Protein phosphorylation patterns during aestivation in the land snailOtala lactea

Protein phosphorylation patterns were investigated in whole tissues and subcellular fractions of active and aestivatingOtala lactea (Müller) (Pulmonata, Helicidae). Measurement of overall protein phosphorylation showed that incorporation of³²P increased until the second day after injection and remained constant for the remaining 4 days of the time course. Comparison of tissues from aestivating and active snails on day 3 showed a decreased protein phosphorylation in aestivating snails (44% of active). No differences in total and protein-associated radioactivity for foot, mantle or haemolymph were observed. Subcellular fractionation of the hepatopancreas localized the changes to plasma membrane, microsomal, and cytosolic fractions: values for aestivating animals were reduced to 71, 37 and 58% of the corresponding active values. Separation of the individual subcellular fractions on isoelectric focusing columns revealed differences in the phosphate incorporation patterns. Plasma membrane from aestivating animal hepatopancreas had a lower overall level of incorporation and fewer radioactive peaks in the pH 7–10 region than did the plasma membrane fraction from active animals. SDS-PAGE analysis of plasma membrane fractions from active and aestivating snails showed a relative decrease in phosphorylation between 60–80 kDa and 30–40 kDa. IEF analysis of cytosolic proteins from aestivating snail hepatopancreas also showed peaks of radioactivity that were apparently shifted by 0.3 pH units toward higher pI values. Increased phosphate incorporation was observed at a peak that corresponded to the pI value for pyruvate kinase in aestivating snails but definite assignment of peaks was not possible. SDS-PAGE analysis of cytosolic proteins showed an aestivation-related decrease in relative protein phosphorylation between 30–35 kDa and 40–45 kDa. A relative increase in phosphorylation during aestivation was observed for proteins between 16–22 kDa. Overall, the data indicate that snails dramatically alter their protein phosphorylation pattern in hepatopancreas during aestivation. (Mol Cell Biochem143: 7–13, 1995)Abbreviations CY cytosol - dpm radioactive disintegrations per minute - IEF isoelectrofocusing - GP glycogen phosphorylase - MC microsomes - MT mitochondria - PAGE polyacrylamide gel electrophoresis - PKF phosphofructokinase - PK pyruvate kinase - PM plasma membrane - SDS sodium dodecyl sulphate     

Subcellular accumulation of Cu in the Antarctic bivalve<Emphasis Type="Italic"> Laternula elliptica</Emphasis> from a naturally Cu-elevated bay of King George Island

Subcellular Cu sequestration was examined in the digestive gland, kidney and gill of the Antarctic bivalve Laternula elliptica collected from a Cu-elevated bay in King George Island. Cu was associated with both the soluble cytosolic and insoluble particulate cell fractions in all three organs, but their relative contributions to Cu sequestration varied with tissue type and the total amount of Cu accumulated. Low-molecular-weight (10–13 kDa) metallothionein-like proteins were the major Cu-binding ligands in the cytosol of all three organs. Significant portions of the cytosolic Cu were also bound to proteins with different molecular weights in the kidney and gill. A strong immunological response to a metallothionein (MT) antibody confirmed the presence of MTs in all three organs. Numerous electron-dense granules, which are likely to be metal-rich, were observed in renal epithelial cells by transmission electron microscopy, suggesting that these granules also play a role in Cu sequestration.     

Molecular characterization and mRNA transcript profile of vitellogenin in Chinese shrimp, Fenneropenaeus chinensis

A full-length cDNA encoding vitellogenin (Vg) was cloned from Chinese shrimp, Fenneropenaeus chinensis using RACE method. The full-length cDNA consist of 7,942 nucleotides including a 7,761 bp open reading frame, which encodes 2,587 amino acid residues. The deduced amino acid sequence showed high (from 94% to 37%) identity with other known crustacean Vgs. In addition, a consensus cleavage site (R-X-K/R-R) recognized by an endopeptidase and a member of subtilisin family of serine protease were identified in the deduced Vg precursor. RT-PCR analysis shown that Vg mRNA can be detected in both ovary and hepatopancreas of vitellogenic females but not in other experimental tissues including muscle, heart, lymph organ, gill, haemocytes and intestine. These results suggest that the Vg gene may be expressed exclusively in mature females, and both ovary and hepatopancreas are the possible tissues for Vg synthesis in F. chinensis. In addition, Vg gene is detected in genomic DNA of both females and males.     

Studies of the metabolism,food consumption and assimilation efficiency of a small carnivore,the weasel (Mustela nivalis L.)

Summary The average respiratory quotient of weasels was 0.73. There were significant differences in average daily metabolic rate (ADMR) between the sexes and six experimental temperatures. As temperature decreased the mean ADMR increased, with a relatively greater rise for females. Regression equations of ADMR on temperature were Y=483.2–9.6X kcal/kg/day for males, and Y=625.3–15.0X kcal/kg/day for females. The minimum metabolic rate measured for males was 6.6 kcal/kg/h, and the maximum 25.7 kcal/kg/h. The relationship between ADMR and body weight varied with different temperatures, but was consistent with the hypothesis that ADMR was proportional to metabolic weight. The energetic costs of activity for my captive weasels were more than 20% of their daily total energy expenditure. Data on metabolism supported the conclusion that long, thin mustelids lose heat faster than normally-shaped mammals of the same weight.The calorific contents of foods, faeces and urine from feeding trials were determined. There were significant differences in the calorific content of faeces between the sexes, and of faeces and urine between diets.Four natural foods (Microtus, Apodemus, rabbit, starling) were offered during feeding trials. Daily food consumption varied between the diets, but small weasels always ate relatively more than large ones. Mean consumption was 0.33 g/g/day for males, and 0.36 g/g/day for females. Weasels on low-bulk diets (rabbit, starling) lost at least as much energy in urine as in faeces. There were significant differences in assimilation efficiency between the sexes and diets. The average efficiency for males was 78.2%, and 79.8% for females. Bulky foods lowered assimilation efficiencies by up to 9%.Daily energy requirements for maintenance calculated from ADMR data were compared with those from feeding trials. On the Microtus diet weasels expended 18.6–30.1% more energy on maintenance than predicted by the ADMR results, whereas on the other diets they expended 2.7–31.4% less energy. Estimates from ADMR data were probably more accurate.     

Heat shock protein induction in the freshwater prawn Macrobrachium malcolmsonii: acclimation-influenced variations in the induction temperatures for Hsp70

The intracellular build-up of thermally damaged proteins following exposure to heat stress results in the synthesis of heat shock proteins (Hsps). In the present study, the upper thermal tolerance and expression of heat shock protein 70 (Hsp70) were examined in juveniles of the freshwater prawn Macrobrachium malcolmsonii that had been acclimated at two different temperatures, i.e. 20 degrees C (group A) and 30 degrees C (group B), in the laboratory for 30 days. Upper thermal tolerance was determined by a standard method. For heat-shock experiments, prawns in groups A and B were exposed to various elevated temperatures for 3 h each, followed by 1 h recovery at the acclimation temperature. Endogenous levels of Hsp70 were determined in the gill, heart, hepatopancreas and skeletal muscle tissues by Western blotting analysis of one dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The critical thermal maximum (CT max) for prawns in groups A and B was 37.7+/-0.27 degrees C and 41.41+/-0.16 degrees C, respectively. In general, Western blotting analysis for Hsp70 revealed one band at the 70 kDa region, containing both constitutive (Hsc70) and inducible (Hsp70) isoforms, in the gill and heart tissues; these were not detected in the hepatopancreas and skeletal muscle tissues. The onset temperature for Hsp70 induction in both gill and heart tissues was 30 degrees C for prawns in group A and 34 degrees C for those in group B. The optimum induction temperatures (at which Hsp70 induction was maximum) were found to be 34 degrees C and 32 degrees C, respectively, in the gill and heart tissues of group A prawns, and 38 degrees C and 36 degrees C, respectively, for group B prawns. These results suggest that the temperature at which acclimation occurs influences both upper thermal tolerance and Hsp70 induction in M. malcolmsonii.     

Precipitation potential as a major factor in the formation of granular sludge in an upflow sludge-blanket reactor for denitrification of drinking water

The effects of the chemical composition of water on granular sludge formation and characteristics in a denitrifying upflow sludge-blanket (USB) reactor were studied. Denitrification of drinking water showed different biomass sludge characteristics when the reactor was fed with groundwater as opposed to surface water. USB reactors fed with groundwater produced granules with good settling characteristics, SVI (sludge volume index) values lower than 30 ml/g, and high reactor biomass concentrations (20–25 g/l), while surface-water-fed reactors exhibited lower biomass concentrations (10–15 g/l) due to poor settling characteristics (SVI values of 50–90 ml/g). Sludge granules from the reactor fed with surface water had a low mineral content of between 10% and 20% as compared to a mineral content of 25%–50% in the groundwater reactor. The larger mineral content in the groundwater-fed reactor was due to a greater precipitation potential, i.e. higher concentrations of calcium and alkalinity present in groundwater combined with the release of alkalinity and subsequent increase in pH caused by biological denitrification. Verification for this phenomenon was established by enriching surface water with calcium and alkalinity, which increased the reactor's precipitation potential from 15 mg/1 to 40 mg/1 (as CaCO₃). The granules obtained from the reactor fed with enriched surface water had a high mineral content of between 40% and 50% and very low SVI values, contributing to improved granule-settling characteristics and reactor stability.     

Development of Apheliuus mali an endoparasitoid of woolly apple aphid, Eriosoma lanigerum at different temperatures

Developmental times for both sexes of Aphelinus mali (Haldeman) (Hymenoptera: Aphelinidae) an endoparasitoid of woolly apple aphid, Eriosoma lanigerum (Hausmann) (Hemiptera: Pemphigidae) were studied at 13, 15, 18, 20, 25 and 30°C and compared with those of E. lanigerum. Mean developmental times ranged from 11.7–53.3 days for males, 11.8–55.4 days for females and 11.8–54.4 days for both sexes combined and were significantly inversely related to temperature. A good linear model fit (r²>0.99) between developmental rate and temperature in the range 13–30°C was observed. Results indicate significant differences between developmental times of males and females at 13, 18, 20, and 25°C but no differences at 15 and 30°C. The notional developmental threshold was 8.3°C for both males and females. Compared with its host, A. mali has a higher lower developmental threshold. An average of 252.8, 256.7 and 254.8 degree-days (DD) above the lower threshold were required by A. mali to complete development from time of oviposition to adult emergence for males, females and both sexes combined, respectively. Field experimentation also indicated that the developmental time of A. mali lags significantly behind that of its aphid host throughout the year. These findings are discussed in relation to its status as a biological control agent for E. lanigerum.     

Spawning ecology of flannelmouth sucker, Catostomus lattipinnis (Catostomidae), in two small tributaries of the lower Colorado River

We report the first published accounts of spawning behavior and spawning site selection of the flannelmouth sucker in two small tributaries of the lower Colorado River in the Grand Canyon, Arizona. Spawning was observed on 20 March 1992 and from 28 March to 10 April 1993 in the Paria River, and from 16 to 19 March 1993 in Bright Angel Creek. Flannelmouth suckers exhibited promiscuous spawning behavior–individual females were typically paired with two or more males for a given event and sometimes changed partners between events. Multiple egg deposits by different females sometimes occurred at one spawning site. Flannelmouth sucker selected substrates from 16 to 32 mm diameter in both streams. Spawning occurred at depths of 10 to 25 cm in the Paria River and 19 to 41 cm in Bright Angel Creek. Mean column water velocities at spawning locations ranged from 0.15 to 1.0 m sec^-1 in the Paria River and from 0.23 to 0.89 m sec^-1 in Bright Angel Creek. Water temperatures recorded during spawning ranged from 9 to 18° C in the Paria River and 13 to 15° C in Bright Angel Creek. Spawning flannelmouth sucker ascended 9.8 km upstream in the Paria River and 1.25 km in Bright Angel Creek. Spawning females (410–580 mm) were significantly larger than spawning males (385–530 mm) in the Paria River. The mean size of spawning fish in the Paria River was significantly smaller than the entire stock, averaged throughout the study period (380–620 mm). However, fish spawning in 1992–1993 averaged 53 mm larger than fish spawning in the same reach of the Paria River in 1981, indicating a shift in the size structure of this stock.     

Maltose binding protein facilitates high-level expression and functional purification of the chemokines RANTES and SDF-1alpha from Escherichia coli

The chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and SDF-1α (stromal cell-derived factor-1α) are important regulators of leukocyte trafficking and homing. Chemokines form insoluble inclusion bodies when expressed in Escherichia coli (E. coli), resulting in low yields of soluble protein. We have developed a novel chemokine expression system that generates a high amount of soluble protein and uses a simple purification scheme. We cloned different types of RANTES and SDF-1α fused to either maltose binding protein (MBP) or glutathione-S-transferase (GST) and expressed the fusion proteins in E. coli under various conditions. We found that the yield of soluble chemokine is influenced by the type of fusion partner. Fusion to MBP resulted in a higher yield of total and soluble chemokine compared to GST. Under optimized conditions, the yield of soluble MBP–RANTES and MBP–SDF-1α was 2.5- and 4.5-fold higher than that of the corresponding GST-fusion protein, respectively. Recombinant chemokine fusion proteins exhibited specific binding activity to chemokine receptors. These results demonstrate that the use of MBP-fusion proteins may provide an approach to generating high yields of soluble and functional chemokines, such as RANTES and SDF-1α.     

Enzymatic Hydrolysis of Proteins from Crustaceans of the Barents Sea

Enzymatic preparations from king crab hepatopancreas were shown to be capable, in principle, of producing protein hydrolysates. Hydrolysis of protein-containing waste of deep-water prawn and king crab occurs most successfully at pH 8.0–8.5 and 50–55°C for 5–6 h in the presence of 6 g enzyme per kg substrate. The total chemical composition of the hydrolysates, the molecular weight distributions of proteins and polypeptides, and the contents of free amino acids were studied in dry hydrolysates.     

镉诱导金属硫蛋白在华溪蟹组织中的表达

采用体外暴露染毒法,研究了不同浓度与时间条件下,镉诱导河南华溪蟹(Sinopotamon henanense)金属硫蛋白(metallothionein,MT)在肝胰腺、肌肉、鳃和卵巢中的表达差异。镉浓度分别为0、14·5mg/L、29mg/L和58mg/L;处理时间依次为1d、3d和5d。利用镉血红蛋白饱和法和火焰原子吸收分光光度法(AAS)测定MT的蛋白含量。结果显示,用不同的染毒浓度和处理时间,镉在组织中诱导产生MT的含量有较大差异,其中肝胰腺MT的诱导量最大,变化规律也最明显;肌肉中也有较大量MT的表达;而鳃和卵巢MT的诱导量均较低。此外,本文分析了镉的浓度与时间梯度对诱导MT表达的影响与毒性效应机制。结论:组织不同,染毒浓度及时间不同,镉诱导MT的表达也不同,具有一定的组织差异性和规律性。     

Cadmium bioaccumulation and detoxification in the gill and digestive gland of the Antarctic bivalve Laternula elliptica

Exposure to a sublethal concentration of cadmium (Cd; 50 microg L(-1)) resulted in significantly increased Cd concentrations in the gill and digestive gland of the Antarctic bivalve Laternula elliptica. Continuous accumulation of Cd in the two organs during the 14-day exposure period was associated with sequestration of Cd to both the soluble cytosolic and insoluble particulate cell fractions. However, the contribution of each cell fraction to Cd sequestration differed between the two organs; in the gill, a larger portion of Cd was associated with the insoluble fraction, while in the digestive gland, both the soluble and insoluble fractions sequestered similar amounts of Cd. Metal-binding components in the insoluble cell fraction were not identified in this study. On the other hand, a metallothionein-like protein (MTLP) was the major Cd-detoxifying component in the soluble cell fraction of the gill and digestive gland. The amount of MTLP increased linearly with exposure time and the amount of Cd accumulated in the tissue, which suggests a potential utility of MTLP as a biomarker for exposure to Cd and possibly other metals.     

A new species of armored sea robin, Paraheminodus kamoharai (Teleostei: Peristediidae), from the Philippines

A new peristediid, Paraheminodus kamoharai, is described from three specimens (106.9–114.8mm SL) collected from the Sulu Sea, the Philippines. The new species is easily distinguished from its congeners (Paraheminodus laticephalus and P. murrayi) in having 33 bony plates in the upper lateral row, the 24–25th to 30th bony plate each in the upper lateral row with a forwardly directed spine, 17–19 gill rakers on the lower limb, a long slender rostral projection (43.4–47.0% of head length), and short upper (41.1–42.3%) and lower jaws (36.5–37.2%).     

Studies on by-products from the industrial extraction of alginate

The content, the chemical composition and some physical-chemical properties of soluble and insoluble dietary fibres from an industrial alginate extraction by-product, flotation cellulose, were measured by two enzymatic gravimetric methods: an adaptation of the AOAC method (standard method) and a physiological method which used conditions closer to those prevailing in the digestive tract (pH, temperature, ionic strength and ionic composition). Total dietary fibres content obtained with the two methods were close (48.4–52.7; 56.3–59.8%) and about 68–95% of them were insoluble. Soluble fibre were essentially composed of uronic acids and were extracted under the simulated gastric conditions.Swelling in water and water absorption in NaCl (154 mM) of insoluble fibres with particle size between 250–500 µm were 21.9 g g^-1 and 3.6 gg^-1, respectively. The content and physical-chemical characteristics of fibres from flotation cellulose are close to those obtained from other plant and algal industrial by-products. Soluble fibre presented low intrinsic viscosity (152 ml g^-1).     

Ascorbic acid status in high-mountain charr,Salvelinus alpinus,in relation to the reproductive cycle

Synopsis Concentrations of total and dehydroascorbic acids and of glutathione were determined in tissues of male and female Arctic charr at different stages of the reproductive cycle. In fish inhabiting an Alpine lake (2344 m above sea level) the ascorbic acid concentrations were significantly higher in the ovary (344 g g^–1 in August) than in the testis (62.6 g g^–1). The ascorbate concentration was significantly higher in liver and kidney of males than in females in the period prior to spawning. This suggests that there was an intensive investment of body ascorbate reserves in the ovary and the results indicated that females deposited approximately 10 times the amount of vitamin C in ovaries than males deposited in testes. However, the endogenous stores cannot account for all ascorbate deposited in ovaries and the remainder must come from food consumed in summer. Since the resident charr of the high-mountain lake had the ascorbate concentration in ovary by one order of magnitude higher than in cultured salmonids it is plausible to speculate that the change in ascorbic acid metabolism reflects the adaptation to the harsh environmental conditions.     

Intrasexual copulation and mate discrimination in a population of Nodilittorina radiata (Gastropoda: Littorinidae)

Intrasexual copulation and mate discrimination by Nodilittorina radiata (Gastropoda: Littorinidae) were studied on a concrete breakwater at Hakodate Bay, southern Hokkaido, Japan. Intrasexual (male–male) copulations were observed in 4.7–21.1% of copulating pairs on the shore. As females were relatively larger than males and males copulated with females larger than themselves, we hypothesized that males choose potential mates larger than themselves. However, two male mates showed no significant size preference in intrasexual copulations, suggesting that males do not choose relatively larger individuals as mates. In a laboratory mate-choice experiment, male N. radiata preferred to mate with females, indicating precopulatory sex identification. They copulated with males, however, at the frequency of 37%, perhaps because of sex misidentification.     

Stoichiometric measurements of 30S and 50S ribosomal proteins from Escherichia coli

Summary Stoichiometric investigations were made for all 21 proteins of the 30S subunit and for 31 proteins of the 50S subunit. While the results for the 30S proteins strongly suggest the existence of two different stoichiometric classes, the so-called fractional (F) and unit (U) proteins with average molar amounts of 0.1–0.5 and 0.8–1.2 per mole 30S respectively, at least one further group is present in isolated 50S particles. Therefore 50S proteins may be subdivided in the following way: a) 9–10 proteins with average molar amounts of 0.2–0.6 (F-proteins), b) 15–16 proteins with 0.8–1.2 copies per 50S particle (U-proteins), c) at least one protein with an average value of 1.8–2.2 copies, the repeated (R) proteins, d) a group of 7 proteins with 1.4–1.7 copies per particle; they can be named fractional repeated (FR) proteins. These findings indicate that 50S ribosomal particles show a more differentiated degree of structural heterogeneity at least in vitro than has been shown for 30S subunits.Paper No. 35 on Ribosomal Proteins. Preceding paper is by E. Deusser and H. G. Wittmann, Nature 238, 269 (1972).     

Zinc and zinc binding substances in the tissues of common carp

Extraordinarily high concentrations of zinc (300–500 μg/(g fresh tissue)) are often found in the digestive tract tissue of common carp Cyprinus carpio, and high zinc concentrations (typically >100 μg/(g fresh tissue)) are also found in the kidney, gill, skeletal tissues, and spleen. In the present study, we found that only about 40% of the zinc in the digestive tract tissue of common carp could be extracted by water. However, 0.01 M citrate buffer, pH 6.2 could extract over 90% of the zinc. Subcellular zinc distribution in the tissues of common carp, grass carp Ctenopharyngodon idellus, silver carp Aristichthys nobilis, and tilapia Oreochromis aureus were compared. It was found that zinc concentrations in the cytosol, microsomal and mitochondrial fractions were approximately the same for all four species, being only about 16, 5, and 4 μg/(g fresh tissue), respectively. However, zinc concentrations in the nuclei/cell debris fraction of common carp tissue were much higher (46–370 μg/(g fresh tissue)) than the <14 μg/(g fresh tissue) found in the other three species. From this we conclude that neither water-soluble zinc proteins nor metallothionein could account for the high levels of zinc found in common carp tissues. A preliminary biochemical investigation suggests that the main zinc binding substance(s) in the nuclei/cell debris fraction of digestive tract tissue of common carp was probably a membrane protein(s).     

Physiological characteristics of the antarctic copepod Calanoides acutus during late summer in the Weddell Sea

Ingestion, respiration and excretion rates as well as lipid and protein body content of the dominant Antarctic copepod Calanoides acutus (CIV to adult females) were studied during the period covering the end of phytoplankton bloom (February) to the beginning of transition to overwintering (March-April). Daily rations measured with gut fluorescence varied from 2.2 to 2.7% in surface C. acutus. Weight-specific respiration and excretion rates in deep C. acutus decreased by a factor of 11 and 3.5–3.8 compared to their surface counterparts. High lipid (up to 455 µg ind^–1) and protein (198 µg ind^–l) content was observed in surface C. acutus CV in February; a month later the animals with similar lipid and protein content were found in the depth (500–1000 m layer). Their lipid reserves were enough to overwinter and probably to ascend, molt and reproduce. At the same time some of the deep CVs had much lower protein and lipid content and could survive only for 4–5 months. Our own and literature data led to the conclusion that females of C. acutus reach adulthood at the age of more than one year while development of males could be completed in one year.