Pollination in Nicotiana alata stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue

After landing on a wet stigma, pollen grains hydrate and germination generally occurs. However, there is no certainty of the pollen tube growth through the style to reach the ovary. The pistil is a gatekeeper that evolved in many species to recognize and reject the self-pollen, avoiding endogamy and encouraging cross-pollination. However, recognition is a complex process, and specific factors are needed. Here the isolation and characterization of a stigma-specific protein from N. alata, NaStEP (N. alata Stigma Expressed Protein), that is homologous to Kunitz-type proteinase inhibitors, are reported. Activity gel assays showed that NaStEP is not a functional serine proteinase inhibitor. Immunohistochemical and protein blot analyses revealed that NaStEP is detectable in stigmas of self-incompatible (SI) species N. alata, N. forgetiana, and N. bonariensis, but not in self-compatible (SC) species N. tabacum, N. plumbaginifolia, N. benthamiana, N. longiflora, and N. glauca. NaStEP contains the vacuolar targeting sequence NPIVL, and immunocytochemistry experiments showed vacuolar localization in unpollinated stigmas. After self-pollination or pollination with pollen from the SC species N. tabacum or N. plumbaginifolia, NaStEP was also found in the stigmatic exudate. The synthesis and presence in the stigmatic exudate of this protein was strongly induced in N. alata following incompatible pollination with N. tabacum pollen. The transfer of NaStEP to the stigmatic exudate was accompanied by perforation of the stigmatic cell wall, which appeared to release the vacuolar contents to the apoplastic space. The increase in NaStEP synthesis after pollination and its presence in the stigmatic exudates suggest that this protein may play a role in the early pollen-stigma interactions that regulate pollen tube growth in Nicotiana.     

Improving the expression of mini-proinsulin in Pichia pastoris

Increased expression of recombinant mini-proinsulin in Pichia pastoris in 2.5 l bioreactors was achieved by increasing the cultivation pH from 5.1 to 6.3, by decreasing the temperature from 28 to 22 degrees C, and by periodical addition of ammonium sulfate and EDTA to the culture broth. Using this procedure, mini-proinsulin reached nearly 0.3 g l(-1) in the culture supernatant after 160 h of growth.     

Evasive maneuvers by secreted bacterial proteins to avoid innate immune responses

To cause disease, bacterial pathogens must first breach physical barriers, such as the mucous membrane that lines organs, and then successfully replicate and disseminate while avoiding destruction by the immune system. Many bacterial pathogens accomplish this by secreting proteins into their host environment, which act to subvert or dampen the expanding immune response. Here, we discuss how bacterial pathogens use an arsenal of secreted virulence proteins to modify the outcome of innate immune activation by altering how the immune system recognizes microbial invaders.     

A screening of plants used in Colombian traditional medicine revealed the anti-inflammatory potential of Physalis angulata calyces

The use of natural products by communities from the Colombian Caribbean region to treat health issues, together with biodiversity and geographical features, constitute a great scenery to develop new therapies based on ethnopharmacological heritage. Here, we investigated the anti-inflammatory potential of 10 commonly used plants in Colombian folk medicine, evaluating their effect on nitric oxide (NO) production by LPS-stimulated RAW 264.7 macrophages. The most active plant was evaluated in vivo using 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced mouse ear edema, along with its effect on the production of pro-inflammatory mediators in vitro. The extract of Physalis angulata L. calyces showed the highest activity. This extract was fractionated and its dichloromethane fraction (DF) was the most active in vitro, inhibiting the production of NO, prostaglandin E₂ (PGE₂), interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and monocyte chemotactic protein (MCP)-1 (CCL2). In vivo, DF showed a significant inhibition of ear edema and myeloperoxidase (MPO) activity, with evident reduction of the leukocyte infiltration into tissue. Our results support the ethnopharmacological use of the selected plants in folk medicine. P. angulata dichloromethane fraction represents a promising source of pharmacological compounds with great potential therapeutic use to treat inflammatory illness.     

The temperature-sensing protein TlpA is repressed by PhoP and dispensable for virulence of Salmonella enterica serovar Typhimurium in mice

TlpA is a temperature-sensing, coiled-coil protein, encoded on the pSLT virulence plasmid of Salmonella enterica serovar Typhimurium. TlpA was previously presumed to play a role in the pathogenicity of Salmonella. Herein we show that TlpA is tightly regulated, differentially expressed in response to environmental and physiological signals, and can be secreted in vitro. Expression of tlpA was found to be repressed in modified minimal medium containing limiting concentrations of Mg2+ and in the stationary phase of growth, but induced in rich LB broth and in response to elevated temperatures. The response regulator PhoP was found to play a key role in the repression of tlpA in conjunction with two other regulators, RpoS and TlpA itself. In addition, we demonstrate that TlpA is dispensable for intracellular proliferation of S. Typhimurium within host cells and for virulence in mice. Based on presented homology of TlpA to the IncP plasmid encoded protein, KfrA, and to SMC family members, a potential function for TlpA is discussed. Cumulatively, our data do not support the previous hypothesis that TlpA plays a role in the pathogenicity of Salmonella per se, but may suggest an alternative function for TlpA unrelated to host infection.