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Biomedical subjects

C Li

Publications and source records attributed to C Li.

At least 937 records · Page 52Linked to original sources
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

Integrated application of capillary HPLC/continuous-flow liquid secondary ion mass spectrometry to discovery stage metabolism studies.

The application of capillary HPLC/continuous-flow liquid secondary ion mass spectrometry (CF-LSIMS) as part of an integrated approach for characterizing discovery stage in vitro metabolites, using a specific inhibitor for 5-lipoxygenase as a model compound, was investigated. CF-LSIMS demonstrated excellent sensitivity in detecting the metabolites in both the positive and the negative ion modes, with a good full-scan mass spectrum obtained when 5 pmol of metabolite was injected onto the capillary column. Strong pseudomolecular ions and key fragment ions were observed in the primary spectra of the parent drug and its three oxidative in vitro metabolites, allowing the site of metabolism to be pinpointed to particular substructures. This technique demonstrated versatility and offered a very rapid screening procedure for metabolite identification.

Animals↗
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↗
PubMed1995

Abrogation of p53 function by transfection of HPV16 E6 gene enhances the resistance of human diploid fibroblasts to ionizing radiation.

In order to examine the role of p53 expression on the sensitivity of cells to radiation-induced reproductive failure, we examined the radiosensitivity of a human diploid fibroblast cell strain (AG1521) before and after transfection with the E6 or E6/E7 genes of human papillomavirus 16 (HPV16)3. HPV E6 binds to p53, promoting its degradation and abrogating wild-type p53 function. AG1521 cells transfected with either E6 or E6/E7 showed no radiation induction of either p53 or WAF1/Cip1. The radioresistance of these cells were significantly increased; the D0 of the survival curves rose from 130 +/- 4 cGy for wild type (neo-transfected) cells to 178-192 cGy for three subclones transfected with E6 alone, and 151-218 cGy for eight subclones transfected with E6/E7. The change in radiosensitivity took place before the process of cellular immortalization and transformation produced by transfection with these genes. Thus, the effect on radiosensitivity appears to be an early effect of the loss of p53 function in non-transformed cells, perhaps related to the loss of the G1 checkpoint and of the capacity for programmed death amongst radiation damaged cells.

Base Sequence↗
PubMed1995

Distribution of calbindin-D28K in the brain of the fetal sheep in late gestation.

Immunocytochemical distribution of calbindin-D28K was examined in the brains of fetal sheep at 100-105 and 120-125 days of gestation (dGA) and at term in labor (term approximately 150 dGA) and compared to adult sheep. Brains were perfused, cut frozen and immunostained as freely floating sections with a polyclonal anti-calbindin-D28K antibody. Light microscopy revealed that calbindin-D28K immunogenicity in fetal cerebellum, rhombencephalon, mesencephalon, diencephalon and telencephalon was overall very similar to that seen in the brains of adult sheep. In addition even though fetal sheep peripheral plasma cortisol concentrations increase 10-20 times over the last 3-4 weeks of gestation to peak during labor and delivery, no glucocorticoid dependent increases in calbindin-D28K in the hippocampal formation could be seen in the sheep fetus as have been demonstrated for the adult rat given exogenous glucocorticoids. It is concluded that: (1) the basic pattern of calbindin-D28K distribution seen in adult sheep brains is established by at least 100 dGA in the sheep fetus; and (2) the pattern of calbindin-D28K expression in fetal sheep hippocampal formation is not related to peripheral plasma glucocorticoid concentrations.

Animals↗
PubMed1995

Tissue-specific expression of rat neutral endopeptidase (neprilysin) mRNAs.

Neutral endopeptidase is a cell surface zinc metal-lopeptidase that regulates the action of a variety of physiologically active peptides. The enzyme exhibits a wide tissue distribution, being most abundant in kidney and lung. Three rat neutral endopeptidase cDNAs with unique 5'-untranslated sequences were isolated. Distribution of the corresponding mRNAs in rat tissues was analyzed by RNase protection assays and by in situ hybridization. In kidney, the type 2b transcript was the major species. In lung and testis, type 1 and type 2b transcripts were expressed in approximately equal amounts, while in brain and spinal cord the type 1 mRNA was the major transcript. These findings were extended by in situ hybridization studies. All three mRNAs were expressed in the proximal tubule of the kidney, with the type 2b transcript giving the strongest signal. In the frontoparietal cortex, expression of the neutral endopeptidase mRNA subtypes was cell- and region-specific. The type 1 transcript was localized to neurons, type 2b mRNA was not detectable, while type 3 mRNA was localized to the oligodendrocytes of the corpus callosum. These results clearly demonstrate that expression of the three neutral endopeptidase mRNAs can be regulated in a cell-specific manner.

Animals↗
PubMed1995

Effects of unilateral cranial sympathectomy either alone or with sensory nerve sectioning on pedicle growth in red deer (Cervus elaphus).

In a previous study (Li et al. [1993], J. Exp. Zool., 267:188-197) sensory nerve sectioning had no effect on the timing of pedicle growth. The aim of the present study was to determine whether sensory nerve sectioning in conjunction with sympathectomy would influence pedicle growth. Twelve intact male red deer calves were allocated to treatment before any pedicle growth as follows: 1) unilateral sensory nerve removal (USX, n = 5), 2) unilateral superior cervical ganglionectomy (SGX, n = 4), or 3) both USX and SGX (SG/USX, n = 3). The calves were observed weekly. In all cases the untreated side was the control. Pedicle initiation was measured with a pedicle detector and after initiation, growth was measured with a ruler. When the treated pedicles reached a length of 60 mm the calves were killed and tissues from the pedicle were examined immunohistochemically for nerves. No large bundles of nerves were observed in the treated pedicle although a few fine fibres were present. All calves grew pedicles. There were no significant differences in the timing of pedicle initiation either within treatment or between treatments. All denervated pedicles grew faster than controls and were consequently higher at examination. The fact that pedicle growth took place despite reduced innervation indicates that a continuous neural connection is not a pre-requisite for normal pedicle growth.

Animals↗
PubMed1995

Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr-virus-related lymphoproliferation.

Reactivation of Epstein-Barr virus (EBV) after bone-marrow transplantation leads in many cases to lymphoproliferative disease that responds poorly to standard therapy and is usually fatal. To prevent or control this complication, we prepared EBV-specific cytotoxic T-lymphocyte (CTL) lines from donor leucocytes and infused them into ten allograft recipients. Three of the patients had shown signs of EBV reactivation, with or without overt lymphoproliferation, and the others received CTL infusions as prophylaxis. No patient developed any complication that could be attributed to the CTL infusions. In the three patients with EBV reactivation, EBV DNA concentrations (measured by semiquantitative polymerase chain reaction [PCR]), which had increased 1000-fold or more, returned to the control range within 3-4 weeks of immunotherapy. The most striking consequence was the resolution of immunoblastic lymphoma in a 17-year-old patient who received four CTL infusions (two 1 x 10(7)/m2 and two 5 x 10(7)/m2). Because the CTL had been genetically marked before infusion, we were able to show by PCR analysis that they persisted for 10 weeks after administration. EBV-specific donor-type T-cell lines seem to offer safe and effective therapy for control of EBV-associated lymphoproliferation.

Adolescent↗
PubMed1995

Conditions for the primary culture of eye imaginal discs from Drosophila melanogaster.

We have established a primary culture system for Drosophila eye imaginal discs. With this system, we were able to obtain neurite outgrowth from intact eye discs, eye disc fragments, and dissociated eye imaginal disc cells. Immunoreactivity to antibody 24B10 indicates that these extending neurites are photoreceptor axons. Three culture media were tested for their ability to support the survival of and neurite extension from eye disc fragments in vitro at 23 degrees C. These, with supplements, were: five parts of Schneider's Drosophila medium with four parts of basal Eagle's medium ("4 + 5"); Leibovitz's L-15 medium (L-15); and Shields and Sang's M3 modified medium (MM3). We obtained the best results with MM3 supplemented with 2% fetal bovine serum (FBS). Eye disc fragments survived in this medium for at least 20 days. Pigmentation in the nonphotoreceptor pigment cells in cultures from the prepupa required the presence of 20-hydroxyecdysone (20-HE) (1 micrograms/ml), whereas neurite outgrowth was seen in the absence of 20-HE. Donor animals had to fall within a range of ages to obtain appropriate eye disc differentiation in vitro. Eye disc from 5-h pupae (P + 5) or older commenced ommachrome synthesis in vitro in a temporal sequence close to that found in vivo, whereas the in vitro synthesis of this pigment was delayed in eye discs from younger flies. Average neurite length was not affected by age among pupae younger than P + 5; but neurite outgrowth from P + 24 was scarce, probably because by this time photoreceptor axons had already grown in vivo and were severed and unable to regenerate in vitro. Eye discs taken from third instar larvae or white prepupae continued their mitotic activity in vitro. Together with the advance of the morphogenetic furrow at the leading edge of retinal development, this observation is consistent with the evidence that pattern formation continues in vitro. Morphogenetic changes were manifested in cultures. Viability tests with calcein AM and ethidium bromide revealed few dead cells in living cultures.

Animals↗
PubMed1995

Developmental and photoregulation of al-1 and al-2, structural genes for two enzymes essential for carotenoid biosynthesis in Neurospora.

The levels of al-1 and al-2 transcripts change dramatically in response to light and development during the formation of Neurospora crassa asexual spores (conidia). al-1 and al-2 mRNAs accumulate throughout conidiation irrespective of lighting conditions initially at low levels. As conidiation proceeds, two increases in albino message accumulation are observed. This developmentally induced photoindependent message accumulation was not observed in Neurospora mutants blocked at relevant stages of conidiation. During conidiation, light induces additional accumulation of al-1 and al-2 gene-specific transcripts; the photoinduced increase in albino gene transcript levels was not observed in two Neurospora mutants, wc-1 and wc-2, that are defective in all physiological photoresponses.

Blotting, Northern↗
PubMed1995

Effects of Ilexonin A on circulatory neuroregulation.

Ilexonin A (IA), a pentacyclic triterpene, has been semisynthesized in china for the first time. It is extracted from the root of Ilicis pubescentis, a commonly used herbal medicine in Guangdong for the treatment of cardiovascular, cerebrovascular and peripheral vascular diseases and heart failure with satisfactory effects. The pharmacokinetic studies indicated that the elimination half-life after oral and i.v. dosing were 17.7 +/- 2.4 h and 22.5 +/- 2.9 h respectively. The total clearance was 4.6 +/- 0.51/h. The bioavailability of IA capsules was 0.39 +/- 0.14 and LD50 was 234 mg/Kg. We have adopted modern techniques, including cellular electrophysiology, isotope tracing methods, molecular biology, electromicroscopy, etc., to probe into the pharmacologic mechanisms of the effects of IA on cardiovascular system. The results indicated that IA can increase the contractility of isolated guinea pig auricular myocardium, attenuate vascular smooth muscle tension induced by noradrenaline in the rabbit aorta. IA can exert a biphasic regulatory effect on arterial blood pressure. IA also can prolong A-V duration of Hiss bundle electrograph (HBE) in rabbits and prolong the action potential duration and the effective refractory period (ERP) of myocardial cells in guinea pigs. The results showed that IA can increase the cAMP content in the smooth muscle of aorta and exert a calcium-blockade effect. Therefore, the peripheral resistance vessels are relaxed and the cardiac afterload is lowered. IA-blocked calcium channels are correlated with both the potential-dependent channel and the receptor operated channel in vascular smooth muscles. IA also increases the cAMP content of myocardium and accelerates the cellular calcium influx and efflux, and this may be responsible for the direct mechanism of the positive inotropic action of IA. Under electron microscopy, it is observed that IA can alleviate the defect of succinate dehydrogenase in the myocardial mitochrondria of rabbit chronic congestive heart failure (CF) model and reduce the microstructural damage of the failed myodardium, therefore the anoxic tolerance of myocardium is increased, the effect of IA on the platelet stretching activity and microstructure in the patients with CF is also studied. It is found that IA can reduce the hypercoagulability of blood, decrease the severity of blood stagnation and improve the status of microcirculation. Effects of IA introventricular and cardiovascular central microinjection (nucleus tractus solitarius, paraventricular nucleus) on arterial blood pressure and heart rate were studied. It demonstrated that IA possess circulatory neuroregular effects by the medium of alpha-receptor and beta-receptor of cardiovascular motoneurons.

Action Potentials↗
PubMed1995

A patient dose verification program using diode detectors.

In this work, we present a comprehensive patient dose verification program using a commercial dual diode detector system for external photon beam radiotherapy. To achieve acceptable accuracy, factors potentially affecting diode sensitivity need to be carefully considered in dose determination. Several authors have shown that diode sensitivity depends on temperature, saturation, linearity, radiation damage, and beam incidence angle. In addition, we found that the use of wedge filters, block trays at a close distance to the detectors, variation in radiation field size, as well as measurement SSDs may affect diode sensitivity significantly. In practice, we use a 5% dose deviation of measured dose from expected patient dose to prompt reevaluation of patient dosimetry. This yields a 90% acceptance rate in clinical patient cases.

Calibration↗
PubMed1995

Production of genetically modified Epstein-Barr virus-specific cytotoxic T cells for adoptive transfer to patients at high risk of EBV-associated lymphoproliferative disease.

EBV-induced lymphoproliferative disease (EBV-LPD) is a disorder most commonly associated with the immunocompromise that follows allogeneic organ transplantation. In patients receiving T cell-depleted bone marrow from HLA-mismatched or HLA-matched unrelated donors, the incidence of EBV-LPD is particularly high, ranging from 5 to 30%. Administration of EBV-specific cytotoxic T lymphocytes may be one means of preventing and treating this disease. We now describe a method that allows the routine and timely preparation of large numbers of such cells to allow their safe administration to bone marrow transplant recipients. We also describe how these cells may be genetically marked before infusion, to determine their fate and disposition in vivo.

Adolescent↗