Quantification of Rat Kisspeptin Using a Novel Radioimmunoassay

Kisspeptin is a hypothalamic peptide hormone that plays a pivotal role in pubertal onset and reproductive function. Previous studies have examined hypothalamic kisspeptin mRNA expression, either through in situ hybridisation or real-time RT-PCR, as a means quantifying kisspeptin gene expression. However, mRNA expression levels are not always reflected in levels of the translated protein. Kisspeptin-immunoreactivity (IR) has been extensively examined using immunohistochemistry, enabling detection and localisation of kisspeptin perikaya in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV). However, quantification of kisspeptin-IR remains challenging. We developed a specific rodent radioimmunoassay assay (RIA) capable of detecting and quantifying kisspeptin-IR in rodent tissues. The RIA uses kisspeptin-10 as a standard and radioactive tracer, combined with a commercially available antibody raised to the kisspeptin-10 fragment. Adult female wistar rat brain samples were sectioned at 300 µm and the ARC and AVPV punch micro-dissected. Brain punches were homogenised in extraction buffer and assayed with rodent kisspeptin-RIA. In accord with the pattern of kisspeptin mRNA expression, kisspeptin-IR was detected in both the ARC (47.1±6.2 fmol/punch, mean±SEM n = 15) and AVPV (7.6±1.3 fmol/punch, mean±SEM n = 15). Kisspeptin-IR was also detectable in rat placenta (1.26±0.15 fmol/mg). Reverse phase high pressure liquid chromatography analysis showed that hypothalamic kisspeptin-IR had the same elution profile as a synthetic rodent kisspeptin standard. A specific rodent kisspeptin-RIA will allow accurate quantification of kisspeptin peptide levels within specific tissues in rodent experimental models.     

1H‐NMR screening for the high‐throughput determination of genotype and environmental effects on the content of asparagine in wheat grain

Free asparagine in cereals is known to be the precursor of acrylamide, a neurotoxic and carcinogenic product formed during cooking processes. Thus, the development of crops with lower asparagine is of considerable interest to growers and the food industry. In this study, we describe the development and application of a rapid ¹H‐NMR‐based analysis of cereal flour, that is, suitable for quantifying asparagine levels, and hence acrylamide‐forming potential, across large numbers of samples. The screen was applied to flour samples from 150 bread wheats grown at a single site in 2005, providing the largest sample set to date. Additionally, screening of 26 selected cultivars grown for two further years in the same location and in three additional European locations in the third year (2007) provided six widely different environments to allow estimation of the environmental (E) and G x E effects on asparagine levels. Asparagine concentrations in the 150 genotypes ranged from 0.32 to 1.56 mg/g dry matter in wholemeal wheat flours. Asparagine levels were correlated with plant height and therefore, due to recent breeding activities to produce semi‐dwarf varieties, a negative relationship with the year of registration of the cultivar was also observed. The multisite study indicated that only 13% of the observed variation in asparagine levels was heritable, whilst the environmental contribution was 36% and the GxE component was 43%. Thus, compared to some other phenotypic traits, breeding for low asparagine wheats presents a difficult challenge.     

The fate of platinum(II) and platinum(IV) anti-cancer agents in cancer cells and tumours

SRIXE mapping has been used to gain insight into the fate of platinum(II) and platinum(IV) complexes in cells and tumours treated with anticancer active complexes to facilitate the development of improved drugs. SRIXE maps were collected of thin sections of human ovarian (A2780) cancer cells treated with bromine containing platinum complexes, cis-[PtCl(2)(3-Brpyr)(NH(3))] (3-Brpyr=3-bromopyridine) and cis,trans,cis-[PtCl(2)(OAcBr)(2)(NH(3))(2)] (OAcBr=bromoacetate), or a platinum complex with an intercalator attached cis-[PtCl(2)(2-[(3-aminopropyl)amino]-9,10-anthracenedione)(NH(3))]. After 24h the complexes appear to be localised in the cell nucleus with a lower concentration in the surrounding cytoplasm. In cells treated with cis-[PtCl(2)(3-Brpyr)(NH(3))] the concentration of bromine was substantially higher than in control cells and the bromine was co-localised with the platinum consistent with the 3-bromopyridine ligand remaining bound to the platinum. The cells treated with cis,trans,cis-[PtCl(2)(OAcBr)(2)(NH(3))(2)] also showed an increased level of bromine, but to a much lesser extent than for those treated with cis-[PtCl(2)(3-Brpyr)(NH(3))] suggestive of substantial reduction of the platinum(IV) complex. Maps were also collected from thin sections of a 4T1.2 neo 1 mammary tumour xenograft removed from a mouse 3h after treatment with cis,trans,cis-[PtCl(2)(OH)(2)(NH(3))(2)] and revealed selective uptake of platinum by one cell.     

Vasospasm is a significant factor in cyclosporine-induced neurotoxicity: Case report

Background

The aetiology of central nervous system lesions observed in cerebral cyclosporine neurotoxicity remains controversial.

Case presentation

We report a 48-year-old woman with a non-severe aplastic anaemia who presented with stroke-like episodes while on cyclosporine treatment.Transcranial Doppler ultrasound revealed severely elevated flow velocities in several cerebral vessels, consistent with vasospasm. Immediately after reducing the cyclosporine dose, the stroke-like episodes disappeared. Only after cyclosporine withdrawal the transcranial Doppler ultrasound abnormalities fully resolved.

Conclusions

This case demonstrates a significant role of vasospasm in the pathway of cyclosporine-induced neurotoxicity. Transcranial Doppler ultrasound is an effective tool for the diagnosis and follow-up of cyclosporine-induced vasospasm.

     

Heme oxygenase 2 of the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 is induced under a microaerobic atmosphere and is required for microaerobic growth at high light intensity

Cyanobacteria, red algae, and cryptomonad algae utilize phycobilin chromophores that are attached to phycobiliproteins to harvest solar energy. Heme oxygenase (HO) in these organisms catalyzes the first step in phycobilin formation through the conversion of heme to biliverdin IXα, CO, and iron. The Synechocystis sp. PCC 6803 genome contains two open reading frames, ho1 (sll1184) and ho2 (sll1875), whose products have in vitro HO activity. We report that HO2, the protein encoded by ho2, was induced in the cells growing under a microaerobic atmosphere [0.2% (v/v) O₂], whereas HO1 was constitutively expressed under both aerobic and microaerobic atmospheres. Light intensity did not have an effect on the expression of both the HOs. Cells, in which ho2 was disrupted, were unable to grow microaerobically at a light intensity of 40 μmol m⁻² s⁻¹, but did grow microaerobically at 10 μmol m⁻² s⁻¹ light intensity. These cells grew normally aerobically at both light intensities. Comparative analysis of complete cyanobacterial genomes revealed that possession of two HOs is common in cyanobacteria. In phylogenetic analysis of their amino acid sequences, cyanobacterial HO1 and HO2 homologs formed distinct clades. HO sequences of cyanobacteria that have only one isoform were most similar to HO1 sequences. We propose that HO2 might be the more ancient HO homolog that functioned under low O₂ tension, whereas the derived HO1 can better accommodate increased O₂ tension in the environment.     

The biosynthesis of kaurenolide diterpenoids by gibberella fujikuroi

The biosynthesis of 7β-hydroxy- and 7β,18-dihydroxy-kaurenolides from ent-kaur-16-en-19-oic acid has been investigated by incubating unlabelled