Reconstitution of holin activity with a synthetic peptide containing the 1-32 sequence region of EJh,the EJ-1 phage holin

Pneumococcal EJ-1 phage holin (EJh) is a hydrophobic polypeptide of 85 amino acid residues displaying lethal inner membrane disruption activity. To get an insight into holin structure and function, several peptides representing the different topological regions predicted by sequence analysis have been synthesized. Peptides were structurally characterized in both aqueous buffer and membrane environments, and their potential to induce membrane perturbation was determined. Among them, only the N-terminal predicted transmembrane helix increased the membrane permeability. This segment, only when flanked by the positive charged residues on its N-terminal side, which are present in the sequence of the full-length protein, folds into a major alpha-helix structure with a transmembrane preferential orientation. Fluorescein quenching experiments of N-terminal-labeled peptide evidenced the formation of oligomers of variable size depending on the peptideto-lipid molar ratio. The self-assembling tendency correlated with the formation of transmembrane pores that permit the release of encapsulated dextrans of various sizes. When analyzed by atomic force microscopy, peptide-induced membrane lesions are visualized as transbilayer holes. These findings are the first evidence for a lytic domain in holins and for the nature of membrane lesions caused by them.     

Salmonella rapidly kill dendritic cells via a caspase-1-dependent mechanism

Dendritic cells provide a critical link between innate and acquired immunity. In this study, we demonstrate that the bacterial pathogen Salmonella enterica serovar Typhimurium can efficiently kill these professional phagocytes via a mechanism that is dependent on sipB and the Salmonella pathogenicity island 1-encoded type III protein secretion system. Rapid phosphatidylserine redistribution, caspase activation, and loss of plasma membrane integrity were characteristic of dendritic cells infected with wild-type Salmonella, but not sipB mutant bacteria. Caspase-1 was particularly important in this process because Salmonella-induced dendritic cell death was dramatically reduced in the presence of a caspase-1-specific inhibitor. Furthermore, dendritic cells obtained from caspase-1-deficient mice, but not heterozygous littermate control mice, were resistant to Salmonella-induced cytotoxicity. We hypothesize that Salmonella have evolved the ability to selectively kill professional APCs to combat, exploit, or evade immune defense mechanisms.     

Arrest of the cell cycle reduces susceptibility of target cells to perforin-mediated lysis

Cytotoxic T lymphocytes secrete a pore-forming cytolysin, perforin, that damages membranes of target cells. They also ligate Fas receptors on target cells and provoke apoptotic death. A20 (B lymphoma) and P815 (mastocytoma) cell lines were examined for their susceptibility to perforin-mediated lysis and to Fas-induced apoptosis after blockade of the cell cycle at the G₁/S interface. Cells were arrested at the G₁/S interface by inhibition of DNA synthesis with thymidine or aphidicolin. Subsequently, the treated cells were incubated either with CTL cytotoxic granules or the Fas-specific monoclonal antibody Jo-2. We show that arrest of the cell cycle at the G₁/S interface markedly reduced the susceptibility of target cells to perforin-mediated lysis. In contrast, growth arrest with thymidine or aphidicolin increased susceptibility of A20 and P815 cells to Fas-mediated apoptosis. Susceptibility to lysis by intact CTLs was not affected significantly by blockade of target cells with aphidicolin or thymidine. When cells surviving exposure to perforin-containing granules were isolated on Ficoll density gradients and cell-cycle profiles were examined by flow cytometry, the ratio of G₁ to G₂cells increased among the survivors exposed to granules in contrast to controls incubated with buffer alone. The data suggest that cells in G₁ phase of the cell cycle are less susceptible to the perforin pathway than cells in G₂and S phases but are more susceptible to the Fas pathway. J. Cell. Biochem. 69:425–435, 1998. © 1998 Wiley-Liss, Inc.     

Annual changes in the copulatory behavior of male rats maintained under constant laboratory conditions

ABSTRACT

The objective of the present study was to evaluate the sexual behavior of male rats kept under constant laboratory conditions for one entire year. A total of 213 sexually-inexperienced, male Wistar rats were maintained in controlled environmental conditions from birth. Depending the month in which they reached the age 3-month-old, the male rats were divided into 12 groups, one for each month of the year, and their sexual behavior was evaluated. Records of their sexual behavior were made from 09:00 to 11:00 hrs am. The following parameters were recorded: mount (latency and number), intromission (latency and number), ejaculation latency, and intromission rate. During the months of March, June, July and September, the rats showed lower mount and intromission latencies than in January, February, April, May and October-to-December. Similarly, in March, June, July and August they had higher copulatory efficiency than in January, February, April and December. Results suggest that male rats exposed to controlled environmental conditions could have endogenous mechanisms that regulate sexual behavior but are independent of seasonal environmental signals. The annual variability in the sexual behavior of male rats maintained under constant laboratory conditions should be considered when planning research and experiments.     

Surface tension induced by sphingomyelin to ceramide conversion in lipid membranes

We have investigated the effect of sphingomyelin (SM) to ceramide enzymatic conversion on lipid bilayers using Giant Unilamellar Vesicles (GUVs). Sphingomyelinase was added externally to GUVs containing various proportions of SM. In situ asymmetrical SM conversion to ceramide reduced the area of one leaflet. In the absence of equilibration of all the lipids between the two leaflets, a mismatch between the two monolayers was generated. The tension generated by this mismatch was sufficient to trigger the formation of membrane defects and total vesicle collapse at relatively low percentage of SM ( approximately 5% mol). The formation of nanometric size defects was visualised by AFM in supported bilayers. Vesicle rupture was prevented in two circumstances: (a) in GUVs containing a mixture of l(d) and l(o) domains and (b) in GUVs containing 5% lyso-phosphatidylcholine. In both cases, the accumulation of enough ceramide (at initial SM concentration of 10%) allowed the formation of ceramide-rich domains. The coupling between the two asymmetrical monolayers and the condensing effect produced by the newly formed ceramide generated a tension that could underlie the mechanism through which ceramide formation induces membrane modifications observed during the late stages of apoptosis.     

Identification of nerve plexi in connective tissues of the sea cucumber Holothuria glaberrima by using a novel nerve-specific antibody

The echinoderm nervous system is one of the least studied among invertebrates, partly because the tools available to study the neurobiology of this phylum are limited. We have now produced a monoclonal antibody (RN1) that labels a nervous system component of the sea cucumber Holothuria glaberrima. Western blots show that our antibody recognizes a major band of 66 kDa and a minor band of 53 kDa. Immunohistological experiments show that, in H. glaberrima, the antibody distinctly labels most of the known nervous system structures and some components that were previously unknown or little studied. A surprising finding was the labeling of nervous plexi within the connective tissue compartments of all organs studied. Double labeling with holothurian neuropeptides and an echinoderm synaptotagmin showed that RN1 labeled most, if not all, of the fibers labeled by these neuronal markers, but also a larger component of cells and fibers. The presence of a distinct connective tissue plexus in holothurians is highly significant since these organisms possess mutable connective tissues that change viscosity under the control of the nervous system. Therefore, the cells and fibers recognized by our monoclonal antibodies may be involved in controlling tensility changes in echinoderm connective tissue.     

Dorsal Raphe Dual Serotonin-Glutamate Neurons Drive Reward by Establishing Excitatory Synapses on VTA Mesoaccumbens Dopamine Neurons

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Biological soil crust influence on germination and rooting of two key species in a Stipa tenacissima steppe

Background and aims

In Mediterranean steppes, Stipa tenacissima tussocks facilitate the establishment of vascular plants. We hypothesized that this effect may partially reflect the indirect interaction between Stipa tenacissima, biological soil crusts (BSC), and seeds.

Methods

We explored the relationship between BSC composition and soil surface conditions (surface roughness and hydrophobicity by using the water drop penetration time test), and seed germination and seedling rooting in a S. tenacissima steppe in southeastern Spain. We explored the causal factors of seed germination at two spatial scales and used SADIE index to represents the soil surface heterogeneity.

Results

Microsites strongly differed in BSC composition and soil surface conditions. Germination of two key species, Pistacia lentiscus and Brachypodium retusum, was not affected by BSC type. In contrast, rooting was lower on soil from open areas covered by BSC than on soil from open areas dominated by bare soil and soil collected under the tussocks. The effect was similar in both species. Lichens were probably responsible for the decrease in rooting.

Conclusions

Our results suggest that lichen cover and the cover of bare soil and mosses may hamper and facilitate rooting, respectively. By affecting seedling rooting, BSC may contribute to the facilitative effect of Stipa tenacissima.