Modulation of tissue-specific immune response to cardiac myosin can prolong survival of allogeneic heart transplants

The role of immune response to tissue-specific Ags in transplant rejection is poorly defined. We have previously reported that transplantation of cardiac allografts triggers a CD4(+) Th1 cell response to cardiac myosin (CM), a major contractile protein of the heart, and that pretransplant activation of proinflammatory CM-specific T cells accelerates rejection. In this study, we show that administration of CM together with IFA (CM/IFA) can prevent acute rejection of an allogeneic heart transplant. Prolongation of cardiac graft survival is associated with activation of CM- and allo-specific T cells secreting type 2 cytokines (IL-4, IL-5) and reduction of the frequency of proinflammatory IFN-gamma-secreting (type 1) alloreactive T cells. Blocking of IL-4 cytokine with Abs abrogates the prolongation. CM/IFA treatment prevents acute rejection of MHC class I-mismatched, but not fully mismatched grafts. However, if donor heart is devoid of MHC class II expression, CM-IFA administration delays rejection of fully allogeneic cardiac transplants. This finding suggests that the effect of CM modulation depends on the type (direct vs indirect) and strength of recipient's CD4(+) T cell alloresponse. Our results underscore the important role of host immunity to tissue-specific Ags in the rejection of an allograft. This study demonstrates that modulation of the immune response to a tissue-specific Ag can significantly prolong cardiac allograft survival, an observation that may have important implications for the development of novel selective immune therapies in transplantation.     

Sinorhizobium fredii HH103 has a truncated nolO gene due to a -1 frameshift mutation that is conserved among other geographically distant S. fredii strains

Strain SVQ121 is a mutant derivative of Sinorhizobium fredii HH103 carrying a transposon Tn5-lacZ insertion into the nolO-coding region. Sequence analysis of the wild-type gene revealed that it is homologous to that of Rhizobium sp. NGR234, which is involved in the 3 (or 4)-O-carbamoylation of the nonreducing terminus of Nod factors. Downstream of nolO, as in Rhizobium sp. NGR234, the noeI gene responsible for methylation of the fucose moiety of Nod factors was found. SVQ121 Nod factors showed lower levels of methylation into the fucosyl residue than those of HH103-suggesting a polar effect of the transposon insertion into nolO over the noel gene. A noeI HH103 mutant was constructed. This mutant, SVQ503, produced Nod factors devoid of methyl groups, confirming that the S. fredii noeI gene is functional. Neither the nolO nor the noeI mutation affected the ability of HH103 to nodulate several host plants, but both mutations reduced competitiveness to nodulate soybean. The Nod factors produced by strain HH103, like those of other S. fredii isolates, lack carbamoyl residues. By using specific polymerase chain reaction primers, we sequenced the nolO gene of S. fredii strains USDA192, USDA193, USDA257, and 042B(s). All the analyzed strains showed the same -1 frameshift mutation that is present in the HH103 nolO-coding region. From these results, it is concluded that, regardless of their geographical origin, S. fredii strains carry the nolO-coding region but that it is truncated by the same base-pair deletion.     

Cloning, isolation and characterization of the Thermotoga maritima KDPG aldolase

The Thermotoga maritima aldolase gene has been cloned into a T7 expression vector and overexpressed in Escherichia coli. The preparation yields 470 UL(-1) of enzyme at a specific activity of 9.4 U mg(-1). During retroaldol cleavage of KDPG, the enzyme shows a k(cat) that decreases with decreasing temperature. A more than offsetting decrease in K(m) yields an enzyme that is more efficient at 40 degrees C than at 70 degrees C. The substrate specificity of the enzyme was evaluated in the synthetic direction with a range of aldehyde substrates. Although the protein shows considerable structural homology to KDPG aldolases from mesophilic sources, significant differences in substrate specificity exist. A preparative scale reaction between 2-pyridine carboxaldehyde and pyruvate provided product of the same absolute configuration as mesophilic enzymes, but with diminished stereoselectivity.     

Comparative cytotoxic and cytoprotective effects of taurohyodeoxycholic acid (THDCA) and tauroursodeoxycholic acid (TUDCA) in HepG2 cell line

This study was performed to compare the effects of two hydrophilic bile acids, taurohyodeoxycholic acid (THDCA) and tauroursodeoxycholic acid (TUDCA), on HepG2 cells. Cytotoxicity was evaluated at different times of exposure by incubating cells with increasing concentrations (50-800 micromol/l) of either bile acid, while their cytoprotective effect was tested in comparison with deoxycholic acid (DCA) (350 micromol/l and 750 micromol/l)-induced cytotoxicity. Culture media, harvested at the end of each incubation period, were analyzed to evaluate aspartate transaminase (AST), alanine transaminase and gamma-glutamyltranspeptidase release. In addition, the hemolytic effect of THDCA and TUDCA on human red blood cells was also determined. At 24 h of incubation neither THDCA nor TUDCA was cytotoxic at concentrations up to 200 and 400 micromol/l. At 800 micromol/l both THDCA and TUDCA induced a slight increase in AST release. At this concentration and with time of exposure prolonged up to 72 h, THDCA and TUDCA induced a progressive increase of AST release significantly (P<0.05) higher than that of controls being AST values for THDCA (2.97+/-0.88 time control value (tcv) at 48 h and 4.50+/-1.13 tcv at 72 h) significantly greater than those of TUDCA (1.50+/-0.20 tcv at 48 h and 1.80+/-0.43 tcv at 72 h) (P<0.01). In cytoprotection experiments, the addition of 50 micromol/l THDCA decreased only slightly (-5%) AST release induced by 350 micromol/l DCA, while the addition of 50 micromol/l TUDCA was significantly effective (-23%; P<0.05). Higher doses of THDCA or TUDCA did not reduce toxicity induced by 350 micromol/l DCA, but were much less toxic than an equimolar dose of DCA alone. At the concentration used in this experimental model neither THDCA nor TUDCA was hemolytic; however at a very high concentration (6 mmol/l) both bile acids induced 5-8% hemolysis. We conclude that bile acid molecules with a similar degree of hydrophilicity may show different cytotoxic and cytoprotective properties.     

Isolation and characterization of a potato cDNA corresponding to a 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene differentially activated by stress

1-Aminocyclopropane-1-carboxylate (ACC) oxidase enzyme catalyses the final step in ethylene biosynthesis, converting 1-aminocyclopropane-1-carboxylic acid to ethylene. A cDNA clone encoding an ACC oxidase, ST-ACO3, was isolated from potato (Solanum tuberosum L.) by differential screening of a Fusarium eumartii infected-tuber cDNA library. The deduced amino acid sequence exhibited similarity to other ACC oxidase proteins from several plants species. Northern blot analysis revealed that the ST-ACO3 mRNA level increased in potato tubers upon inoculation with F. eumartii, as well as after treatment with salicylic acid and indole-3-acetic acid, suggesting a cross-talk between different signalling pathways involved in the defence response of potato tubers against F. eumartii attack.     

Identification of the human salivary cystatin complex by the coupling of high-performance liquid chromatography and ion-trap mass spectrometry

Human salivary cystatins, five major (S, S1, S2, SA, SN) and two minor (C and D), are multifunctional proteins playing a different role in the oral environment. Salivary cystatin SN is able to effectively inhibit lysosomal cathepsins B, C, H and L and cystatin SA inhibits cathepsins C and L in vitro. These activities suggest, particularly for cystatin SN, an important role in the control of proteolytic events in vivo. Differently, cystatins S are involved, together with statherin, in the mineral balance of the tooth. Due to their distinct role, a reliable method for identification and quantification of the different cystatins, as well as of possible truncated and derived forms, could be helpful for the assessment of the status of the oral cavity. To this purpose high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI MS) was applied to the analysis of human saliva obtained from healthy subjects. All known salivary cystatins, with the exception of cystatin C, were detected. Strong evidence was also obtained for the presence in saliva of post-translational modified isoforms of cystatins, which may be related to donor habits. Cystatin SN and cystatins S, S1 and S2 were well separated by HPLC-ESI MS coupling from other components and thus this approach can be successfully applied to their quantification.     

Three maize root-specific genes are not correctly expressed in regenerated caps in the absence of the quiescent center

The quiescent center is viewed as an architectural template in the root apical meristem of all angiosperm and gymnosperm root tips. In roots of Arabidopsis thaliana (L.) Heynh., the quiescent center inhibits differentiation of contacting initial cells and maintains the surrounding initial cells as stem cells. Here, the role of the quiescent center in the development of the maize (Zea mays L.) root cap has been further explored. Three maize root-specific genes were identified. Two of these were exclusively expressed in the root cap and one of them encoded a GDP-mannose-4,6-dehydratase. Most likely these two genes are structural, tissue-specific markers of the cap. The third gene, a putative glycine-rich cell wall protein, was expressed in the cap and in the root epidermis and, conceivably is a positional marker of the cap. Microsurgical and molecular data indicate that the quiescent center and cap initials may regulate the positional and structural expression of these genes in the cap and thereby control root cap development. Received: 22 September 1999 / Accepted: 9 November 1999     

Relationship between T-wave amplitude and oxygen pulse in guinea pigs in hyperbaric helium and hydrogen

Diving isknown to induce a change in the amplitude of the T wave(A_Tw) ofelectrocardiograms, but it is unknown whether this is linked to achange in cardiovascular performance. We analyzed A_Tw in guinea pigs at 10-60atm and 25-36°C, breathing 2%O₂ in either helium (heliox;n = 10) or hydrogen (hydrox;n = 9) for 1 h at each pressure. Coretemperature and electrocardiograms were detected by using implantedradiotelemeters. O₂ consumption rate was measured by using gas chromatography. In a previous study (S. R. Kayar and E. C. Parker. J. Appl.Physiol. 82: 988-997, 1997), we analyzed theO₂ pulse, i.e., theO₂ consumption rate per heartbeat, in the same animals. By multivariate regression analysis, weidentified variables that were significant toO₂ pulse: body surface area,chamber temperature, core temperature, and pressure. In this study,inclusion of A_Tw made asignificantly better model with fewer variables. After normalizing forchamber temperature and pressure, theO₂ pulse increased with increasing A_Tw in heliox(P = 0.001) but with decreasingA_Tw in hydrox(P < 0.001). ThusA_Tw is associated with thedifferences in O₂ pulse foranimals breathing heliox vs. hydrox.

     

Design,synthesis, and docking of highly hypolipidemic agents: Schizosaccharomyces pombe as a new model for evaluating α-asarone-based HMG-CoA reductase inhibitors

A series of α-asarone-based analogues was designed by conducting docking experiments with published crystal structures of human HMG-CoA reductase. Indeed, synthesis and evaluation of this series showed a highly hypocholesterolemic in vivo activity in a murine model, as predicted by previous docking studies. In agreement with this model, the polar groups attached to the benzene ring could play a key role in the enzyme binding and probably also in its biological activity, mimicking the HMG-moiety of the natural substrate. The hypolipidemic action mechanism of these compounds was investigated by developing a simple, efficient, and novel model for determining HMG-CoA reductase inhibition. The partial purification of the enzyme from Schizosaccharomyces pombe allowed for testing of α-asarone- and fibrate-based analogues, resulting in positive and significant inhibitory activity.