Influence of a Reduced CO<Subscript>2</Subscript> Environment on the Secretion Yield,Potency and N-Glycan Structures of Recombinant Thyrotropin from CHO Cells

A consistent increase of approximately 60% in the secretion yield of CHO-derived hTSH was observed by changing cell culture CO2 conditions from 5% CO2 to an air environment. The overall quality of the products obtained under both conditions was evaluated in comparison with a well-known biopharmaceutical (Thyrogen). The N-glycans identified were of the complex type, presenting di-, tri- and tetra-antennary structures, sometimes fucosylated, 86-88% of the identified structures being sialylated at variable levels. The three most abundant structures were monosialylated glycans, representing approximately 69% of all identified forms in the three preparations. The main difference was found in terms of antennarity, with 8-10% more di-antennary structures obtained in the absence of CO2 and 7-9% more tri-antennary structures in its presence. No remarkable difference in charge isomers was observed between the three preparations, the isoelectric focusing profiles showing six distinct bands in the 5.39-7.35 pI range. A considerably different distribution, with more forms in the acidic region, was observed, however, for two native pituitary preparations. All recombinant preparations showed a higher in vivo bioactivity when compared to native hTSH. Different production processes apparently do not greatly affect N-glycan structures, charge isomer distribution or bioactivity of CHO-derived hTSH.     

Molecular Mechanics Calculations of Proteins. Comparison of Different Energy Minimization Strategies

Abstract

A general strategy for performing energy minimization of proteins using the SYBYL molecular modelling program has been developed. The influence of several variables including energy minimization procedure, solvation, dielectric function and dielectric constant have been investigated in order to develop a general method, which is capable of producing high quality protein structures. Avian pancreatic polypeptide (APP) and bovine pancreatic phospholipase A2 (BP PLA2) were selected for the calculations, because high quality X-ray structures exist and because all classes of secondary structure are represented in the structures. The energy minimized structures were evaluated relative to the corresponding X-ray structures. The overall similarity was checked by calculating RMS distances for all atom positions. Backbone conformation was checked by Ramachandran plots and secondary structure elements evaluated by the length on hydrogen bonds. The dimensions of active site in BP PLA2 is very dependent on electrostatic interactions, due to the presence of the positively charged calcium ion. Thus, the distances between calcium and the calcium-coordinating groups were used as a quality index for this protein. Energy minimized structures of the trimeric PLA2 from Indian cobra (N.n.n. PLA2) were used for assessing the impact of protein-protein interactions. Based on the above mentioned criteria, it could be concluded that using the following conditions: Dielectric constant ? = 4 or 20; a distance dependent dielectric function and stepwise energy minimization, it is possible to reproduce X-ray structures very accurately without including explicit solvent molecules.     

Three separate classes of bacterial ice nucleation structures

Studies of the properties of the ice nucleation structure exposed on the surfaces of various bacteria such as Pseudomonas syringae, Erwinia herbicola, or various strains of Ice+ recombinant Escherichia coli have shown that there are clearly three major related but chemically distinct types of structures on these cells. First, the ability of Ice+ cells to nucleate super-cooled D2O has been examined, and it has been found that this ability (relative to the ability of the same cells to nucleate super-cooled H2O) exhibited three characteristic nucleating patterns. The rarest structure, called class A, is found on only a small fraction of cells in a culture, nucleates H2O at temperatures above -4.4 degrees C, and is an effective nucleator of super-cooled D2O. A second class of structure, called class B, is found on a larger portion of the cells, nucleates H2O between -4.8 and -5.7 degrees C, and is a relatively poor nucleator of super-cooled D2O. The class C structure is found on almost all cells and nucleates at -7.6 degrees C or colder. These three classes of structures were also differentiated by their sensitivities to low concentrations of water-miscible organic solvents such as dioxane or dimethyl sulfoxide. Depending on the specific bacterial strain, the addition of these solvents to bacterial suspensions lowered the nucleation activity of the class A structure by 1,000-fold or more. The nucleation activities of class B structures in the same culture were highly resistant to these compounds and were lowered only by 20 to 40%. The class C structures were more sensitive than Class B structures were, and the nucleation activities decreased 70 to 90%. Finally, the pH sensitivity of these three classes of structures was examined. The class A structure was destroyed in buffers at pH 4.5 lower but was stable in buffers at higher pHs. The class B structure was less sensitive to acidic buffers but was destroyed at pH 5.5 or lower and was stable at higher pHs. However, the class C structure was unaffected by incubation in buffers with pHs of 3.5 to 9.0. Suggestions for the actual nucleation structures of the three classes are proposed.     

Enlarged representative set of protein structures.

To reduce redundancy in the Protein Data Bank of 3D protein structures, which is caused by many homologous proteins in the data bank, we have selected a representative set of structures. The selection algorithm was designed to (1) select as many nonhomologous structures as possible, and (2) to select structures of good quality. The representative set may reduce time and effort in statistical analyses.     

我国两株登革2型病毒5′和3′端非编码区序列测定及二级结构分析

 为测定我国两株临床症状、乳鼠神经毒力不同的登革 2型病毒流行株 5′和 3′端非编码区序列 (untranslated region,UTR) ,分析二级结构差异与毒力变化的关系 ,分别从 D2 - 0 4、D2 - 44株感染的 C6/ 36细胞及鼠脑中提取总 RNA.以该 RNA为模板 ,利用 RACE法 ,分别扩增了 D2 - 0 4、D2 -44株的 5′和 3′末端 c DNA片段 .将其分别与 p GEM- T载体连接得到重组质粒 ,测定上述 c DNA插入片段的序列 .用 RNAdraw软件预测 D2 - 0 4、D2 - 44株 5′和 3′端非编码区的二级结构 .D2 - 0 4、D2 -44株 5′端和 3′端非编码区分别有 96和 454个核苷酸 .其中 5′非编码区 59位 C(D2 - 0 4 )→T(D2 -44 ) ,使 D2 - 44二级结构稳定性下降 ;3′端非编码区有 1 5个核苷酸不同 ,其中 T(355)→ A,T(32 6)→ G引起了所在位置二级结构自由能变化 ,且分别位于两个保守序列区 (conserved sequence,CS)CS1、CS2 A.这些位点变化可能与毒力有关 .     

Structures of N-linked oligosaccharides of microsomal glycoproteins from developing castor bean endosperms.

The structures of sugar chains of the glycoproteins from the microsomal fraction of developing castor bean endosperms have been analyzed. The structural analyses were done by a fluorescence method combined with component analysis, exoglycosidase digestions, partial acetolysis, Smith degradation, and 1H-NMR spectroscopy. The estimated structures fell into three categories; the first was oligomannose-type, the second xylomannose-type, the third complex-type. Among these oligosaccharides, Man3Fuc1Xyl1GlcNAc2 (M3FX) and Man6GlcNAc2 (M6B) were the major structures. The structures of Man4GlcNAc2 (M4C) and Man4Xyl1GlcNAc2 (M4X) have also been found in the microsomal glycoproteins of the developing bean endosperms. These results could indicate that the structures of M4C, M4X, and M3FX are formed in the stage of sugar chain processing in the microsomal fraction, in which oligomannose-type sugar chains are modified into complex-type ones by several kinds of processing enzymes.     

Immunocytochemical study of lampbrush chromosomes of the urodele Pleurodeles waltl: Axial granules are recognized by the mitosis-specific monoclonal antibody MPM-2

The lampbrush chromosomes of the urodele Pleurodeles waltl have been studied using the mitosis-specific monoclonal antibody MPM-2. Immunofluorescence studies revealed that MPM-2 stains structures associated with axial granules, numerous other chromomeres, telomeres and certain chiasmata. These structures showed a negative reaction with the anti-DNA monoclonal antibody AC-30-10. In course of meiotic condensation of the chromosomes, in growing and maturating oocytes, the number of such structures associated with the chromosome axis was found to diminish progressively. These granular structures have been found to be formed by fine fibrils about 5 nm in diameter. Immunogold labeling confirmed the results of immunofluorescence studies. MPM-2 was also found to stain two other types of structures observed in association with the lampbrush chromosome axis in P waltl, viz the sphere organelle (only in later stages of oogenesis) and the structure known as ‘M’ which is singular to this material.     

Structural characteristics of protein binding sites for calcium and lanthanide ions

Surveys of X-ray structures of Ca2+-containing and lanthanide ion-containing proteins and coordination complexes have been performed and structural features of the metal binding sites compared. A total of 515 structures of Ca2+-containing proteins were considered, although the final data set contained only 44 structures and 60 Ca2+ binding sites with a total of 323 ligands. Eighteen protein structures containing lanthanide ions were considered with a final data set containing eight structures and 11 metal binding sites. Structural features analysed include coordination numbers of the metal ions, the identity of their ligands, the denticity of carboxylate ligands, and the type of secondary structure from which the ligands are derived. Three general types of calcium binding site were identified in the final data set: class I sites supply the Ca2+ ligands from a continuous short sequence of amino acids; class II sites have one ligand supplied by a part of the amino acid sequence far removed from the main binding sequence; and class III sites are created by amino acids remote from one another in the sequence. The abundant EF-hand type of Ca2+ binding site was under-represented in the data set of structures analysed as far as its biological distribution is concerned, but was adequately represented for the chemical survey undertaken. A turn or loop structure was found to provide the bulk of the ligands to Ca2+, but helix and sheet secondary structures are slightly better providers of bidentate carboxylate ligation than turn or loop structures. The average coordination number for Ca2+ was 6.0, though for EF-hand sites it is 7. The average coordination number of a lanthanide ion in an intrinsic protein Ca2+ site was 7.2, but for the adventitious sites was only 4.4. A survey of the Cambridge Structural Database showed there are small-molecule lanthanide complexes with low coordination numbers but it is likely that water molecules, which do not appear in the electron density maps, are present for some lanthanide sites in proteins. A detailed comparison of the well-defined Ca2+ and lanthanide ion binding sites suggests that a reduction of hydrogen bonding associated with the ligating residues of the binding sites containing lanthanide ions may be a response to the additional positive charge of the lanthanide ion. Major structural differences between Ca2+ binding sites with weak and strong binding affinities were not obvious, a consequence of long-range electrostatic interactions and metal ion-induced protein conformational changes modulating affinities.     

Structural effects of neutral lipids on divalent cation-induced interactions of phosphatidylserine-containing bilayers.

Ca2+ is known to induce the adhesion and collapse of phosphatidylserine (PS) bilayers into dehydrated multilamellar structures. The aim of this study was to examine how that interaction and the resultant structures might be modified by neutral lipid species. A combination of rapid mixing, x-ray diffraction, thin-layer chromatography, density gradient centrifugation, and freeze-fracture electron microscopy was used in conjunction with osmotic stress techniques to characterize the structures formed by the Ca(2+)-induced interaction of multilamellar liposomes and of large unilamellar vesicles. The results showed that dioleoylphosphatidylcholine and dioleoylphosphatidylethanolamine at concentrations of up to approximately 30 mol % are accommodated in a single dehydrated multilamellar structure. Similar results were obtained using mixed PS species isolated from bovine brain. Principally, the data indicate that neutral lipid is both dehydrated during the rapid collapse process of Ca(PS)2 formation and accommodated within this dehydrated structure. The large energies available on formation of the Ca(PS)2 bilayers contribute to the dehydration of neighboring neutral lipids that likely form continuous bilayers with them. Higher concentrations of these neutral lipids modify Ca(2+)-induced bilayer interactions, leading to progressively weaker interactions, larger bilayer separations, and in some cases separation into two structures; phosphatidylethanolamine species favoring nonbilayer structures tended to promote such separation at lower concentrations than bilayer lipids.     

Conversion of the carbohydrate structures of glycoproteins in roots of Raphanus sativus using several glycosidase inhibitors

An attempt was made to convert the N-glycan structures in Raphanus sativus seeds during germination with a view to develop a method for regulating the N-glycan structures using glycosidase inhibitors. The N-glycan structures of glycoproteins in the roots of seedlings germinated for three days were analyzed by hydrazinolysis followed by N-acetylation, pyridylamination and HPLC. Pyridylaminated sugar chains obtained in the absence of the inhibitors had plant type structures consisting of Man(3)FucXylGlcNAc(2)(M3FX), Man(5-9)GlcNAc(2)(high-Man) and GlcNAc(1-2)Man(3)FucXylGlcNAc(2)(GnM3FX and Gn2M3FX). When germinated in the presence of a glucosidase inhibitor (castanospermine or deoxynojirimycin), the amount of glucosyl high-Man-type structure increased and plant growth was inhibited. When germinated in the presence of a mannosidase inhibitor (swainsonine or deoxymannojirimycin), the amount of the high-Man-type structure increased and that of M3FX was low, and the growth was normal. In the presence of 2-acetamido 1, 2 di-deoxynojirimycin, those of GnM3FX and Gn2M3FX increased and the growth was normal. These results show that the N-glycan processing in both the endoplasmic reticulum (ER) and Golgi apparatus can be controlled artificially using glycosidase inhibitors, and that the glucosidase inhibitors could be useful for the study of the function of N-glycans in plants.     

Detailed folding structures of M-lycotoxin-Hc1a and its mutageneses using 2D HP model

Although the hydrophobic-polar (HP) model was proposed a decade ago, it applies almost to no real-case study because of its intense computation. In this study, a 2D HP model was applied to study the folding structures of M-lycotoxin-Hc1a, an antimicrobial peptide, in order to get full pictures of its numerous folding structures. The normalised hydrophobicity index was used to convert M-lycotoxin-Hc1a and its six mutageneses into HP sequences, and then the 2D HP model was used to compute all the possible folding structures (3²⁴ = 282,429,536,481), and finally the normalised hydrophobicity index was used to distinguish the native state. The results showed that M-lycotoxin-Hc1a had 6 and 138 folding structures at their native state with the minimal energy of ? 13 at pH 2 and pH 7 when glycine served as hydrophobic amino acid. When glycine serves as polar amino acid, M-lycotoxin-Hc1a had 12 and 54 folding structures at their native state with the minimal energy of ? 12 and ? 13 at pH 2 and pH 7, respectively. This study advanced the knowledge on how to apply the HP model to real-life study, and how the mutageneses influenced the folding structures of M-lycotoxin-Hc1a, their native states and minimal energy at different pH levels.     

Crystal structure of a covalent intermediate of endogenous human arylsulfatase A

The structures of human arylsulfatase A crystals soaked in solutions containing 4-methylumbelliferyl phosphate and O-phospho-DL-tyrosine have been determined at 2.7- and 3.2-A resolution, respectively. The formylglycine in position 69, a residue crucial for catalytic activity, was unambiguously identified in both structures as forming a covalent bond to the phosphate moiety. A hydroxyl group is present at the Cbeta of residue 69 and the formation of one out of two possible stereomeric forms is strongly favoured. The structures confirm the importance of the gem-diol intermediate in the arylsulfatase's catalytic mechanism. The presence of an apparently stable covalent bond is consistent with the weak phosphatase activity observed for human arylsulfatase A. The structures of the complexes suggest that phosphate ions and phosphate esters inhibit arylsulfatase in non-covalent and covalent modes, respectively. The metal ion present in the active site of arylsulfatase A isolated from human placenta is Ca(2+) and not Mg(2+) as was found in the structure of the recombinant enzyme.     

NMR structure of a DNA duplex containing nucleoside analog 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole and the structure of the unmodified control

The three-dimensional structures of two DNA duplexes d(CATGAGTAC). d(GTACXCATG) (1) and d(CATGAGTAC).d(GTACTCATG) (2), where X represents 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole, were solved using high resolution nuclear magnetic resonance spectroscopy and restrained molecular dynamics. Good convergence was observed between final structures derived from A- and B-form starting geometries for both 1 and 2. Structures of 1 and 2 are right-handed duplexes within the B-form conformational regime. Furthermore, the structures of 1 and 2 are highly similar, with differences in the structures localized to the vicinity of residue 14 (X versus T). The pyrrole group of 1 is in the syn conformation and it is displaced towards the major groove. Furthermore, unlike T14 in 2, the base of X14 has reduced pi-pi stacking interactions with C13 and C15 and the nitro group of X14 is pointing out of the major groove. The structures presented here establish the basis of the thermal data of DNA duplexes containing X and should be informative during the design of improved wild card nucleobase analogs.     

Studies of the solution structure of the bleomycin-A2-zinc complex by means of two-dimensional NMR spectroscopy and distance geometry calculations

Application of various two-dimensional NMR techniques (SECSY, COSY and NOESY) enabled the complete assignment of the 1H-NMR spectrum of the bleomycin-A2-zinc complex in H2O and D2O at pH 6.7. The spectra were interpreted at 277 K as well as at 300 K. Identification of the resonances permitted a vicinal coupling constant analysis which revealed that the conformation around the C alpha and C beta bond of the beta-aminoalanine and beta-hydroxyhistidine residues is fixed. From this finding it was concluded that both amino functions of the beta-aminoalanine fragment and the amide and imidazole groups of the beta-hydroxyhistidine moiety are involved in zinc coordination. Also, for the mannose carbamoyl group and the pyrimidine ring active participation in zinc coordination could be established. NOE data together with the six coordination sites proposed above were used as interpoint distance constraints in distance geometry calculations for the bleomycin-A2-zinc complex in H2O. Sets of ten structures, randomly chosen within the distance constraints, were calculated (with and without the zinc ion). The calculated structures were very similar but in case that the zinc ion was omitted some flexibility was observed, within the distance constraints, in the pyrimidine-aminoalanine region. Because of the great overall similarity between the structures, a reliable representation of the solution conformation of the bleomycin-zinc complex was reached. Surprisingly, no regular symmetry around the zinc ion was found to be present in the generated structures.     

Intracellular Localization of Poliovirus Plus- and Minus-Strand RNA Visualized by Strand-Specific Fluorescent In Situ Hybridization

The time courses of poliovirus plus- and minus-strand RNA synthesis in infected HEp-2 cells were monitored separately, using a quantitative RNase assay. In parallel, viral RNA and proteins were located in situ by confocal microscopy within cells fixed by a protocol determined to retain their native size and shape. Plus- and minus-strand RNAs were visualized by fluorescent in situ hybridization (FISH) with strand-specific riboprobes. The probes were labelled with different fluorochromes to allow for the simultaneous detection of plus- and minus-strand RNA. The FISH experiments showed minus-strand RNA to be present in distinct, regularly sized, round structures throughout the viral replication cycle. Plus-strand RNA was found in the same structures and also in smaller clusters of vesicles. Association of viral RNA with membranes was demonstrated by combining FISH with immunofluorescence (IF) detection of the viral 2B- and 2C-containing P2 proteins, which are known to be markers for virus-induced membranes. At early times postinfection, the virus-induced membranous structures were distributed through most of the cytoplasm, whereas around peak RNA synthesis, both RNA-associated membranous structures migrated to the center of the cell. During this process, the plus- and minus-strand-containing larger structures stayed as recognizable entities, whereas the plus-strand-containing granules coalesced into a juxtanuclear area of membranous vesicles. An involvement of Golgi-derived membranes in the formation of virus-induced vesicles and RNA synthesis early in infection was investigated by IF with 2C- and Golgi-specific antibodies.     

Fourier transform infrared spectroscopic studies of Ca(2+)-binding proteins

The secondary structures of calmodulin and parvalbumin are well established from X-ray diffraction and nuclear magnetic resonance spectroscopic studies, which indicate that these proteins are predominantly alpha-helical in character. Recent infrared studies have nevertheless suggested that the helical structures present in these proteins in solution are not the standard alpha-helix but rather some kind of distorted helices [Trewhella, J., et al. (1989) Biochemistry 28, 1294]. The evidence for this was the unusually low amide I frequency for calmodulin and troponin C in 2H2O solution. The studies presented here, however, suggest that the helical structures in these proteins are not significantly distorted, for two reasons. First, distorted helical structures have weaker hydrogen bonds than the standard alpha-helix and would therefore be expected to absorb at a higher rather than a lower frequency. Second, distorted helical structures would absorb at an unusual frequency in H2O solutions which is not the case for the proteins studied here. The band frequency of these proteins is observed to occur at a frequency observed with other proteins known to contain predominantly alpha-helical structures. Quantitative analysis of the FT-IR spectra of calmodulin (67% alpha-helix) and parvalbumin (68% alpha-helix) in H2O in the presence of Ca2+ gives helical contents similar to those reported by X-ray studies. This raises the question as to why these proteins in H2O show a normal frequency for the presence of alpha-helical structures and an abnormal frequency in 2H2O. Addition of deuterated glycerol to the proteins in 2H2O solutions results in a significant shift of absorbance to higher frequency.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural localization of GP2 in acinar cells of pancreas: presence of GP2 in endocytic and exocytic compartments

GP2 is a glycoprotein found in pancreatic acinar cells. Its subcellular distribution suggests that it may be involved both in exocytosis and endocytosis. Immunocytochemical studies have demonstrated GP2 to be present on the membrane and in the matrix of zymogen granules, on Golgi saccules, on the apical and basolateral surfaces of the plasma membrane, and in the lumina of acini. In addition, this protein was observed in small vacuoles and tubular structures previously identified as "basal lysosomes," "snake-like tubules," and in lysosomes. Because the latter group of structures are involved in endocytosis, it is possible that GP2 may be involved in this phenomenon. GP2 was readily detectable in pancreatic juice and was totally sedimentable by ultracentrifugation, as assessed by Western blot analysis. Induced lysis of isolated zymogen granules also caused release of GP2 in a sedimentable form which, by electron microscopy, appeared as a fibrillar structure. Immunocytochemical localization of amylase was studied in parallel with GP2 and was found in the secretory product to be associated with thread-like structures, presumably the pancreatic thread protein. The physiological significance of these observations is discussed.     

Reconstruction of spatial structure of plant protein phosphatase type-1 and -2A in complex with okadaic acid

The homology modeling, based on known temple structures of Homo sapiens protein phosphatase type-1 and -2A was implemented. The spatial structures of the human protein phosphatases and their plant homologs from Arabidopsis thaliana was predicted. The quality of models was confirmed by conformational analysis and root mean square deviations. The sites of okadaic acid binding in molecules of plant protein phosphatases (type-1 and -2A) were proved by the data of comparative analysis and molecular dynamics.     

Blocked and Methylated 5'-Terminal Cap Structures of Rat Brain Messenger Ribonucleic Acids

Abstract: The presence and identity of 5'-terminal cap structures in rat brain polysomal mRNA were investigated by radiolabeling the mRNA by periodate oxidation and [³H]sodium borohydride reduction or by β -elimination of 5'-terminal nucleoside and incorporation of ³²P in the presence of polynucleotide kinase. The labeled mRNAs were digested with nucleases and the cap structures were isolated and identified by chromatographic and electrophoretic procedures. The results showed that rat brain mRNAs contained cap 1 and cap 2 structures and no caps of the zero type. The proportion of cap 2 was higher than that of cap 1. Both caps had 7-methylguanosine (m⁷G) as the 5'-terminal nucleoside, which was linked to the next nucleoside by an inverted triphosphate bridge, as in other eukaryotic mRNAs. The most prominent nucleoside in the 5'-penultimate position was 6-methyl-2'- O -methyiadenosine [m⁶A(m)] followed by 2'- O -methyladenosine [A(m)], which together contributed to nearly 70% of both cap 1 and cap 2 structures. 2'- O -Methylguanosine [G(m)] accounted for approximately 18%, the rest being made up of 2'- O -methyl-cytidine [C(m)] and 2'- O -methyluridine [U(m)].     

Effects of low temperature and calcium on microfilament structure in flagellates of Physarum polycephalum

Microfilament structures of the ridge and the backbone in Physarum flagellates disintegrated selectively within a few minutes upon cooling by ice-water. The elongated cells concurrently rounded up to spherical or irregular and amoeboid shape. When such rounded cells were warmed to 25 °C, the microfilament structures were reconstructed within 1 min and cells subsequently returned to an elongated shape. Disruption of microfilaments by cytochalasin A also caused the rounding up of cells, indicating that the rounding up resulted from disintegration of microfilament structures. This transformation induced by the cold treatment was retarded by preincubation of the cells with EGTA for 15 min, but addition of EGTA immediately before the onset of the cold treatment was less effective. The effect of EGTA was cancelled by simultaneous addition of excess Ca²⁺. Addition of procaine also inhibited the transformation induced by the cold treatment, while caffeine inhibited the recovery of the elongated shape when returned to 25 °C. Furthermore, addition of A23187 at 25 °C in the presence of Ca²⁺ mimicked the effect of the cold treatment. Thus, intracellular release of Ca²⁺ was suggested to be involved in the transformation induced by the cold treatment. Lability of the microfilament structures at a high concentration of Ca²⁺ was directly proved using Triton-permeabilized cells. Therefore we concluded that low temperature disrupts microfilament structures that are necessary for the maintenance of the elongated cell shape by inducing intracellular Ca²⁺ release. However, microfilament structures in Physarum amoeba cells were affected neither by the cold treatment nor by high Ca²⁺ concentration.