Structure and denaturation of 4-chlorobenzoyl coenzyme A dehalogenase from <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. strain TM-1

The secondary structure of the trimeric protein 4-chlorobenzoyl coenzyme A dehalogenase from Arthrobacter sp. strain TM-1, the second of three enzymes involved in the dechlorination of 4-chlorobenzoate to form 4-hydroxybenzoate, has been examined. E_mM for the enzyme was 12.59. Analysis by circular dichroism spectrometry in the far uv indicated that 4-chlorobenzoyl coenzyme A dehalogenase was composed mostly of α-helix (56%) with lesser amounts of random coil (21%), β-turn (13%) and β-sheet (9%). These data are in close agreement with a computational prediction of secondary structure from the primary amino acid sequence, which indicated 55.8% α-helix, 33.7% random coil and 10.5% β-sheet; the enzyme is, therefore, similar to the 4-chlorobenzoyl coenzyme A dehalogenase from Pseudomonas sp. CBS-3. The three-dimensional structure, including that of the presumed active site, predicted by computational analysis, is also closely similar to that of the Pseudomonas dehalogenase. Study of the stability and physicochemical properties revealed that at room temperature, the enzyme was stable for 24 h but was completely inactivated by heating to 60°C for 5 min; thereafter by cooling at 1°C min⁻¹ to 45°C, 20.6% of the activity could be recovered. Mildly acidic (pH 5.2) or alkaline (pH 10.1) conditions caused complete inactivation, but activity was fully recovered on returning the enzyme to pH 7.4. Circular dichroism studies also indicated that secondary structure was little altered by heating to 60°C, or by changing the pH from 7.4 to 6.0 or 9.2. Complete, irreversible destruction of, and maximal decrease in the fluorescence yield of the protein at 330–350 nm were brought about by 4.5 M urea or 1.1 M guanidinium chloride. Evidence was obtained to support the hypothetical three-dimensional model, that residues W140 and W167 are buried in a non-polar environment, whereas W182 appears at or close to the surface of the protein. At least one of the enzymes of the dehalogenase system (the combined 4-chlorobenzoate:CoA ligase, the dehalogenase and 4-hydroxybenzoyl coenzyme A thioesterase) appears to be capable of association with the cell membrane.

A reexamination of the propensities of amino acids towards a particular secondary structure: classification of amino acids based on their chemical structure

The correlation between the primary and secondary structures of proteins was analysed using a large data set from the Protein Data Bank. Clear preferences of amino acids towards certain secondary structures classify amino acids into four groups: α-helix preferrers, strand preferrers, turn and bend preferrers, and His and Cys (the latter two amino acids show no clear preference for any secondary structure). Amino acids in the same group have similar structural characteristics at their Cβ and Cγ atoms that predicts their preference for a particular secondary structure. All α-helix preferrers have neither polar heteroatoms on Cβ and Cγ atoms, nor branching or aromatic group on the Cβ atom. All strand preferrers have aromatic groups or branching groups on the Cβ atom. All turn and bend preferrers have a polar heteroatom on the Cβ or Cγ atoms or do not have a Cβ atom at all. These new rules could be helpful in making predictions about non-natural amino acids.

Mechanism of zinc(II)-promoted amyloid formation: zinc(II) binding facilitates the transition from the partially α-helical conformer to aggregates of amyloid β protein(1–28)

The amyloidoses are a group of disorders characterized by aberrant protein folding and assembly, leading to the deposition of insoluble protein fibrils (amyloid), which provokes cell dysfunction and later cell death. One of the physiologically relevant environmental factors able to affect the conformation and hence the aggregation properties of amyloidogenic proteins/peptides is metal ions. Zn(II) promotes aggregation of most amyloidogenic peptides/proteins in vitro, including amyloid β protein (Aβ), but the underlying mechanism is not known. To better understand this mechanism the present study focused on the partially α-helical conformer, supposed to be an intermediate in Aβ aggregation. This partially α-helical conformer is stabilized by 10–20% 2,2,2-trifluoroethanol (TFE): therefore, the influence of Zn binding on the aggregation of the amylidogenic model peptide Aβ(1–28) (Aβ28) was investigated at different TFE concentrations. The results showed a synergistic effect of Zn(II) and 10% TFE, i.e., that either Zn or 10% TFE accelerated Aβ28 aggregation on its own, but with them together an at least 10 times promotion of Aβ28 aggregation was observed. Further studies by thioflavin T fluorescence spectroscopy, transmission electron microscopy, and circular dichroism (CD) spectroscopy suggested that the aggregates of Zn-Aβ28 formed in 10%TFE contain a β-sheet secondary structure and are more of the amyloid type. CD spectroscopy indicated that Zn binding disrupted partially the α-helical structure of Aβ28 in TFE. Thus, we propose that the promotion of Aβ28 aggregation by Zn is based on the transformation of the partially α-helical conformer (intermediate) towards the β-sheet amyloid structure by a destabilization of the α-helix in the intermediate. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Endothelial and virgultar cell formations in the mammalian lymph node sinus: endothelial differentiation morphotypes characterized by a special kind of junction (<Emphasis Type="Italic">complexus adhaerens</Emphasis>)

The lymph node sinus are channel structures of unquestionable importance in immunology and pathology, specifically in the filtering of the lymph, the transport and processing of antigens, the adhesion and migration of immune cells, and the spread of metastatic cancer cells. Our knowledge of the cell and molecular biology of the sinus-forming cells is still limited, and the origin and biological nature of these cells have long been a matter of debate. Here, we review the relevant literature and present our own experimental results, in particular concerning molecular markers of intercellular junctions and cell differentiation. We show that both the monolayer cells lining the sinus walls and the intraluminal virgultar cell meshwork are indeed different morphotypes of the same basic endothelial cell character, as demonstrated by the presence of a distinct spectrum of general and lymphatic endothelial markers, and we therefore refer to these cells as sinus endothelial/virgultar cells (SEVCs). These cells are connected by unique adhering junctions, termed complexus adhaerentes, characterized by the transmembrane glycoprotein VE-cadherin, combined with the desmosomal plaque protein desmoplakin, several adherens junction plaque proteins including α- and β-catenin and p120 catenin, and components of the tight junction ensemble, specifically claudin-5 and JAM-A, and the plaque protein ZO-1. We show that complexus adhaerentes are involved in the tight three-dimensional integration of the virgultar network of SEVC processes along extracellular guidance structures composed of paracrystalline collagen bundle “stays”. Overall, the SEVC system might be considered as a local and specific modification of the general lymphatic vasculature system. Finally, physiological and pathological alterations of the SEVC system will be presented, and the possible value of the molecular markers described in histological diagnoses of autochthonous lymph node tumors will be discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Practical Considerations When Using Temperature to Obtain Rate Constants and Activation Thermodynamics of Enzymes with Two Catalytic Steps: Native and N460T-β-Galactosidase (<Emphasis Type="Italic">E. coli</Emphasis>) as Examples

The values of the rate constants and the associated enthalpies and entropies of enzymes with two catalytic steps can be measured by determining the effects of temperature on the k _cat values. Practical considerations that should be taken into account when doing this are presented. The narrow temperature range available with enzymes and the sensitivity of pH to temperature mean that special attention to detail must be taken and this study highlights the assiduousness needed. The necessity of conversion of apparent k _cat to true k _cat values when assays are done with products having pKa values near to the assay pH is shown and the importance of obtaining sufficient data is emphasized. Reasons that non-linear regression should be used to obtain the estimates of rate constants and activation thermodynamic parameters are given. Other precautions and recommendations are also presented. Results obtained by this method for native β-galactosidase (E. coli) and for a β-galactosidase in which a Thr was substituted for Asn-460 were analyzed to demonstrate the valuable mechanistic details of enzymes that can be obtained from studies of this type.

Calculation of energy-deposition distributions and microdosimetric estimation of the biological effect of a <Superscript>9</Superscript>C beam

Among the alternative beams being recently considered for external cancer radiotherapy, ⁹C has received some attention because it is expected that its biological effectiveness could be boosted by the β-delayed emission of two α particles and a proton that takes place at the ion-stopping site. Experiments have been performed to characterise this exotic beam physically and models have been developed to estimate quantitatively its biological effect. Here, the particle and heavy-ion transport code system ( PHITS ) is used to calculate energy-deposition and linear energy transfer distributions for a ⁹C beam in water and the results are compared with published data. Although PHITS fails to reproduce some of the features of the distributions, it suggests that the decay of ⁹C contributes negligibly to the energy-deposition distributions, thus contradicting the previous interpretation of the measured data. We have also performed a microdosimetric calculation to estimate the biological effect of the decay, which was found to be negligible; previous microdosimetric Monte–Carlo calculations were found to be incorrect. An analytical argument, of geometrical nature, confirms this conclusion and gives a theoretical upper bound on the additional biological effectiveness of the decay. However, no explanation can be offered at present for the observed difference in the biological effectiveness between ⁹C and ¹²C; the reproducibility of this surprising result will be verified in coming experiments.

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Type 1 ribosome-inactivating proteins from <Emphasis Type="Italic">Phytolacca dioica</Emphasis> L. leaves: differential seasonal and age expression,and cellular localization

The expression of type 1 ribosome-inactivating proteins (RIPs) in Phytolacca dioica L. leaves was investigated. Fully expanded leaves of young P. dioica plants (up to 3 years old) expressed two novel RIPs, dioicin 1 and dioicin 2. The former was also found in developing leaves from adult P. dioica within about two and a half weeks after leaf development, and the latter continuously synthesized, with no seasonal or ontogenetic constraint. Fully expanded leaves from adult P. dioica expressed four RIPs (PD-Ls1–4) exhibiting seasonal variation. RIPs were localized in the extracellular space, in the vacuole and in the Golgi apparatus of mesophyll cells. Dioicin 1 and dioicin 2 showed rRNA N-β-glycosidase activity and displayed the following properties, respectively: (1) Mr values of 30,047.00 and 29,910.00, (2) pIs of 8.74 and 9.37, and (3) IC₅₀ values of 19.74 (0.658 nM) and 6.85 ng/mL (0.229 nM). Furthermore, they showed adenine polynucleotide glycosylase activity and nicked pBR322 dsDNA. The amino acid sequence of dioicin 2 had 266 amino acid residues, and the highest percentage identity (81.6%) and similarity (84.6%) with PAP-II from Phytolacca americana, while its identity with other RIPs from Phytolaccaceae was around 40%. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Can copper binding to the prion protein generate a misfolded form of the protein?

The native prion protein (PrP) has a two domain structure, with a globular folded α-helical C-terminal domain and a flexible extended N-terminal region. The latter can selectively bind Cu²⁺ via four His residues in the octarepeat (OR) region, as well as two sites (His96 and His111) outside this region. In the disease state, the folded C-terminal domain of PrP undergoes a conformational change, forming amorphous aggregates high in β-sheet content. Cu²⁺ bound to the ORs can be redox active and has been shown to induce cleavage within the OR region, a process requiring conserved Trp residues. Using computational modeling, we have observed that electron transfer from Trp residues to copper can be favorable. These models also reveal that an indole-based radical cation or Cu⁺ can initiate reactions leading to protein backbone cleavage. We have also demonstrated, by molecular dynamics simulations, that Cu²⁺ binding to the His96 and His111 residues in the remaining PrP N-terminal fragment can induce localized β-sheet structure, allowing us to suggest a potential mechanism for the initiation of β-sheet misfolding in the C-terminal domain by Cu²⁺.

Purification and properties of Sandercyanin, a blue protein secreted in the mucus of blue forms of walleye, Sander vitreus

A blue protein present in the mucus coating blue forms of walleye, Sander vitreus, was purified to homogeneity by a combination of ion exchange and hydrophobic interaction chromatography. The purified protein has a molecular mass of 87,850 and is a homotetramer with a subunit molecular mass of 21,836. Solutions of the protein are deep blue in color and show absorbance maxima at 383 and 633 nm, respectively. Acetone treatment of the protein releases a blue chromophore with the spectral characteristics of biliverdin IXα. The results suggest that the blue protein is a new biliprotein that we have termed Sandercyanin. The function of Sandercyanin is unknown, but limited amino acid sequence analysis suggests it is a lipocalin that may be involved in the transport of heme degradation products.

Revealing the anti-HRP epitope in <Emphasis Type="Italic">Drosophila</Emphasis> and <Emphasis Type="Italic">Caenorhabditis</Emphasis>

Antibodies are very often used as specific cell and/or tissue markers. An example of this is anti-horseradish peroxidase (HRP), an antibody raised against a plant glycoprotein, which was shown some twenty-five years ago to specifically stain neural tissue in an animal, Drosophila melanogaster. This peculiar finding was later expanded to other invertebrate species including Caenorhabditis elegans, which were also shown to bear anti-HRP epitopes. Initial experiments indicated that the epitopes recognised by anti-HRP in invertebrates are of carbohydrate nature. Indeed, more recent experiments have characterised relevant core α1-3-fucosylated N-glycan structures that act as epitopes in various model and parasitic organisms. Moreover, a number of enzymes required for the synthesis of such structures have been identified. Over the years, medically-relevant roles of these structures have become apparent as regards allergenicity and immunoregulation. Although major advances have been made in understanding of the underlying mechanisms and structures related to the anti-HRP epitope, the in vivo role of the relevant epitopes in neural and other tissues is yet to be resolved. Current understanding of the anti-HRP epitopes synthesis and their relevance is discussed and elaborated.

The genomic basis of cerebral palsy: a HuGE systematic literature review

Cerebral palsy has been associated with a number of candidate genes. To date, no systematic review has been conducted to synthesise genetic polymorphism associations with cerebral palsy. We apply the HuGE NET guidelines to search PubMed and EMBASE databases for publications investigating single nucleotide polymorphisms (SNPs) and cerebral palsy outcome. 22 papers were identified and are discussed in this review. Candidate genes were grouped as (1) thrombophilic, (2) cytokine, (3) apolipoprotein E or (4) other SNPs, largely related to cardiovascular physiology/pathophysiology and the functioning of the immune system. Of the studies identified, cohorts were usually small, without adequate control and ethnically diverse, making direct comparison between studies difficult. The most promising candidate genes include factor V Leiden, methylenetetrahydrofolate reductase, lymphotoxin-α, tumour necrosis factor-α, eNOS and mannose binding lectin. Large case–control studies are needed to confirm these candidates with attention given to cohort ethnicity, cerebral palsy subtype analysis and possible multiple gene and gene–environment interactions.

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Development of the rhopalial nervous system in <Emphasis Type="Italic">Aurelia</Emphasis> sp.1 (Cnidaria,Scyphozoa)

We examined the development of the nervous system in the rhopalium, a medusa-specific sensory structure, in Aurelia sp.1 (Cnidaria, Scyphozoa) using confocal microscopy. The rhopalial nervous system appears primarily ectodermal and contains neurons immunoreactive to antibodies against tyrosinated tubulin, taurine, GLWamide, and FMRFamide. The rhopalial nervous system develops in an ordered manner: the presumptive gravity-sensing organ, consisting of the lithocyst and the touch plate, differentiates first; the “marginal center,” which controls swimming activity, second; and finally, the ocelli, the presumptive photoreceptors. At least seven bilaterally arranged neuronal clusters consisting of sensory and ganglion cells and their neuronal processes became evident in the rhopalium during metamorphosis to the medusa stage. Our analysis provides an anatomical framework for future gene expression and experimental studies of development and functions of scyphozoan rhopalia. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Personal Experience of Schizophrenia and the Role of <Emphasis Type="Italic">Danwei</Emphasis>: A Case Study in 1990s Beijing

Danwei as a cornerstone of Chinese urban society has received great research attention. The relationship between the Danwei and psychiatric patients, however, remains unclear. This article aims to shed light on the subject with an integrated micro–macro approach. It introduces a historical understanding of mental health in urban China under the “economic state in transition” framework. A detailed case study in clinical sociology is provided to reveal the many social factors affecting the experience of a schizophrenic patient and his significant others. A changing role of the Danwei is hypothesized and validated with qualitative data. The Danwei was shown to have changed significantly before the mid-1990s, yet it still played a major role in urban workers’ lives, including those of psychiatric patients, and even more so in people’s expectations. This lends support to the need for a more responsive public policy to address various social issues brought about by economic reform, with the learning of worldwide experiences including “community care,” “social support” and “social rehabilitation.” Implications for social research, policymaking and professional practice are discussed.

Entomopathogenic fungi as a potential control agent against the lesser mealworm, <Emphasis Type="Italic">Alphitobius diaperinus</Emphasis> in broiler houses

We tested the pathogenicity of 18 Metarhizium anisopliae (Metschn.) Sorokin isolates and 22 Beauveria bassiana (Balsamo) Vuillemin isolates against Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) larvae and adults. The efficacy of the most virulent isolate—M. anisopliae K—was evaluated in containers with a concrete bottom covered with wood shavings, under simulated poultry house conditions. Application of conidia of this isolate to the shavings or directly to the concrete bottom reduced the yield of larvae in 8–15 time compared with the control. In another test, the mortality of mature larvae placed on previously inoculated shavings or bottom reached 80–90% within 14 days, compared with 14% in the control. The residual activity of conidia kept at 28°C retained its initial level during 14 days post-inoculation, but declined after three weeks. Based on our data M. anisopliae has considerable potential for the control of A. diaperinus.

Membrane microdomains from early gastrula embryos of medaka, <Emphasis Type="Italic">Oryzias latipes</Emphasis>, are a platform of E-cadherin- and carbohydrate-mediated cell–cell interactions during epiboly

Formation of membrane microdomain is critical for cell migration (epiboly) during gastrulation of medaka fish [Adachi et al. (Biochem. Biophys. Res. Commun. 358:848–853, 2007)]. In this study, we characterized membrane microdomain from gastrula embryos to understand its roles in epiboly. A cell adhesion molecule (E-cadherin), its associated protein (β-catenin), transducer proteins (PLCγ, cSrc), and a cytoskeleton protein (β-actin) were enriched in the membrane microdomain. Le^X-containing glycolipids and glycoproteins (Le^X-gp) were exclusively enriched in the membrane microdomain. Interestingly, the isolated membrane microdomain had the ability to bind to each other in the presence of Ca²⁺. This membrane microdomain binding was achieved through the E-cadherin homophilic and the Le^X-glycan-mediated interactions. E-cadherin and Le^X-gp were co-localized on the same membrane microdomain, suggesting that these two interactions are operative at the same time. Thus, the membrane microdomain functions as a platform of the E-cadherin- and Le^X-glycan-mediated cell adhesion and signal transduction.

Glycoconjugate histochemistry of the digestive tract of <Emphasis Type="Italic">Triturus carnifex</Emphasis> (Amphibia,Caudata)

Summary In this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal β1,3 GalNAc and (GlcNAc β1,4) _n oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal β1-3 GalNAc, Gal-αGal, and (GlcNAc β1,4) _n oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal β1,3 GalNAc, (GlcNAc β1,4) _n oligomers and fucose linked α1,6 or terminal α1,3 or α1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-αGal, Gal β1-3 GalNAc and (GlcNAc β1,4) _n oligomers; Fuc linked α1,2 to Gal, α1,3 to GlcNAc in (poly)lactosamine chains and α1,6 to GlcNAc in N-linked glycans. Most of these glycoproteins probably corresponds to the H⁺K⁺-ATPase β-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose, (GlcNAc β1,4) _n oligomers and fucose linked α1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier.     

Scanning force microscopy studies of X-ray-induced double-strand breaks in plasmid DNA

We present an analysis of X-ray-induced damage in ΦX174 plasmid DNA, applying doses between D = 250 and 1,500 Gy. To analyse this damage in detail, the distribution of plasmid fragments after irradiation have been determined by scanning force microscopy. The results show that even for the lowest dose of D = 250 Gy, a significant amount of double-strand breaks are observed. For increasing dose, the percentage of small fragments increases and is accompanied by a shortening of the average fragment length from < L> = 1,400 nm for a dose of D = 250 Gy to < L> = 1,080 nm after irradiation with D = 1,500 Gy. The most crucial parameter, the average number of double-strand breaks per broken plasmid (<DSB_b> ) has been determined for the first time for the applied doses. The results show that the average number of DSBs per broken plasmid <DSB_b> increases almost linearly from a value of <DSB_b> = 1.3 after irradiation with D = 250 Gy to <DSB_b> = 1.7 after exposure to D = 1,500 Gy. The presented results show that the amount of DSBs induced by X-ray radiation in plasmid DNA can be calculated with high accuracy by means of scanning force microscopy, providing relevant information regarding the interaction of X-rays with DNA molecules.