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C Li

Publications and source records attributed to C Li.

At least 19 recordsLinked to original sources
PubMed1996

Rab3 reversibly recruits rabphilin to synaptic vesicles by a mechanism analogous to raf recruitment by ras.

GTP activates the interaction between the synaptic vesicle proteins rabphilin and rab3. This raises the question of whether rabphilin is a resident vesicle protein that recruits rab3 in a stage-dependent fashion, or if it is instead an effector protein recruited by rab3. We now show that rabphilin, like rab3, dissociates from synaptic vesicles after exocytosis in a manner requiring both Ca2+ and membrane fusion. Rabphilin interacts with GTP-rab3 via a N-terminal domain comprising a novel Zn2+(-)finger motif, and this interaction is essential for rabphilin binding to synaptic vesicles. Thus, in the same way that ras recruits raf to the plasma membrane, rab3 reversibly recruits rabphilin to synaptic vesicles in a stage-dependent manner. These results reveal an unexpected similarity between the molecular mechanisms by which small G protein function in recruiting effector proteins to membranes during membrane traffic and signal transduction.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Murine laminin B1 gene regulation during the retinoic acid- and dibutyryl cyclic AMP-induced differentiation of embryonic F9 teratocarcinoma stem cells.

Retinoic acid (RA) and cyclic AMP analogs cause the differentiation of F9 embryonic teratocarcinoma stem cells into parietal endoderm, an epithelial cell of the early mouse embryo. Laminin B1 is induced in this differentiation process, but is not transcriptionally activated until 24-48 h after RA addition and is not maximally induced until approximately 72 h. Cyclic AMP analogs enhance this transcriptional activation. Although several DNase I hypersensitive sites (DHSS) were observed in the LAMB1 5 -flanking DNA, one of the sites, DHSS2, was detected only after 72 h of RA treatment. Transient transfections have demonstrated that the DHSS2 region functions as a "late-acting RA-inducible enhancer," and motifs in this enhancer contain the homeobox protein-binding site TTATTAACA. Greater binding is observed at these sites by electrophoretic mobility shift assay when cells are cultured with RA and cyclic AMP analogs versus RA alone, and no binding is seen in extracts from RA-treated F9 RAR gamma-/- cells which lack RAR gamma mRNA and protein. Laminin B1 mRNA is not induced by RA in the RAR gamma-/- cells (Boylan, J. F., Lohnes, D., Taneja, R., Chambon, P., and Gudas, L. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9601-9605). Our data show that these DNA regulatory elements contribute to the transcriptional activation of the LAMB1 gene during the later stages of the differentiation process.

Animals↗
PubMed1996

Dynamic changes in the locus control region of erythroid progenitor cells demonstrated by polymerase chain reaction.

The locus control region (LCR) far upstream of the human beta-like globin genes is defined by the preferential chromatin accessibility/DNase I hypersensitivity of four constituent DNA sites HS4, 3, 2, and 1. In an attempt to understand the mechanism of LCR function during early stages of erythropoiesis, a new polymerase chain reaction (PCR) method has been developed to examine the chromatin structure/DNase I hypersensitivity of the LCR in progenitor cells logistically available in limited cell numbers. In erythroid progenitors as well as in multipotent cells with erythroid potential, hypersensitive sites HS4, 3, 2, and 1 were present and the chromatin structure of the LCR was accessible. Moreover, the chromatin structure of the LCR underwent dynamic changes during erythropoiesis. In early erythroid progenitors, the HS2 site was more accessible than the HS3 site. In more mature erythroid progenitors, HS2 became less accessible than HS3 and the other sites. The results indicate that the transcriptional program of the globin genes is encoded, at least in part, in the chromatin accessibility of the LCR. This globin program was apparently initiated in multipotent cells and maintained in erythroid progenitors. Furthermore, the program could be modulated in response to cellular changes accompanying differentiation of the progenitor cells.

Adult↗
PubMed1996

In vivo measurements of ion transport in long-living CF mice.

The Cftr (Cystic Fibrosis Transmembrane Conductance Regulator) gene codes for an epithelial chloride (C1) channel essential for fluid secretion into the respiratory and gastrointestinal tract and from exocrine glands. Mice lacking CFTR function due to a disruption of Cftr exon 10 or exon 1 (Cftr (m1UNC/m1UNC) or Cftr(m1HSC/m1HFC) mice, respectively) generally suffer from severe gastrointestinal disease resulting in death shortly after birth or at the time of weaning. However, a subgroup of the Cftr(m1HSC/m1HSC) mice have been characterized which exhibit relatively mild intestinal pathology resulting in a noncompromised lifespan compared to the more severely affected Cftr(m1UNC/m1UNC) mice. We compared the ion transport capacity of the intestinal mucosa of the mildly and severely affected CF mice using the in vivo technique of rectal potential difference (PD) measurement and found that the net calcium-activated chloride conductance toward the lumen was much greater in the rectum of mildly affected mice than in the severely affected mice. Hence, the milder phenotype may be related to the expression of a factor which enhances the net calcium-activated chloride conductance into the lumen of the intestinal tract.

Amiloride↗
PubMed1996

Genotypic and phenotypic characterization of two newly established renal cell carcinoma cell lines.

Two new cell lines from human renal cell carcinoma are reported. Primary cell cultures from 75 consecutive cases of nephrectomy and metastatic surgery due to different stages of RCC during 4 years were studied. Two cell cultures could be propagated for more than 50 passages in vitro. HN4 was derived from a grade III clear cell carcinoma. HN51 originated from a metastatic brain lesion of a clear cell carcinoma grade III. Karyotype analysis of HN4 revealed triploidy with a clonal aberration, der(10)t(3;10)(q13;p12). HN51 also had a triploid pattern with different marker chromosomes but without any clonal aberration. Loss of heterozygosity studies revealed no loss of heterozygosity on 3p or other chromosomal markers in HN4 but LOH was found on one 3p marker and one 14q marker in addition to all 17 p and q markers in HN51. In vitro light microscopy showed distinctly different morphology in the two cell lines although they both had a typical epithelial growth pattern. Doubling times in vitro were low but slightly higher for HN51. Repeated tumorigenenic experiments in athymic mice only gave rise to subcutaneous tumors with HN51. On characterization by 2-dimensional gel electrophoresis, the two cell lines exhibited different polypeptide patterns with higher expression of proliferating cell nuclear antigen in HN51 and higher expression of glutathione-S-transferase in HN4 constituting the most prominent differences.

Aged↗
PubMed1996

Long-term restoration of immunity against Epstein-Barr virus infection by adoptive transfer of gene-modified virus-specific T lymphocytes.

Adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTLs) offers safe and effective therapy for certain viral infections and could prove useful in the eradication of tumor cells. Whether or not the infused T cells persist for extended periods, retaining their ability to expand in response to antigenic stimulation, is not known. We now report long-term detection of gene-marked Epstein-Barr virus (EBV)-specific CTLs in immunocompromised patients at risk for the development of EBV lymphoproliferative disease. Infusions of CTLs not only restored cellular immune responses against EBV, but also established populations of CTL precursors that could respond to in vivo or ex vivo challenge with the virus for as long as 18 months. Our findings support wider use of antigen-specific CTLs in adoptive immunotherapy.

Bone Marrow Transplantation↗
PubMed1995

Urokinase-type plasminogen activator-induced monocyte adhesion requires a carboxyl-terminal lysine and cAMP-dependent signal transduction.

Urokinase-type plasminogen activator (u-PA) or its amino-terminal fragment (ATF) containing the u-PA receptor (u-PAR) binding domain, is known to promote monocyte adhesion. In the present study, U937 monocyte adhesion to a plastic surface was used to investigate the mechanism of its promotion by u-PA and ATF. Adhesion was found to be inhibited by cycloheximide or actinomycin D, implicating protein synthesis and gene expression in u-PA-induced monocyte adhesion. Adhesion was prevented by 2'-deoxyadenosine 3'-monophosphate, indicating that a cAMP-dependent pathway of signal transduction was involved. This concept was supported by the complementary finding that u-PA-induced adhesion was greatly promoted by forskolin, cholera toxin, or 8-bromo-cAMP, which by themselves induced little adhesion. Furthermore, similar to many other cAMP-dependent activities, cGMP diminished u-PA-induced adhesion. When u-PA or ATF was treated with immobilized carboxypeptidase B, its proadhesive effect was abolished, implicating the involvement of carboxyl-terminal lysine residues (Lys158 on u-PA and Lys135 on ATF). Moreover, when a carboxyl-terminal lysine analog was added, the proadhesive effect of carboxypeptidase B-treated u-PA or ATF was restored. In conclusion, the present study indicates that u-PA- or ATF-induced monocyte adhesion involves cAMP-dependent signal transduction, which is triggered by u-PAR binding. It is also critically dependent on the presence of a carboxyl-terminal lysine.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

The genetic defect in two well-studied cases of Bernard-Soulier syndrome: a point mutation in the fifth leucine-rich repeat of platelet glycoprotein Ib alpha.

Bernard-Soulier syndrome (B-Ss) is a rare congenital bleeding disorder caused by abnormal giant platelets, thrombocytopenia, and defective glycoprotein (GP) Ib-V-IX, the adhesion receptor for von Willebrand factor (vWF). This report describes the molecular defect in two related individuals with well-established B-Ss whose platelets exhibit decreased GPIb-IX and normal GPV on their surfaces. The GPIb-V-IX genes of the two patients were analyzed by Southern blotting, hetero-duplex analysis, and polymerase chain reaction (PCR) amplification/sequencing. A point mutation was found in codon 129 of the GPIb alpha gene that results in the substitution of proline for leucine in the first position of the fifth leucine-rich glycoprotein repeat of the mature gene product. The mutation (CTC: leucine, wild-type to CCC: proline, mutant) eliminates a Sac I restriction site, facilitating analysis of the mutation in the propositi (both homozygotes), unaffected family members (8 heterozygotes and 8 wild-type), and 58 normal controls (116 wild-type alleles). The status of the genomes was confirmed by the sequencing of platelet cDNA. The mutation does not affect transcription of the Ib-IX genes, as estimated by PCR and Northern blot analysis, but it does inhibit surface expression of the receptor as assessed by transient transfection of mutant and wild-type GPIb alpha genes into mouse Ib beta-IX L cells. Many of the cells (43%) transfected with the normal gene express surface GPIb alpha, whereas untransfected cells and those transfected with the mutant gene lack surface GPIb alpha entirely. Patient platelets were tested both for vWF binding in the presence of ristocetin and for surface GPIb-IX expression. In these instances, despite their inability to agglutinate with ristocetin and vWF, patient platelets exhibit about 40% of normal vWF binding and 40% of normal Ib-IX surface antigens. The results suggest that the described mutation (GPIb alpha: Leu129 -> Pro) affects the conformation of the GPIb-V-IX receptor, alters its availability on platelet surfaces, and causes the observed Bernard-Soulier phenotype.

Alleles↗
PubMed1995

Age estimation from the permanent molar in northeast China by the method of average stage of attrition.

A new method of age estimation using permanent molars, the method of average stage of attrition (ASA), is described. A total of 633 molars, including the first molar (M1) and the second molar (M2) on both jaws, was collected from 57 cadavers and 54 modern dry skulls in northeast China. The attrition condition of the molar crown was analyzed, and a new graduation standard was established. Six linear equations for age estimation were obtained by means of regression analysis. The ASA method gave an estimated age at death from only one molar, either M1 or M2, on either maxilla or mandible. The maximum error of these equations was 4.53 years. The results show that the ASA method can or does reflect the attrition condition of the whole occlusal surface more objectively than some methods using dental wear because the wear degree is estimated by averaging the wear stages of all the cusps rather than of only one or partial cusps.

Adolescent↗
PubMed1995

Comparison of the structure and expression of the human and rat neprilysin (endopeptidase 24.11)-encoding genes.

The existence of a third non-coding exon in the human neprilysin-encoding gene (h-NEP), positionally located as exon 3, has been demonstrated by reverse transcription of RNA from human kidney and lung, coupled with the polymerase chain reaction. Comparison of nucleotide sequences between h-NEP and the rat NEP (r-NEP) genes shows a high degree of sequence conservation within noncoding exons 1 and 2. In contrast, the region of the gene containing exon 3 is highly divergent. Two transcripts derived from exon 2 by alternative splicing, type-2a and type-2b, were demonstrated in human kidney and lung. In contrast, only the type-2b transcript was present in these same tissues in the rat. The type-1 transcript was detected in human kidney, lung and brain, this transcript appearing to be the major species in brain.

Alternative Splicing↗
PubMed1995

Distinct Ca2+ and Sr2+ binding properties of synaptotagmins. Definition of candidate Ca2+ sensors for the fast and slow components of neurotransmitter release.

Ca(2+)-dependent neurotransmitter release consists of at least two components: a major fast component that is insensitive to Sr2+ and a minor slow component that is potentiated by Sr2+ (Goda, Y., and Stevens, C. F. (1994) Proc. Natl. Acad. U. S. A. 91, 12942-12946). These results suggest that at least two Ca2+ sensors act in synaptic vesicle fusion with distinct Ca2+ and Sr2+ binding properties. We have now investigated the relative Ca2+ and Sr2+ binding activities of synaptotagmins to evaluate their potential roles as Ca2+ sensors for the fast and slow components. Our results demonstrate that the first C2 domains of synaptotagmins I, II, III, V, and VII have very similar Ca2+ requirements for phospholipid binding (range of EC50 = 2.6 microM to 5.0 microM), but distinct Sr2+ requirements (EC50 range = 23 microM to 133 microM); synaptotagmins I and II had the lowest Sr2+ affinity, and synaptotagmin III the highest Sr2+ affinity. Purified synaptotagmin I from bovine brain exhibited similar properties as its recombinant first C2 domain, suggesting that the first C2 domain fully accounts for its Ca(2+)-dependent phospholipid binding properties. Sr2+ was unable to trigger syntaxin binding by synaptotagmin I at all concentrations tested, whereas it was effective for synaptotagmin III. These results suggest that different C2 domains have distinct Sr2+ binding properties. They support the hypothesis that synaptotagmins localized on the same vesicle perform distinct functions, with synaptotagmins I and II serving as candidate Ca2+ sensors for the fast component in release and synaptotagmin III for the slow component.

Animals↗
PubMed1995

Complexins: cytosolic proteins that regulate SNAP receptor function.

A family of proteins called complexins was discovered that compete with alpha-SNAP, but not synaptotagmin, for SNAP receptor binding. Complexins I and II are highly homologous hydrophilic proteins that are tightly conserved, with 100% identity among mouse, rat, and human complexin II. They are enriched in neurons where they colocalize with syntaxin and SNAP-25; in addition, complexin II is expressed ubiquitously at low levels. Complexins bind weakly to syntaxin alone and not at all to synaptobrevin and SNAP-25, but strongly to the SNAP receptor-core complex composed of these three molecules. They compete with alpha-SNAP for binding to the core complex but not with other interacting molecules, including synaptotagmin I, suggesting that the complexins regulate the sequential interactions of alpha-SNAP and synaptotagmins with the SNAP receptor during exocytosis.

Adaptor Proteins, Vesicular Transport↗
PubMed1995

Chemokine regulation of human megakaryocytopoiesis.

We have previously shown that platelet factor 4 (PF4), a platelet-specific CXC chemokine, can directly and specifically inhibit human megakaryocyte colony formation. We therefore hypothesized that PF4 might function as a negative autocrine regulator of megakaryocytopoiesis. Herein we present additional studies characterizing the inhibitory effect of CXC chemokines on human megakaryocyte development. We first corroborated our initial studies by showing that recombinant human (rH) PF4, like the native protein, inhibited megakaryocytopoiesis. We then examined the inhibitory properties of other CXC family members. Neutrophil activating peptide-2 (NAP-2), a naturally occurring N-terminally cleaved beta TG peptide, was found to inhibit megakaryocytopoiesis with two to three orders of magnitude greater potency than PF4. Structure function studies showed that an N-terminal mutation, which eliminated NAP-2's neutrophil activating properties (NAP-2E2-->A), also abrogated its ability to inhibit megakaryocyte development. Further investigations of this type demonstrated that a chimeric PF4 protein (AELR/PF4) in which PF4's N-terminus was replaced with the first four amino acids of NAP-2 was also a potent inhibitor of megakaryocytopoiesis. Interleukin (IL)-8, another CXC chemokine, and three CC chemokines (macrophage inhibitory protein-1 alpha [MIP-1 alpha], MIP-1 beta, and C10) also specifically inhibited megakaryocyte colony formation at NAP-2 equivalent doses. CXC and CC chemokine inhibition was additive suggesting that the effects might be mediated through a common pathway. The inhibitory effects of NAP-2 and MIP-1 alpha could not be overcome by adding physiologically relevant amounts of recombinant human megakaryocyte growth and development factor (MGDR) (50 ng/mL) to the cultures. Using Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) based analyses, we documented mRNA expression of IL-8 receptor isoforms alpha and beta in total platelet RNA and in normal human megakaryocytes, respectively. Based on these results, we hypothesize that chemokines play a physiologic role in regulating megakaryocytopoiesis. Because chemokines are elaborated by ancillary marrow cells, both autocrine and paracrine growth control is suggested, the effects of which might be exerted, in part, through alpha and beta IL-8 receptors.

Amino Acid Sequence↗
PubMed1995

Histogenesis of antlerogenic tissues cultivated in diffusion chambers in vivo in red deer (Cervus elaphus).

In a previous study we showed that formation of deer pedicle and first antler proceeded through four ossification pattern change stages: intramembranous, transition, pedicle endochondral, and antler endochondral. In the present study antlerogenic tissues (antlerogenic periosteum, apical periosteum/perichondrium, and apical perichondrial of pedicle and antler) taken from four developmental stages were cultivated in diffusion chambers in vivo as autografts for 42-68 days. The results showed that all the cultivated tissues without exception formed trabecular bone de novo, irrespective of whether they were forming osseous, osseocartilaginous, or cartilaginous tissue at the time of initial implant surgery; in two cases in the apical perichondria from antler group, avascularized cartilage also formed. Therefore, the antlerogenic cells, like the progenitor cells of somatic secondary type cartilage, have a tendency to differentiate into osteoblasts and then form trabecular bone. Consequently, the differentiation pathway whereby antlerogenic cells change from forming osteoblasts to forming chondroblasts during pedicle formation is caused by extrinsic factors. Both oxygen tension and mechanical pressure are postulated to be the factors that cause this alteration of the differentiation pathway.

Animals↗
PubMed1995

Purification and characterization of recombinant cystic fibrosis transmembrane conductance regulator from Chinese hamster ovary and insect cells.

We have developed procedures to purify highly functional recombinant cystic fibrosis transmembrane conductance regulator (CFTR) from Chinese hamster ovary (CHO) cells to high homogeneity. Purification of CHO-CFTR was achieved using a combination of alkali stripping, alpha-lysophosphatidylcholine extraction, DEAE ion-exchange, and immunoaffinity chromatography. Insect CFTR from Sf9 cells was purified using a modification of the method of Bear et al. (Bear, C. E., Li, C., Kartner, N., Bridges, R. J., Jensen, T. J., Ramjeesingh, M. and Riordan, J. R. (1992) Cell 68, 809-818), which included extraction with sodium dodecyl sulfate, hydroxyapatite, and gel filtration chromatography. Characterization of the properties of purified CFTR from both cell sources using a variety of electrophysiological and biochemical assays indicated that they were very similar. Both the purified CHO-CFTR and Sf9-CFTR when reconstituted into planar lipid bilayers exhibited a low pS, chloride-selective ion channel activity that was protein kinase A- and ATP-dependent. Both the purified CHO-CFTR and Sf9-CFTR were able to interact specifically with the nucleotide photoanalogue 8-N3-[alpha-32P]ATP with half-maximal binding at 25 and 50 microM, respectively. These values compare well with those reported for 8-N3-[alpha-32P]ATP binding to CFTR in its native membrane form. Thus CFTR from either insect or CHO cells can be purified to high homogeneity with retention of many of the biochemical and electrophysiological characteristics of the protein associated in its native plasma membrane form. The availability of these reagents will facilitate further investigation and study of the structure and function of CFTR and its interactions with cellular proteins.

Adenosine Triphosphate↗
PubMed1995

Zn2+ potentiates steady-state ATP activated currents in rat nodose ganglion neurons by increasing the burst duration of a 35 pS channel.

Zn2+ and ATP are normally present in serum at low levels and are released from some neurons during synaptic activity. In whole-cell recordings, 10 microM Zn2+ potentiated steady-state currents in rat nodose ganglion nerve cells by 18% in 0.5 microM ATP and 120% in 2 microM ATP. Fluctuation analysis of whole-cell suggested the mechanism was increased burst duration. In single-channel recordings, 10 microM Zn2+ did not increase the ATP-activated channel conductance but did increase the mean burst duration of the 35 pS primary open conductance state. These observations indicate that Zn2+ potentiates the steady-state response to extracellular ATP by increasing the burst duration of the channels.

Adenosine Triphosphate↗
PubMed1995

Ca(2+)-dependent and -independent activities of neural and non-neural synaptotagmins.

Synaptotagmins (Syts) are brain-specific Ca2+/phospholipid-binding proteins. In hippocampal synapses, Syt I is essential for fast Ca(2+)-dependent synaptic vesicle exocytosis but not for Ca(2+)-independent exocytosis. In vertebrates and invertebrates, Syt may therefore participate in Ca(2+)-dependent synaptic membrane fusion, either by serving as the Ca2+ sensor in the last step of fast Ca(2+)-triggered neurotransmitter release, or by collaborating with an additional Ca2+ sensor. While Syt I binds Ca2+ (refs 10, 11), its phospholipid binding is triggered at lower calcium concentrations (EC50 = 3-6 microM) than those required for exocytosis. Furthermore, Syts bind clathrin-AP2 with high affinity, indicating that they may play a general role in endocytosis rather than being confined to a specialized function in regulated exocytosis. Here we resolve this apparent contradiction by describing four Syts, three of which (Syt VI, VII and VIII) are widely expressed in non-neural tissues. All Syts tested share a common domain structure, with a cytoplasmic region composed of two C2 domains that interacts with clathrin-AP2 (Kd = 0.1-1.0 nM) and with neural and non-neural syntaxins. The first C2 domains of Syt I, II, III, V and VII, but not of IV, VI or VIII, bind phospholipids with a similar Ca(2+)-concentration dependence (EC50 = 3-6 microM). The same C2 domains also bind syntaxin as a function of Ca2+ but the Ca(2+)-concentration dependence of Syt I, II and V (> 200 microM) differs from that of Syt III and VII (< 10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗