Evolution of parental care and ovulation behavior in oysters

Approximately half of all living oysters brood offspring in the inhalant chamber of their mantle cavities; the remainder are broadcast spawners which do not engage in parental care of young. Ostreid ovulation involves a complex behavioral sequence that results in the countercurrent passage of newly spawned eggs through the gills (ctenidia) and into the inhalant chamber. We constructed molecular and combined-evidence phylogenetic trees to test hypotheses concerning the directionality of parental care evolution, and the evolutionary significance of the trans-ctenidial ovulation pathway, in the Ostreidae. Representatives of all three ostreid subfamilies, together with gryphaeid and nonostreoidean pterioid outgroups, were sequenced for a 941-nucleotide fragment of the 28S ribosomal gene. Our phylogenetic analyses indicate that (1) the Ostreidae are robustly monophyletic, (2) broadcast spawning and larval planktotrophy are ancestral ostreid traits, (3) trans-ctenidial ovulation predates the evolution of parental care in ostreid lineages, and (4) brooding originated once in the common ancestor of the Ostreinae/Lophinae, involved a modification of the final behavioral step in the ancestral ovulation pathway, and has been retained in all descendent lineages. Our data permit an independent test of fossil-based ostreid phylogenetic hypotheses and provide novel insights into oyster evolution and systematics.     

Pan-tropical distribution of the Jurassic ostreid bivalves

Globally, the Jurassic cemented ostreid bivalves from northern Tibet, SW China, have a pan-tropical distribution pattern between palaeolatitudes of 60° South and North. Ostreid bivalves have tiny to large larvae, and nearly all of them have a cemented mode of life during their adult stage. The distribution pattern and the habits of the Jurassic ostreid bivalves revealed that, in the Jurassic time interval, all seas and oceans were interconnected, and ostreid bivalves were very capable of dispersion through their planktonic larval stage or as pseudoplankton during the post-larval stage, and even were able to cross the palaeo-Pacific Ocean from east to west. However, latitudinal temperature variation limited the distribution of these thermophilous bivalves to the lower and middle latitudes.     

Plasmodium falciparum: isolation and characterization of a 55-kDa protease with a cathepsin D-like activity from P. falciparum

Native electrophoresis followed by imprint digest method using hemoglobin as substrate allowed the detection of parasite hemoglobinase activity at acidic pH (3.9 to 5). This protease was inhibited specifically by pepstatin A and insensitive to other protease inhibitors. The molecular weight determination using modified SDS-PAGE followed by imprint digest method, demonstrated a single area of activity at 55-58 kDa, similar to cathepsin D characterized in eucaryotic cells. The parasitic origin has been shown by radiolabeling experiments with [35S]-methionine. The 55-kDa protein was immunoprecipitated by a rabbit anti-cathepsin D serum.     

A comparison of the cholinesterases of an oyster (Crassostrea virginica) and a clam (Macrocallista nimbosa).

Cholinesterase activities in the hearts and ganglia of an oyster (Crassostrea virginica) and a venerid clam (Macrocallista nimbosa) were measured and compared. Tissue extracts were partially purified by ammonium sulfate fractionation followed by gel column chromatography. Enzymatic activity was assayed spectrophotometrically; substrates were acetyl-, butyryl-, and propionylthiocholine (ATC, BTC, PTC). Kinetic constants characterizing each enzyme were derived. At all substrate concentrations, the hydrolysis rates of both clam enzymes were in the order: BTC greater than PTC greater than ATC. With oyster enzymes the ranking was ATC greater than or equal to PTC greater BTC. The specific activities of oyster heart and ganglion enzymes were similar. In contrast, clam ganglion extracts were 75-100 times more active than clam heart extracts and, with any substrate, had greater activity than either oyster enzyme. All enzyme preparations proved to be homogeneous on the bases of constant substrate activity ratios in successive column fractions, and of intermediate velocities with mixed substrates. Six cholinesterase inhibitors were tested. The specific acetylcholinesterase antagonist, B.W. 62C47, WAS MUCH MORE EFFECTIVE AGAINST OYSTER ENZYMES, WHILE THE SPECIFIC ANTIBUTYRYLCHOLINESTERASE, ISO-OMPA, almost totally inhibited calm enzyme activity, but had little effect on oyster. Eserine was the most effective inhibitor of both enzymes. In conclusion, the enzymes in oyster tissues are acetylcholinesterases, while clam enzymes are butyrylcholinesterases. Nevertheless, clam ganglion esterase is sifficiently active to hydrolyze the physiological substrate, acetylcholine. These results explain the long-observed differences in isolated heart pharmacology between ostreid and venerid bivalves.     

Detection of ostreid herpesvirus-1 (OsHV-1) by PCR using a rapid and simple method of DNA extraction from oyster larvae

A DNA extraction procedure was developed for the detection of ostreid herpesvirus-1 (OsHV-1) using the polymerase chain reaction (PCR) in oyster larvae. The DNA extraction procedure developed was tested on 8 larval samples. Abnormal nuclei with characteristic features associated with OsHV-1 infections were only observed in samples in which the viral DNA was detected by PCR. A previously described competitive PCR method was applied to detect inhibition during PCR reactions. The results show that the method can be used on small amounts of oyster larvae (3 mg) for the detection of OsHV-1 DNA by PCR.     

Improved imprinting technique for study of plant tissues.

The exposed surface of plant tissues is coated with nail varnish. When peeled off, the transparent coating film bears a replica imprint of the tissue. Transparent adhesive tape is used for lifting the film imprint and mounting it on a microslide without curling. By a similar method, an opaque sealant replica with an aluminum foil support may be prepared which can serve as a mold for obtaining a transparent secondary imprint useful for light microscopic studies. The replicas can be stored like herbarium specimens for future use.     

Improved Imprinting Technique for Study of Plant Tissues

The exposed surface of plant tissues is coated with nail varnish. When peeled off, the transparent coating film bean a replica imprint of the tissue. Transparent adhesive tape is used for lifting the film imprint and mounting is on a microslide without curling. By a similar method, an opaque sealant replica with an aluminum foil support may be prepared which can serve as a mold for obtaining a transparent secondary imprint useful for light microscopic studies. The replicas can be stored like herbarium specimens for future use.     

Zinc finger protein ZFP57 requires its co-factor to recruit DNA methyltransferases and maintains DNA methylation imprint in embryonic stem cells via its transcriptional repression domain

Previously, we discovered that ZFP57 is a maternal-zygotic effect gene, and it maintains DNA methylation genomic imprint at multiple imprinted regions in mouse embryos. Despite these findings, it remains elusive how DNA methyltransferases are targeted to the imprinting control regions to initiate and maintain DNA methylation imprint. To gain insights into these essential processes in genomic imprinting, we examined how ZFP57 maintains genomic DNA methylation imprint in mouse embryonic stem (ES) cells. Here we demonstrate that the loss of ZFP57 in mouse ES cells led to a complete loss of genomic DNA methylation imprint at multiple imprinted regions, similar to its role in mouse embryos. However, reintroduction of ZFP57 into Zfp57-null ES cells did not result in reacquisition of DNA methylation imprint, suggesting that the memory for genomic imprinting had been lost or altered in Zfp57-null ES cells in culture. Interestingly, ZFP57 and DNA methyltransferases could form complexes in the presence of KAP1/TRIM28/TIF1β when co-expressed in COS cells. We also found that the wild-type exogenous ZFP57 but not the mutant ZFP57 lacking the KRAB box that interacts with its co-factor KAP1/TRIM28/TIF1β could substitute for the endogenous ZFP57 in maintaining the DNA methylation imprint in ES cells. These results suggest that ZFP57 may recruit DNA methyltransferases to its target regions to maintain DNA methylation imprint, and this interaction is likely facilitated by KAP1/TRIM28/TIF1β.     

Sporadic imprinting defects in Prader-Willi syndrome and Angelman syndrome: implications for imprint-switch models, genetic counseling, and prenatal diagnosis.

The Prader-Willi syndrome (PWS) and the Angelman syndrome (AS) are caused by the loss of function of imprinted genes in proximal 15q. In approximately 2%-4% of patients, this loss of function is due to an imprinting defect. In some cases, the imprinting defect is the result of a parental imprint-switch failure caused by a microdeletion of the imprinting center (IC). Here we describe the molecular analysis of 13 PWS patients and 17 AS patients who have an imprinting defect but no IC deletion. Heteroduplex and partial sequence analysis did not reveal any point mutations of the known IC elements, either. Interestingly, all of these patients represent sporadic cases, and some share the paternal (PWS) or the maternal (AS) 15q11-q13 haplotype with an unaffected sib. In each of five PWS patients informative for the grandparental origin of the incorrectly imprinted chromosome region and four cases described elsewhere, the maternally imprinted paternal chromosome region was inherited from the paternal grandmother. This suggests that the grandmaternal imprint was not erased in the father's germ line. In seven informative AS patients reported here and in three previously reported patients, the paternally imprinted maternal chromosome region was inherited from either the maternal grandfather or the maternal grandmother. The latter finding is not compatible with an imprint-switch failure, but it suggests that a paternal imprint developed either in the maternal germ line or postzygotically. We conclude (1) that the incorrect imprint in non-IC-deletion cases is the result of a spontaneous prezygotic or postzygotic error, (2) that these cases have a low recurrence risk, and (3) that the paternal imprint may be the default imprint.     

Establishment and maintenance of DNA methylation patterns in mouse Ndn: implications for maintenance of imprinting in target genes of the imprinting center

Ndn is located on chromosome 7C, an imprinted region of the mouse genome. Imprinting of Ndn and adjacent paternally expressed genes is regulated by a regional imprinting control element known as the imprinting center (IC). An IC also controls imprint resetting of target genes in the region of conserved synteny on human chromosome 15q11-q13, which is deleted or rearranged in the neurodevelopmental disorder Prader-Willi syndrome. Epigenetic modifications such as DNA methylation, which occur in gametes and can be stably propagated, are presumed to establish and maintain the imprint in target genes of the IC. While most DNA becomes substantially demethylated by the blastocyst stage, some imprinted genes have regions that escape global demethylation and may maintain the imprint. We have now analyzed the methylation of 39 CpG dinucleotide sequences in the 5' end of Ndn by sodium bisulfite sequencing in gametes and in preimplantation and adult tissues. While sperm DNA is completely unmethylated across this region, oocyte DNA is partially methylated. A distinctive but unstable maternal methylation pattern persists until the morula stage and is lost in the blastocyst stage, where low levels of methylation are present on most DNA strands of either parental origin. The methylation pattern is then substantially remodeled, and fewer than half of maternally derived DNA strands in adult brain resemble the oocyte pattern. We postulate that for Ndn, DNA methylation may initially preserve a gametic imprint during preimplantation development, but other epigenetic events may maintain the imprint later in embryonic development.     

Genomic imprinting in Drosophila is maintained by the products of Suppressor of variegation and trithorax group,but not Polycomb group,genes

Genomic imprinting is a form of epigenetic inheritance that is characterized by differential expression of a gene depending on its parental origin. The mini-X chromosome Dp(1;f)LJ9 in Drosophila shows this type of classical imprinting; when transmitted by the maternal parent genes on this chromosome are fully expressed, but when the chromosome is transmitted by the male parent at least three genes are subject to silencing, resulting in a variegated expression pattern. Chemical and environmental modifiers of position-effect variegation have been shown to alter the somatic maintenance of the imprint. To extend these observations, several mutations in chromatin-associated proteins were examined for their effect on imprinting on the Dp(1;f)LJ9 mini-X chromosome. Effects on establishment and maintenance were independently assessed by genetically associating the mutations in chromatin modifiers with the mini-X chromosome in either the parents, where the imprint is established, or the progeny, in which the imprint must be maintained. Nine Suppressor of variegation [ Su(var)] mutations, including alleles of the Su(var)2-5 gene, which encodes the well characterized heterochromatin-associated protein HP1, abolished maintenance but not the establishment of the imprint. Mutant alleles of two genes in the trithorax group ( trx-G), brahma and trithorax, showed a maternal-effect enhancement of the paternal imprint. Surprisingly, however, with the exception of an Enhancer of Polycomb [ E(Pc)] allele, none of the Polycomb-group ( Pc-G) mutations tested affected the imprint. Thus, the maintenance of this imprint relies on the wild-type products of Su(var) and trx-G, but not Pc-G, genes. Finally, none of the mutations tested affected the maintenance of the maternal imprint or the establishment of either the maternal or paternal imprint, suggesting that the maternal and paternal imprints depend on different molecular processes and that imprint establishment and maintenance are independently regulated.     

Skin imprint cytology in the diagnosis of cutaneous T-cell lymphomas. A preliminary report

A study was undertaken to evaluate the utility of the skin imprint technique and the Papanicolaou stain in the diagnosis of cutaneous T-cell lymphomas (CTCL), to better characterize cytologic findings in CTCL and to establish criteria for the cytologic diagnosis of CTCL. Differential cell counts of 17 specimens, including 12 cases of CTCL and 5 of benign skin infiltrates, were performed. There appear to be some significant differences between the groups in terms of cellularity, number of cerebriform mononuclear cells and presence of mitotic figures. The skin imprint technique, using Papanicolaou staining, seems to facilitate recognition of diagnostically important cells. The usefulness of skin imprint cytology resides in its potential for diagnosing early lesions of CTCL when diagnosis by other methods may be difficult.     

Experimentally generated footprints in sand: Analysis and consequences for the interpretation of fossil and forensic footprints

Fossilized footprints contain information about the dynamics of gait, but their interpretation is difficult, as they are the combined result of foot anatomy, gait dynamics, and substrate properties. We explore how footprints are generated in modern humans. Sixteen healthy subjects walked on a solid surface and in a layer of fine‐grained sand. In each condition, 3D kinematics of the leg and foot were analyzed for three trials at preferred speed, using an infrared camera system. Additionally, calibrated plantar pressures were recorded. After each trial in sand, the depth of the imprint was measured under specific sites. When walking in sand, subjects showed greater toe clearance during swing and a 7° higher knee yield during stance. Maximal pressure was the most influential factor for footprint depth under the heel. For other foot zones, a combination of factors correlates with imprint depth, with pressure impulse (the pressure‐time integral) gaining importance distally, at the metatarsal heads and the hallux. We conclude that footprint topology cannot be related to a single variable, but that different zones of the footprint reflect different aspects of the kinesiology of walking. Therefore, an integrated approach, combining anatomical, kinesiological, and substrate‐mechanical insights, is necessary for a correct interpretation. Am J Phys Anthropol, 2010. © 2009 Wiley‐Liss, Inc.     

Cytology of desmoplastic medulloblastoma in imprint smears: a report of 2 cases

BACKGROUND: Desmoplastic medulloblastoma is a rare subtype of medulloblastoma with astroglial differentiation. The cytomorphologic features in intraoperative imprint smears from 2 cases of desmoplastic medulloblastoma are described. CASE REPORTS: A 22-year-old man and 27-year-old woman with a cerebellar tumor underwent craniotomy and tumor resection. The imprint cytologic smears contained cellular zones and nodular hypocellular areas containing astroglial and oligodendrogliallike elements. The cytology was misinterpreted as glial tumors, while the final histologic diagnosis in both cases were desmoplastic medulloblastoma. CONCLUSION: Desmoplastic medulloblastoma shows distinctive cytology in intraoperative smears. However, the occurrence of this rare type in adults and the presence of astroglial elements in imprint smears may cause a cytologic misinterpretation as gliomas.     

Imprint cytology of parathyroid tissue in relation to other tissues of the neck and mediastinum

OBJECTIVE: To retest the hypothesis that imprint cytology may be used to reliably diagnose parathyroid tissue and, if so, to ascertain whether accuracy in this technique may be easily attained. STUDY DESIGN: Imprint preparations from 15 parathyroid, 10 thyroid, 8 lymphoreticular and 2 adipose tissue specimens were assessed blindly by two pathologists, one of whom (pathologist B) had only limited experience with endocrine tissue imprint cytology. RESULTS: Both assessors consistently distinguished parathyroid and thyroid preparations from lymphoreticular and adipose tissue preparations. While there was occasional difficulty in distinguishing between parathyroid and thyroid preparations, this was usually attributable to the scanty nature of the preparations. No single cytologic feature allowed a distinction between parathyroid and thyroid tissue. However, by considering several relatively diagnostic features collectively, pathologist B showed an increase in specificity and sensitivity rates for distinguishing parathyroid from thyroid imprints from 82% to 100% and 57% to 83%, respectively. CONCLUSION: The high accuracy rates and rapid [table: see text] learning curve shown by imprint cytology in distinguishing between different neck or mediastinal tissue types, together with its time- and cost-cutting potential, support a role for the technique in the intraoperative diagnosis of parathyroid tissue.     

Infectious diseases of marine molluscs and host responses as revealed by genomic tools

More and more infectious diseases affect marine molluscs. Some diseases have impacted commercial species including MSX and Dermo of the eastern oyster, QPX of hard clams, withering syndrome of abalone and ostreid herpesvirus 1 (OsHV-1) infections of many molluscs. Although the exact transmission mechanisms are not well understood, human activities and associated environmental changes often correlate with increased disease prevalence. For instance, hatcheries and large-scale aquaculture create high host densities, which, along with increasing ocean temperature, might have contributed to OsHV-1 epizootics in scallops and oysters. A key to understanding linkages between the environment and disease is to understand how the environment affects the host immune system. Although we might be tempted to downplay the role of immunity in invertebrates, recent advances in genomics have provided insights into host and parasite genomes and revealed surprisingly sophisticated innate immune systems in molluscs. All major innate immune pathways are found in molluscs with many immune receptors, regulators and effectors expanded. The expanded gene families provide great diversity and complexity in innate immune response, which may be key to mollusc''s defence against diverse pathogens in the absence of adaptive immunity. Further advances in host and parasite genomics should improve our understanding of genetic variation in parasite virulence and host disease resistance.     

A chromodomain protein, Swi6, performs imprinting functions in fission yeast during mitosis and meiosis

Inheritance of stable states of gene expression is essential for cellular differentiation. In fission yeast, an epigenetic imprint marking the mating-type (mat2/3) region contributes to inheritance of the silenced state, but the nature of the imprint is not known. We show that a chromodomain-containing Swi6 protein is a dosage-critical component involved in imprinting the mat locus. Transient overexpression of Swi6 alters the epigenetic imprint at the mat2/3 region and heritably converts the expressed state to the silenced state. The establishment and maintenance of the imprint are tightly coupled to the recruitment and the persistence of Swi6 at the mat2/3 region during mitosis as well as meiosis. Remarkably, Swi6 remains bound to the mat2/3 interval throughout the cell cycle and itself seems to be a component of the imprint. Our analyses suggest that the unit of inheritance at the mat2/3 locus comprises the DNA plus the associated Swi6 protein complex.     

Retroperitoneal extramedullary anaplastic plasmacytoma masquerading as sarcoma: Report of a case with an unusual presentation and imprint smears

BACKGROUND: Extramedullary plasmacytoma of the retroperitoneum is rare. Furthermore, plasmacytoma with anaplastic features can be confused with high grade sarcoma clinically and histologically, particularly when the initial immunohistochemical tumor markers are negative. However, paying attention to cytologic imprint smears can give valuable clues to the correct diagnosis. CASE: A 73-year-old male was admitted to our hospital with a recent history of back pain. Abdominal computed tomography revealed a large retroperitoneal mass (6.8 x 5.1 cm). The initial pathologic evaluation revealed a high grade pleomorphic neoplasm that failed to express multiple epithelial, mesenchymal, lymphoid and melanoma immunohistochemical markers. Subsequent fresh tissue evaluation with touch imprints and immunophenotypic characterization confirmed the plasma cell origin of the tumor. Thorough retrospective review of the touch imprint smears clearly showed the plasmacytic cytologic features. Features of multiple myeloma were essentially absent. CONCLUSION: Performing cytologic imprint smears on fresh tissue material may help in making the correct diagnosis and is highly recommended.     

Autophagy plays an important role in protecting Pacific oysters from OsHV-1 and Vibrio aestuarianus infections

Recent mass mortality outbreaks around the world in Pacific oysters, Crassostrea gigas, have seriously affected the aquaculture economy. Although the causes for these mortality outbreaks appear complex, infectious agents are involved. Two pathogens are associated with mass mortality outbreaks, the virus ostreid herpesvirus 1 (OsHV-1) and the bacterium Vibrio aestuarianus. Here we describe the interactions between these 2 pathogens and autophagy, a conserved intracellular pathway playing a key role in innate immunity. We show for the first time that autophagy pathway is present and functional in Pacific oysters and plays an important role to protect animals from infections. This study contributes to better understand the innate immune system of Pacific oysters.