Respiration in relation to ion uptake in the crayfish Austropotamobius pallipes (Lereboullet).

1. O2 uptake was determined for periods of 23-46 h in salt-depleted crayfish held in deionized water (DW) or Na-free media at 10 degrees C. These media were replaced by artificial lakewater media (ALW) containing 0-2-0-6 mM Na and O2 uptake was again determined for periods of 24-66 h. 2. During net ion uptake in ALW the metabolic rate was either elevated or depressed. Standard metabolism in ALW altered by amounts equivalent to 0-1 - 15-5% (mean 6-4 (15) +/- 4-4% S. D.) of the metabolic rate measured during salt-depletion. On three occasions the metabolic rate was elevated by 22-0 - 66-7%, but some of this increase may have been due to locomotor activity. 3. The calculated values for thermodynamic work involved in ion transport were 0-056 - 0-268 J/10 g. h at 10 degrees C, or 1-5 - 7-2% of the mean standard metabolic rate. Most of the observed changes in metabolic rate lie within the limits of experimental error (ca. +/- 7%). Hence the energetic cost of ion transport is too small for direct measurement in intact crayfish.     

Reproduction in an Irish lake population of the crayfish Austropotamobius pallipes (Lerehoullet)

SUMMARY. 1. Mature crayfish were collected in 1984 from an alkaline Irish lake before and after the mating season.
2. There was some correlation between frequency of mating and decrease in vasa deferentia weight in captive males, but in the field vasa deferentia weight losses were highly variable. The data indicate that one third of adult males may not have mated in the wild.
3. All females appear to have spawned, whether or not mated, with an average 85% reduction in ovary weights over the season.
4. Pleopodal egg counts directly after laboratory spawning were on average 81% of ovarian counts; in the field, an estimated 50 days after spawning, they were only 61% of estimated ovarian egg numbers.
5. Average ovarian egg size was positively correlated with female size. Also, larger crayfish had a wider range of egg sizes than smaller crayfish.
6. It is suggested that aggression and competition for shelter among brooding females may result in a progressive loss of eggs.     

Histopathological survey of pathogens and commensals of white-clawed crayfish (Austropotamobius pallipes) in England and Wales

A histopathological survey of white-clawed crayfish (Austropotamobius pallipes) from six rivers in England and Wales was conducted between November 2007 and September 2011. A. pallipes bacilliform virus (ApBV) was present in four samples; infected crayfish showed pathological responses ranging from mild hypertrophy of infected nuclei and emargination of chromatin through to loss of architecture of the hepatopancreatic cells. Crayfish were found to be hosts to two different ciliate species, mites, nematodes, digeneans and the microsporidian Thelohania contejeani. The variation in disease presence between populations highlights the importance of conducting appropriate surveys of native crayfish prior to movement of animals to refugia sites for relocation, restocking or breeding programmes.     

Metal-protein binding patterns in the hepatopancreas of the crayfish (Austropotamobius pallipes) during short term cadmium stress

Cd-binding patterns in the hepatopancreas of the crayfish (Austropotamobius pallipes) have been analysed in response to the administration of a single dose of Cd ions. Four main components, with apparent molecular weights of 3-4, 12, 20 and greater than 70 K daltons, have been isolated from soluble cell fractions. The variation in molar metal/protein ratios of these components is discussed in terms of the uptake, transfer and detoxification of Cd in the hepatopancreas. A model is proposed which describes Cd flux at the subcellular level and indicates that metallothionein-like proteins may mediate in this process.     

Comparison of the conversion rates of alpha-linolenic acid (18:3(n - 3)) and stearidonic acid (18:4(n - 3)) to longer polyunsaturated fatty acids in rats.

The delta 6-desaturase reaction is regarded to be the rate-limiting step in the conversion of linoleic acid (18:2(n - 6)) to arachidonic acid (20:4(n - 6)). The same is probably also the case with the conversion of alpha-linolenic acid (18:3(n - 3)) to eicosapentaenoic acid (20:5(n - 3)). However, there are very few in vivo studies that directly compared the conversion rate between 18:3(n - 3) and stearidonic acid (18:4(n - 3)), which is the delta 6-desaturated product of 18:3(n - 3). We compared this rate by feeding rats on a lipid-free diet supplemented with lard (9%, w/w) and 18:3(n - 3) ethyl ester (1%) diet or on a diet containing lard (9%) and 18:4(n - 3) ethyl ester (1%). A lard (10%)-supplemented diet was used as the control diet. The fatty acid compositions of total phospholipids, triglycerides and free fatty acids of both liver and plasma were measured after 1 or 3 weeks on different diets. The molar ratio of 20:5(n - 3) of most lipid fractions was about 2-fold higher in rats fed the 18:4(n - 3)-supplemented diet than in rats fed the 18:3(n - 3)-supplemented diet. 18:4(n - 3) was found in the liver lipid fraction in only a very small amount, even in the 18:4(n - 3)-supplemented groups. Thus, desaturation at C-6 is suggested to be the rate-limiting step in the conversion of 18:3(n - 3) to 20:5(n - 3).     

Polarographic measurement of oxygen uptake by astrocytes in primary cultures using the tissue-culture flask as the respirometer chamber.

Oxygen uptake was measured in primary cultures of astrocytes from the brain hemispheres of newborn DBA mice by the aid of an oxygen electrode inserted directly into the culture flask, i.e. using the flask, completely filled with MEM medium, as the respirometer chamber. The respiration was initially intense (300 mumol per hr per 100 mg protein) but declined somewhat during the 6 hr of measurement, probably due to a depletion of intermediary metabolites released to the large surplus of medium. The respiratory rates were approximately identical in the presence of a CO2/bicarbonate and a HEPES buffer. Exposure to a high concentration of potassium led to a transient stimulation of the oxygen uptake to almost 100%, a response that was very easily observed using the present method. Since no mechanical damage was inflicted upon the cells, culturing could be continued, if so desired, after the measurement.     

Phylogeography of the threatened crayfish (genus Austropotamobius) in Italy: implications for its taxonomy and conservation

A nucleotide sequence analysis of a portion of the mitochondrial large ribosomal subunit was performed to define the phylogeography of the threatened crayfish Austropotamobius (Decapoda; Astacidae) in Italy. We collected 61 specimens from 31 localities across the Italian peninsula. For the phylogenetic inference, we combined the 61 Austropotamobius spp sequences obtained from this study with 18 sequences deposited in GenBank and corresponding to Italian, French, Irish, Swiss, and Slovenian locations. Among the analysed sequences, 34 distinct haplotypes were detected. Our results confirmed the presence of both A. pallipes and A. italicus in the Italian peninsula and the existence within the latter species of a strong intraspecific genetic variation, due to the occurrence of four subspecies with a well-defined geographic distribution. From a conservation viewpoint, Italy, with its high haplotype variability, may be considered a 'hot spot' for the genetic diversity of the European native crayfish Austropotamobius. We suggest that re-introduction programs should be conducted with extreme caution in Italy, since not only the two Austropotamobius species but also the four A. italicus subspecies are genetically and taxonomically separate units and require independent conservation plans.     

Controlled Biomineralization of Magnetite (Fe(inf3)O(inf4)) and Greigite (Fe(inf3)S(inf4)) in a Magnetotactic Bacterium

A slowly moving, rod-shaped magnetotactic bacterium was found in relatively large numbers at and below the oxic-anoxic transition zone of a semianaerobic estuarine basin. Unlike all magnetotactic bacteria described to date, cells of this organism produce single-magnetic-domain particles of an iron oxide, magnetite (Fe(inf3)O(inf4)), and an iron sulfide, greigite (Fe(inf3)S(inf4)), within their magnetosomes. The crystals had different morphologies, being arrowhead or tooth shaped for the magnetite particles and roughly rectangular for the greigite particles, and were coorganized within the same chain(s) in the same cell with their long axes along the chain direction. Because the two crystal types have different crystallochemical characteristics, the findings presented here suggest that the formation of the crystal types is controlled by separate biomineralization processes and that the assembly of the magnetosome chain is controlled by a third ultrastructural process. In addition, our results show that in some magnetotactic bacteria, external environmental conditions such as redox and/or oxygen or hydrogen sulfide concentrations may affect the composition of the nonmetal part of the magnetosome mineral phase.     

Labeling of lipids of retina subcellular fractions by [1-14C]eicosatetraenoate (20:4(n-6)) docosapentaenoate (22:5(n-3)) and docosahexaenoate (22:6(n-3))

(1-14C)-labeled (n-6) eicosatetraenoate, (n-3) docosapentaenoate and (n-3) docosahexaenoate (20:4, 22:5 and 22:6, respectively) are efficiently taken up and actively esterified into the lipids of bovine retina after 2 h incubation. Photoreceptor membranes, mitochondria, microsomes and postmicrosomal supernatants, which display significant differences in phospholipid and fatty acid compositions, are isolated after such incubations to study the labeling of lipids. The lipid classes preferentially labeled with the acids (1) largely differ among and within subcellular fractions, while (2) some common features in the treatment of the three polyenes are observed in each fraction. In all of them, the three acids are actively incorporated in phosphatidylcholine; ethanolamine glycerophospholipid, phosphatidylserine (PS) and phosphatidylinositol (PI) are highly labeled with 22:6, 22:5 and 20:4 respectively; within ethanolamine glycerophospholipid, the three label phosphatidylethanolamine in preference to plasmenylethanolamine. Most of the 14C esterified in mitochondria is in phospholipids. The endoplasmic reticulum produces in addition highly labeled triacylglycerols, also found in cytosol. High levels of 14C-labeled diacylglycerols are observed exclusively in photoreceptor membranes, where the specific radioactivity of PI is very high. The total amounts of 14C incorporated (1) are in general similar within a given fraction for the three polyenes, but (2) largely differ among fractions. The labeling of the highly unsaturated phospholipids of photoreceptor membranes is the lowest, while the postmicrosomal supernatant (whose lipids are relatively the poorest in polyenoic fatty acids) contains most of the labeled lipids isolated from retinas under these conditions. The results indicate that polyunsaturated species of retina phospholipids undergo an active synthesis and turnover, as well as an intense intracellular traffic among membranes.     

Introduction of antigenic determining 2,4-dinitrophenyl residues into 4-thiouridine, N3-(3-L-amino-3-carboxypropyl) uridine and tRNA-Phe from E. coli.

The introduction of antigenic determining 2,4-dinitrophenyl residues into the rare ribonucleosides 4-thiouridine (1a), and N3-(3-L-amino-3-carboxypropyl) uridine (2) as well as into tRNA-Phe from E. coli has been investigated. Alkylation of 1a with omega-bromo-2,4-dinitroacetophenone (3b) gives S-(2,4-dinitrophenacyl)-4-thiouridine (5A). Applying the reaction to the 5'-monophosphate of 1a, 5b is formed, but this product decomposes at pH 7. However, acylation of 2 with 2,4-dinitrobenzoic acid N-hydroxysuccinimide ester (4b) leads to N3-[3-carboxy-3-L-(2,4-dinitrobenzamido)propyl]uridine (6) which is stable in aqueous solution. The latter reaction was used for the introduction of an antigenic determining 2,4-dinitrophenyl residue into tRNA-Phe from E. coli. The modified tRNA-Phe was isolated and by degradation of the molecule with RNase T2 and alkaline phosphatase the nucleoside derivative 6 was obtained and found to be identical with the synthetic product.     

Evidence for peroxisomal retroconversion of adrenic acid (22:4(n-6)) and docosahexaenoic acids (22:6(n-3)) in isolated liver cells

The intracellular localization of the oxidation of [2-14C]adrenic acid (22:4(n-6)) and [1-14C]docosahexaenoic acid (22:6(n-3)) was studied in isolated liver cells. The oxidation of 22:4(n-6) was 2-3-times more rapid than the oxidation of 22:6(n-3), [1-14C]arachidonic acid (20:4(n-6)) or [1-14C]oleic acid (18:1). (+)-Decanoylcarnitine and lactate, both known to inhibit mitochondrial beta-oxidation, reduced the oxidation of 18:1 distinctly more efficiently than with 22:4(n-6) and 22:6(n-3). In liver cells from rats fed a diet containing partially hydrogenated fish oil, the oxidation of 22:6(n-6) and 22:6(n-3) was increased by 30-40% compared with cells from rats fed a standard pellet diet. With 18:1 as substrate, the amount of fatty acid oxidized was very similar in cells from animals fed standard pellets or partially hydrogenated fish oil. Shortened fatty acids were not produced from [5,6,8,9,11,12,14,15-3H]arachidonic acid. In hepatocytes from rats starved and refed 20% fructose, a large fraction of 14C from 22:4 was recovered in 14C-labelled C14-C18 fatty acids. Oxidation of 22:4 thus caused a high specific activity of the extramitochondrial pool of acetyl-CoA. The results suggest that 22:4(n-6) and to some extent 22:6(n-3) are oxidized by peroxisomal beta-oxidation and by this are retroconverted to arachidonic acid and eicosapentaenoic acid.     

An automatic method for determination of urinary 3-methylhistidine: normal values.

A system for automatic analysis of urinary 3-methylhistidine is described, applying ion-exchange chromatography and using an automatic sample injector, a motoric selector valve, and a diode programmer, which controls the analytical system. The method permits a sampling rate of 22 samples/day. 3-Methylhistidine was completely separated from histidine in 37 min whereas 1-methylhistidine was eluted together with ammonia. The 3-methylhistidine concentration was linear up to 150 nmol/ml and no appreciable sample interaction was found at automatic sequential runs. The error, in a single determination based on duplicate samples, was 4.61% and, in duplicated determinations, 3.26%. The mean urinary 3-methylhistidine output was 299.4 ± 23.8 μmol/day in 12 healthy females and 545.5 ± 35.2 μmol/day in 12 healthy males. The 3-methylhistidine excretion was significantly higher in males than in females, when expressed as the absolute daily output or as the estimated ratio to body weight, body surface area, or creatinine.     

Characterization of the self-assembly of meso-tetra(4-sulfonatophenyl)porphyrin (H(2)TPPS(4-)) in aqueous solutions

The aggregation of meso-tetra(4-sulfonatophenyl)porphyrin (H(2)TPPS(4-)) in phosphate solutions was investigated as a function of pH, concentration, time, ionic strength, and solution preparation (either from dilution of a freshly prepared 2 mM stock or by direct preparation of μM solution concentrations) using a combination of complementary analytical techniques. UV-vis and fluorescence spectroscopy indicated the formation of staggered, side-by-side (J-type) assemblies. Their size and self-associative behavior were determined using analytical ultracentrifugation and small-angle X-ray scattering. Our results indicate that in neutral and basic solutions of H(2)TPPS(4-), porphyrin dimers and trimers are formed at micromolar concentrations and in the absence of NaCl to screen any ionic interactions. At these low concentrations and pH 4, the protonated H(4)TPPS(2-) species self-assembles, leading to the formation of particularly stable aggregates bearing 25 ± 3 macrocycles. At higher concentrations, these structures further organize or reorganize into tubular, rod-like shapes of various lengths, which were imaged by cryogenic and freeze-fracture transmission electron microscopy. Micron-scale fibrillar aggregates were obtained even at micromolar concentrations at pH 4 when prepared from dilution of a 2 mM stock solution, upon addition of NaCl, or both.