Biomedical subjects

Xuan Z Ding

Publications and source records attributed to Xuan Z Ding.

3 recordsLinked to original sources

PubMed2006

Geldanamycin prevents hemorrhage-induced ATP loss by overexpressing inducible HSP70 and activating pyruvate dehydrogenase.

Hemorrhage in mice results in decreased ATP levels in the jejunum, lung, kidney, heart, and brain but not in liver tissue lysates, albeit at variable levels and time kinetics. The decreased protein expression and activity of pyruvate dehydrogenase (PDH) accounted for the hemorrhage-induced ATP loss. Treatment with geldanamycin (GA; 1 microg/g body wt), a known inducer of heat shock protein (HSP)70, inhibited the hemorrhage-induced ATP loss in the jejunum, lung, heart, kidney, and brain. GA was found to increase PDH protein, preserve PDH enzymatic activity, and inhibit mucosal injury in jejunum tissues. GA-induced HSP70i was found to form complexes with PDH protein. HSP70 gene transfer into intestinal epithelial cells promoted PDH and ATP levels, whereas HSP70 short interfering RNA limited them. We conclude that agents able to increase the expression of HSP70 and PDH may be of value in reducing pathology resulting from hemorrhage-associated ATP loss.

Adenosine Triphosphate↗

PubMed2006

A high efficiency cloning and expression system for proteomic analysis.

The recent description of the complete genomes of the two most pathogenic species of Brucella opens the way for genome-based analysis of the antigenicity of their proteins. In the present report, we describe a bench-level high-efficiency cloning and expression system (HECES) that allow expression of large numbers of Brucella proteins based on genomic sequence information. Purified proteins are produced with high efficiency in a microarray format conducive to analysis of their sero-reactivity against serum from immunized animals. This method is applicable at either small or large scale of protein processing. While it does not require robotics, the format is amenable to robotic implementation for all aspects of the process and subsequent analysis of protein characteristics. This method will allow selection of new reagents for diagnosis of brucellosis and development of vaccine against Brucella, an important zoonotic disease and biothreat agent.

Automation↗

PubMed2005

Cloning, expression, and purification of Brucella suis outer membrane proteins.

Brucella, an aerobic, nonsporeforming, nonmotile Gram-negative coccobacillus, is a NIH/CDC category B bioterror threat agent that causes incapacitating human illness. Medical defense against the bioterror threat posed by Brucella would be strengthened by development of a human vaccine and improved diagnostic tests. Central to advancement of these goals is discovery of bacterial constituents that are immunogenic or antigenic for humans. Outer membrane proteins (OMPs) are particularly attractive for this purpose. In this study, we cloned, expressed, and purified seven predicted OMPs of Brucella suis. The recombinant proteins were fused with 6-His and V5 epitope tags at their C termini to facilitate detection and purification. The B. suis surface genes were PCR synthesized based on their ORF sequences and directly cloned into an entry vector. The recombinant entry constructs were propagated in TOP 10 cells, recombined into a destination vector, pET-DEST42, then transformed into Escherichia coli BL21 cells for IPTG-induced protein expression. The expressed recombinant proteins were confirmed with Western blot analysis using anti-6-His antibody conjugated with alkaline phosphatase. These B. suis OMPs were captured and purified using a HisGrab plate. The purified recombinant proteins were examined for their binding activity with antiserum. Serum derived from a rabbit immunized intramuscularly with dialyzed cell lysate of Brucella rough mutant WRR51. The OMPs were screened using the rabbit antiserum and purified IgG. The results suggested that recombinant B. suis OMPs were successfully cloned, expressed and purified. Some of the expressed OMPs showed high binding activity with immunized rabbit antiserum.

Animals↗