Change in the developmental fate of the chick optic vesicle from the neural retina to the telencephalon

The forebrain develops into the telencephalon, diencephalon, and optic vesicle (OV). The OV further develops into the optic cup, the inner and outer layers of which develop into the neural retina and retinal pigmented epithelium (RPE), respectively. We studied the change in fate of the OV by using embryonic transplantation and explant culture methods. OVs excised from 10-somite stage chick embryos were freed from surrounding tissues (the surface ectoderm and mesenchyme) and were transplanted back to their original position in host embryos. Expression of neural retina-specific genes, such as Rax and Vsx2 (Chx10), was downregulated in the transplants. Instead, expression of the telencephalon-specific gene Emx1 emerged in the proximal region of the transplants, and in the distal part of the transplants close to the epidermis, expression of an RPE-specific gene Mitf was observed. Explant culture studies showed that when OVs were cultured alone, Rax was continuously expressed regardless of surrounding tissues (mesenchyme and epidermis). When OVs without surrounding tissues were cultured in close contact with the anterior forebrain, Rax expression became downregulated in the explants, and Emx1 expression became upregulated. These findings indicate that chick OVs at stage 10 are bi-potential with respect to their developmental fates, either for the neural retina or for the telencephalon, and that the surrounding tissues have a pivotal role in their actual fates. An in vitro tissue culture model suggests that under the influence of the anterior forebrain and/or its surrounding tissues, the OV changes its fate from the retina to the telencephalon.     

Differentiation of Staphylococcus spp. by terminal-restriction fragment length polymorphism analysis of glyceraldehyde-3-phosphate dehydrogenase-encoding gene

Classical phenotypic and biochemical testing do not lead to correct identification of the distinct Staphylococcus species. Therefore, the aim of our study was to develop a method for the reliable and accurate determination of distinct Staphylococcus species.

In the present study, the 931–934-bp partial sequences of the glyceraldehyde-3-phosphate dehydrogenase-encoding (gap) gene of 28 validly described Staphylococcus species were amplified and sequenced. By using the respective sequence information we performed a terminal-restriction fragment length polymorphism (T-RFLP) analysis. For T-RFLP the partial gap gene was amplified with double-fluorescently labelled primers and digested with the restriction enzymes DdeI, BspHI and TaqI. Distinctive T-RFLP patterns were rendered by the use of capillary electrophoresis with laser-induced fluorescence detection. This molecular method allowed us to identify all 28 Staphylococcus species with high specificity. This was validated by analysis of 34 Staphylococcus epidermidis and 28 Staphylococcus haemolyticus isolates.

These results demonstrate the feasibility and applicability of the T-RFLP method based on the partial gap gene sequences for rapid and accurate species identification.     

Expression and possible functions of the cholinergic system in a murine embryonic stem cell line

The expression of a cholinergic system during embryonic development is a widespread phenomenon. However, no precise function could be assigned to it during early pre-neural stages and there are only few studies that document when it precisely starts to be expressed. Here, we examined the expression of cholinergic components in a murine embryonic stem cell line by RT-PCR, histochemistry, and enzyme activity measurements; the acetylcholine (ACh) content was measured by HPLC. We have demonstrated that embryonic stem cells express ACh, acetylcholine receptors, choline acetyltransferase (ChAT), acetyl- and butyryl-cholinesterase (AChE and BChE). Butyryl-cholinesterase (BChE) expression was higher than AChE. The cholinesterase activity was down-regulated by adding specific inhibitors to culture medium. Inhibition of BChE led to a reduction of proliferation. This is the first demonstration that mouse embryonic stem cells express the full molecular equipment of a cholinergic system. Locally produced ACh might function as an intercellular signal, modulating the proliferation of stem cells.     

Butyrylcholinesterase Is Complexed with Transferrin in Chicken Serum

The function of the enzyme butyrylcholinesterase (BChE) both in serum and in brain is unclear. In serum, BChE has been found complexed with several biomedically relevant proteins, with which it could function in concert. Here, the existence of a similar complex formed between BChE and sero-transferrin from adult chicken serum was elucidated. In order to identify both proteins unequivocally, we improved methods to highly purify the 81-kDa BChE and the coisolated 75-kDa transferrin, which then allowed us to tryptically digest and sequence the resulting peptides. The sequences as revealed for BChE peptides were highly identical to mammalian BChEs. A tight complex formation between the two proteins could be established (a) since transferrin is coisolated along with BChE over three steps including procainamide affinity chromatography, while transferrin alone is not bound to this affinity column, and (b) since imunoprecipitation experiments of whole serum with a transferrin-specific antiserum allows us to detect BChE in the precipitate with the BChE-specific monoclonal antibody 7D11. The possible biomedical implications of a complex between transferrin and BChE which here has been shown to exist in chicken serum are briefly discussed.     

The aryl acylamidase activity is much more sensitive to Alzheimer drugs than the esterase activity of acetylcholinesterase in chicken embryonic brain

The appearance of cholinergic trait often precedes synaptogenesis, indicating the involvement of cholinesterase proteins in nervous system development, particularly so acetylcholinesterase (AChE). In addition to AChE's acclaimed esterase activity, its lesser known non-cholinergic functions have gained much attention, because of AChE protein expression in areas other than cholinergic innervations; one such function could be exerted by its associated aryl acylamidase (AAA) activity. In this study, an attempt has been made in profiling esterase and AAA activities of AChE at different developmental stages of the chick embryo, e.g. at embryonic day 6 (E6), E9, E12, E15 and E18. AAA activity showed a correlated expression with esterase activity at all stages, but the relative ratios of AAA to esterase activity were higher at younger stages. The inhibition of AAA activity was shown to be more sensitive towards Huperzine, Donepezil whereas inhibition of esterase activity was sensitive to Tacrine and DFP. Remarkably, the major Alzheimer drugs- Huperzine and Donepezil, much more strongly inhibited AAA activity of AChE at younger developmental stages whose IC50 values are 0.01 μM and 0.1 μM respectively. In the case of BW284c51, inhibition was more pronounced at older stages and IC50 value was 0.1 μM. Since in Alzheimer's disease (AD), embryonic forms of AChE have been reported to reappear, a possible role of AAA activity in the pathogenesis of AD should be considered.     

Methicillin Susceptible Staphylococcus aureus (MSSA) of Clonal Complex CC398, t571 from Infections in Humans Are Still Rare in Germany

Methicillin-susceptible Staphylococcus aureus (MSSA) attributed to clonal complex (CC) 398 and exhibiting spa-type t571 received attention in Europe and in the USA for being associated with severe infections in humans. As this spa-type is exhibited by livestock-associated (LA) Methicillin-resistant S. aureus (MRSA) as well, it is important to discriminate LA- and human-derived strains by easy to perform, PCR-based methods. MSSA t571 contain phage int3 carrying scn and chp, whereas LA-MRSA t571 lack these markers. In contrast, pathogenicity island SaPIbov5 (detected by PCR bridging vwbbov and scn) is contained by LA-MRSA t571 and absent in the human MSSA subpopulation. Furthermore, MSSA t571 contain erm(T), the particular genomic arrangement of which was assessed by a PCR bridging erm(T) and the adjacent transposase gene. MSSA t571 are rare so far in Germany among isolates from infections in humans (0.14%) as well as among isolates from nasal colonization (0.13%). LA-MRSA t571 are also infrequent among MRSA isolated from carriage at admission to hospitals (0.1%) and also among isolates from infections in humans (0.013%).     

LUMPY: a probabilistic framework for structural variant discovery

Comprehensive discovery of structural variation (SV) from whole genome sequencing data requires multiple detection signals including read-pair, split-read, read-depth and prior knowledge. Owing to technical challenges, extant SV discovery algorithms either use one signal in isolation, or at best use two sequentially. We present LUMPY, a novel SV discovery framework that naturally integrates multiple SV signals jointly across multiple samples. We show that LUMPY yields improved sensitivity, especially when SV signal is reduced owing to either low coverage data or low intra-sample variant allele frequency. We also report a set of 4,564 validated breakpoints from the NA12878 human genome. https://github.com/arq5x/lumpy-sv.     

Crystal structure of the heme d1 biosynthesis enzyme NirE in complex with its substrate reveals new insights into the catalytic mechanism of S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferases

During the biosynthesis of heme d₁, the essential cofactor of cytochrome cd₁ nitrite reductase, the NirE protein catalyzes the methylation of uroporphyrinogen III to precorrin-2 using S-adenosyl-l-methionine (SAM) as the methyl group donor. The crystal structure of Pseudomonas aeruginosa NirE in complex with its substrate uroporphyrinogen III and the reaction by-product S-adenosyl-l-homocysteine (SAH) was solved to 2.0 Å resolution. This represents the first enzyme-substrate complex structure for a SAM-dependent uroporphyrinogen III methyltransferase. The large substrate binds on top of the SAH in a “puckered” conformation in which the two pyrrole rings facing each other point into the same direction either upward or downward. Three arginine residues, a histidine, and a methionine are involved in the coordination of uroporphyrinogen III. Through site-directed mutagenesis of the nirE gene and biochemical characterization of the corresponding NirE variants the amino acid residues Arg-111, Glu-114, and Arg-149 were identified to be involved in NirE catalysis. Based on our structural and biochemical findings, we propose a potential catalytic mechanism for NirE in which the methyl transfer reaction is initiated by an arginine catalyzed proton abstraction from the C-20 position of the substrate.