Binding and detoxification of heavy metals in lower vertebrates with reference to metallothionein

• 1.1. The metabolism of Cu, Zn, Cd and Hg in lower vertebrates is described, using fish as a model.
• 2.2. The main part of this review deals with metallothionein and the role of this protein for the storage and detoxification of these metals.
• 3.3. Factors influencing the bioavailability and probable uptake routes are identified.
• 4.4. The distribution of the metals within the organism is outlined. The distribution between tissues is described and the subcellular distribution discussed with reference to metallothionein.

     

Evidence of presence of a low molecular weight,non-metallothionein-like metal-binding protein in the marine gastropod Nassarius reticulatus L.

• 1.1. A low molecular weight metal-binding protein was found in the snail Nassarius reticulatus cytosol, which was induced in heavy metal contaminated environments.
• 2.2. In our sodium dodecyl sulfate-mercaptoethanol polyacrylamide gel systems it behaved as a protein of 19 kDa mol. wt.
• 3.3. Amino acid composition studies definitely established this protein not to be metallothionein (Mt) like, because it had a much lower level of cysteine and substantial amounts of aromatic amino acids and histidine.
• 4.4. The metal-binding strength of this protein was concluded to be much weaker than that of Mt.
• 5.5. In the crustacean Pagurus bernhardus L. such a protein could not be demonstrated.
• 6.6. In both the snail and the crustacean Zn may inhibit the accumulation of Hg. The premise for studying the induction of the metal-binding Nassarius protein as a supplement to environmental metal monitoring purposes is briefly discussed.

     

Adaptation to cadmium in three populations of the isopod Porcellio scaber

• 1.1. First-generation laboratory animals of three populations of the isopod Porcellio scaber, collected from a reference wood, a zinc smelter area and a lead mine site, were compared with respect to effects of Cd.
• 2.2. All populations reacted differently to Cd-contaminated food: increased Cd concentrations in the food resulted in decreased consumption and growth for the reference isopods; mine isopods were not affected by Cd and for the smelter animals Cd stimulated growth at an intermediate concentration.
• 3.3. Since Cd concentrations in the isopod did not differ between populations, adaptation is probably based on an increased detoxification capacity.
• 4.4. The assimilation of Cd did not affect the Cu or Zn content of the isopods although the adapted isopods regulated their Cu content on a lower level than reference isopods.

     

Is the metal binding protein metallothionein present in the coelenterate Hydra attenuata?

• 1.1. Studies have been performed on untreated and heavy metal treated Hydra attenuata in order to reveal the presence of low mol. wt metal-binding proteins.
• 2.2. A prepared rat metallothionein (Mt) standard, gel permeation, polyacrylamide gel electrophoresis and autoradiographic techniques were used in the experiments.
• 3.3. Our results indicate that H. attenuata, and three species of marine coelenterates, lack metallothionein (Mt) or other metal binding proteins.

     

Digestive proteases in the midgut gland of the atlantic blue crab,Callinectes sapidus

• 1.1. Digestive proteases from the midgut gland of male Atlantic blue crabs, Callinectes sapidus, were investigated. Tentative identities of proteolytic enzymes were determined with synthetic substrates and inhibitors.
• 2.2. Trypsin, chymotrypsin, carboxypeptidase A and B and leucine aminopeptidase activities were found and quantified.
• 3.3. Activity against Succinyl-(Ala)₃-nitroanalide was also found. This as yet unidentified enzyme has a mol. wt of about 26,000 and has elastolytic activity.

     

Crystalline style proteins from bivalve molluscs

• 1.1. Proteins from crystalline styles of twelve species of bivalve mollusc were examined under different gel electrophoresis conditions and stained to reveal both protein and carbohydrate.
• 2.2. Native extracts of styles produced relatively few protein bands, however denaturation with SDS resulted in much more complex zymograms.
• 3.3. All species possessed several prominent high mol wt glycoproteins.
• 4.4. Eulamellibranchia all had a major non-glycosylated protein at approx. 62,000 mol. wt.
• 5.5. Most Filibranchia had a major non-glycosylated protein at 37,000–50,000 mol. wt.
• 6.6. Eulamellibranchia were a much more homogeneous group than the Filibranchia.

     

Effects of aluminium and pH on calcium fluxes,and effects of cadmium and manganese on calcium and sodium fluxes in brown trout (Salmo trutta L.)

• 1.1. Simultaneous measurement of calcium fluxes in brown trout, at low external [Ca] (20 μ mol 1⁻¹), provided evidence of active uptake of Ca from the medium.
• 2.2. At pH 4.5, calcium influx was inhibited and efflux was stimulated.
• 3.3. Cd and Mn, but not Al, at concentrations within the ranges found in acid waters experiencing fish population decline, inhibited calcium influx. Efflux was unaffected.
• 4.4. Cd and Mn stimulated sodium influx and efflux.

     

Variations in response to environmental hypoxia of different colour forms of the shore crab,Carcinus maenas

• 1.1. The oxygen consumption of red and green Carcinus in normoxic and hypoxic sea water was determined, using an oxygen electrode in a sealed respirometer.
• 2.2. The red crabs had significantly higher “excited” oxygen uptake rates and a lower ability to compensate for hypoxia than the green crabs.
• 3.3. Red Carcinus display an emersion response to declining oxygen at lower oxygen tensions than the green crabs.
• 4.4. Mortality of red crabs exposed to prolonged anoxia was much greater.
• 5.5. The relationship of these findings to the zonation of the two colour forms on the shore is discussed.

     

The purification of kallikrein from human whole saliva

• 1.1. A quick and simple procedure is described for purifying kallikrein from human whole saliva. The enzyme has been purified about 2700-fold with a yield of approx. 30%.
• 2.2. The procedure is based on the immediate fractionation of saliva by ion exchange chromatography. This is followed by a combination of affinity and high performance liquid chromatography.
• 3.3. The results indicate that another protein component binds to the enzyme at pH 8.0.
• 4.4. The homogeneity of the enzyme has been demonstrated by gel electrophoresis in the absence as well as in the presence of sodium dodecylsulfate.
• 5.5. A mol. wt of 40,100±1800 has been calculated from gel electrophores is experiments.
• 6.6. Sedimentation equilibrium in an analytical ultracentrifuge gave a mol. wt of 39,700.
• 7.7. The amino acid composition has been determined and it confirms that the enzyme has a low isoelectric point.
• 8.8. The presence of tryptophan has been demonstrated by absorption and fluorescence spectroscopy.

     

Some physical and chemical properties of purified nematocysts of Hydra attenuata pall. (Hydrozoa,cnidaria)

• 1.1. A method for purifying undischarged nematocysts from Hydra and other cnidarians is described.
• 2.2. Isolated cysts (relative densities 1.22–1.24) evaginate their tubular content even after previous dehydration.
• 3.3. The cyst wall is permeable to dyes of mol. wts up to 600,000.
• 4.4. Approximately two-thirds of the cyst's dry wt are soluble proteins. Eighty per cent of them are of low mol. wt and highly anionic, presumably serving as binding sites for Ca²⁺ and Mg²⁺.
• 5.5. The other 20% includes 30 different proteins amongst them toxins and enzymes (phospholipase and little proteases but no collagenase, chitinase or hyaluronidase).

     

The influences of individual variations in metabolic rate and tidal conditions on the response to hypoxia in Carcinus maenas (L.)

• 1.1. The oxygen consumption of crabs in normoxic and hypoxic (50% O₂) seawater was measured directly after collection.
• 2.2. The influences of size and lunar cycles were removed by scaling the data.
• 3.3. Strong negative correlations between low individual levels of O₂ consumption and the ability to compensate for hypoxia were apparent in Wicklow (subtidal) crabs.
• 4.4. Compensation for hypoxia was much greater on the flood tide than on the ebb.
• 5.5. Crabs from Roscoff (intertidal) had lower levels of compensation than those from Wicklow.
• 6.6. Size, sex and condition had no apparent effect upon these relationships.
• 7.7. Crabs acclimated to laboratory conditions have not shown this tidal variation in compensation for hypoxia.

     

Induction of autoantibodies against spermatozoa by injection of allogeneic sperm in the Nile Tilapia,Oreochromis niloticus

• 1.1. The sperm-agglutinating factor (SAF) could be induced in the serum of male Nile tilapias, Oreochromis niloticus, by injection of allogeneic sperm.
• 2.2. Only one class of molecules was demonstrated to be SAF in the serum.
• 3.3. Analysis on purified SAF revealed it to be a tetrameric molecule of IgM with a mol. wt of 760kD.
• 4.4. Cross reaction of the IgM with sperm of other teleost species suggests that sperm-specific surface antigens may be in evolution.

     

A very stable and potent fibrinolytic enzyme found in earthworm Lumbricus rubellus autolysate

• 1.1. The autolysate of earthworms was found to exhibit powerful fibrin and thrombin substrate hydrolyzing activity.
• 2.2. It also showed a clot-forming activity in the fibrinogen- or plasma-added system.
• 3.3. Zymography revealed that there were three active components with mol. wts of 40,000, 21,000 and 15,000 in the autolysate.
• 4.4. The major form with a mol. wt 35,500 (by SDS-PAGE) was further purified. The N-terminal amino acid sequence of this enzyme (16 residues) was similar to that of the swine pancreatic proelastase.

     

Ca-binding glycoprotein from the spicules of the octocoral Leptogorgia virgulata

• 1.1. A Ca-binding component (CaBC) was isolated from the soluble matrix of spicules of the gorgonian Leptogargia virgulata.
• 2.2. The CaBC is a glycoprotein with apparent mol. wt between 70,000 and 100,000, and contains very high levels of aspartate.
• 3.3. The CaBC probably plays an important role in the formation of the coral spicules.

     

Control of mucin molecular forms expression by salivary protease: Differences with caries

• 1.1. A protease activity capable of degradation of the high mol. wt salivary mucus glycoprotein to a low mol. wt glycoprotein form was identified in human submandibular gland secretion.
• 2.2. The protease exhibited optimum activity at pH 7.0–7.4, and gave on SDS-PAGE under reducing conditions two major protein bands of 48 and 53 kDa. The enzyme showed susceptibility to PMSF, α₁antitrypsin, and egg white and soybean inhibitors, a characteristic typical to serine proteases.
• 3.3. The activity of the protease towards the high mol. wt mucus glycoprotein was found to be 3.8-fold higher in submandibular gland secretion of caries-resistant individuals than that of caries-susceptible. Furthermore, the enzyme from both groups displayed greater activity against the mucus glycoprotein of caries-resistant subjects.
• 4.4. Since the low mol. wt salivary mucus glycoprotein form is more efficient in bacterial clearance than the high mol. wt mucin, the enhanced expression of this indigenous salivary protease activity towards mucin may be the determining factor in the resistance to caries.

     

Aerial respiration in the semaphore crab,Heloecius cordiformis,with or without branchial water

• 1.1. Semaphore crabs (Heloecius cordiformis) are active in air at low tide. Their branchial chambers are lined with a vascular epithelium and are expanded above the gills (five pairs) to form air cavities which could function as lungs. Water is continuously circulated over the gills.
• 2.2. The relative contribution made by the gills and lungs to gas exchange in semaphore crabs active in air and circulating branchial water, was determined by measuring oxygen consumption (at 25°C) in crabs with and without branchial water, and in crabs with their lungs subsequently occluded.
• 3.3. Activity levels and VO₂ were unaffected by the absence of branchial water.
• 4.4. With their lungs occluded, VO₂ dropped (on average) by 61% in crabs with branchial water (i.e. gills still functional) and by 81% in crabs without branchial water (gill function impaired).
• 5.5. It is concluded that semaphore crabs are obligate air breathers while active on land, despite carrying water within their branchial chambers. Lung development and gill reduction in land crabs is discussed briefly in relation to “terrestriality”.

     

The effects of heavy metals and metabolic inhibitors on calcium uptake by gills and scales of Fundulus heteroclitus in vitro

• 1.1. Cadmium (Cd) and zinc (Zn) were inhibitory to calcium uptake by isolated gills of Fundulus heteroclitus in vitro. The metals appeared to act by displacing Ca²⁺ ions from protein carriers involved in facilitated diffusion.
• 2.2. In saltwater fish, transport of calcium across the serosal membrane of gill chloride cells is partly energy dependent and is likely mediated by Ca²⁺-ATPase. However, much of the calcium transport through the gill epithelium appears to occur by passive processes.
• 3.3. Cd (10⁻⁵M—10⁻³M) and Zn (10⁻⁷M—10⁻³ M) inhibited calcium uptake by isolated scale patches incubated in a physiological saline.
• 4.4. Cyanide, oubain, and quercetin treatment of scale patches produced results similar to those of the Cd and Zn treatments suggesting that metal-induced inhibition of ATPases may be responsible for reduced calcium transport by scale osteoblasts.

     

Differential expression of salivary mucin bacterial aggregating activity with caries status

• 1.1. The low and high mol. wt mucin forms were isolated from saliva of caries-resistant (CR) and caries-susceptible (CS) individuals, and assessed for their bacterial aggregating potential towards S. mutans and S. sanguis, the common cariogenic microorganisms encountered in the oral cavity.
• 2.2. The high mol. wt mucin from both groups of subjects exhibited similar protein and carbohydrate content, but the level ofcovalently bound fatty acids was significantly lower in the CR group. The mucin from CR group showed only a weak inhibitory potential, and no inhibitory activity was observed with the mucin of CS group.
• 3.3. The low mol. wt mucins from both groups, while displaying compositional similarities, showed a marked variation in the bacterial aggregating activity. With both bacteria, the activity of the mucin from CR group was at least 128-fold greater than that of CS group.
• 4.4. The conversion of the high mol. wt mucin to a low mol. wt form through the action of salivary protease produced in both groups enhancement in mucin's bacterial aggregating capacity. This enhancement was, however, considerably less pronounced in the case of mucin from CS group.
• 5.5. The results for the first time demonstrate that the bacterial aggregating epitope of salivary mucins is expressed to a greater extent in CR individuals, and that this epitope is apparently more accessible to bacteria in the low mol. wt mucin form.

     

Characterization of an acid phosphatase—Glycoprotein from barley seeds

• 1.1. An acid phosphatase from barley seeds germinated for 120 hr, that is not affected by the presence of cycloheximide, is a glycoprotein.
• 2.2. The enzyme has an optimum pH 4.9 and a mol. wt of 57,000 as determined by SDS-gel electrophesis.
• 3.3. The main sugar is galactose amounting to one half of the protein and it is as either mono or disaccharide.
• 4.4. Serine and glycine predominate and hydroxyproline is absent.