The photoreaction center of Rhodospirillum rubrum mutant strain F24.1

Rhodospirillum rubrum strain F24.1 is a spontaneous revertant of nonphototrophic mutant F24 derived from wild-type strain S1. Strain F24 shows no detectable photochemical activity and contains, at most, traces of the photoreaction center polypeptides. Strain F24.1 has a phototrophic growth rate close to that of the wild-type strain (Picorel, R., del Valle-Tascón, S. and Ramírez, J.M. (1977) Arch. Biophys. Biochem. 181, 665–670) but shows little photochemical activity. Light-induced absorbance changes in the near-infrared, photoinduced EPR signals and ferricyanide-elicited absorbance changes indicate that strain F24.1 has a photoreaction center content of 7–8% as compared to strain S1. Polyacrylamide gel electrophoresis of isolated F24.1 chromatophores shows the photoreaction center polypeptides to be present in amounts compatible with this value. Photoreaction center was prepared from strain F24.1 and showed no detectable difference with that of strain S1. It is concluded that strain F24.1 photosynthesis is due entirely to its residual 7–8% of typical photoreaction center.     

Cell Size and Growth Rate Are Modulated by TORC2-Dependent Signals

1. Download high-res image (81KB)
2. Download full-size image

     

Strategy for fluorescent labeling of human acidic fibroblast growth factor without impairment of mitogenic activity: A bona fide tracer

Here we describe, for the first time, the design and characterization of a bona fide fluorescently labeled mutant of the human acidic fibroblast growth factor (aFGF). The aFGF–Cys2 mutant was recombinantly synthesized by substituting the second amino acid with a reactive cysteine whose sulfhydryl group’s side chain reactivity facilitated the covalent binding of a fluorescent probe as a thiolyte monobromobimane. Using a combination of biophysical and functional assays, we found that the fluorescently labeled mutant aFGF is characterized by essentially the same global folding, mitogenic activity, and association behavior with heparin, its physiological activator, as the unlabeled wild-type protein. We used this new tracer protein mutant to determine the association behavior of aFGF with heparin in the presence of high concentrations of albumin that mimicked more closely the plasma medium in which aFGF is naturally located and in which it has evolved to function. By exposing the aFGF–Cys2–heparin complex to increasing concentrations of albumin up to physiological plasma levels, we were able to demonstrate that macromolecular crowding does not affect the stoichiometry of the interaction. In summary, the dimeric aFGF–Cys2–heparin complex might represent a biologically relevant complex in physiological media.     

Prevalence of potentially pathogenic culturable bacteria on eggshells and in cloacae of female Pied Flycatchers in a temperate habitat in central Spain

ABSTRACT Bacteria grow on avian eggshells and thus can potentially cause diseases in developing embryos. Little is known about culturable bacteria colonizing avian eggshells in free‐living birds, with most studies restricted to poultry. Our objective was to examine the culturable bacterial array growing on eggshells during incubation that could negatively affect hatching success of Pied Flycatchers (Ficedula hypoleuca) in a temperate montane habitat in central Spain. Cloacal culturable bacteria of females were also analyzed because bacteria can be vertically transmitted from females to eggs. We used fecal samples as surrogates of cloacal samples due to the small size of sampled birds. We found that eggshells and female cloacae of Pied Flycatchers harbored 24 and 40 bacterial families and species, respectively, but only a few in each clutch and each cloaca. Rod‐shaped gram‐negative bacteria and bacteria in the family Pseudomonadaceae were the most common bacteria on eggshells during early and late incubation and in female cloacae. Although based on small sample sizes, we found that females with rod‐shaped gram‐negative bacteria in their cloacae laid eggs that also had these bacteria, providing possible evidence for vertical transmission. We found no evidence for vertical transmission of Pseudomonadaceae, suggesting a possible environmental source for these bacteria. The prevalence of bacterial morphological types and major taxonomical categories on eggshells did not vary from early to late stages of incubation, providing support for the hypothesis that incubation may have bacteriostatic effects on bacterial proliferation on eggshells. Despite being primary egg invaders in poultry, we detected no effects of culturable Pseudomonadaceae or Pseudomonas luteola on hatching success. Our study represents the first to examine the culturable bacteria growing on the eggshells of a wild bird in a temperate habitat and additional studies based on culture‐independent techniques are required to confirm our results.     

Serological response against myxoma virus and rabbit hemorrhagic disease virus in European wild rabbits using commercial vaccines

We carried out an experimental study to determine the serological response against myxoma virus (MV) and rabbit hemorrhagic disease virus (RHDV) in wild rabbits using commercial vaccines. Seroconversion against MV ranged between 72.7% and 97.2% in animals vaccinated by subcutaneous and intradermal route, respectively, whereas between 75.0% and 77.8% of the animals presented antibodies against RHDV after inoculation with subcutaneous and intradermal vaccines, respectively. Regardless of the inoculation route, vaccination against MV resulted in a significant increase of seropositivity 5 days post-vaccination (dpv), which did not occur in animals vaccinated against RHDV. Furthermore, seroconversion against MV was significantly higher and faster in intradermally vaccinated rabbits as compared to those inoculated subcutaneously due to either the route of application and/or the type of vaccine used. The results indicated that vaccination significantly increased the prevalence of antibodies against MV and RHDV and suggested that the vaccines currently available induce a safe and effective immune response against both diseases in wild rabbits. Vaccination may be a useful management tool to control both viral diseases in field conditions, particularly in wild rabbits captured for translocations and restocking purposes in which a large number of animals are handled. © 2011 The Wildlife Society.     

Phosphorylation of Yeast Hexokinase 2 Regulates Its Nucleocytoplasmic Shuttling

Nucleocytoplasmic shuttling of Hxk2 induced by glucose levels has been reported recently. Here we present evidence that indicates that Hxk2 nucleocytoplasmic traffic is regulated by phosphorylation and dephosphorylation at serine 14. Moreover, we identified the protein kinase Snf1 and the protein phosphatase Glc7-Reg1 as novel regulatory partners for the nucleocytoplasmic shuttling of Hxk2. Functional studies revealed that, in contrast to the wild-type protein, the dephosphorylation-mimicking mutant of Hxk2 retains its nuclear localization in low glucose conditions, and the phosphomimetic mutant of Hxk2 retains its cytoplasmic localization in high glucose conditions. Interaction experiments of Hxk2 with Kap60 and Xpo1 indicated that nuclear import of the S14D mutant of Hxk2 is severely decreased but that the export is significantly enhanced. Conversely, nuclear import of the S14A mutant of Hxk2 was significantly enhanced, although the export was severely decreased. The interaction of Hxk2 with Kap60 and Xpo1 was found to occur in the dephosphorylated and phosphorylated states of the protein, respectively. In addition, we found that Hxk2 is a substrate for Snf1. Mutational analysis indicated that serine 14 is a major in vitro and in vivo phosphorylation site for Snf1. We also provide evidence that dephosphorylation of Hxk2 at serine 14 is a protein phosphatase Glc7-Reg1-dependent process. Taken together, this study establishes a functional link between Hxk2, Reg1, and Snf1 signaling, which involves the regulation of Hxk2 nucleocytoplasmic shuttling by phosphorylation-dephosphorylation of serine 14.     

Excision of a selectable marker gene in transgenic banana using a Cre/lox system controlled by an embryo specific promoter

Antibiotic and herbicide resistance genes have been used in transgene technology as powerful selection tools. Nonetheless, once transgenic events have been obtained their presence is no longer needed and can even be undesirable. In this work, we have developed a system to excise the selectable marker and the cre recombinase genes from transgenic banana cv. ‘Grande Naine’ (Musa AAA). To achieve this, the embryo specific REG-2 promoter was isolated from rice and its expression pattern in banana cell clumps, somatic embryos and regenerated plantlets was characterized by using a pREG2::uidA fusion construct. Subsequently, the REG-2 promoter was placed upstream of the cre gene, conferring Cre functionality in somatic embryos and recombination of lox sites resulting in excision of the selectable marker and cre genes. PCR analysis revealed that 41.7 % of the analysed transgenic plants were completely marker free, results that were thereafter confirmed by Southern blot hybridization. These results demonstrate the feasibility of using developmentally controlled promoters to mediate marker excision in banana. This system does not require any extra handling compared to the conventional transformation procedure and might be useful in other species regenerating through somatic embryogenesis.     

Colitis induced by proteinase-activated receptor-2 agonists is mediated by a neurogenic mechanism

Proteinase-activated receptor-2 (PAR2) activation induces colonic inflammation by an unknown mechanism. We hypothesized that PAR2 agonists administered intracolonically in mice induce inflammation via a neurogenic mechanism. Pretreatment of mice with neurokinin-1 and calcitonin-gene-related peptide (CGRP) receptor antagonists or with capsaicin showed attenuated PAR2-agonist-induced colitis. Immunohistochemistry demonstrated a differential expression of a marker for the type-1 CGRP receptor during the time course of PAR2-agonist-induced colitis, further suggesting a role for CGRP. We conclude that PAR2-agonist-induced intestinal inflammation involves the release of neuropeptides, which by acting on their receptors cause inflammation. These results implicate PAR2 as an important mediator of intestinal neurogenic inflammation.     

Primary cells derived from Tuberous Sclerosis Complex patients show autophagy alteration in the haploinsufficiency state

Tuberous sclerosis complex (TSC) is an autosomal dominant cancer predisposition disorder caused by heterozygous mutations in TSC1 or TSC2 genes and characterized by mTORC1 hyperactivation. TSC-associated tumors develop after loss of heterozygosity mutations and their treatment involves the use of mTORC1 inhibitors. We aimed to evaluate cellular processes regulated by mTORC1 in TSC cells with different mutations before tumor development. Flow cytometry analyses were performed to evaluate cell viability, cell cycle and autophagy in non-tumor primary TSC cells with different heterozygous mutations and in control cells without TSC mutations, before and after treatment with rapamycin (mTORC1 inhibitor). We did not observe differences in cell viability and cell cycle between the cell groups. However, autophagy was reduced in mutated cells. After rapamycin treatment, mutated cells showed a significant increase in the autophagy process (p=0.039). We did not observe differences between cells with distinct TSC mutations. Our main finding is the alteration of autophagy in non-tumor TSC cells. Previous studies in literature found autophagy alterations in tumor TSC cells or knock-out animal models. We showed that autophagy could be an important mechanism that leads to TSC tumor formation in the haploinsufficiency state. This result could guide future studies in this field.     

Glial cells in adult and developing prothoracic ganglion of the hawk moth Manduca sexta

The glial cells of the prothoracic ganglion of the hawk moth Manduca sexta were studied in histological sections of several postembryonic stages and classified according to cell morphology, size, staining properties, and topographical relationships. In general, each glial cell type was found to be confined to one of the major ganglionic domains and each of these domains (i.e., perineurium, cell body rind, glial cover of the neuropil, and neuropil) was found to comprise specific cell types. Some types of glia were recognized in both larval and later stages, but other types were found exclusively from late pupal stages. It is proposed that the higher morphological diversity expressed by the glia of the pharate adult is attained by differentiation of new cell types during metamorphosis. Before the differentiation of new cell types, extensive cell death and cell proliferation seem to occur within some glial subpopulations.