Biomedical subjects

M Z Lai

Publications and source records attributed to M Z Lai.

At least 37 records · Page 2Linked to original sources

PubMed1993

cAMP analogs prevent activation-induced apoptosis of T cell hybridomas.

Activation of T cell hybridomas through their TCR leads to secretion of IL-2, inhibition of proliferation, and apoptosis. The identification of various inhibitors that prevent activation-induced T cell death (AICD) has helped identify several essential events in apoptosis. For example, inhibition of AICD by dexamethasone indicates a connection between these two programmed death pathways. In this study, we have investigated the interaction between the cAMP signal transduction pathway and the activation- or glucocorticoid-induced cell death. cAMP induced DNA fragmentation in thymocytes. T cell hybridomas displayed different sensitivity to cAMP. Regardless of its cAMP sensitivity, programmed cell death promoted by anti-CD3 or Ag in hybridoma was prevented by the presence of cAMP analogs. In contrast, cAMP had no effect on glucocorticoid-induced T cell death. The inhibitory effect of cAMP on AICD was unlikely to be due to quenching of T cell activation signals, because cAMP added 1 h after T cell activation could still prevent cell death. In addition, the increased binding of AP-1, NF-AT, and NF-kappa B during T cell activation was not significantly affected by cAMP. The presence of the inhibitory cAMP-mediated signals, together with the glucocorticoid-induced pathway, suggest there are at least two distinct mechanisms regulating AICD in immature lymphocytes.

Amino Acid Sequence↗

PubMed1992

Isolation and characterization of nuclear proteins that bind to T cell receptor V beta decamer motif.

TCR V beta promoter contains a highly conserved decamer homologous to cAMP response element (CRE). Recent studies have identified this CRE decamer as the dominant transcription-activating element within the TCR V beta promoter. We have isolated cDNA clones, TCR-ATF1 and TCR-ATF2, encoding DNA-binding proteins that recognize this CRE motif. The nucleotide sequence of TCR-ATF1 has not previously been reported, whereas that of TCR-ATF2 was homologous to CRE-BP1, ATF-2, and mXBP. Both TCR-ATF1 and TCR-ATF2 shared a conserved leucine zipper and DNA binding motif with other CRE-binding proteins. TCR-ATF1 and TCR-ATF2 were expressed in all cell lines examined and in mouse embryos as early as 12.5 days. Despite binding to the same CRE motif, TCR-ATF1 and TCR-ATF2 were different from CREB in the fine nucleotide specificity. TCR-ATF bound methylated CRE and CRE mutant M4 (4C----G) that were not recognized by CREB. Additionally, TCR-ATF1 weakly recognized two other single nucleotide mutants of V beta-CRE that were not bound by TCR-ATF2 and CREB. We have further demonstrated that TCR beta-chain expression was immediately activated by cAMP. Such induction is likely mediated through V beta-CRE sequence, because the inclusion of V beta-CRE in a vector with minimum promoter (pBLCAT2) conferred the cAMP inducibility of CAT activity.

Amino Acid Sequence↗

PubMed1992

T cell epitope selection: dominance may be determined by both affinity for major histocompatibility complex and stoichiometry of epitope.

The majority of T cell hybridomas produced in the BALB/c mouse in response to immunization with lambda repressor cI recognize a peptide fragment comprising of residues 12 to 26 (P12-26). Some other parts of the cI (P1-14, P33-48 and P73-88) are defective in generating T cell responses in the BALB/c mouse. P73-88 may be converted into a T cell determinant if a few more amino acid residues are included (P67-88). Together with P46-67 and P80-102, most peptides derived from cI were capable of eliciting T cell responses by themselves in BALB/c mouse. The mechanisms underlying the selection of P12-26 over the other epitopes when lambda repressor was used as immunogen were examined. The dominant response to P12-26 was attenuated by tolerizing with intravenous administration of P12-26. Under such treatment the T cell response to P12-26 was reduced by 80% but there was no enhancement on the responses toward other epitopes. The selection of P12-26 is, thus, unlikely to be due to a competition at the T cell level. It was also found that the dominance of P12-26 was not simply due to a higher affinity of P12-26 for major histocompatibility complex molecules. For example P12-26 binds better to I-Ad molecule than P80-102, but co-injection with equimole of P12-26 only slightly inhibited P80-102-induced T cell response. Instead, it required a few molar excess of P12-26 to effectively block the association of P80-102 with I-Ad molecules and to inhibit the T cell immunity to P80-102. Since epitopes such as P46-67, P67-88 and P80-102 were generated from lambda repressor cI at a lower molar basis than that of P12-26, it is suggested that the dominance of P12-26 was probably generated by such stoichiometry difference, in addition to the higher affinity of P12-26 for I-Ad molecules.

Amino Acid Sequence↗

PubMed1992

Modulation of restricted class II T cell responses by peptides derived from self class II molecule.

We have explored the possibility of using peptides derived from a major histocompatibility complex (MHC) class II (I-Ab) molecule to modulate I-Ab-restricted T cell responses. Six peptides spanning the polymorphic regions of I-Ab were analyzed for competitive binding to the I-Ab molecule, and for efficacies in blocking I-Ab-specific T cell response. Only PB1 (residues 75-91 of beta chain) bound the I-Ab molecule with high affinity. When these MHC-derived peptides were administered simultaneously with antigen, PB1 effectively inhibited I-Ab-restricted T cell responses as well as another peptide PB2 (residues 59-78 of beta chain). PB2 inhibited specific T cell response only when it was administered simultaneously with antigen. Since PB2 is a weak binder of I-Ab, an additional mechanism must account for its inhibitory activity. Both PB1 and PB2 peptides elicited specific T cell responses, indicating that these peptides were not tolerogenic in syngeneic mice. However, the induction of T cells in response to PB1 and PB2 did not increase reactivity to I-Ab. MHC class II-derived peptides thus can be used to regulate T cell responses without the risk of autoreactivity.

Amino Acid Sequence↗

PubMed1990

Restricted V-(D)-J junctional regions in the T cell response to lambda-repressor. Identification of residues critical for antigen recognition.

The T cell response to lambda-repressor is directed to a 15 amino acid peptide (P12-26) of the protein in A/J mice. Previous studies have demonstrated a preferential use of V alpha 2 and V beta 1 amongst the T cell hybridomas specific for P12-26 in the context of I-Ek. By using the polymerase chain reaction, the sequences of a panel of the T cells using V alpha 2 and V beta 1 were determined. A highly conserved alpha-chain V-J junctional sequence was found in six of the eight T cell hybrids. This consensus alpha-chain VJ sequence may be combined with different members of V alpha 2, indicating a more restricted selection on the junctional region than on the V element in these T cells. In contrast, greater diversities were found on the V-D-J region of beta-chains despite the same V beta 1 and J beta 2.1 were used. However, a highly conserved glutamic acid residue was found at the same position of beta-chains where a similar conservation was identified in cytochrome c-specific T cells. The correlation of the TCR sequence with the fine specificities of these T cells suggests that a single amino acid deletion in the V alpha-J alpha region may reduce the P12-26 response and abolish the recognition of an altered peptide [Phe22] P12-26. In addition, three amino acid difference in the V-D-J region of the beta-chain also determine the P12-26 reactivity. Thus the V(D)J junctional regions of both alpha- and beta-chains may be critical for the recognition of the peptide Ag presented by the specific MHC molecule.

Amino Acid Sequence↗

PubMed1990

Efficiency of cytoplasmic delivery by pH-sensitive liposomes to cells in culture.

The intracellular processing of pH-sensitive liposomes composed of cholesterylhemisuccinate (CHEMS) and dioleoylphosphatidylethanolamine (DOPE) by eukaryotic cell lines has been compared to non-pH-sensitive liposomes made of CHEMS and dioleoylphosphatidylcholine (DOPC). The pH-sensitive liposomes can deliver encapsulated fluorescent molecules [calcein, fluoresceinated dextran, fluoresceinated polypeptide, and diphtheria toxin A chain (DTA)] into the cytoplasm. Cytoplasmic delivery can be blocked in the presence of ammonium chloride or EDTA, indicating that the process requires a low-pH environment and the presence of divalent cations. Inhibition of cellular protein synthesis by DTA delivery from the pH-sensitive liposome is orders of magnitude greater than from the non-pH-sensitive liposome composition. The delivery of DTA into the cytoplasm by pH-sensitive liposomes is at least 0.01% of cell-associated liposomal DTA. There is no significant difference in the degradation rate of bovine serum albumin (BSA) or the rate of acidification of pH-sensitive dye, 8-hydroxy-1,3,6-pyrene-trisulfonate (HPTS), when delivered to cells in pH-sensitive and non-pH-sensitive liposomes. Thus the efficiency of cytoplasmic delivery is less than 10% of the cell-associated liposome contents, which is the smallest difference that can be detected by these two assays. Based upon the various assays used to measure liposome content disposition in the cell, we conclude that the efficiency of cytoplasmic delivery by the CHEMS/DOPE liposomes is greater than 0.01% and less than 10% of the cell-associated liposomal contents.

Animals↗

PubMed1989

Identical peptides recognized by MHC class I- and II-restricted T cells.

Previous data from many groups show that both class I and class II-restricted T cells recognize short synthetic peptides in the context of their respective MHC molecules (9-18), all of the peptides described to date are restricted to only a single class of MHC molecules; however, structural homology between the class I and II MHC molecules and the use of similar TCRs by class I and II-restricted T cells suggest that antigen recognition mechanisms are similar in both systems. To directly compare antigen recognition in the two systems, we analyzed peptides for the ability to function in both a class I and II-restricted system and found that seven of seven individual peptides tested stimulate both class I and II-restricted T cell responses. In addition, two of the peptides can function in different species stimulating both human class I and murine class II T cell responses. Thus, the process of T cell recognition of antigen in the context of MHC molecules was highly conserved in evolution not only between the class I and class II MHC systems, but also between the murine and human species.

Animals↗

PubMed1988

T cell receptor gene usage in the response to lambda repressor cI protein. An apparent bias in the usage of a V alpha gene element.

The T cell response to the lambda repressor cI protein is directed to the same region of the protein (residues 12-26) in both BALB/c and A/J mice. A panel of T cell hybridomas specific for P12-26 in the context of either I-Ek or I-Ad have been isolated To further understand the molecular interaction between the TCR and the Ia-P12-26 complex, the primary structures of the TCR of five T cell hybridomas have been determined. Southern and Northern analyses indicate that two members of the V alpha 3 gene family are used by 13 out of 14 I-Ek-restricted T cells. Four different V beta genes are used by these T cell hybridomas, while the majority (8 out of 13) express V beta 1 in combination with the J beta 2.1 element. No clear correlation can be seen in this system between gene usage and MHC restriction. In addition, the fine specificity of I-Ek-restricted T cells to a single amino acid substitution [Phe22/His22]P12-26 is not attributed to the usage of particular V alpha and V beta elements. The V alpha 3 family gene is also used by a few I-Ad-restricted T cells. Interestingly, these I-Ad T cells share a reactivity pattern more similar to that of I-Ek-restricted T cells than other I-Ad-restricted T cells. The nonrandom selection V alpha 3 is also demonstrated by the fact that V alpha 3 is used by P12-26-specific, but not by cytochrome c- or staphylococcal nucleus-specific, I-Ek-restricted T cells. This suggests that although antigen specificity may not be accounted for by either chain of the TCR, the members of V alpha 3 genes may be selected by the antigen (P12-26).

Amino Acid Sequence↗

PubMed1987

T lymphocyte response to bacteriophage lambda repressor cI protein. Recognition of the same peptide presented by Ia molecules of different haplotypes.

The murine T cell response to bacteriophage lambda cI repressor protein has been investigated. Isolation and characterization of class II-restricted T cell hybridomas from BALB/c and A/J mice undergoing a primary response has revealed that a single region of the protein, residues 12-26, is the immunodominant site. Fine specificity analysis using truncated peptides (P12-24 and P15-26) reveals a great deal of heterogeneity at the clonal level of I-Ad-restricted T cells. I-Ek-restricted T cells are less heterogeneous in their reactivity toward P12-24 and P15-26, but show diversity in their responses to peptide analogues with substitution at Tyr22. The specificity difference between T cell hybridomas of I-Ad-restriction and I-Ek-restriction and the inhibition effect of different inactive peptides suggest that the same peptide is presented in different configurations by different Ia molecules. Further, no cross-reactivity can be detected between T cells of these two haplotypes, Ia molecules and Ia bound-peptides.

Amino Acid Sequence↗

PubMed1987

Definition of fibronectin-mediated uptake of gelatinized latex by liver slices and macrophages.

These studies show that both liver slices and macrophages carried out fibronectin concentration-dependent uptake of 125I-labeled gelatin-coated latex (test latex). Lack of phagocytosis of test latex by liver slices was shown directly by electron microscopy and indirectly by trypsin treatment, which caused the release of all test latex taken up in response to fibronectin. Inhibitors of phagocytosis did not alter this uptake. On the other hand, trypsin released only a portion of test latex from macrophages. Inhibitors of phagocytosis did not effect the released radioactive particles from macrophages but greatly reduced the trypsin-resistant radioactivity, taken as representing phagocytized particles. Opsonization of test latex with fibronectin did not require heparin but its association with liver slices occurred only in the presence of heparin. Macrophages, however, readily bound and internalized the opsonized test latex and heparin only potentiated these reactions. Gelatin competed with test latex for fibronectin for opsonization, but did not inhibit binding and phagocytosis of fibronectin-test latex complexes. Finally, soluble fibronectin-gelatin complexes did not compete for binding and phagocytosis of fibronectin-test latex complexes. Thus, fibronectin concentrated on the surface of latex is preferred for interaction with the fibronectin receptor of macrophages. Gelatin, however, was not essential for this reaction, because fibronectin directly coupled to latex was also readily taken up.

Animals↗

PubMed1987

Immunological self, nonself discrimination.

The ability of immunodominant peptides derived from several antigen systems to compete with each other for T cell activation was studied. Only peptides restricted by a given transplantation antigen are mutually competitive. There is a correlation between haplotype restriction, ability to bind to the appropriate transplantation antigen, and ability to inhibit activation of other T cells restricted by the same transplantation antigen. An exception was noted in that a peptide derived from an antigen, bacteriophage lambda cI repressor, binds to the I-Ed molecule in a specific way, yet is not I-Ed-restricted. Comparison of the sequence of the repressor peptide with that of other peptides able to bind to (and be restricted by) I-Ed and a polymorphic region of the I-Ed molecule itself revealed a significant degree of homology. Thus, peptides restricted by a given class II molecule appear to be homologous to a portion of the class II molecule itself. The repressor-derived peptide is identical at several polymorphic residues at this site, and this may account for the failure of I-Ed to act as a restriction element. Comparison of antigenic peptide sequences with transplantation antigen sequences suggests a model that provides a basis for explaining self, nonself discrimination as well as alloreactivity.

Animals↗

PubMed1985

Effects of replacement of the hydroxyl group of cholesterol and tocopherol on the thermotropic behavior of phospholipid membranes.

The role of the hydroxyl groups of cholesterol and tocopherol in mediating their interaction with phospholipid bilayers has been a subject of considerable interest. We have examined this question by using derivatives of cholesterol and tocopherol in which the hydroxyl group is esterified to succinate. The hemisuccinate esters of cholesterol and alpha-tocopherol can be readily incorporated into phospholipid membranes and in fact can by themselves form closed membrane vesicles as demonstrated by the encapsulation of [3H]sucrose. The thermotropic behavior of mixtures containing each succinate ester and phospholipid was studied by differential scanning calorimetry. The effect of cholesteryl hemisuccinate on the thermotropic properties of dipalmitoylphosphatidylcholine and dimyristoylphosphatidylethanolamine is very similar to that of cholesterol. This indicates that the 3 beta-OH is not required for the formation of a cholesterol-phospholipid complex. In mixtures of tocopherol acid succinate and phospholipids the peak transition temperature is progressively shifted to lower temperatures as the mole fraction of alpha-tocopherol succinate is increased, while the enthalpy of the transition is only slightly affected. At a tocopherol succinate/phospholipid molar ratio of 9/1 a phase transition is still detectable. A comparison between tocopherol succinate and tocopherol indicates that the substitution of the hydroxyl group reduces the interaction of tocopherol with phospholipids to a small but measurable extent. Thus, the hydroxyl group of tocopherol is more important than the hydroxyl group of cholesterol in influencing their interactions with phospholipids.

Chemical Phenomena↗

PubMed1985

Acid- and calcium-induced structural changes in phosphatidylethanolamine membranes stabilized by cholesteryl hemisuccinate.

The membrane stabilization effect of cholesteryl hemisuccinate (CHEMS) and the sensitivity of the CHEMS-phosphatidylethanolamine membranes to protons and calcium ions were studied by differential scanning calorimetry, freeze-fracture electron microscopy, and 31P NMR. (1) At neutral pH, the addition of 8 mol % CHEMS to transesterified egg phosphatidylethanolamine (TPE) raised the lamellar-hexagonal transition temperature of TPE by 11 degrees C. Stable bilayer vesicles were formed when the incorporated CHEMS exceeded 20 mol %. (2) At a pH below 5.5, the protonation of CHEMS enhanced the formation of the hexagonal phase (HII) of TPE. At 25 mol % CHEMS the bilayer-hexagonal transition temperature was lowered by 30 degrees C at pH 4.5. (3) The endothermic acid-induced hexagonal hexagonal transition of TPE-CHEMS was suppressed at 35 mol % CHEMS. However, 31P NMR and electron microscopy indicated that a lamellar-hexagonal transition still occurred at this composition. (4) The main transition of TPE was not affected by the protonation of the incorporated CHEMS, indicating that no macroscopic phase separation occurred in TPE-CHEMS mixtures at low pH. (5) In contrast to the HII-promoting effect of H+, the neutralization of the negative charge on TPE-CHEMS by Ca2+ resulted in aggregates that remained in the lamellar structure even at the hexagonal transition temperature of TPE. It is suggested that calcium might form a complex between CHEMS in apposed bilayers. These results are related to the possible biological function of acidic cholesterol esters in biomembranes.

Calcium↗

PubMed1985

On the correlation between HII phase and the contact-induced destabilization of phosphatidylethanolamine-containing membranes.

The abundance of phosphatidylethanolamine (PtdEtn) in biological membranes and the capacity of this lipid to sustain nonbilayer structures have been promoted as evidence for a role of PtdEtn in biological fusion processes. To date there has been no direct evidence of a connection between the kinetics of bilayer destabilization and the polymorphism accessible to PtdEtn. We have developed a model system to examine this point directly using the proton-induced destabilization of PtdEtn/cholesterylhemisuccinate unilamellar liposomes. We find that the initial rate of bilayer mixing rapidly increases with temperature and reaches a maximal level just below the HII-phase transition temperature. The leakage from these liposomes rapidly increases, both in rate and extent, within the HII-phase transition temperature range. Of an even greater significance is that at no temperature is there any mixing of aqueous contents within the liposomes. Thus, these lipids can begin to undergo the lamellar- to HII-phase transition at the stage of two apposed liposomes. However, the nonbilayer structures formed do not cause fusion--i.e., the concomitant mixing of aqueous contents.

Calorimetry↗

PubMed1983

Enzymatic modifications of human plasma fibronectin in relation to opsonizing activity.

Plasma fibronectin is one of the largest plasma proteins (Mr approximately 440 000), comprising two approximately equal polypeptide chains which are held together by a disulfide linkage near the C-terminal end of the molecule. The binding of gelatinized latex beads to liver slices as well as the internalization of these particles by macrophages, in the presence of heparin, is greatly enhanced by fibronectin. The question as to whether the entire covalent structure of fibronectin was necessary for opsonizing activity was approached by limited proteolytic degradations of the molecule. Patterns of controlled digestion with trypsin, cathepsin D, Staphylococcus aureus protease, and plasmin all indicate that the minimal unit necessary for retention of opsonic activity is some large (Mr 200 000 and 190 000) single-chain entity. Treatment with plasmin proved to be the most reliable procedure for generating the active split product which could be readily separated from the inactive, disulfide-containing C-terminal fragment. Incorporation of dansylcadaverine into plasma fibronectin (3.5 mol/mol of protein) by fibronoligase (coagulation factor XIIIa) did not affect the opsonic activity of the protein.

Cathepsin D↗

PubMed1982

Role of aromatic residues in the structure-function relationship of alpha-bungarotoxin.

The conformation of alpha-bungarotoxin and its cyanogen bromide cleaved and nitrated derivatives was studied by circular dichroism (CD). Native toxin contains no helices but some beta forms and possibly beta turns. Its ordered conformation is little affected when the peptide bond between Met-27 and Trp-28 is cleaved; however, the CD due to Trp-28 is abolished. The CNBr-cleaved derivative retains its immunoaffinity toward anti-toxin sera but loses its neurotoxicity toward the acetylcholine receptor. On the basis of both CD and fluorescence spectra, Trp-28 is probably stabilized by a short-range interaction with the carboxylate group of Asp-30. The ordered conformation of the toxin is also unaltered when one of the two tyrosine residues, identified as Tyr-54, is nitrated with tetranitromethane. This Tyr(NO2)-54 derivative possesses both immunoaffinity and neurotoxicity. However, the toxin is denatured and loses its activities when the other tyrosine residue, Tyr-24, is also nitrated in 6 M guanidine hydrochloride, even after the denaturant is removed. Spectrophotometric titration of the toxin indicates that Tyr-54 has a normal apparent dissociation constant (pKa = 9.7) and Tyr-24 ionizes at pH above 11.2. Both tyrosine residues are in a polar environment, but Tyr-24 is not readily accessible to reagents and is stabilized by long-range interactions, probably involving Glu-41.

Amino Acid Sequence↗

PubMed1981

A role for anti-inflammatory agents and cyclic AMP in regulating fibronectin-mediated phagocytosis.

The present study deals with the effects of anti-inflammatory drugs and agents known to elevate intracellular levels of cyclic AMP (cAMP) on plasma fibronectin-mediated (PFn) phagocytosis of radiolabeled, gelatin-coated latex particles (g-Ltx*) by inflammatory macrophages. Monolayers of casein-elicited peritoneal macrophages were preincubated with the specified agents for either 1 or 24 hrs at 37 degrees C prior to the measurements of phagocytosis in the presence of human plasma fibronectin (47 microgram/ml) and heparin (6.7 U/ml). Under these conditions, prostaglandin E1, colchicine, vincristine, and cytochalasin B were all effective in inhibiting g-Ltx* phagocytosis by macrophages in a dose-dependent fashion. More potent inhibition of phagocytosis was manifested by agents known to increase intracellular levels of cAMP in phagocytic cells. Dibutyryl cyclic AMP (dbcAMP), d,1-isoproterenol and aminophylline (10(-5) to 10(-3) M) were all effective in reducing the uptake of g-Ltx* by macrophages. The combination of dbcAMP and aminophylline acted additively. These studies demonstrate that anti-inflammatory drugs and cAMP-elevating agents exert potent inhibitory effects on fibronectin-mediated phagocytosis of gelatin-coated particles by macrophages. Thus, our system provides a suitable in vitro model for further investigations into the humoral regulation of phagocytosis of denatured collagen-coated particles and tissue debris by inflammatory phagocytic cells.

Alprostadil↗

PubMed1980

Fibronectin-mediated uptake of gelatin-coated latex particles by peritoneal macrophages.

The present study demonstrates the ability of plasma fibronectin or cold-insoluble globulin (Clg) to promote the uptake of 125I-labeled, gelatin-coated latex beads (g-Ltx*) by monolayers of peritoneal macrophages (PM). The uptake of g-Ltx* by PM was enhanced by Clg in a concentration-dependent fashion and required the presence of heparin (10 U/ml) as an obligatory cofactor for maximal particle uptake. Treatment of PM monolayers with trypsin (1 mg/ml) for 15 min at 37 degrees C after particle uptake removed less than 15% of the radioactivity incorporated by the monolayers. However, a similar trypsin treatment of the monolayers before the addition of latex particles depressed Clg-dependent uptake by greater than 75%. Pretreatment of PM monolayers with inhibitors of glycolysis effectively reduced the Clg-dependent uptake of latex. Similarly, pretreatment of monolayers with either inhibitors of protein synthesis or agents that disrupt cytoskeletal elements also significantly depressed Clg-dependent particle uptake. Phagocytosis of g-Ltx* by PM in the presence of Clg and heparin was confirmed by electron microscopy. Finally, g-Ltx* could also be effectively opsonized with Clg at 37 degrees C before their addition to the monolayers. These studies suggest that the recognition of g-Ltx* in the presence of Clg required cell surface protein(s) and that subsequent phagocytosis of these particles by PM was energy dependent and required intact intracellular cytoskeleton elements. Thus, PM monolayers provide a suitable system for further studies on the function of Clg in the recognition and phagocytosis of gelatin-coated particles by phagocytic cells.

Animals↗