Triton Shells Research Articles

De novo transcriptome sequencing of triton shell Charonia lampas sauliae: Identification of genes related to neurotoxins and discovery of genetic markers.

Charonia lampas sauliae (triton snails, triton shells or tritons; Mollusca, Caenogastropoda, Littorinimorpha, Ranellidae) is a marine species with a wide distribution. In Korea, this species is listed as vulnerable and is regionally protected as an endangered species. Here, we report the first comprehensive transcriptome dataset of C. lampas sauliae obtained using the Illumina HiSeq 2500 platform. In total, 97.68% of raw read sequences were processed as clean reads. Of the 577,478 contigs obtained, 146,026 sequences were predicted to contain coding regions. About 89.34% of all annotated unigene sequences showed homologous matches to protein sequences in PANM DB (Protostome database). Further, about one-third of the unigene sequences were annotated using the UniGene, Swiss-Prot, Clusters of Orthologous Groups (COG) and Gene Ontology (GO) databases. In total, 190 enzymes were predicted under key metabolic pathways under stood through Kyoto Encyclopedia of Genes and Genomes (KEGG) database annotation. Repetitive elements such as long terminal repeats (LTRs), short interspersed nuclear elements (SINEs), long interspersed nuclear elements (LINEs), and DNA elements were enriched in the unigene sequences. Among the identified transcripts were the channel proteins, some of which were blocked by tetrodotoxin, which is thought to be synthesized by symbiotic bacteria inhabiting the shells. In addition, conotoxin superfamily peptides, such as B-conotoxin, conotoxin superfamily T and alpha-conotoxin, were identified, which may have relevance to biomedical and evolutionary research. A transcriptome-wide search for polymorphic loci identified 21,568 simple sequence repeats (SSRs) in the unigene sequences. Most SSRs were dinucleotides, among which AC/GT was the dominant SSR type. The molecular and genetic resources revealed in this study could be utilized for investigations on the fitness of the species in the marine environment and sustainability in a changing habitat.

Analysis on the outbreak period and cause of <italic>Acanthaster planci</italic> in Xisha Islands in recent 15 years

Outbreaks of the crown-of-thorns starfish Acanthaster planci have received increasing attention due to their negative impacts on coral reefs. However, outbreaks of A. planci in Chinese seas are still poorly understood. Coral reefs in the Xisha Islands (Paracel Islands), South China Sea, have been monitored during 2005 and 2019, including hard coral coverage, larval recruitment rates, density of coral reef fish and the populations of A. planci . This study aims to analyse the regular pattern regarding outbreaks of A. planci in the main islands of Xisha Islands, the causes of outbreaks of the crown-of-thorns starfish Acanthaster planci , and the solutions for controlling the outbreaks of the crown-of-thorns starfish Acanthaster planci . Monitoring coral reef ecosystems on the main islands of Xisha Islands from 2005 to 2019 showed that the cycle of Acanthaster planci outbreaks in Xisha Islands is about 15 years. These 15 years can be divided into two phases, in which the period of A. planci outbreak is about 5 years, and the recovery period of coral reefs is about 10 years. When A. planci outbreak from 2006 to 2010, the coral reef cover rate decreased from more than 60% to less than 5%. During the recovery period from 2011 to 2019, the coverage of coral reefs gradually increased to around 15%, at the same time the coral recruitment and coral reef fish increased. Further analysis showed that the occurrence of A. planci in Xisha Islands is mainly related to typhoon weather, reduction of coral reef fish, global warming and human activities. The passage of the typhoon brought a large number of nutrients, which led to the increase of plankton, leading to the survival rate of larvae of A. planci . Global warming and a decline in coral reef fish could lead to a greater percentage of the eggs of A. planci hatching and surviving. Based on 15-years monitoring data, it is highly possible that Xisha Islands are entering the next outbreak cycle of A. planci , and therefore measures should be taken to prevent the coral reef ecosystem from being destroyed again. Outbreaks of the crown-of-thorns starfish A. planci should be controlled in several ways. Direct control methods including physical removal of individual starfish and cutting individuals into multiple pieces were most often used methods to kill starfish. The most effective methods to control A. planci require injecting individual starfish with lethal chemicals, including sodium bisulfate, powdered citric acid, vinegar and thiosulfate-citrate-bile-sucrose agar (TCBS). Releasing the predator of A. planci of their different life cycle was also an effective way to control A. planci . The Giant Triton Shells ( Charonia tritonis ), the Napoleon Maori Wrasse ( Cheilinus undulatus ), Starry Puffer ( Arathron stellatus ) and Titan Trigger Fish ( Ballistoides viridescens ) were the main predator of the crown-of-thorns starfish A. planci . Moreover, regular monitoring of the starfish A. planci should be conducted in the Xisha Islands for early warning of outbreaks.

Identification of Human Erythrocyte Cytosolic Proteins Associated with Plasma Membrane During Thermal Stress

The influence of thermal stress on the association between human erythrocyte membranes and cytosolic proteins was studied by exposing erythrocyte suspensions and whole blood to different elevated temperatures. Membranes and cytosolic proteins from unheated and heat-stressed erythrocytes were analyzed by electrophoresis, followed by mass spectrometric identification. Four major (carbonic anhydrase I, carbonic anhydrase II, peroxiredoxin VI, flavin reductase) and some minor (heat shock protein 90α, heat shock protein 70, α-enolase, peptidylprolyl cis-trans isomerase A) cytosolic proteins were found to be associated with the erythrocyte membrane in response to in vitro thermal stress. Unlike the above proteins, catalase and peroxiredoxin II were associated with membranes from unheated erythrocytes, and their content increased in the membrane following heat stress. The heat-induced association of cytosolic proteins was restricted to the Triton shells (membrane skeleton/cytoskeleton). Similar results were observed when Triton shells derived from unheated erythrocyte membranes were incubated with an unheated erythrocyte cytosolic fraction at elevated temperatures. This is a first report on the association of cytosolic catalase, α-enolase, peroxiredoxin VI, peroxiredoxin II and peptidylprolyl cis-trans isomerase A to the membrane or membrane skeleton of erythrocytes under heat stress. From these results, it is concluded that specific cytosolic proteins are translocated to the membrane in human erythrocytes exposed to heat stress and they may play a novel role as erythrocyte membrane protectors under stress by stabilizing the membrane skeleton through their interactions with skeletal proteins.

Erythroid spectrin in miceller detergents.

We have studied the interaction of spectrin, the major protein of the erythrocyte cytoskeleton, with four commonly used detergents at concentrations above their critical miceller concentrations (cmc). Fluorescence spectroscopic studies on the emission intensity, steady state polarization, quenching with acrylamide, and time-resolved fluorescence measurements were done with spectrin in anionic detergents, e.g., SDS, deoxycholate, and nonionic detergents, e.g., Triton-X-100 and octylglucoside at concentrations double their respective cmc's. The spectrin-detergent complexes in all four systems have been characterized by far-UV CD and measurements on tryptophan fluorescence in combination with fluorescence of the extrinsic probe, pyrene. Tryptophan fluorescence studies revealed quaternary structural changes due to unzipping of the spectrin subunits in Triton-X-100 without complete dissociation. Both Triton-X-100 and SDS were found to partially denature spectrin indicated by the far-UV CD. Octylglucoside and deoxycholate are shown to have the least structural perturbations on the cytoskeletal protein, rationalizing the use of octylglucoside, in particular and also deoxycholate to be the most effective in preparing cytoskeletal fractions from erythrocytes rather than the Triton-X-100 that has long been used for preparing the Triton shells.

New insights into regulation of erythrocyte shape.

Two ideas explain the mechanism of shape regulation: the lipid bilayer coupled theory and the protein network scaffold theory. Recently, several important articles have been published on the former theory. However, many phenomena argue against the theory, including behavior of ghosts and triton shells, various types of manipulation of proteins, and fixation of the shape by the addition of large reagents outside the cell. Moreover, hereditary spherocytosis shows normal, uneven distribution of phospholipids, and hereditary and artificial defects of a membrane protein show spherocytes or elliptocytes. The liquid state of the lipid layer does not seem to support the shape or mechanical characteristics. On the other hand, all of these phenomena argue for the protein network scaffold theory. Characteristics of each protein and interactions among proteins are now being clarified, but this theory and the author's own ideas still lack decisive evidence.

Zinc deficiency in the rat alters the lipid composition of the erythrocyte membrane Triton shell.

The effect of dietary zinc deficiency on the lipid composition of the erythrocyte membrane Triton shell was determined. Weanling male Wistar rats were fed an egg white-based diet containing < 1.0 mg Zn/kg diet ad libitum. Control rats were either pair-fed or ad libitum-fed the basal diet supplemented with 100 mg Zn/kg diet. A Zn refed group was fed the -Zn diet until day 18 and then pair-fed the +Zn diet until day 21. Dietary Zn deficiency caused an increased cholesterol/phospholipid ratio in Triton shells compared to those from pair-fed controls. Zn deficiency caused a decreased double bond index of fatty acids in phosphatidylinositol (PI) and phosphatidylcholine (PC); there was a decreased proportion of 18:2n-6 and 22:4n-6 in PC and 20:4n-6 in PI as compared to that found in pair-fed controls. All glycerophospholipids that were retained in the shell had a lower double bond index and increased content of 16:0 and/or 18:0 relative to the phospholipid in the intact membrane.

Mycoplasma pneumoniae cytadherence phase-variable protein HMW3 is a component of the attachment organelle.

The subcellular location of the phase-variable cytadherence-accessory protein HMW3 in Mycoplasma pneumoniae has been examined by biochemical and immunoelectron microscopic techniques. Analysis by Western blot (immunoblot) with HMW3-specific antiserum established the presence of this protein within the M. pneumoniae Triton X-100-insoluble fraction or triton shell. Immunogold labeling of Triton-extracted mycoplasmas with affinity-purified antibodies localized HMW3 to the terminal knob on the rodlike extensions of the triton shell, a location that would correspond to the adherence organelle in whole mycoplasmas. Treatment of triton shells with KI resulted in the selective removal of the adherence-accessory proteins HMW1 to HMW4. Analysis of these triton shells by transmission electron microscopy revealed dramatic ultrastructural changes in the filamentous network and core structure. Immunogold labeling of KI-extracted shells reflected the removal of HMW3 from the disrupted tip structure. An examination of ultrathin sections of wild-type cells by transmission electron microscopy following labeling with HMW3-specific antibodies provided further evidence for the nonrandom distribution of HMW3 and its localization to the terminal portion of filamentous cell extensions. Most colloidal gold molecules were associated with the cell interior, but limited peripheral labeling of the terminal region was also observed. Postfixation antibody labeling of whole cells suggested limited exposure of HMW3 on the mycoplasma surface at the tip structure. However, prefixation antibody labeling failed to indicate surface exposure, raising some uncertainty regarding the relationship of HMW3 with the mycoplasma membrane.

TRITON'S TRUMPET: A NEOLITHIC SYMBOL IN ITALY

Summary. The distribution and uses of triton shells in Italy from the Neolithic to modern times are described in detail, along with a summary of examples from the Neolithic and Copper Age of Central Europe and the central and eastern Mediterranean. Attention is then focused upon the triton shell trumpet, which is interpreted both as a Neolithic visual symbol and an oral signal, and suggestions are offered concerning its changing meaning in historical and more recent times.

Heat-induced alterations in monkey erythrocyte membrane phospholipid organization and skeletal protein structure and interactions

Rhesus monkey erythrocytes were subjected to heating at 50°C for 5–15 min, and the heat-induced effects on the membrane structure were ascertained by analysing the membrane phospholipid organization and membrane skeleton dynamics and interactions in the heated cells. Membrane skeleton dynamics and interactions were determined by measuring the Tris-induced dissociation of the Triton-insoluble membrane skeletons (Triton shells), the spectrin-actin extractability at low ionic strength, spectrin self-association and spectrin binding to normal monkey erythrocyte membrane inside-out vesicles (IOVs). The Tris-induced Triton shell dissociation and spectrin-actin extractability were markedly decreased by the erythrocyte heating. Also, the binding of the heated erythrocyte membrane spectrin-actin with the IOVs was much smaller than that observed with the normal erythrocyte spectrin-actin. Further, the spectrin structure was extensively modified in the heated cells, as compared to the normal erythrocytes. Transbilayer phospholipid organization was ascertained by employing bee venom and pancreatic phospholipases A 2, fluorescamine, and Merocyanine 540 as the external membrane probes. The amounts of aminophospholipids hydrolysed by phospholipases A 2 or labeled by fluorescamine in intact erythrocytes considerably increased after subjecting them to heating at 50°C for 15 min. Also, the fluorescent dye Merocyanine 540 readily stained the 15-min-heated cells but not the fresh erythrocytes. Unlike these findings, the extent of aminophospholipid hydrolysis in 5-min-heated cells by phospholipases A 2 depended on the incubation time. While no change in the membrane phospholipid organization could be detected in 10 min, prolonged incubations led to the increased aminophospholipid hydrolysis. Similarly, fluorescamine failed to detect any change in the transbilayer phospholipid distribution soon after the 5 min heating, but it labeled greater amounts of aminophospholipids in the 5-min-heated cells, as compared to normal cells, after incubating them for 4 h at 37°C. These results have been discussed to analyse the role of membrane skeleton in maintaining the erythrocyte membrane phospholipid asymmetry. It has been concluded that both the ATP-dependent aminophospholipid pump and membrane bilayer-skeleton interactions are required to maintain the transbilayer phospholipid asymmetry in native erythrocyte membrane.

Glycophorin C Content of Human Erythrocyte Membrane Is Regulated by Protein 4.1

Human erythrocyte transmembrane sialoglycoprotein, glycophorin C, plays a functionally important role in maintaining erythrocyte shape and regulating membrane material properties, possibly through its interaction with protein 4.1. Moreover, it has previously been shown that membranes deficient in protein 4.1 exhibit decreased content of glycophorin C. To further define the relationship between protein 4.1 and glycophorin C, a series of studies were performed using both protein 4.1- and glycophorin C-deficient erythrocytes. Quantitation by flow cytometry showed that the glycophorin C content of cells totally deficient in protein 4.1 was 9% of normal and that of cells partially deficient in protein 4.1 was 44% of normal. Interestingly, while homozygous glycophorin C-deficient cells had no detectable levels of this sialoglycoprotein, cells from obligate heterozygotes had normal levels. Protein 4.1 content of membranes of these glycophorin C-deficient cells was also normal. These data suggest that glycophorin C may be synthesized in excess by erythroid cells and its membrane content regulated by protein 4.1. To investigate if this regulation is due to association between protein 4.1 and glycophorin C, we examined the retention of glycophorin C in membrane skeletons (Triton shells) prepared from normal membranes, protein 4.1-deficient membranes, and protein 4.1-deficient membranes reconstituted with exogenous protein 4.1. Glycophorin C is retained by Triton shells prepared from normal membranes, whereas Triton shells prepared from protein 4.1-deficient membranes are totally devoid of this sialoglycoprotein. However, reconstitution of protein 4.1-deficient membranes with purified protein 4.1 resulted in retention of glycophorin C with the Triton shells. This finding suggests that protein 4.1 is necessary for association of glycophorin C with the membrane skeleton. Furthermore, these data suggest that through its interaction with glycophorin C, protein 4.1 may play a role in regulating the membrane content of this sialoglycoprotein in mature human erythrocytes.

Glycophorin C content of human erythrocyte membrane is regulated by protein 4.1

Human erythrocyte transmembrane sialoglycoprotein, glycophorin C, plays a functionally important role in maintaining erythrocyte shape and regulating membrane material properties, possibly through its interaction with protein 4.1. Moreover, it has previously been shown that membranes deficient in protein 4.1 exhibit decreased content of glycophorin C. To further define the relationship between protein 4.1 and glycophorin C, a series of studies were performed using both protein 4.1- and glycophorin C-deficient erythrocytes. Quantitation by flow cytometry showed that the glycophorin C content of cells totally deficient in protein 4.1 was 9% of normal and that of cells partially deficient in protein 4.1 was 44% of normal. Interestingly, while homozygous glycophorin C-deficient cells had no detectable levels of this sialoglycoprotein, cells from obligate heterozygotes had normal levels. Protein 4.1 content of membranes of these glycophorin C- deficient cells was also normal. These data suggest that glycophorin C may be synthesized in excess by erythroid cells and its membrane content regulated by protein 4.1. To investigate if this regulation is due to association between protein 4.1 and glycophorin C, we examined the retention of glycophorin C in membrane skeletons (Triton shells) prepared from normal membranes, protein 4.1-deficient membranes, and protein 4.1-deficient membranes reconstituted with exogenous protein 4.1. Glycophorin C is retained by Triton shells prepared from normal membranes, whereas Triton shells prepared from protein 4.1-deficient membranes are totally devoid of this sialoglycoprotein. However, reconstitution of protein 4.1-deficient membranes with purified protein 4.1 resulted in retention of glycophorin C with the Triton shells. This finding suggests that protein 4.1 is necessary for association of glycophorin C with the membrane skeleton. Furthermore, these data suggest that through its interaction with glycophorin C, protein 4.1 may play a role in regulating the membrane content of this sialoglycoprotein in mature human erythrocytes.

Membrane skeleton-bilayer interaction is not the major determinant of membrane phospholipid asymmetry in human erythrocytes

Transbilayer phospholipid distribution, membrane skeleton dissociation/association, and spectrin structure have been analysed in human erythrocytes after subjecting them to heating at 50°C for 15 min. The membrane skeleton dissociation/association was determined by measuring the Tris-induced dissociation of Triton-insoluble membrane skeletons (Triton shells), the spectrin-actin with normal erythrocyte membrane inside-out vesicles (IOVs). The spectrin structure was ascertained by measuring the spectrin dimer-to-tetramer ratio as well as the spectrin tryptophan fluoresence. Both the Tris-induced Triton shell dissociation and the spectrin-actin extractability under low ionic conditions were considerably reduced by the heat treatment. Also, the binding of heated erythrocyte spectrin-actin to IOVs was significantly smaller than the observed with the normal cell spectrin-actin. Further, the quantity of spectrin dimers was appreciably inreased in heat-treated erythrocytes as compared to the normal cells. This change in the spectrin dimer-to-tetramer ratio was accompanied by marked changes in the spectrin tryptophan fluorescence. Inspite of these heat-induced alterations in structure and bilayer interactions of the membrane skeleton, the inside-outside glycerophospholipid distribution remained virtually unaffected in the heat-treated cells, as judged by employing bee venom and pancreatic phospholipase A 2, fluorescamine and Merocyanine 540 as the external membrane probes. These results strongly indicate that membrane bilayer-skeleton interaction is not the major determining the transbilayer phospholipid asymmetry in human erythrocyte membrane.

Electron spin resonance and biochemical studies of the interaction of the polyamine, spermine, with the skeletal network of proteins in human erythrocyte membranes

Spermine ( N, N−bis(aminopropyl)-1,4-butanediamine) is a polyamine thought to be important in several cell regulatory processes. Previous studies had shown that spermine prevented the lateral diffusion of transmembrane proteins in human erythrocyte ghosts (Schindler et al. (1980) Proc. Natl. Acad. Sci. USA 77, 1457–1461). In this paper, we present results of studies on the effect of spermine on erythrocyte membranes by employing electron spin resonance spin-labeling techniques in conjunction with spin labels specific for skeletal proteins, bilayer lipids or cell-surface sialic acid of the membrane and by employing SDS-polyacrylamide gel electrophoresis analysis of extracted spectrin and Triton shells. The major findings are: (1) spermine significantly decreases the segmental motion of protein spin-label binding sites ( P < 0.0001), which are predominantly on cytoskeletal proteins; (2) addition of spermine leads to a significant increase in the rotational motion of spin-labeled terminal sialic acid residues ( P < 0.001), most of which are located on glycophorin A, a result which may be secondarily caused by spermine-induced aggregation of cytoskeletal proteins and the cytoplasmic pole of this transmembrane sialoglycoprotein; (3) spermine completely inhibits the low-ionic strength extraction of spectrin, the major protein of the skeletal network which is attached to the bilayer proteins by two or more connecting proteins; (4) pretreatment of ghosts with spermine followed by Triton extraction resulted in the retention of significantly increased amounts of Band 3 and other skeletal and bilayer proteins including Bands 4.2, 6 and 7 in Triton X-100 shells relative to that of control-treated ghosts. These results suggest that spermine acts both to increase protein-protein interactions in the cytoskeletal protein network and to bridge skeletal and bilayer proteins and are discussed with reference to possible molecular mechanisms by which spermine may influence cell functions.

Dynamic association of band 3 with Triton shells in human erythrocyte ghosts

To test a possibility that free band 3 and ankyrin-linked band 3 are exchanged in situ, band 3 was labeled with 125I, using intact red blood cells and lactoperoxidase. The cytoplasmic surface of this labeled band 3 was considered to be intact. When Triton shells were incubated with Triton supernatants prepared from 125I-labeled intact erythrocytes at 37° C in the presence of Mg-ATP under isotonic conditions, the incorporation of free 125I-labeled band 3 to shells was observed. This incorporation was affected by the presence of Triton X-100 in the incubation mixture, and significantly decreased when the content of Triton X-100 was less than 0.04% (v/v). On the other hand, ankyrin-linked 125I-labeled band 3 was released when shells prepared from 125I-labeled intact erythrocytes were incubated with the Triton supernatants at 37°C under the same condition as when free 125I-labeled band 3 incorporation was observed. These results strongly suggest that free and ankyrin-linked band 3 exchanged with each other in the presence of Triton X-100. A water-soluble 43 kDa fragment of band 3 inhibited the incorporation of free 125I-labeled band 3 to the shells and also inhibited the Mg-ATP-dependent shape change of ghosts in the absence of Triton X-100. Both of these inhibitory effects remained, even after 10 min of heat treatment at 100°C, but drastically decreased by treatment with trypsin. Our results strongly suggest that a dynamic exchange of the free band 3 for ankyrin-linked band 3 may occur in intact erythrocytes, and it may even contribute to the shape change of erythrocytes.

Concanavalin A-induced cap formation in rat ascites hepatoma cells (AH 7974) and the interaction of cytoplasmic proteins with plasma membranes.

Concanavalin A (Con A) induced cap formation in rat ascites hepatoma cells (AH7974). In these Con A-treated cells, the association of cytoplasmic proteins with cell membranes was suggested by observing their Triton shells. The transition from G-actin to F-actin occurred in these cells. The association of membrane lipid with cytoplasmic proteins extracted from AH cells was studied by the isolation of protein-bound liposomes and phase transition release. The analysis of isolated liposomes revealed that many cytoplasmic proteins which specifically associated with liposomes were cytoskeletal elements including F-actins. The association of proteins with liposomes was affected by the lipid composition of the liposomal membrane and by the Ca2+ concentration of the incubation medium. The strong interaction of liposomal membrane with cytoplasmic proteins or isolated cytoskeletal proteins was demonstrated also by phase transition release using carboxy fluorescein-containing liposomes. These experiments showed that there was a strong affinity between lipid membrane and cytoskeletal elements including F-actins and that the amount of F-actin increased due to Con A treatment. The association of the submembranous microfilaments with the cell membrane may contribute to capping of the cells caused by Con A.

Attachment of cytoskeletons to cell membranes in human blood platelets as revealed by the quick-freezing and deep-etching replica method

This study evaluated ultrastructures of cell membrane-cytoskeletal interactions in resting and activated human blood platelets. Some blood platelets were fixed with paraformaldehyde to inhibit activation and the others were activated with thrombin treatment. The replicas of inside-out cell membranes, cell membrane surfaces, and Triton shells were made by the quick-freezing and deep-etching replica method. A geometrical pattern consisting of main hexagons and partial pentagons was revealed to be formed with microfilaments which were attached laterally to cytoplasmic sides of cell membranes in resting platelets. After the activation, cytoskeletal meshworks in the cytoplasm were densely associated with cell membranes, and parallel microfilamentous bundles in filopodia were connected with small polygonal meshworks lying under the cell membrane. The cell membrane-cytoskeletal interactions are essential to keeping a discoid shape at the resting stage, and to changing the shape quickly after the activation.

Detection of the major adhesin P1 in triton shells of virulent Mycoplasma pneumoniae.

Filamentous structures designated Triton shells were obtained from virulent Mycoplasma pneumoniae by treatment with Triton X-100. Monoclonal antibodies directed against M. pneumoniae were used in conjunction with radioimmunoprecipitation and Western blotting to detect immunologically reactive polypeptides in Triton shells. The major adhesin, protein P1, was associated with these structures.

The Characterization of Protein 4.1 Presles, a Shortened Variant of RBC Membrane Protein 4.1

In a previous report (Blood 60:265, 1982), we described a family with an abnormal RBC membrane protein doublet, which we considered a shortened protein 4.1 on the basis of biochemical and genetic data. Using an anti-4.1 monoclonal antibody, we confirm here that the shortened protein derives from protein 4.1. One of the members of the family contemporaneously displayed the 4.1 (-) trait, eg, the heterozygous state of this variety of hereditary elliptocytosis that lacks protein 4.1. The 4.1a/4.1b ratio was low whenever the 4.1 trait was present, regardless of the type of protein 4.1 involved. The RBCs of the compound heterozygote, containing only the shortened species of protein 4.1, made it possible to analyze without interference the contact between shortened protein 4.1 and sialoglycoprotein beta, or glycoconnectin. Shortened protein 4.1 did not alter the amount of glycoconnectin in the ghosts nor did it change its extractability into the Triton shells. Limited proteolysis of shortened polypeptides 4.1a and 4.1b showed that they are sequence related. It is conflicting that the persons carrying the shortened protein 4.1 are devoid of specific clinical and morphological abnormalities, apart from those pertaining to the 4.1- trait, when the latter is present.

Triton X-100 extraction of P815 tumor cells: evidence for a plasma membrane skeleton structure.

It has been shown that a Triton X-100-insoluble protein matrix can be isolated from the plasma membranes of P815 tumor cells and murine lymphoid cells (Mescher, M. F., M. J. L. Jose and S. P. Balk, 1981, Nature (Lond.), 289:139-144). The properties of the matrix suggested that this set of proteins might form a membrane skeletal structure, stable in the absence of the lipid bilayer. Since purification of plasma membrane results in yields of only 20 to 40%, it was not clear whether the matrix was associated with the entire plasma membrane. To determine if a detergent-insoluble structure was present over the entire cell periphery and stable in the absence of the membrane bilayer or cytoskeletal components, we have examined extraction of whole cells with Triton X-100. Using the same conditions as those used for isolation of the matrix from membranes, we found that extraction of intact cells resulted in structures consisting of a continuous layer of protein at the periphery, a largely empty cytoplasmic space, and a nuclear remnant. Little or no lipid bilayer structure was evident in association with the peripheral layer, and no filamentous cytoskeletal structures could be seen in the cytoplasmic space by thin-section electron microscopy. Analysis of these Triton shells showed them to retain approximately 15% of the total cell protein, most of which was accounted for by low molecular weight nuclear proteins. 5'-Nucleotidase, a cell surface enzyme that remains associated with the plasma membrane matrix, was quantitatively recovered with the shells. Included among the polypeptides present in the shells was a set with mobilities identical to those of the set that makes up the plasma membrane matrix. The polypeptide composition of the shells further confirmed that cytoskeletal proteins were present to a very low extent, if at all, after the extraction. The results demonstrate that a detergent-insoluble protein matrix associated with the periphery of these cells forms a continuous, intact macrostructure whose stability is independent of the membrane bilayer or filamentous cytoskeletal elements, and thus has the properties of a membrane skeletal structure. Although not yet directly demonstrated, the results also strongly suggest that this peripheral layer is composed of the previously described set of plasma membrane matrix proteins. This article discusses possible roles for this proposed membrane skeletal structure in stabilizing the membrane bilayer and affecting the dynamics of other membrane proteins.

Ca 2+- and Mg-ATP-dependent shape change of human erythrocyte ghosts and Triton shells

Human erythrocyte membranes (ghosts) prepared from fresh blood changed in shape from spherical to crenated, when suspended in 10 −7–10 −6 M Ca 2+-EGTA buffers. Although the ghosts from long-stored ACD blood (10 weeks) were less sensitive to 10 −7–10 −6 M Ca 2+, the ghosts obtained from this blood after it had been preincubated with adenine and inosine for 3 h at 37 °C were highly sensitive to Ca 2+. When these highly sensitive ghosts were incubated in 10 mM Tris-Cl buffer (pH 7.4) or 1 mM MgCl 2 (pH 7.4) at 0 °C, they gradually lost Ca 2+ sensitivity within 60 min, but they recovered Ca 2+ sensitivity again after re-incubation with 2 mM Mg-ATP for 20 min at 37 °C followed by washing with 1 mM MgCl 2 (pH 7.4). The shape of these highly Ca 2+-sensitive ghosts immediately changed from crenate to disc on addition of 1 mM Mg-ATP even at 6 °C in the presence of 10 −7–10 −6 M Ca 2+. A similar shape change was also observed when ghosts treated with 0.5% Triton X-100 (Triton shells) were used. Triton shells from fresh blood ghosts or from long-stored blood ghosts which had been preincubated with 2 mM Mg-ATP for 20 min at 37 °C shrank immediately in the presence of 10 −6 M Ca 2+ and then swelled on addition of 1 mM Mg-ATP. The specificity to ATP and the dependency on ATP concentration are in agreement with those of the ghost shape change at step 2 (Jinbu, Y. et al., Biochem biophys res commun 112 (1983) 384–390) [18]. These results suggest that cytoskeletal protein phosphorylation enhances sensitivity to Ca 2+ and induces erythrocyte shape change in the presence of physiological concentrations of ATP and Ca 2+.