetylcholine synthesized in synaptosomes increased in parallel with the increase of Na⁺ concentration in the incubation medium. The effect of Na⁺ on the uptake of [^I4C]choline into synaptosomes was dependent on the concentration of choline in the incubation medium.
About 25 per cent of [¹⁴C]acetylcholine synthesized in synaptosomes was released rapidly into the medium by increasing the K⁺ concentration in the medium from 5 m m to 35 m m . The change of Na⁺ concentration hardly affected the release of [¹⁴C]acetylcholine. The effect of K⁺ on the release of [¹⁴C]choline was rather small compared to that on [¹⁴C] acetylcholine. Ouabain promoted the release of [¹⁴C]acetylcholine.     

RNA Synthesis and Processing during Cytodifferentiation in Fetal Rat Pancreas

In fetal rat pancreas cytodifferentiation occurs between day 14 and day 20 of gestation and is accompanied by an exponential increase in the cellular accumulation of tissue specific proteins and an elaboration of the cellular organelles associated with their synthesis and secretion. Evaluation of RNA synthesis by [³H] uridine incorporation into trichloroacetic acid precipitable material showed that during this period the apparent rate of RNA synthesis increased 7.5 fold from 2 × 10³ dpm/μg DNA/h on day 15 to 1.5 × 10⁴ dpm/μg DNA/h on day 19; [³H] leucine uptake showed that the rate of protein synthesis increased about the same extent with the major difference being that the maximum rate of protein synthesis occurred on day 19, one day after the maximum rate of RNA synthesis. The soluble pyrimidine nucleotide pools decreased from 122 pmol/μg DNA on day 14 to 15 pmol/μg DNA on day 16 followed by an increase to 104 pmol/μg DNA on day 19; the purine nucleotide pools decreased from 367 pmol/μg DNA on day 14 to 286 pmol/μg DNA on day 16 and then increased to 635 pmol/μg DNA on day 19. These values roughly paralleled the transitions observed in the rates of RNA and protein synthesis. Agarose-acrylamide slab gel electrophoresis showed an increase in RNA synthesis and an increase in ribosomal RNA synthesis and processing with cytodifferentiation.     

CIRCADIAN RHYTHM OF PHOTOSYNTHETIC OXYGEN EVOLUTION IN KAPPAPHYCUS ALVAREZII (RHODOPHYTA): DEPENDENCE ON LIGHT QUANTITY AND QUALITY

The rate of oxygen evolution of the tropical red alga Kappaphycus alvarezii (Doty) Doty was measured for 6 days in the laboratory using a computer-aided method for long-term recording. In cool white light, Kappaphycus exhibited a robust circadian rhythm of O₂ evolution in the irradiance range of 100 to 1000 μmol photons·m ^{− 2}·s ^{− 1}. With increasing irradiance, the period of the free-running rhythm, τ, decreased in blue and increased in red light but did not change significantly in green light. The accelerating or slowing action of blue or red light, respectively, points to two photoreceptors used in the light transduction pathway of the circadian oscillator controlling oxygen evolution or the light reactions of photosynthesis in Kappaphycus. No significant changes of τ were observed with increasing irradiance in cool white light, possibly due to the additive opposing responses caused by blue and red light.     

Constitutive mineralization of low concentrations of the herbicide linuron by a Variovorax sp. strain

The mineralization of the herbicide linuron at concentrations of μg and mg L⁻¹ was studied in liquid batch experiments with Variovorax sp. strain SRS16. The strain was highly efficient at mineralizing a range of linuron concentrations (0.002–10 mg L⁻¹) with 20–60% of the added ¹⁴C-ring-labeled linuron metabolized to ¹⁴CO₂ within hours to days depending on the initial linuron concentration and incubation period. At mg L⁻¹ linuron concentrations the mineralization activity by SRS16 was inducible and a shift to constitutive mineralization activity was apparent with a reduction in the linuron concentration to μg L⁻¹ levels. This study revealed that strain SRS16 is a promising candidate for bioaugmentation of water or soil resources contaminated with low linuron concentrations.     

Winter Ciliates in a British Columbian Fjord: Six New Species and an Analysis of Ciliate Putative Prey

ABSTRACT. This work provides the first study of North Pacific planktonic ciliates by quantitative protargol staining. Triplicate water bottle samples were collected at a depth of 2 m (above the shallow pycnocline) at six stations in Indian Arm, British Columbia, on February 15, 1990, and February 26, 1991. Thirty-six ciliate species were observed. Six new species are described from protargolstained specimens: Strombidium lynni n. sp., Strombidium taylori n. sp., Strombidium basimorphum n. sp., Slrombidiurn ventropinnum n. sp., Strobilidium undinum n. sp., and Urotricha cyrtonucleata n. sp.
Ciliate abundance varied significantly (ANOVA, α= 0.05) between sampling sites, ranging from 550 to 6,800 cells/liter in 1990 and from 1,800 to 7,900 cells/liter in 1991. Biomass also varied significantly (ANOVA, α= 0.05) ranging from 3.7 × 10⁵ to 3.3 × 10⁶ pg carbon/liter in 1990 and 3.04 × 10⁶− 6.97 × 10⁶ pg carbon/liter in 1991. Putative prey were enumerated in three size fractions (1.5–5 μm, 5–10 μm and 10–25 μm). The source of variation in ciliate abundance and biomass was not identified. Parameters of salinity, temperature, putative prey, chlorophyll a and pycnocline depth did not significantly correlate with ciliate biomass or abundance (α= 0.05).     

Isolation of an inclusion body from sporulating, enterotoxin-positive Clostridium perfringens

Abstract Inclusion bodies (IB) synthesized during sporulation and enterotoxin formation by Clostridium perfringens were isolated. Sporulating cells were lysed by sonication in the presence of protease inhibitors. IB were isolated by centrifugation in linear gradients of sucrose, sodium bromide or sodium diatrizoate and banded at buoyant densities of 1.33–1.36 g/cm³, 1.30–1.34 g/cm³ and 1.33 g/cm³, respectively. Isolated IB were treated with detergent to remove attached cell membrane. They ranged in size from 0.5–1.4 μm long and from 0.2–0.5 μm wide. They were found to be serologically related to purified enterotoxin.