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S Na

Publications and source records attributed to S Na.

At least 19 recordsLinked to original sources

On atomic force microscopy and the constitutive behavior of living cells.

Atomic force microscopy (AFM) is one of many new technologies available to study the mechanical properties and mechanobiological responses of living cells. Despite the widespread usage of this technology, there has been little attempt to develop new theoretical frameworks to interpret the associated data. Rather, most analyses rely on the classical Hertz solution for the indentation of an elastic half-space within the context of linearized elasticity. In contrast, we propose a fully nonlinear, constrained mixture model for adherent cells that allows one to account separately for the contributions of the three primary structural constituents of the cytoskeleton. Moreover, we extend a prior solution for a small indentation superimposed on a finite equibiaxial extension by incorporating in this mixture model for the special case of an initially random distribution of constituents (actin, intermediate filaments, and microtubules). We submit that this theoretical framework will allow an improved interpretation of indentation force-depth data from a sub-class of atomic force microscopy tests and will serve as an important analytical check for future finite element models. The latter will be necessary to exploit further the capabilities of both atomic force microscopy and nonlinear mixture theories for cell behavior.

Biomechanical Phenomena↗

Comparison of scan-angle method and convective cloud differential method in retrieving tropospheric ozone from TOMS.

Tropospheric ozone, derived from the Scan-Angle Method (SAM) and the Convective Cloud Differential (CCD) method, exhibits a noticeable abundance over the South Atlantic, where it is associated with biomass-burning in the austral spring. This feature is also seen in the distribution of carbon monoxide observed from Measurements Of Pollution In The Troposphere (MOPITT). In the boreal burning season, however, the distribution of the results from SAM and MOPITT-CO present an enhancement related to the biomass-burning over North Africa that does not appear in the CCD results. The relationship of the results from SAM and MOPITT-CO is better than those of the results from the CCD and MOPITT-CO for the December-February period. Conversely, the latter relationship is better than the former for the October-November period. The two methods, SAM and CCD, show higher correlation in the southern burning season, but lower correlation in the northern burning season. The influence of biomass burning on ozone amounts is clearly seen in the SAM results of the elevated ozone over northern equatorial Africa during the northern burning season, but is not present in the CCD results.

Africa↗

Elastodynamics and arterial wall stress.

Recent advances in molecular and cell biology have emphasized the fundamental importance of mechanical factors in regulating the structure and function of cells and extracellular matrix in the vasculature. Consequently, there is an ever-greater motivation to calculate accurately the stress and strain fields in the arterial wall and how they change with disease, injury, and clinical treatment. Although there is an extensive literature on arterial mechanics, our understanding is still far from complete. In this paper, we review some of the salient findings with regard to wall properties, suggest some possible improvements in the calculation of wall stress, and identify some unresolved problems for further research.

Arteries↗

The pro domain of beta-secretase does not confer strict zymogen-like properties but does assist proper folding of the protease domain.

beta-Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid beta peptide. BACE is expressed as a precursor protein containing Pre, Pro, protease, transmembrane, and cytosolic domains. A soluble BACE derivative (PreProBACE460) that is truncated between the protease and transmembrane domains was produced by baculovirus-mediated expression. ProBACE460 was purified from conditioned media of infected insect cells using immobilized concanavalin A and immobilized BACE inhibitor, P10-P4' Stat(Val). Furin cleaves ProBACE460 between the Pro and protease regions to generate mature BACE460. The k(cat)/K(m) of ProBACE460 when assayed with a polypeptide substrate is only 2.3-fold less than that of BACE460. This finding and the similar inhibitory potency of P10-P4' Stat(Val) for ProBACE460 and BACE460 suggest that the Pro domain has little effect on the BACE active site. Exposure of ProBACE460 to guanidine denaturation/renaturation results in a 7-fold higher recovery of BACE activity than when BACE460 is similarly treated. The presence of free BACE Pro peptide during renaturation of BACE460 but not ProBACE460 increases recovery of activity. These findings show that the Pro domain in ProBACE460 does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain.

Amino Acid Sequence↗

Enhanced CD4+ T cell proliferation and Th2 cytokine production in DR6-deficient mice.

We have found that DR6, a member of the TNF receptor family, is highly expressed in resting T cells and downregulated in activated T cells. DR6-targeted mutant mice were generated and showed normal development. However, DR6(-/-) CD4(+) T cells hyperproliferated in response to TCR-mediated stimulation and protein antigen challenge. Activated DR6(-/-) CD4(+) T cells exhibited upregulated CD25 expression and enhanced proliferation in response to exogenous IL-2 stimulation. In addition, increased CD28 and reduced CTLA-4 expression were observed in these cells. Enhanced Th2 cytokine production by activated DR6(-/-) CD4(+) T cells was associated with the increased transcription factor NF-ATc in nuclei. DR6, therefore, functions as a regulatory receptor for mediating CD4(+) T cell activation and maintaining proper immune responses.

Abatacept↗

[Isolation and identification of the broad spectrum antimicrobial peptide AP311 produced by Lactobacillus acidophilus].

The heat stable antimicrobial peptide (AP311) produced by Lactobacillus acidophilus was isolated and identified. The AP311 has broad spectrum of inhibition including many Gram-positive and Gram-negative bacteria. The inhibition activity of AP311 was lost upon treatment with trypsin, subtilisin, proteanase K, chymotrypsin and pepsin. Inhibition activity of AP311 was decreased with pH increasing. The AP311 is very stable at acidic condition (pH2-4) even if heating at 100 degrees C for 30 min. But it was inactivated at basic pH (pH12) and the activity was restored completely upon reversion to acidic. The AP311 was not precipitated and inactivated by various organic solvents, except n-butanol. When the precipitate was redissolved in 0.02 mol/L HC1, its activity was restored. Based on its proteinaceous nature, broad spectrum of inhibition, we propose that AP311 should be considered the broad spectrum antimicrobial peptide.

Anti-Bacterial Agents↗

Role of the guanosine triphosphatase Rac2 in T helper 1 cell differentiation.

T helper 1 (TH1) cells mediate cellular immunity, whereas TH2 cells potentiate antiparasite and humoral immunity. We used a complementary DNA subtraction method, representational display analysis, to show that the small guanosine triphosphatase Rac2 is expressed selectively in murine TH1 cells. Rac induces the interferon-gamma (IFN-gamma) promoter through cooperative activation of the nuclear factor kappa B and p38 mitogen-activated protein kinase pathways. Tetracycline-regulated transgenic mice expressing constitutively active Rac2 in T cells exhibited enhanced IFN-gamma production. Dominant-negative Rac inhibited IFN-gamma production in murine T cells. Moreover, T cells from Rac2-/- mice showed decreased IFN-gamma production under TH1 conditions in vitro. Thus, Rac2 activates TH1-specific signaling and IFN-gamma gene expression.

Animals↗

Induction of osteopontin gene expression during mammary gland involution and effects of glucocorticoid on its expression in mammary epithelial cells.

To understand the molecular mechanisms of mammary gland involution, an involution-induced clone was identified from a cDNA library of mouse mammary gland by differential screening. Characterization of a clone by sequencing and northern analysis showed that expression of the osteopontin gene was induced during involution of mouse mammary gland. But induction of the osteopontin gene was not observed in apoptotic HC11 mammary epithelial cells under serum starvation. In HC11 cells, dexamethasone treatment from the seeding stage showed five-fold induction of osteopontin gene expression, but the expression was not changed when dexamethasone was added to confluent cells.

Animals↗

Expression of activated CDC42 induces T cell apoptosis in thymus and peripheral lymph organs via different pathways.

CDC42, a Ras-related small GTP binding protein, is involved in diverse cellular functions in lymphocytes. We generated transgenic mice expressing constitutively active murine CDC42 (Q61L) under the control of the human CD2 promoter. Transgenic mice showed smaller thymi with a dramatic reduction of CD4+CD8+, CD4+ and CD8+ thymocytes and with increase of CD4-CD8- thymocytes at CD25-CD44+ and CD25+ stage. A high percentage of the transgenic thymocytes were apoptotic, explaining the reduction of cellularity and size of the thymus. Mature T cells (TCR alphabeta+) in peripheral lymph organs, spleen and lymph node, were also dramatically reduced, and exhibited massive apoptosis. Expression of Fas and Fas ligand on both thymocytes and peripheral T cells was upregulated in transgenic mice, but the increased apoptosis in the thymus was independent of Fas (CD95), whereas peripheral spleen and lymph node T cell apoptosis was Fas dependent. Thus, activated CDC42 triggers distinct apoptotic pathways in thymocytes and peripheral T cells.

Amino Acid Substitution↗

Use of a recombinant parvovirus to facilitate screening for human melanoma cell clones expressing tetracycline-responsive transactivators.

The tetracycline regulatory (TET) system provides a useful means of controlling foreign gene expression in mammalian cells. Exploiting this system in cultured cells requires the prior isolation, from the cells of interest, of transfectant clones expressing the necessary TET transactivator, tTA, or reverse transactivator, rtTA. We describe a simple screening procedure for identifying transfectant clones expressing a properly regulated transactivator, and the application of this method to isolating clones of human melanoma cells expressing either tTA or rtTA. Clones in multi-well plates are transduced by exposure to a recombinant parvovirus containing a luciferase reporter, under control of a promoter responsive to the TET system transactivators. Transactivation of reporter expression in the presence or absence of doxycycline (DOXY) is determined after one to two days, using a rapid luciferase assay. Screening is easier and more reproducible with this transduction method than with conventional transient transfection of analogous reporter plasmids. Clones of two human melanoma cell lines showing >100-200-fold transactivation after transfection with either tTA or rtTA were readily identified using this method.

Clone Cells↗

[Cloning and expression of the E. coli serine hydroxymethyltransferase gene (glyA)].

The E. coli K12 glyA gene(13 kb), encoding serine hydroxymethyltransferase (SHMT), has been cloned in the plasmid vector pBR329 using insertion inactivation and complementation test. Subcloning of segments of the original insert (13 kb) into plasmids pBR322, pBR329 and pSMY901 established that a 2.6 kb PstI-EcoR fragment carries the glyA gene. The 12 strains of transforments containing recombined plasmid. were obtained. SHMT and glyA gene product level in strains carrying glyA plasmids were different. No SHMT activity was observed in host strains. The glyA gene products for JM109(pSM13), K12(pSM13), K12(pSM14) and K12(pSM15) account for 15.7%, 15.4%, 11.8%, and 9.48% of the total dissoluble cell protein, respectively.

Cloning, Molecular↗

Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice.

Programmed cell death (apoptosis) is a prominent feature of the development of the immune and nervous systems. The identification of the Caenorhabditis elegans cell death gene, ced-3, as a prototype of the interleukin-1beta converting enzyme (ICE) protease family has led to extensive evidence implicating these enzymes in apoptosis. Among the ten or more members of the ICE protease family, CPP32/yama/apopain exhibits the highest similarity to CED-3 in both sequence homology and substrate specificity. To analyse its function in vivo, we generated CPP32-deficient mice by homologous recombination. These mice, born at a frequency lower than expected by mendelian genetics, were smaller than their littermates and died at 1-3 weeks of age. Although their thymocytes retained normal susceptibility to various apoptotic stimuli, brain development in CPP32-deficient mice was profoundly affected, and discernible by embryonic day 12, resulting in a variety of hyperplasias and disorganized cell deployment. These supernumerary cells were postmitotic and terminally differentiated by the postnatal stage. Pyknotic clusters at sites of major morphogenetic change during normal brain development were not observed in the mutant embryos, indicating decreased apoptosis in the absence of CPP32. Thus CPP32 is shown to play a critical role during morphogenetic cell death in the mammalian brain.

Animals↗

D4-GDI, a substrate of CPP32, is proteolyzed during Fas-induced apoptosis.

Apoptosis (programmed cell death) is a fundamental process for normal development of multicellular organisms, and is involved in the regulation of the immune system, normal morphogenesis, and maintenance of homeostasis, ICE/CED-3 family cysteine proteases have been implicated directly in apoptosis, but relatively few of the substrates through which their action is mediated have been identified. Here we report that D4-GDI, an abundant hematopoietic cell GDP dissociation inhibitor for the Ras-related Rho family GTPases, is a substrate of the apoptosis protease CPP32/Yama/Apopain. D4-GDI was rapidly truncated to a 23-kDa fragment in Jurkat cells with kinetics that parallel the onset of apoptosis following Fas cross-linking with agonistic antibody or treatment with staurosporine. Fas- and staurosporine-induced apoptosis as well as cleavage of D4-GDI were inhibited by the ICE inhibitor, YVAD-cmk. D4-GDI was cleaved in vitro by recombinant CPP32 expressed in Escherichia coli to form a 23-kDa fragment. The CPP32-mediated cleavage of D4-GDI was completely inhibited by 1 microM DEVD-CHO, a reported selective inhibitor of CPP32. In contrast, the ICE-selective inhibitors, YVAD-CHO or YVAD-cmk, did not inhibit CPP32-mediated D4-GDI cleavage at concentrations up to 50 microM. N-terminal sequencing of the 23-kDa D4-GDI fragment demonstrated that D4-GDI was cleaved between Asp19 and Ser20 of the poly(ADP-ribose) polymerase-like cleavage sequence DELD19S. These data suggest that regulation by D4-GDI of Rho family GTPases may be disrupted during apoptosis by CPP32-mediated cleavage of the GDI protein.

Alkaloids↗

Genetic probing of the first and second transmembrane helices of the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae.

Structural features of the putative helical hairpin region comprising transmembrane segments 1 (TM1) and 2 (TM2) of the yeast plasma membrane H(+)-ATPase were probed by site-directed mutagenesis. The importance of phenylalanine residues Phe-116, Phe-119, Phe-120, Phe-126, Phe-144, Phe-159, and Phe-163 was explored by alanine replacement mutagenesis. It was found that substitutions at all positions, except Phe-120 and Phe-144, produced viable enzymes, although a range of cellular growth phenotypes were observed like hygromycin B resistance and low pH sensitivity, which are linked to in vivo action of the H(+)-ATPase. Lethal positions Phe-120 and Phe-144, could be replaced with tryptophan to produce viable enzyme, although the F144W mutant was highly perturbed. ATP hydrolysis measurements showed that Km was not significantly altered for most mutant enzymes, whereas Vmax was moderately reduced with two mutants, F144W and F163A, showing less than 50% of the normal activity. Double Phe-->Ala mutations in TM1 and TM2 were constructed to examine whether such substitutions would result in a higher degree of enzyme destabilization. Mutant F116A/F119A was viable and gave a normal phenotype, while F159A/F163A was not viable. Other double mutants, F116A/F159A and F119AF/159A, which are predicted to lie juxtaposed on TM1 and TM2, produced non-functional enzymes. However, a viable F119V/F159A mutant was isolated and showed hygromycin B resistance. These results suggest that double mutations eliminating 2 phenylalanine residues strongly destabilize the enzyme. A putative proline kink at Gly-122/Pro-123 in TM1 is not essential for enzyme action since these residues could be variously substituted (G122A or G122N; P123A, P123G, or P123F) producing viable enzymes with moderate effects on in vitro ATP hydrolysis or proton transport. However, several substitutions produced prominent growth phenotypes, suggesting that local perturbations were occurring. The location of Pro-123 is important because Gly-122 and Pro-123 could not be exchanged. In addition, a double Pro-Pro created by a G122P mutation was lethal, suggesting that maintenance of an alpha-helical structure is important. Other mutations in the hairpin, including modification of a buried charged residue, E129A, were not critical for enzyme action. These data are consistent with the view that the helical hairpin comprising TM1 and TM2 has important structural determinants that contribute to its overall stability and flexibility.

Cell Membrane↗

Essential region for self-replication of Coryneform bacteria plasmid pXZ10145.

A pTSK series of recombinant plasmids were constructed by cloning DNA fragments of pXZ10145 or its deleted deriviate pATN65 into plasmid vector pACYC177 of E. coli. Experiment results of Coryneform bacteria transformation with these pTSK plasmids allowed us to localize the essential region for self-replication on plasmid pXZ10145. The minimal replication region of the pXZ10145 was located on a 1.2kb Nael-Nrul DNA fragment in which only one open reading frame was found. This ORF was believed to be encoded a trans-acting replication factor. The replication origin (oriV) was locate on a 0.3kb NaeI-SalI fragment which was within the ORF region.

Actinomycetales↗

MOP2 (SLA2) affects the abundance of the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae.

The abundance of yeast plasma membrane H(+)-ATPase on the cell surface is tightly regulated. Modifier of pma1 (mop) mutants were isolated as enhancers of the mutant phenotypes of pma1 mutants. mop2 mutations reduce the abundance and activity of Pma1 protein on the plasma membrane without affecting the abundance of other prominent plasma membrane proteins. The MOP2 gene encodes a 108-kDa protein that has previously been identified both as a gene affecting the yeast cytoskeleton (SLA2) (Holtzman, D.A., Yang, S., and Drubin, D. G. (1993) J. Cell Biol. 122, 635-644) and as a gene affecting endocytosis (END4) (Raths, S., Roher, J., Crausaz, F., and Riezman, H. (1993) J. Cell Biol. 120, 55-65). In some strains, MOP2 (SLA2) is essential for cell viability; in others, a deletion mutant is temperature sensitive for growth. mop2 mutations do not reduce the transcription of PMA1 nor do they lead to the accumulation of Pma1 protein in any intracellular compartment. An epitope-tagged MOP2 protein behaves as a plasma membrane-associated protein whose abundance is proportional to its level of gene expression. Over-expression of MOP2 relieved the toxicity caused by the over-expression of PMA1 from a high copy plasmid; conversely, the growth of mop2 strains was inhibited by the presence of a single extra copy of PMA1. We conclude that MOP2 (SLA2) encodes a plasma membrane-associated protein that is required for the accumulation and/or maintenance of plasma membrane H(+)-ATPase on the cell surface.

Base Sequence↗

The factors affecting transformation efficiency of coryneform bacteria by electroporation.

High-voltage electroporation was performed to transfer plasmid DNA of pXZ10145 into different kinds of corynebacteria strains. A number of factors that affected transformation efficiency were investigated. Cells grown in the presence of 4% glycine and harvested in the early exponential growth phase (OD600 was about 0.2) were much more easily transformed. Transformation efficiency up to 8 x 10(6) transformants per microgram of plasmid DNA with homologously derived DNA was obtained. If heterologously derived DNA was used, transformation efficiency was 10(2) to 10(3) times lower than the former one.

Actinomycetales↗