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Biomedical subjects

H Li

Publications and source records attributed to H Li.

At least 1,369 records · Page 76Linked to original sources
PubMed1997

[Histological change of keratinocyte in full-thickness skin autograft and its effect on hyperpigmentation of the graft].

To understand the reason of hyperpigmentation following full-thickness skin autograft, 54 guinea pigs were selected as skin autograft models. The changes of density of epidermal keratinocyte (KC) and the amount and distribution of melanin in the epidermal KC were observed by histological, histochemical and autoradiographic techniques. The results showed: (1) The histological changes of KC were in fact a wound-repair process; (2) The increased melanin in KC coordinated with "multi-overlapping screen" effect, a result of multiple layers of "supranuclear cap", was the direct cause of hyperpigmentation of the graft and (3) The hydrolytic disturbance of melanin in KC and the longer life span of the KC were probably the important reasons for the increase of melanin in the epidermis.

Animals↗
PubMed1997

[Artificial biological skin for preventing adhesion of repaired tendon in white rats].

To study the effect of tendon healing and adhesion after tendon repaired using the artificial biological skin (Biofill) produced in Brazil, we divided randomly 46 rats [correction of mice] into groups, compared the pelvic limbs with themselves, observed by eyes and light microscopy, and made biomechanical and histological detection in 1st, 2nd, 3rd and 4th weeks postsurgically. The results showed that the speed and strength of tendon healing had light affect in three weeks after operation, and had no difference between experimental groups and control groups after 4 weeks. It is indicated that artificial skin "Biofill" can effectively prevent tendon adhesion after tendon repaired and have not obviously influenced in the intrinsic self-healing process of tendon.

Achilles Tendon↗
PubMed1996

Specific cleavage of alpha-fodrin during Fas- and tumor necrosis factor-induced apoptosis is mediated by an interleukin-1beta-converting enzyme/Ced-3 protease distinct from the poly(ADP-ribose) polymerase protease.

Interleukin-1beta-converting enzyme (ICE)/Ced-3 proteases play a critical role in apoptosis. One well characterized substrate of these proteases is the DNA repair enzyme poly(ADP-ribose) polymerase. We report here that alpha-fodrin, an abundant membrane-associated cytoskeletal protein, is cleaved rapidly and specifically during Fas- and tumor necrosis factor-induced apoptosis; this cleavage is mediated by an ICE/Ced-3 protease distinct from the poly(ADP-ribose) polymerase protease. Studies in cells treated with these apoptotic stimuli reveal that both fodrin and poly(ADP-ribose) polymerase proteolysis are inhibited by acetyl-Tyr-Val-Ala-Asp chloromethyl ketone and CrmA, specific inhibitors of ICE/Ced-3 proteases. However, fodrin proteolysis can be distinguished from poly(ADP-ribose) polymerase proteolysis by its relative insensitivity to acetyl-Asp-Glu-Val-Asp aldehyde (DEVD-CHO), a selective inhibitor of a subset of ICE/Ced-3 proteases that includes CPP32. DEVD-CHO protects cells from Fas-induced apoptosis but does not prevent fodrin proteolysis, indicating that cleavage of this protein can be uncoupled from apoptotic cell death. Moreover, purified fodrin is cleaved in vitro by CPP32 (but not by ICE) into fragments of the same size observed in vivo during apoptosis. These findings suggest that fodrin proteolysis in vivo may reflect the activity of multiple ICE/Ced-3 proteases whose partial sensitivity to DEVD-CHO reflects a limited contribution from CPP32, or an ICE/Ced-3 protease less sensitive than CPP32 to DEVD-CHO inhibition.

Apoptosis↗
PubMed1996

Rationalization of the strength of metal binding to human serum transferrin.

A wide range of metal ions of natural, therapeutic, diagnostic and toxic interest are transported by serum transferrin (80 kDa). It is therefore important to understand the factors that control the strength of metal binding. We show here that even though Sc3+ has only slightly larger ionic radius than Fe3+ (0.075 nm versus 0.065 nm), it binds to the C-lobe and N-lobe sites much more weakly: logK1* (bicarbonate-independent binding constant) 14.6 +/- 0.2, logK2* 13.3 +/- 0.3, respectively (10 mM Hepes, 5 mM bicarbonate, 310 K). Preferential binding to the C-lobe was established by 1H-NMR spectroscopy. We show that the strength of binding of divalent and trivalent metal ions to human serum transferrin correlates with metal ion acidity [and therefore with the strength of binding to hydroxide, K1(OH)]. The correlations are of predictive value for a range of other metal ions. The plot of logK1* (human serum transferrin) versus logK1(OH) has a negative intercept consistent with unfavorable entropy effects due to lobe closure of apotransferrin on binding of metal ions. This interpretation was tested by comparison with similar correlations of the strength of metal binding to the enzymes carbonic anhydrase and carboxypeptidase with that for the low-M(r) ligand imidazole. These plots have positive intercepts consistent with the preorganized (entatic) state of these metalloenzymes (favorable entropy effects on metal binding).

Binding Sites↗
PubMed1996

Regulation of cyclooxygenases by protein kinase C. Evidence against the importance of direct enzyme phosphorylation.

Cyclooxygenases (COXs) are key prostaglandin biosynthetic enzymes. While COX-1 expression is largely constitutive, COX-2 is highly regulated by cytokines, growth factors, and tumor promoters, such as the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA). While phosphorylation of transcription factors may regulate COX transcription, the existence of PKC consensus sequences suggests that direct enzyme phosphorylation might also regulate differential expression of the enzymes. Nevertheless, phosphorylation of both human recombinant COX-1 and COX-2 by rat brain PKC in vitro was minimal, as was phosphorylation of peptides based on PKC consensus sequences in COX-1 (less than 4% of the phosphorylation of the PKC-alpha pseudosubstrate peptide). Similarly, phosphorylation of the corresponding COX-2 peptides was not observed using either the phosphocellulose paper absorption method or electrospray mass spectrometry. MEG-01 and NIH 3T3 cells were labeled with [32P]orthophosphate to investigate COX phosphorylation in vivo. COX-2 synthesis was induced by PMA (100 nM) or serum stimulation in NIH 3T3 cells. COX-1 was expressed constitutively in MEG-01 cells. Specific polyclonal antibodies raised against sequences of human COX-1 (Ala24-Cys35) and COX-2 (Asn580-Lys598) were used for immunoprecipitation. Neither COX-1 nor COX-2 was phosphorylated in vivo, irrespective of the presence of a phosphatase inhibitor (1 microM okadaic acid). Although COX-1 and COX-2 are differentially regulated, no differences were observed in terms of susceptibility to phosphorylation by PKC either in vitro or in vivo. Despite regulated expression of COX-2 by PMA and the existence of consensus sequences for PKC phosphorylation, it appears that it is an unfavorable substrate for this enzyme.

3T3 Cells↗
PubMed1996

Rates of shutdown of HIV-1 into latency: roles of the LTR and tat/rev/vpu gene region.

CEM T-cells chronically infected with most HIV-1 isolates gradually cease virus production over a 4-6 week period. This is due to slow shutdown of virus replication in the majority of the cells, leading to latent infections. We identified one HIV-1 isolate (HIV213) which shut down into latency at a rate much slower than most HIV strains, requiring more than 12 weeks for the majority of the cells to become nonproductive. This indicated that genes of the virus influence the rate of shutting down, or alternatively, the length of time chronically infected cells produce virus. The viral gene(s) influencing differential rates of shutdown were mapped using chimeric viruses composed of the HIV213 genome substituted with various restriction fragments from HIV(MCK), which rapidly progresses into latency. We found that the 3' region of the LTR was the major determinant influencing the rate of shutdown, but the tat/rev/vpu region also slightly influenced this phenotype. These data show that at least these two genomic regions can influence the duration of virus production in chronically infected cells and that polymorphisms in these regions result in phenotypically divergent viruses which go into latency at different rates. It is also possible that this viral property may be an important determinant of clinical outcomes.

Base Sequence↗
PubMed1996

Crystal structure of a T-cell receptor beta-chain complexed with a superantigen.

Superantigens (SAgs) are viral or bacterial proteins that act as potent T-cell stimulants and have been implicated in a number of human diseases, including toxic shock syndrome, diabetes mellitus and multiple sclerosis. The interaction of SAgs with the T-cell receptor (TCR) and major histocompatibility complex (MHC) proteins results in the stimulation of a disproportionately large fraction of the T-cell population. We report here the crystal structures of the beta-chain of a TCR complexed with the Staphylococcus aureus enterotoxins C2 and C3 (SEC2, SEC3). These enterotoxins, which cause both toxic shock and food poisoning, bind in an identical way to the TCR beta-chain. The complementarity-determining region 2 (CDR2) of the beta-chain and, to lesser extents, CDR1 and hypervariable region 4 (HV4), bind in a cleft between the two domains of the SAgs. Thus, there is considerable overlap between the SAg-binding site and the peptide/MHC-binding sites of the TCR. A model of a TCR-SAg-MHC complex constructed from the crystal structures of (1) the beta-chain-SEC3 complex, (2) a complex between staphylococcal enterotoxin B (SEB) and an MHC molecule, and (3) a TCR V(alpha) domain, reveals that the SAg acts as a wedge between the TCR and MHC to displace the antigenic peptide away from the TCR combining site. In this way, the SAg is able to circumvent the normal mechanism for T-cell activation by specific peptide/MHC complexes.

Amino Acid Sequence↗
PubMed1996

111Cd NMR studies of the domain specificity of Ag+ and Cu+ binding to metallothionein.

Metal displacement reactions of Cd7MT with Ag+ or Cu+ and interprotein metal exchange reactions between Cd7MT and Ag12MT or Cu12MT were studied by 111Cd NMR. Titration of 111Cd7MT with Ag+ indicates that Ag+ binds preferentially to the beta-domain of the protein to form the metal hybrid species, (Cd4)alpha(Ag6)beta MT. Once the beta-domain is filled, additional Ag+ ions displace Cd2+ from the alpha-domain to form (Ag6)alpha(Ag6)beta MT. The metal displacement reaction is cooperative and the two domains react independently of one another. The (Cd4)alpha(Ag6)beta MT hybrid protein is also formed as the major product of direct interprotein metal exchange between Cd7MT and Ag12MT. Cu+ reacts with Cd7MT in a manner similar to Ag+, with addition of 6 equiv of Cu+ leading to preferential formation of (Cd4)alpha(Cu6)beta-MT, and 12 equiv of Cu+ to formation of (Cu6)alpha(Cu6)beta MT. However, unlike Ag+, Cu+ appears to produce intermediate species that may contain mixed-metal clusters. Interprotein metal exchange between Cu12-MT and Cd7MT leads to the net transfer of Cd2+ into the alpha-domain and Cu+ into the beta-domain. The differential affinities of the two domains for monovalent and divalent metal ions plus the availability of facile pathways for metal exchange may be features that enable MT to function simultaneously in the metabolism of different metal ions.

Animals↗
PubMed1996

HPLC characterization of Ag+ and Cu+ metal exchange reactions with Zn- and Cd-metallothioneins.

Anion exchange HPLC methods were developed to study the metal exchange reactions of ZnMT and CdMT with Ag+ and Cu+. The kinetics of these reactions revealed the existence of a slow interprotein metal redistribution process that follows initial rapid displacement of metal from Zn7MT or Cd7MT by Ag+ or Cu+. Kinetically-favored products of the reaction of Ag+ with Zn7MT in the presence of Chelex resin include Zn4Ag6MT and Ag12MT. Subsequent slow reaction between Zn7MT and Ag12MT leads to the thermodynamically-favored product, Zn4Ag6MT. Analogous behavior was observed for Cd7MT titrated with Ag+ and Zn7MT with Cu+. The use of Chelex resin to remove unbound metal ions was found to influence the reactions of Zn7MT and Cd7MT with Ag+. Upon addition of Ag+ to Zn7MT in the absence of Chelex, a Ag18MT species forms as the kinetically-favored product in addition to Zn4Ag6MT and Ag12MT. Subsequently, Ag18MT was found to donate Ag+ to Zn7MT and Zn4Ag6MT via slow interprotein metal exchange reactions. Chelex resin influences the titration reaction either by limiting formation of Ag18MT and/or by binding the positively charged Ag18MT species and removing it from solution. The formation of Ag18MT in the absence of Chelex explains why more than 12 equiv of Ag+ is required to displace all of the Zn2+ or Cd2+ from Zn7MT and Cd7MT. The HPLC methods developed in this study allow homogeneous preparations to be made of metal-hybrid MT species containing Ag+ in the beta-domain and Cd2+ or Zn2+ in the alpha-domain.

Animals↗
PubMed1996

Activation of an interleukin 1 converting enzyme-dependent apoptosis pathway by granzyme B.

Cytotoxic T lymphocytes (CTL) can induce apoptosis through a granzyme B-based killing mechanism. Here we show that in cells undergoing apoptosis by granzyme B, both p45 pro-interleukin 1 beta converting enzyme (ICE) and pro-CPP32 are processed. Using ICE deficient (ICE -/-) mice, embryonic fibroblasts exhibit high levels of resistance to apoptosis by granzyme B or granzyme 3, while B lymphoblasts are granzyme B-resistant, thus identifying an ICE-dependent apoptotic pathway that is activated by CTL granzymes. In contrast, an alternative ICE-independent pathway must also be activated as ICE -/- thymocytes remain susceptible to apoptosis by both granzymes. In ICE -/- B cells or HeLa cells transfected with mutant inactive ICE or Ich-1S that exhibit resistance to granzyme B, CPP32 is processed to p17 and poly(ADP-ribose) polymerase is cleaved indicating that this protease although activated was not associated with an apoptotic nuclear phenotype. Using the peptide inhibitor Ac-DEVD-CHO, apoptosis as well as p45 ICE hydrolysis are suppressed in HeLa cells, suggesting that a CPP32-like protease is upstream of ICE. In contrast, p34cdc2 kinase, which is required for granzyme B-induced apoptosis, remains inactive in ICE -/- B cells indicating it is downstream of ICE. We conclude that granzyme B activates an ICE-dependent cell death pathway in some cell types and requires a CPP32-like Ac-DEVD-CHO inhibitable protease acting upstream to initiate apoptosis.

Animals↗
PubMed1996

Development of T-cell lines expressing functional HIV-1 envelope glycoproteins for evaluation of immune responses in HIV-infected individuals.

The human T-lymphoid cell line, CEM, was transfected with gp 160 cDNA of human immunodeficiency virus type 1 (HIV-1)pm213. Three clones expressing the envelope glycoproteins (env), designated CEM-213env1, -env4, and -env7, were isolated. These clones expressed high levels of surface gp41 and gp120, as demonstrated by flow cytometry with anti-HIV env monoclonal antibodies. Processing and function of env was shown by induction of syncytia with CD4-expressing HeLa cells and by immunoblot analysis. The env expression resulted in specific down-regulation of surface CD4 levels, supporting the role of HIV env in CD4 modulation. Furthermore, serum samples from nine of nine HIV-1-infected individuals bound specifically to the env-expressing transfectants, substantiating the presence of conserved antigenic determinants. These sera also mediated antibody-dependent cellular cytotoxicity (ADCC) of the env-expressing cell lines. The env-expressing cell lines provide a relevant, safe, and practical model for qualitative and quantitative analysis of humoral and cellular immune responses and their role in HIV-1 pathogenesis and therapy.

Blotting, Western↗
PubMed1996

Durable mixed allogeneic chimerism and tolerance by a nonlethal radiation-based cytoreductive approach.

For over 40 years, the association between hemopoietic chimerism and donor-specific tolerance for allografts has been recognized. However, toxicity associated with lethal conditioning has prevented the clinical application of bone marrow (BM) chimerism to induce tolerance. We previously demonstrated that engraftment could be achieved with less than total recipient myeloablation (700 cGy) and that the incidence of engraftment correlated with the dose of total body irradiation (TBI). Administration of cyclophosphamide (CyP) on Day +2 reduced the minimum TBI dose sufficient to permit engraftment to 500 cGy. In the current study, addition of antilymphocyte globulin (ALG) to the TBI/CyP-based conditioning approach reduced the radiation required for engraftment to < or = 300 cGy. B10 (H-2b) mice conditioned with ALG on day -3, 300 cGy of TBI with transplantation of B10.BR (H-2k) or BALB/c (H-2d) BM on day 0, and CyP on day +2 exhibited evidence of donor chimerism (49.6 +/- 3.7% and 38.2 +/- 2.4%, respectively) in 97% of recipients. ALG eliminated CD4+ and CD8+ cells and decreased NK1.1+ cells in the peripheral circulation at the time of transplantation. Moreover, T and NK cells in the host BM were significantly decreased compared with cells of recipients conditioned with TBI alone. CyP delayed repopulation of host thymocytes, providing time for the establishment of donor chimerism before production of mature T cells. Chimeric animals exhibited stable multilineage chimerism and donor-specific tolerance to skin grafts and in in vitro assays. This model may provide a clinically acceptable approach for the induction of donor-specific transplantation tolerance.

Animals↗
PubMed1996

Molecular analysis of stimulatory anti-thyrotropin receptor antibodies (TSAbs) involved in Graves' disease. Isolation and reconstruction of antibody genes, and production of monoclonal TSAbs.

Anti-thyrotropin (TSH) receptor autoantibodies (TRAbs) have been known to be involved in Graves' disease. To understand the molecular mechanism for pathogenesis of TSAbs in Graves' disease, we isolated and reconstituted the Ig genes of EBV-transformed B cell clones producing monoclonal thyroid stimulating Ab (TSAb) obtained from patients with Graves' disease. The V region genes of Ig heavy (H) and light (L) chains of two TSAb clones, IgG clone B6B7 and IgM clone 101-2, were isolated by the PCR. Nucleotide sequencing analysis revealed that germ-line VH and VK segments widely used for autoantibodies including the previously isolated TRAbs were utilized in the two clones. A significant number of somatic mutations were found in V regions of both clones, indicating the involvement of somatic mutations for the TSAb specificity. Reconstituted Ig H and L chain genes of the two clones were stably introduced into myeloma cells for IgG1 production. IgGs purified from cultured supernatants of both transfectants exhibited significant TSAb activities, while they did not inhibit TSH binding to the receptor. The successful expression of recombinant TSAbs in eukaryotic cells will provide opportunities to apply them to various pathophysiologic, diagnostic and therapeutic investigations in autoimmune thyroid diseases.

Antibodies, Monoclonal↗
PubMed1996

Phenobarbital induction mediated by a distal CYP2B2 sequence in rat liver transiently transfected in situ.

The promoter activities of the genes for cytochrome P450 2B1 (CYP2B1) and cytochrome P450 2C1 (CYP2C1) have been assayed by direct injection of promoter-luciferase chimeric genes into rat liver. Activities of minimal promoters for CYP2C1 and CYP2B1 were detectable in untreated animals but were not increased by treatment of the animals with phenobarbital. After insertion to the 5' side of the minimal promoters of one to three copies of the CYP2B2 sequence from -2318 to -2155, a phenobarbital-responsive element in primary hepatocyte cultures (Trottier, E., Belzil, A., Stoltz, C., and Anderson, A. (1995) Gene (Amst.) 158, 263-268), phenobarbital treatment induced the activity of the CYP2C1 promoter by 5-15-fold and the CYP2B1 promoter by 2.5-5-fold. Mutation of a basal transcription element-like motif and a CCAAT/enhancer binding protein element in the CYP2B1 proximal promoter region reduced expression, but 3-4-fold induction by phenobarbital was retained. Mutation of the "Barbie box," a putative phenobarbital-responsive element (He, J.-S., and Fulco, A. J. (1991) J. Biol. Chem. 266, 7864-7869) in the CYP2B1 proximal promoter did not reduce the relative response to phenobarbital. These results demonstrate that direct injection of DNA into rat liver may be used to assay phenobarbital responsiveness of cytochrome P450 genes. In this system, a distal CYP2B2 element mediates a response to phenobarbital, and proximal elements, including the Barbie box, are not required for the induction.

Animals↗
PubMed1996

Identification of cGMP-dependent protein kinase and its specific substrates in the anterior pituitary.

In the anterior pituitary, cGMP is produced in response to a number of stimuli, but intracellular events distal to cGMP production are obscure. Since cGMP-dependent protein kinase (PKG) is a major effector of cGMP actions in other tissues we have determined whether PKG and its specific substrates might be present and responsive to external signals in the ovine anterior pituitary. Photoaffinity labelling with [32P]cGMP revealed a specific 78 kDa protein in ovine anterior pituitary that comigrated with purified bovine lung PKG-I. PKG in protein extracts from anterior pituitary or cultured anterior pituitary cells was enriched by DEAE ion-exchange chromatography and assayed for activity. Both tissue and cultured cells had a relatively high PKG activity by comparison with aortic smooth muscle (known high activity) and brain (known low activity). Subcellular distribution studies showed that in anterior pituitary, aortic and brain, PKG activity was present in both cytosol and triton-extracted membrane fractions, while in platelets the activity was associated with only the membrane fraction. To determine if this PKG might be responsive to extracellular signals an activity ratio assay was used. Incubation of cultured cells with atrial natriuretic peptide (ANP) and sodium nitroprusside, activators of membrane and cytosolic guanylate cyclases respectively, increased the activity of PKG. To determine events distal to PKG activation, a search for potential substrates of PKG was performed. Few substrates were detectable upon addition of purified PKG to tissue lysates due to the high background activity of endogenous protein kinases in the anterior pituitary. However, 19 substrates of PKG were detected in heat-stable and 14 in acid-soluble protein extracts of the anterior pituitary, in which background phosphorylation was almost abolished. After partial purification through Q-Sepharose ion-exchange chromatography some of these proteins were preferentially phosphorylated by addition of PKG-I, while the others were additionally substrates of exogenous cAMP-dependent protein kinase (PKA) or Ca2+ and phospholipid-dependent protein kinase (PKC). A 132-kDa substrate showed an identical phosphopeptide map to a PKG substrate previously described in vascular smooth muscle and platelets. These data demonstrate for the first time the presence of functional PKG activity and multiple PKG substrates in the anterior pituitary where they may play a role in mediating the intracellular actions of cGMP.

Affinity Labels↗
PubMed1996

(-)-Epigallocatechin gallate, a polyphenolic tea antioxidant, inhibits peroxynitrite-mediated formation of 8-oxodeoxyguanosine and 3-nitrotyrosine.

Reaction with peroxynitrite at pH 7.4 and 37 degrees C was found to increase the 8-oxodeoxyguanosine levels in calf thymus DNA 35- 38-fold. This oxidation of deoxyguanosine, as well as the peroxynitrite-mediated nitration of tyrosine to 3-nitrotyrosine, was significantly inhibited by ascorbic acid, glutathione and (-)-epigallocatechin gallate, a polyphenolic antioxidant present in tea. For 50% inhibition of the oxidation of deoxyguanosine to 8-oxodeoxyguanosine, 1.1, 7.6 or 0.25 mM ascorbate, glutathione or (-)-epigallocatechin gallate, respectively, was required. For 50% inhibition of tyrosine nitration, the respective concentrations were 1.4, 4.6 or 0.11 mM. Thus, (-)-epigallocatechin gallate is a significantly better inhibitor of both reactions than either ascorbate or glutathione. Reaction of (-)-epigallocatechin gallate with peroxynitrite alone resulted in the formation of a number of products. Ultraviolet spectra of two of these suggest that the tea polyphenol and/or its oxidation products are nitrated by peroxynitrite.

8-Hydroxy-2'-Deoxyguanosine↗