Presence and activity of endo-ß-1,4-mannase, an important digestive carbohydrase within the digestive fluid of terrestrial crustaceans.

Within the midgut gland of the Christmas Island red crab, Gecarcoidea natalis, a single transcript for a GH5_10 endo-ß-1,4-mannase had the highest expression out of all of the carbohydrase enzymes (Gan et al. in Mar Biotechnol 20:654-665, 2018). The activity, and potential digestive importance of this hemicellulase, compared with other carbohydrases, has yet to be established. The digestive fluid of G. natalis contained substantial endo-ß-1,4-mannase activities (630 ± 55 (6) nmol reducing sugars. min-1. mg-1 protein). It was present as a single isozyme of 66.3 ± 0.7 kDa (n = 6). Endo-ß-1,4-mannase activities were higher than that for lichenase and endo-ß-1,4-glucanase but lower than that for ß-1,3-glucanase and amylase. The digestive fluid was able to hydrolyse, galactomannan, into its component monosaccharides. Hence, this confirms expression data that this enzyme is one of the most important digestive cellulases/ hemicellulases. Expression of GH5_10 endo-ß-1,4-mannase mRNA was consistent with that of a digestive enzyme, as it was expressed in the digestive midgut gland but not in muscle and gill. Endo-ß-1,4-mannase activities were also present within the digestive fluid of the terrestrial hermit crabs, Coenobita perlatus and Coenobita brevimanus. Endo-ß-1,4-mannase activities (1351 ± 136 (n=3) nmol reducing sugars. min-1 mg-1 protein for C. perlatus. 665 ± 32 n=(5) nmol reducing sugars. min-1 mg-1 protein for C. brevimanus) were higher than that for endo-ß-1,4-glucanase and amylase but were lower than ß-1,3-glucanase activities. Animals within the terrestrial hermit crab family, Coenobitidae consume legume and palm seeds which contain substantial amounts of mannan. Hence, high endo-ß-1,4-mannase activities suggest that digestion of mannan within these species may represent an important source of carbohydrate.

Review: The structure and function of cellulase (endo-ß-1,4-glucanase) and hemicellulase (ß-1,3-glucanase and endo-ß-1,4-mannase) enzymes in invertebrates that consume materials ranging from microbes, algae to leaf litter.

This review discusses the reaction catalysed, and the structure and function of the cellulase, endo-ß-1,4-glucanase and the hemicellulase enzymes, ß-1,3-glucanase and endo-ß-1,4-mannase that are present in numerous invertebrate groups with a diverse range of feeding specialisations. These range from microbial deposit and filter feeders, micro and macrophagous algal feeders, omnivores to herbivorous leaf litter and wood feeders. Endo-ß-1,4-glucanase from glycosyl hydrolase family 9 (GH9) digests cellulose like ß-1,4-glucans from a range of materials. As it hydrolyses crystalline cellulose very slowly, it is a poor cellulase. Where tested, the enzyme has dual endo-ß-1,4-glucanase and lichenase activity. Its presence does not necessarily indicate the ability of an animal to digest cellulose. It only indicates the ability to digest ß-1,4-glucans and its function, which is discussed in this review, should be considered with reference to the substrates present in the diet. ß-1,3-glucanase (laminarinase) belongs to glycosyl hydrolase family 16 (GH16) and hydrolyses ß-1.3-glucans. These polysaccharides are present in the cell walls of algae, protozoans and yeast, and they also occur as storage polysaccharides within protozoans and algae. Depending on their site of expression, these enzymes may function as a digestive enzyme or may be involved in innate immunity. Enzymes present in the digestive fluids or tissues, would be digestive. Haemolymph GH16 proteins may be involved in innate immunity through the activation of the phenol oxidase system. Insect GH16 proteins expressed within the haemolymph have lost their catalytic residues and function as ß-glucan binding proteins. In contrast, crustacean GH16 proteins expressed within the same tissue, have retained the catalytic residues and thus possibly their ß-1,3-glucanase activity. The potential function of which is discussed. Endo-ß-1,4-mannase from glycosyl hydrolase family 5, subfamily 10 (GH5_10) hydrolyses mannan, glucomannan and galactomannan. These hemicelluloses are present in the cell walls of plants and algae and also function as storage polysaccharides within legume and palm seeds. They are digestive enzymes whose high expression in some species suggests they are a major contributor to hemicellulose digestion. They may also provide the animal with substantial amounts of monosaccharides for energy.

Two reads to rule them all: Nanopore long read-guided assembly of the iconic Christmas Island red crab, Gecarcoidea natalis (Pocock, 1888), mitochondrial genome and the challenges of AT-rich mitogenomes.

Despite recent advances in sequencing technology, a complete mitogenome assembly is still unavailable for the gecarcinid land crabs that include the iconic Christmas Island red crab (Gecarcoidea natalis) which is known for its high population density, annual mass breeding migration and ecological significance in maintaining rainforest structure. Using sequences generated from Nanopore and Illumina platforms, we assembled the complete mitogenome for G. natalis, the first for the genus and only second for the family Gecarcinidae. Nine Nanopore long reads representing 0.15% of the sequencing output from an overnight MinION Nanopore run were aligned to the mitogenome. Two of them were >10 kb and combined are sufficient to span the entire G. natalis mitogenome. The use of Illumina genome skimming data only resulted in a fragmented assembly that can be attributed to low to zero sequencing coverage in multiple high AT-regions including the mitochondrial protein-coding genes (NAD4 and NAD5), 16S ribosomal rRNA and non-coding control region. Supplementing the mitogenome assembly with previously acquired transcriptome dataset containing high abundance of mitochondrial transcripts improved mitogenome sequence coverage and assembly reliability. We then inferred the phylogeny of the Eubrachyura using Maximum Likelihood and Bayesian approaches, confirming the phylogenetic placement of G. natalis within the family Gecarcinidae based on whole mitogenome alignment. Given the substantial impact of AT-content on mitogenome assembly and the value of complete mitogenomes in phylogenetic and comparative studies, we recommend that future mitogenome sequencing projects consider generating a modest amount of Nanopore long reads to facilitate the closing of problematic and fragmented mitogenome assemblies.

cDNA sequences of GHF9 endo-ß-1,4-glucanases in terrestrial Crustacea.

This study aimed to sequence and identify a glycosyl hydrolase family 9 (GHF9) endo-ß-1,4-glucanase expressed in the midgut gland of the herbivorous gecarcinid land crab, Gecarcoidea natalis. Hence this would explain the gene responsible for the production of previously purified and characterised endo-ß-1,4-glucanases. Three different transcripts, two complete and one partial were sequenced from cDNA and an open reading frame of 1383bp was produced. Translated, this would produce a putative protein of 460 amino acid residues, including a 16 amino acid residue signal peptide. The mature protein (without signal peptide) is predicted to have a molecular mass of 47.6-47.7kDa; this closely matches the molecular mass (47.4kDa) of one of the three endo-ß-1,4-glucanase/lichenase enzymes purified previously from G. natalis. It is therefore proposed that the gene described here encodes one of the previously characterised enzymes. The presence of multiple transcripts suggests gene duplication. To confirm that the gene is widely expressed within the Crustacea, cDNA encoding a GHF9 endo-ß-1,4-glucanase was also sequenced in diverse crustaceans, the deposit feeding soldier crab, Mictyris platycheles and the terrestrial hermit crabs, Coenobita purlatus and C. brevimanus. An open reading frame of 1356bp was sequence from M. platycheles, while an incomplete open reading frames of 1384 and 1523bp were respectively sequenced from Coenobita brevimanus and C. perlatus. The midgut gland of M. platycheles contained activity (0.704±0.218µmol reducing sugars produced. min-1·mg-1 tissue wet weight) of a 26.3±0.3(5) endo-ß-1,4-glucanase isozyme (determined from activity staining). These species, particularly M. platycheles does not consume and digest significant amounts of plant cellulose. This implies that the ancestral enzyme is not a cellulase, but rather it may be involved in hydrolysing cellulose like polysaccharides within other organisms such as algae.

A glycosyl hydrolase family 16 gene is responsible for the endogenous production of ß-1,3-glucanases within decapod crustaceans.

To identify the gene responsible for the production of a ß-1,3-glucanase (laminarinase) within crustacea, a glycosyl hydrolase family 16 (GHF16) gene was sequenced from the midgut glands of the gecarcinid land crab, Gecarcoidea natalis and the freshwater crayfish, Cherax destructor. An open reading frame of 1098 bp for G. natalis and 1095 bp for C. destructor was sequenced from cDNA. For G. natalis and C. destructor respectively, this encoded putative proteins of 365 and 364 amino acids with molecular masses of 41.4 and 41.5 kDa. mRNA for an identical GHF16 protein was also expressed in the haemolymph of C. destructor. These putative proteins contained binding and catalytic domains that are characteristic of a ß-1,3-glucanase from glycosyl hydrolase family 16. The amino acid sequences of two short 8-9 amino acid residue peptides from a previously purified ß-1,3-glucanase from G. natalis matched exactly that of the putative protein sequence. This plus the molecular masses of the putative proteins matching that of the purified proteins strongly suggests that the sequences obtained encode for a catalytically active ß-1,3-glucanase. A glycosyl hydrolase family 16 cDNA was also partially sequenced from the midgut glands of other amphibious (Mictyris platycheles and Paragrapsus laevis) and terrestrial decapod species (Coenobita rugosus, Coenobita perlatus, Coenobita brevimanus and Birgus latro) to confirm that the gene is widely expressed within this group. There are three possible hypothesised functions and thus evolutionary routes for the ß-1,3-glucanase: 1) a digestive enzyme which hydrolyses ß-1,3-glucans, 2) an enzyme which cleaves ß-1,3-glycosidic bonds within cell walls to release cell contents or 3) an immune protein which can hydrolyse the cell walls of potentially pathogenic micro-organisms.

Digestive enzymes of two brachyuran and two anomuran land crabs from Christmas Island, Indian Ocean.

The digestive ability of four sympatric land crabs species (the gecarcinids, Gecarcoidea natalis and Discoplax celeste and the anomurans, Birgus latro and Coenobita perlatus) was examined by determining the activity of their digestive enzymes. The gecarcinids are detritivores that consume mainly leaf litter; the robber crab, B. latro, is an omnivore that preferentially consumes items high in lipid, carbohydrate and/or protein; C. perlatus is also an omnivore/detritivore. All species possess protease, lipase and amylase activity for hydrolysing ubiquitous protein, lipid and storage polysaccharides (glycogen and starch). Similarly all species possess enzymes such as N-acetyl-ß-D-glucosaminidase, the cellulases, endo-ß-1,4-glucanase and ß-glucohydrolase and hemicellulases, lichenase and laminarinase for the respective hydrolysis of structural substrates chitin, cellulose and hemicelluloses, lichenan and laminarin. Except for the enzyme activities of C. perlatus, enzyme activity could not be correlated to dietary preference. Perhaps others factors such as olfactory and locomotor ability and metabolic status may determine the observed dietary preferences. The digestive fluid of C. perlatus possessed higher endo-ß-1,4-glucanase, lichenase and laminarinase activities compared to that of the other species. Thus, C. perlatus may be efficient at digestion of cellulose and hemicellulose within plant material. Zymography indicated that the majority of protease, lipase, phosphatase, amylase, endo-ß-1,4-glucanase, ß-glucohydrolase and N-acetyl-ß-D-glucosaminidase isozymes were common to all species, and hence were inherited from a common aquatic ancestor. Differences were observed for the phosphatase, lipase and endo-ß-1,4-glucanase isozymes. These differences are discussed in relation to phylogeny and possible evolution to cope with the adoption of a terrestrial diet.

Chemiluminescence evidence supporting the selective role of ligands in the permanganate oxidation of micropollutants.

The selective increase in the oxidation rate of certain organic compounds with permanganate in the presence of environmental "ligands" and reduced species has been ascribed to the different reactivity of the target compounds toward Mn(III), which bears striking similarities to recent independent investigations into the use of permanganate as a chemiluminescence reagent. In spite of the importance of Mn(III) in the light-producing pathway, the dependence of the oxidation mechanism for any given compound on this intermediate could not be determined solely through the emission intensity. However, target compounds susceptible to single-electron oxidation by Mn(III) (such as bisphenol A and triclosan) can be easily distinguished by the dramatic increase in chemiluminescence intensity when a permanganate reagent containing high, stable concentrations of Mn(III) is used. The differences are accentuated under the low pH conditions that favor the chemiluminescence emission due to the greater reactivity of Mn(III) and the greater influence of complexing agents. This study supports the previously postulated selective role of ligands and reducing agents in permanganate oxidations and demonstrates a new approach to explore the chemistry of environmental manganese redox processes.

Isozymes from the herbivorous gecarcinid land crab, Gecarcoidea natalis that possess both lichenase and endo-ß-1,4-glucanase activity.

Three isozymes with both lichenase and endo-ß-1,4-glucanase activity were purified and characterised from the midgut gland of the herbivorous gecarcinid land crab, Gecarcoidea natalis. The three isozymes, termed 1a, 1b and 2, had respective molecular masses of 53 ± 0 (3), 43 ± 0 (3) and 47.4 ± 0(3) kDa. All isozymes possessed similar V(max) values and thus hydrolysed both carboxy methyl cellulose and lichenan equally. Furthermore the chromatography profiles for lichenase activities mirrored that for endo-ß-1,4-glucanase activities suggesting that the same enzyme possessed both activities. Given this, the endo-ß-1,4-glucanase enzymes described for other animals, may, like the isozymes described in this study, may be able to hydrolyse lichenan. However this ability needs to be confirmed. The main digestive function of these isozymes may be to hydrolyse hemicelluloses such as lichenan and mixed beta-D-glucan. All three isozymes randomly hydrolysed internal glycosidic bonds within carboxy methyl cellulose and lichenan to release short oligomers of 4-5 glucose units in length. They also hydrolysed cellotetraose to either two units of cellobiose or cellotriose and glucose. Cellotriose was hydrolysed to cellobiose and glucose. All three enzymes lacked ß-1,4-glucosidase activity as they could not hydrolyse cellobiose.

Accumulation and excretion of morphine by Calliphora stygia, an Australian blow fly species of forensic importance.

This study examined the ability of the forensically important blow fly, Calliphora stygia to actively excrete morphine, thereby maintaining a low morphine level within its body when fed on a diet containing morphine at low (7pmolg(-1)) and high (17.5pmolg(-1)) concentrations. Morphine was accumulated within the bodies of maggots (≈70% within the tissues) at concentrations which were lower than that of the meat (3-24%). The morphine content of the initial developing stages (second and third instar maggots) maintained on the high morphine diet was higher than those on the low morphine diet. Morphine was cleared from the body with negatively exponential kinetics (High morphine group: Morphine (pmolg(-1) wet weight)=8425e(-0.014t). Low morphine group: Morphine (pmolg(-1) wet weight)=2180e(-0.010t)). Clearance constants for morphine by animals in both groups were similar and thus both groups had a similar ability to excrete morphine. The Malpighian tubules of maggots were able to actively secrete morphine using a transport mechanism that transports small type II organic cations, such as morphine and quinine. The rate of morphine secretion by the Malpighian tubules could explain the clearance of the drug by the maggots. As the morphine was transported across the Malpighian tubules cells, a significant proportion was metabolised into a compound that is yet to be fully characterised.

The last piece in the cellulase puzzle: the characterisation of beta-glucosidase from the herbivorous gecarcinid land crab Gecarcoidea natalis.

A 160 kDa enzyme with beta-glucosidase activity was purified from the midgut gland of the land crab Gecarcoidea natalis. The enzyme was capable of releasing glucose progressively from cellobiose, cellotriose or cellotetraose. Although beta-glucosidases (EC 3.2.1.21) have some activity towards substrates longer than cellobiose, the enzyme was classified as a glucohydrolase (EC 3.2.1.74) as it had a preference for larger substrates (cellobiose

Functional morphology of the gastric mills of carnivorous, omnivorous, and herbivorous land crabs.

Terrestrial decapods consume a wide variety of plant and animal material. The potential adaptations of carnivorous, omnivorous, and herbivorous terrestrial crustaceans were studied by examining the functional morphology of the gastric mill. Two closely related species from each feeding preference group were examined to identify which features of the mill were due to phylogeny and which were due to adaptation. The morphology of the gastric mill matched the diet well; the gastric mills of the carnivorous species (Geograpsus grayi and Geograpsus crinipes) possessed a blunt, rounded medial tooth and flattened lateral teeth with a longitudinal grinding groove. These features make them well suited to a carnivorous diet of soft animal tissue as well as hard material, such as arthropod exoskeleton. In contrast, the mill of the herbivorous gecarcinids (Gecarcoidea natalis and Discoplax hirtipes) consisted of a medial tooth with sharp transverse ridges and lateral teeth with sharp interlocking cusps and ridges and no grinding surface. These features would efficiently shred fibrous plant material. The morphology of the mill of the omnivorous coenobitids (Coenobita perlatus and Birgus latro) was more generalized toward a mixed diet. However, the mill of B. latro was more adapted to deal with highly nutritious food items, such as nuts and heavily calcified decapods. Its mill possessed lateral teeth with extended ridges, which sat close to the calcified cardiopyloric valve to form a flattened floor. Hard items trapped in the mill would be crushed against this surface by the medial tooth.

Purification and characterisation of endo-beta-1,4-glucanase and laminarinase enzymes from the gecarcinid land crab Gecarcoidea natalis and the aquatic crayfish Cherax destructor.

Laminarinase and endo-beta-1,4-glucanase were purified and characterised from the midgut gland of the herbivorous land crab Gecarcoidea natalis and the crayfish Cherax destructor. The laminarinase isolated from G. natalis was estimated to have a molecular mass of 41 kDa by SDS-PAGE and 71 kDa by gel filtration chromatography. A similar discrepancy was noted for C. destructor. Possible reasons for this are discussed. Laminarinase (EC 3.2.1.6) from G. natalis had a V(max) of 42.0 micromol reducing sugars produced min(-1) mg protein(-1), a K(m) of 0.126% (w/v) and an optimum pH range of 5.5-7, and hydrolysed mainly beta-1,3-glycosidic bonds. In addition to the hydrolysis of beta-1,3-glycosidic bonds, laminarinase (EC 3.2.1.39) from C. destructor was capable of significant hydrolysis of beta-1,4-glycosidic bonds. It had a V(max) of 19.6 mumol reducing sugars produced min(-1) mg protein(-1), a K(m) of 0.059% (w/v) and an optimum pH of 5.5. Laminarinase from both species produced glucose and other short oligomers from the hydrolysis of laminarin. Endo-beta-1,4-glucanase (EC 3.2.1.4) from G. natalis had a molecular mass of 52 kDa and an optimum pH of 4-7. It mainly hydrolysed beta-1,4-glycosidic bonds, but was also capable of significant hydrolysis of beta-1,3-glycosidic bonds. Two endo-beta-1,4-glucanases, termed 1 and 2, with respective molecular masses of 53+/-3 and 52 kDa, were purified from C. destructor. Endo-beta-1,4-glucanase 1 was only capable of hydrolysing beta-1,4-glycosidic bonds and had an optimum pH of 5.5. Endo-beta-1,4-glucanases from both species produced some glucose, cellobiose and other short oligomers from the hydrolysis of carboxymethyl cellulose.

A review of feeding and nutrition of herbivorous land crabs: adaptations to low quality plant diets.

This paper reviews the nutritional ecology, the digestive physiology, and biochemistry of herbivorous land crabs and the adaptations that they possess towards a diet of plant material. Land crab species that breathe air and forage out of water can be divided into three feeding specialisations: primarily carnivorous, deposit feeders feeding on micro-organisms and organic matter in the sediment, and herbivores consuming mainly plant material and its detritus. The last forms the focus of this review. The diets of the herbivores are low in nitrogen and high in carbon, are difficult to digest since they contain cellulose and hemicellulose, and may disrupt digestion due to the presence of tannins. Herbivorous crustaceans are able to efficiently utilise plant material as their primary nutrient source and are indeed able to meet their nitrogen requirements from it. Herbivorous land crabs display a range of adaptations towards a low nitrogen intake and these are discussed in this review. They also appear to endogenously produce cellulase and hemicellulase enzymes for the digestion of cellulose and hemicellulose. Generalised and specific adaptations allow them to inhibit the potentially negative digestive effects of tannins. To digest plant material, they possess a plastic digestive strategy of high food intake, short retention time, high assimilation of cell contents, and substantial digestion of cellulose and hemicellulose.

Endogenous production of endo-beta-1,4-glucanase by decapod crustaceans.

The potential ability to produce cellulase enzymes endogenously was examined in decapods crustaceans including the herbivorous gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes, the amphibious freshwater crab Austrothelphusa transversa, the terrestrial hermit crab, Coenobita variabilis the parastacid crayfish Euastacus, and the crayfish Cherax destructor. The midgut gland of both G. natalis and D. hirtipes contained substantial total cellulase activities and activities of the cellulase enzymes endo-beta-1,4-glucanase and beta-glucosidase. With the exception of total cellulase and beta-glucosidase from D. hirtipes, the enzyme activities within the midgut gland were higher than those within the digestive juice. Hence, the enzyme activities appear to reside predominantly within midgut gland, providing indirect evidence for endogenous synthesis of cellulase enzymes by this tissue. A 900 bp cDNA fragment encoding a portion of the endo-beta-1,4-glucanase amino acid sequence was amplified by RT-PCR using RNA isolated from the midgut gland of C. destructor, Euastacus, A. transversa and C. variabilis. This provided direct evidence for the endogenous production of endo-beta-1,4-glucanase. The 900 bp fragment was also amplified from genomic DNA isolated from the skeletal muscle of G. natalis and D. hirtipes, clearly indicating that the gene encoding endo-beta-1,4-glucanase is also present in these two species. As this group of evolutionary diverse crustacean species possesses and expresses the endo-beta-1,4-glucanase gene it is likely that decapod crustaceans generally produce cellulases endogenously and are able to digest cellulose.

Presence and properties of cellulase and hemicellulase enzymes of the gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes.

Digestive juice from the herbivorous gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes exhibited total cellulase activity and activities of two cellulase enzymes; endo-beta-1,4-glucanase and beta-1,4-glucosidase. These enzymes hydrolysed native cellulose to glucose. The digestive juice of both species also contained laminarinase, licheninase and xylanase, which hydrolysed laminarin, lichenin and xylan, respectively, to component sugars. The pH optima of beta-1,4-glucosidase, endo-beta-1,4-glucanase and total cellulase from G. natalis were 4-5.5, 5.5 and 5.5-7, respectively. In the digestive juice from D. hirtipes, the corresponding values were 4-7, 5.5-7 and 4-9, respectively. The pH of the digestive juice was 6.69+/-0.03 for G. natalis and 6.03+/-0.04 for D. hirtipes and it is likely that the cellulases operate near maximally in vivo. In G. natalis, total cellulase activity and endo-beta-1,4-glucanase activity were higher than in D. hirtipes, and the former species can thus hydrolyse cellulose more rapidly. beta-1,4-glucosidase from G. natalis was inhibited less by glucono-d-lactone (K(i)=11.12 mmol l(-1)) than was the beta-1,4-glucosidase from D. hirtipes (K(i)=4.53 mmol l(-1)). The greater resistance to inhibition by the beta-1,4-glucosidase from G. natalis may contribute to the efficiency of the cellulase system in vivo by counteracting the effects of product inhibition and possibly dietary tannins. The activity of beta-1,4-glucosidase in the digestive juice of D. hirtipes was higher than that of G. natalis.

Dietary assimilation and the digestive strategy of the omnivorous anomuran land crab Birgus latro (Coenobitidae).

On Christmas Island, Indian Ocean, the diet of robber crabs, Birgus latro (Linnaeus) was generally high in fat, storage polysaccharides or protein and largely comprised fruits, seeds, nuts and animal material. The plant items also contained significant amounts of hemicellulose and cellulose. In laboratory feeding trials, crabs had similar intakes of dry matter when fed artificial diets high in either fat or storage polysaccharide, but intake was lower on a high protein diet. Assimilation coefficients of dry matter (69-74%), carbon (72-81%), nitrogen (76-100%), lipid (71-96%) and storage polysaccharide (89-99%) were high on all three diets. B. latro also assimilated significant amounts of the chitin ingested in the high protein diet ( 93%) and hemicellulose (49.6-65%) and cellulose (16-53%) from the high carbohydrate and high fat diets. This is consistent with the presence of chitinase, hemicellulase and cellulase enzymes in the digestive tract of B. latro. The mean retention time (27.2 h) for a dietary particle marker ((57)Co-labelled microspheres) was longer than measured in leaf-eating land crabs. The feeding strategy of B. latro involves the selection of highly digestible and nutrient-rich plant and animal material and retention of the digesta for a period long enough to allow extensive exploitation of storage carbohydrates, lipids, protein and significant amounts of structural carbohydrates (hemicellulose, cellulose and chitin).

Detection of HOCl-mediated protein oxidation products in the extracellular matrix of human atherosclerotic plaques.

Oxidation is believed to play a role in atherosclerosis. Oxidized lipids, sterols and proteins have been detected in early, intermediate and advanced human lesions at elevated levels. The spectrum of oxidized side-chain products detected on proteins from homogenates of advanced human lesions has been interpreted in terms of the occurrence of two oxidative mechanisms, one involving oxygen-derived radicals catalysed by trace transition metal ions, and a second involving chlorinating species (HOCl or Cl2), generated by the haem enzyme myeloperoxidase (MPO). As MPO is released extracellularly by activated monocytes (and possibly macrophages) and is a highly basic protein, it would be expected to associate with polyanions such as the glycosaminoglycans of the extracellular matrix, and might result in damage being localized at such sites. In this study proteins extracted from extracellular matrix material obtained from advanced human atherosclerotic lesions are shown to contain elevated levels of oxidized amino acids [3,4-dihydroxyphenylalanine (DOPA), di-tyrosine, 2-hydroxyphenylalanine ( o-Tyr)] when compared with healthy (human and pig) arterial tissue. These matrix-derived materials account for 83-96% of the total oxidized protein side-chain products detected in these plaques. Oxidation of matrix components extracted from healthy artery tissue, and model proteins, with reagent HOCl is shown to give rise to a similar pattern of products to those detected in advanced human lesions. The detection of elevated levels of DOPA and o-Tyr, which have been previously attributed to the occurrence of oxygen-radical-mediated reactions, by HOCl treatment, suggests an alternative route to the formation of these materials in plaques. This is believed to involve the formation and subsequent decomposition of protein chloramines.

Inorganic and organic anion transport by insect renal epithelia.

Insect renal organs typically exhibit high rates of transport of inorganic and organic anions, and therefore provide useful models for the study of epithelial anion transport and its control. Isolated Malpighian tubules of some species secrete a volume of iso-osmotic fluid equal to their own volume in 10-15 s, which means that cellular Cl(-) content is exchanged every 3-5 s. Anion transport can also be achieved against extreme thermodynamic gradients. The concentration of K(+) and Cl(-) in the lumen of the Malpighian tubules of some desert beetles approaches or exceeds saturation. A basolateral Na(+):K(+):2Cl(-) cotransporter plays an important role in vectorial ion transport in Malpighian tubules of many species, but there is also evidence for coupling of Cl(-) transport to the movement of a single cationic species (Na(+) or K(+)). Although an apical vacuolar H(+)-ATPase plays a primary role in energizing transepithelial secretion of chloride via channels or cotransporters in the secretory segment of the Malpighian tubule, several different ATPases have been implicated in reabsorption of Cl(-) by the lower Malpighian tubule or hindgut. Chloride transport is known to be controlled by several neuropeptides, amines and intracellular second messengers. Insect renal epithelia are also important in excretion of potentially toxic organic anions, and the transporters involved may play a role in resistance to insecticides of natural or anthropogenic origin.

Rat T cells express neither CD55 nor CD59 and are dependent on Crry for protection from homologous complement.

All human blood cells express decay-accelerating factor (DAF, CD55), CD59, and, with the exception of erythrocytes, membrane cofactor protein (MCP, CD46) to protect themselves from damage by the constant low-level activation of complement in serum. In rats and mice MCP is expressed only in testis, whereas DAF and CD59 are broadly distributed. Rats and mice also express a unique complement regulator, Crry. Previously we have shown that DAF was absent from at least 75% of rat T cells. To further investigate this surprising finding, we assessed the expression levels of DAF, CD59 and Crry on all blood cell types in the rat. We found that Crry was abundantly expressed on all blood cells. CD59 was expressed abundantly on erythrocytes and granulocytes but was absent from all T cellsand platelets and a minority of B cells and NK cells. Double staining and depletion studies showed that T cells in all rat strains tested were DAF-CD59-. Neutralization of Crry using a blocking monoclonal antibody rendered T cells susceptible to lysis by homologous complement, indicating that Crry was solely responsible for protecting DAF-CD59- T cells from complement damage in the rat.

Intracellular purine deposits in the gecarcinid land crab Gecarcoidea natalis.

The terrestrial crab Gecarcoidea natalis stores large amounts of purine in the body. The major component of the purine deposits is urate (85% of the total purines). The other 15% is comprised of hypoxanthine, guanine, and xanthine. Microscopy studies reveal that these urate deposits are located intracellularly in spongy connective tissue cells throughout the body. Urate exists as numerous membrane-bound crystals 1 µm in diameter. Vesicles thought to represent urate vesicles at various stages of development are also present in the cytoplasm of the cell. Few organelles are visible in the urate storage cells, and it is unlikely that the urate is synthesized on site. Crabs (N = 2) fed a high-nitrogen diet have greater numbers of urate storage cells at more connective tissue sites, and the cells are larger (36.3 ± 1.8 µm (mean ± SE) and 44.0 ± 1.4 µm (mean ± SE)) and contain more urate than urate storage cells in animals collected from the field (N = 3) or maintained in the laboratory on a low-nitrogen diet (N = 1). The mean diameter of urate storage cells in animals fed a diet low in nitrogen and field-collected animals ranges from (13.5 ± 0.5 µm (SE) - 22.3 ± 1.0 µm (SE)). This histological study supports a strong correlation between purine accumulation and the nitrogen content of the diet. J. Morphol. 231:101-110, 1997. © 1997 Wiley-Liss, Inc.