Crystallization and preliminary crystallographic analysis of recombinant human P38 MAP kinase.

The recombinant human p38 MAP kinase has been expressed and purified from both Escherichia coli and SF9 cells, and has been crystallized in two forms by the hanging drop vapor diffusion method using PEG as precipitant. Both crystal forms belong to space group P2(1)2(1)2(1). The cell parameters for crystal form 1 are a = 65.2 A, b = 74.6 A and c = 78.1 A. Those for crystal form 2 are a = 58.3 A, b = 68.3 A and c = 87.9 A. Diffraction data to 2.0 A resolution have been collected on both forms.     

Control of Endosperm Proteins in TRITICUM AESTIVUM (Var. Chinese Spring) and AEGILOPS UMBELLULATA by Homoeologous Group 1 Chromosomes

The genetic control of major wheat endosperm proteins by homoeologous group 1 chromosomes has been studied by two-dimensional polyacrylamide gel electrophoresis. The control of at least 15 distinct protein subunits or groups of protein subunits has been allocated to chromosomes 1A, 1B and 1D of Chinese Spring wheat from the analysis of grains of aneuploid genotypes. In addition, six protein subunits have been shown to be controlled by chromosome 1C^u of the related species, Aegilops umbellulata, from studies of wheat lines carrying disomic substitutions of 1C^u chromosomes. On the basis of protein subunit patterns, chromosome 1C^u is more closely related to chromosome 1D of wheat than to chromosomes 1A or 1B.     

Mutagenicity of plant flavonols in the Salmonella/mammalian microsome test: activation of flavonol glycosides by mixed glycosidases from rat cecal bacteria and other sources.

Over 70 naturally occurring and synthetic flavonoids were screened for mutagenicity with 5 tester strains in the Salmonella/mammalian microsome assay: TA1535, TA100, TA1537, TA1538 and TA98. Frameshift mutagenicity was confined to the flavonols (flavon-3-ols) in strain TA98, TA1537 and TA100. The two most mutagenic falvonols, namely, quercetin (3,3',4',5,7-pentahydroxyflavone) and kaempferol (3,4',5,7-tetrahydroxyflavone), exhibiting 12 and 7 revertants/nmol in TA98 respectively, are also the most common flavonols occurring in plants. Other flavonols exhibited less activity (revertants/nmol): galangin (2.0), rhamnetin (0.45), kaempferide (0.24), fisetin (0.14), myricetin (0.12), robinetin (0.06) and morin (0.05). All of these flavonols apparently exhibited significant activation by Aroclor 1254 induced rat-liver microsome preparations (S9). However, subsequent study revealed that only those flavonols either lacking or possessing one B ring hydroxyl group had an absolute requirement for microsomal activation. Alternatively, quercetin with two B-ring OH groups is not activated by microsomal enzymes, but by soluble (S100) enzymes from liver which are apparently constitutive and not subject to the usual chemical induction. 3 flavonol glycosides, namely, quercetrin (quercetin-3-O-rhamnoside), rutin (quercetin-3-O-rutinoside) and robinin (kaempferol-3-O-galactosido-rhamnoside-7-O-rhamnoside), were found to be nonmutagenic. They could, however, be activated by a variety of mixed glycosidases incorporated in the usual pour plate procedure. The most effective enzyme mixtures were obtained from rat cecal bacteria and from the snail Helix pomatia.     

A new strategy for the detection of subtelomeric rearrangements

We present a new strategy for the detection of subtelomeric rearrangements. This approach is based on two hybridizations with different probe sets. The first set consists of microdissected subtelomeric probes (each 5-10 megabases in size) labeled combinatorially employing 7 different fluorochromes. With this set, subtelomeric interchromosomal exchanges can be detected in a 24-color experiment. The second set comprises a second generation of subtelomeric PAC-, P1- and BAC-clones. Probes for p- and q-arms are labeled with two different colors. This second set detects small deletions; in addition it provides regional information, so that translocated material identified by the first probe set can be assigned to the p- or q-arm of a chromosome. The test has been evaluated in a blind study on a series of subtle translocations and deletions.     

Role of glycoprotein PE2 in formation and maturation of the Sindbis virus spike.

Sindbis virus envelope assembly is a multistep process resulting in the maturation of a rigid, highly ordered T=4 icosahedral protein lattice containing 80 spikes composed of trimers of E1-E2 heterodimers. Intramolecular disulfide bonds within E1 stabilize E1-E1 associations required for envelope formation and maintenance of the envelope's structural integrity. The structural integrity of the envelope protein lattice is resistant to reduction by dithiothreitol (DTT), indicating that E1 disulfides which stabilize structural domains become inaccessible to DTT at some point during virus maturation. The development of E1 resistance to DTT occurs prior to the completion of E1 folding and is temporally correlated with spike assembly in the endoplasmic reticulum. From these data we have predicted that in the final stages of spike assembly, E1 intramolecular disulfides, which stabilize the structural integrity of the envelope protein lattice, are buried within the spike and become inaccessible to the reductive activity of DTT. The spike is formed prior to the completion of E1 folding, and we have suggested that PE2 (the precursor to E2) may play a critical role in E1 folding after PE2-E1 oligomer formation has occurred. In this study we have investigated the role of PE2 in E1 folding, oligomer formation, and development of E1 resistance to both protease digestion and reduction by DTT by using a Sindbis virus replicon (SINrep/E1) which allows for the expression of E1 in the presence of truncated PE2. Through pulse-chase analysis of both Sindbis virus- and SINrep/E1-infected cells, we have determined that the folding of E1 into a trypsin-resistant conformation and into its most compact and stable form is not dependent upon association of E1 with PE2. However, E1 association with PE2 is required for oligomer formation, the export of E1 from the endoplasmic reticulum, and E1 acquisition of resistance to DTT.     

Characterisation of the Small RNAs in the Biomedically Important Green-Bottle Blowfly Lucilia sericata

Background

The green bottle fly maggot, Lucilia sericata, is a species with importance in medicine, agriculture and forensics. Improved understanding of this species’ biology is of great potential benefit to many research communities. MicroRNAs (miRNA) are a short non-protein coding regulatory RNA, which directly regulate a host of protein coding genes at the translational level. They have been shown to have developmental and tissue specific distributions where they impact directly on gene regulation. In order to improve understanding of the biology of L. sericata maggots we have performed small RNA-sequencing of their secretions and tissue at different developmental stages.

Results

We have successfully isolated RNA from the secretions of L. sericata maggots. Illumina small RNA-sequencing of these secretions and the three tissues (crop, salivary gland, gut) revealed that the most common small RNA fragments were derived from ribosomal RNA and transfer RNAs of both insect and bacterial origins. These RNA fragments were highly specific, with the most common tRNAs, such as GlyGCC, predominantly represented by reads derived from the 5’ end of the mature maggot tRNA. Each library also had a unique profile of miRNAs with a high abundance of miR-10-5p in the maggot secretions and gut and miR-8 in the food storage organ the crop and salivary glands. The pattern of small RNAs in the bioactive maggot secretions suggests they originate from a combination of saliva, foregut and hindgut tissues. Droplet digital RT-PCR validation of the RNA-sequencing data shows that not only are there differences in the tissue profiles for miRNAs and small RNA fragments but that these are also modulated through developmental stages of the insect.

Conclusions

We have identified the small-RNAome of the medicinal maggots L. sericata and shown that there are distinct subsets of miRNAs expressed in specific tissues that also alter during the development of the insect. Furthermore there are very specific RNA fragments derived from other non-coding RNAs present in tissues and in the secretions. This new knowledge has applicability in diverse research fields including wound healing, agriculture and forensics.     

The zymogen form of acrosin in testicular,epididymal, and ejaculated spermatozoa from ram

The sperm-specific proteinase acrosin (EC 3.4.21.10) is found in spermatozoa as a zymogen. We have looked for different forms of this zymogen in testicular, epididymal, and ejaculated spermatozoa from ram and have compared total sperm extracts made immediately after cell disruption with extracts made later from isolated sperm heads. We have concluded that the autoactivatable zymogen form, known generally as proacrosin, is the only form of acrosin within intact mature ram spermatozoa; no other zymogen form was detected, although lower levels of proacrosin were found in some samples of testicular spermatozoa. From studies of the activation process, it appears that ram proacrosin is truly autoactivatable; no evidence could be found for the involvement of any auxiliary enzyme. Estimations of the molecular weight of proacrosin using gel chromatography (60,000) and SDS-polyacrylamide gel electrophoresis (51,300) indicated that the zymogen is monomeric. Comparison with the molecular weight of ram acrosin (44,000 or 40,000, using the two respective methods) indicated that a single acrosin molecule is derived from each zymogen molecule. The sperm acrosin inhibitor (molecular weight 11,000 or 8,000) was present in testicular spermatozoa as well as in ejaculated spermatozoa; there was no evidence that it was produced as a result of zymogen activation.     

Proton transfers in the photochemical reaction cycle of proteorhodopsin

The spectral and photochemical properties of proteorhodopsin (PR) were determined to compare its proton transport steps to those of bacteriorhodopsin (BR). Static and time-resolved measurements on wild-type PR and several mutants were done in the visible and infrared (FTIR and FT-Raman). Assignment of the observed C=O stretch bands indicated that Asp-97 and Glu-108 serve as the proton acceptor and donor, respectively, to the retinal Schiff base, as do the residues at corresponding positions in BR, but there are numerous spectral and kinetic differences between the two proteins. There is no detectable dark-adaptation in PR, and the chromophore contains nearly entirely all-trans retinal. Because the pK(a) of Asp-97 is relatively high (7.1), the proton-transporting photocycle is produced only at alkaline pH. It contains at least seven transient states with decay times in the range from 10 micros to 200 ms, but the analysis reveals only three distinct spectral forms. The first is a red-shifted K-like state. Proton release does not occur during the very slow (several milliseconds) rise of the second, M-like, intermediate, consistent with lack of the residues facilitating extracellular proton release in BR. Proton uptake from the bulk, presumably on the cytoplasmic side, takes place prior to release (tau approximately 2 ms), and coincident with reprotonation of the retinal Schiff base. The intermediate produced by this process contains 13-cis retinal as does the N state of BR, but its absorption maximum is red-shifted relative to PR (like the O state of BR). The decay of this N-like state is coupled to reisomerization of the retinal to all-trans, and produces a state that is O-like in its C-C stretch bands, but has an absorption maximum apparently close to that of unphotolyzed PR.