Tyrosine phosphorylation of BIT on photic stimulation in the rat retina

BIT is a transmembrane glycoprotein with three immunoglobulin-like domains in its extracellular region and tyrosine phosphorylation sites in its cytosolic region. We have previously shown that BIT was tyrosine phosphorylated in the hypothalamic suprachiasmatic nucleus in response to light exposure during the dark period, and suggested that it was involved in the light entrainment of the circadian clock. To further investigate the function of BIT in the nervous system, we examined the effect of photic stimulation on its tyrosine phosphorylation in the rat retina. It was found that the tyrosine phosphorylation level of BIT in the retina was higher in the light period than in the dark period. In addition, a light stimulation during the dark period resulted in a rapid phosphorylation of BIT and a subsequent association of BIT with SHP-2. The phosphorylation state was quickly reverted when the light was turned off. The light-dependent phosphorylation of BIT was also observed in isolated cultured retinas, and this was blocked by a specific Src-family inhibitor, PP-2. Immunohistochemical study showed that BIT was highly enriched in the inner and outer plexiform layers in the retina, where the immunoreactivity to anti-SHP-2 antibody was also detected. These results suggest that tyrosine phosphorylation of BIT is involved in neuronal transmission in the retina.     

Caries and oral hygiene in children in postwar Novi Travnik (Bosnia and Herzegovina) and Zabok (Croatia)

The study was performed in 1997 and involved school children between the age of 6 and 12 in Novi Travnik, Bosnia and Herzegovina (n = 203) and Zabok, Croatia (n = 132). OHI-S (Simplified Oral Hygiene Index by Green-Vermillion) and DMF (Decayed, Missing, Filled) index were used as main outcome measures. Prewar data were taken from the respective literature. The value of the DMF/dmf (PERMANENT/deciduous teeth) for six-year-olds in Novi Travnik of the period before the war was: d = 5.6, m = 0.4, f= 0.6 and D = 0.3, F = 0.1 and the average DMF index of twelve-year-olds for the same period were 6.5. The DMF/dmf index in 1997 in Novi Travnik was: d = 9.4+/-4.4; m = 0.7 +/-1.1; D = 1.9+/-1.2 and average DMF index of twelve-year-olds was 9.0+/-4.16. The DMF index of twelve-year-olds in Zabok in 1990 was 3.4 and 4.1+/-2.1 in 1997. Total DMF index for all the examined ages in 1997 for Zabok was 6.1+/-3.7 and for the examinees in Novi Travnik 10.5+/-4.1 (p<0.001). Similarly, the OHI-S in 1997 for Zabok was 1.0+/-0.7 whereas 1.7+/-0.7 (p<0.001) in Novi Travnik. In comparison to prewar data, DMF index in 1997 was considerably higher. Increase of DMF index was higher in Novi Travnik than in Zabok, which can be attributed to the war and wartime conditions.     

Review and new insights on Wegener granulomatosis

The purpose of this paper is to present Wegener granulomatosis manifesting in its periocular form. Review with new developments in understanding of the etiopathogenesis, clinical and laboratory findings as well as therapy modalities are shown through a series of patients gathered in collaboration with colleagues from UMC Utrecht, Netherlands. In the period from 1992 until 2004 the group of 54 patients with established diagnosis of Mb. Wegener were observed. 13 patients developed periocular form but only 2 presented it as initial symptoms. Lacrimal stenosis and orbital infiltration were predominant periocular symptoms while nasal manifestations were predominant systemic symptoms of the disease. Different treatment modalities were employed showing that orbital disease is difficult to treat in spite of satisfying systemic answer to immunosuppressives which calls for alternative solutions.     

Patients review: drug-induced movement disorders

Objective of this paper is to review drug-induced movement disorders (D-IMD) treated patients on Department of Neurology in University Hospital Osijek. We reviewed patients treated during 10 years period (from 1992 to 2002). Analysed group consisted of 14 patients. Reasons for hospitalisation were swallowing problems in 6 patients, neuroleptic malignant syndrome (NMS) in 3 patients, stroke in 2 patients, bolus choking in 2 patients, and speech disturbance in 1 patient. Working diagnosis for most of our patients was neurological disease, yet only later D-IMD diagnosis was established excluding primary neurological disease, or as associated disease to basic neurological disorder. Nine patients have diagnosed as Parkinson syndrome, 3 patients as NMS, and 4 as orolingual dyskinesia, either autonomously, or in combination with Parkinson syndrome. D-IMD was most frequently caused by neuroleptics. Thus the small number of patients hospitalised regarding this syndrome on Department of Neurology.     

Self-made frits for nanoscale columns in proteomics

We report here the production of self-made frits for nano-columns. The frits introduce a minor dead volume and can be placed in capillaries with a wide range of diameters (20-250 microm tested) in an extremely simple and low-cost procedure. The obtained columns appear to be comparable to "no-frit" columns with near-ideal chromatographic characteristics. We expect that this frit will be useful for the spotting of gradients onto MALDI plates but also where special ESI set-ups do not allow for "no-frit" solutions.     

Virus–Pesticide interactions in Ixodes persulcatus and Amblyomma hebraeum ticks (Acarina: Ixodidae)

Sublethal doses of the organophosphate Dursban were tested on non-infected and tick-borne encephalitis virus (TBEV)-infected nymphs of Amblyomma hebraeum and Ixodes persulcatus. It was shown that contact with this pesticide enhanced the survival of non-infected ticks, whereas TBEV infection reduced survival. This effect of TBEV infection was greater in I. persulcatus than in A. hebraeum ticks. This phenomenon may be the result of a virus-specific action on nymphs, whereby the virus may enhance activity and thus food reserves may deplete faster. Our data emphasize that the acaricidal action on non-infected ticks and those infected by different tick-borne pathogens may be different.     

Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: a component of the P1/P2/P0 complex.

The P0 protein is part of the ribosomal eukaryotic stalk, which is an elongated lateral protuberance of the large ribosomal subunit involved in the translocation step of protein synthesis. P0 is the minimal portion of the stalk that is able to support accurate protein synthesis. The P0 C-terminal peptide is highly antigenic and a major target of the antibody response in patients with systemic lupus erythematosus and patients suffering chronic heart disease produced by the Trypanosoma cruzi parasite. The T. cruzi P0 (TcP0) protein was cloned into the pRSET A vector and expressed in Escherichia coli fused to a His-tag. The identity of the protein was confirmed by immunoblotting. Due to the formation of inclusion bodies the protein was purified using the following steps: (i) differential centrifugation to separate the inclusion bodies from soluble proteins and (ii) affinity chromatography under denaturing conditions. TcP0 showed high tendency to aggregation during refolding assays. However, TcP0 could be efficiently folded in the presence of a low concentration of SDS. The folding of the protein was confirmed using urea gradient electrophoresis, limited proteolysis, circular dichroism, and tryptophan fluorescence. Native electrophoresis showed that the folded TcP0 (and not a folding intermediate) was the cause of aggregation in the absence of SDS. The protocol described here permitted us to obtain large amounts (up to 30 mg per culture liter) of pure and folded TcP0, a very hydrophobic protein with a high tendency to aggregation.     

Changes in import of non-human primates after ratification of CITES (Washington Convention) in Japan

The total yearly imports of exotic primates into Japan and the countries of origin from 1971 to 1984 were examined. The species, numbers, originating countries, and end-uses of the monkeys imported from 1981 to 1984 were also investigated. After a high plateau of imports which continued until 1980, the total numbers of monkeys imported into Japan were reduced by half from the pre-1980 level of 7,000–8,000/year to a level of 3,000–4,000/year. This drastic decrease appeared to reflect the effect of the Washington Convention ratified in 1980. The majority of species imported wereMacaca fascicularis of Southeast Asian origin, followed bySaimiri sciureus of South American origin. Indonesian and MalaysianM. fascicularis represented the major species in the decrease in monkey imports. However, imports from the Philippines have conversely increased since 1980. Imports ofS. sciureus andCallithrix from South America have remained almost unchanged. These data imply a trend in which experimental monkeys have changed from macaques to smaller New World monkeys.     

Serum Albumin Domain Structures in Human Blood Serum by Mass Spectrometry and Computational Biology

Chemical cross-linking combined with mass spectrometry has proven useful for studying protein-protein interactions and protein structure, however the low density of cross-link data has so far precluded its use in determining structures de novo. Cross-linking density has been typically limited by the chemical selectivity of the standard cross-linking reagents that are commonly used for protein cross-linking. We have implemented the use of a heterobifunctional cross-linking reagent, sulfosuccinimidyl 4,4′-azipentanoate (sulfo-SDA), combining a traditional sulfo-N-hydroxysuccinimide (sulfo-NHS) ester and a UV photoactivatable diazirine group. This diazirine yields a highly reactive and promiscuous carbene species, the net result being a greatly increased number of cross-links compared with homobifunctional, NHS-based cross-linkers. We present a novel methodology that combines the use of this high density photo-cross-linking data with conformational space search to investigate the structure of human serum albumin domains, from purified samples, and in its native environment, human blood serum. Our approach is able to determine human serum albumin domain structures with good accuracy: root-mean-square deviation to crystal structure are 2.8/5.6/2.9 Å (purified samples) and 4.5/5.9/4.8Å (serum samples) for domains A/B/C for the first selected structure; 2.5/4.9/2.9 Å (purified samples) and 3.5/5.2/3.8 Å (serum samples) for the best out of top five selected structures. Our proof-of-concept study on human serum albumin demonstrates initial potential of our approach for determining the structures of more proteins in the complex biological contexts in which they function and which they may require for correct folding. Data are available via ProteomeXchange with identifier PXD001692.High-resolution structures of proteins are essential for understanding cellular processes. Determining protein structures, however, is difficult: protein stability, purity, quantity, and solubility critically affect success. Nuclear magnetic resonance (NMR)¹ spectroscopy can only be applied to proteins of limited size, whereas x-ray crystallography necessitates prior crystallization of the protein. These conditions make structure determination challenging for many proteins of biological relevance. This includes especially proteins that contain intrinsically unstructured or long coiled-coil regions, proteins associated to a membrane (1, 2) or parts of multi-protein complexes (3). New developments to overcome some of these restrictions include x-ray free electron lasers (XFEL) (4), which only require microcrystals, new detectors in cryo-electron microscopy (5) and in-cell NMR (6), which analyzes the structure of small proteins in a cellular context. Further advancements that assist with protein structure determination have included the development of being able to use sparse NMR data, for example using backbone only data (7), and the understanding of evolutionary constraints for predicting protein structure (8).We present a novel approach to obtain structural details of proteins by mass spectrometry. This can be accomplished through cross-linking and mass spectrometry (CLMS) (9–11). Cross-links establish covalent bonds between residue pairs close in space but not necessarily in sequence. This conserves structural information throughout an analysis that follows the standard proteomics workflow. Typically, a bi-functional chemical reagent, the cross-linker, is incubated with a protein of interest. The cross-linker reacts with two residues—often involving the side-chain amine of lysine—that are near each other in the folded protein. A protease such as trypsin is used to degrade the protein. The resulting mix of cross-linked peptides is then analyzed by mass spectrometry and database searching akin to other shotgun proteomics approaches (12). The pairs of cross-linked residues are identified from the mass spectrometric data and provide information on which residues are near each other in the folded protein. This information is represented in the form of distance constraints, deducible from the length of the cross-linking agent.CLMS data has been used to study large multi-protein complexes (13), networks (14) and proteins in whole cells (15). The distance constraints obtained are sparse but complement other structural data in integrated structural biology well (10). Cross-link data allow placing high-resolution structures of individual sub-units in the electron microscopy structure of an assembled multi-protein complex to obtain its quasi-atomic resolution structure, e.g. the proteasome (16). In an alternative approach, genetic site-directed positioning of a photo-reactive group, azide, as part of a phenylalanine analog, was recently used to derive proximity information that allowed modeling of receptor CRF1R bound to its native ligand (17). Young et al. used 15 cross-links to identify the correct fold of bovine basic fibroblast growth factor using threading and homology modeling (18). In a similar study, Singh et al. used eight cross-links to build a monomer homology model of the major capsid protein E of bacteriophage lambda and to derive a pseudoatomic model of the lambda procapsid shell (19). In both of the aforementioned cases, the cross-link information was merely used to verify structural models by threading and homology modeling, and did not significantly impact model building. Prior attempts to leverage cross-linking data in structure determination delivered improvements, however, without leading to high-resolution models (20).Here, we increase the spatial resolution of information obtained through cross-linking by using a highly reactive chemical as a cross-linking agent. This broadens the specificity of cross-linking and thus increases the spatial resolution in conjunction with mass spectrometry. We employ the heterobifunctional chemical cross-linker sulfosuccinimidyl 4,4′-azipentanoate, sulfo-SDA (21), to chemically cross-link a protein, human serum albumin (HSA).We combine the distance constraints provided by cross-linking and mass spectrometry with computational, conformational space search. This approach allows us to generate structural models of HSA domains that correlate highly with the structure of HSA solved by x-ray crystallography. With this method, we show that our pipeline can be used to analyze the structure of HSA domains from HSA not only in it''s purified form, but additionally unpurified and in its native environment, human blood serum.