Fusion and infection of influenza and Sendai viruses as modulated by dextran sulfate: a comparative study

We have directly compared the effect of two types of dextran sulfate with distinct molecular weights (500 kDa and 5 kDa) on the fusion activity and infectivity of both Sendai and influenza viruses, two lipid-enveloped viruses that differ in their routes of entry into target cells. To correlate membrane merging and infectivity MDCK cells were used as targets for the viruses in both approaches. In either case pronounced inhibition of virus–cell interactions by dextran sulfate was only observed at low pH, even though Sendai virus fuses maximally at pH 7.4. Although membrane merging could not be fully abolished, the inhibitory effect was always greater when the higher molecular weight dextran sulfate was used. The presence of this residual fusion activity, that could not be reduced even with high concentrations of agent, suggests that a limited number of binding sites for dextran sulfate may exist on the viral envelopes. The compounds also inhibited fusion of bound virions, and all results could be reproduced using erythrocyte ghosts as target membranes in the fusion assay, instead of MDCK cells. In agreement with these observations only the infectivity of influenza virus (which requires a low pH-dependent step to enter target cells) was affected by dextran sulfate, again the higher molecular weight compound showing a more pronounced inhibitory effect.     

Semaphorin3A enhances endocytosis at sites of receptor-F-actin colocalization during growth cone collapse

Axonal growth cone collapse is accompanied by a reduction in filopodial F-actin. We demonstrate here that semaphorin 3A (Sema3A) induces a coordinated rearrangement of Sema3A receptors and F-actin during growth cone collapse. Differential interference contrast microscopy reveals that some sites of Sema3A-induced F-actin reorganization correlate with discrete vacuoles, structures involved in endocytosis. Endocytosis of FITC-dextran by the growth cone is enhanced during Sema3A treatment, and sites of dextran accumulation colocalize with actin-rich vacuoles and ridges of membrane. Furthermore, the Sema3A receptor proteins, neuropilin-1 and plexin, and the Sema3A signaling molecule, rac1, also reorganize to vacuoles and membrane ridges after Sema3A treatment. These data support a model whereby Sema3A stimulates endocytosis by focal and coordinated rearrangement of receptor and cytoskeletal elements. Dextran accumulation is also increased in retinal ganglion cell (RGC) growth cones, in response to ephrin A5, and in RGC and DRG growth cones, in response to myelin and phorbol-ester. Therefore, enhanced endocytosis may be a general principle of physiologic growth cone collapse. We suggest that growth cone collapse is mediated by both actin filament rearrangements and alterations in membrane dynamics.     

Neutrophils are cytotoxic and growth-inhibiting for neuroblastoma cells with an anti-GD2 antibody but, without cytotoxicity, can be growth-stimulating

Neutrophils and mononuclear cells (MNC) can mediate antibody-dependent cellular cytotoxicity (ADCC) against cancer cells. To study cytotoxicity and growth inhibition of neuroblastoma cells by neutrophils and MNC with chimeric anti-disialoganglioside (GD2) monoclonal antibody (mAb) ch14.18, we developed digital image microscopy scanning (DIMSCAN) assays that measure fluorescence of target cells in 96-well plates after 6–18 h (cytotoxicity assay) or 7 days (growth assay). Neuroblastoma cell lines (GD2-positive: SMS-KCN, SMS-LHN, LA-N-1; GD2-negative: SK-N-SH) were preloaded with calcein acetoxymethyl ester for the cytotoxicity assay or labeled in situ after 7 days of culture with fluorescein diacetate in the growth assay. Fluorescence, as quantified by DIMSCAN, was correlated with neuroblastoma cell number in both assays (100–2000 cells/well). In the cytotoxicity test, both neutrophils and MNC effectively mediated ADCC of GD2-positive but not GD2-negative neuroblastoma cell lines. Cytotoxicity of both neutrophils and MNC increased with effector to target cell (E:T) ratio (5–50:1) and mAb ch.14.18 dose (0.1–10 μg/ml). ADCC of neutrophils, but not MNC, increased with addition of GM-CSF. Neutrophils, especially with rhGM-CSF, significantly suppressed growth of GD2-positive cell lines at a high E:T ratio (50:1) and mAb dose (10 μg/ml). Without antibody, neutrophils inhibited growth of one cell line (LA-N-1) but stimulated growth of two others (SMS-KCN, SMS-LHN). If neuroblastoma cells did not express GD2 (SK-N-SH), neutrophils stimulated growth whether or not antibody was present. Neutrophil culture supernatants increased growth of SK-N-SH, LA-N-1, and SMS-KCN cells, and MNC culture supernatants increased growth of SK-N-SH. In conclusion, neutrophils can mediate cytotoxicity and growth inhibition with a chimeric anti-GD2 antibody but also can promote tumor cell growth if antibody is not present or if GD2 is not expressed. Received: 18 November 1998 / Accepted: 24 September 1999     

Soybean Bowman–Birk Inhibitor Conjugates with Clinical Dextran. Synthesis and Antiproteolytic Activity

Conjugates of the classical soybean Bowman-Birk inhibitor (BBI) with clinical dextran were synthesized. Clinical dextran was preliminarily oxidized with periodate to dialdehydedextran (DAD). The effect of the degree of oxidation of DAD on coupling of the inhibitor was evaluated. The binding of the protein was shown to increase with increasing degree of DAD oxidation (5, 10, 20%). Total coupling of the inhibitor occurred when the degree of oxidation of the dextran was 20%. The BBI-DAD (20%) conjugate contained 13% protein with BBI/DAD molar ratio 1 : 1. The conjugates retained the ability to inhibit trypsin (Ki = 0.2-0.3 nM) and alpha-chymotrypsin (Ki = 15-30 nM). Thus, the coupling of BBI with the polymeric carrier caused practically no decrease in the antiproteolytic activity of the inhibitor.     

Quantification of metabolic activity of cultured plant cells by vital staining with fluorescein diacetate

The metabolic activity of suspension cultures of Sonneratia alba cells was quantified by measurement of the hydrolysis of fluorescein diacetate (FDA). FDA is incorporated into live cells and is converted into fluorescein by cellular hydrolysis. Aliquots (0.1–0.75 g) of S. alba cells were incubated with FDA at a final concentration of 222 μg/ml suspension for 60 min. Hydrolysis was stopped, and fluorescein was extracted by the addition of acetone and quantified by measurement of absorbance at 490 nm. Fluorescein was produced linearly with time and cell weight. Cells of S. alba are halophilic and proliferated well in medium containing 50 and 100 mM NaCl. Cells grown in medium containing 100 mM NaCl showed 2- to 3-fold higher FDA hydrolysis activity than those grown in NaCl-free medium. When S. alba cells grown in medium supplemented with 50 mM NaCl were transferred to fresh medium containing 100 mM mannitol, cellular FDA hydrolysis activity was down-regulated after 4 days of culture, indicating that the moderately halophilic S. alba cells were sensitive to osmotic stress. Quantification of cellular metabolic activity via the in vivo FDA hydrolysis assay provides a simple and rapid method for the determination of cellular activity under differing culture conditions.     

Major signaling pathways in intestinal stem cells

Background

The discovery of markers to identify the intestinal stem cell population and the generation of powerful transgenic mouse models to study stem cell physiology have led to seminal discoveries in stem cell biology.

Scope of review

In this review we give an overview of the current knowledge in the field of intestinal stem cells (ISCs) highlighting the most recent progress on markers defining the ISC population and pathways governing intestinal stem cell maintenance and differentiation. Furthermore we review their interaction with other stem cell related pathways. Finally we give an overview of alteration of these pathways in human inflammatory gastrointestinal diseases.

Major conclusions

We highlight the complex network of interactions occurring among different pathways and put in perspective the many layers of regulation that occur in maintaining the intestinal homeostasis.

General significance

Understanding the involvement of ISCs in inflammatory diseases can potentially lead to new therapeutic approaches to treat inflammatory GI pathologies such as IBD and celiac disease and could reveal the molecular mechanisms leading to the pathogenesis of dysplasia and cancer in inflammatory chronic conditions. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

Divalent Cation Interactions with Na,K-ATPase Cytoplasmic Cation Sites: Implications for the <Emphasis Type="Italic">para</Emphasis>-Nitrophenyl Phosphatase Reaction Mechanism

The interactions of divalent cations with the adenosine triphosphatase (ATPase) and para-nitrophenyl phosphatase (pNPPase) activity of the purified dog kidney Na pump and the fluorescence of fluorescein isothiocyanate (FITC)-labeled pump were determined. Sr²⁺ and Ba²⁺ did not compete with K⁺ for ATPase (an extracellular K⁺ effect). Sr²⁺ and Ba²⁺ did compete with Na⁺ for ATPase (an intracellular Na⁺ effect) and with K⁺ for pNPPase (an intracellular K⁺ effect). These results suggest that Ba²⁺ or Sr²⁺ can bind to the intracellular transport site, yet neither Ba²⁺ nor Sr²⁺ was able to activate pNPPase activity; we confirmed that Ca²⁺ and Mn²⁺ did activate. As another measure of cation binding, we observed that Ca²⁺ and Mn²⁺, but not Ba²⁺, decreased the fluorescence of the FITC-labeled pump; we confirmed that K⁺ substantially decreased the fluorescence. Interestingly, Ba²⁺ did shift the K⁺ dose-response curve. Ethane diamine inhibited Mn²⁺ stimulation of pNPPase (as well as K⁺ and Mg²⁺ stimulation) but did not shift the 50% inhibitory concentration (IC₅₀) for the Mn²⁺-induced fluorescence change of FITC, though it did shift the IC₅₀ for the K⁺-induced change. These results suggest that the Mn²⁺-induced fluorescence change is not due to Mn²⁺ binding at the transport site. The drawbacks of models in which Mn²⁺ stimulates pNPPase by binding solely to the catalytic site vs. those in which Mn²⁺ stimulates by binding to both the catalytic and transport sites are presented. Our results provide new insights into the pNPPase kinetic mechanism as well as how divalent cations interact with the Na pump.     

Translocation and nuclear accumulation of monomer and dimer of HIV-1 Tat basic domain in triticale mesophyll protoplasts

Cellular internalization of cell-penetrating peptide HIV-1 Tat basic domain (RKKRRQRRR) was studied in Triticale cv AC Alta mesophyll protoplasts. Fluorescently labeled monomer (Tat) and dimer (Tat₂) of Tat basic domain efficiently translocated through the plasma membrane of mesophyll protoplast and showed distinct nuclear accumulation within 10 min of incubation. Substitution of first arginine residue with alanine in Tat basic domain (M-Tat) severely reduced cellular uptake of the peptide (3.8 times less than Tat). Tat₂ showed greater cellular internalization than Tat (1.6 times higher). However, characteristics of cellular uptake remained same for Tat and Tat₂. Cellular internalization of Tat and Tat₂ was concentration dependent and non-saturable whereas no significant change in cellular uptake was observed even at higher concentrations of M-Tat. Low temperature (4 °C) remarkably increased cellular internalization of Tat as well as Tat₂ but M-Tat showed no enhanced uptake. Viability test showed that peptide treatment had no cytotoxic effect on protoplasts further indicating involvement of a common mechanism of peptide uptake at all the temperatures. Endocytic inhibitors nocodazole (10 μM), chloroquine (100 μM) and sodium azide (5 mM) did not show any significant inhibitory effect on cellular internalization of either Tat or Tat₂. These results along with stimulated cellular uptake at low temperature indicate that Tat peptide is internalized in the plant protoplasts in a non-endocytic and energy-independent manner. Competition experiments showed that non-labeled peptide did not inhibit or alter nuclear accumulation of fluorescent Tat or Tat₂ suggesting active transport to the nucleus was not involved. Studies in mesophyll protoplasts show that internalization pattern of Tat peptide is apparently similar to that observed in mammalian cell lines.     

Estimation of Förster's distance between two ends of Dps protein from mycobacteria: distance heterogeneity as a function of oligomerization and DNA binding

Dps protein (DNA binding Protein from Starved Cells) from Mycobacterium smegmatis (Ms-Dps) is known to undergo an in vitro irreversible oligomeric transition from trimer to dodecamer. This transition helps the protein to provide for bimodal protection to the bacterial DNA from the free radical and Fenton mediated damages in the stationary state. The protein exists as a stable trimer, when purified from E. coli cells transformed with an over-expression plasmid. Both trimer as well as dodecamer are known to exhibit ferroxidation activity, thus removing toxic hydroxyl radicals in vivo, whereas iron accumulation and non-sequence specific DNA binding activity are found in dodecamer only. This seems to be aided by the positively charged long C-terminal tail of the protein. We used frequency domain phase-modulation fluorescence spectroscopy and Förster Resonance Energy Transfer (FRET) to monitor this oligomeric switch from a trimer to a dodecamer and to elucidate the structure of DNA–Dps dodecamer complex. As Ms-Dps is devoid of any Cysteine residues, a Serine is mutated to Cysteine (S169C) at a position adjacent to the putative DNA binding domain. This Cysteine is subsequently labeled with fluorescent probe and another probe is placed at the N-terminus, as crystal structure of the protein reveals several side-chain interactions between these two termini, and both are exposed towards the surface of the protein. Here, we report the Förster's distance distribution in the trimer and the dodecamer in the presence and absence of DNA. Through discrete lifetime analysis of the probes tagged at the respective regions in the macromolecule, coupled with Maximum Entropy Method (MEM) analysis, we show that the dodecamer, upon DNA binding shows conformational heterogeneity in overall structure, perhaps mediated by a non-specific DNA–protein interaction. On the other hand, the nature of DNA–Dps interaction is not known and several models exist in literature. We show here with the help of fluorescence anisotropy measurements of labeled DNA having different length and unlabeled native dodecameric protein that tandem occupation of DNA binding sites by a series of Dps molecules perhaps guide the tight packing of Dps over DNA backbone.