Phosphate uptake and polyphosphate metabolism of mycorrhizal and nonmycorrhizal roots of pine and of Suillus bovinus at varying external pH measured by in vivo 31P-NMR

 Comparative in vivo ³¹P-NMR analyses of mycorrhizal and nonmycorrhizal roots of Pinus sylvestris and the fungus of Suillus bovinus in pure culture were used to investigate alterations in phosphate metabolism due to changes in external pH in the range 3.5–8.5. All control samples maintained a constant pH in both cytoplasm and vacuole. Mycorrhizal roots and pure fungus, but not nonmycorrhizal roots, transformed accumulated inorganic phosphate into mobile polyphosphate with a medium chain length. Phosphate uptake rates and polyphosphate accumulation responded differently to external pH. In all cases, maximal phosphate uptake occurred at an external pH close to 5.5. At an external pH of 8.5, both roots and fungus showed a distinct lag in phosphate uptake, which was abolished when the external pH was lowered to 7.5. An irreversible effect on phosphate uptake as a consequence of variation in external pH was also observed. The central role of the fungus in regulating mycorrhizal phosphate metabolism is discussed. Accepted: 15 April 1997     

An o-nitrobenzyl scaffold for peptide ligation: synthesis and applications

Chemical ligation approaches facilitate the chemoselective assembly of unprotected peptides in aqueous solution. Here, two photolabile auxiliaries are described that enlarge the applicability of native chemical ligation to non-cysteine targets. The auxiliaries, designed to allow reaction with thioester peptides, generate an amide bond between the two initial fragments. The o-nitrobenzyl tertiary benzylamide that is formed at the ligation junction can be transformed into a native amide group under mild photolysis conditions. The veratryl auxiliary was found to be excessively labile during peptide purification and ligation. However, the auxiliary based on the o-nitrobenzyl group shows all the necessary properties for a general application in routine peptide and protein synthesis. In addition, the auxiliary linked to the N-terminus can be efficiently photolyzed, suggesting a new approach for the generation of photocaged amines. Synthesis, solid phase introduction onto peptide chains, ligation properties and photolysis in water are described, and a careful study of compatibility of the method with potentially fragile peptide side chains is reported.     

Temperature and ligand dependence of conformation and helical order in myosin filaments

Mammalian myosin filaments are helically ordered only at higher temperatures (>20 degrees C) and become progressively more disordered as the temperature is decreased. It had previously been suggested that this was a consequence of the dependence of the hydrolytic step of myosin ATPase on temperature and the requirement that hydrolysis products (e.g., ADP.P(i)) be bound at the active site. An alternative hypothesis is that temperature directly affects the conformation of the myosin heads and that they need to be in a particular conformation for helical order in the filament. To discriminate between these two hypotheses, we have studied the effect of temperature on the helical order of myosin heads in rabbit psoas muscle in the presence of nonhydrolyzable ligands. The muscle fibers were overstretched to nonoverlap such that myosin affinity for nucleotides was not influenced by the interaction of myosin with the thin filament. We show that with bound ADP.vanadate, which mimics the transition state between ATP and hydrolysis products, or with the ATP analogues AMP-PNP or ADP.BeF(x)() the myosin filaments are substantially ordered at higher temperatures but are reversibly disordered by cooling. These results reinforce recent studies in solution showing that temperature as well as ligand influence the equilibrium between multiple myosin conformations [Málnási-Csizmadia, A., Pearson, D. S., Kovács, M., Woolley, R. J., Geeves, M. A., and Bagshaw, C. R. (2001) Biochemistry 40, 12727-12737; Málnási-Csizmadia, A., Woolley, R. J., and Bagshaw, C. R. (2000) Biochemistry 39, 16135-16146; Urbanke, C., and Wray, J. (2001) Biochem. J. 358, 165-173] and indicate that helical order requires the myosin heads to be in the closed conformation. Our results suggest that most of the heads in the closed conformation are ordered, and that order is not produced in a separate step. Hence, helical order can be used as a signature of the closed conformation in relaxed muscle. Analysis of the dependence on temperature of helical order and myosin conformation shows that in the presence of these analogues one ordered (closed) conformation and two disordered conformations with distinct thermodynamic properties coexist. Low temperatures favor one disordered conformation, while high temperatures favor the ordered (closed) conformation together with a second disordered conformation.     

Generalized Crick equations for modeling noncanonical coiled coils

Crick envisaged the alpha-helical coiled coil to result from systematic bending of an alpha-helix such that every seventh residue was structurally equivalent, and he derived equations for the coordinates of the backbone atoms. Crick's predictions were vindicated experimentally and coiled-coil sequences were shown to have hydrophobic residues alternately spaced 3 and 4 residues apart. Nonetheless, in some coiled coils such canonical heptad repeats are interrupted by inserts of 3 or 4 residues generating decad and hendecad motifs. The supercoiling of the coiled coils varies with the sequence pattern, being left- or right-handed in purely heptad-based or hendecad-based motifs, respectively. To model coiled coils with a mixture of motifs, we describe how Crick's equations can be modified for cases where the pitch is not constant. Using the analogy of the bending of a beam, we took the tilt angle to change linearly with distance along the major helix and the pitch of a motif to be affected by neighboring motifs depending on the rigidity of the alpha-helical strands. We tested our approach by fitting the two-, three-, and four-stranded noncanonical coiled coils of GrpE, hemagglutinin, and tetrabrachion. The backbone atoms of the model and crystal structures agreed with root mean square deviations of <1.1 A.     

A new model for the surface arrangement of myosin molecules in tarantula thick filaments

Three-dimensional reconstructions of the negatively stained thick filaments of tarantula muscle with a resolution of 50 A have previously suggested that the helical tracks of myosin heads are zigzagged, short diagonal ridges being connected by nearly axial links. However, surface views of lower contour levels reveal an additional J-shaped feature approximately the size and shape of a myosin head.We have modelled the surface array of myosin heads on the filaments using as a building block a model of a two-headed regulated myosin molecule in which the regulatory light chains of the two heads together form a compact head-tail junction. Four parameters defining the radius, orientation and rotation of each myosin molecule were varied. In addition, the heads were allowed independently to bend in a plane perpendicular to the coiled-coil tail at three sites, and to tilt with respect to the tail and to twist at one of these sites. After low-pass filtering, models were aligned with the reconstruction, scored by cross-correlation and refined by simulated annealing.Comparison of the geometry of the reconstruction and the distance between domains in the myosin molecule narrowed the choice of models to two main classes. A good match to the reconstruction was obtained with a model in which each ridge is formed from the motor domain of a head pointing to the bare zone together with the head-tail junction of a neighbouring molecule. The heads pointing to the Z-disc intermittently occupy the J-position. Each motor domain interacts with the essential and regulatory light chains of the neighbouring heads. A near-radial spoke in the reconstruction connecting the backbone to one end of the ridge can be identified as the start of the coiled-coil tail.     

Risk factors and milk yield losses associated with lameness in Holstein-Friesian dairy cattle

Weekly locomotion scores on a scale of 1 to 5 were used to investigate the relationship between cattle lameness, management systems and the impact of lameness on milk production. The data were 14026 locomotion scores from 248 Holstein-Friesian cows. Cows were managed in two groups, XE (high-concentrate feed and housed indoors all year) and XM (low-concentrate feed and outdoors in summer). Analysis was performed using residual maximum likelihood. Results indicated that the most significant variables affecting locomotion were time of year when the animal was locomotion scored and management group. Cows scored during February and August had increased locomotion problems. Cows in the more intensively managed group had significantly poorer locomotion compared with those in the more extensive group. Older animals were more susceptible to lameness than heifers. Body weight, body condition score and days in milk (DIM) also accounted for significant variation in locomotion score. Poor locomotion was associated with a significant reduction in the milk yield of later lactation cows. There was a significant difference in the shape of the lactation curve depending on whether or not the cow was lame during lactation. Average persistency was greater for the group of cows never lame throughout lactation compared with those lame before 60 DIM.     

Cloning, structural analysis, and mapping of the B30 and B7 multigenic families to the major histocompatibility complex (MHC) and other chromosomal regions

 We present the cloning, structural analysis, and mapping of new members belonging to two multigenic families, the B30-RING finger family and the B7.1-B7.2 family, as well as two genes derived by exon shuffling from members of these families. Eight new members were found and three of them map to the human major histocompatibilitiy complex (MHC) region. Phylogenic and physical mapping analysis allowed us to decipher the evolutionary story of these two multigenic families and to shed light on the evolution of the MHC region. We also show that a deductive analysis can be used to predict the existence of a given gene. Received: 17 February 1997 / Revised: 17 March 1997