Interactions between cytoskeletal components during myoplasm rearrangement in ascidian eggs

Ooplasmic segregation in ascidian eggs consists of two phases of cytoplasmic movement, the first phase is mediated by the microfilament system and the second is mediated by the microtubule system. Recently, two novel proteins, p58 and myoplasmin-C1, which are localized to the myoplasm, were suggested to have important roles in muscle differentiation. In order to analyze the molecular mechanisms underlying ooplasmic segregation, the interactions between actin, tubulin, p58 and myoplasmin-C1 were examined. During the first segregation, microtubule meshwork in the unfertilized egg disappeared. At the second segregation, a novel structure of the microtubules that extended from the sperm aster and localized in the cortical region of the myoplasm was found. Moreover, uniform distribution of the cortical actin filament was observed at the second segregation. During the course of myoplasm rearrangement, p58 and myoplasmin-C1 are colocalized and can form a molecular complex in vitro. This complex of p58 and myoplasmin-C1 is a good candidate for a cytoskeletal component of the myoplasm, and is likely to be involved in the correct distribution of cytoplasmic determinants.     

Characterization of Antigenic Polypeptides Recognized by Monoclonal Antibodies Specific to the Myoplasm of Eggs of the Ascidian Ciona intestinalis

The determinants responsible for the differentiation of ascidian larval muscle cells are thought to be contained within the egg myoplasm. To analyze the macromolecules composing the myoplasm, several hybridoma cell lines which secrete monoclonal antibodies specific to myoplasmic components of Ciona eggs have been established (17). In the present investigation, seven of these myoplasm-specific antigens were characterized according to their molecular features and distribution patterns within the egg cytoplasm. Four of the seven antigenic polypeptides were shown to be components of the cortical cytoplasm, two were related to mitochondria, and one is likely to be a yolk protein. An antigen recognized by IIG6B2 antibody, which inhibited muscle development when injected into fertilized eggs, was a single polypeptide of relative molecular mass about 40,000 and isoelectric point about 5. The antigen was designated myoplasmin-C1 after its characteristic localization. The IIF9E9 antigen was a single 35-kDa polypeptide related to mitochondria and was thus designated myoplasmin-M1. The other five antibodies recognized two or more spots by immunoblotting analysis using two-dimensional gel electrophoresis. All of these myoplasm-specific antigens, except for the IIH10D6 antigen, are likely to be produced by the oocyte itself. Synthesis of IIH10D6 antigen seems to be associated with test cells.     

The myoplasm of ascidian eggs: a localized cytoskeletal domain with multiple roles in embryonic development

The myoplasm of ascidian eggs is a localized cytoplasmic region containing a unique cytoskeletal domain. During ooplasmic segregation, the myoplasm moves first to the vegetal pole and then to the future posterior region of the fertilized egg, where it subsequently enters the muscle cell lineage during cleavage. In the vegetal pole region, the myoplasm defines a developmental center which later controls gastrulation and embryonic axis formation. In the posterior region, the myoplasm defines another developmental center, which specifies muscle cell development. Evidence is described suggesting that the integrity of the myoplasmic cytoskeletal domain is required for normal embryonic functions of the myoplasm.     

Identification of a cytoskeletal protein localized in the myoplasm of ascidian eggs: localization is modified during anural development

The myoplasm of ascidian eggs is a localized cytoskeletal domain that is segregated to presumptive larval tail muscle cells during embryonic development. We have identified a cytoskeletal protein recognized by a vertebrate neurofilament monoclonal antibody (NN18) which is concentrated in the myoplasm in eggs and embryos of a variety of ascidian species. The NN18 antigen is localized in the periphery of unfertilized eggs, segregates with the myoplasm after fertilization, and enters the larval tail muscle cells during embryonic development. Western blots of one-dimensional and two-dimensional gels showed that the major component recognized by NN18 antibody is a 58 x 10(3) Mr protein (p58), which exists in at least three different isoforms. The enrichment of p58 in the Triton X-100-insoluble fraction of eggs and its reticular staining pattern in eggs and embryos suggests that it is a cytoskeletal protein. In subsequent experiments, p58 was used as a marker to determine whether changes in the myoplasm occur in eggs of anural ascidian species, i.e. those exhibiting a life cycle lacking tadpole larvae with differentiated muscle cells. Although p58 was localized in the myoplasm in eggs of four urodele ascidian species that develop into swimming tadpole larvae, this protein was distributed uniformly in eggs of three anural ascidian species. The eggs of two of these anural species contained the actin lamina, another component of the myoplasm, whereas the third anural species lacked the actin lamina. There was no detectible localization of p58 after fertilization or segregation into muscle lineage cells during cleavage of anural eggs. NN18 antigen was uniformly distributed in pre-vitellogenic oocytes and then localized in the perinuclear zone during vitellogenesis of urodele and anural ascidians. Subsequently, NN18 antigen was concentrated in the peripheral cytoplasm of post-vitellogenic oocytes and mature eggs of urodele, but not anural, ascidians. It is concluded that the myoplasm of ascidian eggs contains an intermediate filament-like cytoskeletal network which is missing in anural species that have modified or eliminated the tadpole larva.     

Myoplasmic impedance of the barnacle muscle fiber.

Myoplasmic impedance was measured on a barnacle (Balanus nubilus) single muscle fiber that was placed in a cylindrical cavity to limit the volume and prevent the hydration of the myoplasm. At both ends of the cavity, the myoplasm was in direct contact with an electrolyte solution. When equilibrium with the external medium was reached, the myoplasmic impedance was measured at 10 degrees C with an impedance bridge at 1000 Hz. The results indicated that the myoplasmic impedance of the muscle fiber is mainly resistive. Treating the myoplasm as a suspension of small conductive particles, we deduced the specific conductivity of the contractile filaments kf and their volume fraction rho (kf = 2.78 X 10(-3) omega-1cm-1, and rho = 0.48). The experimental technique permits an estimate of the specific myoplasmic conductivity in vivo (6.27 X 10(-3) omega-1cm-1). Finally, a decrease in the pH of the external solution from 10.1 to 4.0 lowered the myoplasmic conductivity by 16%. This may be considered as indirect evidence that the conductivity of the contractile filaments is associated with the protein counter-ions, since Hinke et al. (1973. Ann. N.Y. Acad. Sci. 204, 274-296.) reported evidence that a lowering of pH decreases the number of counter-ions.     

Calcium Binding and Tension Development in Detergent-Treated Muscle Fibers

The nonionic detergent Brij 58 eliminates irreversibly the capability of the sarcoplasmic reticulum (SR) of skinned crayfish muscle fibers to sequester Ca and to release it under appropriate stimulation. In contrast to deoxycholate (DOC) which causes an irreversible diminution of tension as well, Brij 58 does not affect the contractile proteins. Comparison of the time-course of tension development before and after Brij treatment demonstrates that Ca is accessible to the contractile proteins more rapidly after the SR is destroyed but, nevertheless, much more slowly than is predicted for free diffusion of Ca in the myoplasm. Slowing apparently results because of the presence of ca 1 mmol/kg fiber of myoplasmic Ca-binding sites that remain after Ca uptake of the SR is eliminated. A theoretical model is presented which allows for the effects of binding sites and of an unstirred layer in the vicinity of the fiber on Ca diffusion into the myoplasm.     

Style-specific and developmentally regulated accumulation of a glycosylated thaumatin/PR5-like protein in Japanese pear (Pyrus serotina Rehd.)

The stylar proteins of Japanese pear (Pyrus serotina Rehd.) were analyzed by two-dimensional gel electrophoresis, and a 32-kDa protein with an isoelectric point of 4.8 was found to be a major component in the style. The 32-kDa protein was a soluble glycoprotein which reacted with concanavalin A. The 32-kDa protein specifically accumulated in the style in a developmentally regulated manner, but was not detected in the other floral organs and leaves. An oligonucleotide representing the N-terminal amino acid sequence of the 32-kDa protein was used to amplify a cDNA fragment by polymerase chain reaction (PCR). The generated PCR product was used to screen a style cDNA library. The selected cDNA clone encoded 244 amino acid residues containing the N-terminal sequence of the 32-kDa protein. The N-terminus of the protein was preceded by putative signal peptide of 22 amino acid residues. The 32-kDa protein showed significant homology with the thaumatin/PR5-like proteins, and was named PsTL1 (Pyrus serotina thaumatin-like protein 1). The possible biological role of PsTL1 in the styles is discussed. Received: 27 November 1997 / Accepted: 19 January 1998     

Structure and expression of the gene encoding ribosomal protein S1 from the cyanobacterium Synechococcus sp. strain PCC 6301: striking sequence similarity to the chloroplast ribosomal protein CS1

We isolated a 38 kDa ssDNA-binding protein from the unicellular cyanobacterium Synechococcus sp. strain PCC 6301 and determined its N-terminal amino acid sequence. A genomic clone encoding the 38 kDa protein was isolated by using a degenerate oligonucleotide probe based on the amino acid sequence. The nucleotide sequence and predicted amino acid sequence revealed that the 38 kDa protein is 306 amino acids long and homologous to the nuclear-encoded 370 amino acid chloroplast ribosomal protein CS1 of spinach (48% identity), therefore identifying it as ribosomal protein (r-protein) S1. Cyanobacterial and chloroplast S1 proteins differ in size from Escherichia coli r-protein S1 (557 amino acids). This provides an additional evidence that cyanobacteria are closely related to chloroplasts. The Synechococcus gene rps1 encoding S1 is located 1.1 kb downstream from psbB, which encodes the photosystem 11 P680 chlorophyll a apoprotein. An open reading frame encoding a potential protein of 168 amino acids is present between psbB and rps1 and its deduced amino acid sequence is similar to that of E. coli hypothetical 17.2 kDa protein. Northern blot analysis showed that rps1 is transcribed as a monocistronic mRNA.     

Absorbance signals from resting frog skeletal muscle fibers injected with the pH indicator dye, phenol red

Singly dissected twitch fibers from frog muscle were studied on an optical bench apparatus after micro-injection with the pH indicator dye, phenol red. Dye-related absorbances in myoplasm, denoted by A0(lambda) and A90(lambda), were estimated as a function of wavelength lambda (450 nm less than or equal to lambda less than or equal to 640 nm) with light polarized parallel (0 degrees) and perpendicular (90 degrees) to the fiber axis respectively. At all lambda, A0(lambda) was slightly greater than A90(lambda), indicating that some of the phenol red molecules were bound to oriented structures accessible to myoplasm. The phenol red "isotropic" signal, [A0(lambda) + 2A90(lambda)]/3, a quantity equal to the average absorbance of all the dye molecules independent of their orientation, had a spectral shape that was red-shifted by approximately 10 nm in comparison with in vitro dye calibration curves measured in 140 mM KCl. The red-shifted spectrum also indicates that some phenol red molecules were bound in myoplasm. A quantitative estimate of indicator binding was obtained from measurements of the dye's apparent diffusion constant in myoplasm, denoted by Dapp. The small value of Dapp, 0.37 x 10(-6) cm2 s-1 (at 16 degrees C), can be explained if approximately 80% of the dye was bound to myoplasmic sites of low mobility. To estimate the apparent myoplasmic pH, denoted by pHapp, the isotropic absorbance of phenol red was fitted by in vitro calibration spectra. pHapp was found to be independent of dye concentration (0.2-2 mM), but varied widely (range, 6.8-7.5; mean value, 7.17) among fibers judged from functional characteristics to be normal. When fibers were subjected to acid or alkaline loads by exposure to Ringer's solution containing, respectively, dissolved CO2 or NH3, the changes in pHapp were in agreement with those expected from pH micro-electrode studies. It is concluded that in spite of the several indications for the presence of bound phenol red inside muscle cells, the pHapp signal from the indicator is useful for monitoring changes in myoplasmic pH in response to physiological and pharmacological manipulations.     

Molecular cloning of yeast cytochrome C-like polypeptide expressed in human lung carcinoma: An antigen recogizable by lung cancer-specific human monoclonal antibody

Summary We previously determined the amino acid sequence to the epitope (ATLFKTR) of cytochrome c fromCandida krusei, which is cross-reactive to the lung cancer-specific human monoclonal antibody HB4C5. Here we report that an antigen messenger RNA, which codes for a structure similar to the cytochrome c epitope, is expressed in the human lung adenocarcinoma A549. Sequencing analysis has revealed that this messenger RNA encodes a novel 190 amino acid polypeptide of 21-kDa containing an amino acid sequence (ALLFFT) similar to the cytochrome c epitope, although the total messenger RNA sequence is apparently different from the cytochrome c messenger RNA. Western analysis indicated that an antibody-recognizable 21-kDa antigen which has the same molecular weight as the predicted polypeptide is expressed in the A549 adenocarcinoma. Thein vitro translated product of the antigen messenger RNA and synthesized ALLFFT peptide were both shown to be reactive with the monoclonal antibody, indicating that this protein contains the epitope which enables A549 cells to specifically react with the antibody. The antigen mRNA was not expressed in non-transformed fibroblasts, suggesting that the antigen mRNA expression was associated with cellular transformation. Also in part of the antigen nucleotide sequence, there was a segment that had about 90% homology to the long terminal repeat sequence (no. 297–475) of the human endogenous retrovirus HERV-K10, which was related to the mouse mammary tumor virus.     

Primary Structure, sequence analysis, and expression of the thermostable d - hydantoinase from Bacillus stearothermophilus SD1

The gene coding for the thermostable d-hydantoinase from the thermophilic bacterium Bacillus stearothermophilus SD1 was cloned and its nucleotide sequence was completely determined. The d-hydantoinase protein showed considerable amino acid sequence homology (20–28%) with other hydantoinases and functionally related allantoinases and dihydroorotases. Strikingly the sequence of the enzyme from B. stearothermophilus SD1 exhibited greater than 89% identity with hydantoinases from thermophilic bacteria. Despite the extremely high amino acid homology among the hydantoinases from thermophiles, the C-terminal regions of the enzymes were completely different in both sequence and predicted secondary structure, implying that the C-terminal region plays an important role in determining the biochemical properties of the enzymes. Alignment of the sequence of the d-hydantoinase from B. stearothermophilus SD1 with those of other functionally related enzymes revealed four conserved regions, and five histidines and an acidic residue were found to be conserved, suggesting a close evolutionary relationship between all these enzymes. Received: 20 December 1996 / Accepted: 12 March 1997     

Protein osmotic pressure and the state of water in frog myoplasm

We measured the osmotic pressure of diffusible myoplasmic proteins in frog (Rana temporaria) skeletal muscle fibers by using single Sephadex beads as osmometers and dialysis membranes as protein filters. The state of the myoplasmic water was probed by determining the osmotic coefficient of parvalbumin, a small, abundant diffusible protein distributed throughout the fluid myoplasm. Tiny sections of membrane (3.5- and 12-14-kDa cutoffs) were juxtaposed between the Sephadex beads and skinned semitendinosus muscle fibers under oil. After equilibration, the beads were removed and calibrated by comparing the diameter of each bead to its diameter measured in solutions containing 3-12% Dextran T500 (a long-chain polymer). The method was validated using 4% agarose cylinders loaded with bovine serum albumin (BSA) or parvalbumin. The measured osmotic pressures for 1.5 and 3.0 mM BSA were similar to those calculated by others. The mean osmotic pressure produced by the myoplasmic proteins was 9.7 mOsm (4 degrees C). The osmotic pressure attributable to parvalbumin was estimated to be 3.4 mOsm. The osmotic coefficient of the parvalbumin in fibers is approximately 3.7 mOsm mM(-1), i.e., roughly the same as obtained from parvalbumin-loaded agarose cylinders under comparable conditions, suggesting that the fluid interior of muscle resembles a simple salt solution as in a 4% agarose gel.     

Myoplasmic fixed charges of the barnacle muscle fiber (author's transl)]

The experimental model used to study diffusion and electrical conduction in the cytoplasm of large muscle fibers was adapted to evaluate the myoplasmic density of fixed charges. Membranes of myoplasm were prepared and phi X, the myoplasmic thermodynamically effective charge density, was calculated from the membrane potential (Kamo, N., Toyoshima, Y. and Kobatake, Y. (1971) Kolloid Z.u.Z. Polymère 1061--1068) when these membranes were used as the partition between two electrolyte solutions. The dilution of KCl in the external solutions reduced phi X, which increases with the reduction of the water content in the membrane of myoplasm. With a water content of 73.0 ml/100 g KCl concentration in the external medium equal to 0.15 M, phi X was evaluated to 0.058 equiv/l. The substitution of KCl by NaCl introduces a reduction in phi X of 20--50% depending on E1KCl] in the external solutions. The addition of ATP, Mg2+, and Ca2+ also causes a reduction of phi B by 30--50% according to the experimental conditions. These results indicate that the fraction of counterions dissociated from the myoplasmic macromolecules is reduced when the concentration of the counterions is diminished or when CKl is replaced by Nal. It also suggests a reduction of phi X during muscular contraction.     

Myoplasmic binding of fura-2 investigated by steady-state fluorescence and absorbance measurements.

Binding of the fluorescent Ca2+ indicator dye fura-2 by intracellular constituents has been investigated by steady-state optical measurements. Fura-2's (a) fluorescence intensity, (b) fluorescence emission anisotropy, (c) fluorescence emission spectrum, and (d) absorbance spectra were measured in glass capillary tubes containing solutions of purified myoplasmic proteins; properties b and c were also measured in frog skeletal muscle fibers microinjected with fura-2. The results indicate that more than half, and possibly as much as 85%, of fura-2 molecules in myoplasm are in a protein-bound form, and that the binding changes many properties of the dye. For example, in vitro characterization of the Ca2+-dye reaction indicates that when fura-2 is bound to aldolase (a large and abundant myoplasmic protein), the dissociation constant of the dye for Ca2+ is three- to fourfold larger than that measured in the absence of protein. The problems raised by intracellular binding of fura-2 to cytoplasmic proteins may well apply to cells other than skeletal muscle fibers.     

The H8 antigen of pathogenic Neisseriae

We cloned and sequenced the H8 gene from N. meningitidis FAM18. The predicted amino acid sequence included a consensus lipoprotein signal sequence processing site, consistent with lipid modification that could account for the unusual electrophoretic and solubilization properties of H8. The amino acid sequence was rich in alanine and proline, especially in an imperfectly periodic region near the amino terminus, which encompassed the epitope recognized by available monoclonal antibodies. In a panel of neisserial strains, the presence of DNA homologous to the H8 gene correlated with the expression of an H8 protein. We cloned a gene from N. meningitidis JB515 that was distinct from the H8 gene but encoded a protein also recognized by an anti-H8 monoclonal antibody. Mice were not protected from meningococcemia by passive immunization with such an antibody.     

Bioinformatic analysis of the TonB protein family

TonB is a protein prevalent in a large number of Gram-negative bacteria that is believed to be responsible for the energy transduction component in the import of ferric iron complexes and vitamin B₁₂ across the outer membrane. We have analyzed all the TonB proteins that are currently contained in the Entrez database and have identified nine different clusters based on its conserved 90-residue C-terminal domain amino acid sequence. The vast majority of the proteins contained a single predicted cytoplasmic transmembrane domain; however, nine of the TonB proteins encompass a ∼290 amino acid N-terminal extension homologous to the MecR1 protein, which is composed of three additional predicted transmembrane helices. The periplasmic linker region, which is located between the N-terminal domain and the C-terminal domain, is extremely variable both in length (22–283 amino acids) and in proline content, indicating that a Pro-rich domain is not a required feature for all TonB proteins. The secondary structure of the C-terminal domain is found to be well preserved across all families, with the most variable region being between the second α-helix and the third β-strand of the antiparallel β-sheet. The fourth β-strand found in the solution structure of the Escherichia coli TonB C-terminal domain is not a well conserved feature in TonB proteins in most of the clusters. Interestingly, several of the TonB proteins contained two C-terminal domains in series. This analysis provides a framework for future structure-function studies of TonB, and it draws attention to the unusual features of several TonB proteins. Byron C. H. Chu and R. Sean Peacock contributed equally to this work.     

Ooplasmic segregation of the myoplasmic actin network in stratified ascidian eggs

Summary Ooplasmic segregation in ascidians includes the movement of the myoplasm, a pigmented cytoplasmic region thought to be involved in the determination of the embryonic muscle and mesenchyme cell lineages, into the vegetal hemisphere of the egg. A myoplasmic cytoskeletal domain (MCD), composed of a cortical actin network (the PML) and an underlying filamentous lattice extending deep into the cytoplasm, is present in this region. The MCD gradually recedes into the vegetal hemisphere during ooplasmic segregation. It has been proposed that the segregation of the myoplasm is mediated by the contraction of the PML. To test this possibility we have examined ooplasmic segregation in eggs in which the internal parts of the MCD were separated from the PML by centrifugal force. Transmission and scanning electron microscopy of eggs extracted with Triton X-100 showed that the PML remained intact when the internal portions of the MCD were displaced and stratified by centrifugation. When stratified eggs were fertilized there were no rearrangements of the visible cytoplasmic inclusions, but the cellular deformations and the recession of the PML characteristic of ooplasmic segregation occurred as usual. The results indicate that the recession of the PML occurs independently of the internal constituents of the MCD and suggest that PML contraction is the motive force for ooplasmic segregation.     

Polymorphisms of <Emphasis Type="Italic">β</Emphasis>-<Emphasis Type="Italic">defensin</Emphasis> genes in Valle del Belice dairy sheep

The aim of this work was to study β-defensin 1 (SBD1) and β-defensin 2 (SBD2) genes in Valle del Belice dairy sheep in order to identify polymorphisms that can be utilized as markers of the analyzed genes, and search for the functional effects and roles of the identified polymorphisms (variation of the amino acid sequence of the protein and stability of mRNA molecule). The study was conducted on 300 randomly selected animals belonging to four flocks. A total of seven SNPs were identified, two in SBD1 and five in SBD2. The two SNPs identified in SBD2 coding region, at position 1659 and position 1667, were non-synonymous, leading to amino acid changes in the protein product. Nevertheless, the functional effects predicted by the two SNPs demonstrated that amino acid substitutions may not have effect on β-defensin 2 protein function. Moreover, we demonstrated that SBD2 mutant sequence shows changes in mRNA secondary structure. These results suggest that identified SNPs could play a role in the modulation of the immune response.