Studies on the regioselectivity and kinetics of the action of trypsin on proinsulin and its derivatives using mass spectrometry

Human M-proinsulin was cleaved by trypsin at the R³¹R³²–E³³ and K⁶⁴R⁶⁵–G⁶⁶ bonds (B/C and C/A junctions), showing the same cleavage specificity as exhibited by prohormone convertases 1 and 2 respectively. Buffalo/bovine M-proinsulin was also cleaved by trypsin at the K⁵⁹R⁶⁰–G⁶¹ bond but at the B/C junction cleavage occurred at the R³¹R³²–E³³ as well as the R³¹–R³²E³³ bond. Thus, the human isoform in the native state, with a 31 residue connecting C-peptide, seems to have a unique structure around the B/C and C/A junctions and cleavage at these sites is predominantly governed by the structure of the proinsulin itself. In the case of both the proinsulin species the cleavage at the B/C junction was preferred (65%) over that at the C/A junction (35%) supporting the earlier suggestion of the presence of some form of secondary structure at the C/A junction. Proinsulin and its derivatives, as natural substrates for trypsin, were used and mass spectrometric analysis showed that the k_cat./K_m values for the cleavage were most favourable for the scission of the bonds at the two junctions (1.02 ± 0.08 × 10⁵ s^− 1 M^− 1) and the cleavage of the K²⁹–T³⁰ bond of M-insulin-RR (1.3 ± 0.07 × 10⁵ s^− 1 M^− 1). However, the K²⁹–T³⁰ bond in M-insulin, insulin as well as M-proinsulin was shielded from attack by trypsin (k_cat./K_m values around 1000 s^− 1 M^− 1). Hence, as the biosynthetic path follows the sequence; proinsulin → insulin-RR → insulin, the K²⁹–T³⁰ bond becomes shielded, exposed then shielded again respectively.     

Seed size,number and strategies in annual plants: a comparative functional analysis and synthesis

Background and AimsPlants depend fundamentally on establishment from seed. However, protocols in trait-based ecology currently estimate seed size but not seed number. This can be rectified. For annuals, seed number should simply be a positive function of vegetative biomass and a negative function of seed size.MethodsUsing published values of comparative seed number as the ‘gold standard’ and a large functional database, comparative seed yield and number per plant and per m² were predicted by multiple regression. Subsequently, ecological variation in each was explored for English and Spanish habitats, newly calculated C-S-R strategies and changed abundance in the British flora.Key ResultsAs predicted, comparative seed mass yield per plant was consistently a positive function of plant size and competitive ability, and largely independent of seed size. Regressions estimating comparative seed number included, additionally, seed size as a negative function. Relationships differed numerically between regions, habitats and C-S-R strategies. Moreover, some species differed in life history over their geographical range. Comparative seed yield per m² was positively correlated with FAO crop yield, and increasing British annuals produced numerous seeds. Nevertheless, predicted values must be viewed as comparative rather than absolute: they varied according to the ‘gold standard’ predictor used. Moreover, regressions estimating comparative seed yield per m² achieved low precision.ConclusionsFor the first time, estimates of comparative seed yield and number for >800 annuals and their predictor equations have been produced and the ecological importance of these regenerative traits has been illustrated. ‘Regenerative trait-based ecology’ remains in its infancy, with work needed on determinate vs. indeterminate flowering (‘bet-hedging’), C-S-R methodologies, phylogeny, comparative seed yield per m² and changing life history. Nevertheless, this has been a positive start and readers are invited to use estimates for >800 annuals, in the Supplementary data, to help advance ‘regenerative trait-based ecology’ to the next level.     

Canopy structure of tropical and sub-tropical rain forests in relation to conifer dominance analysed with a portable LIDAR system

Background and Aims

Globally, conifer dominance is restricted to nutient-poor habitats in colder, drier or waterlogged environments, probably due to competition with angiosperms. Analysis of canopy structure is important for understanding the mechanism of plant coexistence in relation to competition for light. Most conifers are shade intolerant, and often have narrow, deep, conical crowns. In this study it is predicted that conifer-admixed forests have less distinct upper canopies and more undulating canopy surfaces than angiosperm-dominated forests.

Methods

By using a ground-based, portable light detection and ranging (LIDAR) system, canopy structure was quantified for old-growth evergreen rainforests with varying dominance of conifers along altitudinal gradients (200–3100 m a.s.l.) on tropical and sub-tropical mountains (Mount Kinabalu, Malaysian Borneo and Yakushima Island, Japan) that have different conifer floras.

Key Results

Conifers dominated at higher elevations on both mountains (Podocarpaceae and Araucariaceae on Kinabalu and Cupressaceae and Pinaceae on Yakushima), but conifer dominance also varied with soil/substrate conditions on Kinabalu. Conifer dominance was associated with the existence of large-diameter conifers. Forests with higher conifer dominance showed a canopy height profile (CHP) more skewed towards the understorey on both Kinabalu and Yakushima. In contrast, angiosperm-dominated forests had a CHP skewed towards upper canopy, except for lowland dipterocarp forests and a sub-alpine scrub dominated by small-leaved Leptospermum recurvum (Myrtaceae) on Kinabalu. Forests with a less dense upper canopy had more undulating outer canopy surfaces. Mixed conifer–angiosperm forests on Yakushima and dipterocarp forests on Kinabalu showed similar canopy structures.

Conclusions

The results generally supported the prediction, suggesting that lower growth of angiosperm trees (except L. recurvum on Kinabalu) in cold and nutrient-poor environments results in a sparser upper canopy, which allows shade-intolerant conifers to co-occur with angiosperm trees either as emergents or as codominants in the open canopy.     

稀有鮈鲫(Gobiocypris rarus)的骨骼特征及系统发育地位

稀有鮈鲫(Gobiocypris rarus)已作为新型实验动物而逐渐成为生物学研究各领域的热门对象,但是,其系统分类位置仍存在争议。本研究制作了稀有鮈鲫的透明骨骼标本,对其骨骼特征进行描述;选取47个形态特征,与鲤科各亚科鱼类的典型特征进行比较,建立了分支分析特征矩阵,并使用PAUP4.0软件中的最大简约法(MP)构建系统发育树。结果显示,稀有鮈鲫和鮈亚科鱼类聚在一起,属于鮈亚科。     

Validation of an innovative method,based on tilt sensing,for the assessment of activity and body position

Since there is less movement during sleep than during wake, the recording of body movements by actigraphy has been used to indirectly evaluate the sleep–wake cycle. In general, most actigraphic devices are placed on the wrist and their measures are based on acceleration detection. Here, we propose an alternative way of measuring actigraphy at the level of the arm for joint evaluation of activity and body position. This method analyzes the tilt of three axes, scoring activity as the cumulative change of degrees per minute with respect to the previous sampling, and measuring arm tilt for the body position inference. In this study, subjects (N?=?13) went about their daily routine for 7 days, kept daily sleep logs, wore three ambulatory monitoring devices and collected sequential saliva samples during evenings for the measurement of dim light melatonin onset (DLMO). These devices measured motor activity (arm activity, AA) and body position (P) using the tilt sensing of the arm, with acceleration (wrist acceleration, WA) and skin temperature at wrist level (WT). Cosinor, Fourier and non-parametric rhythmic analyses were performed for the different variables, and the results were compared by the ANOVA test. Linear correlations were also performed between actimetry methods (AA and WA) and WT. The AA and WA suitability for circadian phase prediction and for evaluating the sleep–wake cycle was assessed by comparison with the DLMO and sleep logs, respectively. All correlations between rhythmic parameters obtained from AA and WA were highly significant. Only parameters related to activity levels, such as mesor, RA (relative amplitude), VL5 and VM10 (value for the 5 and 10 consecutive hours of minimum and maximum activity, respectively) showed significant differences between AA and WA records. However, when a correlation analysis was performed on the phase markers acrophase, mid-time for the 10 consecutive hours of highest (M10) and mid-time for the five consecutive hours of lowest activity (L5) with DLMO, all of them showed a significant correlation for AA (R?=?0.607, p?=?0.028; R?=?0.582, p?=?0.037; R?=?0.620, p?=?0.031, respectively), while for WA, only acrophase did (R?=?0.621, p?=?0.031). Regarding sleep detection, WA showed higher specificity than AA (0.95?±?0.01 versus 0.86?±?0.02), while the agreement rate and sensitivity were higher for AA (0.76?±?0.02 versus 0.66?±?0.02 and 0.71?±?0.03 versus 0.53?±?0.03, respectively). Cohen’s kappa coefficient also presented the highest values for AA (0.49?±?0.04) and AP (0.64?±?0.04), followed by WT (0.45?±?0.06) and WA (0.37?±?0.04). The findings demonstrate that this alternative actigraphy method (AA), based on tilt sensing of the arm, can be used to reliably evaluate the activity and sleep–wake rhythm, since it presents a higher agreement rate and sensitivity for detecting sleep, at the same time allows the detection of body position and improves circadian phase assessment compared to the classical actigraphic method based on wrist acceleration.     

Assessment of post-stroke elbow flexor spasticity in different forearm positions

Abstract

Purpose/Aim: There have been conflicting results regarding which muscle contribute most to the elbow spastic flexion deformity. This study aimed to investigate whether flexor spasticity of the elbow changed according to the position of the forearm, and to determine the muscle or muscles that contributed most to the elbow spastic flexion deformity by clinical examination.

Methods: This study is a single group, observational and cross-sectional study. Sixty patients were assessed for elbow flexor spasticity in different forearm positions (pronation, neutral and supination) with Modified Tardieu Scale. The primary outcome measure was a domain of the Modified Tardieu Scale, the dynamic component of spasticity (spasticity angle).

Results: In general, there was a significant difference between forearm positions regarding spasticity angle (p?<?.001). In pairwise comparisons, median spasticity angles in pronation (70 degrees) and neutral position (60 degrees) were significantly higher than those in supination (57.5 degrees) (adjusted p?<?.001 and adjusted p?=?.003, respectively). However, median spasticity angle in pronation did not differ significantly from those in neutral position in favour of pronation (adjusted p?=?.274).

Conclusions: The severity of spasticity changes according to the elbow position which suggests that the magnitude of contribution of each elbow flexor muscle to spastic elbow deformity is different. Reduction of spasticity from pronation to supination leads us to consider brachialis as the most spastic muscle. Since biceps was suggested to be the least spastic muscle in this study, and also to avoid spastic pronation deformity of the forearm, it should be rethought before performing chemodenervation into biceps muscle.     

Neuromuscular dysfunction with the experimental arm acting as its own reference following eccentric and isometric exercise

Eccentric exercise has been extensively used as a model to study muscle damage-induced neuromuscular impairment, adopting mainly a bilateral matching task between the reference (unexercised) arm and the indicator (exercised) arm. However, little attention has been given to the muscle proprioceptive function when the exercised arm acts as its own reference. This study investigated muscle proprioception and motor control, with the arm acting both as reference and indicator, following eccentric exercise and compared them with those observed after isometric exercise. Fourteen young male volunteers were equally divided into two groups and performed an eccentric or isometric exercise protocol with the elbow flexors of the non-dominant arm on an isokinetic dynamometer. Both exercise protocols induced significant changes in indicators of muscle damage, that is, muscle soreness, range of motion and maximal isometric force post-exercise (p < 0.05–0.001), and neuromuscular function was similarly affected following both protocols. Perception of force was impaired over the 4-day post-exercise period (p < 0.001), with the applied force being systematically overestimated. Perception of joint position was significantly disturbed (i.e., target angle was underestimated) only at one elbow angle on day 4 post-exercise (p < 0.05). The misjudgements and disturbed motor output observed when the exercised arm acted as its own reference concur with the view that they could be a result of a mismatch between the central motor command and an impaired motor control after muscle damage.     

Yellow‐chevroned Parakeet (Brotogeris chiriri) abundance and canopy foraging at a dry forest in western Brazil

I studied Brotogeris chiriri abundance and foraging activity at a dry forest of the Urucum mountains in western Mato Grosso do Sul, Brazil, to evaluate their relationships with food resource production. Brotogeris chiriri abundance sharply increased during the early wet season (mainly October 2001) when it mostly foraged for fleshy fruits. At that time Protium heptaphyllum, one of the most common tree species, bore a large crop of fruits, the arils of which were extensively consumed by B. chiriri. Conversely, only a few parakeets were recorded foraging from the late wet to the late dry season, when dry fruit production predominated. The monthly pattern of parakeet abundance paralleled both its monthly pattern of foraging activity and fleshy fruit availability. Moreover, the variations in foraging activity were highly correlated to fleshy fruit production. Thus, data presented here evidenced the effect of both fruiting pulses and a common tree species that produced a large and ephemeral fruit crop, on the dynamic of a small and mobile canopy forager at a primary dry forest.