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The cellular trafficking pathways that conduct zinc to its sites of binding in functional proteins remain largely unspecified. In this study, the hypothesis was investigated that nonspecific proteomic binding sites serve as intermediates in zinc trafficking. Proteome from pig kidney LLC-PK1 cells contains a large concentration of such sites, displaying an average conditional stability constant of 1010-11, that are dependent on sulfhydryl ligands to achieve high-affinity binding of zinc. As a result, the proteome competes effectively with induced metallothionein for Zn2+ upon exposure of cells to extracellular Zn2+ or during in vitro direct competition. The reaction of added Zn2+ bound to proteome with apo-carbonic anhydrase was examined as a potential model for intracellular zinc trafficking. The extent of this reaction was inversely dependent upon proteome concentration and under cellular conditions thought to be negligible. The rate of reaction was strictly first order in both Zn2+ and apo-carbonic anhydrase, and also considered to be insignificant in cells. Adding the low molecular weight fraction of cell supernatant to the proteome markedly enhanced the speed of this reaction, a phenomenon dependent on the presence of glutathione (GSH). In agreement, inclusion of GSH accelerated the reaction in a concentration-dependent manner. The implications of abundant high-affinity binding sites for Zn2+ within the proteome are considered in relation to their interaction with GSH in the efficient delivery of Zn2+ to functional binding sites and in the operation of fluorescent zinc sensors as a tool to observe zinc trafficking.

A novel Schiff base fluorescent probe 7-Hydroxy-8-(((2-(hydroxymethyl)quinolin-8-yl)imino)methyl)-coumarin (XL) consist of formylcoumarin and aminoquinoline moieties was synthesized for dual detection of Zn

Hemagglutinin (HA) is a transmembrane protein of the influenza A virus and a key component in its life cycle. The protein allows the virus to enter a host cell by recognizing specific glycans attached to transmembrane proteins of the host, which leads to viral endocytosis. In recent years, significant progress has been made in understanding the structural relationship between changes in the HA receptor-binding site (RBS) and the sialylated glycans that bind them. Several mutations were identified in the HA RBS that allows the virus to change host tropism. Their impact on binding the analogs of human and avian receptors was determined with X-ray crystallography. In this article, we provide a short overview of the HA protein structure and briefly discuss the adaptive mutations introduced to different HA subtypes.

Microfluidic paper-based analytical devices (μPADs) fabricated by wax-printing are suitable platforms for the development of simple and affordable molecular diagnostic assays for infectious diseases, especially in resource-limited settings. Paper devices can be modified for biological assays by adding appropriate reagents to the test areas. For this purpose, the use of affinity immobilization strategies can be a good solution for bioactive paper fabrication. This paper describes a methodology to capture labeled-DNA strands and hybrids on paper via the anchoring of antibodies with a fusion protein that combines a family 3 carbohydrate binding module (CBM) from Clostridium thermocellum, with high affinity to cellulose, and the ZZ fragment of the staphyloccocal protein A, which recognizes IgG antibodies via their Fc portion. Antibodies immobilized via CBM-ZZ were able to capture appropriately labeled (biotin, fluorescein) DNA strands and DNA hybrids. The ability of an antibody specific to biotin to discriminate complementary from noncomplementary, biotin-labeled targets was demonstrated in both spot and microchannel assays. Hybridization was detected by fluorescence emission of the fluorescein-labeled DNA probe. The efficiency of the capture of labeled-DNA by antibodies immobilized on paper via the CBM-ZZ construct was significantly higher when compared with a physical adsorption method where antibodies were simply spotted on paper without the intermediation of other molecules. The experimental proof of concept of wax-printed μPADs functionalized with CBM-ZZ for DNA detection at room temperature presented in this study constitutes an important step toward the development of easy to use and affordable molecular diagnostic tests.

International audience; Mutagenesis experiments suggest that Asp79 in cellulase Cel6A (E2) from Thermobifida fusca has a catalytic role, in spite of the fact that this residue is more than 13 Å from the scissile bond in models of the enzyme–substrate complex built upon the crystal structure of the protein. This suggests that there is a substantial conformational shift in the protein upon substrate binding. Molecular mechanics simulations were used to investigate possible alternate conformations of the protein bound to a tetrasaccharide substrate, primarily involving shifts of the loop containing Asp79, and to model the role of water in the active site complex for both the native conformation and alternative low-energy conformations. Several alternative conformations of reasonable energy have been identified, including one in which the overall energy of the enzyme–substrate complex in solution is lower than that of the conformation in the crystal structure. This conformation was found to be stable in molecular dynamics simulations with a cellotetraose substrate and water. In simulations of the substrate complexed with the native protein conformation, the sugar ring in the –1 binding site was observed to make a spontaneous transition from the 4C1 conformation to a twist-boat conformer, consistent with generally accepted glycosidase mechanisms. Also, from these simulations Tyr73 and Arg78 were found to have important roles in the active site. Based on the results of these various MD simulations, a new catalytic mechanism is proposed. Using this mechanism, predictions about the effects of changes in Arg78 were made which were confirmed by site-directed mutagenesis.

Background—Although a large number of allergens have been characterised, the structural, functional, and biochemical features that these molecules have in common, and that could explain their ability to elicit powerful IgE antibody responses, are still uncertain. Recently, there has been considerable interest in the role of the cysteine protease activity of the house dust mite allergen Der p 1 in biasing the immune response in favour of IgE production. Aims—To search for remote homologues of Der p 1 with sequences similar to the 30 conserved amino acids surrounding the catalytic cysteine residue (Cys34). Methods—Potential homologues were analysed by examining their three dimensional structures and multiple sequence alignments using the programs PROPSEARCH, ClustalW, GeneDoc, and Swiss Pdb Viewer. Results—Diverse allergens (for example, the plant cysteine protease papain, the transport protein lipocalin Mus m 1, and the ragweed allergen Amb a 5) have a similar structural motif; namely, a groove resembling the substrate binding groove of Der p 1. The groove is located inside an α–β motif, between an α helix on one side and an antiparallel β sheet on the other side. A similar common motif (a cysteine stabilised α–β fold) can also be found in some toxins and defensins. Conclusion—Allergens of diverse sources have a common structural motif, namely a groove located inside an α–β motif, which could potentially serve as a ligand binding site.

Background—Connective tissue growth factor (CCN2; CTGF) is a newly identified growth factor, which is involved in the regulation of wound repair and fibrosis. Because there is variation among individuals with respect to tissue response to injury, genetic factors might be involved in the final outcome of tissue repair or scarring. For example, polymorphisms in the promoter region of genes, such as those encoding transforming growth factor β1 (TGF-β1), interleukin 10 (IL-10), and tumour necrosis factor α (TNF-α), influence transcriptional responses and are thought to contribute to the dysregulation of these genes in pathological conditions. Aim—To investigate whether the promoter region of the ccn2 (ctgf) gene contains polymorphic sequences that might account for differential expression. Materials/Methods—Seventy seven human DNA samples were sequenced—45 were from healthy controls and 32 were from patients with ischaemic heart disease (IHD)—using M13 tailed sequence specific ccn2 (ctgf) primers for amplification of a 600 bp fragment upstream of the transcription start site. Amplicons were bidirectionally sequenced with a dye primer M13 forward and reverse sequencing kit. Results—A C to G substitution was identified at position −132 in one of the patients with IHD. Moreover, in five of the 32 patients with IHD and in six of the 45 healthy controls, a G to C polymorphism was found at position −447. These substitutions at −132 and −447 are thought to lie within predicted binding domains for the transcription factors Pbx-1 and MZF1, respectively. In addition, insertions at position −43 (G), −47 (C), −71 (G) and a C to T substitution at position −198 were found in all DNA samples compared with the published ccn2 (ctgf) promoter sequence. These corrections do not involve sequences predicted to function as transcription factor binding sites. Conclusion—Sequence analysis of the ccn2 (ctgf) promoter of 77 human DNA samples has revealed corrections and polymorphic sites. The latter lie within putative regulatory elements.

Whatman 3MM paper was chemically modified to generate nickel-charged iminodiacetic acid paper (Ni2+-IDA paper). Bacteria were transformed with Escherichia coli expression plasmids coding for either unmodified proliferating cell nuclear antigen (PCNA) or PCNA containing a genetically engineered polyhistidine tract (his-tag) located at its NH2 terminus. They were then grown, induced, and lysed, and macromolecules were transferred to Ni2+-IDA paper. After exhaustive washing, his-tagged PCNA but not unmodified PCNA remained bound to the paper. Moreover, bound his-tagged PCNA was biochemically active in an in situ DNA synthesis assay with exogenous template-primer and purified calf thymus DNA polymerase delta (pol delta). Ni2+-IDA paper was used to identify a PCNA- point mutant that, relative to wild-type PCNA, promotes increased DNA synthesis by pol delta beyond a model abasic template site. In addition, metal-charged IDA paper promises to be generally useful for functional screening of cells expressing cloned proteins.

Background: 123 I-labelled 2β-carbomethoxy-3β-(4-iodophenyl)-N-(3-fluoropropyl) nortropane ( 123 I-FP-CIT) imaging is a diagnostic tool to help differentiate dementia with Lewy bodies (DLB) from Alzheimer’s disease (AD). However, in animals, cholinesterase inhibitors (ChEi) have been reported to reduce radioligand binding to the striatal dopamine transporter. As ChEi are frequently used in people with dementia, it is important to determine whether their use affects 123 I-FP-CIT uptake in the striatum. Objective: To clarify whether chronic ChEi therapy modulates striatal dopamine transporter binding measured by 123 I-FP-CIT in patients with AD, DLB and Parkinson’s disease with dementia (PDD). Design: Cross sectional study in 99 patients with AD (nine on ChEi, 25 not on ChEi), DLB (nine on ChEi, 19 not on ChEi) and PDD (six on ChEi, 31 not on ChEi) comparing 123 I-FP-CIT striatal binding (caudate, anterior and posterior putamen) in patients receiving compared with those not receiving ChEi, correcting for key clinical variables including diagnosis, age, sex, Mini-Mental State Examination score, severity of parkinsonism and concurrent antidepressant use. Results: As previously described, 123 I-FP-CIT striatal uptake was lower in DLB and PDD subjects compared with those with AD. Median duration of ChEi use was 180 days. 123 I-FP-CIT uptake was not significantly reduced in subjects receiving ChEi compared those not receiving ChEi (mean percentage reduction: AD 4.3%; DLB 0.7%; PDD 6.1%; p = 0.40). ChEi use did not differentially affect striatal 123 FP-CIT uptake between patient groups (p = 0.83). Conclusions: Use of ChEi does not significantly influence the ability of 123 I-FP-CIT imaging to distinguish AD from DLB.

Background: Primary progressive multiple sclerosis (PP-MS) differs from relapsing-remitting or secondary progressive MS (RR/SP-MS) in ways suggesting differences in the pathogenic pathways. Susceptibility to both PP-MS and RR/SP-MS is linked to carriage of the HLA molecule DRB1*1501. Several serologically defined HLA-DR groups (DR1, DR4, DR6, and DR9) occur less often in RR/SP-MS than in controls. Some or all of the HLA-DR molecules encoded by alleles in these serologically defined groups have a negatively charged glutamic acid at residue 71 or 74 of the beta 1 chain (beta 171 / beta 174). Residues at these positions are important in the formation of pocket 4 in the antigen binding site of the HLA-DR molecule. Objectives: To investigate whether the presence of alleles encoding HLA-DR molecules containing glutamic acid at beta 171 / beta 174 correlates with the course of MS. Methods: HLA-DR and HLA-DQ alleles and genotypes were analysed in 121 MS patients (50 with PP-MS) and 109 controls by molecular typing. Results: Alleles encoding HLA-DR molecules containing a glutamic acid at beta 171 / beta 174 occurred less often in patients with RR/SP-MS than in those with PP-MS or controls. In subjects not carrying the DRB1*1501 allele, a much higher proportion of PP-MS patients carried alleles encoding HLA-DR molecules containing a glutamic acid at beta 171 / beta 174 than did RR/SP-MS patients or controls. Conclusions: The amino acid residues involved in determining the shape and charge of pocket 4 of the HLA-DR beta 1 chain could influence the clinical course of MS by determining protection against RR/SP-MS or susceptibility to the development of PP-MS.

Amplification of DNA from whole blood collected on Guthrie card filter paper presents considerable technical obstacles due to the presence of natural PCR inhibitors (protein, heavy metals, heme, and heme degradation products) and low copy number of genomic material. For this purpose we evaluated guanidine thiocyanate‐impregnated filter paper (GT‐903), a DNA collection device designed specifically to bind PCR inhibitors and preserve DNA in an aqueous extractable form. Compared to standard 903, which retains DNA and elutes inhibitors during aqueous extraction, we found GT‐903 retained 90% of protein, hemoglobin, and iron. SDS‐PAGE analysis indicated that the majority of the protein released from standard 903 corresponded to albumin (70‐) and globin (15‐kDa); negligible levels of these proteins were eluted from GT‐903. To evaluate PCR efficiency, we amplified the 491 bp region encoding the cystic fibrosis ΔF508 mutation. Using comparable template, we found GT‐903 amplification more efficient than standard 903 following qualitative (TBE‐PAGE) and quantitative (anti‐dsDNA EIA) determination. We conclude that GT‐903 provides a good DNA collection device and addresses the complications associated with natural PCR inhibitors. J. Clin. Lab. Anal. 11:87–93. © 1997 Wiley‐Liss, Inc.

Terminal differentiation of the keratinocytes (cornification) has been linked to a restricted supply of retinol. Retinol is distributed to target cells by the retinol-binding protein (RBP), which circulates in the plasma in complex with transthyretin (TTR). In this study we have addressed the question of retinol delivery to the epidermis via RBP. Retinol radiobinding assays, affinity chromatography with TTR coupled to Sepharose beads, polyacrylamide gel electrophoresis, and immunoblotting techniques were used to show that epidermal extracts contain retinol binding sites with no affinity for TTR. Immunoreactive RBP was detected in the epidermal extracts. The RBP in the epidermis was in the apoform (without retinol) in contrast to serum where the majority of RBP is in the holoform (with retinol). Epidermal RBP was converted in vitro into the holoform only after addition of 20 times more retinol, which was needed to reconstitute holoforms of RBP in dermal extracts, human buccal mucosal extracts, and delipidized normal serum or purified delipidized RBP. Moreover, several immunoreactive RBP bands (possibly degradation products) were identified in the epidermal but not in dermal extracts. Retinol-binding protein from nonkeratinizing human oral mucosa showed different immunoblotting patterns when compared to epidermal RBP. These results suggest that degradation of RBP within the epidermis may result in a decreased retinol supply to the keratinocytes, and may lead to the cornification of the epidermis.

Endoglucanase CenA of Cellulomonas fimi comprises an N-terminal cellulose-binding domain and a C-terminal catalytic domain joined together by a sequence of 23 proline and threonine residues (the Pro-Thr box). The domains function independently when separated by proteolysis. TnphoA has been used to generate cenA′-′phoA fusions. CenA′-′PhoA fusion polypeptides which contain the entire cellulose-binding domain of CenA bind to cellulose, allowing their purification from periplasmic extracts in a single, facile step. This result has implications for purification or immobilisation of chimeric proteins on a cheap cellulose matrix.