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PubMed1989

Giardia lamblia: the roles of bile, lactic acid, and pH in the completion of the life cycle in vitro.

Large numbers (10(4) to greater than 10(5)/ml) of Type I water-resistant Giardia lamblia cysts were produced in vitro under conditions that are characteristic of the human intestinal lumen. We define Type I cyst morphology as oval shaped, smooth, and refractile, with cyst wall, axostyle, and median body visible in relief by Normarski differential interference contrast optics. Human and porcine bile induced higher levels of encystation than bovine bile at the alkaline pH (7.8) which occurs in the human lower small intestine. High-pressure liquid chromatography analysis showed that the porcine bile had a preponderance of hyocholate, rather than cholate, while bovine bile had less chenodeoxycholate and more deoxycholate than human bile. Lactic acid, a major product of bacterial metabolism in the human colon, further stimulated encystation. Growth of the preencystation culture without bile also increased subsequent encystation. More than 90% of Type I cysts produced with porcine bile plus lactic acid were viable as indicated by the uptake and retention of fluorescein diacetate and exclusion of propidium iodide. Biological activity of in vitro-derived water-resistant cysts was demonstrated by the observation that 1 to 9.5% excysted in vitro. The percentage of excystation was greatly decreased following encystation at pH 7.0 or by omission of bile or lactic acid. This is the first quantitative in vitro demonstration of the complete life cycle of G. lamblia from humans.

Animals↗
PubMedDate not supplied

Treatment and prevention of Pneumocystis carinii pneumonia and further elucidation of the P. carinii life cycle with 1,3-beta-glucan synthesis inhibitor L-671,329.

Two different classes of 1,3-beta-glucan synthesis inhibitors, the echinocandins and papulacandins, have anti-Pneumocystis activity in an immunosuppressed rat model for acute P. carinii pneumonia (PCP). This activity combined with potent anti-Candida activity makes the echinocandins attractive agents for treating both Pneumocystis and candidiasis in the immunocompromised patient. Natural product echinocandin L-671,329 rapidly eliminates greater than 99% of the P. carinii cysts after 4 days of treatment at a dose of 1 mg/kg twice daily while 2-3 weeks of therapy with trimethoprimsulfamethoxazole (TMP-SMZ) or pentamidine was required to achieve the same degree of cyst clearance. Effects of L-671,329, TMP-SMZ and pentamidine on the trophozoite stage of P. carinii were also explored using a P. carinii-specific DNA probe to quantitate organism load. Although L-671,329 was not as effective as the known agents against the trophozoite stage, prophylactic use of L-671,329 at a daily dose of 1 mg/kg prevented the development of cysts and trophozoites in the rat model. The foamy exudate commonly seen in lungs of animals with PCP is also absent in rats receiving L-671,329 prophylaxis. In addition to demonstrating the potential of L-671,329 as a prophylactic agent these studies also help in elucidating the life cycle of P. carinii. The observation that L-671,329 prophylaxis prevents the appearance of trophozoites, while acute therapy does not directly affect trophozoites, provides the first evidence that the cyst stage is required for trophozoite proliferation. The rapid elimination of cysts by L-671,329 in animals with acute PCP also indicates that all cysts are turning over within 4 days since it is the development of new cysts which is prevented with this compound.

Animals↗
PubMed2006

First description of the endogenous life cycle of Hypoderma sinense affecting yaks and cattle in China.

Larvae belonging to five species of Hypoderma spp. (Diptera, Oestridae) cause myiasis in wild and domestic ruminants that is characterized by migrations within deep tissues. In China hypodermosis is one of the most important arthropod diseases affecting ruminants and, moreover, represents a significant zoonosis, with numerous reports of Hypoderma spp. affecting farmers. Recently, a sixth species, Hypoderma sinense Pleske, has been rediscovered but the endogenous migration pathway within the host body is completely unknown and it represents a major constraint for the control of larval infection. In December 2003 a total of 165 larval stages of Hypoderma spp. were collected from different anatomical sites of 40 yaks slaughtered at an abattoir in the province of Gansu, China. The morphological characters and size of the recovered larvae were used to infer migratory routes and 45 specimens were also subjected to a polymerase chain reaction (PCR) assay of cox1 mtDNA and amplicons sequenced. All the larvae molecularly processed were identified as H. sinense and sequence identity was confirmed by a PCR-restriction fragment length polymorphism (PCR-RFLP) tool carried out using BfaI and HinfI endonucleases. The finding of H. sinense larvae only in the oesophagus or both in oesophagus and subcutaneous tissue of 12 and 15 animals, respectively, indicates that H. sinense larvae migrate through the oesophagus similarly to Hypoderma lineatum (De Villers). The description of the endogenous life cycle of H. sinense will help to determine the timing of specific treatment programmes to guarantee the improvement of animal welfare and health, thus resulting in an increase in livestock production in China.

Animals↗
PubMed1999

Mutations in nonconserved domains of Ty3 integrase affect multiple stages of the Ty3 life cycle.

Ty3, a retroviruslike element of Saccharomyces cerevisiae, transposes into positions immediately upstream of RNA polymerase III-transcribed genes. The Ty3 integrase (IN) protein is required for integration of the replicated, extrachromosomal Ty3 DNA. In retroviral IN, a conserved core region is sufficient for strand transfer activity. In this study, charged-to-alanine scanning mutagenesis was used to investigate the roles of the nonconserved amino- and carboxyl-terminal regions of Ty3 IN. Each of the 20 IN mutants was defective for transposition, but no mutant was grossly defective for capsid maturation. All mutations affecting steady-state levels of mature IN protein resulted in reduced levels of replicated DNA, even when polymerase activity was not grossly defective as measured by exogenous reverse transcriptase activity assay. Thus, IN could contribute to nonpolymerase functions required for DNA production in vivo or to the stability of the DNA product. Several mutations in the carboxyl-terminal domain resulted in relatively low levels of processed 3' ends of the replicated DNA, suggesting that this domain may be important for binding of IN to the long terminal repeat. Another class of mutants produced wild-type amounts of DNA with correctly processed 3' ends. This class could include mutants affected in nuclear entry and target association. Collectively, these mutations demonstrate that in vivo, within the preintegration complex, IN performs a central role in coordinating multiple late stages of the retrotransposition life cycle.

Amino Acid Sequence↗
PubMed2001

The sheepshead minnow as an in vivo model for endocrine disruption in marine teleosts: a partial life-cycle test with 17alpha-ethynylestradiol.

The sheepshead minnow (Cyprinodon variegatus Lacépède), an estuarine fish species, was exposed to 17alpha-ethynylestradiol (EE2) at nominal test concentrations of 0.2, 2, 20, 200, 400, 800, 1,600, and 3,200 ng/L. Fish were exposed for up to 59 d, from subadult stages to sexual maturity, under flow-through conditions. The exposure period was followed by an evaluation of reproductive success and survival of progeny. The reproductive success of exposed sheepshead minnows, as determined from data on egg production from two subsequent spawning trials, was reduced in fish exposed to 200 ng/L EE2 and, in one spawning trial, in the 20-ng/L treatment. Hatching success was reduced in the progeny of fish exposed to 200 ng/L EE2, but survival was good among fry that successfully hatched. Histological examination indicated generalized edema, damage to gill epithelia, hepatic toxicity, fibrosis of the testis, and evidence of sex reversal, including testes-ova and spermatagonia-like cells in ovaries. The maximum acceptable toxicant concentration (MATC) for gonadal development in males was within the normal range of EE2 concentrations in sewage treatment plant effluents. The exposure regimen and choice of test organism, combined with histological examination, allowed independent evaluation of ecologically significant acute, reproductive and estrogenic endpoints. Estrogen receptor-mediated effects occurred at concentrations where reproductive effects were measurable under standard reproduction assays. The sheepshead minnow appears to be a sensitive in vivo model for partial life-cycle testing of compounds that have the potential to disrupt the endocrine system as well as reproduction in estuarine and coastal marine fish species.

Animals↗
PubMed2005

Evolution of salamander life cycles: a major-effect quantitative trait locus contributes to discrete and continuous variation for metamorphic timing.

The evolution of alternate modes of development may occur through genetic changes in metamorphic timing. This hypothesis was examined by crossing salamanders that express alternate developmental modes: metamorphosis vs. paedomorphosis. Three strains were used in the crossing design: Ambystoma tigrinum tigrinum (Att; metamorph), wild-caught A. mexicanum (Am; paedomorph), and laboratory Am (paedomorph). Att/Am hybrids were created for each Am strain and then backcrossed to their respective Am line. Previous studies have shown that a dominant allele from Att (met(Att)) and a recessive allele from lab Am (met(lab)) results in metamorphosis in Att/Am hybrids, and met(Att)/met(lab) and met(lab)/met(lab) backcross genotypes are strongly associated with metamorphosis and paedomorphosis, respectively. We typed a molecular marker (contig325) linked to met and found that met(Att)/met(lab) and met(Att)/met(wild) were associated with metamorphosis in 99% of the cases examined. However, the frequency of paedomorphosis was 4.5 times higher for met(lab)/met(lab) than for met(wild)/met(wild). We also found that met(Att)/met(wild) and met(wild)/met(wild) genotypes discriminated distributions of early and late metamorphosing individuals. Two forms of phenotypic variation are contributed by met: continuous variation of metamorphic age and expression of discrete, alternate morphs. We suggest that the evolution of paedomorphosis is associated with genetic changes that delay metamorphic timing in biphasic life cycles.

Age Factors↗
PubMed1989

Life cycle of Hysterothylacium haze (Nematoda: Anisakidae: Raphidascaridinae).

Natural infections with Hysterothylacium haze in the Japanese common goby, Acanthogobius flavimanus, were observed in detail. In gobies in which no worm eggs were deposited, second-stage larvae were found in the digestive tract wall, and third-stage larvae occurred in the digestive tract wall, mesentery, and body cavity, whereas fourth-stage larvae and adults were found in the body cavity. This stage-habitat relationship demonstrates the infectivity of second-stage larvae to the goby and the larval migration. In heavily infected gobies, eggs and all worm stages, from hatched second-stage larvae to adults, often were found together in the body cavity of one individual host, suggesting that hatched second-stage larvae can develop in the body cavity. It was shown experimentally that H. haze develops to the second stage in the egg and does not hatch spontaneously. When a goby was fed the viscera of heavily infected gobies containing eggs and various stages of worms or artificially incubated eggs containing second-stage larvae, second- and third-stage larvae were recovered from the digestive tract wall, and fourth-stage larvae and adults were found in the body cavity. When polychaetes or crustaceans were placed in contact with infected goby viscera or incubated eggs, only second-stage larvae were recovered from the body cavity of the invertebrates. The experimental results were consistent with observations on natural infections and indicate that the direct life cycle of H. haze may involve invertebrates as transport hosts.

Animals↗
PubMed2006

Double-stranded RNA-binding protein E3 controls translation of viral intermediate RNA, marking an essential step in the life cycle of modified vaccinia virus Ankara.

Infection of human cells with modified vaccinia virus Ankara (MVA) activates the typical cascade-like pattern of viral early-, intermediate- and late-gene expression. In contrast, infection of human HeLa cells with MVA deleted of the E3L gene (MVA-DeltaE3L) results in high-level synthesis of intermediate RNA, but lacks viral late transcription. The viral E3 protein is thought to bind double-stranded RNA (dsRNA) and to act as an inhibitor of dsRNA-activated 2'-5'-oligoadenylate synthetase (2'-5'OA synthetase)/RNase L and protein kinase (PKR). Here, it is demonstrated that viral intermediate RNA can form RNase A/T1-resistant dsRNA, suggestive of activating both the 2'-5'OA synthetase/RNase L pathway and PKR in various human cell lines. Western blot analysis revealed that failure of late transcription in the absence of E3L function resulted from the deficiency to produce essential viral intermediate proteins, as demonstrated for vaccinia late transcription factor 2 (VLTF 2). Substantial host cell-specific differences were found in the level of activation of either RNase L or PKR. However, both rRNA degradation and phosphorylation of eukaryotic translation initiation factor-2alpha (eIF2alpha) inhibited the synthesis of VLTF 2 in human cells. Moreover, intermediate VLTF 2 and late-protein production were restored in MVA-DeltaE3L-infected mouse embryonic fibroblasts from Pkr(0/0) mice. Thus, both host-response pathways may be involved, but activity of PKR is sufficient to block the MVA molecular life cycle. These data imply that an essential function of vaccinia virus E3L is to secure translation of intermediate RNA and, thereby, expression of other viral genes.

2',5'-Oligoadenylate Synthetase↗
PubMed1977

Sarcocystis hemionilatrantis (Sp. N.) life cycle in mule deer and coyotes.

Fifteen coyotes (Canis latrans) shed sporulated sporocysts in their feces after eating freshly ground skeletal muscles from a mule deer (Odocoileus hemionus hemionus) infected with microscopic-sized cysts of Sarcocystis. Sporocysts were shed intermittently from 12 to 36 days after ingestion of the infected meat. Sporocyst size averaged 14.4 X 9.3 mum. Eleven mule deer fawns orally inoculated with these sporocysts became infected and 9 of 11 died between post-inoculation days (PID) 27 and 63. Clinical signs of anorexia, weight loss, pyrexia and weakness were evident prior to death. A calf (Bos taurus) and two lambs (Ovis aries) orally inoculated with these sporocysts did not become infected and remained healthy throughout the experiments. Similarly, uninoculated control animals consisting of three mule deer fawns, two lambs and one calf remained healthy during the experiment. Preliminary histologic examinations conducted on selected tissues from all animals revealed microscopic-sized schizogonous stages in macrophages, between muscle fibers and near blood vessels in the esophagus, heart, biceps femoris, semi-membranosus, diaphragm and tongue from seven of eight fawns which died between PID 27 and 39. Developing or mature muscle cysts were not found in fawn tissue until PID 60. Sarcocysts were found in the three infected fawns examined after this time. Muscle cysts or earlier schizont stages were not found in tissues from the inoculated or uninoculated calves and lambs. A single muscle cyst was found in one control fawn; the other two control fawns were negative for both muscle cysts and other schizogonous stages. These results established that the life cycle of this species of Sarcocystis can be completed with coyotes as the definitive host and mule deer as the intermediate host. Based on the demonstrated host specificity and earlier findings, the name Sarcocystis hemionilatrantis is proposed for this parasite of mule deer and coyotes.

Animals↗
PubMed1995

The life cycle of Contracaecum osculatum (Rudolphi, 1802) sensu stricto (Nematoda, Ascaridoidea, Anisakidae) in view of experimental infections.

Hatched, ensheathed third-stage larvae of Contracaecum osculatum, 300-320 microns long, were shown to be infective to copepods, to nauplius larvae of Balanus and to small specimens of fishes (sticklebacks, O-group eelpout). Other fishes such as gobies and small flatfishes became infected by ingesting infected crustaceans. Cod were infected by being given infected small fishes. In the crustacean paratenic hosts, little growth of the larvae occurred, if any. In the liver sinusoids of sticklebacks and gobies the length of most of the unencapsulated third-stage larvae had not even doubled within 6 months of infection. The fate of larvae (max. 2 mm long) given to cod via infected intermediate fish hosts was apparently decided by the size of the larvae only. Small larvae became encapsulated and eventually died in the liver and wall of the gastrointestinal tract. Larger larvae migrated to the liver parenchyma, where some grew to a length of as much as 10 mm. The growth of the larvae in sticklebacks was shown not to be affected by an increase in temperature (infected fish being transferred from 8 degrees to 14 degrees and 20 degrees C), by the intensity of infection and, partly, by the age of infection (e.g. some 2-week-old and 6-month-old larvae were of identical size). In the liver and mesentery of plaice the third-stage larvae developed via copepod paratenic hosts to infectivity (i.e. to more than 4 mm in length), showing that the life cycle may be completed with an optional paratenic invertebrate host and only one intermediate fish host.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2002

Life cycle of MTs: persistent growth in the cell interior, asymmetric transition frequencies and effects of the cell boundary.

Microtubule dynamics were investigated in CHO and NRK cells by novel experimental approaches designed to evaluate the microtubule behavior in the cell interior. These approaches were: (1) laser photobleaching of a path through the centrosome; (2) direct observation of microtubules in centrosome-containing cytoplasts; (3) GFP-CLIP-170 expression as a marker for microtubule plus end growth; and (iv) sequential subtraction analysis. The combination of these approaches allowed us to obtain data where the density of microtubules had previously prevented conventional methods to be applicable. In the steady state, nascent microtubules grew persistently from the centrosome towards the cell margin. Frequently, they arrived at the cell margin without undergoing any transition to the shortening phase. In contrast to the growth of microtubules, shortening of the plus ends from the periphery was non-persistent; that is, rescue was frequent and the extent of shortening showed a distribution of lengths reflecting a stochastic process. The combination of persistent growth and a cell boundary led to a difference in apparent microtubule behavior in the cell interior compared with that near the cell margin. Whereas microtubules in the cell interior showed asymmetric transition frequencies, their behavior near the cell margin showed frequent fluctuations between phases of shortening and growth. Complete microtubule turnover was accomplished by the relatively rare episodes of shortening back to the centrosome. Release from the centrosome with subsequent minus end shortening also occurred but was a minor mechanism for microtubule turnover compared with the plus end pathway. We propose a life cycle for a microtubule which consists of rapid growth from the centrosome to the cell margin followed by an indefinite period of fluctuations of phases of shortening and growth. We suggest that persistent growth and asymmetric transition frequencies serve the biological function of providing a mechanism by which microtubules may rapidly accommodate to the changing shape and advancing edge of motile cells.

Animals↗
PubMed1997

Structures of orbivirus VP7: implications for the role of this protein in the viral life cycle.

BACKGROUND: Bluetongue virus (BTV) is the prototypical virus of the genus orbivirus in the family Reoviridae and causes an economically important disease in domesticated animals, such as sheep. BTV is larger and more complex than any virus for which comprehensive atomic level structural information is available. Its capsid is made primarily from four structural proteins two of which, VP3 and VP7, form a core which remains intact as the virus penetrates the host cell. Each core particle contains 780 copies of VP7. The architecture of the trimeric VP7 molecule has been revealed by crystallographic analysis and is unlike other viral coat proteins reported to date. RESULTS: Two new crystal structures of VP7 have been solved, one (a cleavage product) at close to atomic resolution and the other at lower resolution. The VP7 subunit consists of two domains. The smaller, 'upper', domain is exposed on the core surface and has the beta jelly-roll motif common to many capsid proteins. The second, 'lower', domain is composed of a bundle of alpha helices. The cleavage product comprises the upper domain, which forms a rigid invariant trimeric fragment. The lower resolution structure of the intact molecule indicates that the alpha-helical domain can rotate about the linker to the upper domain to adopt radically different orientations with respect to the threefold axis in the intact protein. CONCLUSIONS: The crystal structures of VP7 reveal a remarkable mix of rigidity and flexibility that may provide insights towards understanding how VP7 interacts with the other capsid proteins of different stoichiometries. These results suggest that substantial conformational changes in VP7 occur at some stage in the viral life cycle. Such changes may be related to the central role that VP7 is likely to play in cell attachment and membrane penetration.

Bluetongue virus↗
PubMed1997

A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle.

Adeno-associated virus (AAV) replication is dependent on two copies of a 145-bp inverted terminal repeat (ITR) that flank the AAV genome. This is the primary cis-acting element required for productive infection and the generation of recombinant AAV (rAAV) vectors. We have engineered a plasmid (pDD-2) containing only 165 bp of AAV sequence: two copies of the D element, a unique sequence adjacent to the AAV nicking site, flanking a single ITR. When assayed in vivo, this modified hairpin was sufficient for the replication of the plasmid vector when Rep and adenovirus (Ad) helper functions were supplied in trans. pDD-2 replication intermediates were characteristic of the AAV replication scheme in which linear monomer, dimer, and other higher-molecular-weight replicative intermediates are generated. Compared to infectious AAV clones for replication, the modified hairpin vector replicated more efficiently independent of size. Further analysis demonstrated conversion of the input circular plasmid to a linear substrate with AAV terminal repeat elements at either end as an initial step for replication. This conversion was independent of both Rep and Ad helper genes, suggesting the role of host factors in the production of these molecules. The generation of these substrates suggested resolution of the modified terminal repeat through a Holliday-like structure rather than replication as a mechanism for rescue. Production of replicative intermediates via this plasmid substrate were competent not only for AAV DNA replication but also for encapsidation, infection, integration, and subsequent rescue from the chromosome when superinfected with Ad and wild-type AAV. These studies demonstrate that this novel 165-bp ITR substrate is sufficient in cis for the AAV life cycle and should provide a valuable reagent for further dissecting the cis sequences involved in AAV replication, packaging, and integration. In addition, this novel plasmid vector can be used as a substrate for both rAAV vector production and synthetic plasmid vector delivery.

DNA, Viral↗
PubMed1976

Cytological and genetic studies of the life cycle of Saccharomycopsis lipolytica.

In the alkane yeast Saccharomycopsis lipolytica (formerly: Candida lipolytica) the variability in the ascospore number is caused by the absence of a correlation between the meiotic divisions and spore wall formation. In four spored yeasts, after meiosis II, a spore wall is formed around each of the four nuclei produced by meiosis II. However, in the most frequently occurring two spored asci of S. lipolytica, the two nuclei are already enveloped by the spore wall after meiosis I due to a delay of meiosis II. This division takes place within the spore during the maturation of the ascus. In this case germination of the binucleate ascospore is not preceded by a mitosis. It follows that the cells of the new haploid clones are mononucleate. In the three spored asci, which occur rarely, only one nucleus is surrounded by a spore wall after meiosis I; the other nucleus undergoes meosis II before the onset of spore wall formation. The result is one bincleate and two mononucleate spores. In the one spored asci the two meiotic divisions occur within the young ascosphore, i.e. spore wall formation starts immediately after development of the ascus. These cytological observations were substantiated by genetic data, which in addition confirmed the prediction that binucleate spores may be heterokaryotic. This occurs when there is a postreduction of at least one of the genes by which the parents of the cross differ. This also explains the high frequency of prototrophs in the progeny of non-allelic auxotrophs since random spore isolates are made without distinguishing between mono- and binucleate spores. The possibility of analysing offspring of binucleate spores by tetrad analysis is discussed. These findings enable us to understand the life cycle of S. lipolytica in detail and we are now in a position to start concerted breeding for strain improvement especially with respect to single cell protein production.

Ascomycota↗
PubMed1999

Inhibition of replication of HIV-1 at both early and late stages of the viral life cycle by single-chain antibody against viral integrase.

Retroviruses including HIV-1 integrates a DNA copy of their RNA genome into cellular DNA of the infected cell. This reaction, essential and unique to replication of retroviruses, is mediated by the viral enzyme, integrase (IN). We constructed a recombinant gene encoding a single-chain, antigen-binding peptide (scAb2-19), which interacted with a carboxyl terminal part of HIV-1 IN. HeLa CD4 cells expressing scAb2-19 localized in either cytoplasmic or nuclear compartment were resistant to HIV-1 infection at an multiplicity of infection (MOI) of 0.25 or 0.063, but the resistance was overcome when MOI was increased to 1. To determine whether this resistance was due to inhibition of the early events, transduction experiments were performed with a replication-incompetent HIV-1 vector carrying bacterial lacZ driven by an internal Tat-independent cytomegalovirus immediate early promoter. Both cytoplasmic and nuclear expressions of scAb2-19 resulted in decrease in the transduction efficiency on HeLa CD4 cells. This implies that an early step of replication--before or during integration--was affected by the scAb2-19. Furthermore, cytoplasmic expression of scAb2-19 did not affect the viral amount released from the cells transfected with HIV-1 infectious clone DNA (pLAI). However, infectivity relative to reverse transcriptase activity was lower for virions released from the 293T cells cotransfected with pLAI and the cytoplasmic scAb2-19 expression plasmid than for those released from the 293T cells transfected with pLAI alone. This implies that scAb2-19 reduced infectivity of released virions by interfering a late step of the viral replication. The single-chain, antigen-binding peptide molecule may prove useful not only for studies of the functions of IN and its role in the viral life cycle but also for developing a gene therapy strategy against AIDS.

Amino Acid Sequence↗
PubMed1988

Iodothyronine 5'-deiodinase activity in the amphibian Rana catesbeiana at different stages of the life cycle.

In a previous study, 5' deiodinase (5'D) activity was detected in skin but not in other tissues of premetamorphic tadpoles. When these tadpoles were stimulated to metamorphic climax by treatment with thyroid hormone, activity was increased in skin and was found also in gut and tail tissue. In the present study, 5'D activity was studied at various stages of spontaneous development from early prometamorphosis through metamorphic climax and including the adult frog. 5'D activity was detected in gut and skin at all stages studied and in metamorphosing tail tissue. It was minimal or absent in all other tissues examined. Activity in prometamorphic skin was comparable to that previously observed in premetamorphic skin but increased significantly during metamorphic climax and remained at this level in the adult. 5'D activity was not observed in tail tissue until resorption commenced and when present could not be correlated with tadpole stage or tail length. In both skin and tail, the 5'D activity exhibited values for Michaelis-Menten constant (Km) for rT3 and T4 in the nanomolar range. During prometamorphosis, the 5'D activity in gut exhibited a value for the rT3 Km in the micromolar range. As the tadpoles entered metamorphic climax, the rT3 Vmax of this activity in gut decreased and a second type of 5'D activity, with a Km for rT3 in the nanomolar range, was detected in this tissue. By stage XXI, only the latter component was observed, and its activity increased significantly during metamorphic climax. In the adult frog, 5'D activity in gut was comparable to that seen at the onset of climax. The adult frog also exhibited T3 5D activity in all tissues studied. These observations, together with those reported in the previous study, indicate that gut and skin are the principal T3-generating organs in anuran amphibia and suggest that increased activity of the 5'D system, at the time in the life cycle that the tissues require increased amounts of thyroid hormone, is an important part of thyroid hormone economy in this species.

Aging↗
PubMed2005

Life-cycle exposure of fathead minnows (Pimephales promelas) to an ethinylestradiol concentration below 1 ng/L reduces egg fertilization success and demasculinizes males.

Forty-eight hours after fertilization, fathead minnow (Pimephales promelas) eggs were exposed to the synthetic estrogen 17alpha-ethinylestradiol (EE2) at nominal concentrations of 0.32 and 0.96 ng/L and measured concentrations of 3.5, 9.6, and 23 ng/L. The fish were observed through the larval, juvenile, and adult stages. Growth, secondary sex characteristics, the liver somatic index, the gonadosomatic index, and fecundity were examined after several lengths of exposure. No significant changes were seen in fry or juvenile growth from 8 to 30 days posthatch (dph). An increase in the ovipositor index (a female secondary sex characteristic) was the most sensitive early response at 60 dph and was seen in fish exposed to EE2 concentrations > or = 3.5 ng/L. Continuation of the EE2 exposure until 150 dph, through maturation and reproduction, allowed measurement of two sensitive end points: decreased egg fertilization and sex ratio (skewed toward females), both of which were significantly affected at the lowest EE2 concentration tested, 0.32 ng/L. The next most sensitive end point was demasculinization (decreased male secondary sex characteristic index) of males exposed to an EE2 concentration of 0.96 ng/L. The effects of low concentrations of EE2 (0.32 and 0.96 ng/L) were manifested in male fish (decreased male sex characteristics and reduced egg fertilization success), whereas female fish showed no changes in the gonadosomatic index. Exposure to higher EE2 concentrations negatively affected females, as shown by a reduced gonadosomatic index at 150 dph in fish exposed to > or =3.5 ng/L EE2. Although there were some end points that showed changes at 60 dph, the reproductive end points and external sex characteristics measured in mature fish at 150 dph were more sensitive, with response thresholds of EE2 ranging from 0.32 to 0.96 ng/L. The concentrations of EE2 that negatively affected fathead minnows were similar to or lower than those detected in many municipal wastewater effluents. In conclusion, life-cycle exposure of fathead minnows proved to be a very sensitive bioassay, and responses were seen at concentrations of less than 1 ng/L, which are environmentally relevant concentrations of EE2.

Animals↗
PubMed2001

Life-cycle toxicity of 4-nonylphenol to medaka (Oryzias latipes).

We studied the chronic effects of 4-nonylphenol (4-NP) on reproductive status of medaka (Oryzias latipes) over two generations of continuous exposure. The exposure study of the parental (F0) medaka was begun on embryos within 24 h postfertilization and continued with monitoring through embryological development, hatching, posthatch survival, growth, sexual differentiation, and reproduction under flow-through exposures to mean measured 4-NP concentrations of 4.2, 8.2, 17.7, 51.5, and 183 microg/L for up to 104 d. Eggs spawned from the F0 fish at 102 and 103 d posthatch were also examined for hatchability, survival after hatching, growth, and sexual differentiation until 60 d posthatch. The 183-microg/L treatment significantly reduced the embryo survival and swim-up success of the F0 fish. The cumulative mortality after swim-up of the F0 fish exposed to 17.7 and 51.5 microg/L were significantly higher than the control mortality. No concentration-related effect of 4-NP was observed on the growth of surviving F0 fish at 60 d posthatch. However, the sex ratio estimated from the appearance of their secondary sex characteristics was skewed toward female in the 51.5-microg/L treatment. Additionally, gonadal histology showed that 20% of the fish in the 17.7-microg/L treatment and 40% in the 51.5-microg/L treatment had testis-ova, indicating that 4-NP affects the gonadal development and survival of medaka at similar concentrations in juveniles. The sex ratio of the F0 fish in the 51.5-microg/L treatment was completely skewed toward female; subsequently, the effects on fecundity and fertility in this generation were monitored at mean measured concentrations of 4.2, 8.2, and 17.7 microg/L from 71 to 103 d posthatch. Fecundity was unaffected by any of the treatments examined. The mean fertility in the 17.7-microg/L treatment was reduced to 76% of that in the controls, although no statistically significant differences were determined. Overall, these results indicate that the lowest-observed-effect concentration (LOEC) and no-observed-effect concentration (NOEC) of 4-NP through the life cycle of the F0 medaka were 17.7 and 8.2 microg/L, respectively. In the F1 medaka, no significant effects were observed on hatching success, posthatch mortality, or growth, but sexual differentiation at 60 d posthatch was affected. Induction of testis-ova in the gonads of the F1 fish was observed in both the 8.2- and the 17.7-microg/L concentrations. The results indicate that 4-NP can have significant effects on reproductive potential of medaka at concentrations as low as 17.7 microg/L.

Animals↗