Stomatal closure by ultraviolet radiation

The effect of ultraviolet radiation (UV) (255–325 nm) on stomatal closure was investigated on tef [ Eragrostis tef (Zucc) Trotter] in the presence of white light (ca 50 ·mol m⁻² s⁻¹). The action spectrum showed that UV (ca 2 ·mol m⁻² s⁻¹, half band width about 10 nm) of 285 nm or shorter wavelengths was very efficient in causing stomatal closure. The effectiveness decreased sharply towards longer wavelengths. Radiation of 313 nm or longer wavelengths was practically without effect. Increasing UV intensity increased stomatal resistance. When stronger white light (5 to 9 times stronger than the one used during irradiation) was administered, stomates re-opened rapidly irrespective of whether the UV was on or off, although a subsequent gradual closing tendency was observed when the UV was on.     

Towards compendia of negative genetic association studies: an example for Alzheimer disease

Most genetic sequence variants that contribute to variability in complex human traits will have small effects that are not readily detectable with population samples typically used in genetic association studies. A potentially valuable tool in the gene discovery process is meta-analysis of the accumulated published data, but in order to be valid these require a sample of studies representative of the true genetic effect and thus hypothetically should include some positive and an abundance of negative reports. A survey of the literature on association studies for Alzheimer disease (AD) from January 2004–April 2005, identified 138 studies, 86 of which reported positive findings other than for apolipoprotein E (APOE), strongly indicative of publication bias. We report here an analysis of 62 genetic markers, tested for association with AD risk as well as for possible effects upon quantitative indices of AD severity (mini-mental state examination scores, age-at-onset, and cerebrospinal fluid (CSF) β-amyloid (Aβ) and CSF tau proteins). Within this set, only modest signals were present that, with the exception of APOE are easily lost when corrections for multiple hypotheses are applied. In isolation, results are thus broadly negative. Genes studied encompass both novel candidates as well as several recently claimed to be associated with AD (e.g. urokinase plasminogen activator (PLAU) and acetyl-coenzyme A acetyltransferase 1 (ACAT1)). By reporting these data we hope to encourage the publication of gene compendia to guide further studies and aid future meta-analyses aimed at resolving the involvement of genes in complex human traits.     

Process optimization for increased yield of surface-expressed protein in Escherichia coli

The autotransporter family of Gram-negative protein exporters has been exploited for surface expression of recombinant passenger proteins. While the passenger in some cases was successfully translocated, a major problem has been low levels of full-length protein on the surface due to proteolysis following export over the cytoplasmic membrane. The aim of the present study was to increase the surface expression yield of the model protein SefA, a Salmonella enterica fimbrial subunit with potential for use in vaccine applications, by reducing this proteolysis through process design using Design of Experiments methodology. Cultivation temperature and pH, hypothesized to influence periplasmic protease activity, as well as inducer concentration were the parameters selected for optimization. Through modification of these parameters, the total surface expression yield of SefA was increased by 200 %. At the same time, the yield of full-length protein was increased by 300 %, indicating a 33 % reduction in proteolysis.     

Identification of New Differentially Methylated Genes That Have Potential Functional Consequences in Prostate Cancer

Many differentially methylated genes have been identified in prostate cancer (PCa), primarily using candidate gene-based assays. Recently, several global DNA methylation profiles have been reported in PCa, however, each of these has weaknesses in terms of ability to observe global DNA methylation alterations in PCa. We hypothesize that there remains unidentified aberrant DNA methylation in PCa, which may be identified using higher resolution assay methods. We used the newly developed Illumina HumanMethylation450 BeadChip in PCa (n = 19) and adjacent normal tissues (n = 4) and combined these with gene expression data for identifying new DNA methylation that may have functional consequences in PCa development and progression. We also confirmed our methylation results in an independent data set. Two aberrant DNA methylation genes were validated among an additional 56 PCa samples and 55 adjacent normal tissues. A total 28,735 CpG sites showed significant differences in DNA methylation (FDR adjusted P<0.05), defined as a mean methylation difference of at least 20% between PCa and normal samples. Furthermore, a total of 122 genes had more than one differentially methylated CpG site in their promoter region and a gene expression pattern that was inverse to the direction of change in DNA methylation (e.g. decreased expression with increased methylation, and vice-versa). Aberrant DNA methylation of two genes, AOX1 and SPON2, were confirmed via bisulfate sequencing, with most of the respective CpG sites showing significant differences between tumor samples and normal tissues. The AOX1 promoter region showed hypermethylation in 92.6% of 54 tested PCa samples in contrast to only three out of 53 tested normal tissues. This study used a new BeadChip combined with gene expression data in PCa to identify novel differentially methylated CpG sites located within genes. The newly identified differentially methylated genes may be used as biomarkers for PCa diagnosis.     

Folding into a beta-hairpin can prevent amyloid fibril formation

The tetrapeptide KFFE is one of the shortest amyloid fibril-forming peptides described. Herein, we have investigated how the structural environment of this motif affects polymerization. Using a turn motif (YNGK) or a less rigid sequence (AAAK) to fuse two KFFE tetrapeptides, we show by several biophysical methods that the amyloidogenic properties are strongly dependent on the structural environment. The dodecapeptide KFFEAAAKKFFE forms abundant thick fibril bundles. Freshly dissolved KFFEAAAKKFFE is monomeric and shows mainly disordered secondary structure, as evidenced by circular dichroism, NMR spectroscopy, hydrogen/deuterium exchange measurements, and molecular modeling studies. In sharp contrast, the dodecapeptide KFFEYNGKKFFE does not form fibrils but folds into a stable beta-hairpin. This structure can oligomerize into a stable 12-mer and multiples thereof, as shown by size exclusion chromatography, sedimentation analysis, and electrospray mass spectrometry. These data indicate that the structural context in which a potential fibril forming sequence is present can prevent fibril formation by favoring self-limiting oligomerization over polymerization.     

Apolipoprotein [a] genotype influences isoform dominance pattern differently in African Americans and Caucasians

Plasma lipoprotein [a] (Lp[a]) concentrations are inversely associated with, and largely determined by, apolipoprotein [a] (apo[a]) gene size, a highly polymorphic trait. We studied if, within an individual, the smaller apo[a] isoform always dominated, whether there was interaction between the two alleles, and whether these features differed between Caucasians and African Americans. We determined apo[a] gene sizes, apo[a] protein sizes and relative amounts, and plasma Lp[a] levels in 430 individuals (263 Caucasians and 167 African Americans). Of the 397 heterozygotes with at least one detectable apo[a] isoform (238 Caucasians and 159 African Americans), the larger allele dominated in 28% of Caucasians and 23% of African Americans, while the smaller allele dominated in 56% of Caucasians and 45% of African Americans. In Caucasians, dominance of the smaller allele increased with Lp[a] levels, from 44% at Lp[a] < or = 30 nM to 81% at Lp[a] >100 nM (P < 0.0001). Dominance by the smaller allele increased with increasing size of the larger allele in both groups but with the smaller allele only in African Americans. There was no interaction between apo[a] alleles within genotypes; one apo[a] isoform level was not associated with the other isoform level, and isoform levels were not affected by the difference in size. More of the dominance pattern was explained by Lp[a] level and apo[a] genotype in African Americans than in Caucasians (29% vs. 13%). Thus, genotype influences isoform-specific Lp[a] levels and dominance patterns differently in African Americans and in Caucasians.