Constitutive expression of the SAP1 gene from willow (Salix discolor) causes early flowering in Arabidopsis thaliana

SAP1-1 and SAP1-2 were isolated from the male reproductive buds of willow (Salix discolor, clone S365). SAP1-1 differs from SAP1-2 based on a few nucleotide substitutions, but the sizes of their full-length cDNAs are identical. The deduced amino acid sequences of SAP1-1 and SAP1-2 were 98% similar and contain the same C-terminal amino acid motif “GYGA” like that of PTAP1-2 from Populus trichocarpa. The expression patterns of SAP1 in various parts of the male reproductive buds of S. discolor implicate this gene in the formation of the inflorescence meristems, bracts, and floral meristems. To characterize the functions of SAP1, we assessed Arabidopsis thaliana transformed with 35S∷SAP1-1. A total of 52 transgenic T₁ lines were obtained, and a 3:1 segregation ratio was obtained in the T₂ generation of each line. In the T₃ generation, five homozygous transgenic lines were obtained, which were used for further analysis. Screening of transgenic lines was greatly facilitated by the detection of GFP expression starting with germinating seeds. Phenotypes of the homozygous transgenic lines included early flowering, conversion of inflorescence branches to solitary flowers, formation of terminal flowers, and formation of flowers with greater number of petals, stamens, and pistils. Northern analysis showed similar expression levels in all five lines. This study provides the first functional analysis of an APETALA1 (AP1)/SQUAMOSA (SQUA) homolog from a dioecious species and suggests that SAP1 is a homolog of the AP1/SQUA gene.     

Photonic immobilization of high‐density protein arrays using Fourier optics

The technique of UV‐light‐assisted immobilization of disulfide containing proteins has been combined with the Fourier‐transforming properties of lenses as well as with a simple millimeter scale feature size spatial mask. The result is a new simple and inexpensive way of creating high‐density protein arrays with feature sizes down to a few hundred nanometers, which represents an improvement of tenfold over existing commercially available high‐density protein arraying methods.     

Interactions of two O-phosphorylresveratrol derivatives with model membranes

The hydrosoluble resveratrol derivative 3-O-phosphorylresveratrol was shown to be more cytotoxic against DU 145 prostate cancer cells than its analog 4'-O-phosphorylresveratrol. In an attempt to unveil the molecular determinants that lye at the root of their different biological effects, here we investigate the interactions of the two resveratrol derivatives with DMPC model membranes by using DSC, membrane permeation/poration assays and molecular dynamics. The results show that the 3-O-derivative interacts with DMPC membranes and diffuses across them. The 4'-O-derivative lies preferentially onto the surface of membrane. The MD simulations provide a molecular interpretation of the experiments and highlight that, in order to maximize the apolar interactions, the 3-O-derivative is embedded in the lipid hydrophobic region. This topographical position of the 3-O resveratrol analog perturbs the liquid-crystalline order of the lipid bilayer promoting membrane curvature and partial lipid loss from the vesicle. This finding reconciles with the lowering of the enthalpy of the lipid phase transition and the ability of the molecule to diffuse across membranes. The present data contribute to explain the different biological activity of the two molecules and evidence that membrane permeability is a key requirement for effective design of resveratrol derivatives to be used for therapeutic purposes.     

Fibrin Clot Structure and Mechanics Associated with Specific Oxidation of?Methionine Residues in Fibrinogen

Using a combination of structural and mechanical characterization, we examine the effect of fibrinogen oxidation on the formation of fibrin clots. We find that treatment with hypochlorous acid preferentially oxidizes specific methionine residues on the α, β, and γ chains of fibrinogen. Oxidation is associated with the formation of a dense network of thin fibers after activation by thrombin. Additionally, both the linear and nonlinear mechanical properties of oxidized fibrin gels are found to be altered with oxidation. Finally, the structural modifications induced by oxidation are associated with delayed fibrin lysis via plasminogen and tissue plasminogen activator. Based on these results, we speculate that methionine oxidation of specific residues may be related to hindered lateral aggregation of protofibrils in fibrin gels.     

A high-fat diet associated with acute schistosomiasis mansoni causes disorganization in splenic architecture in mice

To date, the effect of the changes promoted by hypercholesterolemia and experimental schistosomiasis infection on splenic architecture has remained elusive. In this paper, we compared spleen from control and infected mice fed either high-fat (29% lipids) or standard diet (12% lipids), assessing spleen volume by liquid displacement and splenic disorganization by histopathology, morphometry and stereology. Infected mice showed higher spleen volume than in corresponding uninfected mice (P<0.05). The white pulp compartment was reduced, red pulp and germinal center were enhanced (P<0.01). Microscopic examination showed cellular infiltrates characterized by polymorfonuclear cells, with intensive lymphocytic mitosis and Mott cells. Hemosiderin deposits tended to be in less extent in infected mice compared with uninfected controls. The red pulp compartment showed a significantly (P<0.05) increased average number of megakaryocytes compared with uninfected mice, which may be associated with hematopoietic reconstitution. High-fat fed mice showed larger white pulp than controls (P<0.05). Standard fed mice showed exudative-productive granuloma distributed only sparsely in the red pulp, whereas a tissue reaction characterized by a cell infiltration in high-fat fed mice was found. The results of the present study suggest that there is a significant relationship between high-fat diet intake and splenic disorganization such as a decrease in the numerical density of white pulp and, red pulp and germinal center hyperplasia. Such structural disorganization due to co-morbidites (schistosomiasis and dyslipidemia) may affect the microenvironments of the spleen that are necessary for the generation of immune responses to antigens.     

Susceptibility of a Brazilian wild rodent isolate of Schistosoma mansoni to praziquantel in mice

The therapeutic effects of praziquantel (PZQ) against a Schistosoma mansoni isolate derived from Nectomys squamipes (isolate R) and a susceptible isolate (BH) were analyzed in Swiss mice by fecal egg counting, adult worm reduction and oogram pattern. Infected mice were orally administrated with 62.5mg/kg (group 1), 125mg/kg (group 2), 250mg/kg (group 3) and 500mg/kg (group 4), each dose divided over 3 days (49, 50 and 51 days after infection). The data were analyzed using one-way analysis of variance (ANOVA). In regard to isolate R, no fecal eggs were observed with 250 mg/Kg and 500 mg/kg (p<0.05), whereas BH excretion reached zero with all doses. Mean worm burden reduction was significantly (p<0.05) higher at the two highest concentrations, regardless of isolate. At 62.5mg/kg, the percentage of immature eggs varied from 17% (isolate R) to 38% (isolate BH). At 125 mg/kg, the percentage of immature eggs varied from 20% (isolate R) to 16% (isolate BH). At 250 mg/kg, immature eggs dropped significantly to 1% (isolate R) and 4% (isolate BH). At 500 mg/kg, no immature eggs were found in isolate R, whereas in BH was 8%. No dosage significantly (p>0.05) affected the percentage of mature eggs, regardless of isolate. There was a large increase (p<0.001) in the percentages of dead eggs in all treated groups of 62% and 64% in groups 3 and 4, respectively (isolate R). The percentage of dead eggs rose from 34% (group 1) to 58% (group 3) in isolate BH. Although group 4 showed lowest increase in the percentage of dead eggs (46%), it was higher (p<0.001) compared to the 8% in the control. Our findings indicate that the wild isolate from N. squamipes is susceptible to PZQ.     

A dp53-dependent mechanism involved in coordinating tissue growth in Drosophila

Coordination of growth between and within organs contributes to the generation of well-proportioned organs and functionally integrated adults. The mechanisms that help to coordinate the growth between different organs start to be unravelled. However, whether an organ is able to respond in a coordinated manner to local variations in growth caused by developmental or environmental stress and the nature of the underlying molecular mechanisms that contribute to generating well-proportioned adult organs under these circumstances remain largely unknown. By reducing the growth rates of defined territories in the developing wing primordium of Drosophila, we present evidence that the tissue responds as a whole and the adjacent cell populations decrease their growth and proliferation rates. This non-autonomous response occurs independently of where growth is affected, and it is functional all throughout development and contributes to generate well-proportioned adult structures. Strikingly, we underscore a central role of Drosophila p53 (dp53) and the apoptotic machinery in these processes. While activation of dp53 in the growth-depleted territory mediates the non-autonomous regulation of growth and proliferation rates, effector caspases have a unique role, downstream of dp53, in reducing proliferation rates in adjacent cell populations. These new findings indicate the existence of a stress response mechanism involved in the coordination of tissue growth between adjacent cell populations and that tissue size and cell cycle proliferation can be uncoupled and are independently and non-autonomously regulated by dp53.     

Defective B cell response to TLR9 ligand (CpG-ODN), Streptococcus pneumoniae and Haemophilus influenzae extracts in common variable immunodeficiency patients

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinaemia and antibody deficiency to both T dependent and independent antigens. Patients suffer from recurrent sinopulmonary infections mostly caused by Streptococcus pneumoniae and Haemophilus influenzae, but also gastrointestinal or autoimmune symptoms. Their response to vaccination is poor or absent. In this study we investigated B cell activation induced by the TLR9 specific ligand (CpG-ODN) and bacterial extracts from S. pneumoniae and H. influenzae known to stimulate several TLR. We found that B cells from CVID patients express lower levels of CD86 after stimulation with CpG-ODN, S. pneumoniae and H. influenzae extracts in combination with anti-IgM antibody and also display a lower proliferative index when stimulated with bacterial extracts. Our results point to a broad TLR signalling defect in B lymphocytes from CVID patients that may be related to the hypogammaglobulinaemia and poor response to vaccination characteristic of these patients.