Showing papers in "Comparative Biochemistry and Physiology B in 1996"

Comparative properties of arginases.

Abstract: Arginase is a primordial enzyme, widely distributed in the biosphere and represented in all primary kingdoms. It plays a critical role in the hepatic metabolism of most higher organisms as a cardinal component of the urea cycle. Additionally, it occurs in numerous organisms and tissues where there is no functioning urea cycle. Many extrahepatic tissues have been shown to contain a second form of arginase, closely related to the hepatic enzyme but encoded by a distinct gene or genes and involved in a host of physiological roles. A variety of functions has been proposed for the “extrahepatic” arginases over the last three decades. In recent years, interest in arginase has been stimulated by a demonstrated involvement in the metabolism of the ubiquitous and multifaceted molecule nitric oxide. Molecular biology has begun to furnish new clues to the disparate functions of arginases in different environments and organisms. Comparative studies of arginase sequences are also beginning to elucidate the comparative evolution of arginases, their molecular structures and the nature of their catalytic mechanism. Further studies have sought to clarify the involvement of arginase in human disease. This review presents an outline of the current state of arginase research by giving a comparative overview of arginases and their associated properties.

Cuticular sclerotization in insects

Abstract: The insect cuticle is an extracellular structure covering the total outer surface of the animal and providing protection against harmful influences from the environment. The mechanical properties of cuticles may vary considerably, and pronounced regional differences are generally observed. The properties may also change during development, and it can be assumed that the physical and chemical properties of all cuticular regions tend to be close to the optimal for proper physiological function during all developmental stages. Cuticular regions can be stabilized by the process of sclerotization, whereby o-diphenols are oxidatively incorporated into the material. Our current knowledge of the sclerotization process is reviewed, and it is suggested that the main features of the chemistry of sclerotization probably have been established, and that the major questions now remaining concern the precise regional and temporal control of the process.

Metabolic adaptations supporting anoxia tolerance in reptiles : recent advances

Abstract: Animal survival during severe hypoxia and/or anoxia is enhanced by a variety of biochemical adaptations including adaptations of fermentative pathways of energy production and, most importantly, the ability to sharply reduce metabolic rate by 5-20 fold and enter a hypometabolic state. The biochemical regulation of metabolic arrest is proving to have common molecular principles that extend across phylogenetic lines and that are conserved in different types of arrested states (not only anaerobiosis but also estivation, hibernation, etc.). Our new studies with anoxia-tolerant vertebrates have identified a variety of regulatory mechanisms involved in both metabolic rate depression and in the aerobic recovery process using as models the freshwater turtle Trachemys scripta elegans and garter snakes Thamnophis sirtalis parietalis. Mechanisms include: 1) post-translational modification of cellular and functional proteins by reversible phosphorylation and changes in protein kinase (PKA, PKC) and/or phosphatase activities to regulate this, 2) reversible enzyme binding associations with subcellular structural elements, 3) differential gene expression and/or mRNA translation producing new mRNA variants and new protein products, 4) changes in protease activity, particularly the multicatalytic proteinase complex, and 5) both constitutive and anoxia-induced modifications to cellular antioxidant systems to deal with oxidative stress during the anoxic-aerobic transition of recovery.

The adaptations of fish to extremely alkaline environments

Abstract: The Lake Magadi Tilapia (MT; Oreochromis alcalicus grahami , the Lahontan cutthroat trout (LCT; Oncorhynchus clarki henshawi ) and the tarek (Ct; Chalcalburnus tarichi ) have evolved unique strategies that allow them to overcome problems associated with ammonia excretion (J Amm ) and acid-base regulation in their alkaline environments. In Lake Magadi, Kenya (pH 10), the MT circumvents problems associated with J Amm by excreting virtually all (>90%) of its waste-nitrogen as urea. Base excretion appears to be facilitated by modified seawater-type gill chloride cells, through apical Cl − /HCO 3 − exchangers and an outwardly directed OH − /HCO 3 − /CO 3 = excretion system. The LCT avoids potentially toxic increases in internal ammonia by permanently lowering ammonia production rates following transfer into alkaline (pH 9.4) Pyramid Lake, Nevada, from its juvenile freshwater (pH 8.4) environment. Greater apical exposure of LCT gill chloride cells, presumably the freshwater variety, probably facilitates base excretion by elevating Cl − /HCO 3 − exchange capacity. In Lake Van, Turkey (pH 9.8) high ammonia tolerance enables C. tarichi to withstand the high internal ammonia concentrations that it apparently requires for the facilitation of J Amm . It also excretes unusually high amounts of urea. We conclude that adjustments to nitrogenous waste metabolism and excretion patterns, as well as modifications to gill functional morphology, are necessary adaptations that permit these animals to thrive in environments considered unsuitable for most fishes.

A glucokinase-like enzyme in the liver of atlantic salmon (salmo salar)

Abstract: An enzyme with properties similar to rat liver glucokinase (Hexokinase IV or D) is present in salmon liver in addition to low-Km hexokinase(s). The specific activity of this enzyme increases about 1.6 fold, comparing activities after feeding diets with 25% and 0% digestive energy from starch. The enzyme has a low affinity for glucose, S0.5 = 25.2-26.8 mM (95% confidence interval) and a low activity with fructose, approximately 8% of the activity with glucose. Its molecular mass was estimated to 50.7 +/- 0.6 kDa (SEM. n = 3) by gel filtration, and it displays positive cooperativity with respect to glucose. The Hill constant = 1.73-1.81 (95% confidence interval). The enzyme is competitively inhibited by N-acetyl glucosamine, K(i) approximately 0.28 mM.

Avian eggshell pigments and their variability

Abstract: Eggshell pigment constituents were determined by a high-performance liquid chromatography method. Most of the work was done on whole clutches of red-backed shrike ( Lanius collurio ). In addition to the known pigments (protoporphyrin IX, biliverdin), a new pigment, zinc-containing protoporphyrin IX, was found as well. Its content was highly variable—from 0% to 100%. The content of total protoporphyrin IX (with and without zinc) ranged from 1.72 to 114.84 nmol (average for whole clutches was 39.93 nmol), while the content of zinc-deficient protoporphyrin IX was ND (not detectable) -110.46 nmol (average for whole clutches was 34.02 nmol) and the content of zinc-containing protoporphyrin IX was ND - 32.28 nmol (average for whole clutches was 5.91 nmol). Zinc-containing protoporphyrin IX was absent in 33% of eggs, while zinc-deficient protoporphyrin IX was absent in a single case only (2%). If clutches from relatively “polluted” and “unpolluted” regions were compared, no differences have been found. Based on these findings is the conclusion that the high variability in eggshell pigment content is likely to reflect physiological influences (e.g. order of egg laying and the whole condition of the nesting female) rather than environmental interferences. This conclusion is in agreement with our previous findings. Additional data regarding the pigment content of seven other bird species are also included.

Temperature and pH sensitivity of trypsins from Atlantic salmon (Salmo salar) in comparison with bovine and porcine trypsin.

Abstract: Four differently charged trypsins were purified from pyloric caeca of Atlantic salmon ( Salmo salar ). The isoelectric points of three anionic isoforms were 4.70, 4.60, and 4.55 (anionic trypsin I, II and III, respectively). And for the first time a cationic isoform (isoelectric point above 9.3) has been isolated from a marine species. The apparent molecular weights of all four isoforms were about 25 kDa as determined by SDS-PAGE. The salmon enzymes were inhibited by serine proteinase inhibitors in general and also by specific trypsin inhibitors. Anionic trypsin I and the cationic isoform were further examined. Anionic trypsin 1 showed the typical cold-adaptation features, low pH and temperature stability (also lower Gibb's free energy of GdnHCl-induced unfolding) and high catalytic efficiency as compared to the mammalian trypsins. The cationic isoform did not show these features, but resembled the mammalian trypsins.

Cell signalling by inositol phosphoglycans from different species.

Abstract: The discovery of glycosyl-phosphatidylinositol (GPI) molecules and their products has given new insight into the field of signal transduction. In the last decade a novel mechanism of protein attachment to membranes has emerged, which involves a covalent linkage of the protein to the glycan moiety of a GPI. The discovery that GP1-anchored proteins are ubiquitous throughout the eukaryotes was followed by the observation that uncomplexed GPI molecules are implicated in signal transduction for a diversity of hormones and growth factors. The hydrolysis of free-GPI generates a novel second messenger: the inositol phosphoglycan (IPG). The aim of this article is to review the role of IPG and IPG-like molecules in signal transduction and to discuss future research directions.

Seasonal variations of the lipid content and fatty acid composition of Crassostrea gigas cultured in E1 Grove, Galicia, N.W. Spain

Abstract: Lipid classes and fatty acids of triacylglycerol and phospholipid fractions of introduced Crassostrea gigas from E1 Grove were analysed and compared for 16 months. The fluctuations in the lipid content were correlated to the phytoplankton concentration and sexual cycle. The seasonal variations in lipid content were due largely to changes in triacylglycerols, the major lipid class. Phospholipids and sterols followed a similar seasonal cycle with minor oscillations. Polyunsaturated fatty acid (PUFA) was negatively correlated with temperature and showed a pattern similar to n-3 series with great fluctuations, whereas the n-6 fatty acids had minor fluctuations throughout the year and showed no clear seasonal variation. The content of 20:5n-3 was higher than 22:6n-3 in triacylglycerols and similar in phospholipids. The 20:4n-6 was the major fatty acid of n-6 series and correlated negatively with condition index, temperature and chlorophyll a in the phospholipid fraction. The maturity index correlated positively with 18:4n-3 from phospholipids.

Digestive enzymes in marine species. I. Proteinase activities in gut from redfish (Sebastes mentella), seabream (Sparus aurata) and turbot (Scophthalmus maximus)

Abstract: The proteolytic activities of the digestivetract of three carnivorous fish species ( Sebastes mentella, Scophthalmus maximus, Sparus aurata ) have been studied. The activity found in the stomach showed a pH optimum of 2.0 for all species, whereas this was in the alkaline range (9.5–10.0) in the intestinal extracts. Similar optimal temperature was measured for all species, although higher residual activities were detected at low temperatures in redfish and turbot. Lower E a values were detected in stomach than in intestine. The effect of NaCl concentration on protein digestion differed between stomach and intestine being inhibitory in the former. The main proteolytic enzyme for the acid activity of the stomach of the three species seemed to be a pepsin. However, the enzymatic composition of the intestine was more species specific.

Hypothermia in hypoxic animals : mechanisms, mediators, and functional significance

Abstract: A basic tenet of biology is that body temperature (Tb) has a marked effect on oxygen uptake of resting animals. For most animals, the temperature coefficient (Q10) is >> 2.5; e.g., resting oxygen uptake changes about 11% per degree C change in Tb. An important consequence of this dependence is that hyperthermia could be deleterious for hypoxic animals, particularly for oxygen sensitive organs, e.g., heart and brain. Conversely, a moderate degree of hypothermia could be beneficial during hypoxia. This concept is not new. Forced hypothermia is sometimes used in surgical procedures, particularly for heart and brain surgery. However, in many situations where hypothermia might have benefits, e.g., pediatric intensive care, it is not permitted. This is due in part to dogma and in part to the real and potential disadvantages of hypothermia, even in severely hypoxic animals. Among these in ventricular fibrillation. This is apparently preventable if blood pH is allowed to rise following the "Buffalo Curve." Another important disadvantage, were it to occur, is elevation of oxygen demand due to a thermogenic responses. However, at least in some species, the thermogenic response is blunted during hypoxia; e.g., in young rats. Furthermore, even if a thermogenic response occurs, this takes place primarily in muscles (shivering) and brown fat (non-shivering) and not in the O2-sensitive organs, heart and brain. A third disadvantage, for prolonged hypothermia, might be impairment of the immune response, a serious problem if hypoxia is combined with infection. This paper will review four aspects of behavioral fever and hypothermia: the occurrence among animals, the mechanisms and mediators that might trigger behavioral responses, and the functional significance.

Site-specific fatty acid composition in adipose tissues of several northern aquatic and terrestrial mammals

Abstract: Site-specific differences in fatty acid compositions (by gas-liquid chromatography) were compared in aquatic, semiaquatic and terrestrial mammals: the ringed seals ( Phoca hispida hispida and P. h. botnica ), otter ( Lutra lutra ), raccoon dog ( Nyctereutes procyonoides ), brown bear ( Ursus arctos ) and grey wolf ( Canis lupus ). In addition, we briefly discuss our earlier results for the Canadian beaver ( Castor canadensis ) and muskrat ( Ondatra zibethicus ). In both aquatic and terrestrial species, large amounts of Δ9-monounsaturated fatty acids (MUFAs) and small amounts of saturated fatty acids and exogenous long-chain MUFAs were found in the cold tissues of the extremities. In seals, the poikilothermic outer blubber had these characteristics and differed from the inner blubber. On the other hand, the subcutaneous and inner fat depots of the coated semiaquatic and terrestrial mammals were uniform. In the bare extremities, however, these mammals also had an excess of A9-MUFAs. The degree of Δ9-desaturation in the outer blubber of the seals was significantly correlated with age. The excess of Δ9-MUFAs in the bare extremities of land mammals increased the overall double bond content of these tissues compared with the inner depots. In contrast, due to the large amounts of dietary polyunsaturated fatty acids, this was not found in the aquatic and semiaquatic species. The observed site-specific differences are discussed as possible inherited evolutionary adaptations to low temperature of the tissues.

Isolation of acid peptide fractions from a fish protein hydrolysate with strong stimulatory effect on atlantic salmon (Salmo salar) head kidney leucocytes

Abstract: Medium size (3000 d > Mw > 500 d) peptides from a hydrolysate of emptied stomachs from Atlantic cod (Gadus morhua) were fractionated on an S-Sepharose cation exchange chromatography column. Four distinctly separated acid peptide fractions were used in in vitro stimulatory experiments with head kidney leucocytes from Atlantic salmon (Salmo salar). All four acid peptide fractions promoted strongly elevated oxidative burst reactions in the leucocytes after 2 and 7 days of incubation at concentrations from 1 to 25 μg/ml. The stimulation was equally good, and in most cases better than the stimulation achieved with similar concentrations of lipopolysaccharides from the fish pathogen Aeromonas salmonicida. Visual inspection and pictures of peptide stimulated cells showed strongly enhanced vacuolisation and formation of long stretched out pseudopodes after 7 days of incubation. Acid peptide fractions from fish protein hydrolysate may be useful as adjuvants in fish vaccine and as an immune stimulant in fish feed.

Polyunsaturated fatty acids in neutral lipids and phospholipids of some freshwater insects

Abstract: The fatty acid compositions of total neutral lipids and total polar lipids from eight species of freshwater insects were determined: stonefly nymphs (Plecoptera), beetle larvae (Coleoptera), Chironomidae (Diptera), water boatmen (Corixidae and Notonecta; Heteroptera) and mayfly nymphs (Ecdyonurus venosus, Caenis, Ephemerella; Ephemeroptera). In addition, the compositions of individual phosphoglycerides were deter- mined for four of the species (Plecoptera, Corixidae, Ecdyonurus venosus and Emphemerella). Saturated and monounsaturated fatty acids together represented up to 85% of the fatty acids of total neutral lipids with 16:0 (18-31%) being the most abundant saturated fatty acid and 16:ln-7 (10-28%), 18:ln-9 (6-12%) and 18: ln- 7 (3-12%) the most abundant monounsaturates. Polyunsaturated fatty acids (PUFA) accounted for between 16% and 33% of the total fatty acids of neutral lipids, with 20:5n-3 (4-12%), 18:3n-3 (3-30%) and 18:2n-6 (1-8%) all being major components. Arachidonic acid, 20:4n-6 (0.4-1.0%) and 22:6n-3 were, respectively, minor and insignificant components of total neutral lipids. PUFA were major fatty acids (34-56% of the total) in total polar lipids and in phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphati- dylinositol. The major PUFA present were 20:5n-3 (14-27%) and 18:3n-3 (6-23%). The most abundant n- 6 PUFA, especially in phosphoglycerides from Corixidae, was 18:2n-6 (3-11%). Arachidonic acid, 20:4n-6, was present in all phosphoglycerides accounting for 1-4% of the total fatty acids, except in the phosphatidylinosi- tol of Corixidae where it accounted for 12% of the total. 22:6(n-3) was not present in significant amounts in any phosphoglyceride in any species. 18:ln-9 (8-20%) and 18:ln-7 (2-14%) were the most abundant monoun- saturated fatty acids, especially in phosphatidylethanolamine. 16:0 was abundant in phosphatidylcholine (11- 21%), and 18:0 (17-23%) was abundant in phosphatidylserine. The results are discussed in relation to the functions and origins of PUFA in freshwater insects. COMP BIOCHEM PHYSIOL 114B, 161-170, 1996.

Keyhole limpet hemocyanin: Structural and functional characterization of two different subunits and multimers☆

Abstract: Keyhole limpet hemocyanin (KLH), the large respiratory glycoprotein from the primitive gastropod mollusc, Megathura crenulata, is a potent immunogen used classically as a carrier protein for haptens and more recently in human vaccines and for immunotherapy of bladder cancer. Two KLH isoforms were identified and isolated by high-performance anion exchange chromatography. Subsequent analyses disclosed that these isoforms--designated KLH-A and KLH-B--were composed of distinct subunits that differed in primary structure, molecular weight (KLH-A was 449,000 and KLH-B was 392,000), polymerization/reassociation characteristics, and O2-binding constants (KLH-A had a P50 of 7.32 and KLH-B had a P50 of 2.46). Both subunits appear to be composed of eight oxygen binding domains, and reassociate in solution only with like subunits. These results support the concept that structural and functional heterogeneity is a common feature of molluscan hemocyanins, and provide a rational basis for studying and optimizing the immunostimulatory properties of KLH.

Digestive enzymes in marine species. II. Amylase activities in gut from seabream (Sparus aurata), turbot (Scophthalmus maximus) and redfish (Sebastes mentella)

Abstract: The amylase activity of the digestive tract of three carnivorous fish species (Sparus aurata, Scophthalmus maximus and Sebastes mentella) has been studied. The activity of seabream and turbot showed its maximum at neutral pH (7.0–7.5); meanwhile, the activity of redfish had an optimum pH at 4.5-5.0. The t° function ranged between 35 and 45°C for the three species. The Arrhenius plots of the intestinal activities of seabream and turbot showed breakpoints at temperatures close to those of their physiological activities. High saline concentrations inhibited the activity of seabream and turbot and activated the activity of redfish. Seabream activity was absolutely dependent on calcium ions. On the contrary, redfish activity was only detected in the absence of this metal. Studies carried out by using several effectors suggested that the activities found in these three species are different. Considering our results from a point of view of the environmental conditions of these species, it might be concluded that enzymatic digestion of dietary carbohydrates proceeds at very low rate. Physiological implications are discussed.

Anoxia tolerant animals from a neurobiological perspective

Abstract: This paper discusses the mechanisms for brain anoxia survival seen in crucian carp (Carassius carassius) and a few species of freshwater turtle (Chrysemys and Trachemys species). Comparisons are made with the hypoxic tolerant mammalian neonate brain. In the anoxic tolerant species the basic strategy for anoxia survival appears to be the maintenance of ion gradients, and thereby the avoidance of anoxic depolarization. Important facilitating factors involve having huge glycogen stores, increased blood supply to the brain, the suppression of electrical activity, increased release of inhibitory neuromodulators and neurotransmitters, upregulation of inhibitory neuroreceptors, the down-regulation of excitatory ion conductance and the down-regulation of Ca2+ channels. By contrast, for the mammalian neonate the most important causes of its increased hypoxia tolerance may be just simple consequences of the comparatively undifferentiated state of the brain of the newborn, with its lower energy requirements, slower decline in ATP and lower excitability levels acting to delay depolarization.

Midgut proteinases in three divergent species of Coleoptera

Abstract: Cysteine proteinases have been found in some families of Coleoptera and, based on this, these enzymes were supposed to be characteristic of Coleoptera. To test this hypothesis, we studied midgut homogenates of three phylogenetically distant Coleoptera species: Tenebrio molitor (Tenebrionidae) larvae, Pyrearinus termitilluminans (Elateridae) larvae, and Pheropsophus aequinoctialis (Carabidae) adults. T. molitor display two cysteine proteinases (pHo 6.8) resolved in Superose (FPLC) with Mr 31,000 and 51,000. These enzymes are inhibited by E-64 and pHMB, are activated by EDTA + cysteine and hydrolyze benzoyl-DL-arginine-β-naphthylamide. T. molitor enzymes differ from a cysteine proteinase (Mr 64,000 using Superose) present in the wheat meal ingested by the insect. The cysteine proteinases predominate in the anterior two thirds of T. molitor midgut, probably because they are unstable in the higher luminal pH observed in the posterior third of the midgut. P. termitilluminans and P. aequinoctialis do not display cysteine proteinases, although they have trypsins (Mr 15,000, 25,000 and 41,000 for P. termitilluminans ; Mr 26,000, 33,000 and 52,000 for P. aequinoctialis ) and chymotrypsins (Mr 38,000 and 25,000 for P. aequinoctialis and Mr 15,000 for P. termitilluminans ). Our results, together with literature data, suggest that cysteine proteinases occur in the Cucujiformia ancestor, which corresponds to the ancestor of most Coleoptera which ingest seeds rich in serine proteinase inhibitors.

Estimation of protein digestibility—IV. Digestive proteinases from the pyloric caeca of coho salmon (Oncorhynchus kisutch) fed diets containing soybean meal

Abstract: The goal of this study was to better understand why dietary soybean products are poorly utilized by salmonids. The influence of dietary intake on coho salmon fingerling weight gain and specific properties of pyloric caeca enzymes was investigated. Fingerlings were fed diets containing heated or unheated soybean meal (SBM) or Promoveal™, as 15–25% herring meal replacer, for 8–12 weeks. Fish fed to apparent satiation with diets containing heated SBM replacer gained more weight than those fed unheated SBM at the same level. Fish increased in body weight at the same rate when fed restricted rations containing either 15% SBM replacer that was variously heated up to 20 min, 15% Promoveal™ replacer or the herring meal basal diet. After the experimental diets were fed, digestive proteinases were isolated from the pyloric caeca. Yield of pyloric caeca enzymes (PCE), recovery of trypsin in PCE, soybean trypsin inhibitor (SBTI) sensitivity of PCE trypsin, specific activity of PCE trypsin and in vitro casein digestibility by PCE were determined for each dietary group. Weight gain vs in vitro casein digestibility by PCE was linear for animals fed unheated SBM to apparent satiation (r2 = 0.71, P

Phospholipid composition of erythrocyte membranes and plasma of mammalian blood including Australian marsupials; quantitative 31P NMR analysis using detergent.

Abstract: The phospholipid classes of erythrocyte membranes and plasma from several domestic animals and marsupials were quantified by 31P NMR using detergents. Washed erythrocyte samples were thoroughly haemolysed by tip-sonication and dissolved in sodium cholate; plasma samples were dissolved in Triton X-100. The species studied were: common wombat (Vombatus ursinus), black-striped wallaby (Macropus dorsalis), bandicoot (Isoodon macrocarpus), Eastern grey kangaroo (Macropus giganteus), Tammar wallaby (Macropus eugenii), sheep (Ovis aries), goat (Capra hircus), cattle (Bos taurus), horse (Equus caballus), dog (Canus familiaris) and rabbit (Orytolagus caniculus). There were considerable species variations in the relative abundance of erythrocyte and plasma phospholipid classes. The variations may be attributed to the habitats and diets of the animals as well as to their phylogenetic differences.

On the presence of prophenoloxidase in the hemolymph of the horseshoe crab, Limulus.

Abstract: Melanization, and hence the participation of phenoloxidase, in defense mechanism of arthropods is well established. However, in the living fossil, horseshoe crab, it has been claimed that the prophenoloxidase system widely found in the hemolymph of most arthropods is absent. On the contrary, we present evidence for the presence of a prophenoloxidase system in the hemolymph of Limulus and a method to study its activation. Activation of prophenoloxidase was achieved by treatment with either the anionic detergent, SDS, or the cationic detergent, cetylpyridinium chloride. The detergents seemed to bind to the proenzyme below their critical micellar concentration and induce conformational changes that cause the activation of prophenoloxidase. In addition, a number of fatty acids and phospholipids also activated the prophenoloxidase. Proteases such as trypsin activated the enzyme only marginally. The approximate molecular weight of the proenzyme was found to be 70,000. Substrate specificity studies, product analysis and inhibition experiments revealed that the Limulus enzyme is a typical o-diphenoloxidase. The possible reasons for the failure to detect the phenoloxidase activity by earlier workers are discussed.

Localization and gene expression of glucose transporters in bovine mammary gland

Abstract: Glucose uptake in the mammary gland is a rate-limiting step in milk synthesis To study glucose transporters in the bovine mammary gland, the erythrocyte-type glucose transporter (GLUT1) and the insulinresponsive glucose transporter (GLUT4) proteins were assessed by Western blotting and immunohistochemical staining, using polyclonal antibodies against the C-terminal peptide of GLUT1 and GLUT4 Our results demonstrated that the bovine mammary gland expressed a relatively high level of GLUT1 protein, whereas GLUT4 protein was not detected in the mammary gland of either lactating or dry cows The absence of GLUT4 may indicate that glucose transport is not regulated by insulin in the lactating and dry bovine mammary gland The anti-GLUT1 antibody strongly stained the single layer of epithelial cells of mammary alveoli The expression of GLUT1 mRNA was similar in the mammary gland of late lactation and non-lactating cows However, a smaller molecular weight species (38 kDa) of GLUT1 protein was detected in the mammary gland of nonlactating cows where its abundance in crude membrane preparation was 80% higher than in lactating animals There were no significant differences in GLUT1 mRNA in bovine mammary gland at 118 d and 181 d postpartum, however, GLUT1 protein expression tended to be greater at 118 d postpartum

Apyrase and alpha-glucosidase in the salivary glands of Aedes albopictus.

Abstract: An apyrase and an α-glucosidase were detected in the salivary glands extracts of adult Aedes albopictus. The apyrase is a 61,000 Da secreted protein that hydrolyses ATP and ADP. This protein is synthesized in adults and is preferentially accumulated in the distal lateral lobes of the female salivary glands. The α-glucosidase is a secreted 67,000 Da protein. This enzyme is synthesized during adult life and accumulated in the proximal-lateral lobes of both males and females. The results are discussed and compared with data previously obtained with Aedes aegypti salivary glands.

Molecular cloning of an ecdysone receptor (B1 isoform) homologue from the silkworm, Bombyx mori, and its mRNA expression during wing disc development.

Abstract: We reported the isolation and sequence of a clone encoding a putative ecdysone receptor B1 isoform of the silkworm, Bombyx mori. The predicted open reading frame encoded 543 amino acids, with 51%, 95% and 71% identities with the Drosophila melanogaster ecdysone receptor B1 isoform in the N terminal A/B region, DNA binding domain (C region) and ligand binding domain (E region), respectively. A single 6.2 kb message for the EcR gene was abundant in wing discs and fat bodies at the onset of metamorphosis. At the same stage, however, no or a tiny amount of mRNA was shown in posterior or middle silk glands, respectively. During the final instar, the mRNA expression in wing discs was maximal on the day of wandering. These data suggest the transcription of the Bombyx EcR gene is regulated in tissue-specific and stage-specific manner during metamorphosis.

Cuticular lipid composition of Heliothis virescens and Helicoverpa zea pupae

Abstract: The cuticular lipids of laboratory-reared tobacco budworms, Heliothis virescens (Fabricius), and corn earworms, Helicoverpa zea (Boddie), pupae were characterized. Quantities of cuticular lipid were greater for pupae in diapause than for non-diapausing pupae. Major cuticular lipids of H. virescens pupae were longchain n-alcohols (42%) and aldehydes (41%) with chain lengths of mainly C26, C28 and C24 in decreasing order of abundance. Lesser amounts (2–5%) of wax esters and hydrocarbons were identified. The wax ester fraction was a complex mixture of C32-C52 components composed mainly of C24-C28 n-alcohol and saturated, monounsaturated and diunsaturated fatty acid (C16-C22) moieties. For H. zea, the major fractions were wax esters (38%), hydrocarbons (19%), n-alcohols (17%), diols (16%) and lesser amounts (2–5%) of aldehydes, acetate esters of alcohols and oxoalcohols. Triacontanyl hexadecanoate comprised 85% of the wax ester fraction. The H. zea alcohols, alcohol ester moieties and aldehydes consisted of a homologous series of C32-C52 compounds with the 30 carbon chain length as major constituents. The H. zea diols were C30-C36 even-chain n-alcohols with hydroxyl groups on carbon numbers 11, 12, 13, 14 or 15. Mass spectral analysis indicated the presence of unsaturation in the alkyl chain of the major diol components.

Mouse and rat cystatin C: Escherichia coli production, characterization and tissue distribution.

Abstract: Recombinant mouse (Mus musculus) and rat (Rattus norvegicus) cystatin C were produced by expression in Escherichia coli, isolated and functionally characterized. The mouse and rat inhibitors were both fully active in titrations of papain. Determination of equilibrium constants for dissociation (Ki) for their complexes with the target proteinase, cathepsin B, produced values not largely different from that for human cystatin C (Ki 0.07-0.13 nM). Rabbit antisera against mouse and rat cystatin C were produced and used for improved affinity purification of the recombinant inhibitors. Affinity purified immunoglobulins isolated from the antiserum against mouse cystatin C were used for construction of a sensitive enzyme-linked immunosorbent assay. The assay was used to demonstrate a high degree of immunological cross-reactivity between mouse and rat cystatin C and could be used for cystatin C quantification in mouse and rat tissue homogenates. All tissues analyzed contained cystatin C, with a relative content very similar to that of human tissues. For all species, brain tissue contained the highest cystatin C amounts and liver the lowest, whereas kidney, spleen and muscle tissues were intermediate in content. In the mouse, a notable high cystatin C content in parotid gland tissue was observed. The high degree of similarity in distribution pattern and functional properties for mouse, rat and human cystatin C indicates that a murine model should be relevant for studies of the human disease, hereditary cystatin C amyloid angiopathy.

The fatty acid composition of brain phospholipids from chicken and duck embryos

Abstract: The effects of differences in the fatty acid composition of the lipids of egg yolk on the subsequent levels of arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3) in the total phospholipids and in the isolated phospholipid classes of the embryonic brain were investigated by a comparison of two domesticated avain species, the chicken and the duck. The yolk phospholipids of chicken eggs contained similar proportions of 20:4n-6 and 22:6n-3 (approx. 6% wt/wt of total fatty acids). In marked contrast, the yolk phospholipids of commercially produced duck eggs contained an overwhelming preponderance of 20:4n-6 over 22:6n-3 (approx. 10% cf.1%). These differences between the yolks of the two species were only partly reflected in the fatty acid compositions of the total phospholipids of the embryonic brains at equivalent developmental stages. Typically, the chicken brain phospholipids contained approximate proportions of 20:4n-6 and 22:6n-3 of 8% and 17%, respectively, whereas both these polyunsaturates were present at approx. 11% in the duck samples. The brain phospholipids were resolved into their component phospholipid classes by high performance liquid chromatography. In both species, phosphatidylcholine contained only low levels of 20:4n-6 and 22:6n-3, whereas phosphatidylethanolamine displayed a high content of 22:6n-3. Phosphatidylserine was also rich in 22:6n-3 whereas phosphatidylinositol exhibited a high proportion of 20:4n-6. The results suggest that the relatively low level of 22:6n-3 in the yolk of duck eggs is partly compensated for by an enhanced efficiency in the incorporation of this fatty acid into the brain phospholipids, in comparison with the chicken.

Yolk lipids and their fatty acids in the wild and captive ostrich (Struthio camelus)

Abstract: A comparative study has been made of the major lipid fractions and their fatty acid compositions in the yolk of eggs from ostriches under wild and farmed conditions. There were no differences in the lipid contents and proportions of the lipid fractions between the two groups of yolks. In both groups of yolks triacylglycerol and phospholipid were the major fractions. In the eggs from the wild ostriches, all the lipid fractions displayed substantial concentrations of C18 polyunsaturated fatty acids, triacylglycerol being particularly rich in linolenic acid and phospholipid rich in linoleic acid; phospholipid displayed substantial concentrations also of C20 and C22 polyunsaturates. There were considerable differences in the fatty acid compositions between the yolks. Those from the farmed birds displayed lower proportions of C18 polyunsaturates, particularly linolenic acid, throughout the lipid fractions. Compensatory increases were displayed most obviously in the concentrations of oleic acid and palmitoleic acid as well as other acids. The distinctive and extensive changes in fatty acid composition, particularly relating to the polyunsaturates, are discussed with respect to overall dietary requirements and specificities for embryo metabolism and possible effects on reproductive performance.

Phosphoinositide second messengers in olfaction.

Abstract: Olfactory stimuli (odorants) are detected and recognized by binding to receptors belonging to the G-protein-coupled receptor superfamily. The binding of odorants to some receptors stimulates the activity of an odorant-sensitive phospholipase C (PLC) thereby generating the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 plays a key role in membrane depolarization by binding to a receptor that is itself a cation channel. The formation of DAG is expected to stimulate the activity of protein kinase C (PKC). PKC, together with G-protein-coupled receptor kinases, mediates signal termination by phosphorylation of odorant receptors and possibly other substrates. This review summarizes recent evidence regarding the role of phosphoinositide-derived second messengers in the molecular events underlying olfactory signaling. In addition, the role of calcium as a “third messenger” that provides a mechanism for interaction between phosphoinositide second messengers and components of the cyclic AMP signaling pathway is also discussed.

Antibacterial activity in the coelomocytes of the sea urchin Paracentrotus lividus.

Abstract: Naturally present antibacterial activity directed against Vibrio alginolyticus was demonstrated in coelomocytes lysate (CL) and cell-free coelomic fluid (CF) of the marine echinoderm Paracentrotus lividus. Kinetic analysis revealed that 5 min of contact was enough to induce significant bactericidal effect. Maximum activity required 30 min of contact. Nonsensitive to the effect of trypsin, the activity was almost completely suppressed by incubation with subtilisin. Purified from CL by three successive steps of chromatography (gel filtration, anion exchange, reverse phase), active antibacterial protein appeared as a single polypeptide chain of approximate molecular weight of 60 kDa.