Early development of the cephalic skeleton of Barbus barbus (Teleostei, Cyprinidae)

The inception, and development of the cephalic skeleton of Barbus barbus from hatching to 24 days passes through periods of fast and slow growth; these rates are not the same in different parts of the skull. Trabeculae, parachordal plates, Meckelian cartilages and hyposymplectics are present at hatching. Then the cartilaginous floor of the neurocranium develops, the pars quadrata, the hyoid bars and branchial arches elements appear shortly before the first movable dermal bones, the dentaries, maxillae and opercles. The first bone of the braincase to appear is the parasphenoid; other bones develop subsequently and at the same time: the angular, quadrate, interopercle and fifth ceratobranchial. Later the splanchnocranium continues to develop at a relatively fast rate while the neurocranium shows little growth. The braincase does not begin to close before the 24th day, nor do the first bones of the skull roof appear, while the bucco-pharyngeal apparatus is complete, having the adult shape. The early constitution of the latter structures seems to be linked with the mechanical demands of biological functions such as breathing and feeding.     

Alternative splicing of smooth muscle myosin heavy chains and its functional consequences

The aim of our study was to determine the relation between alternatively spliced myosin heavy chain (MHC) isoforms and the contractility of smooth muscle. The relative amount of MHC with an alternatively spliced insert in the 5′ (amino terminal) domain was determined on the protein level using a peptide-directed antibody (a25K/50K) raised against the inserted sequence (QGPSFAY). Smooth muscle MHC isoforms of both bladder and myometrium but not nonmuscle MHC reacted with a25/50K. Using a quantitative Western-blot approach the amount of 5′-inserted MHC in rat bladder was detected to be about eightfold higher than in normal rat myometrium. The amount of heavy chain with insert was found to be decreased by about 50% in the myometrium of pregnant rats. Although bladder contained significantly more 5′-inserted MHC than myometrium, apparent maximal shortening velocities (Vmax) were comparable, being 0.138 ± 0.012 and 0.114 ± 0.023 muscle length per second of skinned bladder and normal myometrium fibers, respectively. Phosphorylation of myosin light chain 20 induced by maximal Ca²⁺/calmodulin activation was the same in bladder and myometrial fibers. These results suggest that the amount of 5′-inserted MHC is not necessarily associated with contractile properties of smooth muscle. © 1996 Wiley-Liss, Inc.     

Alternative splicing and cycling kinetics of myosin change during hypertrophy of human smooth muscle cells

We investigated in vivo expression of myosin heavy chain (MHC) isoforms, 17 kDa myosin light chain (MLC₁₇), and phosphorylation of the 20 kDa MLC (MLC₂₀) as well as mechanical performance of chemically skinned fibers of normal and hypertrophied smooth muscle (SM) of human myometrium. According to their immunological reactivity, we identified three MHC isoenzymes in the human myometrium: two SM-MHC (SM1 with 204 kDa and SM2 with 200 kDa), and one non-muscle specific MHC (NM with 196 kDa). No cross-reactivity was detected with an antibody raised against a peptide corresponding to a seven amino acid insert at the 25K/50K junction of the myosin head (a-25K/50K) in both normal and hypertrophied myometrium. In contrast, SM-MHC of human myomatous tissue strongly reacted with a-25K/50K. Expression of SM1/SM2/NM (%) in normal myometrium was 31.7/34.7/33.6 and 35.1/40.9/24 in hypertrophied myometrium. The increased SM2 and decreased NM expression in the hypertrophied state was statistically significant (P < 0.05). MHC isoform distribution in myomatous tissue was similar to normal myometrium (35.3/35.3/29.4). In vivo expression of MLC_17a increased from 25.5% in normal to 44.2% in hypertrophied (P < 0.001) myometrium. Phosphorylation levels of MLC₂₀ upon maximal Ca²⁰-calmodulin activation of skinned myometrial fibers were the same in normal and hypertrophied myometrial fibers. Maximal force of isometric contraction of skinned fibers (pCa 4.5, slack-length) was 2.85 mN/mm² and 5.6 mN/mm² in the normal and hypertrophied state, respectively (P < 0.001). Apparent maximal shortening velocity (Vmax_app, extrapolated from the force-velocity relation) of myometrium rose from 0.13 muscle length s ¹ (ML/s) in normal to 0.24 ML/s in hypertrophied fibers (P < 0.001). J. Cell. Biochem, 64:171–181. © 1997 Wiley-Liss, Inc.     

Molecular identification (RAPD) of the eight species of the genus Barbus (Cyprinidae) in the Iberian Peninsula

Random amplified polymorphic DNA (RAPD) analysis has been used to identify the eight Barbus species of the Iberian Peninsula, whose morphological differentiation is difficult. Ten random primers were employed to generate RAPD markers. Different RAPD profiles were observed for the different species. Four species-specific markers were found in B. bocagei , seven in B. comizo , five in B. graellsii , three in B. guiraonis , eight in B. haasi , 13 in B. meridionalis , four in B. microcephalus and four in B. sclateri. Evidence is presented that RAPD markers constitute useful tools for accurate taxonomic identification of Spanish barbels which is one of the first prerequisites in effective conservation programmes.     

The status of the barbel, Barbus barbus (L.) (Teleostei, Cyprinidae), in the United Kingdom

The barbel was originally a fish with a relatively restricted range in the British Isles. However, despite the general environmental degradation of many British rivers, and in contrast with some native freshwater fishes, the barbel is now more widely distributed than it was a century ago. It may even be more abundant numerically.     

Barbus xanthos,a new barbel from the Southern Aegean basin (Teleostei: Cyprinidae)

Barbus xanthos, a new species, is described from the Eşen, Dalaman, Tersakan and Büyük Menderes rivers in south-western Anatolia. It differs from other Barbus species in the adjacent basins by having 53–60 lateral line scales, a weakly ossified last unbranched dorsal-fin ray (about 33–50%), numerous small irregular-shaped black or dark-brown spots smaller than scales, often forming large, black or dark-brown blotches on back and flank in juveniles and adults, and a straight or slightly convex posterior dorsal-fin margin. B. xanthos differs from its most closely related congener, B. pergamonensis, by nine nucleotide substitution sites in the mitochondrial DNA cytochrome oxidase I barcode region.     

Visualization of head-head interactions in the inhibited state of smooth muscle myosin

The structural basis for the phosphoryla- tion-dependent regulation of smooth muscle myosin ATPase activity was investigated by forming two- dimensional (2-D) crystalline arrays of expressed unphosphorylated and thiophosphorylated smooth muscle heavy meromyosin (HMM) on positively charged lipid monolayers. A comparison of averaged 2-D projections of both forms at 2.3-nm resolution reveals distinct structural differences. In the active, thiophosphorylated form, the two heads of HMM interact intermolecularly with adjacent molecules. In the unphosphorylated or inhibited state, intramolecular interactions position the actin-binding interface of one head onto the converter domain of the second head, thus providing a mechanism whereby the activity of both heads could be inhibited.     

Morphologic and genetic variability in the Barbus fishes (Teleostei,Cyprinidae) of Central Italy

Italian freshwaters are highly biodiverse, with species present including the native fishes Barbus plebejus and Barbus tyberinus that are threatened by habitat alteration, fish stocking and invasive fishes, especially European barbel Barbus barbus. In central Italy, native fluvio‐lacustrine barbels are mainly allopatric and so provide an excellent natural system to evaluate the permeability of the Apennine Mountains. Here, the morphologic and genetic distinctiveness was determined for 611 Barbus fishes collected along the Padany–Venetian (Adriatic basins; PV) and Tuscany–Latium (Tyrrhenian basins; TL) districts. Analyses of morphological traits and mitochondrial DNA sequence data explored the natural and anthropogenic factors that have shaped their distribution ranges. Over 100 alien B. barbus were recorded in the Tiber basin (TL district) and Metauro basin (PV district). Comparisons of genetic and morphometric data revealed that morphometric data could identify alien B. barbus from native Barbus, but could not differentiate between B. tyberinus and B. plebejus. Genetic analyses revealed ~50 D‐loop mtDNA haplotypes and identified a distinct Barbus lineage present only in the Vomano River at the southern boundary of PV district. Demographic expansion and molecular variance analyses revealed a lack of geographic structuring across the sampling regions. While the contemporary B. plebejus distribution has been driven primarily by anthropogenic fish translocations, the dispersal of B. tyberinus has been via natural dispersion, including their crossing of the Apennine Mountains via temporary river connectivity. The results also revealed that the Barbus fishes of the mid‐Adriatic region of Europe have a complex pattern of local endemism. To conserve these patterns of genetic uniqueness, especially in the mid‐Adriatic basins, Barbus fishes should be managed by treating them as unique evolutionary units and ceasing translocations of all Barbus fishes between river basins.     

Dynamics of myosin heavy chain isoform transition in the longissimus muscle of domestic and wild pigs during growth: a comparative study

Dynamics of myofiber differentiation/maturation in porcine skeletal muscle is associated with domestication, breeding and rearing conditions. This study was aimed to comparatively elucidate the age-dependent myosin heavy chain (MyHC) isoform expression and transition pattern in domestic and wild pig (WP) skeletal muscle from birth until adulthood. Domestic pigs (DPs) of Large White breed raised in conventional production system were compared with WPs reared in a large hunting enclosure. Muscle samples for immuno/enzyme histochemistry were taken from the longissimus dorsi muscle within 24 h postmortem at 24 to 48 h, 21 to 23 days, 7 months and ~2 years postpartum. Based on the antibody reactivity to MyHCs (NCL-MHCs, A4.74, BF-F3) and succinate dehydrogenase activity, myofibers were classified into I, I/IIa, IIa, IIx and IIb types. In addition, foetal MyHC expression was determined with the use of F158.4C10 antibody. Maturation of the longissimus dorsi muscle in the WP was characterized by an accelerated transformation of the fast to slow MyHC during the first hours postpartum, followed by differentiation towards oxidative myofibers in which type I, IIa and IIx MyHCs predominated. In the DP, the transformation shifted towards glycolytic myofibers that expressed MyHC-IIb. The expression of foetal MyHC was higher in the DP than in the WP at 1 day of age, and the decline in the foetal MyHC during the first 3 weeks was more rapid in the WP than in the DP denoting an accelerated early postnatal muscle maturation in WP than DP piglets. All foetal MyHC-positive myofibers co-expressed IIa isoform, but not vice versa. The intense myofiber hypertrophy was evident from 3 weeks until 7 months of age. In this period, the myofiber cross-sectional area increased up to 10- and 20-fold in the WP and the DP, respectively. In the DP, the hypertrophy of all myofiber types was more pronounced than in the WP, particularly the hypertrophy of IIx and IIb myofibers. To summarize, the comparison between growing DP with wild ancestors showed that genetic selection and rearing conditions lead to substantial changes in the direction and intensity of postnatal MyHC transformation as evidenced by different proportion of individual myofiber types and differences in their hypertrophic potential.     

Hexaploidy in yellowfish species (Barbus, Pisces, Cyprinidae) from southern Africa

Five small-scaled yellow fish (large Burbus spp.) from southern Africa are shown to have modal 148 or 150 chromosomes. Themajority ofcyprinid species have 2N = 50 chromosomes, indicating that the yellow fish karyotype is hexaploid in origin. However, as there is no indication that the species are unisexual or that normal reproduction occurs by any means other than bisexual fertilization, the yellowfish karyotype is considered to have reverted to a diploid condition.     

Morphology and fine structure of Barbus barbus (Teleostei: Cyprinidae) spermatozoa

Morphology and fine structure of Barbus barbus L 1758 spermatozoa were studied using scanning (SEM) and transmission (TEM) electron microscopy. The results confirm that spermatozoa exhibit morphological features typical to all teleost fishes. They are differentiated into a head, a midpiece and a flagellum with the typical '9 + 2' pairs of microtubules. Both dynein arms are present in the flagellum. The spermatozoa have spherical nuclei, 4–6 mitochondria located in the postnuclear cytoplasmic region and centriolar complex (proximal and distal centrioles). Total length, head width, length of midpiece and length of flagellum were measured to be 56.35 ± 7.42, 1.80 ± 0.06, 0.48 ± 0.14 and 54.30 ± 6.97 μm, respectively. Highly significant linear correlation was observed between posterior and anterior width of midpiece (P < 0.01). Principal component analysis (PCA) was used to explore which parameters can explain the individual variation of sperm morphology. About 44% of the total accumulated variance was absorbed by the analysis of the two first components, distinguishing different groups of parameters related to head and midpiece. The lengths of flagellum and head are more isolated; indicating that the individual variation of sperm morphology depends on these two parameters. Comparing the results of this study with information on cyprinids spermatozoa reveals that the number of mitochondria and the length of the flagellum are good characters to characterize spermatozoa of the Cyprinidae in a phylogenetic arrangement.     

Expression of cardiac myosin light chain 2 during embryonic heart development in medaka fish,Oryzias latipes,and phylogenetic relationship with other myosin light chains

Cardiac myosin light chain 2 (MLC‐2) plays a key role in heart development, contraction, and embryo and adult heart maintenance. In some animals, defects in the function of cardiac MLC‐2 cause hypertrophic cardiomyopathy. To illuminate the functions of cardiac MLC‐2 in embryonic heart formation and contraction, and into the evolution of MLC‐2, we characterized the expression and requirement for medaka cardiac MLC‐2 gene in the developing heart. Medaka cardiac MLC‐2 cDNA (mcmlc2) was isolated and its gene expression pattern was determined. The mcmlc2 was found to be expressed in the bilateral cardiac mesoderm, the formed heart tube, and in both the differentiated ventricle and atrium. Knockdown of mcmlc2 function caused severe cardiac disorders, including edema in the atrium and sinus venosus. Using phylogenetic analysis, we found that physiological variations in the MLC‐2 molecules evolved due to amino acid changes in the Ca²⁺ binding domain during molecular evolution. Our findings concerning the function and expression of mcmlc2 are nearly identical with those of other MLC‐2 genes, and our phylogenetic analysis suggests that during evolution, the variations in physiological function within the MLC‐2 gene family have arisen from a change in the amino acids in the Ca²⁺ binding domain in the MLC‐2 molecule.     

Structure and function of muscle myosin

We have studied the primary structures of myosins from chicken muscles in order to clarify the relationship between structure and function of muscle myosin. The primary structures of the various kinds of light chains from chicken muscle myosins have been determined. We also report the primary structure of the 23K fragment of subfragment-1 (S-1) component from the heavy chain of chicken fast skeletal muscle myosin. In addition, antibody was prepared against the 23K fragment. The antibody was found to inhibit the Mg²⁺-ATPase activity and the initial P_i burst of the ATPase in the S-1 component. The antibody suppressed the ATP-induced fluorescence enhancement of S-1, though it did not suppress the binding of ATP to S-1. These results are also discussed.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Phylogenetic analysis of the genus Rasbora (Teleostei: Cyprinidae)

Liao, T. Y., Kullander, S. O. & Fang, F. (2009). Phylogenetic analysis of the genus Rasbora (Teleostei: Cyprinidae).—Zoologica Scripta, 39, 155–176. A phylogenetic analysis was conducted based on 35 cyprinid taxa, including 29 species of Rasbora, and 41 morphological characters. A strict consensus tree from four equally parsimonious trees recovered rasborins as a monophyletic group characterized by (i) presence of dark supra‐anal pigment and subpeduncular streak, (ii) 5–6 branched anal‐fin rays, (iii) dorsal‐fin insertion 1–3 scales behind pelvic‐fin insertion, (iv) lateral process of second vertebra more or less straight, (v) 1–5 more abdominal than caudal vertebrae, (vi) absence of foramen in anterior wall of horizontal limb of the cleithrum, (vii) presence of rasborin process on epibranchial 4, and (viii) interhyal well ossified. Rasbora sensu stricto can be distinguished from all other rasborin genera by the presence of an opercular canal. Four new genera, viz. Brevibora, n. gen., Kottelatia, n, gen., Rasbosoma, n. gen. and Trigonopoma, n. gen., are recognized and described.     

Identification of SNPs,mapping and analysis of allele frequencies in two candidate genes for meat production traits: the porcine myosin heavy chain 2B (MYH4) and the skeletal muscle myosin regulatory light chain 2 (HUMMLC2B)

Myosin is one of the most important skeletal muscle proteins. It is composed of myosin heavy chains and myosin light chains that exist with different isoforms coded by different genes. We studied the porcine myosin heavy chain 2B (MYH4) and the porcine skeletal muscle myosin regulatory light chain 2 (HUMMLC2B) genes. A single nucleotide polymorphism (SNP), identified for each gene, was used for linkage mapping of MYH4 and HUMMLC2B to porcine chromosome (Sscr) 12 and Sscr 3, respectively. The mapping of these two genes was confirmed by using a porcine-rodent radiation hybrid panel, even if for MYH4 the LOD score and the retention fraction were low. Allele frequencies at the two loci were studied in a sample of 307 unrelated pigs belonging to seven different pig breeds. Moreover the distribution of the alleles at these two loci was analysed in groups of pigs with extreme divergent (positive and negative) estimated breeding values (EBV) for four meat production traits that have undergone selection in Italian heavy pigs.     

Biogeography and pattern of gene flow among Barbus species (Teleostei: Cyprinidae) inhabiting the Italian Peninsula and neighbouring Adriatic drainages as revealed by allozyme and mitochondrial sequence data

Genetic relationships among 24 Italian and Adriatic populations of barbs (genus Barbus ) were assessed using electrophoretic analysis of allozymes and mitochondrial DNA sequences of the cytochrome b gene. Results obtained with both markers were concordant, but they were not congruent with the current morphology-based systematics and taxonomy. Populations assigned to the same nominal taxa ( B. caninus , B. petenyi and B. rebeli ), were very divergent in both allozymes and mtDNA, indicating that these populations deserve recognition as different units for conservation and management. On the other hand, the two fluvio-lacustrine taxa considered as distinct species (i.e. B. plebejus and B. tyberinus ) are genetically very close to each other, showing no clear differences at either allozymes or mtDNA. The population of B. caninus from Pellice River carried allozyme alleles and mtDNA specific for B. plebejus , indicating a genetic introgression towards the former species. © 2002 The Linnean Society of London, Biological Journal of the Linnean Society , 2002, 75 , 83–99.     

Refined model of the 10S conformation of smooth muscle myosin by cryo-electron microscopy 3D image reconstruction

The actin-activated ATPase activity of smooth muscle myosin and heavy meromyosin (smHMM) is regulated by phosphorylation of the regulatory light chain (RLC). Complete regulation requires two intact myosin heads because single-headed myosin subfragments are always active. 2D crystalline arrays of the 10S form of intact myosin, which has a dephosphorylated RLC, were produced on a positively charged lipid monolayer and imaged in 3D at 2.0 nm resolution by cryo-electron microscopy of frozen, hydrated specimens. An atomic model of smooth muscle myosin was constructed from the X-ray structures of the smooth muscle myosin motor domain and essential light chain and a homology model of the RLC was produced based on the skeletal muscle S1 structure. The initial model of the 10S myosin, based on the previous reconstruction of smHMM, was subjected to real space refinement to obtain a quantitative fit to the density. The smHMM was likewise refined and both refined models reveal the same asymmetric interaction between the upper 50 kDa domain of the "blocked" head and parts of the catalytic, converter domains and the essential light chain of the "free" head observed previously. This observation suggests that this interaction is not simply due to crystallographic packing but is enforced by elements of the myosin heads. The 10S reconstruction shows additional alpha-helical coiled-coil not seen in the earlier smHMM reconstruction, but the location of one segment of S2 is the same in both.     

Contraction kinetics and myosin isoform composition in smooth muscle from hypertrophied rat urinary bladder

Mechanical properties and isoform composition of myosin heavy and light chains were studied in hypertrophying rat urinary bladders. Growth of the bladder was induced by partial ligation of the urethra. Preparations were obtained after 10 days. In maximally activated skinned preparations from the hypertrophying tissue, the maximal shortening velocity and the rate of force development following photolytic release of ATP were reduced by about 20 and 25%, respectively. Stiffness was unchanged. The relative content of the basic isoform of the essential 17 kDa myosin light chain was doubled in the hypertrophied tissue. The expression of myosin heavy chain with a 7 amino acid insert at the 25K/50K region was determined using a peptide-derived antibody against the insert sequence. The relative amount of heavy chain with insert was decreased to 50%, in the hypertrophic tissue. The kinetics of the cross-bridge turn-over in the newly formed myosin in the hypertrophic smooth muscle is reduced, which might be related to altered expression of myosin heavy or light chain isoforms. © 1996 Wiley-Liss, Inc.