中国林蛙早期胚胎发育过程同工酶的研究

本文采用聚丙烯酰胺凝胶电泳方法,对中国林蛙（Rana chensinensis)早期胚胎发育过程中（受精后0－512h)乳酸脱氢酶（LDH）同工酶和酯酶（EST）同工酶进行了研究,结果表明：1．LDH同工酶在受精卵及早期胚胎发育过程中一直存在,并且在胚胎发育的不同时期差异显著,LDH1同工酶在胚胎发育的各个阶段都占绝对优势,而DH5活性从尾芽期开始增强,LDH2,LDH3,LDH4 3种同工酶的活性较;低同时也发现LDH1,LDH3,LDH4同工酶各亚带也发育阶段的特异性,2．EST同工酶在开口期才开始出现。     

鳙鱼同工酶发育遗传学研究

采用淀粉或聚丙烯酰胺凝胶电泳法分析鳙鱼早期发育阶段(从未受精卵到卵黄吸尽期)及成体不同组织(脑、眼、心、肌、肾、肝)中六种同工酶(LDH,MDH,IDH,ADH,SDH,EST)的分化表达模式。鳙鱼同工酶基因的表达具有明显的组织特异性。早期发育阶段,ADH和SDH均无染色活性;LDH、MDH和IDH具有不同的发育变化谱式,而EST酶谱在整个早期发育阶段均无明显变化。与鲢、草鱼相比,鳙鱼早期发育过程中胚胎Ldh-A基因激活的时间被推迟。上述结果可为鳙鱼种群的生化遗传结构分析以及鳙鱼的人工育种提供基础资料。     

Lactate dehydrogenase in teleosts. The role of LDH-C4 isozyme.

1. Lactate dehydrogenase (LDH) occupies an important position in cell metabolism. 2. Teleosts possess at least three genetic loci coding for lactate dehydrogenase subunits, Ldh-A, Ldh-B and Ldh-c. LDH exists in most tissues in several isozymic forms. 3. The isozyme LDH-C4 is synthesized predominantly in regions of the nervous system concerned with the eye.     

Hypoxia tolerance and partitioning of bimodal respiration in the striped catfish (Pangasianodon hypophthalmus)

Air-breathing fish are common in the tropics, and their importance in Asian aquaculture is increasing, but the respiratory physiology of some of the key species such as the striped catfish, Pangasianodon hypophthalmus Sauvage 1878 is unstudied. P. hypophthalmus is an interesting species as it appears to possess both well-developed gills and a modified swim bladder that functions as an air-breathing organ indicating a high capacity for both aquatic and aerial respiration. Using newly developed bimodal intermittent-closed respirometry, the partitioning of oxygen consumption in normoxia and hypoxia was investigated in P. hypophthalmus. In addition the capacity for aquatic breathing was studied through measurements of oxygen consumption when access to air was denied, both in normoxia and hypoxia, and the critical oxygen tension, Pcrit, was also determined during these experiments. Finally, gill ventilation and air-breathing frequency were measured in a separate experiment with pressure measurements from the buccal cavity. The data showed that P. hypophthalmus is able to maintain standard metabolic rate (SMR) through aquatic breathing alone in normoxia, but that air-breathing is important during hypoxia. Gill ventilation was reduced during air-breathing, which occurred at oxygen levels below 8 kPa, coinciding with the measured Pcrit of 7.7 kPa. The findings in this study indicate that the introduction of aeration into the aquaculture of P. hypophthalmus could potentially reduce the need to air-breathe. The possibility of reducing air-breathing frequency may be energetically beneficial for the fish, leaving more of the aerobic scope for growth and other activities, due to the proposed energetic costs of surfacing behavior.     

Some aspects of isozymes of lactate dehydrogenase, malate dehydrogenase and glucosephosphate isomerase in fish

1. The present paper reports some aspects of the isozymes of LDH, MDH and GPI in fish. 2. In Petromyzontiformes LDH is encoded by a single Ldh-A gene locus. In Myxiniformes and in most vertebrates LDH is encoded by two gene loci, A and B. A third Ldh-C locus is characteristic of the bony fishes Actinopterygii. 3. In fish the MDH isozymes are generally encoded by three gene loci Mdh-M, Mdh-A and Mdh-B. 4. In most diploid bony fish the GPI is controlled by two independent gene loci Gpi-A and Gpi-B. 5. The relationships of isozymes with evolution of vertebrates, tissual specificity, ontogenetic changes, with physiological and metabolic roles are discussed.     

Lactate dehydrogenase (LDH) gene duplication during chordate evolution: the cDNA sequence of the LDH of the tunicate Styela plicata

L-Lactate dehydrogenase (L-LDH, E.C. 1.1.1.27) is encoded by two or three loci in all vertebrates examined, with the exception of lampreys, which have a single LDH locus. Biochemical characterizations of LDH proteins have suggested that a gene duplication early in vertebrate evolution gave rise to Ldh-A and Ldh-B and that an additional locus, Ldh-C arose in a number of lineages more recently. Although some phylogenetic studies of LDH protein sequences have supported this pattern of gene duplication, others have contradicted it. In particular, a number of studies have suggested that Ldh-C represents the earliest divergence among vertebrate LDHs and that it may have diverged from the other loci well before the origin of vertebrates. Such hypotheses make explicit statements about the relationship of vertebrate and invertebrate LDHs, but to date, no closely related invertebrate LDH sequences have been available for comparison. We have attempted to provide further data on the timing of gene duplications leading to multiple vertebrate LDHs by determining the cDNA sequence of the LDH of the tunicate Styela plicata. Phylogenetic analyses of this and other LDH sequences provide strong support for the duplications giving rise to multiple vertebrate LDHs having occurred after vertebrates diverged from tunicates. The timing of these LDH duplications is consistent with data from a number of other gene families suggesting widespread gene duplication near the origin of vertebrates. With respect to the relationships among vertebrate LDHs, our data are not consistent with previous claims that Ldh-C represented the earliest divergence. However, the precise relationships among some of the main lineages of vertebrate LDHs were not resolved in our analyses.      

Pattern of Skeletal Muscle Differentiation in Fish: Molecular Biological Approaches

The initial stages of myogenesis going in myoblasts include the stages of induction, determination, and differentiation. The induction and determination of cells in the myotomes are controlled by morphogenetic signals from neighboring tissues of the notochord and neural tube manifested as expression of genes of Shh and Wnt families, respectively. In fish (at the example of danio), this signal is passed to somite cells neighboring the notochord; later the cells migrate to the embryo surface and differentiate into slow muscle fibers. Synthesis of the main contractile proteins, primarily the components of myosin molecule—heavy chain (MHC) and individual isoforms of light chains (MLC1, MLC2, and MLC3)—are encoded by different genes during different ontogenetic stages. The peptide maps obtained after -chymotrypsin digestion of MHCs from larvae, fast and slow skeletal muscle of loach are different, which points to differences in their primary structure. In addition, considerable differences were revealed in the structure of MLC isoforms at different ontogenetic stages. The definitive fast muscle contained three light chain types, MLC1, MLC2, and MLC3; slow muscle, MLC1 and MLC3; while the larval muscle fibers included a specific larval MLCL in addition to MLC3.     

Musculature in sipunculan worms: ontogeny and ancestral states

SUMMARY Molecular phylogenetics suggests that the Sipuncula fall into the Annelida, although they are morphologically very distinct and lack segmentation. To understand the evolutionary transformations from the annelid to the sipunculan body plan, it is important to reconstruct the ancestral states within the respective clades at all life history stages. Here we reconstruct the ancestral states for the head/introvert retractor muscles and the body wall musculature in the Sipuncula using Bayesian statistics. In addition, we describe the ontogenetic transformations of the two muscle systems in four sipunculan species with different developmental modes, using F-actin staining with fluorescent-labeled phalloidin in conjunction with confocal laser scanning microscopy. All four species, which have smooth body wall musculature and less than the full set of four introvert retractor muscles as adults, go through developmental stages with four retractor muscles that are eventually reduced to a lower number in the adult. The circular and sometimes the longitudinal body wall musculature are split into bands that later transform into a smooth sheath. Our ancestral state reconstructions suggest with nearly 100% probability that the ancestral sipunculan had four introvert retractor muscles, longitudinal body wall musculature in bands and circular body wall musculature arranged as a smooth sheath. Species with crawling larvae have more strongly developed body wall musculature than those with swimming larvae. To interpret our findings in the context of annelid evolution, a more solid phylogenetic framework is needed for the entire group and more data on ontogenetic transformations of annelid musculature are desirable.     

Purification and Properties of the Threespine Stickleback (Gasterosteus aculeatus) Lactate Dehydrogenase LDH-B4 and LDH-C4 Isoenzymes

In the threespine stickleback (Gasterosteus aculeatus) lactate dehydrogenase (LDH, EC 1.1.1.27) is encoded by three loci, Ldh-A, Ldh-B, and Ldh-C. LDH-B₄ isoenzyme restricted its function to eye and brain, while LDH-C₄ isoenzyme functions in the eye. In the Dead Vistula stickleback population, none of LDH loci is polymorphic. The LDH-B₄ and LDH-C₄ isoenzymes from the eye were purified to homogeneity to specific activity of 186 and 229 μmol NADH min⁻¹mg⁻¹, respectively, at 30°C. Some physico-chemical and kinetic properties revealed that eye LDH-C₄ isoenzyme was more thermostable and had a higher affinity to pyruvate than LDH-B₄ isoenzyme. Lower Km for pyruvate of eye LDH-C₄ isoenzyme distinguishes it from fish LDH-C₄ isoenzyme isolated from liver.     

Lactate dehydrogenase from the northern krill Meganyctiphanes norvegica: comparison with LDH from the Antarctic krill Euphausia superba

Electrophoretic polymorphism of lactate dehydrogenase (LDH, EC 1.1.1.27) from abdominal muscle is reported in the northern krill Meganyctiphanes norvegica. In the population, from the Gullmarsfjord (west coast of Sweden), LDH was encoded for by two different Ldh-A* and -B* loci. The isoenzymes were named according to their electrophoretic mobilities. Ldh-A* locus was polymorphic. The allelic frequencies were a=0.99, a'=0.002, a"=0.004, a"'=0.004. The level of LDH polymorphism is low. Most individuals possess the same amount of two LDH homopolymers (LDH-A*(4) and LDH-B*(4)). The Meganyctiphanes norvegica LDH-A*(4) and LDH-B*(4) isoenzymes and the predominant LDH-A*(4) isoenzyme from Euphausia superba were purified to specific activities of 294, 306 and 464 micromol NADH min(-1) mg(-1), respectively. In both species the LDH isoenzymes were separated by chromatofocusing. All three isoenzymes are L-specific tetramers with molecular weight of approximately 160 kDa. Northern krill LDH-A*(4) has higher affinity for pyruvate and lactate and is more thermostable than LDH-B*(4). Both isoenzymes are inhibited significantly by high concentration of pyruvate but not lactate. Antarctic krill isoenzyme exhibits high substrate affinities, high NAD inhibition, high inhibition at 10 mM pyruvate, lack of lactate inhibition, and high heat stability and resembles northern krill LDH-A*(4) isoenzyme.     

Stem cell expression and development of trunk musculature of lesser‐spotted dogfish (Scyliorhinus canicula) reveal differences between sharks and teleosts

This study compares the trunk skeletal muscle anatomy in 870‐ and 2900‐degree‐day‐old lesser‐spotted dogfish larvae (Scyliorhinus canicula) via haematoxylin/eosin staining as well as immunohistochemistry and Western blot analysis. The results showed poorly differentiated muscle formation in the trunk segments in the younger larvae and fully developed skeletal muscle with a division of red and white cells in the older larvae. The stem cell marker PAX7, which is present in all developmental stages of teleost fish, is only expressed in the younger dogfish. The results show the necessity of examining the skeletal muscle development in sharks to understand the evolutional changes from cartilaginous fishes to teleosts.     

Variation of loci encoding homologous enzymes in an evolutionary series of vertebrates

A study of variability of 11 allozyme loci (sAat, G3pdh, Gpi, sIdh, Ldh-A, Ldh-B, sMdh, sMe, sSod, Pgdh, and Sdh) in the evolutionary series of vertebrates from Cyclostomat to Mammalia revealed that (1) in vertebrates, these loci encoding multimeric enzymes are characterized by different heterozygosity levels, the extremes of which (represented by loci Ldh-A and Pgdh) differ from each other more than by a factor of 4; (2) classes of vertebrates markedly differed from one another in genetic variation; lower Tetrapoda are characterized by the highest level of genetic polymorphism, the classes representing the margins of the phyletic line-primitive (Cyclostomata and Chondrchthyes) and advanced (Aves and Mammalia)--have minimum heterozygosity levels, whereas Osteichthyes are characterized by intermediate heterozygosity level; (3) in the evolutionary series of vertebrates, heterozygosity varies rather independently in the groups of loci characterized by low, medium, and high variability. These patterns are explained in the context of intraorganismic factors: integration of mono- and polygenic traits (primarily, body size and ontogeny rate) and evolutionary specialization.     

Testing the evolutionary constraints of metamorphosis: The ontogeny of head shape in Triturus newts

In vertebrates with complex, biphasic, life cycles, larvae have a distinct morphology and ecological preferences compared to metamorphosed juveniles and adults. In amphibians, abrupt and rapid metamorphic changes transform aquatic larvae to terrestrial juveniles. The main aim of this study is to test whether, relative to larval stages, metamorphosis (1) resets the pattern of variation between ontogenetic stages and species, (2) constrains intraspecific morphological variability, and (3) similar to the “hour‐glass” model reduces morphological disparity. We explore postembryonic ontogenetic trajectories of head shape (from hatching to completed metamorphosis) of two well‐defined, morphologically distinct Triturus newts species and their F1 hybrids. Variation in head shape is quantified and compared on two levels: dynamic (across ontogenetic stages) and static (at a particular stage). Our results show that the ontogenetic trajectories diverge early during development and continue to diverge throughout larval stages and metamorphosis. The high within‐group variance and the largest disparity level (between‐group variance) characterize the metamorphosed stage. Hence, our results indicate that metamorphosis does not canalize head shape variation generated during larval development and that metamorphosed phenotype is not more constrained relative to larval ones. Therefore, metamorphosis cannot be regarded as a developmental constraint, at least not for salamander head shape.     

Phylogenetic Analysis of Vertebrate Lactate Dehydrogenase (LDH) Multigene Families

In this paper we analyzed 49 lactate dehydrogenase (LDH) sequences, mostly from vertebrates. The amino acid sequence differences were found to be larger for a human–killifish pair than a human–lamprey pair. This indicates that some protein sequence convergence may occur and reduce the sequence differences in distantly related species. We also examined transitions and transversions separately for several species pairs and found that the transitions tend to be saturated in the distantly related species pair, while transversions are increasing. We conclude that transversions maintain a conservative rate through the evolutionary time. Kimura's two-parameter model for multiple-hit correction on transversions only was used to derive a distance measure and then construct a neighbor-joining (NJ) tree. Three findings were revealed from the NJ tree: (i) the branching order of the tree is consistent with the common branch pattern of major vertebrates; (ii) Ldh-A and Ldh-B genes were duplicated near the origin of vertebrates; and (iii) Ldh-C and Ldh-A in mammals were produced by an independent gene duplication in early mammalian history. Furthermore, a relative rate test showed that mammalian Ldh-C evolved more rapidly than mammalian Ldh-A. Under a two-rate model, this duplication event was calibrated to be approximately 247 million years ago (mya), dating back to the Triassic period. Other gene duplication events were also discovered in Xenopus, the first duplication occurring approximately 60–70 mya in both Ldh-A and Ldh-B, followed by another recent gene duplication event, approximately 20 mya, in Ldh-B. Received: 5 October 2001 / Accepted: 24 October 2001     

两种泥鳅不同核质关系下LDH同工酶基因表达的研究

采用聚丙烯酰胺不连续系统凝胶电泳方法分析了泥鳅和大鳞副泥鳅不同杂交组合不同核质关系下胚发育阶段（0－145h）中乳酸脱氢酶（LDH）同工酶的分化表达谱式,ＬＤＨ同工酶的基因表达随细胞质、细胞核不同及胚胎发育各个时期具有不同的个体发育谱式,以泥鳅卵子为细胞质的Ⅰ、Ⅱ两组,杂交组（Ⅱ）ＬＤＨ同工酶基因在受精后３min至原肠中期雄核基因参与表达与调控,部分基因位点比本交组（Ⅰ）启动与表达的时间要早,两组的管家酶主要来自细胞质。以大鳞副泥鳅为细胞质的Ⅲ、Ⅳ两组,各酶均以细胞质调控为主,其管家酶与泥鳅有很大不同。并具体分析和讨论了不同核质关系ＬＤＨ同工酶基因的表达和调控的时空顺序。     

Morphometrics of gills during growth and development of the air-breathing habit in Colisa fasciatus (Bloch and Schneider)

Gill area and other component parameters of Colisa fasciatus during early life were measured for fish larvae divided into two groups (a) exclusively aquatic and (b) bimodal breathers. Statistical analyses of the data in relation to body size yielded two significantly different straight lines (one for aquatic and other for bimodal breathers) for each parameter. Morphological examinations of gill arches indicated that an increase in the gill area was brought about mainly by an increase in the filament length. The higher slope value (2.41) of gill area in the aquatic phase than that in the bimodal phase ( b = 0.80) is suggestive of a higher weight-specific metabolism in the younger larvae. A heterogenous growth pattern during early ontogenesis of the fish results in intraspecific variation in the gill dimensions which might have been influenced by the acquisition of the air-breathing mechanism in the post-larval stage, besides ecological factors.     

北京鸭LDH同工酶的研究——Ⅲ.LDH座位在北京鸭发生过程中的表现方式

采用电泳结合光密度扫描的方法,对北京鸭肝脏、胸肌、心肌和肾脏4种组织的LDH同工酶进行了发生遗传学分析。发现LDH座位在北京鸭发生过程中有三种表现方式:肝脏表现出B型酶到A型酶的典型转换;胸肌则表现出AB型酶到A型酶的特殊转换;心肌和肾脏始终保持B型酶为主,只表现出量上的增加趋势,同时也发现LDH1同工酶各亚带也具有组织的和发育阶段的特异性。显然这都是由于LDH座位即ldh a和ldh b两个基因的差别表达所造成的。     

草鱼同工酶发育遗传学研究——Ⅱ.早期发育过程中的同工酶分析

采用淀粉凝胶电泳技术研究了草鱼早期发育过程中(受精后0—200小时)6种同工酶系统(LDH、MDH、GDH、ADH、IDH、EST)的表达谱式。除了ADH以外,其余5种同工酶系统均具有明显的发育变化谱式。根据早期发育过程中同工酶的变化谱式及其组织分布,草鱼的同工酶可分为三大类型:(1)在未受精卵及早期发育过程中一直存在,并常有较广泛的组织分布;(2)未受精卵及早期发育过程中均不存在,一般仅分布于少数几种组织中;(3)未受精卵及胚胎发育早期不存在,直到早期发育过程中某一特定时期才开始出现。