At least six different actins are expressed in a higher mammal: An analysis based on the amino acid sequence of the amino-terminal tryptic peptide

The protein chemical characterization of the amino-terminal tryptic peptide of actin from different bovine tissues shows that at least six different actin structural genes are expressed in this mammal.Unique amirio acid sequences are found for actin from skeletal muscle, for actin from heart muscle, for two different actin species from smooth muscle, and for two different actin species typical of non-muscle tissues such as brain and thymus. The presence of more than one actin species in the same tissue (e.g. nonmuscle tissues and smooth muscles) is demonstrated by different amino-terminal peptides which, however, are closely related. The actins from the sarcomeric muscles (e.g. skeletal muscle and heart muscle) show unique but extremely similar amino-terminal peptides. A limited comparison of bovine and avian actins involving smooth and skeletal muscles emphasizes that among higher vertebrates actin divergence involves tissue rather than species specificity.For the lower eukaryotic organism Physarum polycephalum a single actin amino-terminal peptide is found, indicating that only one actin species is present during the plasmodial stage. The amino acid sequence of this peptide although unique reveals a high degree of homology with the corresponding mammalian cytoplasmic actin peptides.Different actin extraction and purification procedures have been compared by the relative yields of the different amino-terminal peptides. The results indicate that the various actin species obtained by the current purification procedures are a true reflection of the actual actins present in the tissue. In addition we compare the resolution provided by either isoelectric focusing analysis of different actins or by the protein chemical characterization of the amino-terminal peptides of different actins. We show that the latter procedure is more suitable for recording changes in actin expression during evolution and differentiation.     

Actin amino-acid sequences. Comparison of actins from calf thymus, bovine brain, and SV40-transformed mouse 3T3 cells with rabbit skeletal muscle actin.

Actin was purified from calf thymus, bovine brain and SV40-transformed mouse 3T3 cells grown in tissue culture. Isoelectric focusing analysis showed the presence of the two actin polypeptides beta and gamma typical for non-muscle actins in all three actins. Tryptic and thermolytic peptides accounting for the complete amino-acid sequence of the cytoplasmic actins were separated and isolated by preparative fingerprint techniques. All peptides were characterized by amino-acid analysis and compared with the corresponding peptides from rabbit skeletal muscle actin. Peptides which differed in amino-acid composition from the corresponding skeletal muscle actin peptides were subjected to sequence analysis in order to localize the amino-acid replacement. The results obtained show that all three mammalian cytoplasmic actins studied contain the same amino-acid exchanges indicating that mammalian cytoplasmic actins are very similar if not identical in amino-acid sequence. The presence of two different isoelectric species beta and gamma in cytoplasmic actins from higher vertebrates is acccounted for by the isolation of two very similar but not identical amino-terminal peptides in all three actin preparations. The nature of the amino-acid replacements in these two peptides not only accounts for the different isoelectric forms but also shows that beta and gamma cytoplasmic actins are the products of two different structural genes expressed in the same cell. The total number of amino-acid replacements so far detected in the comparison of these cytoplasmic actins and skeletal muscle actin is 25 for the beta chain and 24 for the gamma chain. With the exception of the amino-terminal three or four residues, which are responsible for the isoelectric differences, the replacements do not involve charged amino acids. The exchanges are not randomly distributed. No replacements were detected in regions 18--75 and 299--356 while the regions between residues 2--17 and 259--298 show a high number of replacements. In addition documentation for a few minor revisions of the amino acid sequence of rabbit skeletal muscle actin is provided.     

Peptidomic analysis of skin secretions provides insight into the taxonomic status of the African clawed frogs Xenopus victorianus and Xenopus laevis sudanensis (Pipidae)

Peptidomic analysis was used to compare the distribution of host-defense peptides in norepinephrine-stimulated skin secretions from Xenopus victorianus Ahl, 1924 (also described as the subspecies X. laevis victorianus) and Xenopus laevis sudanensis Perret, 1966 with the previously determined distributions in Xenopus laevis (Daudin, 1802) and Xenopus petersii Bocage, 1895. Peptides belonging to the magainin, peptide glycine-leucine-amide (PGLa), and caerulein precursor fragment (CPF) families were purified by reversed-phase HPLC and characterized by electrospray mass spectrometry. Magainin-P2, PGLa-P1, CPF-P1, CPF-P2, and CPF-P3 previously isolated from X. petersii and structurally different from orthologous peptides from X. laevis, were identified in X. victorianus and X. laevis sudanensis skin secretions whereas the corresponding X. laevis peptides were absent. Magainin-1, identical in X. petersii and X. laevis, was also identified in the secretions. Xenopsin-precursor fragment (XPF) peptides, absent from X. petersii but present in X. laevis skin secretions, were not identified in the X. victorianus and X. laevis sudanensis secretions. The data indicate that X. victorianus and X. laevis sudanensis are more closely related to X. petersii than to X. laevis and support separate species status. The study illustrates the value of analysis of host-defense peptides in the evaluation of taxonomic and phylogenetic relationships between closely related frog species.     

A comparison of host-defense peptides in skin secretions of female Xenopus laevis × Xenopus borealis and X. borealis × X. laevis F1 hybrids

Peptidomic analysis was used to compare the diversity of host-defense peptides in norepinephrine-stimulated skin secretions from laboratory-generated female F1 hybrids of Xenopus laevis and Xenopus borealis (Pipidae). Skin secretions of hybrids with maternal X. laevis (X_LB) contained 12 antimicrobial peptides (AMPs), comprising 8 from X. laevis and 4 from X. borealis. Magainin-B1, XPF-B1, PGLa-B1 CPF-B2, CPF-B3 and CPF-B4 from X. borealis and XPF-1, XPF-2, and CPF-6 from X. laevis were not detected and CPF-1 and CPF-7 were present in low concentration. The secretions contained caerulein and caerulein-B1 derived from both parents but lacked X. laevis xenopsin and X. borealis caerulein-B2. Skin secretions of hybrids with maternal X. borealis (X_BL) contained 14 AMPs comprising 6 from X. borealis and 8 from X. laevis. Magainin-B1, XPF-B1, PGLa-B1, CPF-B2, XPF-1, CPF-5, and CPF-7 were absent and CPF-B3, CPF-B4, CPF-1 and CPF-6 were present only in low concentration. Xenopsin and caerulein were identified in the secretions but caerulein-B2 was absent and caerulein-B1was present in low concentration. No peptides were identified in secretions of either X_LB or X_BL hybrids that were not present in the parental species. The data indicate that hybridization between X. laevis and X. borealis results in increased diversity of host-defense peptides in skin secretions but point to extensive AMP gene silencing compared with previously studied female X. laevis × X. muelleri F1 hybrids and no novel peptide expression.     

Studies on the role of actin's N tau-methylhistidine using oligodeoxynucleotide-directed site-specific mutagenesis

The primary structure of all actins except that isolated from Naegleria gruberi contains a unique N tau-methylhistidine (MeHis) at position 73. This modified residue has been implicated as possibly being important for the post-translational processing of actin's amino terminus, the binding of actin to DNase I, and in the polymerization of G-actin. We have investigated the potential role of MeHis in each of these processes by utilizing site-directed mutagenesis to change His-73 of skeletal muscle actin to Arg and Tyr. Wild type and mutant actins were synthesized in vivo, using non-muscle cells transfected with mutant cDNAs, and in vitro by translating mutant RNAs synthesized using SP6 RNA polymerase in a rabbit reticulocyte lysate. We have found that actins containing Arg or Tyr at position 73 undergo amino-terminal processing, bind to DNase I-agarose, and become incorporated into the cytoskeleton of a nonmuscle cell as efficiently as wild type actin. Furthermore, using an in vitro copolymerization assay we have found that although there is no difference between the Arg mutant and the wild type actins, the Tyr mutant has a slightly greater critical concentration for polymerization. These results show that MeHis is not absolutely required for any of these processes.     

Quantification and localization of erythropoietin-receptor-expressing cells in the liver of Xenopus laevis

Erythropoiesis occurs in the African clawed frog, Xenopus laevis and is mediated by erythropoietin (xlEPO), a primary regulator of this process. Previously, we have shown that the xlEPO receptor (xlEPOR), which is expressed by erythroid progenitors that respond to xlEPO, is found predominantly in the liver. The aim of the present study was to determine the dynamics of erythropoiesis in the livers of normal and anemic X. laevis by identifying the number and precise location of mature and immature erythrocytes. We quantified mature and immature erythrocyte numbers by o-dianisidine staining or immunohistochemistry and investigated the dynamics of erythropoiesis in normal, acute hemolytic and blood-loss states by in vivo cell proliferation assays with 5-bromo-2′-deoxyuridine (BrdU). We detected 0.12×10⁸ xlEPOR⁺ BrdU⁺ cells in the liver of the normal X. laevis at 24 h after BrdU injection. Frogs presenting with acute hemolytic anemia and pancytopenia show a 10-fold increase in the number of xlEPOR⁺/BrdU⁺ cells (approximately 1.30×10⁸ cells) in the liver. The xlEPOR⁺ cells are found predominantly on the inner wall of hepatic sinusoids. Hematopoietic progenitors that undergo slow cell cycling were also observed in the hepatic sinusoids. This study clarifies the rate of production of mature and immature erythrocytes per day in the liver of X. laevis and the way that these cell numbers change in response to anemia.     

Cloning and expression of an actin gene in the haemocytes of pearl oyster (Pinctada fucata, Gould 1850)

An actin gene (designated pfact1) of pearl oyster, Pinctada fucata, was cloned from haemocytes by the techniques of homological cloning and rapid amplification of cDNA ends (RACE). The full length of Pfact1 cDNA was 1608 bp in length, having a 5′ untranslated region (UTR) of 82 bp, a 3′ UTR of 395 bp, and an open reading frame (ORF) of 1131 bp encoding a polypeptide of 376 amino acids with a predicted molecular weight of 41.76 kDa and an estimated isoelectric point of 5.29. Sequence analysis revealed that Pfact1 shared high similarity with other actins and was more closely related to vertebrate cytoplastic actins than muscle types. Phylogenetic analysis indicated that molluscan actins could also be generally grouped into two classes: muscle type and cytoplasmic type, although both are similar to vertebrate cytoplastic actins. Fluorescent real-time quantitative RT-PCR was used to examine the expression level of Pfact1 in haemocytes of P. fucata after the challenge of Vibrio alginolyticus, and results showed that Pfact1 exhibited stable expression in all time points, indicating that Pfact1 could be a suitable internal control for gene expression analysis in haemocytes of P. fucata.     

Cytoskeletal actin gene families ofXenopus borealis andXenopus laevis

Summary We have sequenced the coding and leader regions, as well as part of the 3 untranslated region, of aXenopus borealis type 1 cytoskeletal actin gene [defined according to the arrangement of acidic residues at the N-terminus; Vandekerckhove et al. (1981) J Mol Biol 152:413–426]. The encoded amino acid sequence is the same as the avian and mammalian (type 1) cytoskeletal actins, except for an isoleucine at position 10 (as found in the mammalian cytoskeletal actins), and an extra amino acid, alanine, after the N-terminal methionine. Five introns were found, in the same positions as those of the rat and chicken -actin genes. The 5 and 3 untranslated regions resemble those of the human (type 8) cytoskeletal actin gene more closely than the mammalian genes.Primer extension showed that this type 1 gene is transcribed in ovary and tadpole. Sequencing of primer extension products demonstrated two additional mRNA species inX. borealis, encoding type 7 and 8 isoforms. This contrasts with the closely related speciesXenopus laevis, where type 4, 5, and 8 isoforms have been found. The type 7 isoform has not previously been found in any other species. The mRNAs of theX. borealis type 1 and 8 andX. laevis type 5 and 8 isoforms contain highly homologous leaders. TheX. borealis type 7 mRNA has no leader homology with the other mRNA species and, unlike them, has no extra N-terminal alanine codon. The evolutionary implications of these data are discussed.     

Actin of chara giant internodal cells: a single isoform in the subcortical filament bundles and a larger, immunologically related protein in the chloroplasts

Internodal cells of Chara corallina Klein ex. Wild have been studied to determine the number of actin isoforms they contain and whether actin occurs at locations in the cortical cytoplasm outside the filament bundles. A monoclonal antibody to chicken actin is specific for actin in numerous animal cells but binds to two Chara proteins after their separation by two-dimensional polyacrylamide gel electrophoresis. One protein resembles known actins in relative molecular mass (43,000-M_r) and isoelectric point (5.5) while the other is distinctly different (58,000-M_r, isoelectric point = 4.8). Because it is indetectable in cells whose actin bundles have been extracted, the 43,000-M_r protein is assigned to the bundles and concluded to be rare or absent in the remaining cortical cytoplasm. The 58,000-M_r protein, in contrast, does not extract with the actin bundles. It was localized within the chloroplasts by immunofluorescence and by the dependence of proteolysis on the permeabilization of the chloroplast envelope.     

Evaluation and screening of efficient promoters to improve astaxanthin production in Xanthophyllomyces dendrorhous

Astaxanthin is a valuable carotenoid that is widely used in the aquaculture, food, pharmaceutical, and cosmetic industries. Xanthophyllomyces dendrorhous is a carotenoid-synthesizing yeast strain that produces astaxanthin as its main pigment. Although metabolic engineering using gene manipulation is a valuable way to improve astaxanthin production, a gene expression system for X. dendrorhous has been poorly developed. In this study, three known promoters of X. dendrorhous, glycerol-3-phosphate dehydrogenase (gpd) promoter (Pgpd), glucose dehydrogenase (gdh) promoter (Pgdh), and actin (act) promoter (Pact), were evaluated for use in the overexpression of target proteins using green fluorescence protein (GFP) as an expression level indicator protein. The actin promoter, Pact, showed the highest expression level of GFP when compared with Pgpd and Pgdh. Additionally, to obtain new promoters for higher expression of target protein in X. dendrorhous, intracellular GFP intensity was evaluated for 13 candidate promoters. An alcohol dehydrogenase promoter, Padh4, showed more efficient expression of GFP rather than Pact. Overexpression of crtE gene encoding rate-limiting enzyme of carotenoid synthesis under the adh4 promoter yielded an increase in intracellular astaxanthin content of about 1.7-fold compared with the control strain. The promoters identified in this study must be useful for improving carotenoids production in X. dendrorhous.     

A rapid and sensitive method for the detection of histone peptides

Fluorescamine has been used to obtain a peptide map of a mixture of histones (H₃, H₂A, H₂B, and H₄) prepared from oocytes of Xenopus laevis. Fluorescamine was found to be more sensitive than o-phthalaldehyde or ninhydrin-Cd for the detection of peptide fragments obtained from tryptic digestion of oocyte histones of X. laevis and the peptic digestion of the β chain of insulin. Using the β chain of insulin for a comparison, the 8 major peptide fragments could be separated by electrophoresis within 30 min and were detectable at the picomols level. Some 70 peptide spots of X. laevis oocyte histones were resolved, thus permitting the analysis of this complex mixture of polypeptides without the need for prior separation.     

Structural Differences Explain Diverse Functions of Plasmodium Actins

Actins are highly conserved proteins and key players in central processes in all eukaryotic cells. The two actins of the malaria parasite are among the most divergent eukaryotic actins and also differ from each other more than isoforms in any other species. Microfilaments have not been directly observed in Plasmodium and are presumed to be short and highly dynamic. We show that actin I cannot complement actin II in male gametogenesis, suggesting critical structural differences. Cryo-EM reveals that Plasmodium actin I has a unique filament structure, whereas actin II filaments resemble canonical F-actin. Both Plasmodium actins hydrolyze ATP more efficiently than α-actin, and unlike any other actin, both parasite actins rapidly form short oligomers induced by ADP. Crystal structures of both isoforms pinpoint several structural changes in the monomers causing the unique polymerization properties. Inserting the canonical D-loop to Plasmodium actin I leads to the formation of long filaments in vitro. In vivo, this chimera restores gametogenesis in parasites lacking actin II, suggesting that stable filaments are required for exflagellation. Together, these data underline the divergence of eukaryotic actins and demonstrate how structural differences in the monomers translate into filaments with different properties, implying that even eukaryotic actins have faced different evolutionary pressures and followed different paths for developing their polymerization properties.     

A Single Vegetative Actin Isovariant Overexpressed under the Control of Multiple Regulatory Sequences Is Sufficient for Normal Arabidopsis Development

The relative significance of gene regulation and protein isovariant differences remains unexplored for most gene families, particularly those participating in multicellular development. Arabidopsis thaliana encodes three vegetative actins, ACT2, ACT7, and ACT8, in two ancient and highly divergent subclasses. Mutations in any of these differentially expressed actins revealed only mild phenotypes. However, double mutants were extremely dwarfed, with altered cell and organ morphology and an aberrant F-actin cytoskeleton (e.g., act2-1 act7-4 and act8-2 act7-4) or totally root-hairless (e.g., act2-1 act8-2). Our studies suggest that the three vegetative actin genes and protein isovariants play distinct subclass-specific roles during plant morphogenesis. For example, during root development, ACT7 was involved in root growth, epidermal cell specification, cell division, and root architecture, and ACT2 and ACT8 were essential for root hair tip growth. Also, genetic complementation revealed that the ACT2 and ACT8 isovariants, but not ACT7, fully rescued the root hair growth defects of single and double mutants. Moreover, we synthesized fully normal plants overexpressing the ACT8 isovariant from multiple actin regulatory sequences as the only vegetative actin in the act2-1 act7-4 background. In summary, it is evident that differences in vegetative actin gene regulation and the diversity in actin isovariant sequences are essential for normal plant development.     

Hybridization between the African clawed frogs Xenopus laevis and Xenopus muelleri (Pipidae) increases the multiplicity of antimicrobial peptides in skin secretions of female offspring

Peptidomic analysis was used to compare the distribution of host-defense peptides in norepinephrine-stimulated skin secretions from laboratory-generated female F1 hybrids of the common clawed frog Xenopus laevis (Daudin, 1802) and Mueller's clawed frog Xenopus muelleri (Peters, 1844) with the corresponding distribution in skin secretions from the parent species. A total of 18 peptides were identified in secretions from the hybrid frogs. Eleven peptides (magainin-1, magainin-2, CPF-1, CPF-3, CPF-4, CPF-5, CPF-6, CPF-7, XPF-1, XPF-2, and PGLa) were identified in secretions of both the hybrids and X. laevis. Four peptides (magainin-M1, XPF-M1, CPF-M1, and tigerinin-M1) were previously found in skin secretions of X. muelleri but magainin-M2 and CPF-M2 from X. muelleri were not detected. Three previously undescribed peptides (magainin-LM1, PGLa-LM1, and CPF-LM1) were purified from the secretions of the hybrid frogs that were not detected in secretions from either X. laevis or X. muelleri. Magainin-LM1 differs from magainin-2 from X. laevis by a single amino acid substitution (Gly¹³ → Ala) but PGLa-LM1 and CPF-LM1 differ appreciably in structure from orthologs in the parent species. CPF-LM1 shows potent, broad-spectrum antimicrobial activity and is hemolytic. The data indicate that hybridization increases the multiplicity of skin host-defense peptides in skin secretions. As the female F1 hybrids are fertile, hybridization may represent an adaptive strategy among Xenopus species to increase protection against pathogenic microorganisms in the environment.     

Rapid Nucleotide Exchange Renders Asp-11 Mutant Actins Resistant to Depolymerizing Activity of Cofilin,Leading to Dominant Toxicity in Vivo

Conserved Asp-11 of actin is a part of the nucleotide binding pocket, and its mutation to Gln is dominant lethal in yeast, whereas the mutation to Asn in human α-actin dominantly causes congenital myopathy. To elucidate the molecular mechanism of those dominant negative effects, we prepared Dictyostelium versions of D11N and D11Q mutant actins and characterized them in vitro. D11N and D11Q actins underwent salt-dependent reversible polymerization, although the resultant polymerization products contained small anomalous structures in addition to filaments of normal appearance. Both monomeric and polymeric D11Q actin released bound nucleotides more rapidly than the wild type, and intriguingly, both monomeric and polymeric D11Q actins hardly bound cofilin. The deficiency in cofilin binding can be explained by rapid exchange of bound nucleotide with ATP in solution, because cofilin does not bind ATP-bound actin. Copolymers of D11Q and wild type actins bound cofilin, but cofilin-induced depolymerization of the copolymers was slower than that of wild type filaments, which may presumably be the primary reason why this mutant actin is dominantly toxic in vivo. Purified D11N actin was unstable, which made its quantitative biochemical characterization difficult. However, monomeric D11N actin released nucleotides even faster than D11Q, and we speculate that D11N actin also exerts its toxic effects in vivo through a defective interaction with cofilin. We have recently found that two other dominant negative actin mutants are also defective in cofilin binding, and we propose that the defective cofilin binder is a major class of dominant negative actin mutants.     

Actin Heterogeneity in primary embryonic culture cells from Drosophila melanogaster.

We have investigated actin heterogeneity in differentiating primary embryonic cell cultures from Drosophila melanogaster. Proteins labeled with [³⁵S]methionine have been separated using O'Farrell two-dimensional gel electrophoresis. Cultures of heterogeneous cell types synthesize at least three major forms of actin, each differing slightly in isoelectric point. We have used a cell separation technique based on differential cell adhesion in the presence of ethylene glycol-bis(β-aminoethyl ether) N,N′-tetraacetate to prepare cultures either highly enriched for, or highly depleted of, myogenic cells. Postfusion myogenic cells synthesize predominantly the most acidic actin form (actin I), while nonmyogenic cells synthesize almost exclusively the two more basic forms (actins II and III). Synthesis of actins at earlier intervals in myogenic cell differentiation in vitro has also been examined. Immediately prior to the onset of myoblast fusion, the synthesis of actin I represents approximately 40% of total actin synthesis. As myogenic cell differentiation progresses this actin form is synthesized at an increasing rate, relative to actins II and III. Drosophila appears to be quite similar to vertebrates with regard to the number of actin species synthesized, as well as to cell and developmental specificity of actin synthesis.     

The BH4 domain of Bcl-2 orthologues from different classes of vertebrates can act as an evolutionary conserved inhibitor of IP3 receptor channels

Ca²⁺ signalling plays an important role in various physiological processes in vertebrates. In mammals, the highly conserved anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein is an important modulator of the inositol 1,4,5-trisphosphate receptor (IP₃R), i.e. the main intracellular Ca²⁺ - release channel located at the endoplasmic reticulum (ER). The Bcl-2 Homology (BH) 4 domain of Bcl-2 (BH4-Bcl-2) is a critical determinant for inhibiting IP₃Rs, by directly targeting a region in the modulatory domain of the receptor (domain 3). In this paper, we aimed to track the evolutionary history of IP₃R regulation by the BH4 domain of Bcl-2 orthologues from different classes of vertebrates, including Osteichthyes, Amphibia, Reptilia, Aves and Mammalia. The high degree of conservation of the BH4 sequences correlated with the ability of all tested peptides to bind to the domain 3 of mouse IP₃R1 in GST-pull downs and their overall ability to inhibit IP₃-induced Ca²⁺ release (IICR) in permeabilized cells. Nevertheless, the BH4 domains differed in their potency to suppress IICR. The peptide derived from X. laevis was the least potent inhibitor. We identified a critical residue in BH4-Bcl-2 from H. sapiens, Thr7, which is replaced by Gly7 in X. laevis. Compared to the wild type X. laevis BH4-Bcl-2, a “humanized” version of the peptide (BH4-Bcl-2 Gly7Thr), displayed increased IP₃R-inhibitory properties. Despite the differences in the inhibitory efficiency, our data indicate that the BH4 domain of Bcl-2 orthologues from different classes of vertebrates can act as a binding partner and inhibitor of IP₃R channels.     

Peptide-containing fraction from a culture medium of Fusarium sambucinum: composition and biological effects

The culture fluid of the fungus Fusarium sambucinum was investigated for the presence of new peptide-containing bioregulators, previously identified in various mammalian and plant tissues. A fraction containing peptides with molecular weights from 1000 to 2000 Da, which exhibited specific membranotropic activity and a number of physical and chemical properties characteristic of this group of bioregulators, was obtained. The effects of this fraction on the model roller organotypic cultivation of liver tissue of the Pleurodeles waltl newt in vitro were investigated for the first time. This fraction caused the additional activation of pigmented liver cells of newt (analogues to Kupffer cells of the liver of mammals) and provided the maintenance of cell-cell adhesive interactions in tissues. The results show that a new group of peptide bioregulators was present in the culture medium of the fungus F. sambucinum.     

The Complete Amino Acid Sequence of Actins from Bovine Aorta, Bovine Heart, Bovine Fast Skeletal Muscle, and Rabbit, Slow Skeletal Muscle

Complete amino acid sequences for four mammalian muscle actins are reported: bovine skeletal muscle actin, bovine cardiac actin, the major component of bovine aorta actin, and rabbit slow skeletal muscle actin. The number of different actins in a higher mammal for which full amino acid sequences are now available is therefore increased from two to five. Screening of different smooth muscle tissues revealed in addition to the aorta type actin a second smooth muscle actin, which appears very similar if not identical to chicken gizzard actin. Since the sequence of chicken gizzard actin is known, six different actins are presently characterized in a higher mammal.
The two smooth muscle actins—bovine aorta actin and chicken gizzard actin—differ by only three amino acid substitutions, all located in the amino-terminal end. In the rest of their sequences both smooth muscle actins share the same four amino acid substitutions, which distinguish them from skeletal muscle actin. Cardiac muscle actin differs from skeletal muscle actin by only four amino acid exchanges. No amino acid substitutions were found when actins from rabbit fast and slow skeletal muscle were compared.
In addition we summarize the amino acid substitution patterns of the six different mammalian actins and discuss their tissue specificity. The results show a very close relationship between the four muscle actins in comparison to the nonmuscle actins. The amino substitution patterns indicate that skeletal muscle actin is the highest differentiated actin form, whereas smooth muscle actins show a noticeably closer relation to nonmuscle actins. By these criteria cardiac muscle actin lies between skeletal muscle actin and smooth muscle actins.     

Detection of hypoxia in the pulmonary tissues of Xenopus laevis over repeated dives

The oxygen environment in African clawed frogs (Xenopus laevis) continuously changes during their development, which involves a rapid increase in the body size, metamorphosis, and transition to adulthood. Nevertheless, there are limited reports on experimental models that are available for studying fluctuations in the oxygen environment in X. laevis. Thus, this study aimed to develop an experimental model on intermittent hypoxia in X. laevis and evaluate hypoxia and oxidative stress in the same. X. laevis were submerged in water with a dissolved oxygen concentration of 2 mg/L for 30 min; they were then removed from the water and allowed to freely absorb oxygen for 5 min. Immunostaining of pimonidazole-containing frozen tissue sections of the lung and liver using anti-pimonidazole antibodies as the hypoxia probes revealed that more than 95% of the submerged X. laevis cells were pimonidazole positive, providing direct evidence of tissue hypoxia. When the amount of oxidative stress in the lungs and liver was evaluated in terms of the amount of lipid peroxides, the diving group showed a 2.08-fold and 3.20-fold increase over the normal group, respectively. Following hypoxia exposure, the dry-to-wet weight ratios of the lung tissues was 1.27 times higher (p < .05), while the liver tissues was 1.06 times higher (although not significant). Thus, the degree of damage depended on the tissues affected. In the future, we believe that this model will be a promising option for analyzing the physiological responses of X. laevis to hypoxia and oxidative stress.