Vitamin B12-induced alterations in activities of α-glycerophosphate dehydrogenase and malic enzyme in brain of singi fish, Heteropneustes fossilis (Bloch)

Different doses of vitamin B₁₂ (0.25, 0.5, 1, 2 and 4 μg/g, injected intraperitoneally for three consecutive days) altered the activities of mitochondrial-α-glycerophosphate dehydrogenase (α-GPD) and NADP-dependent cytosolic malic enzyme (ME) in the brain of singi fish. The α-GPD activity increased at doses of 0.5, 1, 2 and 4 μg/g vitamin B₁₂. A dose of 0.5 μg/g vitamin B₁₂ induced less activity than higher doses. ME activity increased with 1, 2 and 4 μg/g of vitamin B₁₂/g. The mitochondrial and cytosolic protein content remained unchanged after vitamin B₁₂ administration. Cycloheximide treatment inhibited the vitamin B₁₂-induced increase in α-GPD and ME activity. Thus, vitamin B₁₂ is involved in the induction of some enzymes in fish brain.     

Integrating ecology into macroevolutionary research

On 9 March, over 150 biologists gathered in London for the Centre for Ecology and Evolution spring symposium, 'Integrating Ecology into Macroevolutionary Research'. The event brought together researchers from London-based institutions alongside others from across the UK, Europe and North America for a day of talks. The meeting highlighted methodological advances and recent analyses of exemplar datasets focusing on the exploration of the role of ecological processes in shaping macroevolutionary patterns.     

Decrease in triiodothyronine binding sites in chick embryo erythrocytes during early development

Specific thyroid hormone (TH) binding sites have been detected in nuclei of erythrocytes obtained from developing chick embryos. The binding characteristics and relative affinities for TH analogs were those expected of TH receptors. Nuclear triiodothyronine (T3) saturation analysis was carried out in vitro by incubating intact erythrocytes in M199 medium with 3-200 pM [125I]T3 for 1 hr at 37 degrees C or 20-24 hr at 21 degrees C. Nuclei were obtained by centrifugation after lysing the erythrocytes in a stabilizing buffer containing 0.3% saponin, followed by addition of Triton X-100 (final concentration 0.2%) to minimize the nonspecific binding. Scatchard analysis of equilibrium binding data suggested that the nuclei possess a single class of binding sites. The binding is reversible and the rate of dissociation is temperature dependent. T3 and T4 appear to bind to the same sites, but the affinity of T3 was 16 times greater. Among TH analogs tested, Triac had the highest affinity followed by L-T3, D-T3, Tetrac, L-T4, D-T4, T2, and rT3. Serial studies performed on different days of chick embryogenesis demonstrated a rapid and significant decrease of the erythrocyte nuclear T3 receptor. On Day 5, the number of T3 binding sites was maximal at 1600 +/- 100 per nucleus. The number declined steadily until, by Day 20, it had reached about 60 +/- 10 sites/nucleus. RBC from adult and baby chickens had less than 1% as many binding sites as those from Day 5 embryos. There was no significant change in the affinity of the sites (Kd approximately equal to 20 pM at 37 degrees C). The reason for the loss of T3 binding sites during embryogenesis is not known. Since the plasma level of the TH increases during embryogenesis, this may reflect down regulation. Another possibility is that the change in erythrocyte population which occurs during this period involves production of erythrocytes which contain fewer T3 binding sites.     

PERMANENT GENETIC RESOURCES: Ten novel polymorphic dinucleotide microsatellite loci cloned from the Antarctic fur seal Arctocephalus gazella

Ten new dinucleotide microsatellite loci were isolated from the Antarctic fur seal Arctocephalus gazella. These markers should prove useful for studying the reproductive ecology of Antarctic fur seals and other related pinniped species.     

High levels of interspecific gene flow in an endemic cichlid fish adaptive radiation from an extreme lake environment

Studying recent adaptive radiations in isolated insular systems avoids complicating causal events and thus may offer clearer insight into mechanisms generating biological diversity. Here, we investigate evolutionary relationships and genomic differentiation within the recent radiation of Alcolapia cichlid fish that exhibit extensive phenotypic diversification, and which are confined to the extreme soda lakes Magadi and Natron in East Africa. We generated an extensive RAD data set of 96 individuals from multiple sampling sites and found evidence for genetic admixture between species within Lake Natron, with the highest levels of admixture between sympatric populations of the most recently diverged species. Despite considerable environmental separation, populations within Lake Natron do not exhibit isolation by distance, indicating panmixia within the lake, although individuals within lineages clustered by population in phylogenomic analysis. Our results indicate exceptionally low genetic differentiation across the radiation despite considerable phenotypic trophic variation, supporting previous findings from smaller data sets; however, with the increased power of densely sampled SNPs, we identify genomic peaks of differentiation (F_ST outliers) between Alcolapia species. While evidence of ongoing gene flow and interspecies hybridization in certain populations suggests that Alcolapia species are incompletely reproductively isolated, the identification of outlier SNPs under diversifying selection indicates the radiation is undergoing adaptive divergence.     

Estrous cycle-regulated expression of CYP1B1 mRNA in the rat ovary

CYP1B1, a member of the cytochrome p450 superfamily, is expressed constitutively in the steroidogenic tissues of mammals and is inducible by peptide hormones, cAMP and aromatic hydrocarbon receptor (AHR) ligands. The mechanism of induction of this cytochrome p450 is similar to that for CYP1A1, i.e. through the aromatic hydrocarbon receptor (AHR) signaling pathway. We have recently reported that CYP1B1, but not CYP1A1, is expressed in rat granulosa cells (GC) in the absence of any external stimulus. The induction of CYP1B1 mRNA in rat GC by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vitro was followed by an increase in AHR and estrogen receptor (ER-beta) RNA levels. Estrous cycle-dependent expression of AHR, AHR-nuclear translocator (ARNT) and ER-mRNAs in the rat ovary was reported. We suggest that CYP1B1 may play a major role in the regulation of rat ovarian function/cycle but until now this has been unexplored experimentally. The present study was therefore aimed at examining the expression of CYP1A1, CYP1B1 and ER-mRNA in rat ovarian tissues throughout the estrous cycle to establish any correlation in the expressions of these mRNAs in rat ovary. Total RNA was extracted from the ovary and liver of cycling adult rats and the mRNAs were analyzed using relative RT-PCR with gene-specific primers for the target mRNA and for RPL 19 or S16 primers as an internal control. The results indicated that in the ovary, CYP1B1 mRNA increased significantly on the evening of proestrus and dramatically decreased on the morning of estrus, while ER-mRNA remained unaltered throughout the estrous cycle. CYP1A1 mRNA in the ovary and both CYP1A1 and CYP1B1 mRNAs in the liver were undetectable. That the sudden decrease of ovarian CYP1B1 mRNA on the morning of estrus was not an effect of the LH surge was verified in vitro using our short-term GC culture model. GC prepared from rats super-stimulated with equine chorionic gonadotropin (eCG) were cultured for 6 h with or without LH and TCDD. It was observed that both CYP1A1 and CYP1B1 mRNAs were induced by TCDD with no apparent effect of LH. It is suggested that the high level of CYP1B1 mRNA expression on the evening of proestrus in rat ovary might be involved in metabolism of estrogens to catecholestrogen (a known effect of CYP1B1), and that expression is unaffected in GC by LH.     

Multiplex PCR and Next Generation Sequencing for the Non-Invasive Detection of Bladder Cancer

Background

Highly sensitive and specific urine-based tests to detect either primary or recurrent bladder cancer have proved elusive to date. Our ever increasing knowledge of the genomic aberrations in bladder cancer should enable the development of such tests based on urinary DNA.

Methods

DNA was extracted from urine cell pellets and PCR used to amplify the regions of the TERT promoter and coding regions of FGFR3, PIK3CA, TP53, HRAS, KDM6A and RXRA which are frequently mutated in bladder cancer. The PCR products were barcoded, pooled and paired-end 2 x 250 bp sequencing performed on an Illumina MiSeq. Urinary DNA was analysed from 20 non-cancer controls, 120 primary bladder cancer patients (41 pTa, 40 pT1, 39 pT2+) and 91 bladder cancer patients post-TURBT (89 cancer-free).

Results

Despite the small quantities of DNA extracted from some urine cell pellets, 96% of the samples yielded mean read depths >500. Analysing only previously reported point mutations, TERT mutations were found in 55% of patients with bladder cancer (independent of stage), FGFR3 mutations in 30% of patients with bladder cancer, PIK3CA in 14% and TP53 mutations in 12% of patients with bladder cancer. Overall, these previously reported bladder cancer mutations were detected in 86 out of 122 bladder cancer patients (70% sensitivity) and in only 3 out of 109 patients with no detectable bladder cancer (97% specificity).

Conclusion

This simple, cost-effective approach could be used for the non-invasive surveillance of patients with non-muscle-invasive bladder cancers harbouring these mutations. The method has a low DNA input requirement and can detect low levels of mutant DNA in a large excess of normal DNA. These genes represent a minimal biomarker panel to which extra markers could be added to develop a highly sensitive diagnostic test for bladder cancer.     

Niche divergence facilitated by fine‐scale ecological partitioning in a recent cichlid fish adaptive radiation

Ecomorphological differentiation is a key feature of adaptive radiations, with a general trend for specialization and niche expansion following divergence. Ecological opportunity afforded by invasion of a new habitat is thought to act as an ecological release, facilitating divergence, and speciation. Here, we investigate trophic adaptive morphology and ecology of an endemic clade of oreochromine cichlid fishes (Alcolapia) that radiated along a herbivorous trophic axis following colonization of an isolated lacustrine environment, and demonstrate phenotype‐environment correlation. Ecological and morphological divergence of the Alcolapia species flock are examined in a phylogenomic context, to infer ecological niche occupation within the radiation. Species divergence is observed in both ecology and morphology, supporting the importance of ecological speciation within the radiation. Comparison with an outgroup taxon reveals large‐scale ecomorphological divergence but shallow genomic differentiation within the Alcolapia adaptive radiation. Ancestral morphological reconstruction suggests lake colonization by a generalist oreochromine phenotype that diverged in Lake Natron to varied herbivorous morphologies akin to specialist herbivores in Lakes Tanganyika and Malawi.