Calabaria and the phytogeny of erycine snakes

Two major subgroups of erycine snakes, designated Charina and Eryx , are delimited with a cladistic analysis of 75 morphological characters. The hypotheses of species relationships within the two clades are ( reinhardtii ( bottae, trivirgata )) and ( colubrinus, conicus, elegans, jayakari, muelleri, somalicus ( miliaris ( tataricus ( jaculus, johnii )))), respectively. This pattern of grouping obtains without assuming multistate character additivity. At least 16 synapomorphies indicate that reinhardtii is an erycine and that it is the sister lineage of the ( bottae, trivirgata ) clade. Calabaria and Lichanura are synonymized with Charina for reasons of taxonomic efficiency, and to emphasize the New-Old World geographic distribution of the three species in that assemblage. Further resolution of Eryx species relationships is required before Gongylophis (type species conicus ) can be recognized.     

Behavioural variation in natural populations. V. Morphological correlates of locomotion in the garter snake (Thamnophis radix)

A series of morphologieal and locomotor performance variables was measured in a population of newborn garter snakes to determine whether performance capacity has a significant morphological basis in these animals. Morphological traits measured were body length and mass, number of body and tail vertebrae and numbers of vertebral abnormalities. Locomotor performances included burst and mid-distance speed and distance and time crawled before anti-predator displays were assumed. All performance variables were repeatable in daily replicate trials ( P < 0.001). Individual burst speed, mid-distance speed, and distance crawled were significantly correlated pairwise ( P < 0.01). Most morphological and performance variables had a significant mass dependence (static allometry), although the effects were rather weak ( r ² < 0.1, except for body length): larger animals performed better and had fewer abnormalities. There were significant associations between some morphological traits and locomotor performance. Morphological factors accounted for 19% of the variation in mid-distance speed and 14% of the variation in antipredator behavior by multiple regression analysis. Canonical correlation of all performance and morphological variables simultaneously accounted for 24% of the observed variation in performance. Numbers of body and tail vertebrae (assayed by scale counts) had an interactive effect on speed of locomotion.     

Some Microscopical Observations of Functioning Sieve Tubes of Heracleum using Nomarski Optics

The study of isolated phloem in Heracleum has been extendedto intact functioning sieve tubes. Techniques of phloem dissectioncombined with Nomarski interference optics have been developedto permit useful visual observations, photomicrography, andciné photographs of sieve tubes which are apparentlynormal. In these preparations, plastids and organelles calledby us ‘marker particles’ are visible in rapid bouncingmotion, and the state of dispersal of these particles is relatedto the amount of damage done to the preparations. The movement of the marker particles and their subsequent fixationshows that they are apparently attached to or restrained byan invisible network in situ. The network is very sensitiveto disturbance and readily collapses around the sieve platesto form slime plugs upon damage to the sieve element. The markerparticles do not move through the cell nor across sieve platesin mature Heracleum. In young cells cyclosis is observable alongthe periphery and this suggests that a vacuole may then be present.In undamaged mature cells there was evidence neither of a vacuolenor of trans-cellular tubules of any size optically detectable. The motion of the particles was greater than Brownian movementand appeared to be under some physiological control. Their movementprobably indicated the presence of an operating transport phenomenon,either because solution was moving past them in the sieve tubeor because they were themselves attached to a contractile networkactively in pulsatory motion. Nearby companion and parenchymacells showed normal cytoplasmic streaming. Proposed mechanisms of translocation involving cytoplasmic streamingdo not seem to be applicable to the phloem of Heracleum. Themovement of the marker particles seemed to agree best with amechanism of ‘activated’ mass flow.     

Protein conformational exchange measured by 1H R1ρ relaxation dispersion of methyl groups

Activated dynamics plays a central role in protein function, where transitions between distinct conformations often underlie the switching between active and inactive states. The characteristic time scales of these transitions typically fall in the microsecond to millisecond range, which is amenable to investigations by NMR relaxation dispersion experiments. Processes at the faster end of this range are more challenging to study, because higher RF field strengths are required to achieve refocusing of the exchanging magnetization. Here we describe a rotating-frame relaxation dispersion experiment for ¹H spins in methyl ¹³CHD₂ groups, which improves the characterization of fast exchange processes. The influence of ¹H–¹H rotating-frame nuclear Overhauser effects (ROE) is shown to be negligible, based on a comparison of R _1ρ relaxation data acquired with tilt angles of 90° and 35°, in which the ROE is maximal and minimal, respectively, and on samples containing different ¹H densities surrounding the monitored methyl groups. The method was applied to ubiquitin and the apo form of calmodulin. We find that ubiquitin does not exhibit any ¹H relaxation dispersion of its methyl groups at 10 or 25 °C. By contrast, calmodulin shows significant conformational exchange of the methionine methyl groups in its C-terminal domain, as previously demonstrated by ¹H and ¹³C CPMG experiments. The present R _1ρ experiment extends the relaxation dispersion profile towards higher refocusing frequencies, which improves the definition of the exchange correlation time, compared to previous results.     

Substrate Specificity of and Product Formation by Muconate Cycloisomerases: an Analysis of Wild-Type Enzymes and Engineered Variants

Muconate cycloisomerases play a crucial role in the bacterial degradation of aromatic compounds by converting cis,cis-muconate, the product of catechol ring cleavage, to (4S)-muconolactone. Chloromuconate cycloisomerases catalyze both the corresponding reaction and a dehalogenation reaction in the transformation of chloroaromatic compounds. This study reports the first thorough examination of the substrate specificity of the muconate cycloisomerases from Pseudomonas putida PRS2000 and Acinetobacter “calcoaceticus” ADP1. We show that they transform, in addition to cis,cis-muconate, 3-fluoro-, 2-methyl-, and 3-methyl-cis,cis-muconate with high specificity constants but not 2-fluoro-, 2-chloro-, 3-chloro-, or 2,4-dichloro-cis,cis-muconate. Based on known three-dimensional structures, variants of P. putida muconate cycloisomerase were constructed by site-directed mutagenesis to contain amino acids found in equivalent positions in chloromuconate cycloisomerases. Some of the variants had significantly increased specificity constants for 3-chloro- or 2,4-dichloromuconate (e.g., A271S and I54V showed 27- and 22-fold increases, respectively, for the former substrate). These kinetic improvements were not accompanied by a change from protoanemonin to cis,cis-dienelactone as the product of 3-chloro-cis,cis-muconate conversion. The rate of 2-chloro-cis,cis-muconate turnover was not significantly improved, nor was this compound dehalogenated to any significant extent. However, the direction of 2-chloro-cis,cis-muconate cycloisomerization could be influenced by amino acid exchange. While the wild-type enzyme discriminated only slightly between the two possible cycloisomerization directions, some of the enzyme variants showed a strong preference for either (+)-2-chloro- or (+)-5-chloromuconolactone formation. These results show that the different catalytic characteristics of muconate and chloromuconate cycloisomerases are due to a number of features that can be changed independently of each other.