Sample preparation and imaging of erythrocyte cytoskeleton with the atomic force microscopy

A novel method for the covalent attachment of erythrocytes to glass microscope coverslips that can be used to image intact cells and the cytoplasmic side of the cell membrane with either solid or liquid mode atomic force microscopy (AFM) is described. The strong binding of cells to the glass surface is achieved by the interaction of cell membrane carbohydrates to lectin, which is bound to N-5-azido-2-nitrobenzoyloxysuccinimide (ANBNOS)-coated coverslips (1). The effectiveness of this method is compared with the other commonly used methods of immobilizing intact erythrocytes on glass coverslips for AFM observations. Experimental conditions of AFM imaging of biologic tissue are discussed, and typical topographies of the extracellular and the cytoplasmic surfaces of the plasma membrane in the dry state and in the liquid state are presented. Comparison of the spectrin network of cell age-separated erythrocytes has demonstrated significant loss in the network order in older erythrocytes. The changes are quantitatively described using the pixel height histogram and window size grain analysis.     

Comparative pharmacology of feeding in molluscs

1. This paper reviews the role of transmitters in identified neurons of gastropod molluscs in generating and modulating fictive feeding. 2. In Lymnaea and Helisoma the 3 phase rhythm is generated by sets of interneurons which use acetylcholine for the N1 (protraction) phase, glutamate for the N2 (rasp) phase interneurons. The N3 interneurons are likely to use several different transmitters, of which one is octopamine. 3. In all the species examined, serotonin (5-HT) is released from giant cerebral cells. Other amines, including dopamine and octopamine, are present in the buccal ganglia and all these amines activate or enhance feeding. 4. Nitric oxide (NO), mostly originating from sensory processes, can also activate fictive feeding, but (at least in Lymnaea) may also be released centrally from buccal (B2) and cerebral neurons (CGC). 5. The central pattern generator for feeding is also modulated by peptides including APGWamide, SCP(B) and FMRFamide. 6. There is increasing evidence that most of these transmitters/modulators act on feeding neurons through second messenger systems--allowing them to act as longer-lasting neuromodulators of the feeding network. 7. Many of the transmitters are used in similar ways by each of the gastropods examined so far, so that their function in the CNS seems to have been conserved through evolution.     

Measles Virus DNA Vaccination: Antibody Isotype Is Determined by the Method of Immunization and by the Nature of both the Antigen and the Coimmunized Antigen

Plasmids encoding the measles virus hemagglutinin (HA) and nucleoprotein (NP) proteins inoculated into the skin of BALB/c mice by the gene gun method induced both humoral and cytotoxic lymphocyte class I-restrict- ed immune responses. Although intramuscular immunization induces the immunoglobulin G2a (IgG2a) antibody isotype for both antigens, with gene gun immunization, the NP still generated mainly IgG2a and the major isotype induced by the HA was IgG1. Interestingly, gene gun coimmunization of HA and NP plasmids resulted in a dominant IgG1 HA response and the switching of antibodies generated against the NP to the IgG1 isotype.The initial studies showing that injection of DNA into muscle induces an immune response to the encoded protein opened a new approach to vaccination (for reviews, see references 19 and 22). Recent studies suggest that inoculated muscle cells probably act only as a source of antigen and that immune priming takes place elsewhere in the body (14). For example, excision of an injected muscle a few minutes after DNA inoculation did not affect antibody and cytotoxic T-lymphocyte (CTL) responses (21). Thus, it may be interesting to examine other DNA delivery systems to study how the immune system responds to DNA vaccination. One alternative system involves precipitating DNA onto gold beads which are then propelled into the skin by means of pressurized helium gas (12). When such a system is used, less DNA is required, but unlike the case with intramuscular inoculations, the response is Th2-like, generating immunoglobulin G1 (IgG1) antibodies (17). More recent observations suggest that this is probably due to the mode of inoculation rather than the route (10). We have been studying DNA vaccination against the paramyxovirus measles virus (MV). This disease is one of the primary causes of infant mortality in developing countries, and there is an urgent need for an effective vaccine in infants, as the present live attenuated vaccine is inefficient in the presence of maternal antibodies. Our previous studies established that in a mouse model at least three MV proteins play a role in protection (23). Both glycoproteins, hemagglutinin (HA) and fusion, induce neutralizing antibodies (9, 11), and HA and nucleoprotein (NP) induce CTLs (3, 4), which do not protect against infection but help in recovery (5). In our previous study on DNA vaccination, we showed that intramuscular inoculation of DNAs coding for the MV HA and NP (pV1J-HA and pV1J-NP [6]) induced class I-restricted CTLs and a humoral response corresponding to a Th1 response (6). In the present study, we have extended our observations to compare the same plasmids’ ability to induce an immune response when they are delivered into the skin by a gene gun (Bio-Rad, Ivry sur Seine, France). Gold beads were coated with DNA as follows: approximately 30 mg of gold powder (1.0-μm gold beads; Bio-Rad) was mixed with 100 μl of 0.1 M spermidine (Sigma, L’Isle D’Abeau, France). After sonication, 0.5, 2, or 5 μg of plasmid DNA was added per mg of gold powder, and then 200 μl of 2.5 M CaCl₂ was added to the mixture, with gentle vortexing. Pellets were washed three times and suspended in cold 100% ethanol. Tubes containing dried DNA-coated gold beads were stored at 4°C.

Immune response to MV HA DNA.

Six- to eight-week-old female BALB/c mice (Iffa-Credo, Domaine des Oncins, France) were immunized via the shaved abdominal epidermis one to three times at 21-day intervals with 0.5, 2, or 5 μg of pV1J-HA DNA/mg of gold beads. Two gene gun inoculations (each containing 0.5 mg of gold beads) were given for each dose. The antibody levels measured by enzyme-linked immunosorbent assay, as previously described (6), reached a plateau after two inoculations and did not significantly increase with a third inoculation (result not shown).Our previous studies with intramuscular inoculation established that pV1J-HA induced IgG2a antibodies which are associated with a Th1-type response. When we studied the antibody isotype induced in BALB/c by the gene gun immunization, we observed that it was mainly IgG1 (Fig. ​(Fig.1).1). These data are similar to those described for influenza hemagglutinin by Feltquate et al. (10). The antibody isotype did not vary with time after immunization, number of immunizations, or the amount of plasmid used (data not shown) and was not influenced by genetic background, as pV1J-HA-immunized DBA/2 (H-2^d), C3H (H-2^k), and C57/Black (H-2^b) mice induced mainly the IgG1 isotype (Fig. ​(Fig.11).Open in a separate windowFIG. 1Anti-MV HA isotype of antibodies induced in BALB/c, DBA/2 (H-2^d), C3H (H-2^k), and C57/Black (H-2^b) mice immunized with 0.5, 2, or 5 μg of pV1J-HA by epidermal gene gun. Sera were collected 3 weeks after the immunization. Sera from mice immunized with a control pV1J had means ± standard deviations of 158 ± 198 ng/ml for IgG1 anti-HA antibodies (n = 11) and 10 ± 18 ng/ml for IgG2a anti-HA antibodies (n = 11). Data represent individual animals. To study CTL activity, spleen cells from the immunized mice were stimulated in vitro and analyzed in a cytolytic assay as previously described (6). Despite the apparent Th2-type response, good memory CTL responses were obtained with all protocols used, even when responses were measured just 8 days after a single immunization (Fig. ​(Fig.2),2), and persisted for several months. Open in a separate windowFIG. 2Anti-MV HA and NP CTL response after immunization with pV1J-HA or -NP, respectively. BALB/c mice were immunized with 0.5 (circle), 2 (triangle), or 5 (square) μg of pV1J-HA by epidermal gene gun one (a, d), two (b, e), or three (c, f) times at 3-week intervals. The spleen cells were removed 3 weeks (continuous line) or 8 days (dotted line) after the last immunization. After in vitro stimulation with P815-HA or -NP cells, respectively, lysis was measured on P815-HA or -NP cells, and P815 cells were used as a negative control. The results show the specific lysis of targets at graded effector/target ratios. Each curve represents an individual animal.

Immune response to MV NP DNA.

BALB/c mice were immunized with pV1J-NP with the gene gun and a similar schedule of immunizations. The antibody response with the different number of doses and different plasmid concentrations was similar to that observed for HA, i.e., increased levels after one boost. Similar antibody levels were induced in the range of 0.5 to 5 μg of DNA (data not shown). As was previously shown by intramuscular inoculation (6), the antibody isotype induced was mainly IgG2a (Fig. ​(Fig.3),3), in contrast to the HA results. One explanation for this could be that as the NP is a cytosolic protein and the HA is membrane bound, the potential processing and presentation of the two proteins may be different. However, the same argument would be valid for intramuscular inoculation. Furthermore, it has been reported that gene gun immunization with influenza NP induces a Th2 response (17), so clearly the directed differentiation of T cells is more complicated than a simple distinction between cytosol and membrane-bound proteins. The two methods of immunization (intramuscular versus gene gun) target different cell types, possibly influencing the T-cell response. Furthermore, 9 weeks after immunization, one-third of the 18 mice analyzed showed increased levels of anti-NP IgG1 over IgG2a, regardless of the quantity of DNA injected or the number of inoculations (data not shown). CTL responses were also high, even after a single inoculation (Fig. ​(Fig.2).2). Open in a separate windowFIG. 3Anti-MV NP antibody response as measured by enzyme-linked immunosorbent assay in BALB/c mice immunized with 5 μg of pV1J-NP by epidermal gene gun. Sera were collected 3 weeks after immunization. Each pair of bars represents an individual animal. Sera from mice immunized with a control pV1J had means ± standard deviations of 13 ± 45 ng/ml of IgG1 anti-NP antibodies (n = 11) and 83 ± 276 ng/ml of IgG2a anti-NP antibodies (n = 11).

Coimmunization of HA and NP DNA.

Our results show that when injected by the gene gun, the different MV proteins induce different antibody isotypes. This phenomenon has been suggested to parallel the induction of Th1 and Th2 pathways (1). The pathway taken has been shown to be influenced by the induction of certain cytokines. To determine if coimmunization of these two plasmids would influence the isotype of the antibody response, BALB/c mice were immunized with a mixture of pV1J-HA and pV1J-NP in ratios of 1:1, 4:1, or 1:4 while the total amount of DNA was kept constant (5 μg).Measurement of the anti-HA isotype antibody in mice vaccinated with the different mixtures showed it to be mainly IgG1, similar to that for HA alone (data not shown). In contrast, the anti-NP antibodies switched from the IgG2a to the IgG1 isotype after coimmunization (Fig. ​(Fig.4).4). The proximity of expression of the two antigens was not important in this switching effect, as when pV1J-HA and -NP were inoculated separately in different areas of the skin, the antibody response induced 3 weeks later was the same as that induced when the mixture was inoculated (Fig. ​(Fig.4).4). When analyzed 6 weeks later, only one of six mice showed IgG2a predominance. Open in a separate windowFIG. 4Relationship between the isotype of anti-NP antibodies in sera from mice immunized with 5 μg of pV1J-NP or mixtures of pV1J-HA and pV1J-NP at ratios of 1:1, 4:1, and 1:4 so that the total quantity of DNA/mg gold beads was 5 μg, or pV1J-HA and pV1J-NP injected in different skin area. BALB/c mice were immunized by epidermal gene gun. Sera were collected 3 weeks after immunization. Data are results for individual animals.Cytokines have been used to direct the immune response in several studies. Expression of interleukin-12 either alone or with immunizing antigens can increase protection against microbial pathogens (2) or tumors in animal models (13, 18), in parallel with a Th1 response. Expression or addition of interleukin-4 with the immunogen induces a Th2 response (16, 20). The local concentrations of the cytokines in the initial priming of the immune response are probably critical, as once the T cells have been committed, they cannot be modified. Although some studies have suggested the possibility of Th1 and Th2 switching, a more recent study has shown that once differentiated, T cells cannot switch (15). In agreement with this, Feltquate et al. (10) have shown that initial immunization establishes the Th-cell type of the immune response and that this is not modified by subsequent alternative methods of immunization.Acute viral infections induce a Th1 response, whereas soluble proteins favor a Th2 response (7). When tetanus toxoid was administered 1 day after viral infection, the response to this soluble protein changed from Th2 to Th1 (8). Presumably, this change is due to the domination by the cytokines induced by the viral infection of those produced by the tetanus toxoid. In our studies, we observed that after the coexpression of MV HA and NP, the HA-induced Th2 response was dominant. These observations will obviously have an impact on DNA vaccination, as DNAs coding for several pathogens should ideally be administered concomitantly.     

4-Hydroxybenzoate 3-geranyltransferase from Lithospermum erythrorhizon: purification of a plant membrane-bound prenyltransferase

Geranyldiphosphate:4-hydroxybenzoate 3-geranyltransferase is a regulatory enzyme in the biosynthesis of shikonin, a phytoalexin and pharmaceutical produced by cell cultures of Lithospermum erythrorhizon Sieb. et Zucc.. In Linsmaier-Skoog medium, the activity of this enzyme could be enhanced more than 200-fold by addition of methyl jasmonate, and this culture material was used for the solubilization and purification of the enzyme. Of various detergents examined, digitonin was the most suitable for the solubilization of the enzyme. The solubilized enzyme was purified 800-fold by chromatography over diethylaminoethyl (DEAE)-Sephacel, Heparin-Sepharose, Reactive Green 19-Agarose, and Cholic Acid-Agarose. The purified enzyme required magnesium ions as cofactor and was highly specific for geranyldiphosphate (GPP) and 4-hydroxybenzoate (4HB) as substrates. The K _m values for 4HB and GPP were calculated by the method of Lineweaver and Burk as 18.4 μM and 13.8 μM, respectively. Received: 2 July 1997 / Accepted: 14 October 1997     

The bimodal quasi-static and dynamic elastance of the murine lung.

The double sigmoidal nature of the mouse pressure-volume (PV) curve is well recognized but largely ignored. This study systematically examined the effect of inflating the mouse lung to 40 cm H2O transrespiratory pressure (Prs) in vivo. Adult BALB/c mice were anesthetized, tracheostomized, and mechanically ventilated. Thoracic gas volume was calculated using plethysmography and electrical stimulation of the intercostal muscles. Lung mechanics were tracked during inflation-deflation maneuvers using a modification of the forced oscillation technique. Inflation beyond 20 cm H2O caused a shift in subsequent PV curves with an increase in slope of the inflation limb and an increase in lung volume at 20 cm H2O. There was an overall decrease in tissue elastance and a fundamental change in its volume dependence. This apparent "softening" of the lung could be recovered by partial degassing of the lung or applying a negative transrespiratory pressure such that lung volume decreased below functional residual capacity. Allowing the lung to spontaneously recover revealed that the lung required approximately 1 h of mechanical ventilation to return to the original state. We propose a number of possible mechanisms for these observations and suggest that they are most likely explained by the unfolding of alveolar septa and the subsequent redistribution of the fluid lining the alveoli at high transrespiratory pressure.     

Histamine and H1 -histamine receptors faster venous circulation

The study has analysed the action of histamine in the rabbit venous system and evaluated its potential role in contraction during increased venous pressure. We have found that a great variety exists in histamine sensitivity and H(1) -histamine receptor expression in various types of rabbit veins. Veins of the extremities (saphenous vein, femoral vein, axillary vein) and abdomen (common iliac vein, inferior vena cava) responded to histamine by a prominent, concentration-dependent force generation, whereas great thoracic veins (subclavian vein, superior vena cavas, intrathoracic part of inferior vena cava) and a pelvic vein (external iliac vein) exhibited slight sensitivity to exogenous histamine. The lack of reactivity to histamine was not due to increased activity of nitric oxide synthase (NOS) or heme oxygenase-1. H(1) -histamine receptor expression of veins correlated well with the histamine-induced contractions. Voltage-dependent calcium channels mediated mainly the histamine-induced force generation of saphenous vein, whereas it did not act in the inferior vena cava. In contrast, the receptor-operated channels were not involved in this response in either vein. Tyrosine phosphorylation occurred markedly in response to histamine in the saphenous vein, but not in the inferior vena cava. Histamine induced a prominent ρ kinase activation in both vessels. Protein kinase C and mitogen-activated protein kinase (MAPK) were not implicated in the histamine-induced intracellular calcium sensitization. Importantly, transient clamping of the femoral vein in animals caused a short-term constriction, which was inhibited by H(1) -histamine receptor antagonist in vivo. Furthermore, a significantly greater histamine immunopositivity was detected in veins after stretching compared to the resting state. We conclude that histamine receptor density adapts to the actual requirements of the circulation, and histamine liberated by the venous wall during increased venous pressure contributes to the contraction of vessels, providing a force for the venous return.     

NOX5 variants are functionally active in endothelial cells

NADPH oxidases have been identified as sources of reactive oxygen species (ROS) in vascular cells. In addition to the initially described enzyme containing gp91phox (NOX2), several homologues to NOX2 have been identified. Whereas NOX1, NOX2, and NOX4 are expressed in endothelial cells, a functional role of NOX5 containing additional N-terminal calcium-binding domains of varying sequences has not been reported in these cells. NOX5 protein was found in the endoplasmic reticulum of human microvascular endothelial cells (HMEC-1) and in the vascular wall. HMEC-1 cells expressed NOX5beta and NOX5delta as well as a variant lacking calcium-binding domains (NOX5S). NOX5beta and NOX5S increased basal ROS levels. Ionomycin exclusively enhanced NOX5beta-mediated ROS production. Although p22phox, when overexpressed, interacted with both NOX5 proteins, it was not essential for NOX5-mediated ROS production. NOX5 proteins stimulated endothelial cell proliferation and the formation of capillary-like structures whereas depletion of NOX5 by siRNA prevented these responses to thrombin. These data show that endothelial cells express different NOX5 variants including NOX5S lacking calcium-binding domains. NOX5 proteins are functional, promoting endothelial ROS production, proliferation, and the formation of capillary-like structures and contribute to the endothelial response to thrombin. These findings suggest that NOX5 variants play a novel role in controlling ROS-dependent processes in the vasculature.     

Characterization ofκ-casein and keratin domains in fibrinogen

Summary Following the observation of a close sequence homology between the N-terminal moiety of the -chain of fibrinogen with large parts of-casein, the occurrence of a keratin domain in the middle section of the Achain is suggested.     

Inter‐conversion of catalytic abilities in a bifunctional carboxyl/feruloyl‐esterase from earthworm gut metagenome

Carboxyl esterases (CE) exhibit various reaction specificities despite of their overall structural similarity. In present study we have exploited functional metagenomics, saturation mutagenesis and experimental protein evolution to explore residues that have a significant role in substrate discrimination. We used an enzyme, designated 3A6, derived from the earthworm gut metagenome that exhibits CE and feruloyl esterase (FAE) activities with p-nitrophenyl and cinnamate esters, respectively, with a [(k_cat/K_m)]_CE/[(k_cat/K_m)]_FAE factor of 17. Modelling-guided saturation mutagenesis at specific hotspots (Lys²⁸¹, Asp²⁸², Asn³¹⁶ and Lys³¹⁷) situated close to the catalytic core (Ser¹⁴³/Asp²⁷³/His³⁰⁵) and a deletion of a 34-AA–long peptide fragment yielded mutants with the highest CE activity, while cinnamate ester bond hydrolysis was effectively abolished. Although, single to triple mutants with both improved activities (up to 180-fold in k_cat/K_m values) and enzymes with inverted specificity ((k_cat/K_m)_CE/(k_cat/K_m)_FAE ratio of ∼0.4) were identified, no CE inactive variant was found. Screening of a large error-prone PCR-generated library yielded by far less mutants for substrate discrimination. We also found that no significant changes in CE activation energy occurs after any mutation (7.3 to −5.6 J mol⁻¹), whereas a direct correlation between loss/gain of FAE function and activation energies (from 33.05 to −13.7 J mol⁻¹) was found. Results suggest that the FAE activity in 3A6 may have evolved via introduction of a limited number of ‘hot spot’ mutations in a common CE ancestor, which may retain the original hydrolytic activity due to lower restrictive energy barriers but conveys a dynamic energetically favourable switch of a second hydrolytic reaction.