Three major red hemoproteins, named RpSG I, II (identical with prolixin-S) and III, in the salivary glands of the blood-sucking insect, Rhonius prolixus, show homology in N-terminal amino acid (AA) sequences, and are immunologically related. We focussed on one of these proteins, RpSG-I, in this paper. RpSG-I in fresh salivary gland extract was separated into two components (Ia and Ib) by isoelectric focussing gel electrophoresis. Absorption spectra of RpSG-Ia and Ib showed Soret peaks at 400 nm and 420 nm, respectively, suggesting that they are nitric oxide (NO)-unbound and -bound hemoproteins and function as NO-carriers. RpSG-I is stage-specific in appearance, being absent in 3rd and 4th instar nymphs, appearing and increasing gradually in 5th (last) instar nymphs after engorgement, and present in the adult stage. We purified RpSG-I from salivary gland extract by size exclusion and ion exchange HPLCs. It is a single electrophoretic band with an absorption peak at 400 nm, representing the NO-unbound molecule. Full-size cDNA of RpSG-I was cloned by screening with a specific polyclonal antibody from a salivary gland cDNA library. Sequence analysis of RpSG-I cDNA showed an open reading frame encoding a signal peptide (23 AA) and mature protein (179 AA) of 19,778 daltons. The deduced N-terminal AA sequence of the RpSG-I was identical with that of the hemoprotein reported as nitrophorin-3 (Champagne et al., 1995).

Amino Acid Sequence↗

PubMed1998

Both farnesyltransferase and geranylgeranyltransferase I inhibitors are required for inhibition of oncogenic K-Ras prenylation but each alone is sufficient to suppress human tumor growth in nude mouse xenografts.

The ability of Ras oncoproteins to cause malignant transformation requires their post-translational modifications by prenyl groups. Because K-Ras can be both farnesylated and geranylgeranylated it is not known whether both farnesyltransferase and geranylgeranyltransferase I inhibitors are required for suppressing human tumor growth in whole animals. In this paper we report that oncogenic Ras processing, MAP kinase activation and growth in nude mice are inhibited by the farnesyltransferase inhibitor FTI-276 in H- and N-Ras transformed NIH3T3 cells; whereas in KB-Ras transformed NIH3T3 cells both FTI-276 and the geranylgeranyltransferase I inhibitor GGTI-297 are required for inhibition. Furthermore, human lung A-549 and Calu-1 carcinoma cell lines were found to co-express H-, N- and K-Ras. In Calu-1 cells, the processing of H- and N-Ras is inhibited greatly by FTI-276 but only partially by GGTI-297 whereas K-Ras processing inhibition requires both FTI-276 and GGTI-297. In contrast, in A-549 cells the processing of H- and N-Ras is inhibited only by FTI-276 and K-Ras processing is resistant to co-treatment with FTI-276 and GGTI-297. Yet, the growth in nude mice of A-549 and Calu-1 xenografts, both of which express K-Ras mutations, is inhibited by FTI-276 (80% inhibition) and GGTI-297 (60%). Furthermore, FTI-276 inhibits tumor growth of NIH3T3 cells transformed by a form of oncogenic H-Ras that is exclusively geranylgeranylated and whose processing is resistant to this inhibitor. Taken together, the results demonstrate that both FTase and GGTase I inhibitors are required for inhibition of K-Ras processing but that each alone is sufficient to suppress human tumor growth in nude mice.

3T3 Cells↗

PubMed1998

A proportion of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic antibodies (ANCA) only react with PR3 after cleavage of its N-terminal activation dipeptide.

ANCA directed against PR3 are highly specific for Wegener's granulomatosis and microscopic polyangiitis, and have been implicated in the pathogenesis of small vessel vasculitis. Most PR3-ANCA are directed against conformational epitopes on PR3. This study was designed to determine whether the cleavage of the N-terminal activation dipeptide of PR3 is required for the binding of PR3-ANCA. Recombinant PR3 (rPR3) variants were expressed in the epithelial cell line, 293. As confirmed by radiosequencing, the rPR3 secreted into the 293 cell culture supernatant is N-terminally unprocessed. Two enzymatically inactive rPR3 mutants were expressed in 293 cells: rPR3-S176A and delta-rPR3-S176A. rPR3-S176A contains the N-propetide Ala-2-Glu-1, delta-rPR3-S176A does not. Culture supernatants of rPR3-S176A and delta-rPR3-S176A expressing 293 cells were used as sources of target antigen for PR3-ANCA testing by capture ELISA. Forty unselected consecutive PR3-ANCA+ sera were tested. With delta-rPR3-S176A as antigen all 40 were recognized, compared with only 34 of 40 when rPR3-S176A served as target antigen. The majority of the serum samples contained a mixture of antibodies reacting with epitopes accessible on the mature and on the proform of PR3. In conclusion, the cleavage of the N-terminal activation dipeptide of PR3 is not an absolute requirement for recognition by all PR3-ANCA. However, a substantial proportion of PR3-ANCA recognize (a) target antigen(s) exposed only after the conformational change of PR3 associated with the N-terminal processing. In 15% of sera this PR3-ANCA subset occurred exclusively. PR3-ANCA subtypes can be differentiated using specifically designed rPR3 variants as target antigens, and non-haematopoietic mammalian cells without regulated secretory pathway can be used for their expression.

Antibodies, Antineutrophil Cytoplasmic↗

PubMed1998

Paradoxical early immune activation during acceptance of liver allografts compared with rejection of skin grafts in a rat model of transplantation.

Liver allografts in many animal models are often spontaneously accepted across a complete histocompatibility barrier without requirement for immunosuppression. In contrast, skin allografts are usually rejected, even across minor histocompatibility barriers. To identify the mechanism of liver allograft acceptance we have compared skin rejection with liver acceptance in DA rat strain recipients of PVG donors, a major histocompatibility complex (MHC) class I plus II mismatch. In spite of the established role of draining lymph nodes (LN) in induction of rejection of skin allografts, there was much greater involvement of LN after liver than after skin transplantation. Few donor cells migrated to these organs from transplanted skin but many cells migrated from transplanted liver. There was also a paradoxical increase in interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) mRNA in LN and spleen of liver allograft recipients that greatly exceeded their expression in skin allograft recipients. For example, there were 2. 7+/-1.6x104 molecules of IFN-gamma per 106 molecules of beta-actin mRNA in the LN draining liver allografts 1 day after transplantation compared with 2.0+/-0.3x103 molecules/106 beta-actin in LN draining skin allografts and 8.1+/-1.8x102 molecules/106 beta-actin in LN draining skin isografts. Examination of the graft showed that infiltration and cytokine mRNA up-regulation occurred more slowly in the transplanted skin than in liver but progressed inexorably in skin grafts until rejection. These results show that liver acceptance is associated with a paradoxical marked early activation then subsequent decline of the immune response.

Animals↗

PubMed1998

A comparison of guanosine-quartet inhibitory effects versus cytidine homopolymer inhibitory effects on rat neointimal formation.

Phosphorothioate oligodeoxynucleotides (PS oligos) manifest antisense and G-quartet aptameric inhibitory effects on vascular smooth muscle cell (SMC) proliferation. PS oligo cytidine homopolymers also have nonsequence-specific, non-G-quartet inhibitory effects on in vitro and in vivo SMC proliferation. In this study, we compared the effects of S-dC18 and S-dC28, 18-mer and 28-mer cytidine homopolymers, respectively, which lack guanosines, with those of ZK10, a G-tetrad forming compound, on in vitro SMC proliferation and in vivo neointimal formation. ZK10 significantly inhibited in vitro human aortic SMC proliferation. At the same molar concentration, ZK10 had significantly greater inhibitory potency on SMC proliferation than either S-dC18, S-dC28, or 7DG-ZK10, which is a modified ZK10 with ten 7-deaza guanosine substitutions. ZK10 was significantly more potent than S-dC18 and S-dC28 in inhibiting PDGF-induced in vitro SMC migration. S-dC18, S-dC28, and ZK10 treatment significantly reduced the intima/media area ratio after rat carotid artery balloon injury compared with the values of the control groups. ZK10 was a more potent inhibitor of neointimal formation than the same chain length S-dC18. ZK10 formed higher-order structures, as shown on gel electrophoresis, in contrast to S-dC28 and 7DG-ZK10. Therefore, the 18-mer ZK10 has comparable in vivo SMC inhibitory effects to the 28-mer S-dC28, a fact that may have ramifications for the development of optimal PS oligos to inhibit angioplasty restenosis.

Angioplasty, Balloon↗

PubMed1998

Mechanical evaluation of a new minimum-contact plate for internal fracture fixation.

OBJECTIVE: To determine whether the mechanical properties (stiffness and strength) of a new design of internal fixation plate, with reduced bone-to-plate contact area for reducing disturbance to the bone blood supply at the fracture site, are comparable with those of commonly used fracture fixation plates. DESIGN: Four-point bending and torsion tests were conducted to compare the new minimum contact plate (MCP) with two established internal fixation plates: the dynamic compression plate (DCP) and the limited contact dynamic compression plate (LC-DCP). The bending stiffness and strength were determined according to the International Standards Organization bending test for bone plates, and the torsional stiffness and torque to failure were calculated from torsional loading data with the plates attached to synthetic bones. RESULTS: The new MCP plate was significantly stiffer than the other two plates and stronger than the DCP plate in bending (p < 0.01). In torsion, the MCP plate was marginally stiffer (p < 0.11) and significantly stronger (p < 0.01) than the LC-DCP plate and not as stiff and strong as the DCP plate (p < 0.01). CONCLUSIONS: Mechanically, the MCP plate has adequate stiffness and strength for clinical application because it is at least as stiff and strong as one of the commonly used plates under both bending and torsional loading conditions.

Animals↗

PubMed1998

Mechanical performance of an axially mobile plate for fracture fixation.

BACKGROUND: Emphasis of the advantages of secondary bone healing over primary bone healing has led to a trend of moving from rigid fixation to less-rigid fixation. A reduction in axial stiffness of fixators has been shown to be beneficial to bone healing, and many external fixators have been designed that incorporate axial dynamization. METHODS AND RESULTS: A new axially mobile plate has been designed for long-bone fracture fixation, to give the bone fragments a degree of axial mobility while maintaining bending and torsional stability after fixation. Mechanical bending and torsion tests have been conducted on the axially mobile plate and also on the conventional dynamic compression plate and the limited-contact dynamic compression plate. The results have shown that the new plate is stiffer and stronger in both bending and torsion than at least one of these conventional plates. CONCLUSION: The axially mobile plate has adequate mechanical properties and can be taken forward to clinical trails.

Biomechanical Phenomena↗

PubMed1998

Coupled B-snake grids and constrained thin-plate splines for analysis of 2-D tissue deformations from tagged MRI.

Magnetic resonance imaging (MRI) is unique in its ability to noninvasively and selectively alter tissue magnetization and create tagged patterns within a deforming body such as the heart muscle. The resulting patterns define a time-varying curvilinear coordinate system on the tissue, which we track with coupled B-snake grids. B-spline bases provide local control of shape, compact representation, and parametric continuity. Efficient spline warps are proposed which warp an area in the plane such that two embedded snake grids obtained from two tagged frames are brought into registration, interpolating a dense displacement vector field. The reconstructed vector field adheres to the known displacement information at the intersections, forces corresponding snakes to be warped into one another, and for all other points in the plane, where no information is available, a C1 continuous vector field is interpolated. The implementation proposed in this paper improves on our previous variational-based implementation and generalizes warp methods to include biologically relevant contiguous open curves, in addition to standard landmark points. The methods are validated with a cardiac motion simulator, in addition to in-vivo tagging data sets.

Computer Simulation↗

PubMed1998

Focusing of therapeutic ultrasound through a human skull: a numerical study.

A numerical model was developed which can use digitized layer interfaces to calculate ultrasound wave absorption, diffraction, reflection, and refraction. This model was used to evaluate the feasibility of ultrasound therapy and surgery through a human skull. A digitized human skull profile was obtained from magnetic resonance (MR) images and used to calculate the ultrasound field in the brain of a volunteer from a spherically curved phased array. With no phase correction, the focus of the array was shifted and defocused. The phased array technique was used to correct focal shift, reduce side lobes, and enhance focal amplitude. The optimum source element width was estimated for each frequency to obtain a near optimium focus, and an appropriate frequency range for transskull ultrasound therapy and surgery was determined. Acoustic pressure amplitude on the skull surfaces was examined, and it was shown that the skull heating problem could be overcome. Despite high attenuation, complex interface shape, and nonuniform thickness of a human skull, a sharply focused transskull ultrasound field can be generated for noninvasive ultrasound therapy and surgery in the brain.

Humans↗

PubMed1998

A cytochrome cbb3 (cytochrome c) terminal oxidase in Azospirillum brasilense Sp7 supports microaerobic growth.

Spectral analysis indicated the presence of a cytochrome cbb3 oxidase under microaerobic conditions in Azospirillum brasilense Sp7 cells. The corresponding genes (cytNOQP) were isolated by using PCR. These genes are organized in an operon, preceded by a putative anaerobox. The phenotype of an A. brasilense cytN mutant was analyzed. Under aerobic conditions, the specific growth rate during exponential phase (mu(e)) of the A. brasilense cytN mutant was comparable to the wild-type specific growth rate (m(e) of approximately 0.2 h-1). In microaerobic NH4+-supplemented conditions, the low respiration of the A. brasilense cytN mutant affected its specific growth rate (mu(e) of approximately 0.02 h-1) compared to the wild-type specific growth rate (mu(e) of approximately 0.2 h-1). Under nitrogen-fixing conditions, both the growth rates and respiration of the wild type were significantly diminished in comparison to those under NH4+-supplemented conditions. Differences in growth rates and respiration between the wild type and the A. brasilense cytN mutant were less pronounced under these nitrogen-fixing conditions (mu(e) of approximately 0.03 h-1 for the wild type and 0.02 h-1 for the A. brasilense cytN mutant). The nitrogen-fixing capacity of the A. brasilense cytN mutant was still approximately 80% of that determined for the wild-type strain. This leads to the conclusion that the A. brasilense cytochrome cbb3 oxidase is required under microaerobic conditions, when a high respiration rate is needed, but that under nitrogen-fixing conditions the respiration rate does not seem to be a growth-limiting factor.

Aerobiosis↗

PubMed1998

The Bacillus subtilis DinR binding site: redefinition of the consensus sequence.

Recently, the DinR protein was established as the cellular repressor of the SOS response in the bacterium Bacillus subtilis. It is believed that DinR functions as the repressor by binding to a consensus sequence located in the promoter region of each SOS gene. The binding site for DinR is believed to be synonymous with the formerly identified Cheo box, a region of 12 bp displaying dyad symmetry (GAAC-N4-GTTC). Electrophoretic mobility shift assays revealed that highly purified DinR does bind to such sites located upstream of the dinA, dinB, dinC, and dinR genes. Furthermore, detailed mutational analysis of the B. subtilis recA operator indicates that some nucleotides are more important than others for maintaining efficient DinR binding. For example, nucleotide substitutions immediately 5' and 3' of the Cheo box as well as those in the N4 region appear to affect DinR binding. This data, combined with computational analyses of potential binding sites in other gram-positive organisms, yields a new consensus (DinR box) of 5'-CGAACRNRYGTTYC-3'. DNA footprint analysis of the B. subtilis dinR and recA DinR boxes revealed that the DinR box is centrally located within a DNA region of 31 bp that is protected from hydroxyl radical cleavage in the presence of DinR. Furthermore, while DinR is predominantly monomeric in solution, it apparently binds to the DinR box in a dimeric state.

Amino Acid Sequence↗

PubMed1998

Selective regulation of apoptosis: the cytotoxic lymphocyte serpin proteinase inhibitor 9 protects against granzyme B-mediated apoptosis without perturbing the Fas cell death pathway.

Cytotoxic lymphocytes (CLs) induce caspase activation and apoptosis of target cells either through Fas activation or through release of granule cytotoxins, particularly granzyme B. CLs themselves resist granule-mediated apoptosis but are eventually cleared via Fas-mediated apoptosis. Here we show that the CL cytoplasmic serpin proteinase inhibitor 9 (PI-9) can protect transfected cells against apoptosis induced by either purified granzyme B and perforin or intact CLs. A PI-9 P1 mutant (Glu to Asp) is a 100-fold-less-efficient granzyme B inhibitor that no longer protects against granzyme B-mediated apoptosis. PI-9 is highly specific for granzyme B because it does not inhibit eight of the nine caspases tested or protect transfected cells against Fas-mediated apoptosis. In contrast, the P1(Asp) mutant is an effective caspase inhibitor that protects against Fas-mediated apoptosis. We propose that PI-9 shields CLs specifically against misdirected granzyme B to prevent autolysis or fratricide, but it does not interfere with homeostatic deletion via Fas-mediated apoptosis.

Amino Acid Sequence↗

PubMed1998

Cigarette smoke increases amosite asbestos fiber binding to the surface of tracheal epithelial cells.

Binding of asbestos fibers to the cell surface appears to be important in the initiation of intracellular signaling events as well as in initiation of particle uptake by the cell. We have previously shown that cigarette smoke increases the uptake of asbestos fibers by tracheal epithelial cells in explant culture. Whether smoke acts by increasing surface binding of fibers is not known. In this study, we exposed rat tracheal explants to air or cigarette smoke and then to a suspension of amosite asbestos. Explants were harvested after 1 or 24 h of dust exposure and washed by repeated dips in culture medium to remove loosely bound fibers, and the number of fibers adhering to the apical cell surfaces was determined by scanning electron microscopy. Smoke-exposed explants retained significantly more surface fibers than air-exposed explants. After four washes, binding levels were similar at 1 and 24 h. The smoke effect was still present when incubations were carried out at 4 degrees C, but binding was decreased approximately 25%. Preincubation of the asbestos fibers with iron chloride to increase surface iron increased fiber binding in both air- and smoke-exposed explants, whereas preincubation of the fibers with the iron chelator deferoxamine decreased binding after air exposure and completely eliminated the smoke effect. Inclusion of mannitol or catalase in the medium or preincubation of the explants with GSH produced decreases in binding of 10-25% in air-exposed explants and generally greater decreases in smoke-exposed explants. We conclude that 1) amosite binding is a very rapid process that does not require active cellular metabolism, 2) cigarette smoke increases adhesion of fibers to the epithelial surfaces, and 3) iron on the asbestos fiber appears to play an important role in binding, probably through an active oxygen species-mediated process.

Animals↗

PubMed1998

Transcription activation by the human estrogen receptor subtype beta (ER beta) studied with ER beta and ER alpha receptor chimeras.

We have studied the two estrogen receptor (ER) subtypes, ER alpha and ER beta, and chimeric constructs with ER alpha and ER beta to examine the bioactivities of these receptors and their responses to estrogen and antiestrogen ligands. Transcriptional activity of ER beta is highly dependent on cell/promoter context and on the nature of the ligand. ER beta activated significant levels of transcription in response to estrogens in certain cell types, but showed only moderate activity compared with ER alpha in others. Antiestrogens such as tamoxifen and 2-phenylbenzofuran, which show some agonistic activity with ER alpha, exhibit no agonistic activity with ER beta. Alteration of the amino-terminal A/B receptor domain can result in a dramatic change in cell type- and ligand-specific transcriptional activity of ER beta. Upon replacing the A/B domain of ER beta with the A/B domain of ER alpha, this receptor chimera not only exhibits an improved transcriptional response to estrogens, but also is now able to activate transcription upon treatment with these antiestrogens. As antiestrogen agonism was lacking in ER beta and the ER beta/alpha chimera containing the amino-terminal A/B domain of ER beta fused to domains C through F of ER alpha, but was restored in an ER alpha/beta chimera containing the A/B domain of ER alpha, antiestrogen agonism was shown to depend on the A/B domain (activation function-1-containing region) of ER alpha. Together, these results indicate that the differences in the amino-terminal regions of ER alpha and ER beta contribute to the cell- and promoter-specific differences in transcriptional activity of these receptors, and their ability to respond to different ligands, thus providing a mechanism for differentially regulated transcription by these two ERs.

Amino Acid Sequence↗