Differences between larval and pupal hemocytes of the tobacco hornworm, Manduca sexta, determined by monoclonal antibodies and density centrifugation

Insect hemocytes play a major role in developmental processes where they disassociate and rebuild metamorphosing tissues while undergoing physiological changes themselves. We identified hemocyte changes from the last larval to the beginning of the pupal stage of the tobacco hornworm, Manduca sexta. Larval and pupal hemocytes behaved differently in a 40% Percoll density gradient. Larval granular cells were found in almost all density layers, pupal granular cells were abundant in high density layers; larval plasmatocytes occurred in dense layers, pupal plasmatocytes became enriched in less dense layers of the gradient. Using a panel of monoclonal antibodies generated against purified hemocytes, several different antibody binding patterns were identified. Quantitative differences in staining intensities were observed more often than qualitative changes, e.g. a loss or a gain of staining. Both phenomena were related to both plasmatocytes and granular cells. The distribution of the corresponding antigens in tissues was tested on cross sections of larvae and pupae as well as in Western blot analyses using organ homogenates. Several antibodies were specific for hemocytes only, among which two antibodies bound to molecules of the hematopoietic organ. Other antibodies had an additional reactivity to other tissues, mainly to the basal lamina.     

The human antimicrobial peptide LL-37 transfers extracellular DNA plasmid to the nuclear compartment of mammalian cells via lipid rafts and proteoglycan-dependent endocytosis

Antimicrobial peptides, such as LL-37, are found both in nonvertebrates and vertebrates, where they represent important components of innate immunity. Bacterial infections at epithelial surfaces are associated with substantial induction of LL-37 expression, which allows efficient lysis of the invading microbes. Peptide-mediated lysis results in the release of bacterial nucleic acids with potential pathobiological activity in the host. Here, we demonstrate that LL-37 targets extracellular DNA plasmid to the nuclear compartment of mammalian cells, where it is expressed. DNA transfer occurred at physiological LL-37 concentrations that killed bacterial cells, whereas virtually no cytotoxic or growth-inhibitory effects were observed in mammalian cells. Furthermore, LL-37 protected DNA from serum nuclease degradation. LL-37.DNA complex uptake was a saturable time- and temperature-dependent process and was sensitive to cholesterol-depleting agents that are known to disrupt lipid rafts and caveolae, as shown by flow cytometry. Confocal fluorescence microscopy studies showed localization of internalized DNA to compartments stained by cholera toxin B, a marker of lipid rafts, but failed to demonstrate any co-localization of internalized DNA with caveolin-positive endocytotic vesicles. Moreover, LL-37-mediated plasmid uptake and reporter gene expression were strictly dependent on cell surface proteoglycans. We conclude that the human antimicrobial peptide LL-37 binds to, protects, and efficiently targets DNA plasmid to the nuclei of mammalian cells through caveolae-independent membrane raft endocytosis and cell surface proteoglycans.     

Reduced mobilization of Rev-responsive element-deficient lentiviral vectors

Infection of cells transduced with a lentiviral vector by human immunodeficiency virus (HIV) could lead to packaging of the lentiviral vector RNA into HIV particles and unintended transfer of the vector. To prevent this, the Rev-responsive element (RRE) of an HIV-1 vector was functionally replaced by a heterologous RNA element (MS2). Providing Rev fused to an MS2 binding protein allowed efficient vector production. Mobilization of the vector from infected target cells was below the level of detection and at least 10(3)- to 10(4)-fold lower than for the RRE-containing vector. Thus, RRE-deficient lentiviral vectors provide a novel approach to reduce the risk of vector mobilization.     

ADPβS evokes microglia activation in the rabbit retina <Emphasis Type="Italic">in vivo</Emphasis>

To investigate whether stimulation of purinergic P2Y(1) receptors modulates the activation of microglial and Müller glial cells in the rabbit retina in vivo, adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS; 2 mM in 100 mul saline), a non-hydrolyzable ADP analogue, was intravitreadly applied into control eyes or onto retinas that were experimentally detached from the pigment epithelium. Both retinal detachment and application of ADPssS onto control retinas induced phenotype alterations of the microglial cells (decrease of soma size, retraction of cell processes) and had no influence on microglial cell density. ADPssS application onto detached retinas accelerated the process retraction and resulted in a strongly decreased density of microglial cells. The effects of ADPssS on microglia density and phenotype in detached retinas were partially reversed by co-application of the selective inhibitor of P2Y(1) receptors, MRS-2317 (3 mM in 100 mul saline). ADPssS apparently did not influence Müller cell gliosis, as determined by electrophysiological and calcium imaging records. It is concluded that rabbit retinal microglial cells express functional P2Y(1) receptors in vivo, and that activation of these receptors stimulates phenotype alterations that are characteristical for microglia activation.     

Shear treatment of starter culture medium improves separation behavior of Streptococcus thermophilus cells

A central step in the production of starter cultures is the separation of the cells from the fermentation medium, which is usually achieved by disk centrifuges. In case of microorganisms which produce exopolysaccharides (e.g., various strains of lactic acid bacteria), the properties of the respective exopolysaccharides may interfere with this separation step. By using six strains of Streptococcus thermophilus the hypothesis was tested that a shear treatment of the fermented culture medium improves subsequent cell separation markedly. Depending on the type of exopolysaccharides (freely present in the medium, or as capsules around the cells) an energy input of up to 2.5 kJ/mL generated with an Ultra‐Turrax affected cell chain length of the strains and viscosity of fermentation medium differently. For bacteria producing capsular exopolysaccharides, space‐ and time‐resolved centrifugation experiments revealed an increase of sedimentation velocity after shear treatment. In general, viability of the microorganisms, detected by flow cytometry measurements and fermentation experiments, was not affected by the shearing procedure. The results therefore indicate that strain‐targeted shearing is helpful to improve the separability of cells from the fermented media.     

Population profiles of a stable, commensalistic bacterial culture grown with toluene under sulphate-reducing conditions.

BACKGROUND: Most bacteria present in nature are not culturable in pure culture by means of classic cultivation methods (Pace NR, 1997, Science 276:734-740; Amann RI et al., 1995, Microbiol Rev 59:143-169.). However, it was recently shown that most aerobic heterotrophic bacteria could grow only on artificial media when other micro-organisms are present (Kaeberlein T et al., 2002, Science 296:1127-1129). Because the sulphate reducer Desulfobacula toluolica DSM 7467 and a bacterium (strain MV1) identified as Cellulosimicrobium sp. were not culturable unaccompanied, flow cytometry was used to highlight the strains' relation within the consortium. METHODS: DNA patterns were used to provide strain-specific information about population proliferation dynamics. Cells were grown anaerobically and fed with toluene under sulphate-reducing conditions. RESULTS: Oxidation of toluene occurred only in association with sulphate reduction and growth of D. toluolica. A characteristic chromosomal pattern, with at least six subpopulations of D. toluolica, appeared during the stationary phase, and asymmetric cell division was detected. The accompanying strain MV1 grew repeatedly to a high percentage of the culture only in certain growth phases of D. toluolica independently of the feeding substrate toluene. CONCLUSIONS: A commensalistic relation between the two strains is suggested. The repeated rapid and frequent changes of the quantities within the community subsets are indicative of very flexible adaptations to changing environmental conditions, reflecting the need for modulated cell states and the ability to use every available source of carbon and energy for survival.     

Characterization of the specificity of binding ofMoluccella laevis lectin to glycosphingolipids

The specificity ofMoluccella laevis lectin was investigated by analysing its binding to glycosphingolipids separated on thin-layer chromatograms or adsorbed on microtitre wells. The binding activity of the lectin was highest for glycosphingolipids with terminal -linkedN-acetylgalactosamine, both in linear structures, as the Forssman glycosphingolipid, GalNAc3GalNAc3Gal4Gal4Glc1Cer, and in branched structures, as glycosphingolipids with the blood group A determinant, GalNAc3(Fuc2)Gal. In addition, the lectin bound, though considerably more weakly, to linear glycosphingolipids with terminal -linked galactose. When considering the use of theM. laevis lectin for biochemical and medical purposes this cross-reactivity may be of importance. Nomenclature: The glycosphingolipid nomenclature follows the recommendations by the IUPAC-IUB Commission on Biochemical Nomenclature (CBN for Lipids:Eur J Biochem (1977)79:11–21,J Biol Chem (1982)257:3347–51, andJ Biol Chem (1987)262:13–18). It is assumed that Gal, Glc, GlcNAc, GalNAc, and NeuAc are of thed-configuration, Fuc of thel-configuration, and all sugars present in the pyranose form.     

Prp4 Kinase Grants the License to Splice: Control of Weak Splice Sites during Spliceosome Activation

The genome of the fission yeast Schizosaccharomyces pombe encodes 17 kinases that are essential for cell growth. These include the cell-cycle regulator Cdc2, as well as several kinases that coordinate cell growth, polarity, and morphogenesis during the cell cycle. In this study, we further characterized another of these essential kinases, Prp4, and showed that the splicing of many introns is dependent on Prp4 kinase activity. For detailed characterization, we chose the genes res1 and ppk8, each of which contains one intron of typical size and position. Splicing of the res1 intron was dependent on Prp4 kinase activity, whereas splicing of the ppk8 intron was not. Extensive mutational analyses of the 5’ splice site of both genes revealed that proper transient interaction with the 5’ end of snRNA U1 governs the dependence of splicing on Prp4 kinase activity. Proper transient interaction between the branch sequence and snRNA U2 was also important. Therefore, the Prp4 kinase is required for recognition and efficient splicing of introns displaying weak exon1/5’ splice sites and weak branch sequences.     

Fast direct melting of brackish sea-ice samples results in biologically more accurate results than slow buffered melting

Sea-ice samples intended for biological analyses, e.g., chlorophyll-a, cell enumeration of algae and protozoa and primary production, are affected by the sampling and sample processing methods. In this study, we compared different sample processing methods by melting Baltic Sea ice samples in different ways (direct melting, buffered melting in filtered seawater (FSW) and buffered melting in artificial seawater at two different salinities with added nutrients) at two temperatures [+4 °C and room temperature (RT)]. We show that sea-ice samples intended for most commonly used biological analyses can be melted without the addition of FSW. In particular, adding artificial seawater should be avoided. To minimize biological processes, such as growth, death, predation and pigment degradation, the melting should be done rapidly at RT preferably by gently shaking the sample to keep the melt cool.