Eight amino acid residues in transmembrane segments of yeast glucose transporter Hxt2 are required for high affinity transport

Hxt2 and Hxt1 are high affinity and low affinity facilitative glucose transporter paralogs of Saccharomyces cerevisiae, respectively, that differ at 75 amino acid positions in their 12 transmembrane segments (TMs). Comprehensive analysis of chimeras of these two proteins has previously revealed that TMs 1, 5, 7, and 8 of Hxt2 are required for high affinity glucose transport activity and that leucine 201 in TM5 is the most important in this regard of the 20 amino acid residues in these regions that differ between Hxt2 and Hxt1. To evaluate the importance of the remaining residues, we systematically shuffled the amino acids at these positions and screened the resulting proteins for high affinity and high capacity glucose transport activity. In addition to leucine 201 (TM5), four residues of Hxt2 (leucine 59 and leucine 61 in TM1, asparagine 331 in TM7, and phenylalanine 366 in TM8) were found to be important for such activity. Furthermore, phenylalanine 198 (TM5), alanine 363 (TM8), and either valine 316 (TM7) or alanine 368 (TM8) were found to be supportive of maximal activity. Construction of a homology model suggested that asparagine 331 interacts directly with the substrate and that the other identified residues may contribute to maintenance of protein conformation.     

Blood Lactate/ATP Ratio,as an Alarm Index and Real-Time Biomarker in Critical Illness

Objective

The acute physiology, age and chronic health evaluation (APACHE) II score and other related scores have been used for evaluation of illness severity in the intensive care unit (ICU), but there is still a need for real-time and sensitive prognostic biomarkers. Recently, alarmins from damaged tissues have been reported as alarm-signaling molecules. Although ATP is a member of the alarmins and its depletion in tissues closely correlates with multiple-organ failure, blood ATP level has not been evaluated in critical illness. To identify real-time prognostic biomarker of critical illness, we measured blood ATP levels and the lactate/ATP ratio (ATP-lactate energy risk score, A-LES) in critically ill patients.

Methods and Results

Blood samples were collected from 42 consecutive critically ill ICU patients and 155 healthy subjects. The prognostic values of blood ATP levels and A-LES were compared with APACHE II score. The mean ATP level (SD) in healthy subjects was 0.62 (0.19) mM with no significant age or gender differences. The median ATP level in severely ill patients at ICU admission was significantly low at 0.31 mM (interquartile range 0.25 to 0.44) than the level in moderately ill patient at 0.56 mM (0.38 to 0.70) (P<0.01). Assessment with ATP was further corrected by lactate and expressed as A-LES. The median A-LES was 2.7 (2.1 to 3.3) in patients with satisfactory outcome at discharge but was significantly higher in non-survivors at 38.9 (21.0 to 67.9) (P<0.01). Receiver operating characteristic analysis indicated that measurement of blood ATP and A-LES at ICU admission are as useful as APACHE II score for prediction of mortality.

Conclusion

Blood ATP levels and A-LES are sensitive prognostic biomarkers of mortality at ICU admission. In addition, A-LES provided further real-time evaluation score of illness severity during ICU stay particularly for critically ill patients with APACHE II scores of ≥20.0.     

Morphological and mechanical properties of muscle and tendon in highly trained sprinters

The purpose of this study was to investigate muscle and tendon properties in highly trained sprinters and their relations to running performance. Fifteen sprinters and 15 untrained subjects participated in this study. Muscle thickness and tendon stiffness of knee extensors and plantar flexors were measured. Sprinter muscle thickness was significantly greater than that of the untrained subjects for plantar flexors, but not for knee extensors (except for the medial side). Sprinter tendon stiffness was significantly lower than that of the untrained subjects for knee extensors, but not for plantar flexors. The best official record of a 100-m race was significantly correlated to the muscle thickness of the medial side for knee extensors. In conclusion, the tendon structures of highly trained sprinters are more compliant than those of untrained subjects for knee extensors, but not for plantar flexors. Furthermore, a thicker medial side of knee extensors was associated with greater sprinting performance.     

On the Relationship between Food Composition and Serine Dehydrase Activity in Rat Liver

Relationship between serine dehydrase activity in rat liver and dietary protein and carbohydrate levels, using casein and wheat starch, was investigated. Between 10% to 50% of dietary protein levels the enzyme activity sharply increased with increase of protein level. The enzyme activity was also increased by the restriction of carbohydrate intake, even when protein intake was equal in amount. Apparently enzyme activity is controlled by dietary protein level and also by carbohydrate level. In the case of high protein or low carbohydrate diet these changes may be mainly acting for utilization of protein as caloric source.     

Crystal structure of squid rhodopsin with intracellularly extended cytoplasmic region

G-protein-coupled receptors play a key step in cellular signal transduction cascades by transducing various extracellular signals via G-proteins. Rhodopsin is a prototypical G-protein-coupled receptor involved in the retinal visual signaling cascade. We determined the structure of squid rhodopsin at 3.7A resolution, which transduces signals through the G(q) protein to the phosphoinositol cascade. The structure showed seven transmembrane helices and an amphipathic helix H8 has similar geometry to structures from bovine rhodopsin, coupling to G(t), and human beta(2)-adrenergic receptor, coupling to G(s). Notably, squid rhodopsin contains a well structured cytoplasmic region involved in the interaction with G-proteins, and this region is flexible or disordered in bovine rhodopsin and human beta(2)-adrenergic receptor. The transmembrane helices 5 and 6 are longer and extrude into the cytoplasm. The distal C-terminal tail contains a short hydrophilic alpha-helix CH after the palmitoylated cysteine residues. The residues in the distal C-terminal tail interact with the neighboring residues in the second cytoplasmic loop, the extruded transmembrane helices 5 and 6, and the short helix H8. Additionally, the Tyr-111, Asn-87, and Asn-185 residues are located within hydrogen-bonding distances from the nitrogen atom of the Schiff base.     

Structure-activity relationship on (+/-)-nantenine derivatives in antiserotonergic activities in rat aorta

A series of nine (+/-)-nantenine derivatives were synthesized and assayed for their pharmacological activities by using tension in aorta and binding experiments in rat brain membrane. Replacing a methyl group with a hydrogen ((+/-)-nornantenine) and an ethyl group at a nitrogen atom ((+/-)-ethylnornantenine) or introducing a hydroxyl group at the alpha/beta position of C-4 or displacement of a methoxy moiety at the C-1 position with a hydroxyl ((+/-)-domesticine) of (+/-)-nantenine decreased the affinity. Moreover, changing a methyl group of (+/-)-domesticine to hydrogen at a nitrogen atom ((+/-)-nordomesticine) caused loss of the activities. These results suggest that a methyl group at a nitrogen atom and a methoxy moiety at C-1 play important roles in the development of the antiserotonergic activity. Molecular modeling analysis of the interaction between the 5-HT2A receptor and (+/-)-nantenine suggested that electron lone pairs of N-6 and of the oxygen atom of the methoxy group at C-1 are important in forming a hydrogen bond to Asp155 and Asn343 of the 5-HT2A receptor, respectively.     

Identification of the binding sites and selectivity of sarpogrelate,a novel 5-HT2 antagonist,to human 5-HT2A, 5-HT2B and 5-HT2C receptor subtypes by molecular modeling

The aim of the present study was to investigate the binding sites interactions and the selectivity of sarpogrelate to human 5-HT(2) receptor family (5-HT(2A), 5-HT(2B) and 5-HT(2C) receptor subtypes) using molecular modeling. Rhodopsin (RH) crystal structures were used as template to build structural models of the human serotonin-2A and -2C receptors (5-HT(2A)R, 5-HT(2C)R), whereas for 5-HT(2B)R, we used our previously published three-dimensional (3D) models based on bacteriorhodopsin (BR). Sarpogrelate, a novel 5-HT(2)R antagonist, was docked to the receptors. Molecular dynamics (MD) simulations produced the strongest interaction for 5-HT(2A)R/sarpogrelate complex. Upon binding, sarpogrelate constraints aromatic residues network (Trp(3.28), Phe(5.47), Trp(6.48), Phe(6.51), Phe(6.52) in 5-HT(2A)R; Phe(3.35), Phe(6.51), Trp(7.40) in 5-HT(2B)R; Trp(3.28), Phe(3.35), Phe(5.47), Trp(6.48), Phe(6.51), Phe(6.52) in 5-HT(2C)R) in a stacked configuration, preventing activation of the receptor. The models suggest that the structural origin of the selectivity of sarpogrelate to 5-HT(2A)R vs both 5-HT(2B)R and 5-HT(2C)R comes from the following results: (1) The tight interaction between the antagonist and the transmembrane domain (TMD) 3. Asp(3.32) neutralizes the cationic head and interacts simultaneously with carboxylic group hydrogen of the antagonist molecule. (2) Due to steric hindrance, Ser(5.46) (vs Ala(5.46) in 5HT(2B) and 5HT(2C)) prevents sarpogrelate to enter deeply inside the hydrophobic core of the helix bundle and to interact with Pro(5.50). (3) The side chain of Ile(4.56) (vs Ile(4.56) in 5HT(2B)R and Val(4.56) in 5HT(2C)R) constraints sarpogrelate to adjust its position by translating toward the strongly attractive Asp(3.32). These results are in good agreement with binding affinities (pKi) of sarpogrelate for 5-HT(2) receptor family expressed in transfected cell.