An Update on the Functional Molecular Immunology (FIMM) Database

Data on the major histocompatibility complex, T-cell epitopes, B-cell epitopes, antigens and diseases are heterogeneous and scattered among different databases and the literature. Since it has become increasingly difficult to obtain an integrated view of functional immune response components, we have developed and updated over several years the Functional molecular IMMunology (FIMM) database (http:// research.i2r.a-star.edu.sg/fimm/). FIMM contains integrated expert-curated data on protein antigens, and on human immunological receptors that recognise and bind them in healthy or disease states. Interfaces with multiple, intuitive query options and query reports provide immunologists with prioritised information that aids data interpretation, vaccine target discovery and immune disease research.     

Characterization of the hamster genomic fragment cloned by TAR cloning technology with interspecific sequence information

CHO (Chinese Hamster ovary) cells are widely used for biotechnology and biomedical purposes, and now the EST library database of CHO cells is built. Based on this, the construction of the hamster genome library is under exertion. Though the transformation-associated recombination (TAR) cloning method is accounted as an innovative cloning technology without the construction of the genome library in human and mouse, there has been no trial to isolate the genomic fragment from hamster genome by TAR cloning. In this study, approximately 31 kb of hamster genomic fragment was isolated from the normal human/hamster mono-chromosomal somatic cell line (UV5HL9-5B) using universal hooks of rodent repeats sequence of B1 and B2 by TAR cloning. This fragment was analyzed by bioinformatics tools related to the genome alignment for the similarity analysis among rodent and primate, and was classified into rodents by phylogenetic analysis. One putative gene was found in this region which has homology with the human c14orf4 gene. A zinc finger protein domain was found in the translated hamster ORF. Therefore, we suggest that TAR cloning technique can be applied in CHO cells using mouse genomic information, and it can lead to the establishment of the hamster genome database.     

Ultra-high resolution HLA genotyping and allele discovery by highly multiplexed cDNA amplicon pyrosequencing

ABSTRACT: BACKGROUND: High-resolution HLA genotyping is a critical diagnostic and research assay. Current methods rarely achieve unambiguous high-resolution typing without making population-specific frequency inferences due to a lack of locus coverage and difficulty in exon-phase matching. Achieving high-resolution typing is also becoming more challenging with traditional methods as the database of known HLA alleles increases. RESULTS: We designed a cDNA amplicon-based pyrosequencing method to capture 94% of the HLA class I open-reading-frame with only two amplicons per sample, and an analogous method for class II HLA genes, with a primary focus on sequencing the DRB loci. We present a novel Galaxy server-based analysis workflow for determining genotype. During assay validation, we performed two GS Junior sequencing runs to determine the accuracy of the HLA class I amplicons and DRB amplicon at different levels of multiplexing. When 116 amplicons were multiplexed, we unambiguously resolved 99%of class I alleles to four- or six-digit resolution, as well as 100% unambiguous DRB calls. The second experiment, with 271 multiplexed amplicons, missed some alleles, but generated high-resolution, concordant typing for 93% of class I alleles, and 96% for DRB1 alleles. In a third, preliminary experiment we attempted to sequence novel amplicons for other class II loci with mixed success. CONCLUSIONS: The presented assay is higher-throughput and higher-resolution than existing HLA genotyping methods, and suitable for allele discovery or large cohort sampling. The validated class I and DRB primers successfully generated unambiguously high-resolution genotypes, while further work is needed to validate additional class II genotyping amplicons.     

Cell penetrating peptide-modified pharmaceutical nanocarriers for intracellular drug and gene delivery

Cell-penetrating peptides (CPPs) including TAT peptide (TATp) have been successfully used for intracellular delivery of a broad variety of cargos including various nanoparticulate pharmaceutical carriers (liposomes, micelles, nanoparticles). Here, we will consider the main results in this area, with a special emphasis on TATp-mediated delivery of liposomes and DNA. We will also address the development of "smart" stimuli-sensitive nanocarriers, where cell-penetrating function can be activated by the decreased pH only inside the biological target minimizing thus the interaction of drug-loaded nanocarriers with nontarget cells.     

Viability Testing of Orchid Seed and the Promotion of Colouration and Germination

This study reports the ability of Fusarium to induce orchidseed colouration and germination. The in vitro bioassay germinationtest, using a Fusarium isolate from the protocorm of Cypripediumreginae, was compared with standard chemical procedures of triphenyltetrazolium chloride (TTC) and acid fuchsin (AC) for testingseed viability. With Cypripedium reginae, Cypripedium parviflorumand Platanthera grandiflora, the efficiency of the bioassaywas similar to that of the TTC and AC procedures. However, thebioassay was more appropriate for estimating embryo viabilityafter a prolonged seed pretreatment (more than 2 h) in 10% sodiumhypochlorite, a surface sterilant often used to enhance germinationof terrestrial species. We also obtained in vitro Cypripediumreginae seed germination induction and protocorm formation bythe same Fusarium isolate. This is the first confirmation ofBernard's early reports that orchid fusaria could stimulateseed germination (Bernard N. 1990.Révue Généralede Botanique12 : 108–120). However, the importance ofthe non-mycorrhizal Fusarium fungus in promoting germinationseems to be relatively minor compared to that of specificRhizoctoniaorchid mycorrhizas. Our results are discussed in light of thecurrent North American strategy on orchid conservation methodswhich proposes the use of symbiotic germination.Copyright 2000Annals of Botany Company Orchid, Cypripedium, Platanthera, seed, Fusarium, bioassay, staining, viability, germination, protocorm, mycorrhiza     

Affinity recovery of eight HER2-binding affibody variants using an anti-idiotypic affibody molecule as capture ligand

Affibody molecules generated by combinatorial protein engineering to bind the human epidermal growth factor receptor 2 (HER2) have in earlier studies proven to be promising tracers for HER2-mediated molecular imaging of cancer. Amino acid extensions either at the N- or C-terminus of these Z(HER2) affibody molecules, have been successfully employed for site-specific radiolabeling of the tracer candidates. Hexahistidyls or other tags, which would be convenient for recovery purposes, should be avoided since they could negatively influence the tumor targeting efficacy and biodistribution properties of the tracer. Using a new ?-lactamase-based protein fragment complementation assay (PCA), an affibody molecule was isolated which bound a Z(HER2) affibody molecule with sub-micromolar affinity, but not unrelated affibody molecules. This suggests that the interacting area include the HER2-binding surface of Z(HER2). This novel anti-idiotypic affibody molecule Z(E01) was produced in Escherichia coli, purified, and chemically coupled to a chromatography resin in order to generate an affibody-based affinity column, suitable for recovery of different variants of Z(HER2) affibody molecules, having a common binding surface for HER2. Eight such Z(HER2) affibody molecules, designed for future radioimaging investigations, having different C-terminal peptide extensions aimed for radioisotope ((??m)Tc)-chelation, were successfully produced and recovered in a single step to high purity using the anti-idiotypic affibody ligand for the affinity purification. These results clearly suggest a potential for the development of anti-idiotypic affibody-based resins for efficient recovery of related variants of a target protein that might have altered biochemical properties, thus avoiding the cumbersome design of specific recovery schemes for each variant of a target protein.     

Hen egg white lysozyme fibrillation: a deep‐UV resonance Raman spectroscopic study

Amyloid fibrils are associated with numerous degenerative diseases. The molecular mechanism of the structural transformation of native protein to the highly ordered cross‐β structure, the key feature of amyloid fibrils, is under active investigation. Conventional biophysical methods have limited application in addressing the problem because of the heterogeneous nature of the system. In this study, we demonstrated that deep‐UV resonance Raman (DUVRR) spectroscopy in combination with circular dichroism (CD) and intrinsic tryptophan fluorescence allowed for quantitative characterization of protein structural evolution at all stages of hen egg white lysozyme fibrillation in vitro. DUVRR spectroscopy was found to be complimentary to the far‐UV CD because it is (i) more sensitive to β ‐sheet than to α ‐helix, and (ii) capable of characterizing quantitatively inhomogeneous and highly light‐scattering samples. In addition, phenylalanine, a natural DUVRR spectroscopic biomarker of protein structural rearrangements, exhibited substantial changes in the Raman cross section of the 1000‐cm^–1 band at various stages of fibrillation. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Molecular Co-occupancy Identifies Transcription Factor Binding Cooperativity In Vivo

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Standardized Aronia melanocarpa extract regulates redox status in patients receiving hemodialysis with anemia

Molecular and Cellular Biochemistry - The aim of our study was to investigate the effects of one-month consumption of polyphenol-rich standardized Aronia melanocarpa extract (SAE) on redox status...     

Two Metabolic Fuels,Glucose and Lactate,Differentially Modulate Exocytotic Glutamate Release from Cultured Astrocytes

Neurochemical Research - Astrocytes have a prominent role in metabolic homeostasis of the brain and can signal to adjacent neurons by releasing glutamate via a process of regulated exocytosis....