Lysophosphatidic acid receptor-selective effects on Jurkat T cell migration through a Matrigel model basement membrane

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) from platelets and mononuclear phagocytes mediate T cell functions through endothelial differentiation gene-encoded G protein-coupled receptors (Edg Rs) specific for LPA (Edg-2, -4, and -7) or S1P (Edg-1, -3, -5, -6, and -8). Jurkat leukemic T cells with the SV40 virus large T Ag (Jurkat-T cells) express Edg-3>-2>-4 Rs, as assessed by RT-semiquantitative PCR and Western blots with anti-Edg R mAbs. Jurkat-T cells expressing predominantly Edg-2 R (Jurkat-T-2 cells) and Edg-4 R (Jurkat-T-4 cells) were developed by cotransfection with the respective sense plasmids and a mixture of antisense plasmids for the other Edg Rs, and hygromycin selection. Migration of Jurkat-T-4 cells, but not Jurkat-T-2 cells, through a layer of Matrigel on a 5-um pore polycarbonate filter was stimulated up to 5-fold by 10(-9) to 10(-6) M LPA and by 30-300 ng/ml of anti-Edg-4 R Ab, but not anti-Edg-2 R Ab. LPA and anti-Edg-4 R Ab also enhanced by up to 4-fold the expression of matrix metalloproteinase by Jurkat-T-4 cells, but not Jurkat-T-2 cells, as assessed by cleavage of [(3)H]-type IV human collagen in the Matrigel. Enhancement of matrix metalloproteinase-dependent trans-Matrigel migration of Jurkat-T cells by the chemokine RANTES was suppressed by anti-Edg-2 R Abs, but was stimulated by anti-Edg-4 R Abs. The opposite effects of Edg-2 and Edg-4 LPA receptors on trans-Matrigel migration and some other T cell functions provide receptor-selective mechanisms for regulation of T cell recruitment and immune contributions.     

An antagonist IL-15/Fc protein prevents costimulation blockade-resistant rejection.

IL-15 is a powerful T cell growth factor (TCGF) with particular importance for the maintenance of CD8(+) T cells. Because costimulation blockade does not result in universal tolerance, we hypothesized that "escape" from costimulation blockade might represent a CD8(+) and IL-15/IL-15R(+)-dependent process. For this analysis, we have used an IL-15 mutant/Fcgamma2a protein, a potentially cytolytic protein that is also a high-affinity receptor site specific antagonist for the IL-15Ralpha receptor protein, as a therapeutic agent. The IL-15-related fusion protein was used as monotherapy or in combination with CTLA4/Fc in murine islet allograft models. As monotherapies, CTLA4/Fc and an IL-15 mutant/Fcgamma2a were comparably effective in a semiallogeneic model system, and combined treatment with IL-15 mutant/Fcgamma2a plus CTLA4/Fc produced universal permanent engraftment. In a fully MHC-mismatched strain combination known to be refractory to costimulation blockade treatment, combined treatment with both fusion proteins proved to be highly effective; >70% of recipients were tolerized. The analysis revealed that the IL-15 mutant/Fc treatment confers partial protection from both CD4(+) and CD8(+) T cell graft infiltration. In rejections occurring despite CTLA4/Fc treatment, concomitant treatment with the IL-15 mutant/Fcgamma2a protein blocked a CD8(+) T cell-dominated rejection processes. This protection was linked to a blunted proliferative response of alloreactive T cells as well silencing of CTL-related gene expression events. Hence, we have demonstrated that targeting the IL-15/IL-15R pathway represents a new and potent strategy to prevent costimulation blockade-resistant CD8(+) T cell-driven rejection.     

Developmental expression and stress induction of glutathione S-transferase in the spruce budworm, Choristoneura fumiferana

Developmental and stress-induced expression of Choristoneura fumiferana glutathione S-transferase (CfGST) mRNA and protein were examined using Northern blots and Western blots. High levels of CfGST mRNA and protein were detected in 1st instar larvae and diapausing 2nd instar larvae. Expression of CfGST gradually decreased during larval development from 3rd to 5th instar, after which the expression increased once again, reaching peak levels in 6th instar larvae. CfGST mRNA and protein were undetectable in the pupal stage. Exposure to low temperature did not induce an increase in CfGST expression. Feeding on balsam fir foliage resulted in an increase in the expression of CfGST as compared to larvae that fed on artificial diet. The bacterial insecticide, Bacillus thuringiensis delta-endotoxin (Bt), the non-steroidal ecdysone analog, tebufenozide, and the synthetic pyrethroid, permethrin, induced the expression of CfGST mRNA in 5th instar larvae, whereas the chitin synthesis inhibitor, diflubenzuron, did not have any such effect. These results suggest that CfGST plays an important role in detoxifying various allelochemicals and insecticides in the spruce budworm. The developmental expression pattern strongly suggests that in addition to detoxification, CfGST might be involved in other functions.     

A recombinant glycine receptor fragment forms homo-oligomers distinct from its GABA(A) counterpart

The ligand-gated ion channel receptor superfamily includes receptors for glycine, GABA, acetylcholine and serotonin. Whereas the acetylcholine and serotonin receptors mediate excitory neurotransmissions, both glycine and GABA(A) receptors are inhibitory. In this study, a fragment of the human glycine receptor alpha1 subunit, consisting of residues Ala165-Met291 (numbering based on the precursor protein), was hyperexpressed for the first time in Escherichia coli. This fragment is highly homologous in sequence to the corresponding fragment of the GABA(A) receptor. The recombinant fragment was found to have stable beta-rich secondary structure, similar to that found for the homologous GABA(A) receptor fragment, and ordered tertiary packing, suggesting a stable structural domain. Results from laser scattering studies suggest that the fragment forms trimers in solution. In addition, SDS-induced changes in secondary structure were found to occur prior to changes in oligomerization status, suggesting that oligomerization was secondary structure dependent. A study of quaternary structure using single particle analysis electron microscopy (EM) also suggested that the fragment formed homo-trimers. One trimer measures approximately 7.5 nm in diameter with a central cavity approximately 1.5 nm across. This is the first EM study on a single domain of the glycine receptor and the result is in contrast to the pentameric assembly of the equivalent GABA(A) receptor fragment reported by us earlier. The fact that this fragment alone could form oligomers in vitro suggests that amino acid residues within this segment may be involved in the oligomerization of the glycine receptor in vivo. Furthermore, the finding that two cousin receptor fragments form distinct quaternary structures indicates that sequence similarity does not necessarily imply quaternary structure similarity and, hence, care must be taken when applying a structure model derived from studies of individual receptors to the whole ligand-gated ion channel superfamily.     

Herpes simplex virus-induced keratitis: evaluation of the role of molecular mimicry in lesion pathogenesis

Viruses are suspected but usually unproven triggering factors in autoimmunity. One favored mechanism to explain the role of viruses in the genesis of autoimmunity is molecular mimicry. An immunoinflammatory blinding lesion called herpetic stromal keratitis (HSK) that follows ocular infection with herpes simplex virus (HSV) is suggested to result from a CD4(+) T-cell response to a UL6 peptide of HSV that cross-reacts with a corneal autopeptide shared with the immunoglobulin G2a(b) (IgG2a(b)) isotype. The present report reevaluates the molecular mimicry hypothesis to explain HSK pathogenesis. Our results failed to reveal cross-reactivity between the UL6 and IgG2a(b) peptides or between peptide reactive T cells and HSV antigens. More importantly, animals infected with HSV failed to develop responses that reacted with either peptide, and infection with a recombinant vaccinia UL6 vector failed to cause HSK, in spite of generating UL6 reactivity. Other lines of evidence also failed to support the molecular mimicry hypothesis, such as the failure to affect HSK severity upon tolerization of susceptible BALB/c and B-cell-deficient mice with IgG2a(b) or UL6 peptides. An additional study system revealed that HSK could be induced in mouse strains, such as the OT2 x RAG1(-/-) mice (T cell receptor transgenic recognizing OVA(323-339)) that were unable to produce CD4(+) T-cell responses to any detectable HSV antigens. Our results cast doubt on the molecular mimicry hypothesis as an explanation for the pathogenesis of HSK and indicate that if autoimmunity is involved its likely proceeds via a bystander activation mechanism.     

On the dispersion index of a Markovian molecular clock

The number of nucleotide substitutions accumulated in a gene or in a lineage is an important random variable in the study of molecular evolution. Of particular interest is the ratio of the variance to the mean of that random variable, often known as the dispersion index. Because nucleotide substitution is most commonly modeled by a continuous-time four-state Markov chain, this paper provides a systematic method of computing the dispersion indices exhibited by a continuous-time four-state Markov chain. Using this method along with computer algebra and Monte Carlo simulation, this paper offers partially proven conjectures that were supported by thorough computer experiments. It is believed that the Tamura model, the equal-input model and the Takahata-Kimura model always exhibit dispersion indices less than 2. It is also believed that a general four-state model can be chosen to exhibit a dispersion index of any desired magnitude, although the chance of a randomly chosen such model exhibiting a dispersion index greater than 2 is as small as about 2%. Relevance of these findings to the neutral theory is discussed.     

SeO(2) induces apoptosis with down-regulation of Bcl-2 and up-regulation of P53 expression in both immortal human hepatic cell line and hepatoma cell line

An immortal human hepatic cell line HL-7702 and human hepatoma cell line SMMC-7721 were treated with 3-30 microM SeO(2). SeO(2) at 30 microM markedly inhibited cell proliferation and viability, and prompted apoptosis of both normal hepatic and hepatoma cells after 48h treatment. SeO(2) could also down-regulate the Bcl-2 level, greatly in HL-7702 and slightly in SMMC-7721 cells, but up-regulate wild type P53 level a little in HL-7702 and significantly in SMMC-7721 cells. The Bcl-2/P53 value was closely correlated with the apoptotic rate as well as SeO(2) concentrations.     

Amyloplast sedimentation dynamics in maize columella cells support a new model for the gravity-sensing apparatus of roots

Quantitative analysis of statolith sedimentation behavior was accomplished using videomicroscopy of living columella cells of corn (Zea mays) roots, which displayed no systematic cytoplasmic streaming. Following 90 degrees rotation of the root, the statoliths moved downward along the distal wall and then spread out along the bottom with an average velocity of 1.7 microm min(-1). When statolith trajectories traversed the complete width or length of the cell, they initially moved horizontally toward channel-initiation sites and then moved vertically through the channels to the lower side of the reoriented cell where they again dispersed. These statoliths exhibited a significantly lower average velocity than those sedimenting on distal-to-side trajectories. In addition, although statoliths undergoing distal-to-side sedimentation began at their highest velocity and slowed monotonically as they approached the lower cell membrane, statoliths crossing the cell's central region remained slow initially and accelerated to maximum speed once they reached a channel. The statoliths accelerated sooner, and the channeling effect was less pronounced in roots treated with cytochalasin D. Parallel ultrastructural studies of high-pressure frozen-freeze-substituted columella cells suggest that the low-resistance statolith pathway in the cell periphery corresponds to the sharp interface between the endoplasmic reticulum (ER)-rich cortical and the ER-devoid central region of these cells. The central region is also shown to contain an actin-based cytoskeletal network in which the individual, straight, actin-like filaments are randomly distributed. To explain these findings as well as the results of physical simulation experiments, we have formulated a new, tensegrity-based model of gravity sensing in columella cells. This model envisages the cytoplasm as pervaded by an actin-based cytoskeletal network that is denser in the ER-devoid central region than in the ER-rich cell cortex and is linked to stretch receptors in the plasma membrane. Sedimenting statoliths are postulated to produce a directional signal by locally disrupting the network and thereby altering the balance of forces acting on the receptors in different plasma membrane regions.     

Fast repair of hydroxy radical purine deoxynucleotide adducts by phenylpropanoid glycosides and their derivatives from Chinese herbs

DNA damaged by oxygen radicals has been implicated as a causative event in a number of degenerative diseases, including cancer and aging. So it is very significant to look for ways in which either oxygen radicals are scavenged prior to DNA damage or damaged DNA is repaired to supplement the cells' inadequate repair capacity. The repair activities and reaction mechanism of phenylpropanoid glycosides (PPGs) and their derivatives, isolated from Chinese folk medicinal herbs, towards both dGMP-OH* adducts and dAMP-OH* adducts were studied with the pulse radiolytic technique. On pulse irradiation of nitrous oxide saturated 2 mM dGMP or dAMP aqueous solution containing one of the PPGs or their derivatives, the transient absorption spectra of the hydroxyl adduct of dGMP or dAMP decayed with the formation of that of phenoxyl radicals of PPGs or their derivatives within several decades of microseconds after electron pulse irradiation. The result indicated that dGMP or dAMP hydroxyl adducts can be repaired by PPGs or their derivatives. The rate constants of the repair reactions were deduced to be 0.641-1.28 x 10(9) M(-1) s(-1) for dGMP-OH* and 0.2-0.491 x 10(9) M(-1) s(-1) for dAMP-OH*, which positively correlated to the number of phenolic hydroxyl groups in the glycoside structure. A deeper understanding of this new repair mechanism may help researchers to design strategies to prevent and/or intervene more effectively in free radical related diseases.     

Stable restoration of the sarcoglycan complex in dystrophic muscle perfused with histamine and a recombinant adeno-associated viral vector

Limb-girdle muscular dystrophies 2C-F represent a family of autosomal recessive diseases caused by defects in sarcoglycan genes. The cardiomyopathic hamster is a naturally occurring model for limb-girdle muscular dystrophy caused by a primary deficiency in delta-sarcoglycan. We show here that acute sarcolemmal disruption occurs in this animal model during forceful muscle contraction. A recombinant adeno-associated virus vector encoding human delta-sarcoglycan conferred efficient and stable genetic reconstitution in the adult cardiomyopathic hamster when injected directly into muscle. A quantitative assay demonstrated that vector-transduced muscle fibers are stably protected from sarcolemmal disruption; there was no associated inflammation or immunologic response to the vector-encoded protein. Efficient gene transduction with rescue of the sarcoglycan complex in muscle fibers of the distal hindlimb was also obtained after infusion of recombinant adeno-associated virus into the femoral artery in conjunction with histamine-induced endothelial permeabilization. This study provides a strong rationale for the development of gene therapy for limb-girdle muscular dystrophy.