Hormonal aspects of oögenesis in the flies Phormia regina and Sarcophaga bullata

The corpora allata (CA) and median neurosecretory cells (MNC) of Phormia regina and Sarcophaga bullata become active with increasing age of the fly, on a diet of sugar alone. To prevent or retard oögenesis the CA or MNCs must be removed shortly after emergence, with subsequent protein meals. Topical JH application partially compensates for CA or MNC removal. This shows that the MNC activate the CA, and not vice versa. The trauma of either operation slightly depresses egg development.Injection of ecdysone into both species in the stage of initial yolk deposition causes the primary oöcytes to degenerate. This leads to development of the penultimate oöcytes. Older and younger egg stages are not sensitive to ecdysone. In P. regina the application of JH to females with developing primary oöcytes stimulates yolk deposition in the penultimate oöcytes.     

Cryptic species and systematics of the hynobiid salamanders of the Liua–Pseudohynobius complex: Molecular and phylogenetic perspectives

Using mitochondrial DNA sequencing and allozyme electrophoresis, we examined 18 populations of the Liua–Pseudohynobius complex, endemic to China. Based on their phylogenetic affiliation and exhibited fixed allelic differences, the complex comprises at least six species, two of which are previously unknown cryptic species. The complex is clearly divided into two groups, genus Liua including Liua shihi and Liua tsinpaensis, and genus Pseudohynobius including Pseudohynobius flavomaculatus, Pseudohynobius shuichengensis and the two new species. The previously often used genus name Ranodon is inappropriate, because the type species of the genus, Ranodon sibricus, is distantly related to this complex. The species diversity among Chinese hynobiid salamanders are far from being recognized and further effort should be directed at extensive field collection in central and western China.     

Spiroplasma infection in Drosophila melanogaster: What is the advantage of killing males?

Male-killing bacteria are maternally inherited agents that cause death of sons of infected females. Their transmission rate is commonly high but imperfect and also sensitive to different environmental factors. Therefore, the proportion of infected females should be reduced in each generation. In order to explain male-killers spread and persistence in host population, a mechanism resulting in the relative increase of infected females must outweigh the losses caused by the imperfect transmission. The resource release hypothesis states that the males’ death results in increased resources available to sibling females which would otherwise be used by their male siblings. Infected females are then expected: to be larger than uninfected females in natural populations; or to have higher viability; or to have shorter development times; or any combination of these outcomes. Here, we tested the resource release hypothesis by measuring body size of infected and uninfected wild-caught Drosophila melanogaster females and carried out other fitness related measures in the laboratory. Wild-caught infected females produced more daughters than uninfected females in their first days in the laboratory. However, although no significant difference in viability was found in a controlled experiment with infected and uninfected flies from a standard laboratory strain, there was a decrease in development time probably mediated by reduced competition. Fitness effects conditioned by the host genetic background are pointed out as a possible explanation for this difference between wild and laboratory flies. Our findings are discussed in the context of the resource advantage hypothesis.     

Molecular pathogenesis of Wilson disease: haplotype analysis, detection of prevalent mutations and genotype–phenotype correlation in Indian patients

Wilson disease (WD) is an autosomal recessive disorder caused by defects in the copper-transporting P-type ATPase gene (ATP7B) resulting in the accumulation of copper in the liver and the brain. We identified prevalent mutations in the ATP7B of Indian WD patients and attempted to correlate those with the disease phenotype. Patients from 62 unrelated families and their first-degree relatives comprising 200 individuals were enrolled in this study. Three dinucleotide repeat markers flanking WD locus and a few intragenic SNPs were used to determine the genotypes and construct haplotypes of the patients. Seven recurring haplotypes accounting for 58% of the total mutant chromosomes were identified, and four underlying defects in the ATP7B representing 37% of WD chromosomes were detected. In addition, five other rare mutations were characterized. Thus a total of nine mutations including five novel changes were identified in the ATP7B of WD patients. Interestingly, homozygotes for different mutations that would be expected to produce similar defective proteins showed significant disparity in terms of organ involvement and severity of the disease. We also observed WD patients with neurological symptoms with little or no manifestation of hepatic pathogenesis. In one WD family, the proband and a sib had remarkably different phenotypes despite sharing the same pair of mutant chromosomes. These findings suggest a potential role for yet unidentified modifying loci for the observed phenotypic heterogeneity among the WD patients.     

Qualitative and quantitative determination of major saponins in Paris and Trillium by HPLC-ELSD and HPLC–MS/MS

High-performance liquid chromatographic (HPLC) with evaporative light scattering detection (ELSD) and HPLC with electrospray ionization multistage tandem mass spectrometry (HPLC–ESI-MSⁿ) were used to identify and quantify steroid saponins in Paris and Trillium plants. The content of the known saponins such as Paris I, II, III, V, VI, VII, H, gracillin and protodioscin in Paris and Trillium plants was determined simultaneously using the developed HPLC-ELSD method. Furthermore, other 12 steroid saponins were identified by HPLC–ESI(+/−)-MSⁿ detection. In the end, a developed analytical procedure was proved to be a reliable and rapid method for the quality control of Paris and Trillium plants. In addition, the alternative resources for Paris yunnanensis used as a traditional Chinese medicine were discovered according to the hierarchical clustering analysis of the saponin fraction of these plants.     

Toxoplasma gondii infections in chickens – performance of various antibody detection techniques in serum and meat juice relative to bioassay and DNA detection methods

Chickens, especially if free-range, are frequently exposed to Toxoplasma gondii, and may represent an important reservoir for T. gondii. Poultry products may pose a risk to humans, when consumed undercooked. In addition, chickens are regarded as sensitive indicators for environmental contamination with T. gondii oocysts and have been used as sentinels. The aim of the present study was to determine the suitability of commonly used antibody detection methods, i.e. the modified agglutination test (MAT), IFAT and ELISA to detect T. gondii-infected chickens. Samples of experimentally and naturally infected chickens were used. The infection state of all chickens was determined by Magnetic-Capture (MC-) real-time PCR (RT PCR). Naturally exposed chickens were additionally examined by mouse bioassay and conventional RT PCR on acidic pepsin digests (PD-RT PCR). Blood serum and meat juice of various sources were tested for antibodies to T. gondii. In naturally infected chickens, there was substantial agreement between the mouse bioassay and MC-RT PCR or the mouse bioassay and conventional PD-RT PCR. PD-RT PCR was slightly more sensitive than MC-RT PCR, as all (26/26) bioassay-positive chickens also tested positive in at least one of the tissues tested (heart, drumstick). By MC-RT PCR, 92.3% (24/26) of the naturally infected bioassay-positive chickens were positive. The diagnostic sensitivity of MC-RT PCR was clearly related to the organ examined. Based on a quantitative assessment of the MC-RT PCR results in experimentally infected chickens, brain and heart tissues harbored an at least 100?times higher parasite concentration than breast, thigh or drumstick musculature. In naturally infected chickens, only three out of 24 birds, which were MC-RT PCR-positive in heart samples, also tested positive in drumstick musculature. Under experimental conditions, the agreement between MC-RT PCR and the serological techniques revealed 100% diagnostic sensitivity and specificity. Under field conditions, examinations of sera by ELISA, IFAT and MAT showed good performance in identifying chickens that were positive in either a mouse bioassay, MC-RT PCR, or PD-RT PCR as illustrated by diagnostic sensitivities of 87.5%, 87.5% and 65.2%, respectively, and diagnostic specificities of 86.2%, 82.8% and 100%, respectively. The examination of meat juice samples from breast, drumstick or heart musculature revealed similar or even better results in the ELISA. The results in the MAT with meat juice from breast musculature were less consistent than those of ELISA and IFAT because a number of negative chickens tested false-positive in the MAT. The MAT performed similar to ELISA and IFAT when applied to test meat juice samples collected from heart, thigh or drumstick musculature.     

Evaluation of a rapid molecular screening approach for the detection of toxigenic Clostridium difficile in general and subsequent identification of the tcdC Δ117 mutation in human stools

We have developed and validated a rapid molecular screening protocol for toxigenic Clostridium difficile, that also enables the identification of the hypervirulent epidemic 027/NAP1 strain. We describe a multiplex real-time PCR assay, which detects the presence of the tcdA and tcdB genes directly in stool samples. In case of positive PCR results, a separate multiplex real-time PCR typing assay was performed targeting the tcdC gene frame shift mutation at position 117. We prospectively compared the results of the screening PCR with those of a cytotoxicity assay (CTA), and a rapid immuno-enzyme assay for 161 stool samples with a specific request for diagnosis of C. difficile infection (CDI). A total of 16 stool samples were positive by CTA. The screening PCR assay confirmed all 16 samples, and gave a PCR positive signal in eight additional samples. The typing PCR assay detected the tcdC Δ117 mutation in 2/24 samples suggesting the presence of the epidemic strain in these samples. This was confirmed by PCR ribotyping and sequencing of the tcdC gene. Using CTA as the “gold standard”, the sensitivity, specificity, positive predictive value, and negative predictive value, for the screening PCR were 100%, 94.4%, 66.7%, and 100%, respectively. In conclusion, PCR may serve as a rapid negative screening assay for patients suspected of having CDI, although the low PPV hamper the use of PCR as a standalone test. However, PCR results may provide valuable information for patient management and minimising the spread of the epidemic 027/NAP1 strain.     

Molecular phylogenetic analyses based on the nuclear rRNA genes and the intron–exon structures of the nuSSU rRNA gene in Dictyocatenulata alba (anamorphic Ascomycota)

Molecular phylogenies inferred from the nuclear small subunit rRNA gene (nuSSU), nuclear large subunit rRNA gene D1/D2 region (nuLSU), and ITS-5.8S rRNA gene (ITS) indicated that five cultures of the lichenized hyphomycete Dictyocatenulata alba isolated from Japan form a monophyletic clade with high bootstrap support, and a close relationship to the Ostropomycetidae (Lecanoromycetes, Pezizomycotina, Ascomycota). Insertion sequences were found in the nuSSU of all isolates [e.g., nine insertions in the strain JCM 5358 (Japan Collection of Microorganisms)], some of which were group I introns. Five new insertion positions were found among the D. alba isolates. Using BLAST, none of the insertion sequences of D. alba were closely related to those of fungi or other organisms deposited in public DNA databases. Insertion positions were similar in some isolates, and two positions were common to all isolates. Although all phylogenetic analyses based on nuSSU, nuLSU, and ITS revealed the monophyly of D. alba, the isolates were divided into two (in the nuSSU tree) or three (in the nuLSU and ITS trees) groups. Based on the phylogenetic analyses and the intron–exon structures, the five isolates identified as D. alba belong to three cryptic species and therefore D. alba should be considered a species complex. The very slow-growing, tough agar colonies of the isolates, the occurrence of the species on both slightly lichenized and nonlichenized surfaces of trees, or pebbles (rarely on soil), suggest that the members of the D. alba complex may be lichenized. The photobiont was not clearly identified in this study.     

Acute toxicity,critical body residues,Michaelis–Menten analysis of bioaccumulation,and ionoregulatory disturbance in response to waterborne nickel in four invertebrates: Chironomus riparius, Lymnaea stagnalis, Lumbriculus variegatus and Daphnia pulex

We investigated the bioaccumulation and acute toxicity (48 h or 96 h) of Ni in four freshwater invertebrate species in two waters with hardness of 40 (soft water) and 140 mg L^− 1 as CaCO₃ (hard water). Sensitivity order (most to least) was Lymnaea stagnalis > Daphnia pulex > Lumbriculus variegatus > Chironomus riparius. In all cases water hardness was protective against acute Ni toxicity with LC50 values 3–3.5 × higher in the hard water vs. soft water. In addition, higher water hardness significantly reduced Ni bioaccumulation in these organisms suggesting that competition by Ca and Mg for uptake at the biotic ligand may contribute to higher metal resistance. CBR50 values (Critical Body Residues) were less dependent on water chemistry (i.e. more consistent) than LC50 values within and across species by ~ 2 fold. These data support one of the main advantages of the Tissue Residue Approach (TRA) where tissue concentrations are generally less variable than exposure concentrations with respect to toxicity. Whole body Ni bioaccumulation followed Michaelis–Menten kinetics in all organisms, with greater hardness tending to decrease B_max with no consistent effect on K_d. Across species, acute Ni LC50 values tended to increase with both K_d and B_max values — i.e. more sensitive species exhibited higher binding affinity and lower binding capacity for Ni, but there was no correlation with body size. With respect to biotic ligand modeling, log K_NiBL values derived from Ni bioaccumulation correlated well with log K_NiBL values derived from toxicity testing. Both whole body Na and Mg levels were disturbed, suggesting that disruption of ionoregulatory homeostasis is a mechanism of acute Ni toxicity. In L. stagnalis, Na depletion was a more sensitive endpoint than mortality, however, the opposite was true for the other organisms. This is the first study to show the relationship between Na and Ni.     

Exploration of structure–function relationships in Escherichia coli cystathionine γ-synthase and cystathionine β-lyase via chimeric constructs and site-specific substitutions

Cystathionine γ-synthase (CGS) and cystathionine β-lyase (CBL) share a common structure and several active-site residues, but catalyze distinct side-chain rearrangements in the two-step transsulfuration pathway that converts cysteine to homocysteine, the precursor of methionine. A series of 12 chimeric variants of Escherichia coli CGS (eCGS) and CBL (eCBL) was constructed to probe the roles of two structurally distinct, ~ 25-residue segments situated in proximity to the amino and carboxy termini and located at the entrance of the active-site. In vivo complementation of methionine-auxotrophic E. coli strains, lacking the genes encoding eCGS and eCBL, demonstrated that exchange of the targeted regions impairs the activity of the resulting enzymes, but does not produce a corresponding interchange of reaction specificity. In keeping with the in vivo results, the catalytic efficiency of the native reactions is reduced by at least 95-fold, and α,β versus α,γ-elimination specificity is not modified. The midpoint of thermal denaturation monitored by circular dichroism, ranges between 59 and 80 °C, compared to 66 °C for the two wild-type enzymes, indicating that the chimeric enzymes adopt a stable folded structure and that the observed reductions in catalytic efficiency are due to reorganization of the active site. Alanine-substitution variants of residues S32 and S33, as well as K42 of eCBL, situated in proximity to and within, respectively, the targeted amino-terminal region were also investigated to explore their role as determinants of reaction specificity via positioning of key active-site residues. The catalytic efficiency of the S32A, S33A and the K42A site-directed variants of eCBL is reduced by less than 10-fold, demonstrating that, while these residues may participate in positioning S339, which tethers the catalytic base, their role is minor.     

The site of regulation of light capture in Symbiodinium: Does the peridinin–chlorophyll a–protein detach to regulate light capture?

Dinoflagellates from the genus Symbiodinium form symbiotic associations with cnidarians including corals and anemones. The photosynthetic apparatuses of these dinoflagellates possess a unique photosynthetic antenna system incorporating the peridinin–chlorophyll a–protein (PCP). It has been proposed that the appearance of a PCP-specific 77 K fluorescence emission band around 672–675 nm indicates that high light treatment results in PCP dissociation from intrinsic membrane antenna complexes, blocking excitation transfer to the intrinsic membrane-bound antenna complexes, chlorophyll a–chlorophyll c₂–peridinin–protein-complex (acpPC) and associated photosystems (Reynolds et al., 2008 Proc Natl Acad Sci USA 105:13674–13678).We have tested this model using time-resolved fluorescence decay kinetics in conjunction with global fitting to compare the time-evolution of the PCP spectral bands before and after high light exposure. Our results show that no long-lived PCP fluorescence emission components appear either before or after high light treatment, indicating that the efficiency of excitation transfer from PCP to membrane antenna systems remains efficient and rapid even after exposure to high light. The apparent increased relative emission at around 675 nm was, instead, caused by strong preferential exciton quenching of the membrane antenna complexes associated with acpPC and reaction centers. This strong non-photochemical quenching (NPQ) is consistent with the activation of xanthophyll-associated quenching mechanisms and the generally-observed avoidance in nature of long-lived photoexcited states that can lead to oxidative damage. The acpPC component appears to be the most strongly quenched under high light exposure suggesting that it houses the photoprotective exciton quencher.     

Relationship between GSTM1 and GSTT1 polymorphisms and schizophrenia: A case–control study in a Tunisian population

There is substantial evidence found in the literature that supports the fact that the presence of oxidative stress may play an important role in the pathophysiology of schizophrenia. The glutathione S-transferases (GSTs) forms one of the major detoxifying groups of enzymes responsible for eliminating products of oxidative stress. Interindividual differences observed in the metabolism of xenobiotics have been attributed to the genetic polymorphism of genes coding for enzymes involved in detoxification. Thus, in this study we investigated the association of glutathione S-transferase Mu-1 (GSTM1) and glutathione S-transferase theta-1 (GSTT1) gene deletion polymorphisms and schizophrenia in a Tunisian population. A case–control study including 138 schizophrenic patients and 123 healthy controls was enrolled. The GSTM1 and GSTT1 polymorphisms were analyzed by multiplex polymerase chain reaction (PCR). No association was found between the GSTM1 genotype and schizophrenia, whereas the prevalence of the GSTT1 active genotype was significantly higher in the schizophrenic patients (57.2%) than in the controls (45.5%) with (OR = 0.6, IC 0.37–0.99, p = 0.039). Thus, we noted a significant association between schizophrenia and GSTT1 active genotype. Furthermore, the combination of the GSTM1 and GSTT1 null genotypes showed a non-significant trend to an increased risk of schizophrenia. The present finding indicated that GSTT1 seems to be a candidate gene for susceptibility to schizophrenia in at least Tunisian population.     

Phototaxis in Drosophila: R1–6 input and interaction among ocellar and compound eye receptors

All 3 photoreceptor types in the compound eye of Drosophila can evoke positive phototaxis. Here we describe input from R1–6 receptors which are very sensitive. Previous reports in this series of studies described input from R7 and R8, the other less sensitive receptors. Here we studied fast-walking phototaxis using extremely dim stimuli. We also studied input from the simple ocellar eyes. Thus, we can now summarize a complete synthesis of inputs and interactions among all compound eye and ocellar receptor types. Receptor-deficient mutants were used to establish receptor-specific input. Two other findings are presented: (1) eye colour pigments affect the spectral sensitivity for phototaxis; and (2) the ocelli interact to facilitate input from the compound eye receptor types. Possible mechanisms of receptor interaction are discussed in the light of these findings of positive input from all photoreceptor types in Drosophila.     

Low C18 to C20 fatty acid elongase activity and limited conversion of stearidonic acid, 18:4(n–3), to eicosapentaenoic acid, 20:5(n–3), in a cell line from the turbot, Scophthalmus maximus

The TF cell line, derived from a top predatory, carnivorous marine teleost, the turbot (Scophthalmus maximus), is known to have a limited conversion of C₁₈ to C₂₀ polyunsaturated fatty acids (PUFA). To illuminate the underlying processes, we studied the conversions of stearidonic acid, 18:4(n–3), and its elongation product, 20:4(n–3), in TF cells and also in a cell line, AS, derived from Atlantic salmon (Salmo salar), by adding unlabelled (25 μM), U-¹⁴C (1 μM) or deuterated (d5; 25 μM) fatty acids. Stearidonic acid, 18:4(n–3), was metabolised to 20:5(n–3) in both cells lines, but more so in AS than in TF cells. Δ5 desaturation was more active in TF cells than in AS cells, whereas C₁₈ to C₂₀ elongation was much reduced in TF as compared to AS cells. Only small amounts of docosahexaenoic acid (22:6(n–3)) were produced by both cell lines, although there was significant production of 22:5(n–3) in both cultures, especially when 20:4(n–3) was supplemented. We conclude that limited elongation of C₁₈ to C₂₀ fatty acids rather than limited fatty acyl Δ5 desaturation accounts for the limited rate of conversion of 18:3(n–3) to 20:5(n–3) in the turbot cell line, as compared to the Atlantic salmon cell line. The results can account for the known differences in conversions of C₁₈ to C₂₀ PUFA by the turbot and the Atlantic salmon in vivo.     

Peace Propaganda and Biomedical Experimentation: Influential Uses of Radioisotopes in Endocrinology and Molecular Genetics in Spain (1947–1971)

A political discourse of peace marked the distribution and use of radioisotopes in biomedical research and in medical diagnosis and therapy in the post-World War II period. This occurred during the era of expansion and strengthening of the United States’ influence on the promotion of sciences and technologies in Europe as a collaborative effort, initially encouraged by the policies and budgetary distribution of the Marshall Plan. This article follows the importation of radioisotopes by two Spanish research groups, one in experimental endocrinology and one in molecular biology. For both groups foreign funds were instrumental in the early establishment of their laboratories. The combination of funding and access to previously scarce radioisotopes helped position these groups at the forefront of research in Spain.     

The impact of renin–angiotensin system,angiotensin І converting enzyme (insertion/deletion), and angiotensin ІІ type 1 receptor (A1166C) polymorphisms on breast cancer survival in Iran

Introduction

Several proteins of renin–angiotensin system (RAS) have been implicated in the process of growth promotion or inhibition of breast tissue and cancer cells. This study aimed to investigate the association between angiotensin I converting enzyme (ACE) insertion/deletion (I/D) and angiotensin receptor-1 (AGTR1) A1166C polymorphisms and survival of 110 women with breast cancer.

Materials and methods

The I/D and A1166C polymorphisms were evaluated by using Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) in 110 breast cancer patients who had been treated between 2007 and 2009. Genomic DNA was extracted from a Formalin-Fixed Paraffin-Embedded (FFPE) tissue of breast cancer sample blocks. All the potential clinical and pathological prognostic variables were analyzed to establish the impact of I/D and A1166C polymorphisms on disease-free and overall survival rates. Disease-free and overall survival rates were the primary endpoints of the study.

Results

The ACE (I/D) polymorphism was associated with 3-year disease-free survival. Disease-free survival in DD carriers was significantly increased compared to ID plus II carriers (HR = 4.75; 95% CI, 1.39–16.24; p = 0.013). No significant association was found between AGTR1 (A1166C) and 3-year disease-free survival (p = 0.233). Also, the ACE (I/D) and AGTR1 (A1166C) polymorphisms were not associated with breast cancer overall survival.

Conclusion

The ACE (I/D) polymorphism was associated with 3-year disease-free survival of the women with breast cancer. Besides, disease-free survival in DD carriers was significantly increased compared to ID plus II carriers.