Search PubMed⌕ Search

Biomedical subjects

H Li

Publications and source records attributed to H Li.

At least 19 recordsLinked to original sources
PubMed1997

Production of the novel C-C chemokine MCP-4 by airway cells and comparison of its biological activity to other C-C chemokines.

Monocyte chemotactic protein-4 (MCP-4) is a newly identified C-C chemokine with potent eosinophil chemoattractant properties. We describe studies of its biological activity in vitro to induce chemotaxis of peripheral blood eosinophils and to induce histamine release from IL-3-primed peripheral blood basophils. MCP-4 and eotaxin caused a similar rise in eosinophil intracytoplasmic Ca2+ and complete cross-desensitization. MCP-4 also abolished the eosinophil Ca2+ response to MCP-3 and partially desensitized the response to macrophage inflammatory protein-1alpha. MCP-4 activated cell migration via either CCR2b or CCR3 in mouse lymphoma cells transfected with these chemokine receptors. MCP-4 inhibited binding of 125I-eotaxin to eosinophils and CCR3-transfected cells and inhibited 125I-MCP-1 binding to CCR2b-transfectants. MCP-4 mRNA was found in cells collected in bronchoalveolar lavage of asthmatic and nonasthmatic subjects and was prominently expressed in human lung and heart. MCP-4 mRNA was expressed in several human bronchial epithelial cell lines after cytokine stimulation. Pretreatment of BEAS-2B epithelial cells with the glucocorticoid budesonide inhibited MCP-4 mRNA expression. These features make MCP-4 a candidate for playing a role in eosinophil recruitment during allergic respiratory diseases.

Amino Acid Sequence↗
PubMed1997

Patterns of hemopoietic reconstitution in nonobese diabetic mice: dichotomy of allogeneic resistance versus competitive advantage of disease-resistant marrow.

Complete replacement of the immune system via allogeneic bone marrow transplantation is sufficient to prevent diabetes in the nonobese diabetic (NOD) mouse model. In the present study we examined whether mixed allogeneic reconstitution would be sufficient to interrupt the autoimmune process with respect to occurrence of overt diabetes, as well as preexisting autoimmune insulitis. NOD mice were lethally irradiated and reconstituted with a mixture of NOD and B10.BR marrow. A relative resistance to allogeneic bone marrow engraftment was noted in NOD recipients of the mixed bone marrow inoculum, compared with disease-resistant controls. Moreover, unlike disease-resistant controls, all animals that initially repopulated as mixed donor/host chimeras became predominantly allogeneic by 4 mo, suggesting a competitive advantage for long term engraftment for disease-resistant marrow. All but one mouse in the group that engrafted with allogeneic marrow remained free of diabetes for the entire follow-up period (n = 22). Moreover, in all animals examined, virtually all islets were free of insulitis. In contrast, 74% of NOD mice that received similar conditioning and failed to engraft with donor marrow developed acute diabetes and intra-islet insulitis was present in all animals examined. These data suggest that NOD mice exhibit a relative resistance to engraftment compared with disease-resistant recipients. Conversely, animals that initially repopulated as a mixture of syngeneic and donor marrow become converted to virtually all donor by 4 mo. These data provide additional support that a defective stem cell is responsible for autoimmune diabetes in this experimental model.

Animals↗
PubMed1997

PL48: a novel gene associated with cytotrophoblast and lineage-specific HL-60 cell differentiation.

The cDNA for a novel gene, PL48, isolated by subtractive hybridization between undifferentiated human term cytotrophoblast and differentiating cytotrophoblast, has been cloned and sequenced. PL48 contains an open reading frame coding for a 537-amino acid protein, has multiple potential PKC, casein kinase II, and cAMP/cGMP-dependent kinase phosphorylation sites, and N-linked glycosylation sites. It is not present in a wide variety of proliferating cancer cells, but PL48 mRNA shows marked expression during cytotrophoblast and granulocyte lineage-specific HL-60 promyelocytic cell differentiation induced by DMSO.

Amino Acid Sequence↗
PubMed1997

Purification, visualization, and biophysical characterization of Kv1.3 tetramers.

The voltage-gated K+ channel of T-lymphocytes, Kv1.3, was heterologously expressed in African Green Monkey kidney cells (CV-1) using a vaccinia virus/T7 hybrid expression system; each infected cell exhibited 10(4) to 5 x 10(5) functional channels on the cell surface. The protein, solubilized with detergent (3-[cholamidopropyl)dimethylammonio]-1-propanesulfonic acid or cholate), was purified to near-homogeneity by a single nickel-chelate chromatography step. The Kv1.3 protein expressed in vaccinia virus-infected cells and its purified counterpart are both modified by a approximately 2-kDa core-sugar moiety, most likely at a conserved N-glycosylation site in the external S1-S2 loop; absence of the sugar does not alter the biophysical properties of the channel nor does it affect expression levels. Purified Kv1.3 has an estimated size of approximately 64 kDa in denaturing SDS-polyacrylamide electrophoresis gels, consistent with its predicted size based on the amino acid sequence. By sucrose gradient sedimentation, purified Kv1.3 is seen primarily as a single peak with an approximate mass of 270 kDa, compatible with its being a homotetrameric complex of the approximately 64-kDa subunits. When reconstituted in the presence of lipid and visualized by negative-staining electron microscopy, the purified Kv1.3 protein forms small crystalline domains consisting of tetramers with dimensions of approximately 65 x 65 A. The center of each tetramer contains a stained depression which may represent the ion conduction pathway. Functional reconstitution of the Kv1.3 protein into lipid bilayers produces voltage-dependent K+-selective currents that can be blocked by two high affinity peptide antagonists of Kv1.3, margatoxin and stichodactylatoxin.

Animals↗
PubMed1997

The human homolog of the rodent immediate early response genes, PC4 and TIS7, resides in the lung cancer tumor suppressor gene region on chromosome 3p21.

Recently, human chromosome band 3p21.3 was shown to undergo overlapping homozygous deletions in several small cell lung cancer lines further defining a putative tumor suppressor gene(s) region. We report the cloning and mutational analysis of a novel human gene, SKMc15, from the commonly homozygously deleted region in three small cell lung cancer lines (NCI-H1450, NCI-H740, GLC20). It has 11 exons ranging in size from 50 to 541 bp with an open reading frame of 442 amino acids. The gene covers 7 to 10 kb of genomic DNA; the message of 1.8 to 2 kb is expressed in all analyzed fetal and adult human and mouse tissues including heart, brain, placenta, lung liver, skeletal muscle, kidney, testis and pancreas and in small cell and non-small cell cancer lines. The intron/exon boundaries were used to analyze the gene for mutations by exon PCR-SSCP sequencing in 60 small cell lung cancer cell lines. No loss-of-function mutations were detected. The cDNA sequence has high homology, 75% at the protein level, to the rat early response gene PC4 and its murine homolog TIS7. In addition, the known partial sequence of the putative mouse interferon beta2 (64 amino acids) gene is highly conserved in PC4/TIS7 (94%) and in SKMc15 (83%) at the amino acid level. The sequence TAAAT, which is thought to be involved in mRNA degradation, is present in the 3' UTR of SKMc15 and in the 3' UTR of PC4 and TIS7 genes.

Amino Acid Sequence↗
PubMed1997

Peripheral benzodiazepine receptor in cholesterol transport and steroidogenesis.

Steroidogenesis begins with the metabolism of cholesterol to pregnenolone by the inner mitochondrial membrane cytochrome P450 side-chain cleavage (P450scc) enzyme. The rate of steroid formation, however, depends on the rate of cholesterol transport from intracellular stores to the inner mitochondrial membrane and loading of P450scc with cholesterol. In previous in vitro studies, we demonstrated that a key element in the regulation of cholesterol transport is the mitochondrial peripheral-type benzodiazepine receptor (PBR). We also showed that the polypeptide diazepam binding inhibitor (DBI), an endogenous PBR ligand, stimulates cholesterol transport and promotes loading of cholesterol to P450scc in vitro, and that its presence is vital for hCG-induced steroidogenesis by Leydig cells. Based on these data and the observations that i) the mitochondrial PBR binding and topography are regulated by hormones; ii) the 18-kDa PBR protein is functionally coupled to the mitochondrial contact site voltage-dependent anion channel protein; iii) the 18-kDa PBR protein is a channel for cholesterol, as shown by molecular modeling and in vitro reconstitution studies; iv) targeted disruption of the PBR gene in steroidogenic cells dramatically reduces the ability of the cells to transport cholesterol in the mitochondria and produce steroids; v) endocrine disruptors, with known anisteroidogenic effect, inhibit PBR ligand binding; and vi) in vivo reduction of adrenal PBR expression results in reduced circulating glucocorticoid levels, we conclude that PBR is an indispensable element of the steroidogenic machinery.

Animals↗
PubMed1997

The structure of the cytochrome p450BM-3 haem domain complexed with the fatty acid substrate, palmitoleic acid.

The substrate-bound structures of two cytochrome P450s, P450cam and P450eryF, are known. While these structures reveal important features that control substrate specificity, the problem of how conformational changes allow for substrate entry and product release remains unsolved. The structure of the haem domain of the bacterial fatty acid hydroxylase, P450BM-3, previously was solved in the substrate-free form. Unlike the substrate-bound P450cam and P450eryF structures, the substrate access channel is open in substrate-free P450BM-3. Here we present the X-ray structure of P450BM-3 at 2.7 A bound with a fatty acid substrate, palmitoleic acid. A comparison of the substrate-bound and -free forms reveals major conformational differences and provides the first detailed picture of substrate-induced conformational changes in a P450.

Amino Acid Sequence↗
PubMed1997

Nitrate-reducing bacteria on rat tongues.

Nitrite-producing bacteria (NPB) were isolated from tongues of laboratory rats. The most commonly found nitrite-producing organism was Staphylococcus sciuri, followed by Staphylococcus intermedius, Pasteurella spp., and finally Streptococcus spp. Both morphometric quantification of bacteria on tongue sections and enumeration of culturable bacteria (CFU) showed an increase in the density of bacteria towards the posterior tongue. Up to 65% of bacteria were located in the deep clefts on the posterior tongue. The proportion of culturable NPB in the total culturable microbial population increased from 6% (10(5) CFU cm-2) on the anterior tongue to 65% (10(7) CFU cm-2) on the posterior tongue. Different species compositions of NPB were found on different tongue sections with S. intermedius populations decreasing and S. sciuri and Pasteurella populations increasing towards the posterior tongue. Nitrite production was sensitive to oxygen, and significant nitrite production was only detected on the posterior tongue where the majority of bacteria are situated in deep clefts in the tongue surface. This study suggests the importance of bacteria in nitrite production, from nitrate, on the tongue. Nitrite produced on the tongue may subsequently form nitric oxide in the acidic environment of the stomach. Because of the antimicrobial properties of nitric oxide, a key role for nitrate-reducing tongue bacteria in host animal defense against food-borne pathogens in proposed.

Animals↗
PubMed1997

Relationship of Treponema denticola periplasmic flagella to irregular cell morphology.

Treponema denticola is an anaerobic, motile, oral spirochete associated with periodontal disease. We found that the periplasmic flagella (PFs), which are located between the outer membrane sheath and cell cylinder, influence its morphology in a unique manner. In addition, the protein composition of the PFs was found to be quite complex and similar to those of other spirochetes. Dark-field microscopy revealed that most wild-type cells had an irregular twisted morphology, with both planar and helical regions, and a minority of cells had a regular right-handed helical shape. High-voltage electron microscopy indicated that the PFs, especially in those regions of the cell which were planar, wrapped around the cell body axis in a right-handed sense. In those regions of the cell which were helical or irregular, the PFs tended to lie along the cell axis. The PFs caused the cell to form the irregular shape, as two nonmotile, PF-deficient mutants (JR1 and HL51) were no longer irregular but were right-handed helices. JR1 was isolated as a spontaneously occurring nonmotile mutant, and HL51 was isolated as a site-directed mutant in the flagellar hook gene flgE. Consistent with these results is the finding that wild-type cells with their outer membrane sheath removed were also right-handed helices similar in shape to JR1 and HL51. Purified PFs were analyzed by two-dimensional gel electrophoresis, and several protein species were identified. Western blot analysis using antisera to Treponema pallidum PF proteins along with N-terminal amino acid sequence analysis indicated T. denticola PFs are composed of one class A sheath protein of 38 kDa (FlaA) and three class B proteins of 35 kDa (FlaB1 and FlaB2) and one of 34 kDa (FlaB3). The N-terminal amino acid sequences of the FlaA and FlaB proteins of T. denticola were most similar to those of T. pallidum and Treponema phagedenis. Because these proteins were present in markedly reduced amounts or were absent in HL51, PF synthesis is likely to be regulated in a hierarchy similar to that found for flagellar. synthesis in other bacteria.

Amino Acid Sequence↗
PubMed1997

Regulation of rat testis gonocyte proliferation by platelet-derived growth factor and estradiol: identification of signaling mechanisms involved.

To determine what factors regulate gonocyte proliferation in newborn rats, we first examined the expression of several signal transduction molecules by immunocytochemistry in 3-day-old rat testis sections. We found that gonocytes specifically expressed the iota and zeta isoforms of protein kinase (PK) C (PKC) and the phosphatidylinositol 3-kinase (PI3-K). Because both the zeta PKC and PI 3-K have been shown to play a role in platelet-derived growth factor (PDGF)-induced cell proliferation, we examined the effects of PDGF on gonocytes. For this, we developed a method to obtain highly purified and viable gonocytes in culture. After enzymatic digestion, differential adhesion, and two successive gradient fractionations, the gonocyte suspension obtained was over 90% pure, as assessed by light microscopy. The viability of cultured gonocytes exceeded 90% after 48 h in the presence of 2.5% FBS used as a survival factor. Immunodetection studies showed that isolated gonocytes expressed zeta PKC, PI 3-K, and the PDGF receptor. Treatment with 10 ng/ml PDGF induced a 4-fold increase of bromodeoxyuridine incorporation into gonocytes (from 5% proliferative gonocytes under basal conditions to 20% in the presence of PDGF). Because neonatal Sertoli cells secrete high levels of the growth promoting steroid, 17 beta-estradiol, we also tested its effect and found that it induced gonocyte proliferation at a level comparable with that of PDGF and that this effect was blocked by the estrogen receptor antagonist, ICI 164384. The combination of PDGF and estradiol, however, was not additive, suggesting that their effects were mediated by common molecular target(s). These results demonstrate that PDGF and estradiol activate gonocyte proliferation in vitro, suggesting that they may act as the physiological regulators of gonocyte development in vivo.

Animals↗
PubMed1997

A single morphogenetic field gives rise to two retina primordia under the influence of the prechordal plate.

Two bilaterally symmetric eyes arise from the anterior neural plate in vertebrate embryos. An interesting question is whether both eyes share a common developmental origin or they originate separately. We report here that the expression pattern of a new gene ET reveals that there is a single retina field which resolves into two separate primordia, a suggestion supported by the expression pattern of the Xenopus Pax-6 gene. Lineage tracing experiments demonstrate that retina field resolution is not due to migration of cells in the median region to the lateral parts of the field. Removal of the prechordal mesoderm led to formation of a single retina both in chick embryos and in Xenopus explants. Transplantation experiments in chick embryos indicate that the prechordal plate is able to suppress Pax-6 expression. Our results provide direct evidence for the existence of a single retina field, indicate that the retina field is resolved by suppression of retina formation in the median region of the field, and demonstrate that the prechordal plate plays a primary signaling role in retina field resolution.

Amino Acid Sequence↗
PubMed1997

RNA apatamers for yeast ribosomal protein L32 have a conserved purine-rich internal loop.

Two in vitro selection experiments were conducted to determine the RNA sequence requirements for binding ribosomal protein L32 (RPL32) from Saccharomyces cerevisiae. To preserve the wild-type stem-internal loop-stem fold, only a limited portion of the RNA comprising the internal loop region was randomized. Most of the selected RNAs have secondary structures similar to that of the wild-type, and four purines on both sides of the internal loop are highly conserved. Indeed, a pair of 5'-GA-3' dinucleotides is found in all but one of the stem-loop-stem L32 aptamers and these conserved purines may contact the protein directly or form a necessary RNA secondary or tertiary structure. These aptamers have a potential G:U pair bordering the loop adjacent to the conserved GAs, but a cytidine replaces a phylogenetically conserved adenosine at one loop position in many of the selected RNAs. In model RNAs, the cytidine-bearing variant binds protein slightly more strongly than does the wild-type RNA. That the seven-member, 2 + 5 internal loop is important for protein binding is reinforced by the finding that the position, but not the size, of the loop is variable. A minority of the RNA aptamers has three consecutive uridines and may fold into a similar structure, but with the internal loop inverted.

Base Sequence↗
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↗