Family B heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) play important roles in carbohydrate metabolism. Recent structures of family B GPCR-Gs protein complexes reveal a disruption in the α-helix of transmembrane segment 6 (TM6) not observed in family A GPCRs. To investigate the functional impact of this structural difference, we compared the structure and function of the glucagon receptor (GCGR; family B) with the β2 adrenergic receptor (β2AR; family A). We determined the structure of the GCGR-Gs complex by means of cryo–electron microscopy at 3.1-angstrom resolution. This structure shows the distinct break in TM6. Guanosine triphosphate (GTP) turnover, guanosine diphosphate release, GTP binding, and G protein dissociation studies revealed much slower rates for G protein activation by the GCGR compared with the β2AR. Fluorescence and double electron-electron resonance studies suggest that this difference is due to the inability of agonist alone to induce a detectable outward movement of the cytoplasmic end of TM6.

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Summary Reasons for performing study Lungeing is often part of the clinical lameness examination. The difference in movement symmetry, which is a commonly employed lameness measure, has not been quantified between surfaces. Objectives To compare head and pelvic movement symmetry between surfaces and reins during lungeing. Study design Quantitative gait analysis in 23 horses considered sound by their owners. Methods Twenty‐three horses were assessed in‐hand and on the lunge on both reins on hard and soft surfaces with inertial sensors. Seven movement symmetry parameters were quantified and used to establish 2 groups, namely symmetrical (n = 9) and forelimb‐lame horses (n = 14), based on values from straight‐line assessment. Movement symmetry values for left rein measurements were side corrected to allow comparison of the amount of movement symmetry between reins. A mixed model (P<0.05) was used to study effects on movement symmetry of surface (hard/soft) and rein (inside/outside with respect to movement symmetry on the straight). Results In forelimb‐lame horses, surface and rein were identified as significantly affecting all head movement symmetry measures (rein, all P<0.0001; surface, all P<0.042). In the symmetrical group, no significant influence of surface or rein was identified for head movement symmetry (rein, all P>0.245; surface, all P>0.073). No significant influence of surface or rein was identified for any of the pelvic movement symmetry measures in either group. Conclusions While more symmetrical horses showed a consistent amount of movement symmetry across surfaces/reins, horses objectively quantified as lame on the straight showed decreased movement symmetry during lungeing, in particular with the lame limb on the inside of a hard circle. The variation within group questions straight‐line movement symmetry as a sole measure of lameness without quantification of movement symmetry on the lunge, ideally on hard and soft surfaces to evaluate differences between reins and surfaces. In future, thresholds for lungeing need to be determined using simultaneous visual and objective assessment.

Swainsonine, an indolizidine alkaloid, found in plants of the genus Swainsona, has been shown to be a strong inhibitor in vitro of the alpha-D-mannosidase activity in normal human fibroblasts. Therefore, inhibition of alpha-D-mannosidase activity in extracts of harvested cells grown with swainsonine in the medium has been used to follow the association of the alkaloid with normal human fibroblasts in culture. Swainsonine that could not be removed by extensive washing became associated with the cells within 1 min, and it is concluded that the alkaloid is internalized rapidly by the cells. The amount of swainsonine taken up into the cells depended on the length of time in contact and the concentration of swainsonine in the medium, but at 37 degrees C a plateau of internalized swainsonine occurred after 2 hr with extracellular concentrations of swainsonine of 100 microM or greater. At lower concentrations of swainsonine the rate of uptake was found to be temperature-dependent, increasing greatly at 20 degrees C. The rapidity and temperature sensitivity of the uptake, together with the observation that mannose or mannose-6-phosphate did not prevent the association, suggest that swainsonine enters the cells by permeation rather than by endocytosis. When swainsonine is withdrawn from the culture medium, there is a decrease with time of cell-associated swainsonine. The kinetics of uptake and release of swainsonine and its slightly basic nature make it likely that swainsonine is concentrated initially in the lysosomes. This rapid, but reversible, concentration of swainsonine in lysosomes would be consistent with the observed effects of the toxin in vivo.