RhoA, RhoB and RhoC GTPases are over 85% identical at the amino acid level, with RhoA and RhoC differing at only one residue (43) across the initial two-thirds of their sequences. A putative regulatory distinction between the molecules is their capacity to be uniquely activated by guanine nucleotide exchange factors (GEFs). We hypothesize that variation of amino acid residue 43 between RhoA/B (valine) and RhoC (isoleucine) impacts GEF activity. Direct participation of residue 43 in GEF-catalyzed exchange was confirmed by the observation that mutation of this position to a threonine reduced GEF-catalyzed nucleotide exchange activity in vitro (Vav2, XPLN, GEFT, Dbl and Dbs) and greatly depressed RhoA and RhoC GTP-loading profiles in cell lysates. Using a residue swap approach, substitution of RhoA Val 43 with an Ile was found to significantly promote basal nucleotide exchange activity and enhance GTP-loading in cells. Substitution of Val 43 with an Ile in RhoB negatively affected nucleotide exchange in vitro. Substitution of RhoC Ile 43 with a Val increased GEF-catalyzed exchange in vitro. In addition, RhoC-I43V was more efficacious at driving ovarian cancer cell invasion through matrigrel than wild-type RhoC, RhoC-I43T, wild-type RhoA, RhoA-V43I or RhoA-V43T GTPases. These findings suggest that a divergence between RhoA/B and RhoC at residue 43 impacts basal and GEF-stimulated nucleotide exchange activity.
The purpose of this retrospective review is to evaluate the safety and efficacy of the bronchial blockers (BBs) used in thoracic anesthesia. We enrolled 302 patients who had a BB placed to achieve one-lung ventilation (OLV). Variables recorded from the anesthetic record included type of device used, type and side of surgery, specific indications for OLV, Mallampati score, route of intubation, and complications related to the use of BBs. The BBs used include the Arndt Wire-guided, Univent, Cohen Flexi-tip, Fogarty catheter, and Fuji. The majority of BBs placed were Arndt (n = 156) or Univent (n = 131). BBs were used significantly more often in thoracoscopic procedures than in thoracotomies (P < 0.01). Of the 251 patients, 216 (86%) had a Mallampati score of I/II and 35 (14%) had a score of III/IV. There were no identified complications related to BBs. In summary, BBs can be safely used to achieve OLV and offer advantages for OLV in specific situations.
RhoA, RhoB, and RhoC GTPases share over 80% sequence identity but diverge in cellular function. In previous work, we hypothesized that variation at amino acid residue 43 between RhoA/B (valine) and RhoC (isoleucine) was a structural impediment to XPLN activation of RhoC. In this work, we have systematically studied a putative role of Rho residue 43 as a structural determinant of guanine nucleotide exchange factor (GEF) activity. Specifically, we performed in vitro analysis of GEF activity (VAV2, XPLN, Dbs, GEFT, and Dbl) against both normal RhoA/B/C proteins and those mutated at residue 43. Substitution of val43 with an isoleucine in RhoA reduced 40‐50% of GEF catalyzed exchange, except for GEFT (reduced only 10‐20%). Substitution of val43 with an isoleucine in RhoB had variable effect on exchange, with Vav2 and GEFT modestly sensitive (10% decrease) and XPLN‐stimulated exchange reduced 80%. On the other hand, substitution of ile43 with a valine in RhoC promoted all GEF catalyzed exchange (1.3‐4.0 fold increases). Substitution of a polar threonine at residue 43 significantly reduced all RhoA GEF exchange 60‐80%, nearly eliminated RhoB GEF exchange (80‐100% decrease), but had little to no affect on RhoC exchange, with the exception of GEFT (reduced 75%). In support, RhoA V43T displayed a reduced phenotype when expressed in NIH3T3 fibroblasts, whereas constitutively active RhoA V43T, 63L was phenotypical equivalent to RhoA 63L. Together, these findings support the hypothesis that a divergence at residue 43 between RhoA/B and RhoC is a structural determinant of GEF activity and selectivity. This work was supported with funds from the RJ McElroy Trust.
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