Expression of Drosophila Ten-a,a dimeric receptor during embryonic development

Ten-a and Ten-m are the two Drosophila members of the newly discovered Ten-m family of dimeric type II transmembrane proteins. Here, we report complete cDNA cloning and protein expression patterns of Ten-a. The Ten-a protein, a dimeric receptor of about 500 kDa is mainly expressed on axons of the embryonic central nervous system and on muscle attachment sites.     

Outbreak of hind limb paralysis in young CFW Swiss Webster mice

An outbreak of paralysis among 16- to 20-week-old CFW Swiss Webster sentinel mice developed in one of our barrier facilities. Two months after arrival and over a period of four weeks, six of 400 mice purchased from an approved vendor, developed progressive hind limb paralysis without other clinical signs of disease. On the basis of the histopathologic changes and negative serologic test results, lymphoblastic lymphoma causing compression of the spinal cord was diagnosed. There were two leading features to this outbreak: its unusual epidemiologic presentation, and the localization of the lesions principally in the lumbar muscles. A presumptive diagnosis of retroviral infection with Abelson's murine leukemia virus (A-MuLV) was established on the basis of histopathologic and immunohistochemical findings. Little is known about retroviral status in many commercial colonies, and few users report presence of spontaneous lymphomas. This report points out complications derived from commercially available animals that carry endogenous retroviruses. It also emphasizes the need of diagnosing and reporting clusters of hind limb paralysis or lymphomas in mice to assess the prevalence and relevance of retroviral infections in commercial colonies.     

Kinetics of apoptotic markers in exogeneously induced apoptosis of EL4 cells

We investigated the time-dependence of apoptotic events in EL4 cells by monitoring plasma membrane changes in correlation to DNA fragmentation and cell shrinkage. We applied three apoptosis inducers (staurosporine, tubericidine and X-rays) and we looked at various markers to follow the early-to-late apoptotic events: phospholipid translocation (identified through annexin V-fluorescein assay and propidium iodide), lipid package (via merocyanine assay), membrane fluidity and anisotropy (via fluorescent measurements), DNA fragmentation by the fluorescence-labeling test and cell size measurements. The different apoptotic inducers caused different reactions of the cells: staurosporine induced apoptosis most rapidly in a high number of cells, tubercidine triggered apoptosis only in the S phase cells, while X-rays caused a G2/M arrest and subsequently apoptosis. Loss of lipid asymmetry is promptly detectable after one hour of incubation time. The phosphatidylserine translocation, decrease of lipid package and anisotropy, and the increase of membrane fluidity appeared to be based on the same process of lipid asymmetry loss. Therefore, the DNA fragmentation and the cell shrinkage appear to be parallel and independent processes running on different time scales but which are kinetically inter-related. The results indicate different signal steps to apoptosis dependent on inducer characteristics but the kinetics of "early-to-late" apoptosis appears to be a fixed program.     

Analysis of interactions between domains of a small heat shock protein,Hsp30 of Neurospora crassa

The alpha-crystallin-related, small heat shock proteins (sHsps), despite their overall variability in sequence, have discrete regions of conserved sequence that are involved in structural organization, as well as nonconserved regions that may perform similar roles in each protein. Recent X-ray diffraction analyses of an archeal and a plant sHsp have revealed both similarities and differences in how they are organized, suggesting that there is variability, particularly in the oligomeric organization of sHsps. As an adjunct to crystallographic analysis of sHsp structure, we employed the yeast 2-hybrid system to detect interactions between peptide regions of the sHsp of Neurospora crassa, Hsp30. We found that the conserved alpha-crystallin domain can be divided into N-terminal and C-terminal subdomains that interact strongly with one another. This interaction likely represents the tertiary contacts of the monomer that were visualized in the crystallographic structures of MjHsp16.5 and wheat Hsp16.9. The conserved sHsp monomeric fold is apparently determined by these regions of conserved sequence. We found that the C-terminal portion of the alpha-crystallin domain also interacts with itself in 2-hybrid assays; however, this interaction requires peptide extension into the semiconserved carboxyl tail. This C-terminal association may represent a principal contact site between dimers that contributes to higher-order assembly, as seen for the crystallized sHsps.     

Monoamine oxidase: radiotracer development and human studies

Monoamine oxidase (MAO) is an integral protein of outer mitochondrial membranes and occurs in neuronal and nonneuronal cells in the brain and in peripheral organs. It oxidizes amines from both endogenous and exogenous sources, thereby influencing the concentration of neurotransmitter amines as well as many xenobiotics. It occurs in two subtypes, MAO A and MAO B, which are different gene products and have different substrate and inhibitor specificities. Both MAO A and B can be imaged and quantified in the living human brain using positron emission tomography (PET) and radiotracers labeled with carbon-11. PET studies have been carried out to measure the effects of age, MAO inhibitor drugs, tobacco smoke exposure, and other factors on MAO activity in the human brain.     

Cultivation of Mycobacterium bovis BCG in bioreactors

The Mycobacterium bovis BCG vaccine for commercial use is classically produced as surface pellicles by culture on synthetic medium. Under these conditions, reproducibility of the cultures and quality assessment are hampered by slow growth of the bacilli, the formation of bacterial aggregates and a high proportion of dead bacilli after processing and final formulation of the vaccine. Here, we established dispersed cultures of M. bovis BCG in synthetic media in small-scale bioreactors. These cultures allow recording and adjusting of culture parameters and give rise to single bacilli with a high degree of live bacteria. In the murine model, bioreactor-grown M. bovis BCG exhibited slightly stronger replication and persistence than the vaccine produced under the classical conditions. The protective efficacy against challenge with M. tuberculosis was identical for both vaccine preparations.     

Manufacturing DNA microarrays from unpurified PCR products

For the production of DNA microarrays from PCR products, purification of the the DNA fragments prior to spotting is a major expense in cost and time. Also, a considerable amount of material is lost during this process and contamination might occur. Here, a protocol is presented that permits the manufacture of microarrays from unpurified PCR products on aminated surfaces such as glass slides coated with the widely used poly(L-lysine) or aminosilane. The presence of primer molecules in the PCR sample does not increase the non-specific signal upon hybridisation. Overall, signal intensity on arrays made of unpurified PCR products is 94% of the intensity obtained with the respective purified molecules. This slight loss in signal, however, is offset by a reduced variation in the amount of DNA present at the individual spot positions across an array, apart from the considerable savings in time and cost. In addition, a larger number of arrays can be made from one batch of amplification products.     

The C-terminal region of Escherichia coli UvrC contributes to the flexibility of the UvrABC nucleotide excision repair system

Nucleotide excision repair in Escherichia coli involves formation of the UvrB–DNA complex and subsequent DNA incisions on either site of the damage by UvrC. In this paper, we studied the incision of substrates with different damages in varying sequence contexts. We show that there is not always a correlation between the incision efficiency and the stability of the UvrB–DNA complex. Both stable and unstable UvrB–DNA complexes can be efficiently incised. However some lesions that give rise to stable UvrB–DNA complexes do result in a very low incision. We present evidence that this poor incision is due to sterical hindrance of the damage itself. In its C-terminal region UvrC contains two helix–hairpin–helix (HhH) motifs. Mutational analysis shows that these motifs constitute one functional unit, probably folded as one structural unit; the (HhH)₂ domain. This (HhH)₂ domain was previously shown to be important for the 5′ incision on a substrate containing a (cis-Pt)·GG adduct, but not for 3′ incision. Here we show that, mainly depending on the sequence context of the lesion, the (HhH)₂ domain can be important for 3′ and/or 5′ incision. We propose that the (HhH)₂ domain stabilises specific DNA structures required for the two incisions, thereby contributing to the flexibility of the UvrABC repair system.     

Rapid purification and crystal structure analysis of a small protein carrying two terminal affinity tags

Small peptide tags are often fused to proteins to allow their affinity purification in high-throughput structure analysis schemes. To assess the compatibility of small peptide tags with protein crystallization and to examine if the tags alter the three-dimensional structure, the N-terminus of the chicken alpha-spectrin SH3 domain was labeled with a His6 tag and the C-terminus with a StrepII tag. The resulting protein, His6-SH3-StrepII, consists of 83 amino-acid residues, 23 of which originate from the tags. His6-SH3-StrepII is readily purified by dual affinity chromatography, has very similar biophysical characteristics as the untagged protein domain and crystallizes readily from a number of sparse-matrix screen conditions. The crystal structure analysis at 2.3 A resolution proves native-like structure of His6-SH3-StrepII and shows the entire His6 tag and part of the StrepII tag to be disordered in the crystal. Obviously, the fused affinity tags did not interfere with crystallization and structure analysis and did not change the protein structure. From the extreme case of His6-SH3-StrepII, where affinity tags represent 27% of the total fusion protein mass, we extrapolate that protein constructs with N- and C-terminal peptide tags may lend themselves to biophysical and structural investigations in high-throughput regimes.