Allozyme electrophoresis as a tool for distinguishing different zooxanthellae symbiotic with giant clams

The taxonomy of zooxanthellae in marine invertebrate symbioses is not well understood owing mainly to their lack of reliable morphological differences. Nevertheless, previous work using protein and DNA electrophoreses has set the stage for advancing our taxonomic understanding of cnidarian zooxanthellae. Here we present the use of allozymes as genetic markers for distinguishing algal isolates from tridacnid hosts. Zooxanthellae from seven Tridacna and Hippopus species were isolated and maintained in axenic clonal cultures over many generations. Of 16 enzyme systems, α- and β-esterase (EST), esterase-F (EST-F), glucose phosphate isomerase (GPI), and malate dehydrogenase (MDH) were found suitable polymorphic markers of genetic differences among clonal cultures. Of 39 clonal isolates, 97% were found to be genetically distinguishable. This high extent of genetic variation in zooxanthellae within and between clam species was unexpected, and is difficult to explain based solely on the general notion of asexual reproduction in symbiotic zooxanthellae. Our results are also consistent with the occurrence of sexual reproduction in clam zooxanthellae. The close genetic similarity of the symbionts of Tridacna gigas, the largest and fastest-growing clam species, and the difficulty of initiating their clonal cultures in the given nutrient medium, compared with the symbionts of other clam species, are further indicative of possibly distinct algal symbionts in T. gigas. These findings are discussed in light of current taxonomic understanding of these organisms.     

Molecular identification of Rab7 (ApRab7) in Aiptasia pulchella and its exclusion from phagosomes harboring zooxanthellae

The establishment and maintenance of the intracellular association between marine cnidarians and their symbiotic microalgae is essential to the well being of coral reef ecosystems; however, little is known concerning its underlying molecular mechanisms. In light of the critical roles of the small GTPase, Rab7, as a key regulator of vesicular trafficking, we cloned and characterized the Rab7 protein in the endosymbiosis system between the sea anemone, Aiptasia pulchella and its algal symbiont, Symbiodinium spp. The Aiptasia homologue of Rab7 proteins, ApRab7 is 88% identical to human Rab7 protein and contains all Rab-specific signature motifs. Results of EGFP reporter analysis, protein fractionation, and immunocytochemistry support that ApRab7 is located in late endocytic and phagocytic compartments and is able to promote their fusion. Significantly, the majority of phagosomes containing live symbionts that either have taken long residency in, or were newly internalized by Aiptasia digestive cells did not contain detectable levels of ApRab7, while most phagosomes containing either heat-killed or photosynthesis-impaired symbionts were positive for ApRab7 staining. Overall, our data suggest that live algal symbionts persist inside their host cells by actively excluding ApRab7 from their phagosomes, and thereby, establish and/or maintain an endosymbiotic relationship with their cnidarian hosts.     

Molecular cloning of Rab5 (ApRab5) in Aiptasia pulchella and its retention in phagosomes harboring live zooxanthellae

The intracellular association of symbiotic dinoflagellates (zooxanthellae) with marine cnidarians is the very foundation of the highly productive and diversified coral reef ecosystems. To reveal its underlying molecular mechanisms, we previously cloned ApRab7, a Rab7 homologue of the sea anemone Aiptasia pulchella, and demonstrated its selective exclusion from phagosomes containing live zooxanthellae, but not from those containing either dead or photosynthesis-impaired algae. In this study, Rab5 was characterized, due to its key role in endocytosis and phagocytosis acting upstream of Rab7. The Aiptasia Rab5 homologue (ApRab5) is 79.5% identical to human Rab5C and contains all Rab-specific signature motifs. Subcellular fractionation study showed that ApRab5 is mainly cytosolic. EGFP reporter and phagocytosis studies indicated that membrane-associated ApRab5 is present in early endocytic and phagocytic compartments, and is able to promote their fusion. Significantly, immunofluorescence study showed that the majority of phagosomes containing either resident or newly internalized live zooxanthellae were labeled with ApRab5, while those containing either heat-killed or photosynthesis-impaired algae were mostly negative for ApRab5 staining whereas the opposite was observed for ApRab7. We propose that active phagosomal retention of ApRab5 is part of the mechanisms employed by live zooxanthellae to: (1) persist inside their host cells and (2) exclude ApRab7 from their phagosomes, thereby, establishing and/or maintaining an endosymbiotic relationship with their cnidarian hosts.     

Brown ring disease in clams

Brown Ring Disease (BRD) in the manila clam, Ruditapes philippinarum is a bacterial disease which perturbs the calcification process. This disease occurs in wild and reared clam populations of France, Spain, and occasionally Italy, but has never been reported in Japan from where this species originates. BRD has also been detected in another clam species (Ruditapes decussatus) from France and Spain. Following Koch's classical postulates for pathogen identification, a bacterial strain was identified as the agent responsible for this disease. This bacterium was characterized as belonging to the genus Vibrio and has been termed Vibrio P1 or VP1. The macroscopic sign which characterises this disease is a conchiolin deposit adhering to the inner surface of the shell. To recover from the disease, the clams regenerate their shells by covering the deposit with calcified secretions much like the nacrezation process. Disease and recovery stages have been established for use in epidemiological and experimental studies. Tissue lesions are not systematically observed in diseased clams. Alterations of the digestive gland and the mantle are uniquely detected in the more severe stages of the disease. In all diseased clams, however, the periostracal lamina shows alterations: It is invaded by cell debris and bacteria, and areas of darker melanin-like pigmentation are observed. This disorganized periostracal lamina is not a good substrate for the biomineralization process and, therefore, accumulates forming a deposit on the inner face of the shell. Host-pathogen interactions have been focused at two levels: the internal defense system and the external embedding mechanism. Changes in the hemolymph were observed after challenge with VP1. In the early stages of the disease, an elevation in both circulating hemocytes and peptidase levels appeared; however, a subsequent depression of these parameters was recorded as the disease progressed. An external defense process such as bacterial embedding within periostracal lamina layers is described. Melanization of the periostracal lamina has also been suggested to be an active defense response against bacteria. These new defense processes in Brown Ring Diseased clams could complement the classical hemolymphatic system and, therefore, may act to limit bacterial proliferation within the tissues.     

Simian virus 40 maturation in cells harboring mutants deleted in the agnogene

The predominant leader region of the late 16 S mRNAs of simian virus 40 encodes a histone-like, 61-amino acid, DNA-binding protein called the agnoprotein or LP1. To test the hypothesis that this protein facilitates assembly of viral minichromosomes into virions, we have studied the synthesis of virions in cells infected with mutants deleted in this region of the SV40 genome. We found that 220 S mature virions, indistinguishable from those of wild type, were produced in cells infected with these mutants. As in wild-type-infected cells, no assembly intermediates other than 75 S chromatin were observed. However, data obtained from both steady-state and pulse-chase labeling experiments indicated that cells infected with agnogene deletion mutants produced virions more slowly than cells infected with wild-type virus. Taken together with data showing that similar levels of virion proteins were present in the wild-type- and mutant-infected cells, these findings strongly suggest that LP1 plays a role in expediting virion assembly.     

Study of insulin resistance in cybrid cells harboring diabetes-susceptible and diabetes-protective mitochondrial haplogroups

AimThis study aims to elucidate the independent role of mitochondria in the pathogenesis of insulin resistance (IR).MethodsCybrids derived from 143B osteosarcoma cell line and harboring the same nuclear DNA but different mitochondrial haplogroups were studied. Cybrid B4 (the major diabetes-susceptible haplogroup in Chinese population), cybrid D4 (the major diabetes-resistant haplogroup in Chinese population) and cybrid N9 (the diabetes-resistant haplogroup in Japanese population) were cultured in a medium containing 25 mM glucose and stimulated with 0 μM, 0.1 μM, and 1.0 μM insulin. We compared the insulin activation of PI3K–Akt (glucose uptake) and ERK–MAPK (pro-inflammation) signaling pathways, intracellular and mitochondrial oxidative stress (DCF and MitoSOX Red), and their responses to the antioxidant N-acetylcysteine (NAC).ResultsUpon insulin treatment, the translocation of cytoplasmic GLUT1/GLUT4 to the cell membrane in cybrid D4 and N9 cells increased significantly, whereas the changes in B4 cells were not or less significant. On the contrary, the ratio of insulin-induced JNK and P38 to Akt phosphorylation was significantly greater in cybrid B4 cells than in cybrid D4 and N9 cells. The levels of DCF and MitoSOX Red, which are indicative of the oxidative stress, were significantly higher in the B4 cells in basal conditions and after insulin treatment. Following treatment with the antioxidant NAC, cybrid B4 cells showed significantly reduced insulin-induced phosphorylation of P38 and increased GLUT1/GLUT4 translocation to the cell membrane, suggesting that NAC may divert insulin signaling from pro-inflammation to glucose uptake.ConclusionsMitochondria play an independent role in the pathogenesis of IR, possibly through altered production of intracellular ROS.     

RNA-mediated restoration of mitochondrial function in cells harboring a Kearns Sayre Syndrome mutation

Mutations in mitochondrial DNA (mtDNA) generate multi-system disorders due to failure of ATP production. A cybrid containing a 1.9-kb mtDNA deletion from a patient with Kearns Sayre Syndrome is respiration-defective and grows glycolytically. When treated with a ribonucleoprotein (RNP) complex of polycistronic RNA 1 (pcRNA1) containing mtDNA-encoded genes and a multi-subunit carrier complex R8, full-length pcRNA1 was transported to mitochondria. Translation of the pcRNA1-encoded mRNAs was observed in mitochondria from RNP-treated cells. Respiration of the cybrid was rescued to approximately 90% of normal within hours, switching the cells to aerobic growth. These findings have implications for the development of effective mitochondrial gene therapy.     

Presence of four toxins in red tide infested clams and cultured Gonyaulax tamarensis cells.

Four toxins have been isolated by Sephadex G-15 and high pressure ion exchange chromatography from the soft shell clams, $\text{Mya arenaria}$, which were collected during 1972 and 1974 red tide outbreaks on the New England coast. One of the toxins was saxitoxin and the rest seem to be new toxins. The same toxins were isolated from the extract of cultured $\text{Gonyaulax tamarensis}$.     

Same stem cells but different: Isogenic embryonic stem cells harboring different p53 mutants

Comment on: Wei QX, et al. Cell Cycle 2011; 10:1261-70.     

Insulin producing cells established using non-integrated lentiviral vector harboring PDX1 gene

AIM: To investigate reprogramming of human adipose tissue derived stem cells into insulin producing cells using non-integrated lentivirus harboring PDX1 gene.METHODS: In this study, human adipose tissue derived stem cells(hADSCs) were obtained from abdominal adipose tissues by liposuction, selected by plastic adhesion, and characterized by flow cytometric analysis.Human ADSCs were differentiated into adipocytes and osteocytes using differentiating medium to confirm their multipotency. Non-integrated lentiviruses harboring PDX1(Non-integrated LV-PDX1) were constructed using specific plasmids(pLV-HELP, pMD2G, LV-105-PDX1-1).Then, hADSCs were transduced with non-integrated LVPDX1. After transduction, ADSCsPDX1+were cultured in high glucose DMEM medium supplement by B27, nicotinamide and βFGF for 21 d. Expressions of PDX1 andinsulin were detected at protein level by immunofluorescence analysis. Expressions of PDX1, neurogenin3(Ngn3), glucagon, glucose transporter2(Glut2) and somatostatin as specific marker genes were investigated at mRNA level by quantitative RT-PCR. Insulin secretion of hADSCsPDX1+in the high-glucose medium was detected by electrochemiluminescence test. Human ADSCsPDX1+were implanted into hyperglycemic rats.RESULTS: Human ADSCs exhibited their fibroblast-like morphology and made colonies after 7-10 d of culture.Determination of hADSCs identified by FACS analysis showed that hADSCs were positive for mesenchymal cell markers and negative for hematopoietic cell markers that guaranteed the lack of hematopoietic contamination. In vitro differentiation of hADSCs into osteocytes and adipocytes were detected by Alizarin red and Oil red O staining and confirmed their multilineage differentiation ability. Transduced hADSCs+PDX1became round and clusters in the differentiation medium. The appropriate expression of PDX1 and insulin proteins was confirmed using immunocytochemistry analysis.Significant expressions of PDX1, Ngn3, glucagon, Glut2and somatostatin were detected by quantitative RTPCR. hADSCsPDX1+revealed the glucose sensing ability by expressing Glut2 when they were cultured in the medium containing high glucose concentration. The insulin secretion of hADSCsPDX1+in the high glucose medium was 2.32 μU/mL. hADSCsPDX1+implantation into hyperglycemic rats cured it two days after injection by reducing blood glucose levels from 485 mg/dL to the normal level.CONCLUSION: Human ADSCs can differentiate into IPCs by non-integrated LV-PDX1 transduction and have the potential to be used as a resource in type 1 diabetes cell therapy.     

Dimethylsulfoniopropionate in giant clams (Tridacnidae)

The tridacnid clams maintain symbiotic associations with certain dinoflagellates (termed zooxanthellae). Tridacnids are thus candidates to have high tissue concentrations of dimethylsulfoniopropionate (DMSP), a tertiary sulfonium compound that is not synthesized by animals but is commonly produced by dinoflagellates. This study establishes that DMSP is about an order of magnitude more concentrated in the light-exposed and shaded mantle and gills of Tridacna maxima and T. squamosa than in any other known animal tissues. The DMSP concentration in the light-exposed, siphonal mantle--the location of most zooxanthellae--is an inverse function of body size, paralleling an inverse relation between apparent density of zooxanthellae (measured as pheophytin concentration) and body size. The shaded mantle and gills are high in DMSP despite having low densities of zooxanthellae, indicating that high DMSP concentrations occur in molluscan tissue, not just in algal cells. DMSP is almost an order of magnitude less concentrated in the adductor muscle than in other tissues. The high DMSP concentrations found in tridacnids, by providing abundant substrate for formation of volatile dimethylsulfide, probably explain the peculiar tendency of tridacnids to rapidly develop offensive odors and tastes after death: a serious problem for their exploitation as food. Tridacnids are the one group of animals in which DMSP concentrations are high enough in some tissues to be in the range capable of perturbing enzyme function at high physiological temperatures. Thus, tridacnids may require enzyme forms adapted to DMSP.     

Osimertinib for lung cancer cells harboring low-frequency EGFR T790M mutation

Osimertinib, a third-generation EGFR tyrosine kinase inhibitor, shows significant benefit among patients with EGFR T790M mutation at disease progression. We analyzed the whole exome sequence of 48 samples obtained from 16 lung cancer patients with a longitudinal follow-up: treatment-naïve-baseline primary tumors positive for EGFR activating-mutations, paired re-biopsies upon disease progression but negative for EGFR T790M mutation based on qPCR, and their matched normal blood samples. Our Next generation sequencing (NGS) analysis identified an additional set of 25% re-biopsy samples to harbor EGFR T790M mutation occurring at a low-allele frequency of 5% or less, undetectable by conventional qPCR-based assays. Notably, the clinical utility of osimertinib among patients harboring low-allele frequency of EGFR T790M in tissue biopsy upon disease progression remains less explored. We established erlotinib-resistant PC-9R cells and twenty single-cell sub-clones from erlotinib-sensitive lung cancer PC-9 cells using in vitro drug-escalation protocol. NGS and allele-specific PCR confirmed the low-allele frequency of EGFR T790M present at 5% with a 100-fold higher resistance to erlotinib in the PC-9R cells and its sub-clones. Additionally, luciferase tagged PC-9, and PC-9R cells were orthotopically injected through the intercostal muscle into NOD-SCID mice. The orthotopic lung tumors formed were observed by non-invasive bioluminescence imaging. Consistent with in vitro data, osimertinib, but not erlotinib, caused tumor regression in mice injected with PC-9R cells, while both osimertinib and erlotinib inhibited tumors in mice injected with PC-9 cells. Taken together, our findings could extend the benefit of osimertinib treatment to patients with low EGFR T790M mutation allele frequency on disease progression.     

Double-immunolabeling systems for phenotyping of immune cells harboring bovine viral diarrhea virus

Optimal staining conditions were defined for simultaneous detection of bovine viral diarrhea virus (BVDV) and mononuclear leukocyte surface antigens in tissue sections and cytospins. Because of the extreme lability of the virus antigens and the variable stability of the epitopes on the cell differentiation antigens, cryopreservation had to be used. This method gives slightly sub-optimal preservation of morphology. However, the specificity and sensitivity of the immunolabeling ensured reliable identification of the double-labeled cells, i.e., the phenotypic identification of virus-infected cells within the immune system.     

Temporal variation in gametogenic cycles of vesicomyid clams

Summary

Sulfide-rich cold seeps in Monterey Canyon support dense communities of vesicomyid clams, which harbor chemosynthetic bacterial endosymbionts. Because these animals rely upon non-photosynthetic food sources, their life histories may be decoupled from seasonal phytoplankton productivity. We examined this hypothesis by investigating temporal changes in gametogenesis in two vesicomyid clam species. Clams were collected from two cold seeps (600 m and 900 m depths), and their gonads were removed and processed for histological analyses. Oocyte diameters and relative abundances of tissue types were measured in thin sections taken across the gonad. Mean oocyte diameters of Calyptogena kilmeri increased from August to November, 1994, but did not change by March, 1995. Relative proportions of ovarian tissue types (reproductive vs. somatic) also changed during this period, indicating reproductive seasonality. Analyses of Calyptogena pacifica tissues showed no significant change within a 3-month period. Existence of seasonal cycles for vesicomyid clams suggests that either (1) chemosynthetic production is seasonal or (2) reproductive output is tied to factors other than food production, such as synchrony of spawning to maximize fertilization success, or larval survival.     

Experimental challenges of wild Manila clams with Perkinsus species isolated from naturally infected wild Manila clams

Manila clams, Ruditapes philippinarum, are widely harvested in the coastal waters in Japan. However, there have been significant decreases in the populations of Manila clams since the 1980s. It is thought that infection with the protozoan Perkinsus species has contributed to these decreases. A previous study demonstrated that high infection levels of a pure strain of Perkinsus olseni (ATCC PRA-181) were lethal to hatchery-raised small Manila clams, however, the pathogenicity of wild strain Perkinsus species to wild Manila clam is unclear. To address this, we challenged large (30-40mm in shell length) and small (3-15mm in shell length) wild Manila clams with Perkinsus species isolated from naturally infected wild Manila clams. We report high mortalities among the small clams, but not among the large ones. This is the first report to confirm the pathogenicity of wild isolate of Perkinsus species to wild Manila clams.