Hybridization between two serranids, the coney (Cephalopholis fulva) and the creole-fish (Paranthias furcifer), at Bermuda

Intergeneric hybridization between the epinepheline serranids Cephalopholis fulva and Paranthias furcifer in waters off Bermuda was investigated by using morphological and molecular characters. Putative hybrids, as well as members of each presumed parent species, were analyzed for 44 morphological characters and screened for genetic variation at 16 nuclear allozyme loci, two nuclear (n)DNA loci, and three mitochondrial (mt)DNA gene regions. Four of 16 allozyme loci, creatine kinase (CK-B*), fumarase (FH*), isocitrate dehydrogenase (ICDH-S*), and lactate dehydrogenase (LDH-B*), were unique in C. fulva and P. furcifer. Restriction fragments of two nuclear DNA intron regions, an actin gene intron and the second intron in the S7 ribosomal protein gene, also exhibited consistent differences between the two presumed parent species. Restriction fragments of three mtDNA regions—ND4, ATPase 6, and 12S/16S ribosomal RNA—were analyzed to identify maternal parentage of putative hybrids. Both morphological data and nuclear genetic data were found to be consistent with the hypothesis that the putative hybrids were the result of interbreeding between C. fulva and P. furcifer. Mean values of 38 morphological characters were different between presumed parent species, and putative hybrids were intermediate to presumed parent species for 33 of these characters. A principal component analysis of the morphological and meristic data was also consistent with hybridization between C. fulva and P. furcifer. Thirteen of 15 putative hybrids were heterozygous at all diagnostic nuclear loci, consistent with F1 hybrids. Two putative hybrids were identified as post-F1 hybrids based on homozygosity at one nuclear locus each. Mitochondrial DNA analysis showed that the maternal parent of all putative hybrid individuals was C. fulva. A survey of nuclear and mitochondrial loci of 57 C. fulva and 37 P. furcifer from Bermuda revealed no evidence of introgression between the parent species mediated by hybridization.

Diversities in hepatic HIF-1, IGF-I/IGFBP-1, LDH/ICD, and their mRNA expressions induced by CoCl2 in Qinghai-Tibetan plateau mammals and sea level mice

Ochotona curzoniae and Microtus oeconomus are the native mammals living on the Qinghai-TibetanPlateau of China. The molecular mechanisms of their acclimatization to the Plateau-hypoxia remain unclear. Expressions of hepatic hypoxia-inducible factor (HIF)-1 alpha, insulin-like growth factor-I (IGF-I)/IGF binding protein (BP)-1(IGFBP-1; including genes), and key metabolic enzymatic genes [lactate dehydrogenase (LDH)-A/isocitrate dehydrogenase (ICD)] are compared in Qinghai-Tibetan- Plateau mammals andsea- level mice after injection of CoCl2 (20, 40, or 60 mg/ kg) and normobaric hypoxia (16.0% O-2, 10.8% O-2, and 8.0% O-2) for 6 h, tested by histochemistry, Western blot analysis, ELISA, and RT-PCR. Major results are CoCl2 markedly increased 1) HIF-1 alpha only in mice, 2) hepatic and circulatory IGF-I in M. oeconomus, 3) hepatic IGFBP-1 in mice and O. curzoniae, and 4) LDH-A but reduced ICD mRNA in mice (CoCl2 20 mg/kg) but were unchanged in the Tibetan mammals. Normobaric hypoxia markedly 1) increased HIF-1 alpha and LDH-A mRNA in mice and M. oeconomus (8.0% O-2) not in O. curzoniae, and 2) reduced ICD mRNA in mice and M. oeconomus (8.0% O-2) not in O. curzoniae. Results suggest that 1) HIF-1 alpha responsiveness to hypoxia is distinct in lowland mice and plateau mammals, reflecting a diverse tolerance of the three species to hypoxia; 2) CoCl2 induces diversities in HIF-1, IGF-I/IGFBP-1 protein or genes in mice, M. oeconomus, and O. curzoniae. In contrast, HIF-1 mediates IGFBP-1 transcription only in mice and in M. oeconomus (subjected to severe hypoxia); 3) differences in IGF-I/IGFBP-1 expressions induced by CoCl2 reflect significant diversities in hormone regulation and cell protection from damage; and 4) activation of anaerobic glycolysis and reduction of Krebs cycle represents strategies of lowland-animals vs. the stable metabolic homeostasis of plateau- acclimatized mammals.

Genetic Studies Identify Critical Biomarkers and Refine the Classification of Malignant Gliomas

Gliomagenesis is driven by a complex network of genetic alterations and while the glioma genome has been a focus of investigation for many years; critical gaps in our knowledge of this disease remain. The identification of novel molecular biomarkers remains a focus of the greater cancer community as a method to improve the consistency and accuracy of pathological diagnosis. In addition, novel molecular biomarkers are drastically needed for the identification of targets that may ultimately result in novel therapeutics aimed at improving glioma treatment. Through the identification of new biomarkers, laboratories will focus future studies on the molecular mechanisms that underlie glioma development. Here, we report a series of genomic analyses identifying novel molecular biomarkers in multiple histopathological subtypes of glioma and refine the classification of malignant gliomas. We have completed a large scale analysis of the WHO grade II-III astrocytoma exome and report frequent mutations in the chromatin modifier, alpha thalassemia mental retardation x-linked (ATRX), isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2), and mutations in tumor protein 53 (TP53) as the most frequent genetic mutations in low grade astrocytomas. Furthermore, by analyzing the status of recurrently mutated genes in 363 brain tumors, we establish that highly recurrent gene mutational signatures are an effective tool in stratifying homogeneous patient populations into distinct groups with varying outcomes, thereby capable of predicting prognosis. Next, we have established mutations in the promoter of telomerase reverse transcriptase (TERT) as a frequent genetic event in gliomas and in tissues with low rates of self renewal. We identify TERT promoter mutations as the most frequently mutated gene in primary glioblastoma. Additionally, we show that TERT promoter mutations in combination with IDH1 and IDH2 mutations are able to delineate distinct clinical tumor cohorts and are capable of predicting median overall survival more effectively than standard histopathological diagnosis alone. Taken together, these data advance our understanding of the genetic alterations that underlie the transformation of glial cells into neoplasms and we provide novel genetic biomarkers and multi – gene mutational signatures that can be utilized to refine the classification of malignant gliomas and provide opportunity for improved diagnosis.

Radiolabeled inhibitors as probes for imaging mutant IDH1 expression in gliomas: Synthesis and preliminary evaluation of labeled butyl-phenyl sulfonamide analogs.

INTRODUCTION: Malignant gliomas frequently harbor mutations in the isocitrate dehydrogenase 1 (IDH1) gene. Studies suggest that IDH mutation contributes to tumor pathogenesis through mechanisms that are mediated by the neomorphic metabolite of the mutant IDH1 enzyme, 2-hydroxyglutarate (2-HG). The aim of this work was to synthesize and evaluate radiolabeled compounds that bind to the mutant IDH1 enzyme with the goal of enabling noninvasive imaging of mutant IDH1 expression in gliomas by positron emission tomography (PET). METHODS: A small library of nonradioactive analogs were designed and synthesized based on the chemical structure of reported butyl-phenyl sulfonamide inhibitors of mutant IDH1. Enzyme inhibition assays were conducted using purified mutant IDH1 enzyme, IDH1-R132H, to determine the IC50 and the maximal inhibitory efficiency of the synthesized compounds. Selected compounds, 1 and 4, were labeled with radioiodine ((125)I) and/or (18)F using bromo- and phenol precursors, respectively. In vivo behavior of the labeled inhibitors was studied by conducting tissue distribution studies with [(125)I]1 in normal mice. Cell uptake studies were conducted using an isogenic astrocytoma cell line that carried a native IDH1-R132H mutation to evaluate the potential uptake of the labeled inhibitors in IDH1-mutated tumor cells. RESULTS: Enzyme inhibition assays showed good inhibitory potency for compounds that have iodine or a fluoroethoxy substituent at the ortho position of the phenyl ring in compounds 1 and 4 with IC50 values of 1.7 μM and 2.3 μM, respectively. Compounds 1 and 4 inhibited mutant IDH1 activity and decreased the production of 2-HG in an IDH1-mutated astrocytoma cell line. Radiolabeling of 1 and 4 was achieved with an average radiochemical yield of 56.6 ± 20.1% for [(125)I]1 (n = 4) and 67.5 ± 6.6% for [(18)F]4 (n = 3). [(125)I]1 exhibited favorable biodistribution characteristics in normal mice, with rapid clearance from the blood and elimination via the hepatobiliary system by 4 h after injection. The uptake of [(125)I]1 in tumor cells positive for IDH1-R132H was significantly higher compared to isogenic WT-IDH1 controls, with a maximal uptake ratio of 1.67 at 3 h post injection. Co-incubation of the labeled inhibitors with the corresponding nonradioactive analogs, and decreasing the normal concentrations of FBS (10%) in the incubation media substantially increased the uptake of the labeled inhibitors in both the IDH1-mutant and WT-IDH1 tumor cell lines, suggesting significant non-specific binding of the synthesized labeled butyl-phenyl sulfonamide inhibitors. CONCLUSIONS: These data demonstrate the feasibility of developing radiolabeled probes for the mutant IDH1 enzyme based on enzyme inhibitors. Further optimization of the labeled inhibitors by modifying the chemical structure to decrease the lipophilicity and to increase potency may yield compounds with improved characteristics as probes for imaging mutant IDH1 expression in tumors.

Presence of alternative lengthening of telomeres mechanism in patients with glioblastoma identifies a less aggressive tumor type with longer survival.

Patients with glioblastoma (GBM) have variable clinical courses, but the factors that underlie this heterogeneity are not understood. To determine whether the presence of the telomerase-independent alternative lengthening of telomeres (ALTs) mechanism is a significant prognostic factor for survival, we performed a retrospective analysis of 573 GBM patients. The presence of ALT was identified in paraffin sections using a combination of immunofluorescence for promyelocytic leukemia body and telomere fluorescence in situ hybridization. Alternative lengthening of telomere was present in 15% of the GBM patients. Patients with ALT had longer survival that was independent of age, surgery, and other treatments. Mutations in isocitrate dehydrogenase (IDH1mut) 1 frequently accompanied ALT, and in the presence of both molecular events, there was significantly longer overall survival. These data suggest that most ALT+ tumors may be less aggressive proneural GBMs, and the better prognosis may relate to the set of genetic changes associated with this tumor subtype. Despite improved overall survival of patients treated with the addition of chemotherapy to radiotherapy and surgery, ALT and chemotherapy independently provided a survival advantage, but these factors were not found to be additive. These results suggest a critical need for developing new therapies to target these specific GBM subtypes.

Isoenzymatic polymorphism in Citrus spp. and Poncirus trifoliata (L.) Raf. (Rutaceae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A parallel future for Ocean research?

[EN] Today, science is difficult to pursue because funding is so tenuous. In such a financial climate, researchers need to consider parallel alternatives to ensure that scientific research can continue. Based on this thinking, we created BIOCEANSolutions, a company born of a research group. A great variety of environmental regulations and standards have emerged over recent years with the purpose of protecting natural ecosystems. These have enabled us to link our research to the market of environmental management. Marine activities can alter environmental conditions, resulting in changes in physiological states, species diversity, abundance, and biomass in the local biological communities. In this way, we can apply our knowledge, to plankton ecophysiology and biochemical oceanography. We measure enzyme activities as bio-indicators of energy metabolism and other physiological rates and biologic-oceanographic processes in marine organisms. This information provides insight into the health of marine communities, the stress levels of individual organisms, and potential anomalies that may be affecting them. In the process of verifying standards and complying with regulations, we can apply our analytic capability and knowledge. The main analyses that we offer are: (1) the activity of the electron transport system (ETS) or potential respiration (Φ), (2) the physiological measurement of respiration (oxygen consumption), (3) the activity of Isocitrate dehydrogenase (IDH), (4) the respiratory CO2 production, and (5) the activity of Glutamate dehydrogenase (GDH) and (6) the physiological measurement of ammonium excretion. In addition, our experience in a productive research group allows us to pursue and develop technical-experimental activities such as marine and freshwater aquaculture, oceanographic field sampling, as well as providing guidance, counseling, and academic services. In summary, this new company will permit us to create a symbiosis between public and private sectors that serve clients and will allow us to grow and expand as a research team.

Planktonic potential CO2 emission calculation: preliminary results by applying an adapted enzymatic methodology to marine ecosystems

[EN]Isocitrate Dehydrogenase (IDH) is a key enzyme in the Krebs cycle, being responsible for the production of one of the three CO2 molecules related to cellular respiration. In order to measure the potential CO2 production linked to the marine planktonic community we have adapted an enzymatic methodology. Preliminary results show that different proportions of autotrophs, heterotrophs and mixotrophs and their metabolic pathways, lead to different relationships between potential CO2 emission and potential O2 consumption during cellular respiration. Although more experiments need to be made, this methodology is leading to a better understanding of cellular respiration in marine samples and their impact on the food chain, vertical Carbon flux and the current sequestering capacity for anthropogenic CO2.

Starvation's role in plankton metabolism

[EN]Starvation at all scales of plankton from archaea to medusae is the prevailing condition in marine ecosystems. Such nutrient-limitation will shift the physiological state in these organisms with accompanying changes in their physiology and biochemistry. Here, we review our laboratory’s progress in documenting these changes associated with starvation in a range of marine organisms. Specifically, we focused on respiration, ammonium excretion, CO2 production, RQ, respiratory ETS activity, isocitrate dehydrogenase and glutamate dehydrogenase activity in the mysid, Leptomysis lingvura, a dinoflagellate, Oxyrrhis marina and two bacteria, Vibrio natriegens, and Pseudomonas nautica

Sarcomatous evolution of oligodendroglioma ("oligosarcoma"): confirmatory report of an uncommon pattern of malignant progression in oligodendroglial tumors

By analogy to gliosarcoma, the neologism "oligosarcoma" is to describe an uncommon form of biphasic central nervous system tumor composed of contiguous neuroepithelial and mesenchymal elements, each of which individually meet the criteria of oligodendroglioma and sarcoma, respectively. By virtue of its distinctive genotype (codeletion 1p/19q), oligodendroglioma is a particularly inviting paradigm to test the assumption that such mixed tumors are clonally derived from a glial primary. We observed this constellation in a 41-year-old male who underwent two resection procedures for a recurring right frontal tumor at five years' interval. On imaging, both lesions were contrast-enhancing, and measured 7 cm × 7 cm × 6.8 cm and 7 cm × 6.5 cm × 4cm, respectively. Following the first operation, temozolomide monotherapy was administered. Whereas initial histology showed conventional anaplastic oligodendroglioma, the recurrence consisted mostly of a fibrosarcoma-like, fascicular neoplasm that was immunoreactive for vimentin, smooth muscle actin, S100 protein, and focally epithelial membrane antigen. In between, a subset of otherwise indistinguishable spindle cells expressed GFAP, and focally merged with residues of oligodendroglioma. Molecular testing for loss of heterozygosity confirmed codeletion of 1p/19q in both the primary tumor and the sarcomatous recurrence. Similarly, generalized immunoreactivity for the mutant R132H form of isocitrate dehydrogenase in both lesions indicated an identical mutation of the IDH1 gene. By the above standards, biologically consistent "oligosarcomas" are felt to be exceedingly rare, and possibly participate of a nosologically heterogeneous group of combined glial/mesenchymal lesions that may also include iatrogenically induced second malignancies as well as true collision tumors.

Polymorphisms, linkage and mapping of four enzyme loci in the fish genus Xiphophorus (Poeciliidae).

Electrophoretic variants at four additional enzyme loci--two esterases (Est-2, Est-3), retinal lactate dehydrogenase (LDH-1) and mannose phosphate isomerase (MPI)--among three species and four subspecies of fish of the genus Xiphophorus were observed. Electrophoretic patterns in F1 hybrid heterozygotes confirmed the monomeric structures of MPI and the esterase and the tetrametric structure of LDH in these fishes. Variant alleles of all four loci displayed normal Mendelian segregation in backcross and F2 hybrids. Recombination data from backcross hybrids mapped with Haldane's mapping function indicate the four loci to be linked as Est-2--0.43--Est3--0.26--LDH-1--0.19--MPI. Significant interference was detected and apparently concentrated in the Est-3 to MPI region. No significant sex-specific differences in recombination were observed. This group (designated linkage group II) was shown to assort independently from the three loci of linkage group I (adenosine deaminase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase) and from glyceraldehyde-3-phosphate dehydrogenase and two isocitrate dehydrogenase loci. Evidence for conservation of the linkage group, at least in part, in other vertebrate species is presented.

Three linked enzyme loci in fishes: implications in the evolution of vertebrate chromosomes.

A three-point linkage group comprised of loci coding for adenosine deaminase (ADA), glucose-6-phosphate dehydrogenase (G6PDH), and 6-phospho-gluconate dehydrogenase (6PGD) is described in fish of the genus Xiphophorus (Poeciliidae). The alleles at loci in this group were shown to assort independently from the alleles at three other loci--isocitrate dehydrogenase 1 and 2, and glyceraldehyde-3-phosphate dehydrogenase 1. Alleles at the latter three loci also assort independently from each other. Data were obtained by observing the segregation of electrophoretically variant alleles in reciprocal backcross hybrids derived from crosses between either X. helleri guentheri or X. h. strigatus and X. maculatus. The linkage component of chi2 was significant (less than 0.01) in all crosses, indicating that the linkage group is conserved in all populations of both species of Xiphophorus examined. While data from X. h. guentheri backcrosses indicate the linkage relationship ADA--6%--G6PDH--24%--6PGD, and ADA--29%--6PGD (30% when corrected for double crossovers), data from backcrosses involving strigatus, while supporting the same gene order, yielded significantly different recombination frequencies. The likelihood of the difference being due to an inversion could not be separated from the possibility of a sex effect on recombination in the present data. The linkage of 6PGD and G6PDH has been shown to exist in species of at least three classes of vertebrates, indicating the possibility of evolutionary conservation of this linkage.

Combined ATRX/IDH1 immunohistochemistry predicts genotype of oligoastrocytomas

AIMS To assess whether in oligoastrocytomas ATRX deficiency - as a surrogate of the alternative lengthening of telomeres (ALT) pathway - has a role in predicting the presence or absence of loss of heterozygosity of 1p and 19q (LOH), the genetic signature of oligodendroglial differentiation and a favourable prognostic marker. METHODS AND RESULTS A series of 54 oligoastrocytomas were investigated by immunohistochemistry as well as microsatellite analysis for LOH 1p19q. Genetic findings were correlated with morphological assessment. CONCLUSIONS ATRX deficiency was mutually exclusive with LOH. Conversely, ATRX-proficient tumours immunoreactive for R132H-mutant isocitrate dehydrogenase 1 (IDH1) showed a high rate (85%) of LOH. A more oligodendroglioma-like morphology was associated with a higher rate of LOH even in the morphologically ambiguous group of oligoastrocytomas. Our findings support the concept that oligoastrocytomas represent a morphological grey zone rather than a group of truly "mixed" or "intermediate" tumours. More precise classification of diffuse gliomas may also improve grading of borderline cases. We propose an immunohistochemical algorithm for classification of morphologically ambiguous diffuse gliomas. This article is protected by copyright. All rights reserved.

Heterogeneity of Mitochondrial Protein Biogenesis during Primary Leaf Development in Barley1

The natural developmental gradient of light-grown primary leaves of barley (Hordeum vulgare L.) was used to analyze the biogenesis of mitochondrial proteins in relation to the age and physiological changes within the leaf. The data indicate that the protein composition of mitochondria changes markedly during leaf development. Three distinct patterns of protein development were noted: group A proteins, consisting of the E1 β-subunit of the pyruvate dehydrogenase complex, ORF156, ORF577, alternative oxidase, RPS12, cytochrome oxidase subunits II and III, malic enzyme, and the α- and β-subunits of F1-ATPase; group B proteins, consisting of the E1 α-subunit of the pyruvate dehydrogenase complex, isocitrate dehydrogenase, HSP70A, cpn60C, and cpn60B; and group C proteins, consisting of the four subunits of the glycine decarboxylase complex (P, H, T, and L proteins), fumarase, and formate dehydrogenase. All of the proteins increased in concentration from the basal meristem to the end of the elongation zone (20.0 mm from the leaf base), whereupon group A proteins decreased, group B proteins increased to a maximum at 50 mm from the leaf base, and group C proteins increased to a maximum at the leaf tip. This study provides evidence of a marked heterogeneity of mitochondrial protein composition, reflecting a changing function as leaf cells develop photosynthetic and photorespiratory capacity.

In Vitro Reconstitution of Electron Transport from Glucose-6-Phosphate and NADPH to Nitrite1

An NADPH-dependent NO2−-reducing system was reconstituted in vitro using ferredoxin (Fd) NADP+ oxidoreductase (FNR), Fd, and nitrite reductase (NiR) from the green alga Chlamydomonas reinhardtii. NO2− reduction was dependent on all protein components and was operated under either aerobic or anaerobic conditions. NO2− reduction by this in vitro pathway was inhibited up to 63% by 1 mm NADP+. NADP+ did not affect either methyl viologen-NiR or Fd-NiR activity, indicating that inhibition was mediated through FNR. When NADPH was replaced with a glucose-6-phosphate dehydrogenase (G6PDH)-dependent NADPH-generating system, rates of NO2− reduction reached approximately 10 times that of the NADPH-dependent system. G6PDH could be replaced by either 6-phosphogluconate dehydrogenase or isocitrate dehydrogenase, indicating that G6PDH functioned to: (a) regenerate NADPH to support NO2− reduction and (b) consume NADP+, releasing FNR from NADP+ inhibition. These results demonstrate the ability of FNR to facilitate the transfer of reducing power from NADPH to Fd in the direction opposite to that which occurs in photosynthesis. The rate of G6PDH-dependent NO2− reduction observed in vitro is capable of accounting for the observed rates of dark NO3− assimilation by C. reinhardtii.