In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.

Despite recent advances in the chemotherapy of chronic hepatitis B (CHB), an effective viral suppression after cessation of therapy has not yet been achieved. To investigate whether hepatitis B virus (HBV)-specific T-cell responses are inducible and can contribute to the viral suppression after cessation of the therapy, we conducted a proof-of-concept study with a DNA vaccine comprising of most HBV genes plus genetically engineered interleukin-12 DNA (IL-12N222L) in 12 CHB carriers being treated with lamivudine (LAM). When the ex vivo and/or cultured IFN-g enzyme-linked immunospot (ELISPOT) assay was performed, the detectable HBV-specific IFN-g secreting T-cell responses were observed at the end of treatment and during a follow-up. These type 1T-cell responses, particularly CD4 + memory T-cell responses could be maintained for at least 40 weeks after the therapy and correlated with virological responses, but not with alanine aminotransferase elevation. Moreover, DNA vaccination under LAM treatment appeared to be well-tolerated and showed 50% of virological response rate in CHB carriers. Thus, a combination therapy of the DNA vaccine with chemotherapy may be one of new immunotherapeutic methods for the cure of CHB. Gene Therapy ( Patients with chronic HBV infection who showed remission also develop vigorous CTL and strong type 1T helper (Th1) immune responses that are comparable to those in patients who have a selflimited disease. 4 In contrast, the CTL and Th1 responses are undetectable or relatively weak in patients with chronic HBV infection. 3,5 Lamivudine (LAM) and adefovir dipivoxil as nucleoside analogues can suppress HBV replication effectively during treatment period, 6,7 but their use is limited by the high risk of viral relapse upon discontinuation even after long-term treatment.2 A restoration of HBV-specific CD4 + and CD8 + T-cell responses by LAM monotherapy was previously observed, but these T-cell responses were not only transient during treatment, but were also undetectable or very weak at the end of 1-year treatment. 8,9 It was recently reported that the inverse correlation between the number of antigen-specific interferon (IFN)-g producing CD4 + T cells and serum HBV DNA was observed during the treatment of LAM with recombinant interleukin-12 (IL-12) protein, but not detectable after the treatment. 10Therefore, further studies are needed to elucidate the relationship between T-cell responses and the suppression of viral relapse after stopping the therapy.DNA vaccine has the advantage of inducing both humoral and cellular immune responses, especially Th1 and CTL responses. HBV DNA vaccine was shown to induce strong T-cell responses, leading to the suppression of viral replication in HBV transgenic mice.11 In contrast, DNA immunization induced very weak T-cell

Hundreds of proteins involved in signaling pathways contain a Ca(2+)-dependent membrane-binding motif called the C2-domain. However, no small C2-domain proteins consisting of a single C2-domain have been reported in animal cells. We have isolated two cDNA clones, OsERG1a and OsERG1b, that encode two small C2-domain proteins of 156 and 159 amino acids, respectively, from a fungal elicitor-treated rice cDNA library. The clones are believed to have originated from a single gene by alternative splicing. Transcript levels of the OsERG1 gene are dramatically elevated by a fungal elicitor prepared from Magnaporthe grisea or by Ca(2+) ions. The OsERG1 protein produced in Escherichia coli binds to phospholipid vesicles in a Ca(2+)-dependent manner and is translocated to the plasma membrane of plant cells by treatment with either a fungal elicitor or a Ca(2+) ionophore. These results suggest that OsERG1 proteins containing a single C2-domain are involved in plant defense signaling systems.

Volcano-type ulcerations in Behçet's colitis showed a less favorable response to medical treatment, a more frequent requirement for surgery, and more frequent recurrences than the geographic and aphthous types of ulcerations.

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A relationship between radiological characteristics and molecular signatures in AO/AOAs was shown. It is believed that radiological characteristics have prognostic value as a surrogate marker for molecular characteristics.

Lysinoalanine, N6-(DL-2-amino-2-carboxyethyl)-L-lysine, an unusual amino acid implicated as a renal toxic factor in rats, has been found in proteins of home-cooked and commercial foods and ingredients. Although it has been reported to occur in both edible and nonfood proteins only after alkali treatment, it has now been identified in food proteins that had not been subjected to alkali. Lysinoalanine is generated in a variety of proteins when heated under nonalkaline conditions.

Objective-To compare MR signal characteristics of contrast agent-labeled apoptotic and viable human mesenchymal stem cells (hMSCs) in matrix associated stem cell implants (MASI).Methods-hMSCs were labeled with FDA approved ferumoxides nanoparticles. One group (A) remained untreated while a second group (B) underwent Mitomycin C-induced apoptosis induction. Viability of group A and apoptosis of group B was confirmed by Caspase-assays and TUNEL stains. Labeled viable hMSCs, unlabeled viable hMSCs, labeled apoptotic hMSCs and unlabeled apoptotic hMSCs (n=7 samples each) in an agarose scaffold were implanted into cartilage defects of porcine patellae specimens and underwent MR imaging at 7T, using T1 weighted SE sequences, T2-weighted SE sequences and T2*-weighted GE sequences. Signal to noise ratios (SNR) of the implants were calculated and compared between different experimental groups using linear mixed regression models (LMM).Results-Ferumoxides-labeled hMSCs provided a strong negative T2 and T2*-enhancement. Corresponding SNR data of labeled hMSCs were significantly lower compared to unlabeled controls (p<0.05). Apoptosis induction resulted in a significant signal decline of ferumoxides-labeled hMSC transplants on short TE T2-weighted sequences. SNR data of labeled apoptotic hMSCs were significantly lower compared to labeled viable hMSCs (p<0.05).Conclusion-Apoptosis of transplanted ferumoxides-labeled stem cells in cartilage defects can be visualized non-invasively by a significant signal decline on T2-weighted MR images. The described MR signal characteristics may serve as a non-invasive outcome measure for the assessment of MASI therapies in clinical practice. Additional studies are needed to further enhance the observed differences between viable and apoptotic cells, e.g. by further optimizing the applied MR pulse sequence parameters or by determining more robust T2-relaxation times.