Movement of DNA across the chloroplast envelope: Implications for the transfer of promiscuous DNA

Little is known about the mechanistic basis for the movement of promiscuous nucleic acids across cell membranes. To address this problem we sought conditions that would permit the entry of plasmid DNA into isolated, intact pea chloroplasts. DNA uptake did not occur normally, but was induced by hypotonic treatments, by incubation with millimolar levels of Mg²⁺, or by heat shock at 42 °C. These results are consistent with DNA movement being permitted by conditions that transiently alter the permeability of the chloroplast envelope. Plant cells are subject to osmotic tensions and/or conditions inducing polymorphic changes in the membranes, such as those used in the present study, under several environmental stresses. In an evolutionary time frame, these phenomena may provide a mechanism for the transfer of promiscuous nucleic acids between organelles.Abbreviations PEG polyethylene glycol - T-DNA transferred DNA To Dan Arnon; a superb scientist whose findings opened up enormous fields of research in photosynthesis, and a good friend.     

Targeting HIV-1 envelope glycoprotein trimers to B cells by using APRIL improves antibody responses

An HIV-1 vaccine remains elusive, in part because various factors limit the quantity and quality of the antibodies raised against the viral envelope glycoprotein complex (Env). We hypothesized that targeting Env vaccines directly to B cells, by fusing them to molecules that bind and activate these cells, would improve Env-specific antibody responses. Therefore, we fused trimeric Env gp140 to A PRoliferation-Inducing Ligand (APRIL), B-cell Activating Factor (BAFF), and CD40 Ligand (CD40L). The Env-APRIL, Env-BAFF, and Env-CD40L gp140 trimers all enhanced the expression of activation-induced cytidine deaminase (AID), the enzyme responsible for inducing somatic hypermutation, antibody affinity maturation, and antibody class switching. They also triggered IgM, IgG, and IgA secretion from human B cells in vitro. The Env-APRIL trimers induced higher anti-Env antibody responses in rabbits, including neutralizing antibodies against tier 1 viruses. The enhanced Env-specific responses were not associated with a general increase in total plasma antibody concentrations, indicating that the effect of APRIL was specific for Env. All the rabbit sera raised against gp140 trimers, irrespective of the presence of CD40L, BAFF, or APRIL, recognized trimeric Env efficiently, whereas sera raised against gp120 monomers did not. The levels of trimer-binding and virus-neutralizing antibodies were strongly correlated, suggesting that gp140 trimers are superior to gp120 monomers as immunogens. Targeting and activating B cells with a trimeric HIV-1 Env-APRIL fusion protein may therefore improve the induction of humoral immunity against HIV-1.     

S100 immunoreactivity is increased in reactive astrocytes of the visual pathways following a mechanical lesion of the rat occipital cortex

After demonstration of the paracrine action of glial neurotrophic factors, gliosis has also been considered to be related to neuronal trophism and plasticity rather than solely a repair event following brain injury. S100 is a Ca2+ binding protein, present mainly in astrocytes, that exerts paracrine trophic effects on several neuronal populations. This study analyses the presence of S100 protein by means of immunohistochemistry combined with stereology in the reactive glial cells of the rat visual pathways following a lesion of the visual cortex. Adult male Wistar rats were submitted to a unilateral aspiration of the occipital cortex or to a sham operation. One week later the rats were killed and their brain processed for immunochemistry. Single antibody immunohistochemistry was performed for the visualization of glial fibrillary acidic protein (GFAP, a marker for astrocytes), OX-42 (a marker for microglia) and S100 protein. Double immunofluorescence procedures were applied for co-localization of the S100/GFAP and S100/OX-42. An optical dissector, point interceptors and rotators were used to quantify the degree of glial activation and the changes in the S100 immunoreactivity. We observed an intense microglial and astroglial reaction in addition to an increased S100 immunoreactivity in the occipital cerebral cortex, geniculate nucleus and hippocampus ipsilateral to the lesion. In the ipsilateral superior colliculus, an intense astroglial activation was accompanied by an up-regulation of S100 immunoreactivity. Double-immunofluoresence revealed an increased S100 immunoreactivity in reactive astrocytes, but not in the reactive microglia. Evidence has therefore been obtained that after mechanical trauma, the astroglial S100 protein participates in the trophism and plasticity of the injured visual pathways.     

De novo deletion of 1q31.1-q32.1 in a patient with developmental delay and behavioral disorders

We describe the case of a 6-year-old boy with a de novo deletion of the long arm of chromosome 1 encompassing band 1q31.1-q32.1, minor facial anomalies, mild developmental delay, and behavioral disorders. His postnatal karyotype was normal. Using array-comparative genomic hybridization, we identified and characterized a de novo 1q interstitial deletion of about 15.6 Mb, which partially overlaps those of other reported cases. We considered the gene content of the deleted region in an attempt to compare the clinical features of our patient with these other cases, even though they were not characterized molecularly in detail. The most remarkable difference was the absence of microcephaly. To the best of our knowledge, this is the first report of a de novo 1q31.1-q32.1 deletion. Moreover, it illustrates how molecular delineation associated with fine clinical characterization can improve the genotype-phenotype correlations of classical cytogenetic abnormalities.     

Tumor promoter phorbol-12-myristate-13-acetate induces poly(ADP)-ribosylation in fibroblasts.

The tumor promoter phorbol-12-myristate-13-acetate (PMA) causes an increase in pol(ADP)-ribosylation in mouse and human fibroblasts via the intermediate formation of active oxygen. In contrast to poly(ADP)-ribosylation induced by the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine, de novo RNA and protein synthesis are required and the accumulation of the polymer occurs in the absence of detectable DNA strand breakage. Our results suggest a mechanism for PMA-induced modulation of chromatin structure and gene expression.     

Near-ultraviolet sensitivity of skin fibroblasts of patients with bloom's syndrome

Near-ultraviolet survival of colony forming ability was compared between fibroblast strains from normal individuals and seven strains from Bloom's Syndrome patients using monochromatic light at 313 nm. Six BS strains possessed abnormal survival properties. Two strains lacked the low dose shoulder, which is characteristic for normal fibroblasts, but their overall sensitivity was in the normal range. Four BS strains were hypersensitive and possessed biphasic survival curves with a sharp initial drop. They mimick in culture the characteristic solar sensitivity of BS patients.