A MICROSCOPIC TECHNIQUE TO MEASURE MESOPHYLL SUCCULENCE

Mesophyll succulence (S_m), the ratio of water content to chlorophyll content of a cell or tissue, has been proposed as an index of CAM potential. In plant tissues where all cells contain chloroplasts, S_m may be measured easily. If water tissue (water-storing parenchyma lacking chloroplasts) is present, however, severe technical difficulties arise. We have developed a comparable index, morphological mesophyll succulence (S_mm), which allows rapid determination of vacuole to chloroplast volumes in cells. There is a high correlation between the results obtained by these two techniques. Advantages of the microscopic technique include: more rapid determinations, fewer equipment needs, ability to be applied in the field, and no need to physically separate water tissues from photosynthetic tissues.     

The TGFbeta/Smad 3-signaling pathway is involved in butyrate-mediated vitamin D receptor (VDR)-expression

Previously, we demonstrated the pivotal role of the vitamin D receptor (VDR) in mediating the butyrate-induced differentiation in colon cancer cells. Smad 3, a downstream component of transforming growth factor-beta (TGFbeta) signaling, has been shown to act as a coactivator of VDR and to possibly regulate the vitamin D signaling pathway. In this study, we demonstrate a distinct impact of the TGFbeta/Smad 3-signaling pathway in the butyrate-mediated VDR expression and induction of differentiation. Butyrate treatment resulted in a significant induction of the phosphorylation level of Smad 3, while the combination of butyrate and a specific TGFbeta1-antibody or a TGFbeta-receptor inhibitor considerably diminished the butyrate-induced upregulation of VDR expression. Using a specific inhibitor, we were also able to demonstrate an involvement of the p38 MAPK in the increase of Smad 3 phosphorylation following butyrate treatment, thus opening the view to further elucidate possible mechanisms mediating the upregulation of VDR expression following butyrate treatment in colon cancer cells.     

A flexible approach for highly multiplexed candidate gene targeted resequencing

We have developed an integrated strategy for targeted resequencing and analysis of gene subsets from the human exome for variants. Our capture technology is geared towards resequencing gene subsets substantially larger than can be done efficiently with simplex or multiplex PCR but smaller in scale than exome sequencing. We describe all the steps from the initial capture assay to single nucleotide variant (SNV) discovery. The capture methodology uses in-solution 80-mer oligonucleotides. To provide optimal flexibility in choosing human gene targets, we designed an in silico set of oligonucleotides, the Human OligoExome, that covers the gene exons annotated by the Consensus Coding Sequencing Project (CCDS). This resource is openly available as an Internet accessible database where one can download capture oligonucleotides sequences for any CCDS gene and design custom capture assays. Using this resource, we demonstrated the flexibility of this assay by custom designing capture assays ranging from 10 to over 100 gene targets with total capture sizes from over 100 Kilobases to nearly one Megabase. We established a method to reduce capture variability and incorporated indexing schemes to increase sample throughput. Our approach has multiple applications that include but are not limited to population targeted resequencing studies of specific gene subsets, validation of variants discovered in whole genome sequencing surveys and possible diagnostic analysis of disease gene subsets. We also present a cost analysis demonstrating its cost-effectiveness for large population studies.     

DNA polymerases provide a canon of strategies for translesion synthesis past oxidatively generated lesions

Deducing the structure of the DNA double helix in 1953 implied the mode of its replication: Watson-Crick (WC) base pairing might instruct an enzyme, now known as the DNA polymerase, during the synthesis of a daughter stand complementary to a single strand of the parental double helix. What has become increasingly clear in the last 60 years, however, is that adducted and oxidatively generated DNA bases are ubiquitous in physiological DNA, and all organisms conserve multiple DNA polymerases specialized for DNA synthesis opposite these damaged templates. Here, we review recent crystal structures depicting replicative and bypass DNA polymerases encountering two typical lesions arising from the oxidation of DNA: abasic sites, which block the replication fork, and the miscoding premutagenic lesion 7,8-dihydro-8-oxoguanine (8-oxoG).     

M domains couple the ClpB threading motor with the DnaK chaperone activity

The AAA(+) chaperone ClpB mediates the reactivation of aggregated proteins in cooperation with the DnaK chaperone system. ClpB consists of two AAA domains that drive the ATP-dependent threading of substrates through a central translocation channel. Its unique middle (M) domain forms a coiled-coil structure that laterally protrudes from the ClpB ring and is essential for aggregate solubilization. Here, we demonstrate that the conserved helix 3 of the M domain is specifically required for the DnaK-dependent shuffling of aggregated proteins, but not of soluble denatured substrates, to the pore entrance of the ClpB translocation channel. Helix 3 exhibits nucleotide-driven conformational changes possibly involving a transition between folded and unfolded states. This molecular switch controls the ClpB ATPase cycle by contacting the first ATPase domain and establishes the M domain as a regulatory device that acts in the disaggregation process by coupling the threading motor of ClpB with the DnaK chaperone activity.     

A lysine-free mutant of epidermal growth factor as targeting moiety of a targeted toxin

AimsElevated levels of epidermal growth factor (EGF) receptor are observed on several human tumors, e.g. cervical carcinoma and mamma carcinomas. The natural ligand EGF is an alternative to established antibodies and tyrosine kinase inhibitors for targeting EGF receptor-overexpressing tumor cells for therapy. Conjugations of compounds to EGF lack the necessary homogeneity for an intended application, since several amino acids may react with the chemical linker.Main methodsWe designed an EGF variant (EGF^RR) in which the two lysines were substituted with arginine (K28R and K48R). EGF^RR was fused to the protein toxin saporin to obtain a model protein for detailed analyses on EGF receptor binding and on both the enzymatic activity of saporin and the cytotoxicity of the fusion protein.Key findingsThe mutation decreased the enzymatic activity of saporin 2.3-fold and the binding of EGF^RR retained its specificity for EGF receptor while increasing the Kd 5.5-fold. In spite of these differences the cytotoxicity of the fusion protein was unchanged in comparison to a fusion protein with EGF both when applied alone and in combination with cytotoxicity augmenting saponin.SignificanceWe conclude that EGF^RR retained its ability to bind with high specificity to EGF receptor and is thus suitable for a number of chemical linkage applications such as targeting drugs or dyes to EGF receptor-expressing cells.     

Foraging activity of Rhinolophus hipposideros on the Island of Herrenchiemsee, Upper Bavaria

We studied the foraging behaviour of Rhinolophus hipposideros on the island “Herrenchiemsee” in Lake Chiemsee (Upper Bavaria) during summer 2001. The island offers extensively managed woodlands, highly structured open landscapes and a broad reed belt around the shore. On average the flight activity of the 6 radio tracked females outside the roost lasted 229 min per night. The home range size varied between 6.8 and 62.7 ha (mean 25.2 ha). The size of the activity centres varied between 2.8 and 8.2 ha (mean 5.3) and all except one were located almost exclusively in woodland. Within woodlands the bats did not select for specific spatial structures (different age classes of the stands or canopy densities). Only two bats regularly foraged in additional habitats outside woodlands. One of these bats used orchards and tree rows; the other foraged over artificial ponds and gardens adjoining to its woodland foraging area. We never found the bats foraging over the lake or the reed belt. Longer linear landscape elements as tree lines were used during commuting flights but there was no indication of a continuous foraging activity along these elements. Two females left the island to forage on the mainland in August after the fledging of juveniles. To reach the mainland shore, the bats had to fly at least 1.2 km across the lake.

Assuming that most foraging flights on the island occur in woodlands, a bat density in this habitat type of 0.7 bats/ha can be calculated.     

Isolation and characterization of the novel polyadenylate- and polyuridylate-degrading acid endoribonuclease V from calf thymus

A novel endoribonuclease was detected and purified to homogeneity from calf thymus. The homogeneity was checked by analysis in polyacrylamide gels (both in the presence and in the absence of sodium dodecyl sulfate) as well as by isoelectric focusing. This nuclease activity, which is called Endoribonuclease V, cleaves poly(A) and poly(U); other single- or double-stranded synthetic polyribo- as well as polydeoxyribonucleotides are not degraded. Endoribonuclease V cleaves poly(A) to create first oligoribonucleotides and ultimately 3'-AMP; no P-2':3'-Ado degradation products were detected. The enzyme has a pH optimum of 5.8, an isoelectric point of pH 6.3, a molecular weight of 52,300, and requires neither monovalent nor divalent cations. The enzyme activity is not inhibited by N-ethylmaleimide.