Characterization of iodothyronine sulfotransferase activity in the cytosol of Rana catesbeiana tadpole tissues

We have investigated the sulfation of thyroid hormones (THs) in the cytosol from Rana catesbeiana tadpole tissues. Sulfation of 3,3′,5-triiodothyronine (T₃) by the liver cytosol, which was dependent on protein amount, incubation time, and temperature, suggested the presence of TH sulfotransferases (SULTs) in the liver. The apparent Michaelis-Menten constant (K_m) of the liver cytosol was 0.22 μM for T₃, and the apparent maximum velocity (V_max) of the liver cytosol was 7.65 pmol/min/mg protein for T₃. Iodothyronine sulfating activity in the liver cytosol was increased in tadpoles at premetamorphic (stages IX-X) and metamorphic climax (stage XX) stages, and in adult frogs. The substrate preference of iodothyronine sulfation for the liver cytosol from tadpoles (stage X) was: 3,3′,5′-triiodothyronine > T₃ > 3,3′,5,5′-tetraiodothyroacetic acid > 3,3′,5-triiodothyroacetic acid, T₄, 3-iodothyronine > 3,5-diiodothyronine. Several halogenated phenols were potent inhibitors (IC₅₀ = 0.15-0.21 μM). The substrate preference for T₃ was gradually lost by the onset of metamorphic climax stages. These enzymatic characteristics of iodothyronine sulfation in the liver cytosol from tadpoles resembled those of mammalian phenol SULTs, except that the tadpole cytosol had a higher affinity (one or two orders of magnitude) for T₃ than mammalian SULTs. These results suggested that an enzyme homologous to mammalian phenol SULT (SULT1) may be involved in TH metabolism in tadpoles.     

Cane toad toxicity: An assessment of extracts from early developmental stages and adult tissues using MDCK cell culture

Extracts of the cane toad (Bufo [Chaunus] marinus) adversely affected the growth of Mardin-Darby canine kidney (MDCK) cells during culture. In a similar manner to ouabain treatment, application of toad extracts over a 24 h period resulted in high levels of cytotoxicity, as indicated by cell detachment, increased membrane permeability and loss of mitochondrial function. Cell viability and growth were unchanged for controls (PBS) and increased with the application of Limnodynastes peronii tadpole and adult frog extracts. We investigated the general cytotoxicity of cane toad developmental stages (e.g. eggs, embryonic hatchlings, tadpoles and post-metamorphic toadlets) as well as selected adult tissues (e.g. skin, gut, liver). Our results showed that pre-metamorphic cane toad aqueous extracts used at 1 mg/ml on MDCK cells generated cytotoxicity levels comparable to ouabain treatment (3 μM). After normalisation, extracts from 2-3-month-old toadlets appeared less toxic than pre- and early metamorphic stages. Adult tissues revealed a gradient of cytotoxicity levels ranging from non-toxic brain to highly toxic dorsal skin extracts.     

Cortisol is necessary for seawater tolerance in larvae of a marine teleost the summer flounder

Larval-stage summer flounder (Paralichthys dentatus) were immersed in the corticosteroid-receptor blocker RU486 to test the effects of cortisol deficiency on salinity tolerance. Premetamorphic larvae held at 10 (near isosmotic) or 30 (hyperosmotic) parts per thousand ( per thousand) seawater survived well over 5d in 0, 0.012, or 0.12 microM RU486. However, at concentrations of 1.2 or 3.6 microM RU486, mortality was significantly greater for larvae in 30 per thousand compared to larvae in 10 per thousand. In a separate experiment, the ability of RU486 to inhibit tolerance to hyperosmotic medium (30 per thousand) was confirmed; immersion at 1.2 microM RU486 induced mortality of larvae in the metamorphic climax stage held at 30 per thousand, but not 0 or 10 per thousand. Mortality due to RU486 in pre- or prometamorphic stage larvae was prevented by concurrent immersion in cortisol at concentrations approximately 10-200 times greater than RU486, indicating that the action of RU486 was specific to antagonism of cortisol. The efficacy of 1.2 microM RU486 in reducing survival in 30 per thousand was found to be stage-dependent and exhibited the following hierarchy for fastest time to 50% mortality: prometamorphosis>metamorphic climax>premetamorphosis. In a 5-d pretreatment of pre- or prometamorphic larvae by immersion in 20 microM cortisol and/or 0.12 microM RU486 at 30 per thousand, only RU486 had a limited effect on decreasing survival when larvae were challenged with abrupt exposure to 50 per thousand. In total, the results evidence for the first time a necessary role for cortisol in seawater tolerance of a larval marine teleost.     

Differentiation of the segmented tubular nephron and excretory duct during lamprey metamorphosis

Summary The tubular portion of the adult lamprey nephron differentiates into various morphologically distinct segments during the seven stages of metamorphosis.At stage 1, a rudimentary nephron unit (RNU) originates from a nephrogenic mass attached to the peritoneum and elongates to become associated at its distal end with the archinephric (excretory) duct. The rapidly dividing cells show little sign of differentiation. Separation of the RNU from the peritoneum in stage 2 is first accompanied by widening of lateral intercellular spaces and then eventually by the formation of a small lumen at the proximal end. Cells surrounding a portion of this lumen show early signs of ciliogenesis and hence are located in the position of the presumptive neck segment. The distal end of the stage 2 RNU contains an electron-dense material in an enlarged intercellular space and the cells make non-junctional contact with the archinephric duct. The larval cells of the latter structure are either lost during degeneration and desquamation or are transformed into adult cells. By the end of stage 3 replication of basal bodies and formation of cilia has resulted in the appearance of a ciliated neck segment while numerous microvilli extending into a narrow lumen mark the position of the proximal segment. Furthermore,most of the distal portion of the newly formed tubule possesses a lumen containing a compact mass of material likely secreted by the cells during lumen formation. This presumptive distal segment terminates in the transforming epithelium of the archinephric duct as a presumptive connecting or collecting segment. Fully differentiated neck and proximal segments are present in stage 4 with the latter having a complete pparatus for endocytosis but the distal segment shows only early signs of differentiation of the smooth tubular network. This network appears to arise as an expanded surface area of the plasma membrane. Stage 5 is characterized by elongation of the proximal portion of the tubule, expansion of the tubular network in distal cells, and the identification of a collecting segment. The last two stages show no further major differentiation of the segments. The early differentiation of tubular segments in the adult lamprey nephron likely reflects the immediate physiological needs of this organism as the larval kidney undergoes regression.The potential of the developing kidney of adult lampreys as a mource of information on morphogenesis of microvilli, the endocytotic apparatus, mitochondria, and a smooth tubular network is discussed. It appears that the archinephric duct and non-nephrogenic mesenchyme may play some role in tubulogenesis in lampreys.     

免疫系统的原始功能

本文通过分析动物免疫系统的功能，认为其原始功能为调控变态发育，即免疫细胞原为对幼体进行改造使其完成变态而在成体形成后被诱导自灭的某细胞，由于系统发育过程中幼体标志的大量歧化，使得在个体实际发育中冗余了不少未被自身成体诱导自灭的有关细胞，从而进化出了免疫功能，并在与病原生物等抗原性物质的相互作用中不断发展，此认识可系统解释名种现象的起源。     

Cholangiocyte apoptosis is an early event during induced metamorphosis in the sea lamprey, Petromyzon marinus L

Background

Research in biliary atresia has been hindered by lack of a suitable animal model. Lampreys are primitive vertebrates with distinct larval and adult life cycle stages. During metamorphosis the biliary system of the larval lamprey disappears. Lamprey metamorphosis has been proposed as a model for biliary atresia. We have begun to explore cellular events during lamprey metamorphosis by assessing for cholangiocyte apoptosis.

Materials and Methods

Sea lamprey larvae were housed under controlled environmental conditions. Premetamorphic larvae were induced to undergo metamorphosis by exposure to 0.01% KClO₄. Animals were photographed weekly, and the stage of metamorphosis was assigned based upon external features. Livers were harvested and processed for routine histology and immunohistochemistry. DNA fragmentation was detected using deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays and cholangiocytes were identified with antibodies to cytokeratin-19. Percent TUNEL+ cholangiocytes at different stages of metamorphosis was determined.

Results

The percentage of TUNEL+ cholangiocytes was 10% in premetamorphic (stage 0) lamprey (n = 6), 51% at stage 1 (n = 6), 40% at stage 2 (n = 5), 18% at stage 3 (n = 5), and 9% stage 4 (n = 4). Routine hemotoxylin and eosin stained paraffin-embedded tissue sections revealed frequent apoptotic bodies at stages 3 and 4 of metamorphosis without histologic evidence of necrosis.

Conclusions

DNA fragmentation is identified at the earliest stages of metamorphosis during induced metamorphosis in lampreys. Additional studies are necessary to validate this potentially valuable animal model.     

A reliable,non-invasive technique for measuring growth in tadpoles exposed to salt

The use of chemical de-icers raises salt levels in roadside streams and ponds, which has adverse effects on tadpole development. Experiments on the effects of de-icers on tadpole development are often hampered by difficulties measuring body size without introducing handling stress that may skew results or cause unintended mortality. We have found a linear relationship between surface area and body mass in tadpoles that is unaffected by exposure to salt. Measuring surface area is therefore a suitable technique whose use should be encouraged when investigating the effects of salt exposure on tadpole growth and development.