Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Phloem loading of sucrose: involvement of membrane ATPase and proton transport

p-Chloromercuribenzenesulfonic acid markedly inhibited sucrose accumulation into sugar beet source leaves without inhibiting hexose accumulation. The site of inhibition is proposed to be the plasmalemma ATPase, since the ATPase-mediated H⁺ efflux was completely inhibited by p-chloromercuribenzenesulfonic acid under conditions where intracellular metabolism, as measured by photosynthesis and hexose accumulation, was unaffected. Fusicoccin, a potent activator of active H⁺/K⁺ exchange, stimulated both active sucrose accumulation and proton efflux in the sugar beet leaf tissue. These data provide strong evidence for the phloem loading of sucrose being coupled to a proton transport mechanism driven by a vectorial plasmalemma ATPase.     

The Mechanical Behaviour of Chondrocytes Predicted with a Micro-structural Model of Articular Cartilage

The integrity of articular cartilage depends on the proper functioning and mechanical stimulation of chondrocytes, the cells that synthesize extracellular matrix and maintain tissue health. The biosynthetic activity of chondrocytes is influenced by genetic factors, environmental influences, extracellular matrix composition, and mechanical factors. The mechanical environment of chondrocytes is believed to be an important determinant for joint health, and chondrocyte deformation in response to mechanical loading is speculated to be an important regulator of metabolic activity. In previous studies of chondrocyte deformation, articular cartilage was described as a biphasic material consisting of a homogeneous, isotropic, linearly elastic solid phase, and an inviscid fluid phase. However, articular cartilage is known to be anisotropic and inhomogeneous across its depth. Therefore, isotropic and homogeneous models cannot make appropriate predictions for tissue and cell stresses and strains. Here, we modelled articular cartilage as a transversely isotropic, inhomogeneous (TI) material in which the anisotropy and inhomogeneity arose naturally from the microstructure of the depth-dependent collagen fibril orientation and volumetric fraction, as well as the chondrocyte shape and volumetric fraction. The purpose of this study was to analyse the deformation behaviour of chondrocytes using the TI model of articular cartilage. In order to evaluate our model against experimental results, we simulated indentation and unconfined compression tests for nominal compressions of 15%. Chondrocyte deformations were analysed as a function of location within the tissue. The TI model predicted a non-uniform behaviour across tissue depth: in indentation testing, cell height decreased by 43% in the superficial zone and between 11 and 29% in the deep zone. In unconfined compression testing, cell height decreased by 32% in the superficial zone, 25% in the middle, and 18% in the deep zones. This predicted non-uniformity is in agreement with experimental studies. The novelty of this study is the use of a cartilage material model accounting for the intrinsic inhomogeneity and anisotropy of cartilage caused by its microstructure.     

Cumulative effects of fire on a tussock pampa grassland

Abstract. In order to test for cumulative effects of fire on Paspalum quadrifarium‐dominated grasslands (‘pajonal’), we analysed the impact of single and repeated fires on the community structure and post‐fire recovery of canopy after a final, simultaneous fire event. Nine plots were defined within a homogeneous pajonal stand, and treatments of low (LF), medium (MF) and high frequency (HF) of fire were defined by the application of one, two or four cold‐season burns, respectively, along a 6‐yr period. Both burned and unburned plots were exposed to grazing by cattle during the summer following the first and the third years of that period. High cattle preference for burned sites conditioned fire temperature and vegetation responses to the following burning events. Cumulative effects between successive burning events were observed for the cover of basal area of the dominant and other sprouting species, the cover and thickness of the litter layer, the seed bank size of the principal recruiter species, and the floristic composition. While light interception by the canopy was positively related to fire frequency during the early growth season, further growth of P. quadrifarium determined a greater light interception in LF than in MF and HF. These patterns of light interception were associated with a faster occupation of the inter‐tussock areas by opportunistic species in plots subjected to frequent fires (HF and MF) than in plots with low fire frequency (LF), and a more lasting regrowth of P. quadrifarium in the LF plot than in the HF ones. High fire frequencies reduced the dominance of P. quadrifarium. Percent of species classified as subordinated graminoids or forbs did not vary among treatments. However, the abundance of different forb species was differentially favoured by contrasting frequencies of fire, describing some coarse relationships between their specific responses and their dispersal strategies.     

Pathway of Phloem unloading of sucrose in corn roots

The pathway of phloem unloading and the metabolism of translocated sucrose were determined in corn (Zea mays) seedling roots. Several lines of evidence show that exogenous sucrose, unlike translocated sucrose, is hydrolyzed in the apoplast prior to uptake into the root cortical cells. These include (a) presence of cell wall invertase activity which represents 20% of the total tissue activity; (b) similarity in uptake and metabolism of [¹⁴C]sucrose and [¹⁴C]hexoses; and (c) randomization of ¹⁴C within the hexose moieties of intracellular sucrose following accumulation of [¹⁴C] (fructosyl)sucrose. Conversely, translocated sucrose does not undergo apoplastic hydrolysis during unloading. Asymmetrically labeled sucrose ([¹⁴C](fructose)sucrose), translocated from the germinating kernels to the root, remained intact indicating a symplastic pathway for unloading. In addition, isolated root protoplasts and vacuoles were used to demonstrate that soluble invertase activity (V_max = 29 micromoles per milligram protein per hour, K_m = 4 millimolar) was located mainly in the vacuole, suggesting that translocated sucrose entered via the symplasm and was hydrolyzed at the vacuole prior to metabolism.     

Transport and metabolism of 1'-fluorosucrose, a sucrose analog not subject to invertase hydrolysis

The novel sucrose derivative 1′-fluorosucrose (α-d-glucopyranosyl-β- d-1-deoxy-1-fluorofructofuranoside) was synthesized in order to help define mechanisms of sucrose entry into plant cells. Replacement of the 1′-hydroxyl by fluorine very greatly reduces invertase hydrolysis of the derivative (hydrolysis at 10 millimolar 1′-fluorosucrose is less than 2% that of sucrose) but does not reduce recognition, binding, or transport of 1′-fluorosucrose by a sucrose carrier. Transport characteristics of 1′-fluorosucrose were studied in three different tissues. The derivative is transported by the sucrose carrier in the plasmalemma of developing soybean cotyledon protoplasts with a higher affinity than sucrose (K_m 1′-fluorosucrose 0.9 millimolar, K_m sucrose 2.0 millimolar). 1′-Fluorosucrose is a competitive inhibitor of sucrose uptake with an apparent K_i also of 0.9 millimolar, while the K_i of sucrose competition of 1′-fluorosucrose uptake was 2.0 millimolar. Thus, both sugars are recognized at the same binding site in the plasmalemma. Both sucrose and 1′-fluorosucrose show very similar patterns of phloem translocation from an abraded leaf surface through the petiole indicating that recognition of 1′-fluorosucrose by sucrose carriers involved in phloem loading is likely as well.     

Tyrosine phosphorylation of Munc18‐1 inhibits synaptic transmission by preventing SNARE assembly

Tyrosine kinases are important regulators of synaptic strength. Here, we describe a key component of the synaptic vesicle release machinery, Munc18‐1, as a phosphorylation target for neuronal Src family kinases (SFKs). Phosphomimetic Y473D mutation of a SFK phosphorylation site previously identified by brain phospho‐proteomics abolished the stimulatory effect of Munc18‐1 on SNARE complex formation (“SNARE‐templating”) and membrane fusion in vitro. Furthermore, priming but not docking of synaptic vesicles was disrupted in hippocampal munc18‐1‐null neurons expressing Munc18‐1_Y473D. Synaptic transmission was temporarily restored by high‐frequency stimulation, as well as by a Munc18‐1 mutation that results in helix 12 extension, a critical conformational step in vesicle priming. On the other hand, expression of non‐phosphorylatable Munc18‐1 supported normal synaptic transmission. We propose that SFK‐dependent Munc18‐1 phosphorylation may constitute a potent, previously unknown mechanism to shut down synaptic transmission, via direct occlusion of a Synaptobrevin/VAMP2 binding groove and subsequent hindrance of conformational changes in domain 3a responsible for vesicle priming. This would strongly interfere with the essential post‐docking SNARE‐templating role of Munc18‐1, resulting in a largely abolished pool of releasable synaptic vesicles.