Biomedical subjects

Xing Jian Lou

Publications and source records attributed to Xing Jian Lou.

5 recordsLinked to original sources

PubMed2006

Role of charged residues in the S1-S4 voltage sensor of BK channels.

The activation of large conductance Ca(2+)-activated (BK) potassium channels is weakly voltage dependent compared to Shaker and other voltage-gated K(+) (K(V)) channels. Yet BK and K(V) channels share many conserved charged residues in transmembrane segments S1-S4. We mutated these residues individually in mSlo1 BK channels to determine their role in voltage gating, and characterized the voltage dependence of steady-state activation (P(o)) and I(K) kinetics (tau(I(K))) over an extended voltage range in 0-50 microM [Ca(2+)](i). mSlo1 contains several positively charged arginines in S4, but only one (R213) together with residues in S2 (D153, R167) and S3 (D186) are potentially voltage sensing based on the ability of charge-altering mutations to reduce the maximal voltage dependence of P(O). The voltage dependence of P(O) and tau(I(K)) at extreme negative potentials was also reduced, implying that the closed-open conformational change and voltage sensor activation share a common source of gating charge. Although the position of charged residues in the BK and K(V) channel sequence appears conserved, the distribution of voltage-sensing residues is not. Thus the weak voltage dependence of BK channel activation does not merely reflect a lack of charge but likely differences with respect to K(V) channels in the position and movement of charged residues within the electric field. Although mutation of most sites in S1-S4 did not reduce gating charge, they often altered the equilibrium constant for voltage sensor activation. In particular, neutralization of R207 or R210 in S4 stabilizes the activated state by 3-7 kcal mol(-1), indicating a strong contribution of non-voltage-sensing residues to channel function, consistent with their participation in state-dependent salt bridge interactions. Mutations in S4 and S3 (R210E, D186A, and E180A) also unexpectedly weakened the allosteric coupling of voltage sensor activation to channel opening. The implications of our findings for BK channel voltage gating and general mechanisms of voltage sensor activation are discussed.

Amino Acids↗

PubMed2005

Micropillar array chip for integrated white blood cell isolation and PCR.

We report the fabrication of silicon chips containing a row of 667 pillars, 10 by 20 microm in cross-section, etched to a depth of 80 microm with adjacent pillars being separated by 3.5 microm. The chips were used to separate white blood cells from whole blood in less than 2 min and for subsequent PCR of a genomic target (eNOS). Chip fluid dynamics were validated experimentally using CoventorWare microfluidic simulation software. The amplicon concentrations were determined using microchip capillary electrophoresis and were >40% of that observed in conventional PCR tubes for chips with and without pillars. Reproducible on-chip PCR was achieved using white blood cell preparations isolated from whole human blood pumped through the chip.

Cell Separation↗

PubMed2004

Surface effects on PCR reactions in multichip microfluidic platforms.

We evaluated the compatibility of several common plastics, commercially available plastic tubing and disposable syringes which might be useful in the construction of microfluidic platforms with respect to the polymerase chain reaction (PCR). A simple and inexpensive plastic test module was constructed in order to evaluate some of the construction plastics. We also investigated the effect of addition of PEG 8000 to PCR reaction mixtures on the compatibility of materials. These studies identified several common plastics, plastic tubing, and disposable syringes which were compatible with the PCR reaction.

Equipment Design↗

PubMed2004

Increased amplification efficiency of microchip-based PCR by dynamic surface passivation.

Surface passivation is critical for effective PCR using silicon-glass chips. We tested a dynamic polymer-based surface passivation method. Polyethylene glycol 8000 (PEG 8000) or polyvinylpyrrolidone 40 (PVP-40) applied at 0.75% (w/v) in the reaction mixture produced significant surface passivation effects using either native or SiO2-precoated silicon-glass chips. PCR amplification was achieved from human genomic DNA as a template as well as from human lymphocytes. The dynamic surface passivation effect of PEG 8000 remained similar under both conditions. Dynamic surface passivation offers a simple and cost-effective method to make microfabricated silicon-glass chips PCR friendly. It can also be used in combination with static passivation (silicon oxide surface layer) to further improve PCR performance using silicon-glass PCR chips.

Cell Separation↗

PubMed2004

Mutation detection using ligase chain reaction in passivated silicon-glass microchips and microchip capillary electrophoresis.

The ligase chain reaction (LCR) following PCR is one of the most sensitive and specific methods for detecting mutations, especially single nucleotide polymorphisms (SNPs). Performing LCR in microchips remains a challenge because of the inhibitory effect of the internal surfaces of silicon-glass microchips. We have tested a dynamic polymer-based surface passivation method for LCR conducted in oxide-coated silicon-glass microchips. The combination of polyvinylpyrrolidone 40 (PVP-40) at 0.75% (w/v) with an excess of the ligase produced successful LCR in the silicon-glass microchips, with yields of ligated primers comparable to reactions performed in conventional reaction tubes. Ligated primers were detected and quantified simply and conveniently using microchip capillary electrophoresis.

DNA Mutational Analysis↗