Comparison of helix interactions in membrane and soluble alpha-bundle proteins

Helix-helix interactions are important for the folding, stability, and function of membrane proteins. Here, two independent and complementary methods are used to investigate the nature and distribution of amino acids that mediate helix-helix interactions in membrane and soluble alpha-bundle proteins. The first method characterizes the packing density of individual amino acids in helical proteins based on the van der Waals surface area occluded by surrounding atoms. We have recently used this method to show that transmembrane helices pack more tightly, on average, than helices in soluble proteins. These studies are extended here to characterize the packing of interfacial and noninterfacial amino acids and the packing of amino acids in the interfaces of helices that have either right- or left-handed crossing angles, and either parallel or antiparallel orientations. We show that the most abundant tightly packed interfacial residues in membrane proteins are Gly, Ala, and Ser, and that helices with left-handed crossing angles are more tightly packed on average than helices with right-handed crossing angles. The second method used to characterize helix-helix interactions involves the use of helix contact plots. We find that helices in membrane proteins exhibit a broader distribution of interhelical contacts than helices in soluble proteins. Both helical membrane and soluble proteins make use of a general motif for helix interactions that relies mainly on four residues (Leu, Ala, Ile, Val) to mediate helix interactions in a fashion characteristic of left-handed helical coiled coils. However, a second motif for mediating helix interactions is revealed by the high occurrence and high average packing values of small and polar residues (Ala, Gly, Ser, Thr) in the helix interfaces of membrane proteins. Finally, we show that there is a strong linear correlation between the occurrence of residues in helix-helix interfaces and their packing values, and discuss these results with respect to membrane protein structure prediction and membrane protein stability.     

Anesthetic effects on the structure and dynamics of the second transmembrane domains of nAChR α4β2

Channel functions of the neuronal α4β2 nicotinic acetylcholine receptor (nAChR), one of the most widely expressed subtypes in the brain, can be inhibited by volatile anesthetics. Our Na⁺ flux experiments confirmed that the second transmembrane domains (TM2) of α4 and β2 in 2:3 stoichiometry, (α4)₂(β2)₃, could form pentameric channels, whereas the α4 TM2 alone could not. The structure, topology, and dynamics of the α4 TM2 and (α4)₂(β2)₃ TM2 in magnetically aligned phospholipid bicelles were investigated using solid-state NMR spectroscopy in the absence and presence of halothane and isoflurane, two clinically used volatile anesthetics. ²H NMR demonstrated that anesthetics increased lipid conformational heterogeneity. Such anesthetic effects on lipids became more profound in the presence of transmembrane proteins. PISEMA experiments on the selectively ¹⁵N-labeled α4 TM2 showed that the TM2 formed transmembrane helices with tilt angles of 12° ± 1° and 16° ± 1° relative to the bicelle normal for the α4 and (α4)₂(β2)₃ samples, respectively. Anesthetics changed the tilt angle of the α4 TM2 from 12° ± 1° to 14° ± 1°, but had only a subtle effect on the tilt angle of the (α4)₂(β2)₃ TM2. A small degree of wobbling motion of the helix axis occurred in the (α4)₂(β2)₃ TM2. In addition, a subset of the (α4)₂(β2)₃ TM2 exhibited counterclockwise rotational motion around the helix axis on a time scale slower than 10^- 4 s in the presence of anesthetics. Both helical tilting and rotational motions have been identified computationally as critical elements for ion channel functions. This study suggested that anesthetics could alter these motions to modulate channel functions.     

High-Resolution Orientation and Depth of Insertion of the Voltage-Sensing S4 Helix of a Potassium Channel in Lipid Bilayers

The opening and closing of voltage-gated potassium (Kv) channels are controlled by several conserved Arg residues in the S4 helix of the voltage-sensing domain. The interaction of these positively charged Arg residues with the lipid membrane has been of intense interest for understanding how membrane proteins fold to allow charged residues to insert into lipid bilayers against free-energy barriers. Using solid-state NMR, we have now determined the orientation and insertion depth of the S4 peptide of the KvAP channel in lipid bilayers. Two-dimensional ¹⁵N correlation experiments of macroscopically oriented S4 peptide in phospholipid bilayers revealed a tilt angle of 40° and two possible rotation angles differing by 180° around the helix axis. Remarkably, the tilt angle and one of the two rotation angles are identical to those of the S4 helix in the intact voltage-sensing domain, suggesting that interactions between the S4 segment and other helices of the voltage-sensing domain are not essential for the membrane topology of the S4 helix. ¹³C-³¹P distances between the S4 backbone and the lipid ³¹P indicate a ∼ 9 Å local thinning and 2 Å average thinning of the DMPC (1,2-dimyristoyl-sn-glycero-3-phosphochloline)/DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) bilayer, consistent with neutron diffraction data. Moreover, a short distance of 4.6 Å from the guanidinium C^ζ of the second Arg to ³¹P indicates the existence of guanidinium phosphate hydrogen bonding and salt bridges. These data suggest that the structure of the Kv gating helix is mainly determined by protein-lipid interactions instead of interhelical protein-protein interactions, and the S4 amino acid sequence encodes sufficient information for the membrane topology of this crucial gating helix.     

Solid-state NMR ensemble dynamics as a mediator between experiment and simulation

Solid-state NMR (SSNMR) is a powerful technique to describe the orientations of membrane proteins and peptides in their native membrane bilayer environments. The deuterium (²H) quadrupolar splitting (DQS), one of the SSNMR observables, has been used to characterize the orientations of various single-pass transmembrane (TM) helices using a semistatic rigid-body model such as the geometric analysis of labeled alanine (GALA) method. However, dynamic information of these TM helices, which could be related to important biological function, can be missing or misinterpreted with the semistatic model. We have investigated the orientation of WALP23 in an implicit membrane of dimyristoylglycerophosphocholine by determining an ensemble of structures using multiple conformer models with a DQS restraint potential. When a single conformer is used, the resulting helix orientation (tilt angle (τ) of 5.6 ± 3.2° and rotation angle (ρ) of 141.8 ± 40.6°) is similar to that determined by the GALA method. However, as the number of conformers is increased, the tilt angles of WALP23 ensemble structures become larger (26.9 ± 6.7°), which agrees well with previous molecular dynamics simulation results. In addition, the ensemble structure distribution shows excellent agreement with the two-dimensional free energy surface as a function of WALP23's τ and ρ. These results demonstrate that SSNMR ensemble dynamics provides a means to extract orientational and dynamic information of TM helices from their SSNMR observables and to explain the discrepancy between molecular dynamics simulation and GALA-based interpretation of DQS data.     

Membrane phase behavior of Escherichia coli during desiccation, rehydration, and growth recovery

The membrane lipid bilayer is one of the primary cellular components affected by variations in hydration level, which cause changes in lipid packing that may have detrimental effects on cell viability. In this study, Fourier transform infrared (FTIR) spectroscopy was used to quantify changes in the membrane phase behavior, as identified by membrane phase transition temperature (T_m), of Escherichia coli during desiccation and rehydration. Extensive cell desiccation (1 week at 20%-40% RH) resulted in an increase in T_m from 8.4 ± 1.7 °C (in undried control samples) to 16.5 ± 1.3 °C. Fatty acid methyl ester analysis (FAME) on desiccated samples showed an increase in the percent composition of saturated fatty acids (FAs) and a decrease in unsaturated FAs in comparison to undried control samples. However, rehydration of E. coli resulted in a gradual regression in T_m, which began approximately 1 day after initial rehydration and plateaued at 12.5 ± 1.8 °C after approximately 2 days of rehydration. FAME analysis during progressive rehydration revealed an increase in the membrane percent composition of unsaturated FAs and a decrease in saturated FAs. Cell recovery analysis during rehydration supported the previous findings that showed that E. coli enter a viable but non-culturable (VBNC) state during desiccation and recover following prolonged rehydration. In addition, we found that the delay period of approximately 1 day of rehydration prior to membrane reconfiguration (i.e. decrease in T_m and increase in membrane percent composition of unsaturated FAs) also preceded cell recovery. These results suggest that changes in membrane structure and state related to greater membrane fluidity may be associated with cell proliferation capabilities.     

An activation gating switch in Kv1.2 is localized to a threonine residue in the S2-S3 linker

The activation properties of Kv1.2 channels are highly variable, with reported half-activation (V_1/2) values ranging from ∼−40 mV to ∼+30 mV. Here we show that this arises because Kv1.2 channels occupy two distinct gating modes (“fast” and “slow”). “Slow” gating (τ_act = 90 ± 6 ms at +35 mV) was associated with a V_1/2 of activation of +16.6 ± 1.1 mV, whereas “fast” gating (τ_act = 4.5 ± 1.7 ms at +35 mV) was associated with a V_1/2 of activation of −18.8 ± 2.3 mV. It was possible to switch between gating modes by applying a prepulse, which suggested that channels activate to a single open state along separate “fast” and “slow” activation pathways. Using chimeras and point mutants between Kv1.2 and Kv1.5 channels, we determined that introduction of a positive charge at or around threonine 252 in the S2-S3 linker of Kv1.2 abolished “slow” activation gating. Furthermore, dialysis of the cytoplasm or excision of cell-attached patches from cells expressing Kv1.2 channels switched gating from “slow” to “fast”, suggesting involvement of cytoplasmic regulators. Collectively, these results demonstrate two modes of activation gating in Kv1.2 and specific residues in the S2-S3 linker that act as a switch between these modes.     

Self-assembly of a simple membrane protein: coarse-grained molecular dynamics simulations of the influenza M2 channel

The transmembrane (TM) domain of the M2 channel protein from influenza A is a homotetrameric bundle of α-helices and provides a model system for computational approaches to self-assembly of membrane proteins. Coarse-grained molecular dynamics (CG-MD) simulations have been used to explore partitioning into a membrane of M2 TM helices during bilayer self-assembly from lipids. CG-MD is also used to explore tetramerization of preinserted M2 TM helices. The M2 helix monomer adopts a membrane spanning orientation in a lipid (DPPC) bilayer. Multiple extended CG-MD simulations (5 × 5 μs) were used to study the tetramerization of inserted M2 helices. The resultant tetramers were evaluated in terms of the most populated conformations and the dynamics of their interconversion. This analysis reveals that the M2 tetramer has 2× rotationally symmetrical packing of the helices. The helices form a left-handed bundle, with a helix tilt angle of ∼16°. The M2 helix bundle generated by CG-MD was converted to an atomistic model. Simulations of this model reveal that the bundle's stability depends on the assumed protonation state of the H37 side chains. These simulations alongside comparison with recent x-ray (3BKD) and NMR (2RLF) structures of the M2 bundle suggest that the model yielded by CG-MD may correspond to a closed state of the channel.     

Structure and conformation of the disulfide bond in dimeric lung surfactant peptides SP-B1-25 and SP-B8-25

Raman spectroscopy was used to determine the conformation of the disulfide linkage between cysteine residues in the homodimeric construct of the N-terminal alpha helical domain of surfactant protein B (dSP-B_1-25). The conformation of the disulfide bond between cysteine residues in position 8 of the homodimer of dSP-B_1-25 was compared with that of a truncated homodimer (dSP-B_8-25) of the peptide having a disulfide linkage at the same position in the alpha helix. Temperature-dependent Raman spectra of the S-S stretching region centered at ∼ 500 cm^− 1 indicated a stable, although highly strained disulfide conformation with a χ(CS-SC) dihedral angle of ± 10° for the dSP-B_1-25 dimer. In contrast, the truncated dimer dSP-B_8-25 exhibited a series of disulfide conformations with the χ(CS-SC) dihedral angle taking on values of either ± 30° or 85± 20°. For conformations with χ(CS-SC) close to the ± 90° value, the Raman spectra of the 8-25 truncated dimers exhibited χ(SS-CC) dihedral angles of 90/180° and 20-30°. In the presence of a lipid mixture, both constructs showed a ν(S-S) band at ∼ 488 cm^− 1, corresponding to a χ(CS-SC) dihedral angle of ± 10°. Polarized infrared spectroscopy was also used to determine the orientation of the helix and β-sheet portion of both synthetic peptides. These calculations indicated that the helix was oriented primarily in the plane of the surface, at an angle of ∼ 60-70° to the surface normal, while the β structure had ∼ 40° tilt. This orientation direction did not change in the presence of a lipid mixture or with temperature. These observations suggest that: (i) the conformational flexibility of the disulfide linkage is dependent on the amino acid residues that flank the cysteine disulfide bond, and (ii) in both constructs, the presence of a lipid matrix locks the disulfide bond into a preferred conformation.     

DFT study of the intrinsic conformations of [2Fe-2S-4(SCH3)] clusters and their influence on exchange coupling

We have determined the equilibrium conformations of the diiron(III) cluster [2Fe-2S-4(SCH₃)]²⁻ using density functional theory. The conformers have dihedral Fe-Fe-S-C angles of ∼0° and ±120°. The relative energies of the conformers can be accurately parameterized with a small number of side-chain repulsion parameters. Of the 17 conformers identified on the basis of the ideal values for the dihedrals, 10 conformers are stable in both the ferromagnetic and broken symmetry state for the cluster. The exchange coupling constants for the seven energetically lowest conformers are predicted to belong to a narrow range, 150 cm⁻¹ ? J ? 178 cm⁻¹. The cluster conformers found in proteins do not coincide with any of the intrinsic ones, due to distortion of one of the dihedral angles under the influence of the protein scaffold.     

Thermodynamic analysis of hydration in human serum heme-albumin

Ferric human serum heme-albumin (heme-HSA) shows a peculiar nuclear magnetic relaxation dispersion (NMRD) behavior that allows to investigate structural and functional properties. Here, we report a thermodynamic analysis of NMRD profiles of heme-HSA between 20 and 60 °C to characterize its hydration. NMRD profiles, all showing two Lorentzian dispersions at 0.3 and 60 MHz, were analyzed in terms of modulation of the zero field splitting tensor for the S = ⁵/₂ manifold. Values of correlation times for tensor fluctuation (τ_v) and chemical exchange of water molecules (τ_M) show the expected temperature dependence, with activation enthalpies of −1.94 and −2.46 ± 0.2 kJ mol⁻¹, respectively. The cluster of water molecules located in the close proximity of the heme is progressively reduced in size by increasing the temperature, with ΔH = 68 ± 28 kJ mol⁻¹ and ΔS = 200 ± 80 J mol⁻¹ K⁻¹. These results highlight the role of the water solvent in heme-HSA structure-function relationships.     

Characterization of Membrane Protein Interactions by Isothermal Titration Calorimetry

Understanding the structure, folding, and interaction of membrane proteins requires experimental tools to quantify the association of transmembrane (TM) helices. Here, we introduce isothermal titration calorimetry (ITC) to measure integrin αIIbβ3 TM complex affinity, to study the consequences of helix–helix preorientation in lipid bilayers, and to examine protein-induced lipid reorganization. Phospholipid bicelles served as membrane mimics. The association of αIIbβ3 proceeded with a free energy change of − 4.61 ± 0.04 kcal/mol at bicelle conditions where the sampling of random helix–helix orientations leads to complex formation. At bicelle conditions that approach a true bilayer structure in effect, an entropy saving of > 1 kcal/mol was obtained from helix–helix preorientation. The magnitudes of enthalpy and entropy changes increased distinctly with bicelle dimensions, indicating long-range changes in bicelle lipid properties upon αIIbβ3 TM association. NMR spectroscopy confirmed ITC affinity measurements and revealed αIIbβ3 association and dissociation rates of 4500 ± 100 s^− 1 and 2.1 ± 0.1 s^− 1, respectively. Thus, ITC is able to provide comprehensive insight into the interaction of membrane proteins.     

Effects of trehalose-loaded liposomes on red blood cell response to freezing and post-thaw membrane quality

We are investigating the use of liposomes, which are synthetic, microscopic vesicles, for the intracellular delivery of trehalose into mammalian cells. This study focuses on the effects trehalose-containing liposomes improve the recovery and membrane quality of human RBCs following cryopreservation. Unilamellar liposomes consisting of a lipid bilayer composed of DPPC, PS and cholesterol (60:30:10 mol%) were synthesized using an extrusion method. Liposome-treated RBCs (l-RBCs) were resuspended in either physiological saline, 0.3 M trehalose or liposome solution, then cooled with slow (0.95 ± 0.02 °C/min), medium (73 ± 3 °C/min) and fast (265 ± 12 °C/min) cooling rates and storage in liquid nitrogen, followed by a 37 °C thawing step. RBC post-thaw quality was assessed using percent recovery, RBC morphology, PS and CD47 expression. Liposome treatment did not adversely affect the RBC membrane. Post-thaw recovery of l-RBCs was significantly higher (66% ± 5% vs 29% ± 4%) compared to control RBCs (c-RBC, p = 0.003). Medium and high cooling rates resulted in significantly higher cell recovery compared to a slow cooling rate (p = 0.039 and p = 0.041, respectively). The recovery of l-RBCs frozen in liposome solution and trehalose solution was significantly higher than that of l-RBCs frozen in NaCl solution for all three cooling rates (p = 0.021). Flow cytometry and morphology assessment showed that liposome treatment resulted in improved post-thaw membrane quality. There was no statistically significant difference in the post-thaw recovery between RBCs treated with liposomes containing trehalose in their aqueous core and RBCs treated with liposomes containing saline in their aqueous core (p = 0.114). Liposome treatment significantly improves the recovery and membrane integrity of RBCs following low temperature exposure.     

Effects on capacitance by overexpression of membrane proteins

Functional Channelrhodopsin-2 (ChR2) overexpression of about 10⁴ channels/μm² in the plasma membrane of HEK293 cells was studied by patch-clamp and freeze-fracture electron microscopy. Simultaneous electrorotation measurements revealed that ChR2 expression was accompanied by a marked increase of the area-specific membrane capacitance (C_m). The C_m increase apparently resulted partly from an enlargement of the size and/or number of microvilli. This is suggested by a relatively large C_m of 1.15 ± 0.08 μF/cm² in ChR2-expressing cells measured under isotonic conditions. This value was much higher than that of the control HEK293 cells (0.79 ± 0.02 μF/cm²). However, even after complete loss of microvilli under strong hypoosmolar conditions (100 mOsm), the ChR2-expressing cells still exhibited a significantly larger C_m (0.85 ± 0.07 μF/cm²) as compared to non-expressing control cells (0.70 ± 0.03 μF/cm²). Therefore, a second mechanism of capacitance increase may involve changes in the membrane permittivity and/or thickness due to the embedded ChR2 proteins.     

Spermatozoa from the maned wolf (Chrysocyon brachyurus) display typical canid hyper-sensitivity to osmotic and freezing-induced injury,but respond favorably to dimethyl sulfoxide

We assessed the influences of medium osmolality, cryoprotectant and cooling and warming rate on maned wolf (Chrysocyon brachyurus) spermatozoa. Ejaculates were exposed to Ham’s F10 medium (isotonic control) or to this medium plus NaCl (350–1000 mOsm), sucrose (369 and 479 mOsm), 1 M glycerol (1086 mOsm) or dimethyl sulfoxide (Me₂SO, 1151 mOsm) for 10 min. Each sample then was diluted back into Ham’s medium and assessed for sperm motility and plasma membrane integrity. Although glycerol and Me₂SO had no influence (P > 0.05), NaCl and sucrose solutions affected sperm motility (P < 0.05), but not membrane integrity. Motility of sperm exposed to <600 mOsm NaCl or sucrose was less (P < 0.05) than fresh ejaculate, but comparable (P > 0.05) to the control. As osmolality of the NaCl solution increased, motility decreased to <5%. In a separate study, ejaculates were diluted in Test Yolk Buffer containing 1 M glycerol or Me₂SO and cooled from 5 °C to −120 °C at −57.8 °C, −124.2 °C or −67.0 °C/min, frozen in LN₂, thawed in a water bath for 30 s at 37 °C or 10 s at 50 °C, and then assessed for motility, plasma- and acrosomal membrane integrity. Cryopreservation markedly (P < 0.05) reduced sperm motility by 70% compared to fresh samples. Higher (P < 0.05) post-thaw motility (20.0 ± 1.9% versus 13.5 ± 2.1%) and membrane integrity (51.2 ± 1.7% versus 41.5 ± 2.2%) were observed in samples cryopreserved in Me₂SO than in glycerol. Cooling rates influenced survival of sperm cryopreserved in glycerol with −57.8 °C/min being advantageous (P < 0.05). The findings demonstrate that although maned wolf spermatozoa are similar to domestic dog sperm in their sensitivity to osmotic-induced motility damage, the plasma membranes tolerate dehydration, and the cells respond favorably to Me₂SO as a cryoprotectant.     

Lateral pressure dependence of the phospholipid transmembrane diffusion rate in planar-supported lipid bilayers

The dependence of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) flip-flop kinetics on the lateral membrane pressure in a phospholipid bilayer was investigated by sum-frequency vibrational spectroscopy. Planar-supported lipid bilayers were prepared on fused silica supports using the Langmuir-Blodgett/Langmuir-Schaeffer technique, which allows precise control over the lateral surface pressure and packing density of the membrane. The lipid bilayer deposition pressure was varied from 28 to 42 mN/m. The kinetics of lipid flip-flop in these membranes was measured by sum-frequency vibrational spectroscopy at 37°C. An order-of-magnitude difference in the rate constant for lipid translocation (10.9 × 10⁻⁴ s⁻¹ to 1.03 × 10⁻⁴ s⁻¹) was measured for membranes prepared at 28 mN/m and 42 mN/m, respectively. This change in rate results from only a 7.4% change in the packing density of the lipids in the bilayer. From the observed kinetics, the area of activation for native phospholipid flip-flop in a protein-free DPPC planar-supported lipid bilayer was determined to be 73 ± 12 Å²/molecule at 37°C. Significance of the observed activation area and potential future applications of the technique to the study of phospholipid flip-flop are discussed.     

Packing analysis of carbohydrates and polysaccharides. V. Crystal structures of two polymorphs of pachyman triacetate

The crystal structures of two polymorphic forms of pachyman triacetate, the fully acetylated derivative of a naturally occuring β-(1 → 3)-D -glucan, were determined by a combination of stereochemical and x-ray diffraction analysis. The two polymorphs could be obtained depending on the temperature and the degree of stretching of film specimens of the substance: polymorph I resulted from stretching 25–50% at 125°C and polymorph II resulted from further stretching to 300% at 215°C. Both polymorphs had previously been shown to have sixfold helical chain conformations, but of unequal pitch. Subsequent detailed structure refinement performed with bond lengths, bond angles, conformational angles, and helix-packing parameters as refinement variables, and the simultaneous minimization of packing and conformational energy and the crystallographic R-factor as refinement criteria, resulted in a complete determination of the two crystal structures. Pachyman triacetate I was found to be a right-handed helix packing with antiparallel polarity and space group P2₁2₁2₁ symmetry (unit-cell parameters a = 11.0, b = 19.0, c (fiber repeat) = 22.38 Å). The acetate groups were nearly planar and the O(2) and O(4) acetates were oriented in such a fashion that the carbonyl double-bond nearly eclipsed the corresponding C—H bond of the ring. The O(6) was in the tg position and its acetate was oriented in such a fashion that the bond sequence C(6)—O(6)—C(6C)—C(6M) was nearly trans-planar, with the carbonyl double-bond bisecting the tetrahedral angle formed by C(6) and its two hydrogens. The final R = 0.221. Pachyman triacetate II was similarly found to be a right-handed helix, but packing as a 50:50 mixture of parallel and antiparallel polarities (unit-cell parameters a = 11.49, b = 20.13, c (fiber repeat) = 18.6 Å). The acetate positions in pachyman triacetate II were substantially the same as in pachyman triacetate I. The final R for the 50:50 mixture was 0.234. Probable reasons for the change in packing polarities are discussed, as are the difficulties encountered in the structure refinement of acetate derivatives.     

Conformational behavior of α-d-mannopyranosyl-(1→6)-α,β-d-mannose complexed with two mannose-binding plant lectins, Allium sativam agglutinin I and concanavalin A, using NMR and molecular modeling techniques

Herein, we report the intrinsic conformational preferences of α-d-Manp-(1→6)-α,β-d-Manp, (1) in the free state and as two (ASAI and ConA) lectin-bound forms. NMR spectroscopy and molecular dynamics techniques are used as 3D-structural determination tools. In free form disaccharide 1 displays a fair amount of conformational freedom, with one major (?/ψ 95 ± 30°/195 ± 20°) and one minor (95 ± 30°/70 ± 20°) conformations around the glycosidic linkage and around the ω angle, both the gg and gt rotamers are almost equally populated. This is a first report of a three-dimensional structure of 1 bound with ASAI. Both lectins recognize a major ?/ψ 95 ± 30°/200 ± 30° conformer with the ligand showing more flexibility in the binding site of ConA. Comparison of the mode of binding of the two lectins explains the differences in observed specificities.     

Evaporative water loss and energy metabolic in two small mammals,voles (Eothenomys miletus) and mice (Apodemus chevrieri), in Hengduan mountains region

Evaporative water loss (EWL) and energy metabolism were measured at different temperatures in Eothenomys miletus and Apodemus chevrieri in dry air. The thermal neutral zone (TNZ) of E. miletus was 22.5–30 °C and that of A. chevrieri was 20–27.5 °C. Mean body temperatures of the two species were 35.75±0.5 and 36.54±0.61 °C. Basal metabolic rates (BMR) were 1.92±0.17 and 2.7±0.5 ml O₂/g h, respectively. Average minimum thermal conductance (C_m) were 0.23±0.08 and 0.25±0.06 ml O₂/g h °C. EWL in E. miletus and A. chevrieri increased with the increase in temperature; the maximal EWL at 35 °C was 4.78±0.6 mg H₂O/g h in E. miletus, and 5.92±0.43 mg H₂O/g h in A. chevrieri. Percentage of evaporative heat loss to total heat production (EHL/HP) increased with the increase in temperature; the maximal EHL/HP was 22.45% at 30 °C in E. miletus, and in A. chevrieri it was 19.96% at 27.5 °C. The results may reflect features of small rodents in the Hengduan mountains region: both E. miletus and A. chevrieri have high levels of BMR and high levels of total thermal conductance, compared with the predicted values based on their body masses, while their body temperatures are relatively low. EWL plays an important role in temperature regulation.     

Thermopreference,tolerance and metabolic rate of early stages juvenile Octopus maya acclimated to different temperatures

Thermopreference, tolerance and oxygen consumption rates of early juveniles Octopus maya (O. maya; weight range 0.38–0.78 g) were determined after acclimating the octopuses to temperatures (18, 22, 26, and 30 °C) for 20 days. The results indicated a direct relationship between preferred temperature (PT) and acclimated temperature, the PT was 23.4 °C. Critical Thermal Maxima, (CTMax; 31.8±1.2, 32.7±0.9, 34.8±1.4 and 36.5±1.0) and Critical Thermal Minima, (CTMin; 11.6±0.2, 12.8±0.6, 13.7±1.0, 19.00±0.9) increased significantly (P<0.05) with increasing acclimation temperatures. The endpoint for CTMax was ink release and for CTMin was tentacles curled, respectively. A thermal tolerance polygon over the range of 18–30 °C resulted in a calculated area of 210.0 °C². The oxygen consumption rate increased significantly α=0.05 with increasing acclimation temperatures between 18 and 30 °C. Maximum and minimum temperature quotients (Q₁₀) were observed between 26–30 °C and 22–26 °C as 3.03 and 1.71, respectively. These results suggest that O. maya has an increased capability for adapting to moderate temperatures, and suggest increased culture potential in subtropical regions southeast of México.     

Effect of storage temperature on survival and infectivity of Steinernema rarum (OLI strain) (Rhabditida: Steinernematidae)

Nematode strains of the entomopathogenic family Steinernematidae differ in their ability to infect insects at different temperatures. Survival and infectivity of infective juveniles (IJs) of Steinernema rarum (OLI) were studied after their storage at 23 ± 2 °C and at 5 ± 1 °C. Survival at 23 ± 2 °C was always above 95%. At 5 ± 1 °C, survival decreased at week 5, but infectivity did the same after week 2. Unlike other steinernematids, both infectivity and survival of IJs would be higher for S. rarum (OLI) when stored at 23 ± 2 °C.