Early pattern of calcification in the dorsal carapace of the blue crab, Callinectes sapidus

The pattern of calcium carbonate deposition was observed in the dorsal carapace of premolt (D2-D3) and early postmolt (0-48 h) blue crabs, Callinectes sapidus, using scanning (SEM) and transmission (TEM) electron microscopy. Samples of dorsal carapace for SEM were quick-frozen in liquid nitrogen, subsequently lyophilized, and viewed using secondary and backscattered electrons as well as X-ray maps of calcium. Pieces of lyophilized cuticle were also embedded in epoxy resin and subsequently sectioned and viewed with TEM and SEM. Fresh pieces of dorsal carapace for TEM were also fixed in 2.5% glutaraldehyde in phosphate buffer followed by postfixation in 1% OsO4 in cacodylate buffer. Calcium concentrations were determined using atomic absorption spectrophotometry and quantitative X-ray microanalysis. Calcium accumulation began in the cuticle at 3 h postmolt at the epicuticle/exocuticle boundary and at the distal and proximal margins of the interprismatic septa (IPS). The bidirectional calcification of the IPS continued until the two fronts met at 5-8 h postmolt. The roughly hexagonal walls of the IPS formed a honeycomb-like structure that resulted in a rigid cuticle. The walls of the canal containing sensory neurons also calcified at 3 h, thereby imparting rigidity to the structure and additional strength to the cuticle. Examination of thin sections of lyophilized cuticle and fixed cuticle revealed that the first mineral deposited is more soluble than calcite and is probably amorphous calcium carbonate. The amorphous calcium carbonate is transformed to calcite along a front that follows the original deposition and is probably controlled by a specialized matrix within the IPS. Since amorphous calcium carbonate is isotropic, it would also make the mineral in the exocuticle stronger by an equal distribution of mechanical stress.     

Calcium transport from Malpighian tubules to puparial cuticle ofMusca autumnalis

Summary The transport of calcium from mineralized granules stored in the Malpighian tubules to the puparium of the face fly,Musca autumnalis De Geer, was studied. Calcium was transported directly from the tubules to the cuticle via the hemolymph. Little, if any, calcium entered the hindgut or other tissues during or prior to transport. A total of approximately 0.8 mg of calcium per larva was transported, beginning at the wandering stage; peak hemolymph concentrations occurred at anterior retraction. Hemolymph calcium levels subsequently decreased as puparial calcium increased. Puparial mineralization utilized most of the minerals stored during the larval stage, with lesser amounts of minerals being recovered in the adult or excreted. Deposition of mineral salts in the cuticle was accompanied by an increase in cuticular pH from 7.0 to 8.4. The house fly,Musca domestica L., which contains much lower concentrations of minerals in the puparial cuticle, exhibited no increase in cuticular pH during pupariation. Biomineralization of the face fly puparial cuticle appears to occur, in part, as a result of ionic equilibria involving calcium and magnesium phosphates and carbonates, which have relatively low solubility products at alkaline pH.Contribution No. 87-237-J from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan, Kansas. Cooperative investigation between Agricultural Research Service, U.S. Department of Agriculture and the Kansas Agricultural Experiment Station. K.J.K. is a research chemist and adjunct professor at U.S. Grain Marketing Research Laboratory and Kansas State University, respectively. Mention of a proprietary product in this paper does not imply its approval by the USDA to the exclusion of other products that may also be suitable. Reprint requests to A.B. Broce     

BINDING SITES FOR INORGANIC IONS AND UREA ON ISOLATED CUTICULAR MEMBRANE SURFACES

Binding sites for calcium and chloride ions, and urea on cuticular membrane surfaces were identified by microautoradiography. There was no localization of binding on either the smooth outer or irregular inner surfaces of the astomatous tomato fruit cuticle, and urea was bound to a lesser degree than inorganic ions. Calcium and chloride binding sites on surfaces of green onion leaf cuticles were closely associated with stomatal pores and above the periclinal cell walls. Binding of calcium was more pronounced than of chloride. These results are discussed in light of possible sites of entry for foliar applied nutrients.     

Novel protein inhibits in vitro precipitation of calcium carbonate.

Organic molecules both coexist and interact with inorganic crystal lattices in biomineralizing tissues. Mineral precipitation and crystal morphology are tightly regulated by the actions of these molecules. Polyacrylamide gel electrophoresis studies on water soluble extracts from the cuticle of Callinectes sapidus (Atlantic blue crab) reveal the presence, in unmineralized nascent premolt cuticle, of proteins which are absent in the mineralized postmolt cuticle. In the present studies, homogenates from both premolt and postmolt C. sapidus cuticles have been tested for their effect on the in vitro precipitation of calcium carbonate. The role of protein in this process was determined by heat pretreatment and trypsin pretreatment of the cuticle homogenates prior to the precipitation assay. The results from these experiments indicate that proteins, with molecular weights of approximately 75,000 and between 10,000 and 20,000, concentrated in the C. sapidus premolt cuticle, inhibit calcium carbonate precipitation in vitro. The inhibitory activity of these proteins appears to be a result of specific interactions since trypsin, myoglobin, and ovalbumin are not inhibitory. The presence of lower amounts of these inhibitory proteins in C. sapidus postmolt cuticle may be responsible for the subsequent mineralization of this tissue.     

Organic and inorganic compound variations in haemolymph, epidermal tissue and cuticle over the molt cycle in Scylla serrata (Decapoda)

Analysis of organic and inorganic compounds in haemolymph, epidermal tissue and cuticle, allowed the correlation of their content variation during the molt cycle in Scylla serrata (Decapoda). The extrusion of carbohydrate, chitin, H(2)O- and NaOH-soluble proteins of the epidermal tissue from D2 stage on suggested an early formation of the organic fraction in the new cuticle. The additional secretion of HCl-soluble protein, glycosaminoglycans, calcium, phosphorus and sulfur in D2-D3 stages suggests mineral nucleation shortly before or at ecdysis. This is consistent with the dominant content of proteins and chitin associated with peaks of carbohydrates and GAG-sulfur groups detected in the cuticle immediately after ecdysis. Furthermore, the maximal phosphorus content at this moment suggests calcium, magnesium and manganese phosphate deposits. After an intense accumulation of calcium, chitin and sharp decrease of protein from A to B stages, a constant rate of their deposition in intermolt and dissolution in premolt occurred. The variations concerning proteins, carbohydrates, glycosaminoglycans, calcium, magnesium and sulfur in the haemolymph, before and after ecdysis, suggest a transitory phenomenon for calcium binding and releasing. Other major elements such as, sodium, potassium and chloride may function as osmotic regulators in the haemolymph and in epidermal compartments. The copper profile presented an indicator role for variations of osmolality over the molt cycle.     

Review of conformation-specific affinity purification methods for plasma vitamin K-dependent proteins

There are seven known vitamin K-dependent proteins in blood. These proteins require calcium ion for expressing their full biological activities. Calcium ion also induces conformational changes in this class of proteins. Taking advantage of the ligand induced conformational changes, a number of unique approaches of affinity chromatography have been developed. These methodologies have been very useful tools for both the purification and for understanding the structure–function relationships of this class of proteins. One method is the use of metal ion dependent immunoaffinity chromatography. The antigen can be dissociated from the antibodies with either the removal or addition of calcium ion under physiological conditions. The other method is pseudoaffinity chromatography. This method uses conventional ion-exchange or hydrophobic resin and manipulates the mobilities of the proteins on these resins by the presence or absence of calcium ions. Researchers working with other calcium binding proteins or other proteins that are known to undergo ligand induced conformational changes may benefit from the experience of these unique conformation-specific affinity chromatography approaches.     

Evidence for various calcium sites in human hair shaft revealed by sub-micrometer X-ray fluorescence

New information about calcium status in human scalp hair shaft, deduced from X-ray micro-fluorescence imaging, including its distribution over the hair section, the existence of one or several binding-types and its variation between people, is presented. The existence of two different calcium types is inferred. The first one corresponds to atoms (or ions) easily removable by hydrochloric acid, located in the cortex (granules), in the cuticle zone and also in the core of the medulla, which can reasonably be identified as calcium soaps. The second type consists of non-easily removable calcium atoms (or ions) that are located in the medulla wall, probably also in the cuticle, and rather uniformly in the cortex; these calcium atoms may be involved in Ca(2+)-binding proteins, and their concentration is fairly constant from one subject to another. In addition to its nonuniform distribution across the hair section, the second striking feature of the first type calcium content is its high variability from one subject to another, by up to a factor 10. We expect this information to be useful for analyzing in more detail the relationship between hair calcium and environmental and medical factors.     

Protein kinase C interaction with calcium: a phospholipid-dependent process

The calcium-binding properties of calcium- and phospholipid-dependent protein kinase C (PKC) were investigated by equilibrium dialysis in the presence and the absence of phospholipids. Calcium binding to PKC displayed striking and unexpected behavior; the free proteins bound virtually no calcium at intracellular calcium concentrations and bound limited calcium (about 1 mol/mol of PKC) at 200 microM calcium. However, in the presence of membranes containing acidic phospholipids, PKC bound at least eight calcium ions per protein. The presence of 1 microM phorbol dibutyrate (PDBu) in the dialysis buffer had little effect on these calcium-binding properties. Analysis of PKC-calcium binding by gel filtration under equilibrium conditions gave similar results; only membrane-associated PKC bound significant amounts of calcium. Consequently, PKC is a member of what may be a large group of proteins that bind calcium in a phospholipid-dependent manner. The calcium concentrations needed to induce PKC-membrane binding were similar to those needed for calcium binding (about 40 microM calcium at the midpoint). However, the calcium concentration required for PKC-membrane binding was strongly influenced by the phosphatidylserine composition of the membranes. Membranes with higher percentages of phosphatidylserine required lower concentrations of calcium. These properties suggested that the calcium sites may be generated at the interface between PKC and the membrane. Calcium may function as a bridge between PKC and phospholipids. These studies also suggested that calcium-dependent PKC-membrane binding and PKC function could be regulated by a number of factors in addition to calcium levels and diacylglycerol content of the membrane.     

Antimicrobial activity of the Anseriform outer eggshell and cuticle

The avian eggshell is a complex, multifunctional biomineral composed of a calcium carbonate mineral phase and an organic phase of lipids and proteins. The outermost layer of the eggshell, the eggshell cuticle, is an organic layer of variable thickness composed of polysaccharides, hydroxyapatite crystals, lipids and glycoprotein. In addition to regulating gas exchanges, the eggshell cuticle may contain antimicrobial elements. In this study, we investigated the antimicrobial activity of eggshell cuticle and outer eggshell protein extracts from four Anseriform species: wood duck (Aix sponsa), hooded merganser (Lophodytes cucullatus), Canada goose (Branta canadensis) and mute swan (Cygnus olor). Cuticle and outer eggshell protein was extracted by urea or HCl treatment of eggs. C-type lysozyme, ovotransferrin and an ovocalyxin-32-like protein were detected in all extracts. Cuticle and outer eggshell protein extracts inhibited the growth of Staphylococcus aureus, Escherichia coli D31, Pseudomonas aeruginosa and Bacillus subtilis. The presence of active antimicrobial proteins within the avian cuticle and outer eggshell suggests a role in antimicrobial defense. Protein extracts from the cavity nesting hooded merganser were especially potent. The unique environmental pressures exerted on cavity-nesting species may have led to the evolution of potent antimicrobial defenses.     

The calcium abnormality in cystic fibrosis mitochondria: Relative role of respiration and ATP hydrolysis

Calcium uptake by mitochondria isolated from skin fibroblasts of patients with cystic fibrosis and controls was studied in the presence and absence of inhibitors. Since mitochondrial calcium accumulation may be supported by ATP hydrolysis or respiration, inhibitors of each were used to characterize the basis of previously described alterations in calcium uptake by mitochondria from patients with cystic fibrosis. Calcium uptake measurements under the influence of oligomycin and antimycin A suggest that the increased calcium uptake by mitochondria from patients with cystic fibrosis is related to altered respiratory system activity. Binding constants of calcium to the carrier system in mitochondria were not different between genotypes.     

Association of alpha-phosphatidylinositol-specific phospholipase C with phospholipid vesicles.

The alpha isoform of phosphatidylinositol-specific phospholipase C (alpha-PI-PLC, Mr 62,000) was purified from bovine brain. Enzyme activity was dependent on calcium, sodium cholate and showed the anticipated specificity for the phosphatidylinositols. Calcium interaction with this protein, investigated by gel filtration chromatography, showed no detectable binding at calcium concentrations adequate to activate the enzyme. Association of alpha-PI-PLC with phospholipid vesicles was studied by light scattering, fluorescence energy transfer and gel-filtration chromatography. The enzyme readily associated with vesicles of high charge density, with vesicles of crude acidic phospholipids and with PIP2. Interaction was characterized by a rapid association followed by slower addition of more protein to the phospholipid. Complexes containing 20-30 percent protein (by weight) were readily obtained. Calcium had only a small effect on this interaction. The protein-phospholipid complexes appeared to bind less calcium than a similar amount of phospholipid alone. Thus, alpha-PI-PLC did not appear to be a calcium-binding protein in either its free or membrane-associated states. Although alpha-PI-PLC showed the highest propensity to bind to phospholipids, a number of other proteins also associated with phospholipids under the conditions used. Thus, whether or not the observed interaction of alpha-PI-PLC with membranes was specific and biologically important or whether it was a process common to many proteins, was not known. Knowledge of this interaction may enhance our understanding of possible mechanisms for protein-membrane interactions in general.     

Zinc-dependent interaction between dishevelled and the Drosophila Wnt antagonist naked cuticle

During Drosophila development, the naked cuticle (nkd) gene attenuates wingless/Wnt signaling through a negative feedback loop mechanism. Fly and vertebrate Nkd proteins contain a putative calcium-binding EF-hand motif, the EFX domain, that interacts with the basic/PDZ region of the Wnt signal transducer, dishevelled (Dsh). Here we show that Dsh binding by Drosophila Nkd in vitro is mediated by the EFX domain as well as an adjacent C-terminal sequence. In vivo data suggest that both of these regions contribute to the ability of Nkd to antagonize Wnt signaling. Mutations in the Nkd EF-hand designed to eliminate potential ion binding affected Nkd-Dsh interactions in the yeast two-hybrid assay but not in the glutathione S-transferase pull-down assay. Addition of the chelating agent EDTA abolished the in vitro Nkd-Dsh interaction. Surprisingly zinc, but not calcium, was able to restore Nkd-Dsh binding, suggesting a zinc-mediated interaction. Calcium 45- and zinc 65-blotting experiments show that Nkd is a zinc-binding metalloprotein. The results further clarify how Nkd may antagonize Wnt signaling via interaction with Dsh, and identify a novel zinc-binding domain in Drosophila Nkd that collaborates with the conserved EFX domain to bind Dsh.     

Calcium binding capacity and calcium sensitive protein content in denervated frog muscles

Total and ionic calcium content, calcium binding capacity of sarcoplasmic proteins and calcium insensitive proteins were examined in atrophying leg muscles of frog after 1-5 months period of denervation. Different muscles showed different levels of atrophy and the total calcium content varied with reference to the type of muscle. Ionic calcium levels doubled in the gastrocnemius muscle after three months denervation. Calcium binding capacity of proteins and calcium insensitive proteins decreased rapidly up to four months after denervation in the gastrocnemius muscle. However no significant changes in the levels of calcium binding capacity and calcium insensitive proteins were found with reference to the type of muscle. Since total calcium content remains constant and wet muscle mass (expressed as atrophy) decreased markedly, an apparent increase in calcium concentration occurs in each muscle on denervation.     

Mass spectrometrical analysis of cuticular proteins from the wing of Hebemoia glaucippe (Linnaeus, 1758) (Lepidoptera: Pieridae)

Although several insect cuticular genes and proteins are annotated and an arthropod cuticular database is available, mass spectrometrical data on cuticular proteins and their post-translational modifications are limited. Wings from Hebemoia glaucippe were analyzed by scanning electron microscopy or homogenized, proteins were extracted and run on 2DE. In-gel digestion was carried out by using trypsin, chymotrypsin and Asp-N and subsequently the resulting peptides and post-translational modifications were identified by ion trap tandem mass spectrometry (nano-LC-ESI-MS/MS; HCT). A complex wing skeleton and the cuticle of H. glaucippe were demonstrated. Cuticle protein 18.6, isoform A, pupal cuticle protein, cuticular protein CPR59A and two putative proteins, putative cuticular protein B2DBJ and putative cuticle protein CPG31 with two expression forms were identified. Two phosphorylation sites on the same peptide, T213 and S214, were identified on putative cuticle protein CPG31, quinone formation was observed at Y76 on cuticular protein CPR59A probably indicating the presence of post-translational modifications. The results may be relevant for the interpretation of mechanoelastic and physical properties of these proteins. Along with the extraordinary architecture the proteinaceous matrix is probably representing or allowing the unusual aerodynamic function of the butterfly wing. Moreover, the results may be important for mechanisms of insecticide and drought resistance.     

Identification of a novel hepatic calcium binding protein

Calcium binding activity in the 100,000 X g supernatant of bovine liver has been isolated by a procedure involving DEAE cellulose and Sephadex G-100 chromatography. In addition to calmodulin, two new high affinity calcium binding proteins have been identified. On gel filtration chromatography these proteins migrate with apparent molecular weights of 83,700 and 51,400; whereas by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the two proteins migrate identically with Mr 63,000. In the presence of millimolar Mg2+, both proteins bind up to one mol Ca2+/mol protein. Half-maximal binding occurs at approximately 0.1 microM Ca2+. Amino acid compositional analysis reveals that both proteins are acidic, and contain about 40% glx and asx. Peptide mapping procedures suggest that these proteins may be highly homologous or multiple forms of a single protein. The results show the existence of calcium binding protein(s) other than calmodulin in hepatic cytosol.     

Immunochemical evidence for the transport of haemolymph protein into the cuticle of Manduca sexta

Larvae and pupae of Manduca sexta were utilized to determine whether haemolymph proteins can traverse the epidermal cell and enter the newly deposited pupal cuticle in an unaltered state. The proteins examined were those that function in carrying dopamine (or a dopamine metabolite) from the haemolymph into the cuticle. Radiotracer studies and electrophoretic analysis suggested that the haemolymph carrier proteins were indeed transported into the cuticle. Antibodies against the haemolymph carrier proteins reacted with proteins extracted from the cuticle. Further work demonstrated that proteins extracted from the cuticle are immunologically similar to the haemolymph carrier proteins.     

Evaluation of no-wash calcium assay kits: enabling tools for calcium mobilization

The no-wash calcium assay kits developed by Molecular Devices Corporation have greatly enhanced the throughput of cell-based calcium mobilization high-throughput screening (HTS) assays and enabled screening using nonadherent cells. The fluorescent imaging plate reader (FLIPR) Calcium 3 Assay Kit, optimal for targets that have proteins or peptides as agonists, has 2 potential drawbacks: 1) a significant downward spike in fluorescence signal upon liquid transfer that can be the same magnitude as the agonist response, making data analysis difficult; and 2) medium removal is required for some targets, which essentially reintroduces a wash step. Several no-wash products were introduced in 2005. The authors compare the Fluo-4 NW Calcium Assay Kit and the BD Calcium Assay Kit with the FLIPR Calcium 3 Assay Kit using human native rhabdomyosarcoma cells expressing the urotensin-II receptor (UT). The BDtrade mark Calcium Assay Kit gives the best performance in the true no-wash mode, in which both agonist and antagonist activity are easily quantified. Although these new products provide additional options for measuring calcium mobilization, the different results observed with each kit, using the UT receptor as an example, suggest that one should characterize all dyes against each target in a systematic way prior to choosing one for HTS.     

Calcium-dependent phosphorylation of specific synaptosomal fraction proteins: Possible role of phosphoproteins in mediating neurotransmitter release

Calcium ions caused a marked increase in the level of endogenous phosphorylation of specific proteins from synaptosomal fractions prepared from rat cerebral cortex. The levels of phosphorylation of these specific proteins were dependent upon the presence of calcium and regulated by small changes in the concenrration of calcium ions. The effect of calcium was independent of ATP concentration over a wide range of concentrations. The results are compatible with the hypothesis that some of the effects of calcium on synaptic transmission might be mediated by the effect of calcium on the phosphorylation of specific synaptosomal proteins.     

Voltage-gated calcium channels and disease

Voltage-gated calcium channels are a family of integral membrane calcium-selective proteins found in all excitable and many nonexcitable cells. Calcium influx affects membrane electrical properties by depolarizing cells and generally increasing excitability. Calcium entry further regulates multiple intracellular signaling pathways as well as the biochemical factors that mediate physiological functions such as neurotransmitter release and muscle contraction. Small changes in the biophysical properties or expression of calcium channels can result in pathophysiological changes leading to serious chronic disorders. In humans, mutations in calcium channel genes have been linked to a number of serious neurological, retinal, cardiac, and muscular disorders.     

Calcium translocations during the moulting cycle of the semiterrestrial isopod <Emphasis Type="Italic">Ligia hawaiiensis</Emphasis> (Oniscidea,Crustacea)

Terrestrial isopods moult first the posterior and then the anterior half of the body. During the moulting cycle they retain a significant fraction of cuticular calcium partly by storing it in sternal CaCO₃ deposits. We analysed the calcium content in whole Ligia hawaiiensis and the calcium distribution between the posterior, the anterior ventral, and the anterior dorsal cuticle during four stages of the moulting cycle. The results indicate that: (1) overall, about 80% of the calcium is retained and 20% is lost with the exuviae, (2) in premoult 68% of the calcium in the posterior cuticle is resorbed (23% moved to the anterior ventral cuticle, 17% to the anterior dorsal cuticle, and the remaining 28% to internal tissues), (3) after the posterior moult 83% of the calcium in the anterior cuticle is shifted to the posterior cuticle and possibly to internal storage sites, (4) following the anterior moult up to 54% of the calcium in the posterior cuticle is resorbed and used to mineralise the new anterior cuticle. ⁴⁵Ca-uptake experiments suggest that up to 80% of calcium lost with the anterior exuviae may be regained after its ingestion. Whole body calcium of Ligia hawaiiensis is only 0.7 times that of the fully terrestrial isopods. These terrestrial species can retain only 48% of whole body calcium, suggesting that the amount of calcium that can be retained by shifting it between the anterior and posterior integument is limited. We propose that fully terrestrial Oniscidea rely to a larger degree on other calcium sources like internal stores and uptake from the ingested exuviae.