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Biomedical subjects

X M Yin

Publications and source records attributed to X M Yin.

At least 37 records · Page 2Linked to original sources
PubMed1999

Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while BCL-XL prevents this release but not tumor necrosis factor-R1/Fas death.

"BH3 domain only" members of the BCL-2 family including the pro-apoptotic molecule BID represent candidates to connect with proximal signal transduction. Tumor necrosis factor alpha (TNFalpha) treatment induced a caspase-mediated cleavage of cytosolic, inactive p22 BID at internal Asp sites to yield a major p15 and minor p13 and p11 fragments. p15 BID translocates to mitochondria as an integral membrane protein. p15 BID within cytosol targeted normal mitochondria and released cytochrome c. Immunodepletion of p15 BID prevents cytochrome c release. In vivo, anti-Fas Ab results in the appearance of p15 BID in the cytosol of hepatocytes which translocates to mitochondria where it releases cytochrome c. Addition of activated caspase-8 to normal cytosol generates p15 BID which is also required in this system for release of cytochrome c. In the presence of BCL-XL/BCL-2, TNFalpha still induced BID cleavage and p15 BID became an integral mitochondrial membrane protein. However, BCL-XL/BCL-2 prevented the release of cytochrome c, yet other aspects of mitochondrial dysfunction still transpired and cells died nonetheless. Thus, while BID appears to be required for the release of cytochrome c in the TNF death pathway, the release of cytochrome c may not be required for cell death.

Amino Acid Sequence↗
PubMed1998

BID, a proapoptotic BCL-2 family member, is localized to mouse chromosome 6 and human chromosome 22q11.

BID is a proapoptotic member of the BCL-2 family of cell death regulators. BID shares sequence homology with other members of the family within a single alpha-helical domain, BH3. BH3 is required for BID to interact with BCL-2 and BAX, as well as for its function as a death agonist. We have isolated and characterized mouse Bid and human BID genomic clones. The sequence for BID is encoded within five exons. We used interspecific backcross analysis to localize Bid to the distal region of mouse chromosome 6 near the Atp6e locus. Fluorescence in situ hybridization analysis localized human BID to a syntenic human region, chromosome 22q11, close to the BCR-1 gene.

Animals↗
PubMed1997

Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic BCL-2.

The BCL-2 family of proteins is composed of both pro- and antiapoptotic regulators, although its most critical biochemical functions remain uncertain. The structural similarity between the BCL-XL monomer and several ion-pore-forming bacterial toxins has prompted electrophysiologic studies. Both BAX and BCL-2 insert into KCl-loaded vesicles in a pH-dependent fashion and demonstrate macroscopic ion efflux. Release is maximum at approximately pH 4.0 for both proteins; however, BAX demonstrates a broader pH range of activity. Both purified proteins also insert into planar lipid bilayers at pH 4.0. Single-channel recordings revealed a minimal channel conductance for BAX of 22 pS that evolved to channel currents with at least three subconductance levels. The final, apparently stable BAX channel had a conductance of 0.731 nS at pH 4. 0 that changed to 0.329 nS when shifted to pH 7.0 but remained mildly Cl- selective and predominantly open. When BAX-incorporated lipid vesicles were fused to planar lipid bilayers at pH 7.0, a Cl--selective (PK/PCl = 0.3) 1.5-nS channel displaying mild inward rectification was noted. In contrast, BCL-2 formed mildly K+-selective (PK/PCl = 3.9) channels with a most prominent initial conductance of 80 pS that increased to 1.90 nS. Fusion of BCL-2-incorporated lipid vesicles into planar bilayers at pH 7.0 also revealed mild K+ selectivity (PK/PCl = 2.4) with a maximum conductance of 1.08 nS. BAX and BCL-2 each form channels in artificial membranes that have distinct characteristics including ion selectivity, conductance, voltage dependence, and rectification. Thus, one role of these molecules may include pore activity at selected membrane sites.

Animals↗
PubMed1996

BID: a novel BH3 domain-only death agonist.

The BCL-2 family of proteins consists of both antagonists (e.g., BCL-2) and agonists (e.g., BAX) that regulate apoptosis and compete through dimerization. The BH1 and BH2 domains of BCL-2 are required to heterodimerize with BAX and to repress cell death; conversely, the BH3 domain of BAX is required to heterodimerize with BCL-2 and to promote cell death. To extend this pathway, we used interactive cloning to identify Bid, which encodes a novel death agonist that heterodimerizes with either agonists (BAX) or antagonists (BCL-2). BID possesses only the BH3 domain, lacks a carboxy-terminal signal-anchor segment, and is found in both cytosolic and membrane locations. BID counters the protective effect of BCL-2. Moreover, expression of BID, without another death stimulus, induces ICE-like proteases and apoptosis. Mutagenesis revealed that an intact BH3 domain of BID was required to bind the BH1 domain of either BCL-2 or BAX. A BH3 mutant of BID that still heterodimerized with BCL-2 failed to promote apoptosis, dissociating these activities. In contrast, the only BID BH3 mutant that retained death promoting activity interacted with BAX, but not BCL-2. This BH3-only molecule supports BH3 as a death domain and favors a model in which BID represents a death ligand for the membrane-bound receptor BAX.

Amino Acid Chloromethyl Ketones↗
PubMed1995

Reactive oxygen species and the regulation of cell death by the Bcl-2 gene family.

The maintenance of homeostasis in normal tissues reflects a balance between cell proliferation and cell death. Bcl-2 inaugurated a new category of oncogenes, regulators of cell death. The Bcl-2 gene was identified at the chromosomal breakpoint of t(14;18) bearing B cell lymphomas. Bcl-2 proved unique by blocking programmed cell death rather than promoting proliferation. In adults, Bcl-2 is topographically restricted to progenitor cells and longlived cells but is much more widespread in the developing embryo. Transgenic mice that overexpress Bcl-2 demonstrate extended cell survival, and progress to high grade lymphomas. Bcl-2 has been localized to mitochondria, endoplasmic reticulum and nuclear membranes, also the sites of reactive oxygen species generation. Bcl-2 does not appear to influence the generation of oxygen free radicals but does prevent oxidative damage to cellular constituents including lipid membranes. Bcl-2 deficient mice complete embryonic development but undergo fulminant lymphoid apoptosis of thymus and spleen. Moreover, they demonstrate two unexpected pathologies resulting from cell death, polycystic kidney disease and hair hypopigmentation. The latter is a potential oxidant injury from the melanin biosynthetic pathway. A family of Bcl-2 related genes is emerging that includes Bax, a conserved homolog that heterodimerizes in vivo with Bcl-2 and promotes cell death. The ratio of family members, such as Bcl-2/Bax, determines the survival or death of cells following an apoptotic stimulus.

Amino Acid Sequence↗
PubMed1994

BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax.

Bcl-2 was isolated from the t(14;18) chromosomal breakpoint in follicular B-cell lymphoma. Bcl-2 has the unique oncogenic role of extending cell survival by inhibiting a variety of apoptotic deaths. An emerging family of Bcl-2-related proteins share two highly conserved regions referred to here as Bcl-2 homology 1 and 2 (BH1 and BH2) domains (Fig. 1). This includes Bax which heterodimerizes with Bcl-2 and when overexpressed counteracts Bcl-2. We report here that site-specific mutagenesis of Bcl-2 establishes the two domains as novel dimerization motifs. Substitution of Gly 145 in BH1 domain or Trp 188 in BH2 domain completely abrogated Bcl-2's death-repressor activity in interleukin-3 deprivation, gamma-irradiation and glucocorticoid-induced apoptosis. Mutations that affected Bcl-2's function also disrupted its heterodimerization with Bax, yet still permitted Bcl-2 homodimerization. These results establish a functional role for the BH1 and BH2 domains and suggest Bcl-2 exerts its action through heterodimerization with Bax.

Amino Acid Sequence↗
PubMed1993

Bcl-2 functions in an antioxidant pathway to prevent apoptosis.

Bcl-2 inhibits most types of apoptotic cell death, implying a common mechanism of lethality. Bcl-2 is localized to intracellular sites of oxygen free radical generation including mitochondria, endoplasmic reticula, and nuclear membranes. Antioxidants that scavenge peroxides, N-acetylcysteine and glutathione peroxidase, countered apoptotic death, while manganese superoxide dismutase did not. Bcl-2 protected cells from H2O2- and menadione-induced oxidative deaths. Bcl-2 did not prevent the cyanide-resistant oxidative burst generated by menadione. Two model systems of apoptosis showed no increment in cyanide-resistant respiration, and generation of endogenous peroxides continued at an inherent rate that was unaltered by Bcl-2. Following an apoptotic signal, cells sustained progressive lipid peroxidation. Overexpression of Bcl-2 functioned to suppress lipid peroxidation completely. We propose a model in which Bcl-2 regulates an antioxidant pathway at sites of free radical generation.

Acetylcysteine↗
PubMed1992

The lineage relationship between virgin and memory B cells.

J11d is expressed on a variety of hemopoietic cells, including B cells. In previous studies, we have shown that B cells are heterogeneous with respect to their levels of J11d expression and that memory B cells are contained within the J11dlo B cell population. In this report, we have further examined the relationship between memory precursors and memory cells. We have found that the ability of J11dlo B cells from unprimed mice to respond to primary antigenic stimulation was generally low; the lowest response occurred following immunization with soluble antigen in vitro. Following adoptive transfer into irradiated mice and repeated stimulation of these mice with antigen, J11dlo cells generated memory responses which were similar to those generated by J11dlo memory cells obtained from primed mice. In contrast, J11dhi B cells from unprimed mice gave rise to both primary and memory responses, but the memory responses were lower than those generated by J11dlo cells. Although the expression of J11d on both J11dhi and J11dlo cells from unprimed mice decreased after adoptive transfer, J11dlo, as compared to J11dhi, virgin cells proliferated more rapidly. These results suggest that memory responses are elaborated predominantly from J11dlo virgin cells. Taken together with previous results, our studies indicate that, J11dlo B cells are different from J11dhi B cells. Hence, they are either memory precursors or are memory cells.

Animals↗
PubMed1992

[Antagonistic effect of electro-acupuncture analgesia with Ca2+ injection into habenula could be reversed by gallamine triethiodide].

.1 mol/L CaCl2 0.5 microliters, 0.06 mol/L ACh 0.5 microliters, 5.4 x 10(-3) mol/L gallamine triethiodide (cholinergic nicotinic receptor blocker) 0.5 microliter and 14.4 x 10(-3) mol/L atropine (cholinergic muscarinic receptor blocker) 0.5 microliter were injected through bilateral intracranial cannulae in rat habenula. Pain threshold was measured by the latency of tail-flick reflex elicited by radiant heat exposure before and after intracerebral injection. CaCl2 significantly reduced the basic pain threshold and weakened the effect of the acupuncture analgesia. ACh apparently antagonized the effect of acupuncture analgesia. Gallamine triethiodide could recover the pain threshold almost to the raised level by acupuncture, but atropine only strengthened the effect on pain threshold weakly and briefly. The results suggest that the antagonistic effect of Ca2+ may be mediated via ACh in habenula.

Acupuncture Analgesia↗
PubMed1991

Changes in expression of J11d on murine B cells during activation and generation of memory.

J11d is an antigen expressed on a variety of hemopoietic cells including B cells. In this report we describe the expression of J11d on functional subpopulations of B cells from adult mice of different ages or at different stages of an immune response. In addition we explore the relationship between the expression of J11d and that of other markers characteristic of memory B cells. Our results show that expression of J11d on B cells is related to the immunologic history of the host. Thus, J11d on B cells from normal mice is decreased as the mice age and are thereby exposed to environmental antigens. Down-regulation of J11d on B cells does not occur in germfree or nude mice but does occur when nude mice are reconstituted with T cells and/or are immunized. Since B cells expressing low levels of J11d accumulate following exposure to antigens, we also determined whether memory cells reside in the J11dlo cell population. To this end, we studied primed mice and found that expression of J11d on antigen-specific B cells increased during the first week after immunization and decreased thereafter. Increases in expression of J11d after activation also occurred in B cells stimulated with lipopolysaccharide (LPS) in vitro. However, antigen-specific B cells from long-term primed mice which expressed lower levels of J11d and antigen-specific receptors of higher affinity also expressed lower densities of sIgM and included most sIgG+ cells. Furthermore, these antigen-specific J11dlo B cells transferred a memory response, whereas J11dhi cells did not. Taken together, these data indicate that the level of expression of J11d on B cells distinguishes virgin (J11dhi) from memory (J11dlo) cells in mice.

Age Factors↗
PubMed1991

IL-4 down-regulates the expression of J11d on murine B cells.

T cell-derived IL-4 has many effects on murine B cells, including the up-regulation of class II antigens and the induction of isotype switching. The development of memory B cells and the decreased expression of J11d antigens on these cells are influenced by T cells. In this report, we determined whether the decreased expression of J11d can also be mediated by T cell-derived lymphokines and, in particular, IL-4. We found that IL-4 can down-regulate the expression of J11d on both large and small B cells, that this effect becomes significant after 48 hr of culture and occurs at doses of IL-4 that are similar to those required to up-regulate murine class II MHC antigens encoded by I-region alpha genes (IA). Anti-IL-4 antibody completely blocks this effect but IFN-gamma does not. Other lymphokines (IL-1, IL-2, IL-3, IL-5, IL-6) neither induce a decrease in J11d nor alter the ability of IL-4 to down-regulate J11d expression. The decrease in J11d expression on B cells is not due to a preferential survival of cells expressing lower levels of J11d, although IL-4 has a more pronounced effect on these B cells. Finally, the down-regulation of J11d and up-regulation of IA by IL-4 occurs on all inducible B cells.

Animals↗
PubMed1991

Memory B and T cells.

Three remarkable and unique features of the immune system are specificity, diversity, and memory. Immunological memory involves both T and B cells and results in a secondary antibody response that is faster, of higher affinity, and results in the secretion of non-IgM isotypes of Ig. In this review we discuss the properties of memory T and B cells, their specific receptors, and the events which occur both in the nucleus and on the cell surface during generation and activation of these cells. Although memory T and B cells use different mechanisms to elaborate memory, there are a number of interesting analogies: lymphokines vs antibodies and affinity maturation of B cell antigen receptors vs upregulation of adhesion molecules on T cells. Finally, we discuss the importance of these cells in health and disease and suggest what impact additional information about these cells might have on the manipulation of the immune response.

Animals↗
PubMed1990

Functional and ontogenetic analysis of murine CD45Rhi and CD45Rlo CD4+ T cells.

CD4+ murine T cell clones, TH1 and TH2, can be distinguished by both functional responses and by their patterns of lymphokine secretion. Recently, a mAb, 23G2, which reacts with a subset of CD45 molecules (CD45R), has been reported to bind differentially to clones of TH1 and TH2 cells. In the present study, normal splenic T cells were analyzed for differences in 23G2-reactivity and were separated into two populations based on their density of CD45R (CD45Rhi and CD45Rlo). The CD45Rhi cells secrete more IL-2 than IL-4 after stimulation in vitro; the reverse is true for the CD45Rlo cells. Because neither population secretes only IL-2 or IL-4, we were unable to classify cells as TH1 or TH2. In vivo and in vitro analyses of the CD45Rhi and CD45Rlo cells suggest a lineage relationship between the two subsets that correlates with the degree of Ag exposure and the state of maturation of the mice. In newborn mice and mice raised under sterile conditions, splenic CD4+ T cells are predominantly CD45Rhi. Under conditions of increased antigenic exposure and maturation of the mice, CD45Rlo cells develop; after long term priming in vivo, the majority of specific Ag-reactive cells are CD45Rlo. Adoptive transfer studies using BALB/c nu/nu recipients demonstrate that CD45Rhi cells become CD45Rlo cells and that the recall response (IgG) to specific Ag is mediated by CD45Rlo cells. Taken together, these data indicate that the level of expression of CD45R on CD4+ T cells distinguishes virgin (CD45Rhi) from primed/memory (CD45Rlo) T cells in normal mice.

Animals↗
PubMed1989

Protection analysis (or "footprinting") of specific protein-DNA complexes in crude nuclear extracts using methidiumpropyl-EDTA-iron (II).

Endonuclease protection or "footprinting" analysis is a powerful technique for identifying the nucleotides involved in a protein-DNA interaction. DNase I is the most often employed endonucleolytic agent; however, this endonuclease does not exhibit the true nonsequence-specific cleavage desired for this type of analysis. Methidiumpropyl-EDTA (MPE) is a synthetic DNA intercalator that cleaves DNA in the presence of ferrous ion and oxygen. Cleavage by MPE exhibits no sequence specificity, a characteristic that makes this reagent better suited for protection analysis. Here we report a generally applicable technique for MPE protection (or "footprinting") analysis of specific DNA-protein complexes from a crude nuclear extract. We have used this method to identify the nucleotides of the immunoglobulin (Ig) heavy chain promoter region that are involved in complex formation with a protein that binds the octameric sequence ATGCAAAT, and we compare our results to those obtained previously using DNase I.

Base Sequence↗
PubMed1988

A conserved heptamer upstream of the IgH promoter region octamer can be the site of a coordinate protein-DNA interaction.

Immunoglobulin genes contain a conserved eight base sequence element 5' to the site of transcription initiation. This octamer can serve as a site for the binding of nuclear proteins which are presumably involved in the cell type specific expression of this family of genes. In studying the binding of nuclear proteins to this conserved sequence element, we have detected a protein interaction that involves, in addition to the octamer, nucleotides which are immediately upstream. We have characterized this additional contact as a sequence specific interaction with a heptameric sequence element (CTCATGA) that is conserved among Ig heavy chain promoters. Protein binding to the heptamer is unique in that it is dependent upon the proximity and orientation of, as well as protein interaction with, the conserved octamer.

Base Sequence↗