Enzymatic analysis of the effect of naturally occurring Leu138Pro mutation identified in SHV β-lactamase on hydrolysis of penicillin and ampicillin

<p>Abstract</p> <p>Background</p> <p>The aim of this study was to analyze the significance of leucine to proline substitution at position 138(Leu138Pro) on the hydrolysis of penicillin and ampicillin that we identified in the <it>bla</it>_SHVgene of clinical <it>Escherichia coli </it>swine isolate.</p> <p>Results</p> <p>Kinetic analysis of the mutant proteins showed that <it>K</it>_{<it>m </it>}value of the purified L138P mutant was comparatively higher than SHV-1, SHV-33 and SHV-33(L138P) enzyme for penicillin and ampicillin. Docking simulation of the SHV-1 and SHV-(L138P) enzymes also confirmed that β-lactamases preferred penicillin to ampicillin and the SHV-1 had a higher binding affinity for antibiotics compared to the SHV-(L138P) and other mutants.</p> <p>Conclusions</p> <p>Our result demonstrated that L138P has a reduced role in penicillin and ampicillin hydrolyzing properties of SHV β-lactamases. These naturally occurring mutations rendering reduced function of the existing protein could trigger the emergence or acquisition of more effective alternative mechanisms for β-lactam hydrolysis.</p

Substitution of Heavy Complementarity Determining Region 3 (CDR-H3) Residues Can Synergistically Enhance Functional Activity of Antibody and Its Binding Affinity to HER2 Antigen

To generate a biobetter that has improved therapeutic activity, we constructed scFv libraries via random mutagenesis of several residues of CDR-H3 and -L3 of hu4D5. The scFv clones were isolated from the phage display libraries by stringent panning, and their anti-proliferative activity against HER2-positive cancer cells was evaluated as a primary selection criterion. Consequently, we selected AH06 as a biobetter antibody that had a 7.2-fold increase in anti-proliferative activity (IC50: 0.81 nM) against the gastric cancer cell line NCI-N87 and a 7.4-fold increase in binding affinity (K-D : 60 pM) to HER2 compared to hu4D5. The binding energy calculation and molecular modeling suggest that the substitution of residues of CDR-H3 to W98, F100c, A101 and L102 could stabilize binding of the antibody to HER2 and there could be direct hydrophobic interactions between the aromatic ring of W98 and the aliphatic group of I613 within HER2 domain IV as well as the heavy and light chain hydrophobic interactions by residues F100c, A101 and L102 of CDR-H3. Therefore, we speculate that two such interactions were exerted by the residues W98 and F100c. A101 and L102 may have a synergistic effect on the increase in the binding affinity to HER2. AH06 specifically binds to domain IV of HER2, and it decreased the phosphorylation level of HER2 and AKT. Above all, it highly increased the overall level of p27 compared to hu4D5 in the gastric cancer cell line NCI-N82, suggesting that AH06 could potentially be a more efficient therapeutic agent than hu4D5.OAIID:RECH_ACHV_DSTSH_NO:T201620640RECH_ACHV_FG:RR00200001ADJUST_YN:EMP_ID:A002901CITE_RATE:2.67DEPT_NM:화학생물공학부EMAIL:[email protected]_YN:YCONFIRM:

Early prediction of molecular remission by monitoring BCR-ABL transcript levels in patients achieving a complete cytogenetic response after imatinib therapy for posttransplantation chronic myelogenous leukemia relapse

Imatinib induces a high complete cytogenetic response (CCR) rate in relapsed chronic myelogenous leukemia. By analyzing minimal residual disease (MRD) under the levels of CCR, we tried to assess the molecular response after imatinib therapy. By using real-time quantitative reverse transcriptase-polymerase chain reaction (Q-RT-PCR), MRD was evaluated in 23 patients (3 in cytogenetic relapse, 6 in chronic phase, 9 in accelerated phase, and 5 in blast crisis) who were treated with standard-dose imatinib for relapsed chronic myelogenous leukemia after allogeneic stem cell transplantation. With a median therapy time of 399 days (range, 35–817 days), 19 (83%) patients achieved a CCR. Meanwhile, 11 (58%) of them achieved a molecular remission (MR), which was associated with improved survival. The Q-RT-PCR data were compared according to the best response (MR, n = 11; CCR, n = 8) in the patients achieving a CCR. The BCR-ABL/ABL ratios were similar in 2 groups at 3 months but were significantly different at 6 months (median, 0.0000012 for MR and 0.00022 for CCR; P = .003). The probability of a subsequent MR was significantly higher in patients with a lower BCR-ABL/ABL ratio at 6 months (100% for <0.0001 versus 33% for ≥0.0001; P = .006) or a greater reduction in the level between 3 and 6 months (log-reduction ≥1.0;, 100%; <1.0, 17%; P = .003). Q-RT-PCR is a reliable method for monitoring MRD: the early trends in the BCR-ABL/ABL ratio may be clinically useful in discriminating patients who will achieve an MR from those who will remain in CCR

Graphene quantum dots as anti-inflammatory therapy for colitis

While graphene and its derivatives have been suggested as a potential nanomedicine in several biomimetic models, their specific roles in immunological disorders still remain elusive. Graphene quantum dots (GQDs) may be suitable for treating intestinal bowel diseases (IBDs) because of their low toxicity in vivo and ease of clearance. Here, GQDs are intraperitoneally injected to dextran sulfate sodium (DSS)-induced chronic and acute colitis model, and its efficacy has been confirmed. In particular, GQDs effectively prevent tissue degeneration and ameliorate intestinal inflammation by inhibiting T(H)1/T(H)17 polarization. Moreover, GQDs switch the polarization of macrophages from classically activated M1 to M2 and enhance intestinal infiltration of regulatory T cells (T-regs). Therefore, GQDs effectively attenuate excessive inflammation by regulating immune cells, indicating that they can be used as promising alternative therapeutic agents for the treatment of autoimmune disorders, including IBDs.

Adult Hippocampal Neurogenesis Can Be Enhanced by Cold Challenge Independently From Beigeing Effects

In this study, we investigated the effects of cold challenge on adult hippocampal neurogenesis (AHN) and hippocampal gene expression and whether these are mediated by beigeing of peripheral fat tissues. Cold challenge (6 ± 2°C) for 1 and 4 weeks was found to induce beigeing effects in inguinal white adipose tissue based on hematoxylin and eosin staining as well as uncoupled protein-1 immunohistochemical staining. In the hippocampus, cold challenge for 1 or 4 weeks increased dentate gyrus neurogenesis and expression of genes related to AHN, including notch signaling, G protein-coupled receptor signaling, and adrenergic beta receptor-1. However, this enhancement of neurogenesis and gene expression by cold challenge was not shown by administration of CL 316,243, which induces peripheral beigeing similar to cold challenge but does not cross the blood–brain barrier. These results suggest that cold challenge promotes AHN and central expression of AHN-related, signaling, and β1-adrenergic receptors genes, and that peripheral beigeing by itself is not sufficient to mediate these effects. Considering the increase in AHN and gene expression changes, cold challenge may offer a novel approach to hippocampal modulation

Camptodactyly, Arthropathy, Coxa vara, Pericarditis (CACP)Syndrome: A Case Report

The camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP) is characterized by congenital or early-onset camptodactyly, childhood-onset noninflammatory arthropathy associated with synovial hyperplasia. Some patients have pro-gressive coxa vara deformity and/or noninflammatory pericardial effusion. CACP is inherited as an autosomal recessive mode and the disease gene is assigned to a 1.9-cM interval on human chromosome 1q25-31. We describe a 10-yr-old boy who has typical features of CACP without familial association