Renal organs of cephalopods: a habitat for dicyemids and chromidinids

The renal organs of 32 species of cephalopods (renal appendage of all cephalopods, and renal and pancreatic appendages in decapods) were examined for parasite fauna and for histological comparison. Two phylogenetically distant organisms, dicyemid mesozoans and chromidinid ciliates, were found in 20 cephalopod species. Most benthic cephalopods (octopus and cuttlefish) were infected with dicyemids. Two pelagic cephalopod species, Sepioteuthis lessoniana and Todarodes pacificus, also harbored dicyemids. Chromidinid ciliates were found only in decapods (squid and cuttlefish). One dicyemid species was found in branchial heart appendages of Rossia pacifica. Dicyemids and chromidinids occasionally occurred simultaneously in Euprymna morsei, Sepia kobiensis, S. peterseni, and T. pacificus. The small-sized cephalopod species, Idiosepius paradoxus and Octopus parvus, harbored no parasites. Comparative histology revealed that the external surface of renal organs varies morphologically in various cephalopod species. The small-sized cephalopod species have a simple external surface. In contrast, the medium- to large-sized cephalopod species have a complex external surface. In the medium- to large-sized cephalopod species, their juveniles have a simple external surface of the renal organs. The external surface subsequently becomes complicated as they grow. Dicyemids and chromidinids attach their heads to epithelia or insert their heads into folds of renal appendages, pancreatic appendages, and branchial heart appendages. The rugged and convoluted external surface provides a foothold for dicyemids and chromidinids with a conical head. They apparently do not harm these tissues of their host cephalopods.     

Molecular markers comparing the extremely simple body plan of dicyemids to that of lophotrochozoans: insight from the expression patterns of Hox, Otx, and brachyury

SUMMARY Because of their extremely simple body plan, dicyemids have long been the subject of phylogenetic controversy, regarding whether their body plan reflects their primitiveness or a degeneration from complex metazoans. Several studies have argued that the simple body plan of dicyemids are likely secondarily derived from higher lophotrochozoan animals, as a result of their endoparasitic, or endosymbiotic, lifestyle in the cephalopod kidney. To clarify the evolution of their simple body plan, we investigated the developmental expression patterns of three important regulatory genes, the central type Hox gene ( DoxC ), otx , and brachyury homologs in the dicyemid mesozoa, Dicyema orientale. DoxC was expressed in the trunk and tail of the asexually developing vermiform embryo, with clear anterior boundaries. Do-otx was expressed in the vegetal pole cells of the developing infusoriform embryos, suggesting that the invagination in infusoriform embryo is homologous to the gastrulation of other metazoans. Do-bra is expressed in the presumptive ventral cells, which are ventral to the opening of the urn cavity. The expression of Do-bra suggests that the urn cavity opening of the infusoriform embryo is comparable to the stomodium of trochophore larvae. These gene expression patterns provide molecular clues to trace the evolutionary history of degeneration in the dicyemid embryogenesis and life cycle from those of ancestral lophotrochozoan animals.     

The expression of tubulin and tektin genes in dicyemid mesozoans (Phylum: Dicyemida)

Dicyemid mesozoans (Phylum Dicyemida) are endoparasites (or endosymbionts) that typically are found in the renal sac of benthic cephalopod mollusks such as octopuses and cuttlefishes. Adult dicyemids likely adhere to the renal appendage of hosts via cilia of calotte peripheral cells. These cilia seem to be continuously worn away in the interaction between the dicyemids and the epidermal cells of host renal appendages. We cloned 4 cDNAs and genes, alpha-tubulin, beta-tubulin, tektin B, and tektin C, which are thought to play a key role in ciliogenesis, from Dicyema japonicum, and studied expression patterns of these genes by whole-mount in situ hybridization. We detected coexpression of these genes in the calotte peripheral cells, but not in the trunk peripheral cells. This suggests that regeneration and turnover of cilia continuously occur in the calotte. In vermiform and infusoriform embryos, we also detected coexpression patterns of these genes, which might correlate with ciliogenesis during the embryogenesis. We also predicted the secondary structure and the coiled-coil regions of dicyemid tektins.     

Progenesis in dicyemids

Dicyemids (Phylum Dicyemida) are the most common and characteristic endosymbiont living in the renal sac of benthic cephalopod molluscs. Precocious development of a hermaphroditic gonad occurs in the larvae and smaller juveniles of 40 dicyemid species from 17 cephalopod species so far and is the usual phenomenon in dicyemids. Based on the developmental and morphological features of precocious individuals, progenesis (a form of heterochrony) is the appropriate term for such precocious development. In general, progenetic individuals have much lower fecundity than normal ones because of their smaller body size, and therefore, it appears to be a disadvantageous reproductive trait. Nonetheless, the number of progenetic individuals consists of 30%–50% of the population, a relatively large proportion suggesting that the presence of progenetic individuals probably plays an important role in life history strategy. Precocious development significantly reduces growth time and enables early maturation. Progenetic individuals are common in short-living cephalopod species, in which precocious development seems appropriate for dicyemids, enabling fast larval release before the end of the host's life span.     

Gene conversion-like sequence transfers between transgenic antibody V genes are independent of RAD54

Homology-based Ig gene conversion is a major mechanism for Ab diversification in chickens and the Rad54 DNA repair protein plays an important role in this process. In mice, although gene conversion appears to be rare among endogenous Ig genes, Ab H chain transgenes undergo isotype switching and gene conversion-like sequence transfer processes that also appear to involve homologous recombination or gene conversion. Furthermore, homology-based DNA repair has been suggested to be important for somatic mutation of endogenous mouse Ig genes. To assess the role of Rad54 in these mouse B cell processes, we have analyzed H chain transgene isotype switching, sequence transfer, and somatic hypermutation in mice that lack RAD54. We find that Rad54 is not required for either transgene switching or transgene hypermutation. Furthermore, even transgene sequence transfers that are known to require homology-based recombinations are Rad54 independent. These results indicate that mouse B cells must use factors for promoting homologous recombination that are distinct from the Rad54 proteins important in homology-based chicken Ab gene recombinations. Our findings also suggest that mouse H chain transgene sequence transfers might be more closely related to an error-prone homology-based somatic hypermutational mechanism than to the hyperconversion mechanism that operates in chicken B cells.     

Rapid, transient, and dose-dependent expression of hsp70 messenger RNA in the rat brain after morphine treatment

Induction of Hsp70 in the brain has been reported after intake of drugs of abuse like amphetamine and lysergic acid diethylamide. In this investigation, gene expression of Hsp70 and other heat shock genes in the rat brain was studied in response to morphine. Twenty milligrams per kilogram morphine intraperitoneally resulted in a marked induction of Hsp70 messenger RNA (mRNA) expression in the frontal cortex with a maximum increase of 13.2-fold after 2 hours. A moderate increase of Hsp27 mRNA expression (6.7-fold) could be observed after 4 hours, whereas mRNA expression of Hsp90 and of the constitutive Hsc70 did not exceed a mean factor of 1.8-fold during the 24 hours interval. The increase in Hsp70 mRNA was dose dependent, showing a significant elevation after doses ranging from 10 to 50 mg/kg morphine. In situ hybridization revealed enhanced Hsp70 mRNA expression mainly in cortical areas, in the hippocampus, in the paraventricular and supraoptic nuclei of the hypothalamus, in the locus coeruleus, as well in the pineal body. The double in situ hybridization technique revealed increased Hsp70 mRNA expression mainly in VGLUT1-positive neurons and to a lesser extent in olig1-positive oligodendroglia. Immunohistochemistry revealed a marked increase of Hsp70 protein in neuronal cells and blood vessels after 12 hours. In contrast to animal experiments, morphine did not increase Hsp70 mRNA expression in vitro in micro-opioid receptor (MOR1)-expressing human embryonic kidney 293 cells, suggesting no direct MOR1-mediated cellular effect. To exclude a body temperature-related morphine effect on Hsp70 mRNA expression, the temperature was recorded. Five to 20 mg/kg resulted in hyperthermia (maximum 40.6 degrees), whereas a high dose (50 mg/kg) that produced the highest mRNA induction, showed a clear hypothermia (minimum 37.2 degrees C). These findings argue against the possibility that Hsp70 induction by morphine is caused by its effect on body temperature. It may be speculated that increased expression of Hsp70 after morphine application protects brain structures against potentially hazardous effects of opiates.     

Meta-analysis of heat- and chemically upregulated chaperone genes in plant and human cells

Molecular chaperones are central to cellular protein homeostasis. In mammals, protein misfolding diseases and aging cause inflammation and progressive tissue loss, in correlation with the accumulation of toxic protein aggregates and the defective expression of chaperone genes. Bacteria and non-diseased, non-aged eukaryotic cells effectively respond to heat shock by inducing the accumulation of heat-shock proteins (HSPs), many of which molecular chaperones involved in protein homeostasis, in reducing stress damages and promoting cellular recovery and thermotolerance. We performed a meta-analysis of published microarray data and compared expression profiles of HSP genes from mammalian and plant cells in response to heat or isothermal treatments with drugs. The differences and overlaps between HSP and chaperone genes were analyzed, and expression patterns were clustered and organized in a network. HSPs and chaperones only partly overlapped. Heat-shock induced a subset of chaperones primarily targeted to the cytoplasm and organelles but not to the endoplasmic reticulum, which organized into a network with a central core of Hsp90s, Hsp70s, and sHSPs. Heat was best mimicked by isothermal treatments with Hsp90 inhibitors, whereas less toxic drugs, some of which non-steroidal anti-inflammatory drugs, weakly expressed different subsets of Hsp chaperones. This type of analysis may uncover new HSP-inducing drugs to improve protein homeostasis in misfolding and aging diseases.     

Analysis of SHPRH functions in DNA repair and immunoglobulin diversification

During replication, bypass of DNA lesions is orchestrated by the Rad6 pathway. Monoubiquitination of proliferating cell nuclear antigen (PCNA) by Rad6/Rad18 leads to recruitment of translesion polymerases for direct and potentially mutagenic damage bypass. An error-free bypass pathway may be initiated via K63-linked PCNA polyubiquitination by Ubc13/Mms2 and the E3 ligase Rad5 in yeast, or HLTF/SHPRH in vertebrates. For the latter two enzymes, redundancy with a third E3 ligase and alternative functions have been reported. We have previously shown that the Rad6 pathway is involved in somatic hypermutation of immunoglobulin genes in B lymphocytes. Here, we have used knockout strategies targeting expression of the entire SHPRH protein or functionally significant domains in chicken DT40 cells that do not harbor a HLTF ortholog. We show that SHPRH is apparently redundant with another E3 ligase during DNA damage-induced PCNA modification. SHPRH plays no substantial role in cellular resistance to drugs initiating excision repair and the Rad6 pathway, but is important in survival of topoisomerase II inhibitor treatment. Removal of only the C-terminal RING domain does not interfere with this SHPRH function. SHPRH inactivation does not substantially impact on the overall efficacy of Ig diversification. Redundancy of E3 ligases in the Rad6 pathway may be linked to its different functions in genome maintenance and genetic plasticity.     

Cloning of chitinase-like protein1 cDNA from dicyemid mesozoans (Phylum: Dicyemida)

Dicyemid mesozoans are endoparasites found in the renal sacs of benthic cephalopods. Adult dicyemids insert the distinct anterior region, termed a "calotte," into renal tubules of the host. We cloned cDNA encoding chitinase-like protein from the dicyemid Dicyema japonicum (Dicyema-clp 1), and also cloned the gene fragment corresponding to the cDNA. Dicyema-clp1 has the hydrophobic amino acid-rich region, but not the chitin-binding domains at the C terminus. Analyses using the SignalP prediction program suggest this hydrophobic amino acid-rich region is the anchor sequence to plasma membranes. The putative catalytic site in glyco18 domain exhibited 1 substitution from aspartic acid to asparagine. The gene fragment had short 9 introns (22-26 bp), and the coding sequence consisted of 10 exons (30-233 bp). Specific and strong expression of Dicyema-clpl was detected in the calotte of vermiform stages by whole mount in situ hybridization. N-acetyl-D-glucosamine was detected on the outer surface of both peripheral cells of dicyemids and epidermal cells of host renal appendages. Dicyema-clp appears to be associated with N-acetyl-D-glucosamine in the interface between dicyemid peripheral cells and epidermal cells of the host renal appendage, and possibly aids in adhering the calotte to host epidermal cells.     

Heat shock protein 70 is able to prevent heat shock-induced resistance of target cells to CTL

Heat shock or transfection with heat shock protein 70 (Hsp70) genes has been shown to protect tumor cell lines against immune mechanisms of cytotoxicity. We have reported previously that heat shock confers resistance to CTL in the rat myeloma cell line Y3 that is Hsp70 defective. Evidence is now presented that Hsp70 is able to prevent the induction of the resistant phenotype. In Con A-stimulated lymphocytes and in lymphocyte x Y3 somatic cell hybrid clones a severe, non-Hsp70-inducing heat shock elicits resistance to CTL in contrast to a heat shock that results in Hsp70 expression. Thus, Hsp70 expression appears to be negatively associated with the development of resistance. Furthermore, loading of Y3 cells with recombinant Hsp70 protein before heat shock is able to prevent resistance. Because apoptosis induced in Y3 cells by heat shock is not affected, Hsp70 appears to interfere selectively with the CTL-induced lethal pathway that is found to be calcium but not caspase dependent. It is suggested that after heat shock Hsp70 enhances the CTL-induced apoptotic pathway by chaperoning certain proteins in the target cell that are involved in the execution of cell death. Thus, although shown to confer protection against many cytotoxic mechanisms, Hsp70 does not appear to be generally cytoprotective. This observation could also be of relevance when interpreting the effectiveness of tumor immunity.     

Induction and regulation of the 26-kDa protein in the absence of synthesis of beta-interferon mRNA in human cells

The expression of the gene coding for the 26-kDa protein coinduced with human beta-interferon (HuIFN-beta) in human fibroblasts has been measured by cytoplasmic dot hybridization in WISH cells. The production of the 26-kDa-protein mRNA is not induced by poly(I).poly(C) but maximally induced by cycloheximide alone. In contrast, HuIFN-beta is induced by poly(I).poly(C) and not by cycloheximide. WISH cells showed in addition a low constitutive level of 26-kDa-protein mRNA prior to induction. These results were confirmed by sizing the RNAs by Northern blot analysis. Pretreatment with partially purified or pure IFN-beta has only a slight effect on 26-kDa protein mRNA production. We have also determined the kinetics of induction and the amount of inducer required for an optimal induction of the 26-kDa-protein mRNA in WISH cells. This mRNA was thus maximally induced in WISH cells in the absence of detectable IFN-beta; it represents about 0.05% of poly(A)-rich mRNA in cycloheximide-induced WISH cells. We had already found that the 26-kDa-protein does not share the general characteristics of interferons. These results suggest that HuIFN-beta and the 26-kDa-protein genes are differently regulated.