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2002-06-01·Hybridoma and Hybridomics

Development of a Second Generation Monoclonal Single Chain Variable Fragment Antibody against Venezuelan Equine Encephalitis Virus: Expression and Functional Analysis

Article

作者: Alvi, A.Z. ; Fulton, R.E. ; Nagata, L.P. ; Chau, D. ; Suresh, M.R.

We have generated a single chain variable fragment (ScFv) antibody from a well-characterized monoclonal antibody (MAb) against Venezuelan equine encephalitis virus (VEE), by cloning variable regions of the heavy (V(H)) and the light (V(L)) chain antibody genes, connected by a DNA linker, in phagemid expression vector pCANTAB 5 E. MAb 1A4A1 was successfully cloned as a ScFv in Escherichia coli strain TG-1 and expressed as a approximately 30 kDa ScFv protein which was functional in recognizing VEE by ELISA. Results were reproduced in Escherichia coli strain HB2151 where the same clone, designated A116, was expressed primarily as soluble periplasmic protein. The 30 kDa A116 antibody displayed weak binding specificity to VEE antigen. Sequence analysis revealed a frame shift in the N-terminal region of the V(L) domain, upstream to the complementarity-determining region 1 (CDR1), as the probable cause of reduced activity. The protein sequence of A116 was highly homologous to published murine ScFv protein sequences except in the region of the identified frame shift.

2002-04-01·British Journal of Pharmacology

Stimulation of Ca²⁺ influx through ATP receptors on rat brain synaptosomes: identification of functional P2X₇ receptor subtypes

Article

作者: Gong, Wenrong ; Lundy, Paul M. ; Mi, Lei ; Vair, Cory ; Sawyer, Thomas W. ; Frew, Robert ; Hamilton, Murray G.

ATP receptors of the P2X class have previously been identified on autonomic nerve endings and on a limited population of CNS neurons.

In the present study P2X receptors on mammalian cortical synaptosomes have been identified by a variety of functional and biochemical studies. In choline buffer ATP analogues caused concentration/time dependent Ca2+ influx. Relative to the effects caused by ATP, benzoylbenzoyl ATP (BzATP) was about seven times more active than ATP while 2‐me‐S‐ATP and ATPγS were much less active. α,β‐me‐ ATP and β,γ‐me‐ATP were virtually inactive. In sucrose buffer, relative to choline buffer, the activity of BzATP was more than doubled while activity in sodium buffer was reduced. Moreover, the P2X antagonists PPADS or Brilliant Blue G both significantly attenuated influx. These observations suggest the presence of P2X receptors on synaptosomes which subserve Ca2+ influx. This activity profile of the ATP analogues and the response to blocking agents are characteristic of responses of P2X7 receptors.

Influx was unaffected by the VSCC inhibitors ω‐CTx‐MVIIC and (−) 202 – 791, indicating that ATP induced Ca2+ influx occurred primarily through P2X receptors.

P2X7 receptor protein was identified by Western blotting and immunohistochemical staining. Purified preparations were devoid of significant concentrations of GFAP or the microglial marker OX‐42 but contained greatly enriched amounts of syntaxin and SNAP 25.

The various pharmacological and biochemical studies were all consistent with the presence of functional P2X7 receptors.

British Journal of Pharmacology (2002) 135, 1616–1626; doi:10.1038/sj.bjp.0704624

2002-01-14·40th AIAA Aerospace Sciences Meeting & Exhibit

Detonation waves in dust media

作者: Zhang, F.

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