Hatching in the pike Esox lucius L.: Evidence for a single hatching enzyme and its immunocytochemical localization in specialized hatching gland cells

Antibodies against purified hatching enzyme (HE) from the pike, Esox lucius L., have been used to examine different aspects of the presence of the enzyme in the ontogeny of this teleostean fish. Immunochemical analysis indicates that the two proteolytic enzymes which occur in the hatching medium arise from a single protease, HE itself. The second proteolytic fraction found in gel filtration of hatching medium could be a heterogeneous population of complexes of HE with digestion fragments of its natural substrate, the zona radiata. Immunofluorescence microscopy by means of anti-HE antibodies demonstrates that HE is localized in the so-called hatching gland cells (HGCs). The HGCs in pike appear as oval to round cells 10–15 μm in diameter containing granules of 1.5–2.3 μm. They are found interspersed between the periderm and the presumptive epidermis. The number of HGCs and their granule content increase significantly until the 35-somite stage to reach about 1200 and 30, respectively. From then on these numbers do not change until hatching in the 66-somite stage. The distribution of the HGCs over the embryo also changes, probably since HGC precursors in the yolk sac differentiate to HGCs later than their counterparts in the head region. The immunocytochemical procedure further shows that HE can be detected from the 10-somite stage on. Discrete hatching gland remnant bodies, phagocytized by epidermal cells, are observed in larval stages until 3–7 days after emergence of the embryo.     

Changes of DNA ligases in chick neural retina as a function of age

In the course of chick neural retina development, several forms of DNA ligase have been found. During embryonic life the major DNA ligase activity that is found at seven days is form I (8.2 S) which gradually decreases and disappears by 14 days after incubation, whereas form II (6.2 S) increases to reach a maximum at the time of hatching. Form II then decreases reaching a constant level by Day 7 and from that time new slow sedimenting forms also appear (forms III and IV). Form III(2 S) is first detectable at seven days and increases up to 90 days, whereas form IV (3 S) is the only form detected in the 17- and 18-month-old and also in the 5-year-old birds. These four forms display different elution patterns on phosphocellulose column chromatography. They also differ in their thermal stability and sensitivity towards N-ethylmaleimide.     

Changes of DNA Ligases in Chick Neural Retina as a Function of Age

In the course of chick neural retina development, several forms of DNA ligase have been found. During embryonic life the major DNA ligase activity that is found at seven days is form I (8.2 S) which gradually decreases and disappears by 14 days after incubation, whereas form II (6.2 S) increases to reach a maximum at the time of hatching. Form II then decreases reaching a constant level by Day 7 and from that time new slow sedimenting forms also appear (forms III and IV). Form III (2 S) is first detectable at seven days and increases up to 90 days, whereas form IV (3 S) is the only form detected in the 17- and 18-month-old and also in the 5-year-old birds. These four forms display different elution patterns on phosphocellulose column chromatography. They also differ in their thermal stability and sensitivity towards N-ethylmaleimide.     

Changes in the second stage juveniles of Globodeva rostochiensis prior to hatching in response to potato root diffusate

cAMP levels in eggs of G. rostochiensis and the diameter of the nucleolus of the nucleus within the dorsal pharyngeal gland cell of the second stage juvenile have been measured as indicators of the response of the nematode to the hatching stimulus in potato root diffusate. The nucleolus increased from 2.72 ± 0.103 μm for unhatched individuals to 3.28 ± 0.14 μm and 3.88 ± 0.15 μm after soaking eggs in potato root diffusate for 3 and 4 days respectively. Juveniles expressed from unstimulated eggs in water to potato root diffusate for 4–5 days showed a similar increase in size of the nucleolus to 3.94 ±0.15 μm but those released into water for this time had smaller nucleoli of 3.20 ± 0.98 μm. The change in diameter of the nucleolus is probably related to the accumulation of secretions in this gland cell before hatching. Preliminary results with dibutyryl analogues of CAMP and cGMP showed some inhibition of hatch in 10% potato root diffusate. Theophylline had a similar effect but NaF was dissimilar in that the effect of this inhibitor was not reversible. A standard radioimmunoassay showed that significant changes in cAMP levels occurred in the unhatched juveniles within cysts after treatment with potato root diffusate for 2.5 or 8 h compared with values for cysts kept in water. This change occurs before other known responses of the juveniles to potato root diffusate and it defines the period of interest for future work on the initial action of hatching factor.     

葡萄碘泡虫、似葡萄碘泡虫和茄形碘泡虫的鉴别及系统发育

葡萄碘泡虫Myxobolus acinosus Nie & Li, 1973、似葡萄碘泡虫Myxobolus pseudoacinosus Guo, et al., 2018和茄形碘泡虫Myxobolus toyamai Kudo, 1917形态非常相似, 有着共同的宿主和相同的寄生部位, 是病原鉴定中容易混淆的种。文章基于形态学和18S rRNA基因信息对三者进行了鉴别和分子系统学研究。成熟孢子形态特征的比较分析显示, 三者形态存在显著差异。葡萄碘泡虫与似葡萄碘泡虫18S rDNA序列相似度为98.4—98.8%, 遗传距离为0.013—0.020; 葡萄碘泡虫与茄形碘泡虫18S rDNA序列相似度为96.1—97.2%, 遗传距离为0.038—0.042; 似葡萄碘泡虫和茄形碘泡虫18S rDNA序列相似度为96.4—97.6%, 遗传距离为0.033—0.040。18S rDNA序列比对显示, 葡萄碘泡虫含有15个关键变异位点, 可将该虫与似葡萄碘泡虫和茄形碘泡虫区分; 似葡萄碘泡虫含有5个关键变异位点, 可将该虫与葡萄碘泡虫和茄形碘泡虫区分; 茄形碘泡虫含有33个关键变异位点可将该虫与葡萄碘泡虫和似葡萄碘泡虫区分。18S rRNA二级结构V4区的E23-2构型可将葡萄碘泡虫与似葡萄碘泡虫和茄形碘泡虫区分, 而V7区的H43构型可将茄形碘泡虫与葡萄碘泡虫和似葡萄碘泡虫区分。以上表明, 三者无论在形态上还是在遗传上均具有独立物种的特征。系统发育分析显示, 葡萄碘泡虫、似葡萄碘泡虫和茄形碘泡虫为系统树中分化较晚的一支。     

尖形碘泡虫(黏体门:碘泡虫科)的重描述及其分子系统学研究

研究基于形态和分子信息重描述了寄生于嘉陵江重庆段鲫(Carassius auratus Linnaeus)鳃部和胆囊的尖形碘泡虫(Myxobolus acutus Wu and Chen, 1987),并获得了该虫体的18S rDNA和ITS1 rDNA序列。尖形碘泡虫成熟孢子壳面观呈梨形,前端稍尖,后端钝圆,缝面观呈宽纺锤形。孢子长(13.6±0.9)μm [(11.4—15.3)μm],宽(10.2±0.9)μm [(7.5—12.8)μm],厚(7.6±0.6)μm [(6.9—8.3)μm]。两梨形极囊开口处紧靠并位于孢子前端,极囊大小不等,大极囊长(6.2±0.4)μm [(5.1—7.5)μm],宽(3.8±0.4)μm [(2.8—4.7)μm],极丝盘绕5—8圈,小极囊长(2.7±0.4)μm [(1.7—3.7)μm],宽(1.4±0.2)μm [(0.9—1.9)μm],极丝盘绕2—3圈。基于18S rDNA为分子标记的系统发育分析显示:尖形碘泡虫与中华单极虫(Thelohanellus sinensis)有最近的亲缘关系,两物种形成的进化支与贝壳碘泡虫(M. mu...     

Purification and characterization of heterologously expressed nitrilases from filamentous fungi

Nitrilases from Aspergillus niger CBS 513.88, A. niger K10, Gibberella moniliformis, Neurospora crassa OR74A, and Penicillium marneffei ATCC 18224 were expressed in Escherichia coli BL21-Gold (DE3) after IPTG induction. N. crassa nitrilase exhibited the highest yield of 69,000 U L(-1) culture. Co-expression of chaperones (GroEL/ES in G. moniliformis and P. marneffei; GroEL/ES and trigger factor in N. crassa and A. niger CBS 513.88) enhanced the enzyme solubility. Specific activities of strains expressing the former two enzymes increased approximately fourfold upon co-expression of GroEL/ES. The enzyme from G. moniliformis (co-purified with GroEL) preferred benzonitrile as substrate (K(m) of 0.41 mM, V(max) of 9.7 μmol min(-1) mg(-1) protein). The P. marneffei enzyme (unstable in its purified state) exhibited the highest V(max) of 7.3 μmol min(-1) mg(-1) protein in cell-free extract, but also a high K(m) of 15.4 mM, for 4-cyanopyridine. The purified nitrilases from A. niger CBS 513.88 and N. crassa acted preferentially on phenylacetonitrile (K(m) of 3.4 and 2.0 mM, respectively; V(max) of 10.6 and 17.5 μmol min(-1) mg(-1) protein, respectively), and hydrolyzed also (R,S)-mandelonitrile with higher K(m) values. Significant amounts of amides were only formed by the G. moniliformis nitrilase from phenylacetonitrile and 4-cyanopyridine.     

Inorganic carbon acquisition in two green marine Stichococcus species

The mechanism of inorganic carbon (C(i)) uptake was examined in the marine green microalgae Stichococcus cylindricus and Stichococcus minor. External carbonic anhydrase (CA) activity was not detected in either species, by potentiometric assay or by mass spectrometry. Photosynthetic characteristics of C(i) uptake indicate that both species have high apparent affinity for CO(2) with a low K(1/2) (CO(2)) of about 10 μm. The O(2) evolution rates in light exceeded the spontaneous CO(2) formation rate by 2.5-fold in both species, which thus have active bicarbonate uptake. Mass spectrometric monitoring of CO(2) and O(2) fluxes showed that rates of O(2) evolution exceeded those of CO(2) depletion by about three- and twofold in S. minor and S. cylindricus, respectively, and also showed, in cells photosynthesizing at pH 8.2, a rapid depletion of CO(2) upon illumination to a CO(2) compensation concentration of 15.42 and 12.03 μm in S. minor and S. cylindricus, respectively. Both species also exhibit active CO(2) uptake: addition of bovine CA at CO(2) compensation concentration caused a rapid rise in CO(2) as the CO(2) -HCO(3) (-) equilibrium was restored. Accumulation of unfixed C(i) by cells at pH 8.2 was calculated to be 84.33 mm in S. cylindricus, and 30.37 mm in S. minor to give internal accumulations of 23- and 8-fold, respectively, compared to the external C(i) concentration.     

Stimulation of de novo methylation following limited proteolysis of mouse ascites DNA methylase

The ability of mouse Krebs II ascites cell DNA methylase to add methyl groups to native, unmethylated DNA (de novo activity) is stimulated by limited proteolysis. The affinity of the enzyme for DNA is not altered by this treatment but the rate of reaction is increased so that 40% or more of methylatable sites are methylated within 4.5 h. The activation is associated with a decrease in size of the enzyme to 6.2 S.     

Cloning, expression, characterization and inhibition studies on trypanothione synthetase, a drug target enzyme, from Leishmania donovani

Trypanothione synthetase, a validated drug target, synthesizes trypanothione from glutathione and spermidine. Here we report the gene cloning, expression, characterization and inhibition studies of trypanothione synthetase from Leishmania donovani (LdTryS). The purified recombinant LdTryS enzyme obeyed Michaelis-Menten kinetics. High substrate inhibition was observed with glutathione (K(m)=33.24 μm, k(cat)=1.3 s(-1), K(i)=866 μm). The enzyme shows simple hyperbolic kinetics with fixed glutathione concentration and with other substrates limiting K(m) values for Mg. ATP and spermidine of 14.2 μm and 139.6 μm, respectively. LdTryS was also screened for inhibitors. Tomatine, conessine, uvaol and betulin were identified as inhibitors of the enzyme and were tested for leishmanicidal activity. Finally, the effect of LdTryS inhibitors on redox homeostasis of the parasite gives a broader picture of their action against leishmaniasis.     

Complete development of the northeast Pacific arminacean nudibranch Janolus fuscus

Early life-history stages of the nudibranch Janolus fuscus (superfamily Arminoidea) were reared in the laboratory and collected from the field to document embryology, larval morphology, and pre-and post-metamorphic growth. This species produces Type B egg masses containing an average of 23,063 viable embryos. The spiral holoblastic cleavage pattern resembled that of other nudibranchs, with early cell divisions occurring roughly at 4-h intervals at 11-13 °C. Embryos progress through a gastrula stage with cellular extensions covering the blastopore and a trochophore-like stage before hatching as veliger larvae after 10-18 days. Veligers ranged in size from 125-153.8 μm at hatching, with size being positively correlated with the duration of encapsulated development. After a 36-41-day larval period, some veligers ceased growing at 266 μm and showed signs of approaching competence. Four larvae settled and two metamorphosed into 280-μm juveniles on the bryozoan prey of the adults, Bugula pacifica, 46 and 54 days post-hatching. Average growth of pre-ovipositional (<19 mm) J. fuscus was more rapid (8.79% per day) than growth of ovipositional individuals (3.52% per day). Growth continued to a maximum of 57 mm in the laboratory, with an estimated total lifespan of about 5 months. These data, which agree with concurrent field observations, suggest a subannual life cycle.     

鲤肠道寄生岳阳碘泡虫新种的形态特征及系统发育分析

在洞庭湖岳阳地区开展鱼类寄生虫调查中,发现一种寄生于鲤Cyprinus carpio L.肠道的黏孢子虫。该黏孢子虫的孢囊呈白色,椭圆形,大小为(1.0±0.2) mm (0.8—1.2 mm)。成熟孢子具有壳瓣,壳面观近似圆形,后端有4—6个“V”形褶皱;缝面观呈纺锤形,缝脊直而粗;孢质均匀,含有一个嗜碘泡;孢子长(9.8±0.6) μm (9.6—10.0 μm),孢子宽(8.2±0.3) μm (8.0—8.5μm),孢子厚(7.3±0.1) μm (7.0—7.5 μm);2个极囊梨形,位于孢子顶端,大小相等,呈“八”字形;极囊长(4.4±0.4) μm (3.8—5.1 μm),宽(2.7±0.2) μm (2.2—3.2 μm),极丝4—5圈。该黏孢子虫与肠膜碘泡虫、丑陋圆形碘泡形态特征非常相似,但其极囊/孢子小于1/2;与文献已报道的鲤肠道寄生北京碘泡虫和鲤肠碘泡虫相比较,其在孢子形态、孢子和极囊大小方面分别存在明显差异。基于该黏孢子虫18S rDNA基因序列(GenBank登录号KY203795)比对分析,该黏孢子虫与山东碘泡虫相似率最高,仅为96%。系统发育分析发现,该黏孢子虫与山东碘泡虫、倪李碘泡虫、住心碘泡虫、Myxobolus encephalicus、Sphaerospora molnari、多涅茨尾孢虫和Henneguya zikaweiensis聚为独立分支,和其他已报道的黏孢子虫亲缘关系较远。综合形态学和18S rDNA基因序列数据,文章报道的鲤肠道寄生黏孢子虫为碘泡虫属一新物种,将其命名为岳阳碘泡虫。     

东海剧毒卡尔藻的形态特征及其系统进化分析

利用光学显微镜、荧光显微镜、扫描电镜及分子生物学等方法, 对分布于我国东海海域的剧毒卡尔藻（Karlodinium veneficum）藻株（LAMB090611）的形态特征和显微结构进行了描述, 并探讨了其分子系统进化关系。该藻株细胞长11.1-18.7 μm, 平均值为（14.2±1.8） μm, 宽8.2-14.7 μm, 平均值为（10.8±1.5） μm。细胞形态结构特征为： 上下锥体积基本相同; 顶沟短而直; 腹孔明显; 纵沟延伸至上锥; 横沟错位距离约占细胞总长的28%-38%; 含有2或4个不规则形态的叶绿体; 细胞核位于中部或下锥。此藻种的暴发可引发有害赤潮（harmful algal bloom）。当前加强有害赤潮的预防和监测工作是减少危害的有效途径, 而对引发赤潮原因种的准确识别和鉴定则是基础和关键。     

Partial purification and properties of diacylglycerol kinase from rat liver cytosol

Diacylglycerol:ATP kinase(EC 2.3.1.-) was highly purified (more than 2000-fold) from rat liver cytosol. The specific activity of the obtained enzyme was about 1.5 μmol phosphatidate formed/mg of protein/min. The purification procedures included ammonium sulfate fractionation, DEAE-cellulose chromatography, gel filtration on Sephadex G-200, and finally affinity chromatography on ATP-agarose. The activities of diacylglycerol:GTP kinase and monoacylglycerol:ATP kinase were copurified throughout the procedures, forming a single peak together with diacylglycerol: ATP kinase. Furthermore, these kinase activities showed a single peak when the highly purified enzyme was analyzed by a sucrose density gradient centrifugation and polyacrylamide gel electrophoresis. The three kinase activities are, therefore, most likely catalyzed by a single enzyme. The kinase showed an apparent molecular weight of 121,000 on gel filtration and sedimented at 5.1 S in a sucrose gradient centrifugation. The apparent K_m values were 170 μm for ATP, 540 μm for GTP, and 3.0 μm for diacylglycerol. A number of nucleoside triphosphates and diphosphates competitively inhibited the kinase, in particular the activity utilizing GTP. Among the nucleotides tested, ADP was the most potent inhibitor (the apparent K_i:50 μm for diacylglycerol:ATP kinase and 42 μm for diacylglycerol:GTP kinase). The kinase required Mg²⁺ and deoxycholate for its activity, and the optimal pH was 8.0–8.5. No dependence on added phospholipids was observed.     

The purification and properties of histidinol dehydrogenase from Neurospora crassa

1. A procedure is described for the purification of l-histidinol dehydrogenase (l-histidinol-NAD oxidoreductase, EC 1.1.1.23) from Neurospora crassa. 2. The enzyme, as purified, has a sedimentation coefficient, S(20), of 7.1s and a molecular weight of 81 000. Considerable variation is possible in the state of polymerization of the enzyme, giving rise to observed molecular weights from 40 000 to 240 000. 3. Several kinetic parameters of the enzyme have been determined. The enzyme is maximally active at pH9.8; the K(m) (NAD) is 13.0x10(-5)m and K(m) (histidinol) is 8.2x10(-6)m. The enzyme is highly specific, does not oxidize a range of amino alcohols and other aliphatic alcohols nor reduce NADP and has no demonstrable affinity for histidine. The turnover number is 49 moles of NAD reduced/min./mole of enzyme (mol.wt. 40 000).     

An Ultrastructural Study of First- and Second-Generation Merogony in the Coccidian Sarcocystis tenella1

ABSTRACT. Sporocysts of the coccidian Sarcocystis tenella were originally isolated in the feces of a coyote. Sporocysts used for inoculation of lambs were obtained from experimentally infected dogs. At 14, 16, and 19 days postinoculation (DPI) of lambs with the sporocysts, various developmental stages of first-generation meronts were found within cells located between the endothelium and internal elastic membrane of mesenteric arteries. At 19, 21, and 25 DPI, second-generation merogony occurred in cells associated with capillaries and arterioles of kidney glomeruli and convoluted tubules. Meronts of both generations were bounded by a double pellicular membrane and were situated free in the host cell cytoplasm. Merozoites formed by endopolygeny that involved multiple intranuclear spindles of a single, large irregular nucleus. First-generation meronts measured 22.6 × 17.1 μm (19–28.7 × 7.5–24 μm) and contained 120–240 merozoites, which measured 7.1 × 1.6 μm (4.8–7.5 × 1.3–1.8 μm). Corresponding values for second-generation meronts were 13.2 × 9.2 μm (8.3–15 × 7–13.5 μ), 32–80, and 5.8 × 1.7 μm (5.6–6.2 × 1.4–2.2 μm).     

Extended incubation affects larval morphology, hatching success and starvation resistance in a terrestrially spawning fish, Galaxias maculatus (Jenyns 1842)

The effect of extended incubation (delayed hatching) on larval morphology in the terrestrially spawning common galaxias Galaxias maculatus was investigated by inducing larvae to hatch 1 and 2 weeks after the normal 2 week incubation period. After 1 week of extended incubation, larvae were larger (longer in standard length, L(S), and greater in body depth) compared to controls (larvae that experienced normal incubation durations). After 2 weeks of extended incubation, larvae were smaller (shorter in L(S) and smaller in body depth) than larvae that experienced 1 week of extended incubation. Furthermore, eye area increased while yolk-sac size decreased monotonically with increasing incubation duration. These results suggest that larvae experiencing long periods of extended incubation are using somatic tissue to meet their metabolic demands. Larvae that experienced 2 weeks of extended incubation succumbed to starvation sooner than control larvae, but hatching success was not significantly different. Temperature mediated the effect of extended incubation on the morphology of larvae at hatching, most likely, through its effects on developmental rate and efficiency of yolk utilization. This study demonstrates some of the consequences of terrestrial spawning with extended incubation, which will assist in determining why this intriguing behaviour has evolved several times in a diverse range of taxa.     

Plastid and cytosolic phosphofructokinases from the developing endosperm of ricinus communis: II. Comparison of the kinetic and regulatory properties of the isoenzymes

A steady-state kinetic analysis of plastid phosphofructokinase at pH 8.2 is consistent with the enzyme having a sequential reaction mechanism. Cytosolic phosphofructokinase probably has a similar mechanism. At pH 7.0 plastid phosphofructokinase shows cooperative binding of fructose 6-phosphate and is inhibited by higher concentrations of ATP. In contrast cytosolic phosphofructokinase shows normal kinetics at both pH 8.2 and 7.0 with respect to fructose 6-phosphate and is not inhibited by ATP. In the case of plastid phosphofructokinase the affinity for fructose 6-phosphate increases as the pH is raised from 7 to 8.2 whereas cytosolic phosphofructokinase is affected in an opposite manner. Phosphate is the principal activator of plastid phosphofructokinase since the cooperative kinetics toward fructose 6-phosphate are shifted toward Michaelis-Menten kinetics by 1 mm sodium phosphate and this concentration of phosphate relieves the inhibition by ATP. Both isoenzymes are inhibited by phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate at pH 7.2. Plastid phosphofructokinase is most strongly inhibited by phosphoenol pyruvate with the I_0.5 value varying from 0.08 to 0.5 μm depending on substrate concentrations; phosphate reverses this inhibition. In contrast cytosolic phosphofructokinase is much less inhibited by phosphoenolpyruvate with an I_0.5 approximately 1000-fold higher. Cytosolic phosphofructokinase is powerfully inhibited by 3-phosphoglycerate with an I_0.5 value of 60 μm and this appears to be the principal regulator of this isoenzyme. The two isoenzymes of phosphofructokinase in the endosperm appear, therefore, to be regulated differently. Plastid phosphofructokinase is inhibited by phosphoenolpyruvate and ATP and is activated by phosphate; whereas the cytosolic enzyme is inhibited principally by 3-phosphoglycerate and this inhibition is only partially relieved by phosphate. Some of the differences reported previously for phosphofructokinases from different plant tissues may, therefore, be due to varying ratios of the cytosolic and plastid isoenzymes.     

Characterization of hatching-associated changes in the sea urchin fertilization envelope

The embryo of the sea urchin Strongylocentrotus purpuratus hatches from the fertilization envelope (FE) via synthesis and secretion of a hatching enzyme and by ciliary activity. Although the basic characteristics of the hatching enzyme are known, little is understood about changes in the FE during hatching. We have studied the biochemical changes in FEs during hatching. Polyacrylamide gel analysis revealed an increasingly complex polypeptide spectrum of the extractable fraction of FEs isolated during development. Immunoblotting of these polypeptides (using antiserum against the soluble polypeptides extracted from FEs isolated at 30 minutes postinsemination) revealed a decrease in the soluble FE components during hatching. Immunochemical analysis of hatching medium showed a strong correlation between the soluble FE components released and the hatching interval. Immunoblotting of hatching media indicated the presence of soluble FE polypeptides of similar and lower molecular weights than those obtained for extracts of FEs. These results imply that the hatching-associated changes in the FE of S purpuratus occur via proteolysis of FE components, which are derived from the paracrystalline protein fraction, a subset of cortical granule proteins.