The melibiose permease of Salmonella typhimurium (MelB St ) catalyzes symport of melibiose with Na + , Li + , or H + , and bioinformatics analysis indicates that a conserved Gly117 (helix IV) is part of the Na + -binding site. We mutated Gly117 to Ala, Pro, Trp, or Arg; the effects on melibiose transport and binding of cosubstrates depended on the physical−chemical properties of the side chain. Compared with WT MelB St , the Gly117 → Ala mutant exhibited little difference in either cosubstrate binding or stimulation of melibiose transport by Na + or Li + , but all other mutations reduced melibiose active transport and efflux, and decreased the apparent affinity for Na + . The bulky Trp at position 117 caused the greatest inhibition of melibiose binding, and Gly117 → Arg yielded less than a 4-fold decrease in the apparent affinity for melibiose at saturating Na + or Li + concentration. Remarkably, the mutant Gly117 → Arg catalyzed melibiose exchange in the presence of Na + or Li + , but did not catalyze melibiose translocation involving net flux of the coupling cation, indicating that sugar is released prior to release of the coupling cation. Taken together, the findings are consistent with the notion that Gly117 plays an important role in cation binding and translocation.

ContextLow-grade appendiceal mucinous neoplasm (LAMN) and appendiceal adenocarcinoma are known to cause the majority of pseudomyxoma peritonei (PMP, i.e. mucinous ascites); however, recognition and proper classification of these neoplasms can be difficult despite established diagnostic criteria.ObjectiveTo determine the pathological diagnostic concordance for appendix neoplasia and related lesions during patient referral to an academic medical center specialized in treating patients with PMP.DesignThe anatomic pathology laboratory information system was searched to identify cases over a two-year period containing appendix specimens with mucinous neoplasia evaluated by an outside pathology group and by in-house slide review at a single large academic medical center during patient referral.Results161 cases containing appendix specimens were identified over this period. Forty-six of 161 cases (28.6%) contained appendiceal primary neoplasia or lesions. Of these, the originating pathologist diagnosed 23 cases (50%) as adenocarcinoma and 23 cases (50%) as LAMN; however, the reference pathologist diagnosed 29 cases (63.0%) as LAMN, 13 cases (28.3%) as adenocarcinoma, and 4 cases (8.7%) as ruptured simple mucocele. Importantly, for cases in which the originating pathologist rendered a diagnosis of adenocarcinoma, the reference pathologist rendered a diagnosis of adenocarcinoma (56.5%, 13 of 23), LAMN (39.1%, 9 of 23), or simple mucocele (4.3%, 1 of 23). The overall diagnostic concordance rate for these major classifications was 71.7% (33 of 46) with an unweighted observed kappa value of 0.48 (95% CI, 0.27–0.69), consistent with moderate interobserver agreement. All of the observed discordance (28.3%) for major classifications could be attributed to over-interpretation. In addition, the majority of LAMN cases (65.5%) had potential diagnostic deficiencies including over-interpretation as adenocarcinoma and lacking or discordant risk stratification (i.e. documentation of extra-appendiceal neoplastic epithelium).ConclusionsAppendiceal mucinous lesions remain a difficult area for appropriate pathological classification with substantial discordance due to over-interpretation in this study. The findings highlight the critical need for recognition and application of diagnostic criteria regarding these tumors. Recently published consensus guidelines and a checklist provided herein may help facilitate improvement of diagnostic concordance and thereby reduce over-interpretation and potential overtreatment. Further studies are needed to determine the extent of this phenomenon and its potential clinical impact.

Melibiose permease of Salmonella typhimurium (MelBSt) catalyzes melibiose/cation (H+, Na+ or Li+) symport. The transport mechanism is still poorly understood. Threading model indicates that a conserved Gly117 (helix IV) is part of the Na+‐binding site. Recently, mutation analyses suggested that Gly117 plays an important role in cation binding and translocation in MelBSt. In this study, we observed that Gly117→Cys MelBSt mutant drastically inhibited cell growth in the presence of melibiose in a concentration‐dependent fashion. The mutant G117C increased Vmax value for melibiose transport by >3 fold with little changes in Km and protein expression. A spontaneous mutation Pro148→Leu was isolated from mutant G117C. Strikingly, the double mutant G117C/P148L grew at a similar rate in the absence or presence of melibiose and also largely decreased the Vmax value, with 2‐fold lower than obtained from WT. A good correlation between the Vmax values and the cell‐growth rates in the presence of melibiose indicates that the Na+/melibiose symport dissipates cell membrane potential. With a largely elevated turnover number of melibiose transport, it is likely that the cotransported Na+ may collapse the membrane potential and inhibit cell growth.This work was supported by Texas Norman Hackerman Advanced Research Program 010674‐0034‐2009 to LG.