A comprehensive approach to visual and functional assessment of experimental vascular lesions in vivo

The role of metabolic factors derived from cardiac muscle in the development of reactive hyperemia after brief occlusions of the coronary circulation seems to be well established. However, the contribution of occlusion-induced changes in hemodynamic forces to eliciting reactive hyperemia is less known. We hypothesized that in isolated coronary arterioles changes in intraluminal pressure and flow, during and after release of occlusion (O/R), themselves via activating intrinsic mechanosensitive mechanisms, elicit release of vasoactive factors resulting in reactive dilations. Thus in isolated coronary arterioles (diameter: 88 +/- 8 microm) changes in diameter to changes in pressure or pressure plus flow (P+F) during and after a brief period (30, 60, and 120 s) of O/R of cannulating tube were measured by videomicroscopy. In response to both types of O/R, diameter first decreased, then, subsequently increased during occlusions. When only pressure was changed (from 80-10-80 mmHg), after release of occlusion, peak dilations increased as a function of the duration of occlusions. After flow was established (30 microl/min), O/R elicited changes in both pressure and flow (from 80-10-80 mmHg and from 0 to 30 microl/min). In these conditions, after the release of occlusions, not only the peak but also the duration of reactive dilation increased significantly as a function of the length of occlusions. The dilations during, and peak dilations after occlusions both in pressure and P+F protocols were significantly reduced by the inhibition of NO synthase with Nomega-nitro-L-arginine-methyl-ester (L-NAME) or by endothelium removal, whereas duration of postocclusion dilations were reduced by L-NAME or by endothelium removal only in P+F protocols. Furthermore, in both protocols, catalase significantly reduced the peak but not the duration of reactive dilations. Thus, mechanosensitive mechanisms that are sensitive to deformation, pressure, stretch, and wall shear stress elicit release of NO and H2O2, resulting in reactive dilation of isolated coronary arterioles.     

A single amino-acid polymorphism in pocket A of HLA-A*6602 alters the auxiliary anchors compared with HLA-A*6601 ligands

In this study we have sequenced peptides eluted from a truncated recombinant HLA-A*6602 molecule, and compared their features with data reported for peptides presented in the A*6601 molecule. A striking change in the amino-acid binding preferences was observed at peptide position P1, which interacts with pocket A of the HLA peptide-binding region. For A*6601, aspartic acid and glutamic acid, both of which possess polar acidic side-chains, have been described as auxiliary anchors. This is in marked contrast to A*6602, where we observed serine, which has a neutral polar side-chain, as auxiliary anchor at P1. Accordingly, this shift in the physico-chemical properties of the auxiliary anchor may be best explained by the HLA amino-acid polymorphism at position 163, where arginine (hydrophilic, alkaline) in A*6601 has been replaced by glutamic acid in A*6602. This amino-acid exchange results in a shift towards higher acidity in pocket A, apparently resulting in the loss of preference for acidic auxiliary anchors, and leading to the preference for the neutral amino acid serine. The change of the auxiliary anchor residue at P1 is likely to alter the spectrum of peptides presented by A*6602 compared with A*6601, which may result in allogenicity in the case of a mismatch in allogeneic stem cell transplantation.     

Differential antiviral response of endothelial cells after infection with pathogenic and nonpathogenic hantaviruses

Hantaviruses represent important human pathogens and can induce hemorrhagic fever with renal syndrome (HFRS), which is characterized by endothelial dysfunction. Both pathogenic and nonpathogenic hantaviruses replicate without causing any apparent cytopathic effect, suggesting that immunopathological mechanisms play an important role in pathogenesis. We compared the antiviral responses triggered by Hantaan virus (HTNV), a pathogenic hantavirus associated with HFRS, and Tula virus (TULV), a rather nonpathogenic hantavirus, in human umbilical vein endothelial cells (HUVECs). Both HTNV- and TULV-infected cells showed increased levels of molecules involved in antigen presentation. However, TULV-infected HUVECs upregulated HLA class I molecules more rapidly. Interestingly, HTNV clearly induced the production of beta interferon (IFN-beta), whereas expression of this cytokine was barely detectable in the supernatant or in extracts from TULV-infected HUVECs. Nevertheless, the upregulation of HLA class I on both TULV- and HTNV-infected cells could be blocked by neutralizing anti-IFN-beta antibodies. Most strikingly, the antiviral MxA protein, which interferes with hantavirus replication, was already induced 16 h after infection with TULV. In contrast, HTNV-infected HUVECs showed no expression of MxA until 48 h postinfection. In accordance with the kinetics of MxA expression, TULV replicated only inefficiently in HUVECs, whereas HTNV-infected cells produced high titers of virus particles that decreased after 48 h postinfection. Both hantavirus species, however, could replicate equally well in Vero E6 cells, which lack an IFN-induced MxA response. Thus, delayed induction of antiviral MxA in endothelial cells after infection with HTNV could allow viral dissemination and contribute to the pathogenesis leading to HFRS.     

Actin control over microtubules suggested by DISTORTED2 encoding the Arabidopsis ARPC2 subunit homolog

In Arabidopsis, based on the randomly misshapen phenotype of leaf epidermal trichomes, eight genes have been grouped into a 'DISTORTED' class. Three of the DIS genes, WURM, DISTORTED1 and CROOKED have been cloned recently and encode the ARP2, ARP3 and ARPC5 subunits respectively, of a conserved actin modulating ARP2/3 complex. Here we identify a fourth gene, DISTORTED2 as the Arabidopsis homolog of the ARPC2 subunit of the ARP2/3 complex. Like other mutants in the complex dis2 trichomes also display supernumerary, randomly localized cortical actin patches. In addition dis2 trichomes possess abnormally clustered endoplasmic microtubules near sites of actin aggregation. Since microtubules are strongly implicated in the establishment and maintenance of growth directionality in higher plants our observations of aberrant microtubule clustering in dis2 trichomes suggests a convincing explanation for the randomly distorted trichome phenotype in dis mutants. In addition, the close proximity of microtubule clusters to the arbitrarily dispersed cortical actin patches in the dis mutants provides fresh insights into cytoskeletal interactions leading us to suggest that in higher plants microtubule arrangements directed towards the establishment and maintenance of polar growth-directionality are guided by cortical actin behavior and organization.     

Long-term inhibition by auxin of leaf blade expansion in bean and Arabidopsis

The role of auxin in controlling leaf expansion remains unclear. Experimental increases to normal auxin levels in expanding leaves have shown conflicting results, with both increases and decreases in leaf growth having been measured. Therefore, the effects of both auxin application and adjustment of endogenous leaf auxin levels on midrib elongation and final leaf size (fresh weight and area) were examined in attached primary monofoliate leaves of the common bean (Phaseolus vulgaris) and in early Arabidopsis rosette leaves. Aqueous auxin application inhibited long-term leaf blade elongation. Bean leaves, initially 40 to 50 mm in length, treated once with alpha-naphthalene acetic acid (1.0 mm), were, after 6 d, approximately 80% the length and weight of controls. When applied at 1.0 and 0.1 mm, alpha-naphthalene acetic acid significantly inhibited long-term leaf growth. The weak auxin, beta-naphthalene acetic acid, was effective at 1.0 mm; and a weak acid control, benzoic acid, was ineffective. Indole-3-acetic acid (1 microm, 10 microm, 0.1 mm, and 1 mm) required daily application to be effective at any concentration. Application of the auxin transport inhibitor, 1-N-naphthylphthalamic acid (1% [w/w] in lanolin), to petioles also inhibited long-term leaf growth. This treatment also was found to lead to a sustained elevation of leaf free indole-3-acetic acid content relative to untreated control leaves. Auxin-induced inhibition of leaf growth appeared not to be mediated by auxin-induced ethylene synthesis because growth inhibition was not rescued by inhibition of ethylene synthesis. Also, petiole treatment of Arabidopsis with 1-N-naphthylphthalamic acid similarly inhibited leaf growth of both wild-type plants and ethylene-insensitive ein4 mutants.     

Expression of a soluble glycine binding domain of the NMDA receptor in Escherichia coli

Glycine is an essential co-agonist of the excitatory N-methyl-D-aspartate (NMDA) receptor. The glycine binding site of this subtype of ionotropic glutamate receptors is formed by the S1 and S2 regions of the NR1 subunit. Here, different S1S2 fusion proteins were expressed and purified from Escherichia coli cultures, and refolding protocols were established allowing the production of 30 mg of soluble S1S2 fusion protein from 1 liter bacterial culture. After affinity purification and renaturation, two of the fusion proteins (S1S2 and S1S2-V1) bound the competitive glycine site antagonist [3H]MDL105,519 with K(d) values of 9.35 and 3.9 nM, respectively. In contrast, with three other constructs (S1S2M, S1S2-V2, and -V3) saturable ligand binding could not be obtained. These results redefine the S1S2 domains required for high-affinity glycine binding. Furthermore, our high-affinity binding proteins may be used for the large-scale production of the glycine binding core region for future structural studies.     

Lack of negative charge in the E46Q mutant of photoactive yellow protein prevents partial unfolding of the blue-shifted intermediate

The long-lived light-induced intermediate (pB) of the E46Q mutant (glutamic acid is replaced by glutamine at position 46) of photoactive yellow protein (PYP) has been investigated by NMR spectroscopy. The ground state of this mutant is very similar to that of wild-type PYP (WT), whereas the pB state, formed upon illumination, appears to be much more structured in E46Q than in WT. The differences are most striking in the N-terminal domain of the protein. In WT, the side-chain carboxylic group of E46 is known to donate its proton to the chromophore upon illumination. The absence of the carboxylic group near the chromophore in the E46Q mutant prohibits the formation of a negative charge at this position upon formation of pB. This prevents the partial unfolding of the mutant, as evidenced from NMR chemical shift comparison and proton/deuterium (H/D) exchange studies.     

The glutathione synthetase of Schizosaccharomyces pombe is synthesized as a homodimer but retains full activity when present as a heterotetramer

Glutathione synthetase was overexpressed as a histidine-tagged protein in Schizosaccharomyces pombe and purified by two-step affinity chromatography. The recovered enzyme occurred in two different forms: a homodimeric protein consisting of two identical 56-kDa subunits and a heterotetrameric protein composed of two 32-kDa and two 24-kDa subfragments. Both forms are encoded by the GSH2 gene. The 56-Da protein corresponds to the complete GSH2 open reading frame, while the subfragments are produced following the cleavage of this larger protein by a metalloprotease. A stable homodimer was obtained by site-directed mutagenesis to remove the protease cleavage site, and this showed normal activity. A structural model of the fission yeast glutathione synthetase was produced, based on the x-ray coordinates of the human enzyme. According to this model the interacting domains of the proteolytic subfragments are strongly entangled. The subfragments were therefore coexpressed as independent proteins. These subfragments assembled correctly to yield functional heterotetramers with equivalent activity to the wild type enzyme. Furthermore, a permuted version of the protein was created. This also showed normal levels of glutathione synthetase activity. These data provide novel insight into the mechanisms of protein folding and the structure and evolution of the glutathione synthetase family.     

The C terminus (amino acids 75-94) and the linker region (amino acids 42-54) of the Ca2+-binding protein S100A1 differentially enhance sarcoplasmic Ca2+ release in murine skinned skeletal muscle fibers

S100A1, a Ca2+-binding protein of the EF-hand type, is most highly expressed in striated muscle and has previously been shown to interact with the skeletal muscle sarcoplasmic reticulum (SR) Ca2+ release channel/ryanodine receptor (RyR1) isoform. However, it was unclear whether S100A1/RyR1 interaction could modulate SR Ca2+ handling and contractile properties in skeletal muscle fibers. Since S100A1 protein is differentially expressed in fast- and slow-twitch skeletal muscle, we used saponin-skinned murine Musculus extensor digitorum longus (EDL) and Musculus soleus (Soleus) fibers to assess the impact of S100A1 protein on SR Ca2+ release and isometric twitch force in functionally intact permeabilized muscle fibers. S100A1 equally enhanced caffeine-induced SR Ca2+ release and Ca2+-induced isometric force transients in both muscle preparations in a dose-dependent manner. Introducing a synthetic S100A1 peptide model (devoid of EF-hand Ca2+-binding sites) allowed identification of the S100A1 C terminus (amino acids 75-94) and hinge region (amino acids 42-54) to differentially enhance SR Ca2+ release with a nearly 3-fold higher activity of the C terminus. These effects were exclusively based on enhanced SR Ca2+ release as S100A1 influenced neither SR Ca2+ uptake nor myofilament Ca2+ sensitivity/cooperativity in our experimental setting. In conclusion, our study shows for the first time that S100A1 augments contractile performance both of fast- and slow-twitch skeletal muscle fibers based on enhanced SR Ca2+ efflux at least mediated by the C terminus of S100A1 protein. Thus, our data suggest that S100A1 may serve as an endogenous enhancer of SR Ca2+ release and might therefore be of physiological relevance in the process of excitation-contraction coupling in skeletal muscle.