Nighttime evaporative demand induces plasticity in leaf and root hydraulic traits

Increasing evidence suggests that nocturnal transpiration rate (TR_N) is a non‐negligible contributor to global water cycles. Short‐term variation in nocturnal vapor pressure deficit (VPD_N) has been suggested to be a key environmental variable influencing TR_N. However, the long‐term effects of VPD_N on plant growth and development remain unknown, despite recent evidence documenting long‐term effects of daytime VPD on plant anatomy, growth and productivity. Here we hypothesized that plant anatomical and functional traits influencing leaf and root hydraulics could be influenced by long‐term exposure to VPD_N. A total of 23 leaf and root traits were examined on four wheat (Triticum aestivum) genotypes, which were subjected to two long‐term (30 day long) growth experiments where daytime VPD and daytime/nighttime temperature regimes were kept identical, with variation only stemming from VPD_N, imposed at two levels (0.4 and 1.4 kPa). The VPD_N treatment did not influence phenology, leaf areas, dry weights, number of tillers or their dry weights, consistently with a drought and temperature‐independent treatment. In contrast, vein densities, adaxial stomata densities, TR_N and cuticular TR, were strongly increased following exposure to high VPD_N. Simultaneously, whole‐root system xylem sap exudation and seminal root endodermis thickness were decreased, hypothetically indicating a change in root hydraulic properties. Overall these results suggest that plants ‘sense’ and adapt to variations in VPD_N conditions over developmental scales by optimizing both leaf and root hydraulics.     

ClpP: a distinctive family of cylindrical energy-dependent serine proteases

Processes maintaining protein homeostasis in the cell are governed by the activities of molecular chaperones that mainly assist in the folding of polypeptide chains and by a large class of proteases that regulate protein levels through degradation. ClpP proteases define a distinctive family of cylindrical, energy-dependent serine proteases that are highly conserved throughout bacteria and eukaryota. They typically interact with ATP-dependent AAA+ chaperones that bind and unfold target substrates and then translocate them into ClpP for degradation. Structural and functional studies have provided a detailed view of the mechanism of function of this class of proteases.     

New Insights on Neuronal Alterations in Jimpy Mutant Brain

In spite of abundant data on oligodendrocyte abnormalities in dysmyelinated jimpy brain, little is known about the axonal damage and the expression of neuronal genes. Recent findings indicate that Nogo-A, oligodendrocyte-myelin glycoprotein (OMgp), and myelin-associated glycoprotein (MAG) inhibit axonal growth by binding a common receptor, the Nogo-A receptor (NgR)-p75 complex. In order to evaluate neuronal modifications in the absence of myelin and in the presence of abnormal oligodendrocytes at different developmental stages, the expression of these inhibitory proteins and their receptors was investigated in jimpy mutant brain. Despite the decrease in oligodendrocyte number at P15 and P25 in jimpy, Nogo-A and OMgp mRNA levels are not significantly different compared with control, suggesting an overexpression of neuronal Nogo-A and OMgp in mutant. Double immunolabeling for Nogo-A and neurofilaments shows strong axonal staining of Nogo-A in jimpy and its down-regulation in oligodendrocytes. The current data raise questions about functions of Nogo-A other than neurite growth inhibition in the CNS. No significant changes in NgR mRNA levels were observed in jimpy, where the increase in p75 level can be correlated with the cell death of oligodendrocytes. In the paranodal region, the cell adhesion molecule neurofascin glial isoform NFN155 mRNA level is reduced by 40% whereas neuronal form NFN186 is up-regulated. These results may explain the failure of paranodal region organization, even with normal level of CASPR (paranodin) mRNA detected in jimpy brain.     

Chaperone networks in bacteria: analysis of protein homeostasis in minimal cells

The prevention of aberrant behavior of proteins is fundamental to cellular life. Protein homeostatic processes are present in cells to stabilize protein conformations, refold misfolded proteins, and degrade proteins that might be detrimental to the cell. Molecular chaperones and proteases perform a major role in these processes. In bacteria, the main cytoplasmic components involved in protein homeostasis include the chaperones trigger factor, DnaK/DnaJ/GrpE, GroEL/GroES, HtpG, as well as ClpB and the proteases ClpXP, ClpAP, HslUV, Lon, and FtsH. Based on recent genome sequencing efforts, it was surprising to find that the Mycoplasma, a genus proposed to include a minimal form of cellular life, do not contain certain major members of the protein homeostatic network, including GroEL/GroES. We propose that, in mycoplasmas, there has been a fundamental shift towards favoring processes that promote protein degradation rather than protein folding. The arguments are based on two different premises: (1) the regulation of stress response in Mycoplasma and (2) the unique characteristics of the Mycoplasma proteome.     

Lipid raft-dependent and -independent signaling through HLA-DR molecules

Lipid rafts are plasma membrane microdomains that are highly enriched in signaling molecules and that act as signal transduction platforms for many immune receptors. The involvement of these microdomains in HLA-DR-induced signaling is less well defined. We examined the constitutive presence of HLA-DR molecules in lipid rafts, their possible recruitment into these microdomains, and the role of these microdomains in HLA-DR-induced responses. We detected significant amounts of HLA-DR molecules in the lipid rafts of EBV(+) and EBV(-) B cell lines, monocytic cell lines, transfected HeLa cells, tonsillar B cells, and human monocytes. Localization of HLA-DR in these microdomains was unaffected by the deletion of the cytoplasmic domain of both the alpha and beta chains. Ligation of HLA-DR with a bivalent, but not a monovalent, ligand resulted in rapid tyrosine phosphorylation of many substrates, especially Lyn, and activation of ERK1/2 MAP kinase. However, the treatment failed to induce further recruitment of HLA-DR molecules into lipid rafts. The HLA-DR-induced signaling events were accompanied by the induction of cell-cell adhesion that could be inhibited by PTK and Lyn but not ERK1/2 inhibitors. Disruption of lipid rafts by methyl-beta-cyclodextrin (MbetaCD) resulted in the loss of membrane raft association with HLA-DR molecules, inhibition of HLA-DR-mediated protein tyrosine phosphorylation and cell-cell adhesion. MbetaCD did not affect the activation of ERK1/2, which was absent from lipid rafts. These results indicate that although all the HLA-DR-induced events studied are dependent on HLA-DR dimerization, some require the presence of HLA-DR molecules in lipid rafts, whereas others do not.     

Lyn- and ERK-mediated vs. Ca2+-mediated neutrophil O2- responses with thermal injury

We evaluated thedependency of neutrophil O production on PTK-Lyn andMAPK-ERK1/2 in rats after thermal injury. Activation of PTK-Lyn wasassessed by immunoprecipitation. Phosphorylation of ERK1/2 was assessedby Western blot analysis. O production was measuredby isoluminol-enhanced luminometry. Imaging technique was employed tomeasure neutrophil [Ca²⁺]_i in individualcells. Thermal injury caused marked upregulation of Lyn and ERK1/2accompanying enhanced neutrophil O production.Treatment of rats with PTK blocker (AG556) or MAPK blocker (AG1478)before burn injury caused complete inhibition of the respective kinaseactivation. Both AG556 and AG1478 produced an ~66% inhibition inO production. Treatment with diltiazem (DZ) producedan ~37% inhibition of O production withoutaffecting Lyn or ERK1/2 activation with burn injury. Ca²⁺mobilization was upregulated with burn injury but not affected bytreatment of burn rats with AG556. Unlike the partial inhibition ofburn-induced O production by AG556, AG1478, or DZ,platelet-activating factor antagonist (PAFa) treatment of burn ratsproduced near complete inhibition of O production.PAFa treatment also blocked activation of Lyn. The findings suggestthat the near complete inhibition of O production byPAFa was a result of blockade of PTK as well as Ca²⁺signaling. Overall, our studies show that enhanced neutrophil O production after thermal injury is a result ofpotentiation of Ca²⁺-linked and -independent signalingtriggered by inflammatory agents such as PAF.

     

Improved immunomatrix methods to detect protein:protein interactions

Immunoprecipitation (IP) and coimmunoprecipitation (co-IP) are key techniques for studying protein-protein interactions. These methods utilize immobilized Protein A or Protein G to isolate antibody-bound target antigens. The main disadvantage of traditional IP and co-IP is that the conditions used to elute the precipitated antigen also release the antibody thus contaminating the antigen and destroying the antibody support. To overcome these problems, we describe two methods to generate a reusable antibody support by cross-linking the antibody to immobilized Protein A or Protein G, or by coupling it directly to the resin (see Scheme 1). Antibody cross-linking can be done in 1 h while antibody coupling requires 4 h. IP or co-IP is accomplished by incubating the antibody resin with the protein sample. Washes and elutions are carried out in a spin column to reduce resin loss and decrease assay time. Target proteins are eluted with 0.1 M glycine (pH 2.8) and the resin-bound antibody is re-equilibrated in phosphate-buffered saline (PBS) for reuse. Our studies have demonstrated that the immobilization efficiency for the antibody coupling method was similar for several species of antibody. Furthermore, we illustrate that using both methods of antibody immobilization yield IP and co-IP results similar to traditional protocols but eliminate the antibody heavy and light chain contamination.     

Mek2 is dispensable for mouse growth and development

MEK is a dual-specificity kinase that activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase upon agonist binding to receptors. The ERK/MAP kinase cascade is involved in cell fate determination in many organisms. In mammals, this pathway is proposed to regulate cell growth and differentiation. Genetic studies have shown that although a single Mek gene is present in Caenorhabditis elegans, Drosophila melanogaster, and Xenopus laevis, two Mek homologs, Mek1 and Mek2, are present in the mammalian cascade. The inactivation of the Mek1 gene leads to embryonic lethality and has revealed the unique role played by Mek1 during embryogenesis. To investigate the biological function of the second homolog, we have generated mice deficient in Mek2 function. Mek2 mutant mice are viable and fertile, and they do not present flagrant morphological alteration. Although several components of the ERK/MAP kinase cascade have been implicated in thymocyte development, no such involvement was observed for MEK2, which appears to be nonessential for thymocyte differentiation and T-cell-receptor-induced proliferation and apoptosis. Altogether, our findings demonstrate that MEK2 is not necessary for the normal development of the embryo and T-cell lineages, suggesting that the loss of MEK2 can be compensated for by MEK1.