Insights on the development, kinetics, and variation of photoinhibition using chlorophyll fluorescence imaging of a chilled, variegated leaf

The effect of chilling on photosystem II (PSII) efficiency was studied in the variegated leaves of Calathea makoyana, in order to gain insight into the causes of chilling-induced photoinhibition. Additionally, a relationship was revealed between (chilling) stress and variation in photosynthesis. Chilling treatments (5 degrees C and 10 degrees C) were performed for different durations (1-7 d) under a moderate irradiance (120 micromol m-2 s-1). The individual leaves were divided into a shaded zone and two illuminated, chilled zones. The leaf tip and sometimes the leaf base were not chilled. Measurements of the dark-adapted Fv/Fm were made on the different leaf zones at the end of the chilling treatment, and then for several days thereafter to monitor recovery. Chilling up to 7 d in the dark did not affect PSII efficiency and visual appearance, whereas chilling in the light caused severe photoinhibition, sometimes followed by leaf necrosis. Photoinhibition increased with the duration of the chilling period, whereas, remarkably, chilling temperature had no effect. In the unchilled leaf tip, photoinhibition also occurred, whereas in the unchilled leaf base it did not. Whatever the leaf zone, photoinhibition became permanent if the mean value dropped below 0.4, although chlorosis and necrosis were associated solely with chilled illuminated tissue. Starch accumulated in the unchilled leaf tip, in contrast to the adjacent chilled irradiated zone. This suggests that photoinhibition was due to a secondary effect in the unchilled leaf tip (sink limitation), whereas it was a direct effect of chilling and irradiance in the chilled illuminated zones. The PSII efficiency and its coefficient of variation showed a unique negative linearity across all leaf zones and different tissue types. The slope of this curve was steeper for chilled leaves than it was for healthy, non-stressed leaves, suggesting that the coefficient of variation may be an important tool for assessing stress in leaves.     

Disruption of diacylglycerol kinase delta (DGKD) associated with seizures in humans and mice

We report a female patient with a de novo balanced translocation, 46,X,t(X;2)(p11.2;q37)dn, who exhibits seizures, capillary abnormality, developmental delay, infantile hypotonia, and obesity. The 2q37 breakpoint observed in association with the seizure phenotype is of particular interest, because it lies near loci implicated in epilepsy in humans and mice. Fluorescence in situ hybridization mapping of the translocation breakpoints showed that no known genes are disrupted at Xp11.2, whereas diacylglycerol kinase delta (DGKD) is disrupted at 2q37. Expression studies in Drosophila and mouse suggest that DGKD is involved in central nervous system development and function. Electroencephalographic assessment of Dgkd mutant mice revealed abnormal epileptic discharges and electrographic seizures in three of six homozygotes. These findings implicate DGKD disruption by the t(X;2)(p11.2;q37)dn in the observed phenotype and support a more general role for DGKD in the etiology of seizures.     

Compromised intestinal lipid absorption in mice with a liver-specific deficiency of liver receptor homolog 1

Bile acids (BAs) are water-soluble end products from cholesterol metabolism and are essential for efficient absorption of dietary lipids. By using targeted somatic mutagenesis of the nuclear receptor liver receptor homolog 1 (LRH-1) in mouse hepatocytes, we demonstrate here that LRH-1 critically regulates the physicochemical properties of BAs. The absence of LRH-1 and subsequent deficiency of Cyp8b1 eliminate the production of cholic acid and its amino acid conjugate taurocholic acid and increase the relative amounts of less amphipathic BA species. Intriguingly, while the expression of Cyp8b1 is almost extinguished in the livers of mice that lack LRH-1, the expression of the rate-limiting enzyme of BA synthesis, i.e., Cyp7a1, remains unchanged. The profound remodeling of the BA composition significantly reduces the efficacy of intestinal absorption of lipids and reuptake of BAs and facilitates the removal of lipids from the body. Our studies unequivocally demonstrate a pivotal role for LRH-1 in determining the composition of BAs, which, in turn has major consequences on whole-body lipid homeostasis.     

The discovery of potent, selective, and orally bioavailable hNK1 antagonists derived from pyrrolidine

SAR studies on amides, ureas, and vinylogous amides derived from pyrrolidine led to the discovery of several potent hNK(1) antagonists. One particular vinylogous amide (45b) had excellent potency, selectivity, pharmacokinetic profile, and functional activity in vivo. An in vivo rhesus macaque brain receptor occupancy PET study for compound 45b revealed an estimated Occ(90) approximately 300 ng/ml.     

Protein-liposome conjugates using cysteine-lipids and native chemical ligation

Liposomes have become popular drug delivery vehicles and have more recently also been applied as contrast agents for molecular imaging. Most current methods for functionalization of liposomes with targeting proteins rely on reactions of amine or thiol groups at the protein exterior, which generally result in nonspecific conjugation at multiple sites on the protein. In this study, we present native chemical ligation (NCL) as a general method to covalently couple recombinant proteins in a highly specific and chemoselective way to liposomes containing cysteine-functionalized phospholipids. A cysteine-functionalized phospholipid (Cys-PEG-DSPE) was prepared and shown to readily react with the MESNA thioester of EYFP, which was used as a model protein. Characterization of the EYFP-liposomes using fluorescence spectroscopy showed full retention of the fluorescent properties of conjugated EYFP and provides a lower limit of 120 proteins per liposome. The general applicability of NCL was further tested using CNA35, a collagen-binding protein recently applied in fluorescent imaging of collagen. NCL of CNA35 thioester yielded liposomes containing approximately 100 copies of CNA35 per liposome. The CNA35-liposomes were shown to be fully functional and bind collagen with a 150-fold higher affinity compared to CNA35. Our results show that NCL is an attractive addition to existing conjugation methods that allows direct, covalent, and highly specific coupling of recombinant proteins to liposomes and other lipid-based assemblies.     

Recent decrease in chinstrap penguin (<Emphasis Type="Italic">Pygoscelis antarctica</Emphasis>) populations at two of Admiralty Bay’s islets on King George Island,South Shetland Islands,Antarctica

We examined the breeding populations of chinstrap penguins (Pygoscelis antarctica) on Chabrier Rock and Shag Island within Admiralty Bay, King George Island, South Shetland Islands, Antarctica from 2002 to 2004. When comparing our results to historic data from 1979, we found an overall decline of 57% in the last 25 years, mirroring the population trend of this species in other regions of the Antarctic Peninsula. Our results are discussed in relation to factors hypothesized to be driving the declines found at other sites, as well as the importance of consistent annual censuses to accurately determine population trends.     

Introgression browser: high‐throughput whole‐genome SNP visualization

Breeding by introgressive hybridization is a pivotal strategy to broaden the genetic basis of crops. Usually, the desired traits are monitored in consecutive crossing generations by marker‐assisted selection, but their analyses fail in chromosome regions where crossover recombinants are rare or not viable. Here, we present the Introgression Browser (iBrowser ), a bioinformatics tool aimed at visualizing introgressions at nucleotide or SNP (Single Nucleotide Polymorphisms) accuracy. The software selects homozygous SNPs from Variant Call Format (VCF) information and filters out heterozygous SNPs, multi‐nucleotide polymorphisms (MNPs) and insertion–deletions (InDels). For data analysis iBrowser makes use of sliding windows, but if needed it can generate any desired fragmentation pattern through General Feature Format (GFF) information. In an example of tomato (Solanum lycopersicum) accessions we visualize SNP patterns and elucidate both position and boundaries of the introgressions. We also show that our tool is capable of identifying alien DNA in a panel of the closely related S. pimpinellifolium by examining phylogenetic relationships of the introgressed segments in tomato. In a third example, we demonstrate the power of the iBrowser in a panel of 597 Arabidopsis accessions, detecting the boundaries of a SNP‐free region around a polymorphic 1.17 Mbp inverted segment on the short arm of chromosome 4. The architecture and functionality of iBrowser makes the software appropriate for a broad set of analyses including SNP mining, genome structure analysis, and pedigree analysis. Its functionality, together with the capability to process large data sets and efficient visualization of sequence variation, makes iBrowser a valuable breeding tool.