Trans-SILAC: sorting out the non-cell-autonomous proteome

Non-cell-autonomous proteins are incorporated into cells that form tight contacts or are invaded by bacteria, but identifying the full repertoire of transferred proteins has been a challenge. Here we introduce a quantitative proteomics approach to sort out non-cell-autonomous proteins synthesized by other cells or intracellular pathogens. Our approach combines stable-isotope labeling of amino acids in cell culture (SILAC), high-purity cell sorting and bioinformatics analysis to identify the repertoire of relevant non-cell-autonomous proteins. This 'trans-SILAC' method allowed us to discover many proteins transferred from human B to natural killer cells and to measure biosynthesis rates of Salmonella enterica proteins in infected human cells. Trans-SILAC should be a useful method to examine protein exchange between different cells of multicellular organisms or pathogen and host.     

Involvement of intestinal inducible nitric oxide synthase (iNOS) in the early stages of murine salmonellosis

Local induction of inducible nitric oxide synthase (iNOS) and apoptosis was examined in the intestine of mice infected with virulent Salmonella enterica serovar Enteritidis 5694 (S. enteritidis) and its attenuated derivative mutant E/1/3. Both, intestinal iNOS mRNA expression and iNOS activity showed a peak at 4 h only in animals receiving the virulent S. enteritidis. Aminoguanidine treatment abrogated intestinal epithelial damage produced by virulent S. enteritidis and diminished apoptosis at the tips of the villi. Unlike the virulent strain, mutant E/1/3 induced massive iNOS expression in Peyer's patches, these findings may be related to its protective capacity. Our results suggest that intestinal iNOS participates in the early response to intestinal infection and that the final effect depends on the nature of the insult.     

The Susceptibility of Different Species and Stages of Ticks to Entomopathogenic Fungi

Boophilus annulatus, Hyalomma excavatum and Rhipicephalus sanguineus ticks were shown to be susceptible to different entomopathogenic fungi under laboratory conditions. Comparative results of bioassays using five different fungal species showed that some strains of Metarhizium anisopliae are highly pathogenic against various tick stages tested. In contrast to their activity against insects, fungi also affected tick eggs. All tested tick stages including those feeding on a host were found to be susceptible to these fungi, except for adult H. excavatum ticks, which were relatively resistant. This revised version was published online in July 2006 with corrections to the Cover Date.     

Bacterial Cellulose-Binding Domain Modulates in Vitro Elongation of Different Plant Cells

Recombinant cellulose-binding domain (CBD) derived from the cellulolytic bacterium Clostridium cellulovorans was found to modulate the elongation of different plant cells in vitro. In peach (Prunus persica L.) pollen tubes, maximum elongation was observed at 50 μg mL⁻¹ CBD. Pollen tube staining with calcofluor showed a loss of crystallinity in the tip zone of CBD-treated pollen tubes. At low concentrations CBD enhanced elongation of Arabidopsis roots. At high concentrations CBD dramatically inhibited root elongation in a dose-responsive manner. Maximum effect on root hair elongation was at 100 μg mL⁻¹, whereas root elongation was inhibited at that concentration. CBD was found to compete with xyloglucan for binding to cellulose when CBD was added first to the cellulose, before the addition of xyloglucan. When Acetobacter xylinum L. was used as a model system, CBD was found to increase the rate of cellulose synthase in a dose-responsive manner, up to 5-fold compared with the control. Electron microscopy examination of the cellulose ribbons produced by A. xylinum showed that CBD treatment resulted in a splayed ribbon composed of separate fibrillar subunits, compared with a thin, uniform ribbon in the control.     

Thermal limitations of <Emphasis Type=Italic>Metarhizium anisopliae</Emphasis> efficacy: selection for application on warm-blooded vertebrates

Temperature is one of the main obstacles for on-host applications of entomopathogenic fungi for ectoparasite control. The effects of temperatures typical of the body surfaces of warm-blooded animals on the germination, growth and virulence of four strains of Metarhizium anisopliae toward engorged Rhipicephalus (Boophilus) annulatus females were evaluated. The M. anisopliae strains studied can be divided according to their thermal characteristics: (1) strains which germinate (90–100%), grow and infect ticks similarly at 25, 30 and 35°C; and (2) strains which recover their ability to germinate relatively quickly following a thermal shock (37 or 40°C for 6–48 h) before incubation at a favorable temperature. These latter strains could recover their infectivity after a short thermal shock (6 h at 37–40°C), but not after more prolonged exposure to these temperatures (48–72 h). These two thermal characteristics do not interact, but reflect the efficacy of strains used to control ectoparasites on warm-blooded vertebrates.     

TGF-β1 induces the expression of type I collagen and SPARC,and enhances contraction of collagen gels,by fibroblasts from young and aged donors

Fibroblasts have a major role in the synthesis and reorganization of extracellular matrix that occur during wound repair. An impaired biosynthetic or functional response of these cells to stimulation by growth factors might contribute to the delayed wound healing noted in aging. We, therefore, compared the responses of dermal fibroblasts from young and elderly individuals (26, 29, 65, 89, 90, and 92 years of age) to transforming growth factor-β1 (TGF-β1) with respect to: (1) the synthesis of type I collagen and SPARC (two extracellular matrix proteins that are highly expressed by dermal fibroblasts during the remodeling phase of wound repair) and (2) the contraction of collagen gels, an in vitro assay of wound contraction. With the exception of one young donor, all cultures exposed for 44 hours to 10 ng/ml TGF-β1 exhibited a 1.6- to 5.5-fold increase in the levels of secreted type 1 collagen and SPARC, relative to untreated cultures, and exhibited a 2.0- to 6.2-fold increase in the amounts of the corresponding mRNAs. Moreover, the dose-response to TGF-β1 (0.1–10 ng/ml), as determined by synthesis of type I collagen and SPARC mRNA, was as vigorous in cells from aged donors as in cells from a young donor. In assays of collagen gel contraction, fibroblasts from all donors were stimulated to a similar degree by 10 ng/ml TGF-β1. In conclusion, cells from both young and aged donors exhibited similar biosynthetic and contractile properties with exposure to TGF-β1. It therefore appears that the impaired wound healing noted in the aged does not result from a failure of their dermal fibroblasts to respond to this cytokine. © 1994 Wiley-Liss, Inc.     

The benthic phase of the life cycle ofParapenaeus longirostris (Crustacea,Decapoda, Penaeidae) along the Mediterranean coast of Israel

The population ofP. longirostris along the Mediterranean coast of Israel spends the benthic phase of its life cycle (from body size over 15 mm) on muddy bottom deeper than 45 m. New age groups are recruited within the depth zone of 45–300 m and migrate in both inshore and offshore directions. Inshore migration is limited by unsuitable sandy ground. The limiting line for offshore migration was not found.An age group could be detected during one year within a body size range of TL = 40 mm to TL = 84.5 mm for males and to TL = 102.5 mm for females. Reproductive activity in shallow water, down to a depth of 73 m, takes place during the whole year, while in depths of 150–300 m there is an arrest of reproductive activity from June to August.     

Learning and control of exploration primitives

Animals explore novel environments in a cautious manner, exhibiting alternation between curiosity-driven behavior and retreats. We present a detailed formal framework for exploration behavior, which generates behavior that maintains a constant level of novelty. Similar to other types of complex behaviors, the resulting exploratory behavior is composed of exploration motor primitives. These primitives can be learned during a developmental period, wherein the agent experiences repeated interactions with environments that share common traits, thus allowing transference of motor learning to novel environments. The emergence of exploration motor primitives is the result of reinforcement learning in which information gain serves as intrinsic reward. Furthermore, actors and critics are local and ego-centric, thus enabling transference to other environments. Novelty control, i.e. the principle which governs the maintenance of constant novelty, is implemented by a central action-selection mechanism, which switches between the emergent exploration primitives and a retreat policy, based on the currently-experienced novelty. The framework has only a few parameters, wherein time-scales, learning rates and thresholds are adaptive, and can thus be easily applied to many scenarios. We implement it by modeling the rodent’s whisking system and show that it can explain characteristic observed behaviors. A detailed discussion of the framework’s merits and flaws, as compared to other related models, concludes the paper.     

PCNA ubiquitination is important, but not essential for translesion DNA synthesis in mammalian cells

Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism in which specialized low-fidelity DNA polymerases bypass replication-blocking lesions, and it is usually associated with mutagenesis. In Saccharomyces cerevisiae a key event in TLS is the monoubiquitination of PCNA, which enables recruitment of the specialized polymerases to the damaged site through their ubiquitin-binding domain. In mammals, however, there is a debate on the requirement for ubiquitinated PCNA (PCNA-Ub) in TLS. We show that UV-induced Rpa foci, indicative of single-stranded DNA (ssDNA) regions caused by UV, accumulate faster and disappear more slowly in Pcna(K164R/K164R) cells, which are resistant to PCNA ubiquitination, compared to Pcna(+/+) cells, consistent with a TLS defect. Direct analysis of TLS in these cells, using gapped plasmids with site-specific lesions, showed that TLS is strongly reduced across UV lesions and the cisplatin-induced intrastrand GG crosslink. A similar effect was obtained in cells lacking Rad18, the E3 ubiquitin ligase which monoubiquitinates PCNA. Consistently, cells lacking Usp1, the enzyme that de-ubiquitinates PCNA exhibited increased TLS across a UV lesion and the cisplatin adduct. In contrast, cells lacking the Rad5-homologs Shprh and Hltf, which polyubiquitinate PCNA, exhibited normal TLS. Knocking down the expression of the TLS genes Rev3L, PolH, or Rev1 in Pcna(K164R/K164R) mouse embryo fibroblasts caused each an increased sensitivity to UV radiation, indicating the existence of TLS pathways that are independent of PCNA-Ub. Taken together these results indicate that PCNA-Ub is required for maximal TLS. However, TLS polymerases can be recruited to damaged DNA also in the absence of PCNA-Ub, and perform TLS, albeit at a significantly lower efficiency and altered mutagenic specificity.     

A new β-lactoglobulin-based vector targets luciferase cDNA expression to the mammary gland of transgenic mice

A -lactoglobulin (BLG)/luciferase gene vector (p907), composed of a luciferase intronless gene inserted between the second and sixth BLG exons was constructed. Stable transfections of CID-9 cells with this vector, as well as with a series of additional vectors, were performed to define regulatory regions within the BLG sequence, and the contribution of the SV40 polyadenylation (PA) site to luciferase expression. A relatively low level of luciferase activity was supported by vector p907. It was partially rescued by vector p906, in which the BLG 3 region, downstream of the luciferase cDNA, was replaced with the SV40 PA site. Flanking the SV40 region of vector p906, at its 3 end, with BLG sequences of exon 6/intron 6/exon 7 and the 3 region of the gene resulted in vector p904. This vector supported the highest luciferase activity, 10 times or 2.5 times higher than that measured in cells transfected with vectors p907 and p906, respectively. The induced activity supported by vector p904 is attributed to interaction between the SV40 PA site and elements of the distal part of the BLG 3 flanking sequences. The BLG 5 regulatory region of vector p904 encompasses a 3-kb promoter sequences. Deletion of 935 bp of its proximal end resulted in a 60% decrease in luciferase activity. Reduced activity was also seen with vector p915 lacking sequences of exon 1/intron 1/exon 2. This decrease could not be rescued with heterologous sequences of insulin intron 1, inserted upstream of the luciferase cDNA. Two sets of transgenic mice carrying vectors p907 and p904 were generated. Vector p907 supported only marginal luciferase activity in the mammary gland of all transgenic mice tested and luciferase RNA could not be detected by northern analysis. In contrast, 50% of the transgenic mice carrying vector p904 expressed luciferase RNA in the mammary gland and tissue-specific, hormonal-dependent activity was determined. However, the new p904 vector was not able to insulate the transgene from surrounding host DNA sequences, as reflected by its copy number-independent manner of expression. Nevertheless, vector p904 may represent a valuable tool for the expression of cDNAs in the mammary gland of transgenic animals.