Blood group A determinants with mono- and difucosyl type 1 chain in human erythrocyte membranes

Application of a monoclonal antibody defining monofucosyl type 1 chain A (AH21) revealed the presence of a glycolipid having the same thin-layer chromatography mobility as Aa but showing a clear reactivity with AH21. This glycolipid was detectable in Lea-b- erythrocytes but not in Lea+b- or Lea-b+ erythrocytes. Another monoclonal antibody defining difucosyl type 1 chain A (HH3) detected the presence of a glycolipid component reacting with this antibody in Lea-b+ erythrocytes but not in Lea+b- or Lea-b- erythrocytes. The component defined by monoclonal antibody AH21 and that defined by HH3 were isolated and characterized by 1H NMR spectrometry and methylation analysis as having the structures (Formula: see text) The 1H NMR spectra of these glycolipid antigens were characterized by resonances for anomeric protons that are identical with those of glycolipids with type 1 chain previously isolated but distinctively different from those of type 2 chain analogues. Resonances reflecting ceramide composition are characteristic for these antigens from human erythrocytes and are distinguishable from those of the same antigen from other sources.     

Rapid visualisation of microarray copy number data for the detection of structural variations linked to a disease phenotype

Whilst the majority of inherited diseases have been found to be caused by single base substitutions, small insertions or deletions (<1Kb), a significant proportion of genetic variability is due to copy number variation (CNV). The possible role of CNV in monogenic and complex diseases has recently attracted considerable interest. However, until the development of whole genome, oligonucleotide micro-arrays, designed specifically to detect the presence of copy number variation, it was not easy to screen an individual for the presence of unknown deletions or duplications with sizes below the level of sensitivity of optical microscopy (3-5 Mb). Now that currently available oligonucleotide micro-arrays have in excess of a million probes, the problem of copy number analysis has moved from one of data production to that of data analysis. We have developed CNViewer, to identify copy number variation that co-segregates with a disease phenotype in small nuclear families, from genome-wide oligonucleotide micro-array data. This freely available program should constitute a useful addition to the diagnostic armamentarium of clinical geneticists.     

Chemical characterization of a blood group H type pentaglycosylceramide of human small intestine

A blood group H type pentaglycosylceramide was isolated in relatively large amounts from human adult small intestine (52mg from one individual) and human meconium (fetal origin). The structure was made likely by mass spectrometry and NMR spectroscopy of non-degraded permethylated and permethylated-LiAlH₄-reduced glycolipid and by degradaton to be Fucα1 → 2GAlβ 1 → 3GlcNAcβ 1 → 3GAlβ 1 → 4Glcβ 1 → l Cer. The ceramide was composed mainly of phytosphingosine and 2-hydroxy 16–24 carbon fatty acids. This novel type 1 chain species (Galβ 1 → 3GlcNAc) was not accompanied by the type 2 chain isomer (Galβ 1 → 4GlcNAc) which in contrast is the sole species in human erythrocyte and dog small intestine.     

Prolonged pulsatile release of gonadotropin-releasing hormone from the guinea pig hypothalamus in vitro

The sexually mature mammal secretes luteinizing hormone in a pulsatile fashion. This is presumed to depend on the intermittent release of hypothalamic gonadotropin- releasing hormone (GnRH). The isolated guinea pig hypothalamus has been studied because, in this species, as in primates, the pulse generator appears to reside within the medial basal hypothalamus. The basal 2 mm of guinea pig hypothalami were rapidly removed and perifused at 37 degrees C with Krebs-Ringer solution containing 20 mM bacitracin gassed with 95% O2, 5% CO2. The eluates were sampled at 15 and 5 min intervals and pulsatile patterns of GnRH were consistently observed for periods up to 72 h. There was no difference in GnRH levels from hypothalami of intact and ovariectomized animals. Simultaneous measurement of TRH and somatostatin disclosed independent pulses of both neurohormones which did not coincide with GnRH, indicating that the peaks were secretory episodes not artefacts generated by varying perifusion rates. The hypothalami disclosed no histologic evidence of necrosis when examined after 20 h perifusion.     

Photo-accumulation of the P+QB- radical pair state in purple bacterial reaction centres that lack the QA ubiquinone

Photo-excitation of membrane-bound Rhodobacter sphaeroides reaction centres containing the mutation Ala M260 to Trp (AM260W) resulted in the accumulation of a radical pair state involving the photo-oxidised primary electron donor (P). This state had a lifetime of hundreds of milliseconds and its formation was inhibited by stigmatellin. The absence of the Q_A ubiquinone in the AM260W reaction centre suggests that this long-lived radical pair state is P⁺Q_B⁻, although the exact reduction/protonation state of the Q_B quinone remains to be confirmed. The blockage of active branch (A-branch) electron transfer by the AM260W mutation implies that this P⁺Q_B⁻ state is formed by electron transfer along the so-called inactive branch (B-branch) of reaction centre cofactors. We discuss how further mutations may affect the yield of the P⁺Q_B⁻ state, including a double alanine mutation (EL212A/DL213A) that probably has a direct effect on the efficiency of the low yield electron transfer step from the anion of the B-branch bacteriopheophytin (H_B⁻) to the Q_B ubiquinone.     

Evaluating the efficacy of seasonal grazing and livestock exclusion as restoration tools for birds in riparian habitat of the Okanagan Valley,British Columbia,Canada

Riparian habitat supports the highest density and diversity of songbirds in Western North America despite covering less than 1% of the land area. Widespread destruction and degradation of riparian habitat, especially by livestock grazing, has led to habitat restoration efforts. In 2000, restoration activities in the form of permanent and seasonal exclusion of livestock from riparian areas were initiated to improve habitat for the endangered Western Yellow‐breasted Chat (Icteria virens auricollis) population, which is dependent on early successional shrub habitat for nesting, in the Okanagan Valley of British Columbia, Canada. We assessed the effectiveness of livestock exclusion by examining temporal changes in the abundance, richness, and breeding performance of birds in restoration and reference sites. The abundance of W. Yellow‐breasted Chats significantly increased between 2002 and 2013 in areas where restoration activities occurred. However, restoration did not have significant effects on the abundance, richness, or breeding performance of other riparian birds at the restoration sites independent of temporal changes that occurred at reference sites. Our results provide evidence that limiting livestock grazing in temperate riparian areas can lead to recovery of endangered riparian songbirds that rely on early successional shrub habitat but may have limited effects on common species that are not strictly reliant on this habitat.     

Opsin stability and folding: the role of Cys185 and abnormal disulfide bond formation in the intradiscal domain

The structure in the extracellular, intradiscal domain of rhodopsin surrounding the Cys110–Cys187 disulfide bond has been shown to be important for correct folding of this receptor in vivo. Retinitis pigmentosa misfolding mutants of the apoprotein opsin (such as P23H) misfold, as defined by a deficiency in ability to bind 11-cis retinal and form rhodopsin. These mutants also possess an abnormal Cys185–Cys187 disulfide bond in the intradiscal domain. Here, by mutating Cys185 to alanine, we eliminate the possibility of forming this abnormal disulfide bond and investigate the effect of combining the C185A mutation with the retinitis pigmentosa mutation P23H. Both the P23H and P23H/C185A double mutant suffer from low expression and poor 11-cis retinal binding. Our data suggest that misfolding events occur that do not have an absolute requirement for abnormal Cys185–Cys187 disulfide bond formation. In the detergent-solubilised, purified state, the C185A mutation allows formation of rhodopsin at wild-type (WT) levels, but has interesting effects on protein stability. C185A rhodopsin is less thermally stable than WT, whereas C185A opsin shows the same ability to regenerate rhodopsin in detergent as WT. Purified C185A and WT opsins, however, have contrasting 11-cis retinal binding kinetics. A high proportion of C185A opsin binds 11-cis retinal with a slow rate that reflects a denatured state of opsin reverting to a fast-binding, open-pocket conformation. This slower rate is not observed in a stabilising lipid/detergent system, 1,2-dimyristoyl-sn-glycero-3-phosphocholine/Chaps, in which C185A exhibits WT (fast) retinal binding. We propose that the C185A mutation destabilises the open-pocket conformation of opsin in detergent resulting in an equilibrium between correctly folded and denatured states of the protein. This equilibrium can be driven towards the correctly folded rhodopsin state by the binding of 11-cis retinal.