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L Li

Publications and source records attributed to L Li.

At least 1,603 records · Page 89Linked to original sources
PubMed1994

Neonatal stress transiently alters the development of hippocampal oxytocin receptors.

The development of brain oxytocin (OXT) receptors was examined following the mild stress of daily, 20 min separations of infant rats from their mothers (repeated separation condition) or in undisturbed controls. Changes in OXT receptors were characterized in cell membrane preparations, using the OXT receptor ligand [125I]d(CH2)5[Tyr(Me)2Thr4Tyr-NH9(2)]-ornithine vasotocin ([125I]OTA), from rats at 4, 8, 14, 22 postnatal days of age or as adults. In the hippocampus of control animals, [125I]OTA binding was highest at day 4 or 8 and declined thereafter. Repeated separation decreased the Bmax of [125I]OTA binding in whole hippocampus at day 8, an effect that did not persist into adulthood. This effect was found to be confined to the rapidly proliferating, dorsal hippocampus. It has been suggested that brain OXT is involved in both affiliative/social and stress-related behaviors. While the specific function of OXT receptors in hippocampus is currently unknown, mild stress to the infant and the disruption of infant-mother contact transiently alters the normal development of this system.

Animals↗
PubMed1994

Neuron-specific expression of the synapsin II gene is directed by a specific core promoter and upstream regulatory elements.

Synapsin II is an abundant peripheral membrane protein of synaptic vesicles that is expressed exclusively in neuronal cells. Here we report the isolation and characterization of the 5'-terminal region of the murine synapsin II gene. Primer extension and S1 nuclease protection analysis show that synapsin II gene transcription is initiated from a unique site. The synapsin II gene promoter contains no canonical TATA or CAAT boxes but has putative binding sites for the transcription factors Sp1, AP2, and NGFIA. This promoter is embedded in a large G+C-rich domain with characteristics of a CpG island. Transfection experiments using synapsin II-luciferase fusion genes demonstrate that the 5'-flanking sequence functions as a strong promoter in neuronal but not in nonneuronal cells. Deletion analysis reveals the presence of a neuron-specific core promoter (-79 to 153) and, upstream, two positive and one negative regulatory elements. The 5'-terminal region of the murine synapsin I gene was also cloned and sequenced. Although there is no extensive sequence homology between the 5'-flanking regions of the synapsin I and II genes, comparison analysis has identified two regions of homologous sequences, which may be involved in determining neuron specificity of the core promoters of these two genes.

Amino Acid Sequence↗
PubMed1994

Evidence for NH2- and COOH-terminal interactions in rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

The pH kinetic behavior of several rat fructose-2,6-bisphosphatase forms was analyzed. The bisphosphatase maximal velocity of the hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was optimal at pH 5, but decreased to 12% of the optimal value in the pH range 7.0-7.5. This decrease depended on deprotonation of a group with a pK of 5.7. In contrast, the separate bisphosphatase domain, a 30-amino acid COOH-terminal truncated form (CT30) of the liver enzyme, and the skeletal muscle bifunctional enzyme exhibited pH-insensitive maximal velocities which were 5-10-fold higher than that of the bisphosphatase of the liver bifunctional enzyme at pH 7.0-7.5. The pK values of the C-2 and C-6 phosphoryl groups were 6.0 and 5.75, respectively, as determined by 31P NMR. Analysis of log kcat/Km versus pH profiles revealed two pK values, one at 6.1, which probably is a substrate pK, and the other at 8.4, which represents an enzyme group. Protein kinase-catalyzed phosphorylation of the liver isoform activated the bisphosphatase, and the pK of the group seen in the kcat profile was increased from 5.7 to 6.4. However, phosphorylation of the CT30 mutant had no effect on the bisphosphatase. The data indicate that NH2- and COOH-terminal interactions in the liver bifunctional enzyme affect the pH dependence of the fructose-2,6-bisphosphatase and its activation by phosphorylation.

Amino Acid Sequence↗
PubMed1994

A role for destabilizing amino acid replacements in light-chain amyloidosis.

Light-chain (L-chain) amyloidosis is characterized by deposition of fibrillar aggregates composed of the N-terminal L-chain variable region (VL) domain of an immunoglobulin, generally in individuals overproducing a monoclonal L chain. In addition to proteolytic fragmentation and high protein concentration, particular amino acid substitutions may also contribute to the tendency of an L chain to aggregate in L-chain amyloidosis, although evidence in support of this has been limited and difficult to interpret. In this paper we identify particular amino acid replacements at specific positions in the VL domain that are occupied at frequencies significantly higher in those L chains associated with amyloidosis. Analysis of the structural model for the VL domain of the Bence-Jones protein REI suggests that these positions play important roles in maintaining domain structure and stability. Using an Escherichia coli expression system, we prepared single-point mutants of REI VL incorporating amyloid-associated amino acid replacements that are both rare and located at structurally important positions. These mutants support ordered aggregate formation in an in vitro L-chain fibril formation model in which wild-type REI VL remains soluble. Moreover, the ability of these sequences to aggregate in vitro correlates well with the extent to which domain stability is decreased in denaturant-induced unfolding. The results are consistent with a mechanism for the disease process in which the VL domain, either before or after proteolytic cleavage from the L-chain constant region domain, unfolds by virtue of one or more destabilizing amino acid replacements to generate an aggregation-prone nonnative state.

Amino Acid Sequence↗
PubMed1994

Specific association between the human DNA repair proteins XPA and ERCC1.

Processing of DNA damage by the nucleotide-excision repair pathway in eukaryotic cells is most likely accomplished by multiprotein complexes. However, the nature of these complexes and the details of the molecular interactions between DNA repair factors are for the most part unknown. Here, we demonstrate both in vivo, using the two-hybrid system, and in vitro, using recombinant proteins, that the human repair factors XPA and ERCC1 specifically interact. In addition, we report an initial determination of the domains in ERCC1 and XPA that mediate this interaction. These results suggest that XPA may play a role in the localization or loading of an incision complex, composed of ERCC1 and possibly other repair factors, onto a damaged site.

DNA Damage↗
PubMed1994

Assignment of xeroderma pigmentosum group C (XPC) gene to chromosome 3p25.

The human gene XPC (formerly designated XPCC), which corrects the repair deficiency of xeroderma pigmentosum (XP) group C cells, was mapped to 3p25. A cDNA probe for Southern blot hybridization and diagnostic PCR analyses of hybrid clone panels informative for human chromosomes in general and portions of chromosome 3 in particular produced the initial results. Fluorescence in situ hybridization utilizing both a yeast artificial chromosome DNA containing the gene and XPC cDNA as probes provided verification and specific regional assignment. A conflicting assignment of XPC to chromosome 5 is discussed in light of inadequacies in the exclusive use of microcell-mediated chromosome transfer for gene mapping.

Animals↗
PubMed1994

Functional analysis of human alpha 1(I) procollagen gene promoter. Differential activity in collagen-producing and -nonproducing cells and response to transforming growth factor beta 1.

To gain a further understanding of the regulation of human type I collagen gene expression under physiologic and pathologic conditions, we characterized 5.3 kilobase pairs (kb) of the human alpha 1(I) procollagen gene promoter. A series of deletion constructs containing portions of the alpha 1(I) procollagen 5'-flanking region (with end points from -5.3 kb to -84 base pairs (bp)) ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were transiently transfected into NIH/3T3 cells. Maximal CAT activity was observed with constructs having 5' end points from -804 to -174 bp. A further 5' deletion to -84 bp caused a marked reduction in CAT activity. Cells transfected with plasmids containing longer 5'-flanking fragments of the alpha 1(I) procollagen gene (-2.3 or -5.3 kb) showed reduced CAT activity compared with the -804 bp construct. The activity of the alpha 1(I) procollagen promoter was much lower in cells that do not normally express type I collagen (HeLa cells) compared with collagen-producing NIH/3T3 cells. The CAT activity of deletion constructs containing longer 5' regions than -84 bp was increased by approximately 2-fold in NIH/3T3 cells treated with transforming growth factor beta 1 (TGF beta 1). Electrophoretic mobility shift assays indicated that protein-DNA complex formation with a probe corresponding to the -170 to -80 bp fragment of the alpha 1(I) procollagen promoter was markedly enhanced in nuclear extracts prepared from TGF beta 1-treated fibroblasts as compared with untreated fibroblasts. The DNA binding activity stimulated by TGF beta 1 was specific for an Sp1-like sequence at positions -164 to -142 bp in the promoter. These results demonstrate that 1) there are both positive and negative cis-acting regulatory elements in the human alpha 1(I) procollagen promoter, 2) these regulatory regions function differently in collagen-producing and -nonproducing cells, 3) the alpha 1(I) procollagen promoter contains TGF beta 1-responsive sequences located between -174 and -84 bp from the transcription start site, and 4) TGF beta 1 caused marked stimulation of the DNA binding activity of a nuclear factor interacting with an Sp1-like binding site located within a region encompassing -164 to -142 bp of the alpha 1(I) procollagen promoter.

3T3 Cells↗
PubMed1994

Antitumor agents. 150. 2',3',4',5',5,6,7-substituted 2-phenyl-4-quinolones and related compounds: their synthesis, cytotoxicity, and inhibition of tubulin polymerization.

As part of our continuing search for potential anticancer drug candidates in the 2-phenyl-4-quinolone series, we have synthesized a series of 6,7-methylenedioxy-substituted and unsubstituted 2-phenyl-4-quinolones, as well as related compounds. Their in vitro inhibition of human tumor cell lines and tubulin polymerization is reported. In general, a good correlation was found between cytotoxicity and inhibition of tubulin polymerization. Compounds 7, 9, 13, 16, 22, 23, 36, and 37 showed potent inhibitory effects in both assays. All rigid analogs (47-49) and trimethoxy-substituted compounds showed little or no activity. Substitution at the 4'-position also resulted in compounds with little or no activity, except for hydroxyl or methyl groups at this position. Further investigation is underway to determine if substitution at the 3'-position will result in compounds with increased activity.

Animals↗
PubMed1994

Genomic cloning and chromosomal localization of HRY, the human homolog to the Drosophila segmentation gene, hairy.

The Drosophila hairy gene encodes a basic helix-loop-helix protein that functions in at least two steps during Drosophila development: (1) during embryogenesis, when it partakes in the establishment of segments, and (2) during the larval stage, when it functions negatively in determining the pattern of sensory bristles on the adult fly. In the rat, a structurally homologous gene (RHL) behaves as an immediate-early gene in its response to growth factors and can, like that in Drosophila, suppress neuronal differentiation events. Here, we report the genomic cloning of the human hairy gene homolog (HRY). The coding region of the gene is contained within four exons. The predicted amino acid sequence reveals only four amino acid differences between the human and rat genes. Analysis of the DNA sequence 5' to the coding region reveals a putative untranslated exon. To increase the value of the HRY gene as a genetic marker and to assess its potential involvement in genetic disorders, we sublocalized the locus to chromosome 3q28-q29 by fluorescence in situ hybridization.

Amino Acid Sequence↗
PubMed1994

Isolation and characterization of a TATA-less promoter for the human beta 3 integrin gene.

Proper expression of the human platelet fibrinogen receptor is necessary for the maintenance of normal hemostasis. This receptor is formed by the heterodimer alpha IIb beta 3, a prototypic member of the integrin family of adhesive molecules. beta 3 is also expressed in other tissues with alpha v as the vitronectin receptor. It was not possible to study the basis for tissue-specific expression of this gene, because the beta 3 gene promoter had not been isolated previously. We have now isolated a 6.0-kb human genomic DNA fragment containing 2.0 kb of sequence 5' to the beta 3 ATG start codon. This clone also contains sequence encoding the signal peptide of the immature beta 3 protein and 3.0 kb of 3' intronic sequence. Primer extension and RNase protection studies of poly A+ RNA from a human erythroleukemia (HEL) cell line indicated a major transcription start site 30 bp upstream of the ATG start codon. In an orientation-dependent manner, a 584-bp fragment 5' to the start codon promotes expression of the chloramphenicol acetyl transferase (CAT) reporter gene in K562 cells. CAT expression from this beta 3 promoter is fivefold above expression from a "promoter-less" control CAT construct. This beta 3 promoter lacks TATA and CAAT cis-acting elements, but there are two Sp1 sites flanking the transcription start site. Other potential transcription factor binding sites are also identified. Phorbol esters (TPA), which increase beta 3 transcription in K562 cells, stimulated transcription from the 584-bp 5' beta 3 region. The isolation of this beta 3 promoter region should permit a more detailed analysis of its transcriptional regulation.

Amino Acid Sequence↗
PubMed1994

The FET3 gene of S. cerevisiae encodes a multicopper oxidase required for ferrous iron uptake.

S. cerevisiae accumulate iron by a process requiring a ferrireductase and a ferrous transporter. We have isolated a mutant, fet3, defective for high affinity Fe(II) uptake. The wild-type FET3 gene was isolated by complementation of the mutant defect. Sequence analysis of the gene revealed the presence of an open reading frame coding for a protein with strong similarity to the family of blue multicopper oxidoreductases. Consistent with the role of copper in iron transport, growth of wild-type cells in copper-deficient media resulted in decreased ferrous iron transport. Addition of copper, but not other transition metals (manganese or zinc), to the assay media resulted in the recovery of Fe(II) transporter activity. We suggest that the catalytic activity of the Fet3 protein is required for cellular iron accumulation.

Amino Acid Sequence↗
PubMed1994

Identification of autoantibody epitopes of glutamic acid decarboxylase in stiff-man syndrome patients.

Stiff-man syndrome is a neurologic disorder characterized by progressive rigidity of skeletal muscles. Deficiency of the neurotransmitter gamma-aminobutyric acid and autoantibodies to glutamic acid decarboxylase (GAD), the enzyme synthesizing gamma-aminobutyric acid, are closely associated with the disorder, although the relevant antigenic epitopes have not been identified. In the present study, sera from two patients with SMS was used in an immunoblotting assay with recombinant GAD67 (M(r) 67,000) and GAD65 (M(r) 65,000) isoforms to test whether SMS sera can recognize specific epitopes. We found that both SMS sera recognized the GAD65, but not the GAD67, isoform. Using 13 different synthetic GAD peptides to block the autoantibodies, two GAD65 epitopes were identified. One epitope recognized by both patients' sera, was blocked by the peptide representing amino acid residues 354-368. In one patient only, blocking was also observed by a peptide representing residues 390-402, which includes the binding site of the GAD cofactor, pyridoxal 5'-phosphate. A single amino acid substitution in GAD65 at position 401 (leucine to proline) and representing the analogous GAD67 sequence in this region significantly reduced the peptide's inhibitory effect. These findings suggest that SMS GAD autoantibodies share distinct GAD65 linear epitopes and that some SMS patients' autoantibodies may block the active site, explaining SMS GABA deficiency.

Adult↗
PubMed1994

Antigen recognition by an antibody light chain.

A monoclonal antibody to vasoactive intestinal polypeptide (VIP) was reduced and alkylated and its light and heavy chains were purified by denaturing gel filtration. Following renaturation, the light chain displayed sequence-specific binding of VIP. The specific VIP binding activity of several fractions spanning the light chain peak recovered from the gel filtration column was constant, the light chain was electrophoretically homogeneous, the VIP binding activity was precipitated by anti-light chain antibody but not anti-heavy chain antibody and the activity remained associated with a light chain fraction recovered by resolutive chromatography on a hydroxylapatite column. N-terminal amino acid sequencing of the light and heavy chain fractions confirmed the purity of these proteins and suggested that the VL and VH regions belonged to kappa-family II and gamma-family III, respectively. The VIP-binding affinity of the light chain was only 5-fold lower than that of the parent antibody and the light chain did not bind unrelated peptides. These observations suggest that light chains display structural characteristics necessary for high affinity antigen binding.

Amino Acid Sequence↗
PubMed1994

Thymic peptide modulates glutathione redox cycle and antioxidant enzymes in macrophages.

The effect of a 6-kDa thymic peptide (TP) on the oxidative burst of the murine macrophage cell line J774 was determined. TP was incubated with J774 cells at 37 degrees C and 5% CO2 for 18 h, oxidative burst was triggered by zymosan, and chemiluminescence was amplified by luminol and measured in an automated luminometer. TP exhibited a concentration-dependent suppression of oxidative burst. To study the mechanisms involved in TP's suppression of oxidative burst, its effect on the glutathione redox cycle and antioxidant enzymes was investigated. J774 cells were incubated with varying concentrations of TP for 18 h, washed, resuspended in phosphate-buffered saline, and sonicated to obtain cell lysate. Biochemical assays were performed with the lysate. TP was shown to increase the level of glutathione, and activities of glutathione-peroxidase and glutathione-reductase, indicating its ability to modulate the glutathione redox cycle. The activity of antioxidant enzyme superoxide dismutase was enhanced significantly by TP treatment while catalase activity remained unaffected. These results suggest that TP possesses an antioxidant property and therefore may be involved in the regulation of free radical mediated reactions.

Animals↗
PubMed1994

Application of commercial assays to detect IgG antibodies to hepatitis C virus in urine: a pilot study from autopsy cases.

Hepatitis C virus (HCV), the main cause of non-A, non-B hepatitis in the United States and possibly in the world, is believed to be transmitted primarily through parenteral exposure. Many screening and supplemental tests are available to detect antibodies to HCV in serum. The ability to use commercial assays to detect antibodies to HCV in urine was investigated in this study. A total of 229 serum/urine matched samples were collected sequentially from forensic autopsy cases examined at the Office of the Chief Medical Examiner, State of Maryland. Testing was performed using the Ortho, Innogenetics, and Abbott second generation HCV screening tests and the INNO-LIA HCV Ab supplemental assay. Sample volumes were increased for urine testing. Forty-six of 229 serum samples were positive by screening and confirmed by supplemental tests. The urine samples produced positive results on 44-45 of the same 46 by screening tests and all 46 positives by the supplemental test. There were no false positive samples using urine when compared with the serum pairs. The one false negative sample using urine was still nonreactive when the urine volume was increased to 200 microliters using the screening tests. Generally, five times the serum volume was required for the screening tests to be optimal for urine samples. The urine samples were stored under different conditions prior to testing to determine the influence on antibody stability in urine.

Adult↗
PubMed1994

The synergistic antitumor effect of recombinant interleukin-1 and low-dose of cyclophosphamide in tumor-bearing mice.

Intraperitoneal (i.p.) treatment of MOPC104E ascitic tumor-bearing BALB/c mice with interleukin-1 (IL-1) followed by a low dose of cyclophosphamide (CPA) resulted in synergistic prolongation of their survival time. This antitumor effect was abolished when administration of CPA preceded that of IL-1. The combined i.p. therapy also eradicated subcutaneous (s.c.) tumors, indicating a systemically operating antitumor mechanism. In Winn assay, splenocytes from MOPC104E-bearing mice treated with the combined therapy completely suppressed the growth of MOPC104E cells, but not that of another syngeneic tumor cell line, RL female-8 cells. This tumor-neutralizing activity was completely abrogated by treatment with anti-asialo-GM1 or anti-Thy1.2 and complement, and reduced by treatment with anti-Lyt2.2 and complement. Treatment of splenocytes with 1-leucine methyl ester (Leu0Me), which depletes natural killer (NK) cells and macrophages in vitro, did not affect the neutralizing activity.

Animals↗
PubMed1994

Neurotrophic agents prevent motoneuron death following sciatic nerve section in the neonatal mouse.

We have examined the ability of different neurotrophic and growth factors to prevent axotomy-induced motoneuron cell death in the developing mouse spinal cord. After postnatal unilateral section of the mouse sciatic nerve, most motoneuron (MN) loss occurs in the lateral motor column of the fourth lumbar segment (L4). Significant axotomy-induced cell death occurred after surgery performed on or before postnatal day (PN) 5. In contrast, no significant cell loss was found when axotomy was performed after PN10. Axotomy on PN2 or PN5 resulted in a 44% loss of L4 motoneurons by 7 days, and a 66% loss of motoneurons by 10 days postsurgery. Implantation of gelfoam presoaked in various neurotrophic factors at the lesion site rescued axotomized motoneurons. Nerve growth factor (NGF), neurotrophin-4/5 (NT-4/5) and ciliary neurotrophic factor (CNTF) rescued 20%-30% of motoneurons, whereas brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and insulin-like growth factor 1 (IGF-1) rescued virtually all motoneurons from axotomy-induced death. By contrast, platelet-derived growth factor (PDGF)-AA, PDGF-AB, basic fibroblast growth factor (bFGF), and interleukin (IL-6) were ineffective on motoneuron survival following axotomy. NGF, BDNF, NT-3, IGF-1, and CNTF also prevented axotomy-induced atrophy of surviving motoneurons. These data show that mouse lumbar motoneurons continue to be vulnerable to axotomy up to about 1 week after birth and that a number of trophic agents, including the neurotrophins, CNTF, and IGF-1, can prevent the death of these neurons following axotomy.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗