Complementary chimeric isoforms reveal Dscam1 binding specificity in vivo

Dscam1 potentially encodes 19,008 ectodomains of a cell recognition molecule of the immunoglobulin (Ig) superfamily through alternative splicing. Each ectodomain, comprising a unique combination of three variable (Ig) domains, exhibits isoform-specific homophilic binding in?vitro. Although we have proposed that the ability of Dscam1 isoforms to distinguish between one another is crucial for neural circuit assembly, via a process called self-avoidance, whether recognition specificity is essential in?vivo has not been addressed. Here we tackle this issue by assessing the function of Dscam1 isoforms with altered binding specificities. We generated pairs of chimeric isoforms that bind to each other (heterophilic) but not to themselves (homophilic). These isoforms failed to support self-avoidance or did so poorly. By contrast, coexpression of complementary isoforms within the same neuron restored self-avoidance. These data establish that recognition between Dscam1 isoforms on neurites of the same cell provides the molecular basis for self-avoidance.     

High-level production and covalent immobilization of Providencia rettgeri penicillin G acylase (PAC) from recombinant Pichia pastoris for the development of a novel and stable biocatalyst of industrial applicability

A complete, integrated process for the production of an innovative formulation of penicillin G acylase from Providencia rettgeri(rPAC(P.rett))of industrial applicability is reported. In order to improve the yield of rPAC, the clone LN5.5, carrying four copies of pac gene integrated into the genome of Pichia pastoris, was constructed. The proteinase activity of the recombinant strain was reduced by knockout of the PEP4 gene encoding for proteinase A, resulting in an increased rPAC(P.rett) activity of approximately 40% (3.8 U/mL vs. 2.7 U/mL produced by LN5.5 in flask). A high cell density fermentation process was established with a 5-day methanol induction phase and a final PAC activity of up to 27 U/mL. A single step rPAC(P.rett) purification was also developed with an enzyme activity yield of approximately 95%. The novel features of the rPAC(P.rett) expressed in P.pastoris were fully exploited and emphasized through the covalent immobilization of rPAC(P.rett). The enzyme was immobilized on a series of structurally correlated methacrylic polymers, specifically designed and produced for optimizing rPAC(P.rett) performances in both hydrolytic and synthetic processes. Polymers presenting aminic functionalities were the most efficient, leading to formulations with higher activity and stability (half time stability >3 years and specific activity ranging from 237 to 477 U/g (dry) based on benzylpenicillin hydrolysis). The efficiency of the immobilized rPAC(P.rett) was finally evaluated by studying the kinetically controlled synthesis of beta-lactam antibiotics (cephalexin) and estimating the synthesis/hydrolysis ratio (S/H), which is a crucial parameter for the feasibility of the process.     

Resistance of two planarian species to UV-irradiation

The aim of this work was to determine the effects of 20, 25 and 30 minute UV-irradiation periods lambda = 253.5 nm to two planarian species Dugesia tigrina (Gir.) and Polycelis felina (Daly.). In vivo, UV light effects have been reported to affect intracellular receptors and disrupt simple behaviour. The effects of UV-rays on mortality and behavior as well as morphological, cytological and histological changes in the two planarian species were assessed, and the course and the dynamics of regenerative processes were compared between them. Experimental populations of Dugesia tigrina and Polycelis felina species were maintained in laboratory conditions at room temperature. Mortality, behavioral and morphological changes were monitored daily by means of a light stereomicroscope. For cytological and histopathological analysis, planarians were fixed in Bouine fixative on the first, second, third, fifth and seventh day after exposure to UV-irradiation, respectively. They were embedded in paraffin, cut on a microtome, stained with toluidin blue and embedded in Canada-balsam. UV-rays caused mortality, behavioral, morphological, cytological and histological changes in each planarian species. In regeneration of damaged body parts reticular cells and neoblasts played the main role. Neoblasts as totipotent cells extremely increased in number in the area of damaged tissue, immediately after UV-exposure. Dugesia tigrina was more sensitive to UV-rays than Polycelis felina due to possession of less pigmented cells. The course of regeneration in both species was similar. Most individuals of both species regenerated in 5 to 12 days after UV-irradiation.     

Bilateral MR volumetry of the amygdala in chronic PTSD patients

Posttraumatic stress disorder (PTSD) patients experience symptoms which implicate dysfunction of emotional memory circuits, and possible damage of the amygdala. Laterality differences in activity of the amygdala have been reported in PTSD patients, with presumed adaptive plasticity in the hippocampus and the amygdala. The aim of this study was to investigate possible interhemispheric differences of amygdalar volume in chronic PTSD patients, with calculation of right-to-left volume ratios. Bilateral magnetic resonance (MR) volumetry was applied in 11 chronic PTSD patients. The mean right amygdalar volume of our patients was significantly smaller than the left one (p = 0.031), with the right-to-left volume ratio of 0.96 +/- 0.06. This tendency towards smaller right amygdala may be an acquired effect as a result of stress-induced plasticity, however we can not exclude the possibility of a predisposing condition.     

Endogenously nitrated proteins in mouse brain: links to neurodegenerative disease

Increased abundance of nitrotyrosine modifications of proteins have been documented in multiple pathologies in a variety of tissue types and play a role in the redox regulation of normal metabolism. To identify proteins sensitive to nitrating conditions in vivo, a comprehensive proteomic data set identifying 7792 proteins from a whole mouse brain, generated by LC/LC-MS/MS analyses, was used to identify nitrated proteins. This analysis resulted in the identification of 31 unique nitrotyrosine sites within 29 different proteins. More than half of the nitrated proteins that have been identified are involved in Parkinson's disease, Alzheimer's disease, or other neurodegenerative disorders. Similarly, nitrotyrosine immunoblots of whole brain homogenates show that treatment of mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an experimental model of Parkinson's disease, induces an increased level of nitration of the same protein bands observed to be nitrated in brains of untreated animals. Comparing sequences and available high-resolution structures around nitrated tyrosines with those of unmodified sites indicates a preference of nitration in vivo for surface accessible tyrosines in loops, a characteristic consistent with peroxynitrite-induced tyrosine modification. In addition, most sequences contain cysteines or methionines proximal to nitrotyrosines, contrary to suggestions that these amino acid side chains prevent tyrosine nitration. More striking is the presence of a positively charged moiety near the sites of nitration, which is not observed for non-nitrated tyrosines. Together, these observations suggest a predictive tool of functionally important sites of nitration and that cellular nitrating conditions play a role in neurodegenerative changes in the brain.     

Synthesis, characterization, and cytotoxicity of trimethylplatinum(IV) complexes with 2-thiocytosine and 1-methyl-2-thiocytosine ligands

The reaction of [PtMe₃(MeOH)(bpy)][BF₄] (1) with the thionucleobases 2-thiocytosine (SCy, 2) and 1-methyl-2-thiocytosine (1-MeSCy, 3) resulted in the formation of the complexes [PtMe₃(bpy)(SCy-κS)][BF₄] (4) and [PtMe₃(bpy)(1-MeSCy-κS)] [BF₄] (5), respectively. The complexes were characterized by ¹H and ¹³C NMR spectroscopy as well as by single-crystal X-ray analyses of 4 · MeOH and 5. In 4 · MeOH two strong hydrogen bonds (N4-H?N3′: N4?N3′ 2.976(7) Å) between the thiocytosine ligands give rise to base pairing thus forming dinuclear cations [{PtMe₃(bpy)(SCy-κS)}₂]²⁺. In both complexes the platinum atom is octahedrally coordinated [PtC₃N₂S] by three methyl ligands, the 2,2′-bipyridine ligand and the κS coordinated nucleobase (configuration index: OC-6-33). The structural investigations gave evidence that the sulfur atoms of the nucleobase ligands in 4 · MeOH and 5 have to be regarded as sp³ and sp² hybridized, respectively. Thus, the ligand in 4 · MeOH has to be considered as the deprotonated thiol-amino form of thiocytosine being reprotonated at N1. In complex 5 the 1-MeSCy is coordinated in its thione-amino form. DFT-calculations of the base-paired dinuclear cation in 4 as well as of 4 itself gave proof of the strength of the hydrogen bond (8.5 kcal/mol) and exhibited that cation-anion interactions influence the conformation of the complex. In vitro cytotoxicity studies of 4 and 5 using nine different human tumor cell lines revealed moderate cytotoxic activity.     

Solution Structure,Determined by Nuclear Magnetic Resonance,of the b30-82 Domain of Subunit b of Escherichia coli F1Fo ATP Synthase

Subunit b, the peripheral stalk of bacterial F₁F_o ATP synthases, is composed of a membrane-spanning and a soluble part. The soluble part is divided into tether, dimerization, and δ-binding domains. The first solution structure of b30-82, including the tether region and part of the dimerization domain, has been solved by nuclear magnetic resonance, revealing an α-helix between residues 39 and 72. In the solution structure, b30-82 has a length of 48.07 Å. The surface charge distribution of b30-82 shows one side with a hydrophobic surface pattern, formed by alanine residues. Alanine residues 61, 68, 70, and 72 were replaced by single cysteines in the soluble part of subunit b, b22-156. The cysteines at positions 61, 68, and 72 showed disulfide formation. In contrast, no cross-link could be formed for the A70C mutant. The patterns of disulfide bonding, together with the circular dichroism spectroscopy data, are indicative of an adjacent arrangement of residues 61, 68, and 72 in both α-helices in b22-156.ATP synthesis by oxidative phosphorylation or photophosphorylation is a multistep membrane-located process that provides the bulk of cellular energy in eukaryotes and many prokaryotes. The majority of ATP synthesis is accomplished by the enzyme ATP synthase (EC 3.6.1.34), also called F₁F_o ATP synthase, which, in its simplest form, as in bacteria, is composed of eight different subunits (α₃, β₃, γ, δ, ɛ, a, b₂, and c_9-12). This multisubunit complex is divided into the F₁ headpiece, α₃:β₃, and a membrane-embedded ion-translocating part known as F_o, to which F₁ is attached by a central and a peripheral stalk (1, 5, 25). ATP is synthesized or hydrolyzed on the α₃:β₃ hexamer, and the energy provided for or released during that process is transmitted to the membrane-bound F_o sector, consisting of subunits a and c and part of subunit b (30, 31). The energy coupling between the two active domains occurs via the stalk part(s) (6). The central stalk is made of subunits γ and ɛ, and the peripheral stalk is formed by subunits δ and b. The peripheral stalk, which lies at the edge of the multisubunit assembly of the F₁F_o ATP synthase, acts as a stator to counter the tendency of the α₃:β₃ hexamer to follow the rotation of the central stalk and the attached c-ring, and to anchor the membrane-embedded a subunit (17, 36).In Escherichia coli, subunit b with its 156 residues extends with its soluble part (b_sol; b21-156) from the top of the F₁ sector down, into, and across the membrane, where it is associated with subunit a (2, 15, 32, 34). The 156-residue b subunit has been divided into four functional domains (28). They are, in order from the N to the C terminus; the membrane domain, the tether region, the dimerization domain, and the δ-binding domain. The structure of the synthesized 33-residue peptide comprising the N-terminal membrane-spanning region has been solved by ¹H NMR, showing an α-helical feature (14). The crystallographic structure of the major part of the dimerization domain, b62-122, revealed an α-helix with a length of 9.0 nm (12). Most recently, the NMR solution structure of the very C-terminal segment, b140-156, which interacts with the C terminus of subunit δ (δ91-177), has been determined by NMR spectroscopy (26). This molecule adopts a stable helix formation in solution with a flexible tail between amino acid residues 140 and 145. SAXS (26) and analytical ultracentrifuge studies have indicated that the soluble domain of subunit b (b21-156, b22-156) is dimeric in solution (12). So far, no high-resolution structure of the tether domain, including residues 25 to 52, or the N-terminal segment of the dimerization domain, which is formed by residues 53 to 122, is available (14).Here, we have turned our attention to the production and purification of residues 30 to 82 of subunit b (b30-82) from E. coli F₁F_o ATP synthase, which forms the remaining unsolved structural segment of subunit b. The structural features of this segment have been determined in solution using NMR spectroscopy. The introduction of a cysteine residue into b22-156 at four positions resulted in different intersubunit disulfide patterns, giving insight into the proximity of the residues.     

Job satisfaction among medical doctors in one of the countries in transition: experience from Croatia

Our aim was to explore and compare the job satisfaction between family physicians and hospital specialists in Split, Croatia. The survey was carried out in 2005 and 2006. A validated questionnaire was composed of two parts: 92 statements and questions about job satisfaction in the form of a Lickert scale (range 1-5) and eight questions concerning demographic issues. The questionnaire was completed and returned by 165 hospital specialists from the University Hospital and by 131 family physicians from the Split County. Response rate for family physicians was 39.81% and 41.46% for hospital specialists. Hospital doctors were divided in two groups: internal and surgical. There were no significant differences between family physicians and hospital specialists in total job satisfaction (F = 1.02; p = 0.41). Family physicians were more satisfied with their workplace conditions than internal medicine specialists (19.37 +/- 4.23 vs. 17.37 +/- 4.59; F = 5.93; p = 0.003), and less satisfied with the possibilities for postgraduate training than surgeons (5.27 +/- 1.90 vs. 6.59 +/- 2.07; F = 9.26; p < 0.001). Global job satisfaction was rather low but does not differ between the three medical groups. Disparities were observed in some segments (opportunity for further training and academic advancement, vacation, and salary). The reason for the family physician's relative satisfaction may be due to stable working conditions, independence in organizing work schedules and personal responsibility.     

Ultrasound evaluation of extracranial carotid artery lesions in Parkinsonian patients

The purpose of this investigation was to determine the atherosclerotic changes in patients with vascular parkinsonism and in patients with idiopathic Parkinson's disease, in order to evaluate the possible influence of the extracranial pathology of carotid arteries in developing lacunar cerebral infarcts. Degree of stenosis and plaque morphology of the extracranial part of carotids in both group of patients were evaluated by color Doppler flow imaging ultrasound investigation and the results were compared. We selected two matched groups of patients with parkinsonism: 22 patients with vascular parkinsonism, and 28 with idiopathic Parkinson's disease.The atherosclerotic changes found in patients with Parkinson's disease showed mild carotid lesions with mostly stable calcified plaques and lesser risk for embolic cerebral intravascular events contrary to the higher degree of carotid stenosis found in patients with vascular parkinsonism with mostly mixed plaques prone to embolization. Therefore, we suggest performing ultrasonographic examination of the extracranial part of carotid arteries in all patients with parkinsonism to assess risk of vascular accidents originating from carotid lesions. That would enable adequate treatment of parkinsonism and prevent further occurrence of intracranial vascular changes.