Endothelial, cardiac muscle and skeletal muscle exhibit different viscous and elastic properties as determined by atomic force microscopy

This study evaluated the hypothesis that, due to functional and structural differences, the apparent elastic modulus and viscous behavior of cardiac and skeletal muscle and vascular endothelium would differ. To accurately determine the elastic modulus, the contribution of probe velocity, indentation depth, and the assumed shape of the probe were examined. Hysteresis was observed at high indentation velocities arising from viscous effects. Irreversible deformation was not observed for endothelial cells and hysteresis was negligible below 1 μm/s. For skeletal muscle and cardiac muscle cells, hysteresis was negligible below 0.25 μm/s. Viscous dissipation for endothelial and cardiac muscle cells was higher than for skeletal muscle cells. The calculated elastic modulus was most sensitive to the assumed probe geometry for the first 60 nm of indentation for the three cell types. Modeling the probe as a blunt cone–spherical cap resulted in variation in elastic modulus with indentation depth that was less than that calculated by treating the probe as a conical tip. Substrate contributions were negligible since the elastic modulus reached a steady value for indentations above 60 nm and the probe never indented more than 10% of the cell thickness. Cardiac cells were the stiffest (100.3±10.7 kPa), the skeletal muscle cells were intermediate (24.7±3.5 kPa), and the endothelial cells were the softest with a range of elastic moduli (1.4±0.1 to 6.8±0.4 kPa) depending on the location of the cell surface tested. Cardiac and skeletal muscle exhibited nonlinear elastic behavior. These passive mechanical properties are generally consistent with the function of these different cell types.     

Correlated behavior implicates stromules in increasing the interactive surface between plastids and ER tubules

Stromules are extended by plastids but the underlying basis for their extension and retraction had not been understood until recently. Our live-imaging aided observations on coincident plastid stromule branching and ER tubule dynamics open out new areas of investigation relating to these rapid subcellular interactions. This addendum provides a testable hypothesis on the formation of stromules, which argues against the need for new membrane incorporation and suggests that stromal extensions might result from a remodeling of the plastid envelope membrane in an ER aided manner.Key words: stromules, plastids, endoplasmic reticulum, fluorescent proteins, subcellular interactions, FNR-EGFP, RFP-ERThe extension and retraction of stromules (stroma-filled tubules) from both chlorophyll containing and achlorophyllous plastids is well established for diverse plant species.¹ Many different conditions such as increased subcellular redox stress,² symbiotic interactions,^3–5 elevated temperatures,⁶ viral infection⁷ and alterations in plastid size and density^8,9 have been associated with stromule formation. Stromules extended from different plastids have been observed as forming connecting bridges^10–13 through which an exchange of proteins has been demonstrated.^{10,11,14–16} The latter observations strongly suggested stromules as transient communication channels between plastids for exchanging metabolites.^10,11 The connectivity of plastids via stromules also led to the suggestion that plant cells might contain a plastidom, defined as a well-connected plastid-compartment.¹² However, as pointed out by Netasan and co-workers¹⁷ most stromules do not appear to interconnect plastids and thus the movement of macromolecules between plastids might not be their sole function. In concurrence with this viewpoint the observations of Gunning¹⁸ and Lütz and Engel¹⁹ suggest that stromules have a role in increasing plastid interactions with mitochondria and peroxisomes. Thus a more generalized and acceptable statement is that stromules serve to increase the plastid stromal surface area that is exposed to the subcellular environment.Notably, the largest membrane surface area within a cell is provided by the endoplasmic reticulum (ER). Transmission electron micrographs often suggest a close proximity between plastids and the ER^20,21 and the presence of a chloroplast envelope associated-ER has been demonstrated.²² However, studies aimed at uncovering possible dynamic interactions between stromules and the ER in living plant cells had not been carried out. Our recent work²³ investigated this possible relationship by simultaneously visualizing stromules (labeled using a ferredoxin NADP(H) oxidoreductase (FNR) transit peptide fused to enhanced GFP; hereafter referred to as FNR-EGFP) and the ER (highlighted using a chimeric red fluorescent protein (RFP) carrying a basic chitinase signal sequence and an ER retention sequence; referred to as RFP-ER). Our observations clearly identified the ER as providing the nearest set of membranes with which the plastid envelope can interact. The observations are most pertinent for the narrow cortical sleeve where the relatively large sized plastids (including chloroplasts) are pressed against the cell boundary (plasma membrane and comparatively rigid cell wall) on one side and a turgid vacuole on the inner side. This space is also traversed by the cortical ER mesh created by constantly reorganizing ER-tubules. As shown in Figure 1A the side of a plastid pressed against the plasma membrane is surrounded by a loose mesh of ER tubules while the side pressed close to the vacuolar membrane (Fig. 1B) is free of the ER cradle. The cortically located plastids display strong behavioral correlations between their stromules and the neighboring ER tubules (Fig. 1C–E). Notably stromules form triangular junctions that appear very similar to the 3-way junctions displayed by the cortical ER. The three-way junctions of the ER are cisternal locations from which tubules are extended to create ER polygons. Stromule branches are extended from the stromal triangles in a very similar manner along comparable angles. Interestingly the extension and retraction of stromules and their branches occurs in tandem with contiguous ER tubules. These observations on the coincident behavior of the two organelles indicate that stromule branching and dynamic behavior might rely upon the creation of multiple membrane contact sites (MCSs) with the ER. The MCSs could aid in stretching stromules along ER tubules while loss of contact between the two membranes could cause rapid stromule retraction. While the possibility of identifying MCSs on the stromule envelope is being explored further by us our initial observations evoke a long-standing but very relevant question. Where does the membrane for forming stromules come from?Open in a separate windowFigure 1Confocal laser scanning microscopy based imaging of living Nicotiana benthamiana cells co-expressing FNR-EGFP (labels stromules) and RFP-ER (labels ER), 3D isosurface rendering and a model based on the observations. (A) 3D iso-surface rendering of a chloroplast facing the plasma membrane side shows the plastid situated in a loose cradle of ER tubules. (B) The 3D rendered chloroplast ER reconstruction seen in (A) observed from the vacuolar side after rotation by 180° shows the chloroplast appears smooth and free of the ER mesh. (C–E) Sequential frames from a time-lapse movie depict a FNR-EGFP labeled stromule undergoing a branching event. Note that in (C) the stromule extending along an ER channel with possible contacts at several points along the stromule length. (D) depicts a stromal triangle (arrowhead) that forms a branch initial. (E) depicts a branched stromule with the branches aligned with the ER (size bar = 5 µm). (F) A plastid exhibiting a short, wide stroma-filled area after stromule retraction has occured. (G) The same plastid shown in (F) without the FNR-EGFP labeled stroma (arrowhead) provides an appreciation of the “mobile jacket” created by the stromal contents around the chlorophyll containing thylakoids (size bar = 5 µm). (H and I) A diagrammatic depiction based on our observations and pertinent literature. (H) The narrow cortical region between the vacuolar membrane and the cell boundary is shared by large plastids and a dynamic ER. A depiction of the loose stroma filled jacket (arrowhead) of a plastid along with contiguous ER. Note the relative positions of the vacuolar membrane and the plasma membrane. The plastid size cannot increase isotropically within the narrow confines of the cortex and thus stromule formation along ER tubules is favored (Arrow pointing direction of stromule extension along the ER). (I) Multiple contact sites might be created between the extending stromule and the neighboring ER tubules. The diagrammatic depiction emphasizes the remodeling of the loose plastid jacket for stromule extension along ER. The schematic does not depict the strong possibility that both organelles might share an F-actin based mechanism for their extension.Stromule extension visibly enlarges the area occupied by the stromal contents of a plastid. The general conclusion of stromules increasing the plastid surface is clearly validated. Indeed Hanson and coworkers¹ calculated that for a model plastid of 3 µm diameter extending a 0.2 µm diameter and 10 µm long stromule the stromule represents about 20% of the overall plastid envelope surface area. A point that remains unclear is whether stromule extension involves a net increase in plastidic membrane or merely involves a remodeling of the existing membrane into an elongated shape. In the first scenario stromule formation would involve the creation of fresh membrane vesicles and their incorporation into an existing bi-layered plastid coat. This would be followed by their dispersal into inner and outer plastid envelopes along with complete complements of protein import and export machinery. Currently there is no experimental evidence to support this scenario. Moreover, this viewpoint does not consider observations of stromule retraction at all. If new membranes have been added during stromule extension then what happens to them during the retraction stage? Further, live imaging clearly shows that the time involved in sporadic stromule extension, branching and retraction is in the order of seconds to minutes. Such short periods do not favor the viewpoint involving creation of new membrane and its incorporation into stromules.Interestingly, observations of plastids in living cells often suggest that they have a rather wobbly form than a tight compact shape. The unstable form suggests the presence of relaxed membranes enveloping the main plastid body (Fig. 1F and G). These loose fitting membranes have been described as a “mobile jacket”^1,24 whose presence and irregular protrusions can be clearly distinguished from long stromules using a shape index.⁶ Hanson and co-workers¹ speculated that the “mobile jacket” might be stretched out and could thus provide the extra membrane needed for stromule formation. The notion is supported by the work of Gunning, which shows stromules retracting into short amorphous protrusions.¹⁸ Our observations of short protrusions or “beaks”²³ being created sporadically on a relatively regular-appearing plastid surface support these ideas. According to our observations one or two of the beaks might develop into long stromules that would be extended along neighboring ER tubules (Fig. 1H and I).Moreover, in vitro experiments on sucrose filled liposomes and giant vesicles^25,26 are particularly interesting in this context. Plasmolytic shrinkage of the sucrose filled vesicles creates states in which excess membrane surrounds a small volume. Over time the membrane exhibits small protrusions that are very similar in shape to the protrusions formed by plastid. Taken together the above-mentioned observations support behavior that might be expected upon a reshaping of already existing membranes. Notably the membrane-remodeling scenario for the formation of a stromule does not involve an increase in the net membrane of a plastid but it does achieve an increase in the surface area over which plastid stromal contents can interact with their surroundings. The viewpoint is also able to explain stromule elasticity that is suggested by their rapid extension and retraction.The new observations and ensuing discussion suggesting stromules being formed by stretching of the plastid envelope and identifying the ER as an interactive membrane partner in the process lay down the foundation for a number of other questions. These include investigations relating to the role of stromules in metabolite import and export between the two organelles, understanding the relationship between stromule and the ER behavior in relation to the underlying actin cytoskeleton as well as assessing the specificity of myosin motors that might be involved in these dynamic processes.     

IgY antibodies of chicken do not bind staphylococcal binder of immunoglobulin (Sbi) from Staphylococcus aureus

The immunoglobulin (Ig) binding proteins of Staphylococcus aureus namely staphylococcal protein A (SpA) and staphylococcal binder of immunoglobulin (Sbi) are responsible for false positives during immunoassays. Avian IgY antibodies were reported to have no affinity to SpA and thus are safe for use in immunoassays. However, the behaviour of Sbi with IgY was not reported. The purpose of the present study is to evaluate the interactions between IgY antibodies and Sbi protein from different S. aureus strains. Initially, heterologous cloning and expression of complete sbi gene in Escherichia coli was undertaken. Recombinant Sbi protein was utilized to generate polyclonal anti-Sbi IgY and anti-Sbi antibodies in chicken and BALB/c mice respectively. Indirect ELISA and Western blotting were performed to evaluate the reactivity of anti-Sbi antibodies. Non-reducing PAGE followed by Western blotting and double-antibody sandwich dot-ELISA were performed to analyze the reactivity of IgY antibodies with recombinant Sbi and native Sbi from S. aureus strains. To avoid the possible interference of enzyme-conjugated secondary antibodies from mammalian sources, mouse anti-Sbi revealing antibodies were labeled with biotin so that streptavidin-HRP was used as developing reagent for chromogenic reaction. Sbi was highly immunogenic in chicken and mouse with antibody titers of 1:128,000 and 1:64,000 dilutions respectively. We observed that unimmunized IgY antibodies showed no affinity to either recombinant Sbi or native Sbi from S. aureus strains in Western blotting and double antibody sandwich ELISA. In view of these observations, we recommend that IgY antibodies are safe and free from false positives due to SpA and Sbi in immunoassays involving detection of S. aureus antigens/exotoxins.     

Ebola virus entry requires the host-programmed recognition of an intracellular receptor

Ebola and Marburg filoviruses cause deadly outbreaks of haemorrhagic fever. Despite considerable efforts, no essential cellular receptors for filovirus entry have been identified. We showed previously that Niemann-Pick C1 (NPC1), a lysosomal cholesterol transporter, is required for filovirus entry. Here, we demonstrate that NPC1 is a critical filovirus receptor. Human NPC1 fulfills a cardinal property of viral receptors: it confers susceptibility to filovirus infection when expressed in non-permissive reptilian cells. The second luminal domain of NPC1 binds directly and specifically to the viral glycoprotein, GP, and a synthetic single-pass membrane protein containing this domain has viral receptor activity. Purified NPC1 binds only to a cleaved form of GP that is generated within cells during entry, and only viruses containing cleaved GP can utilize a receptor retargeted to the cell surface. Our findings support a model in which GP cleavage by endosomal cysteine proteases unmasks the binding site for NPC1, and GP-NPC1 engagement within lysosomes promotes a late step in entry proximal to viral escape into the host cytoplasm. NPC1 is the first known viral receptor that recognizes its ligand within an intracellular compartment and not at the plasma membrane.     

Analysis of acyclic nucleoside modifications in siRNAs finds sensitivity at position 1 that is restored by 5′-terminal phosphorylation both in vitro and in vivo

Small interfering RNAs (siRNAs) are short, double-stranded RNAs that use the endogenous RNAi pathway to mediate gene silencing. Phosphorylation facilitates loading of a siRNA into the Ago2 complex and subsequent cleavage of the target mRNA. In this study, 2′, 3′ seco nucleoside modifications, which contain an acylic ribose ring and are commonly called unlocked nucleic acids (UNAs), were evaluated at all positions along the guide strand of a siRNA targeting apolipoprotein B (ApoB). UNA modifications at positions 1, 2 and 3 were detrimental to siRNA activity. UNAs at positions 1 and 2 prevented phosphorylation by Clp1 kinase, abrogated binding to Ago2, and impaired Ago2-mediated cleavage of the mRNA target. The addition of a 5′-terminal phosphate to siRNA containing a position 1 UNA restored ApoB mRNA silencing, Ago2 binding, and Ago2 mediated cleavage activity. Position 1 UNA modified siRNA containing a 5′-terminal phosphate exhibited a partial restoration of siRNA silencing activity in vivo. These data reveal the complexity of interpreting the effects of chemical modification on siRNA activity, and exemplify the importance of using multiple biochemical, cell-based and in vivo assays to rationally design chemically modified siRNA destined for therapeutic use.     

Pectin esterase in relation to leaf abscission in coleus and phaseolus

Pectin esterase (PE) activities in abscission zones, other portions of leaves, and adjacent stem tissues were compared in attached leaves and abscissing petioles (previously debladed) of Coleus blumei Benth. and Phaseolus vulgaris L., cv. Canadian Wonder. Earlier findings of Osborne in bean were confirmed and changes in PE activity in coleus were shown to resemble those in bean in some respects. In both plants PE was lower in the distal portion of abscission zones of abscissing petioles than in that portion of attached leaves but this difference was not as large or as consistently clear-cut in coleus as in bean. The general level of PE activity was an order of magnitude lower and changes associated with abscission were smaller in coleus than in bean. Auxin treatment of debladed petioles of coleus prevented abscission and resulted in small increases in PE activity in abscission zones and most of the other regions sampled. The largest increase was observed in the stem tissue adjacent to the attached leaf opposite the debladed, auxin treated one.     

Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis

The RD1 genomic region is present in virulent strains of Mycobacterium tuberculosis (MTB), missing from the vaccine strain M. bovis BCG, and its importance to virulence has been established experimentally. Based on in silico analysis, it has been suggested that RD1 may encode a novel secretion system, but the mechanism by which this region affects virulence is unknown. Here we examined mutants disrupted in five individual RD1 genes. Both in vitro and in vivo, each mutant displayed an attenuated phenotype very similar to a mutant missing the entire RD1 region. Genetic complementation of individual genes restored virulence. Attenuated mutants could multiply within THP-1 cells, but they were unable to spread to uninfected macrophages. We also examined export of two immunodominant RD1 proteins, CFP-10 and ESAT-6. Export of these proteins was greatly reduced or abolished in each attenuated mutant. Again, genetic complementation restored a wild-type phenotype. Our results indicate that RD1 genes work together to form a single virulence determinant, and argue that RD1 encodes a novel specialized secretion system that is required for pathogenesis of MTB.     

Phosphorylation of IRS1 at Serine 307 in Response to Insulin in Human Adipocytes Is Not Likely to be Catalyzed by p70 Ribosomal S6 Kinase

The insulin receptor substrate-1 (IRS1) is phosphorylated on serine 307 (human sequence, corresponding to murine serine 302) in response to insulin as part of a feedback loop that controls IRS1 phosphorylation on tyrosine residues by the insulin receptor. This in turn directly affects downstream signaling and is in human adipocytes implicated in the pathogenesis of insulin resistance and type 2 diabetes. The phosphorylation is inhibited by rapamycin, a specific inhibitor of mammalian target of rapamycin (mTOR) in complex with raptor (mTORC1). The mTORC1-downstream p70 ribosomal protein S6 kinase (S6K1), which is activated by insulin, can phosphorylate IRS1 at serine 307 in vitro and is considered the physiological protein kinase. Because the IRS1 serine 307-kinase catalyzes a critical step in the control of insulin signaling and constitutes a potential target for treatment of insulin resistance, it is important to know whether S6K1 is the physiological serine 307-kinase or not. We report that, by several criteria, S6K1 does not phosphorylate IRS1 at serine 307 in response to insulin in intact human primary adipocytes: (i) The time-courses for phosphorylation of S6K1 and its phosphorylation of S6 are not compatible with the phosphorylation of IRS1 at serine 307; (ii) A dominant-negative construct of S6K1 inhibits the phosphorylation of S6, without effect on the phosphorylation of IRS1 at serine 307; (iii) The specific inhibitor of S6K1 PF-4708671 inhibits the phosphorylation of S6, without effect on phosphorylation of IRS1 at serine 307. mTOR-immunoprecipitates from insulin-stimulated adipocytes contains an unidentified protein kinase specific for phosphorylation of IRS1 at serine 307, but it is not mTOR or S6K1.     

Reliability of surface EMG during sustained contractions of the quadriceps.

The purpose of this study was to determine test-retest reliability for median frequency (MDF) and amplitude of surface EMG during sustained fatiguing contractions of the quadriceps. Twenty-two healthy subjects (11 males and 11 females) were tested on two days held one week apart. Surface EMG was recorded from rectus femoris (RF), vastus lateralis (VL) and vastus medialis (VM) during sustained isometric contractions at 80% and 20% of maximal voluntary contraction (MVC) held to exhaustion. Quadriceps fatigue was described using four measures for both MDF and amplitude of EMG: initial, final, normalized final and slope. For both MDF and amplitude, the initial, final and normalized EMG showed moderate to high reliability for all three muscle groups at both contraction levels (ICC=0.59-0.88 for MDF; ICC=0.58-0.99 for amplitude). Slope of MDF and amplitude was associated with a large degree of variability and low ICCs for the 80% but not the 20% MVC. MDF and amplitude of EMG during sustained contractions of the quadriceps are reproducible; normalized final values of MDF and amplitude show better reliability than slope.     

Antimicrobial studies,synthesis and characterization of novel 1-nitro-10H-phenothiazine bearing sulfone/nucleoside moieties

The current research article involves one pot synthesis of novel substituted 1-nitro-10H-phenothiazines via Smiles rearrangement. These substituted phenothiazines undergo oxidation to yield 10H-phenothiazine-5,5-dioxides (sulfones) while on treatment with β-D-ribofuranose-1-acetate-2,3,5-tribenzoate yield ribofuranosides. These compounds were screened for their antimicrobial vitalities (in vitro) against selected strains of bacteria and fungi. The characterization of synthesized compounds was done by elemental and spectral studies.