Electronic structure and transport properties of fullerene nanotubules.
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Journal Article↗
Biomedical subjects
W Li
Publications and source records attributed to W Li.
Characterization of a protein kinase C-delta (PKC-delta) ATP binding mutant. An inactive enzyme that competitively inhibits wild type PKC-delta enzymatic activity.
To investigate the function of protein kinase C (PKC)-delta, we mutated its ATP binding site by converting the invariant lysine in the catalytic domain (amino acid 376) to an arginine. Expression vectors containing wild type and mutant PKC-delta cDNAs were generated either with or without an influenza virus hemagglutinin epitope tag. After expression in 32D cells by transfection, the PKC-delta ATP binding mutant (PKC-delta K376R) was not able to phosphorylate itself or the PKC-delta pseudosubstrate region-derived substrate, indicating that PKC-delta K376R was an inactive enzyme. PKC activity was inhibited by 67% in 32D cells coexpressing both PKC-delta wild type (PKC-delta WT) and PKC-delta K376R when compared to 32D cells expressing only PKC-delta WT. Mixture of PKC-delta WT and PKC-delta K376R kinase sources in vitro also reduced the enzymatic activity of PKC-delta WT. These results suggest that PKC-delta K376R competes with PKC-delta WT and inhibits PKC-delta WT phosphorylation of its in vitro substrate. While PKC-delta WT overexpressed in 32D cells demonstrated 12-O-tetradecanoylphorbol-13-acetate (TPA)-dependent translocation from the cytosolic to the membrane fraction, PKC-delta K376R was exclusively localized in the membrane fraction even prior to TPA stimulation. Unlike PKC-delta WT which was phosphorylated on tyrosine residue(s) only after TPA treatment, PKC-delta K376R was constitutively phosphorylated on tyrosine residue(s). Although exposure of PKC-delta WT transfectants to TPA induced 32D monocytic differentiation, the 32D/PKC-delta K376R transfectants were resistant to TPA-induced differentiation. Thus, expression of active PKC-delta is required to mediate 32D monocytic differentiation in response to TPA stimulation.
Adenosine Triphosphate↗
Differential requirement of a motif within the carboxyl-terminal domain of alpha-platelet-derived growth factor (alpha PDGF) receptor for PDGF focus forming activity chemotaxis, or growth.
To determine the molecular basis for the transforming function of platelet-derived growth factor (PDGF)-A in NIH/3T3 cells, we have constructed chimerae consisting of the extracellular domain of the human CSF-1R (fms) linked to the cytoplasmic domain of the alpha PDGF receptor (alpha R) containing a series of deletion or point mutations. The ability of fms/alpha R chimerae to mediate CSF-1-dependent anchorage-independent growth, focus formation, and chemotaxis of NIH/3T3 cells was then examined. Our results provide evidence that a domain encompassing amino acid residues 977-1024 of the alpha PDGFR is required for ligand-dependent focus formation, but not chemotaxis or anchorage-independent growth, and that tyrosine residues within this domain constitute the major binding site for phospholipase C gamma. Therefore, our findings suggest that: (i) the focus forming function of alpha PDGFR correlates well with the ability of the receptor to bind phospholipase C gamma, and (ii) the mechanism of focus formation mediated by alpha PDGFR may be distinguished from that required for chemotaxis or anchorage-independent growth.
3T3 Cells↗
Function of protein kinase A in hedgehog signal transduction and Drosophila imaginal disc development.
Reduced protein kinase A (PKA) activity in anterior imaginal disc cells leads to cell-autonomous induction of decapentaplegic (dpp), wingless (wg), and patched (ptc) transcription that is independent of hedgehog (hh) gene activity. The resulting nonautonomous adult wing and leg pattern duplications are largely due to induced dpp and wg expression and resemble phenotypes elicited by ectopic hh expression. Inhibition of PKA in anterior cells close to the posterior compartment can substitute for hh activity to promote growth of imaginal discs, whereas overexpression of PKA can counteract transcriptional induction of ptc by hh in these cells. PKA therefore appears to be an integral component of the mechanism by which hh regulates the expression of key patterning molecules in imaginal discs.
Animals↗
Discriminating compact nonnative structures from the native structure of globular proteins.
Prediction of the native tertiary structure of a globular protein from the primary sequence will require a potential energy model that can discriminate all nonnative structures from the native structure(s). A successful model must distinguish not only alternate structures that are very nonnative but also alternate structures that are compact and near-native. We describe here a method, based on molecular dynamics simulation, that allows generation of hundreds of compact alternate structures that are arbitrarily close to the native structure. In this way, a significant amount of conformational space in the neighborhood of the native structure can be sampled and these alternate structures can be used as a stringent test of protein folding models. We have used two sets of these alternate structures generated for six crystallographically characterized small globular proteins (1200 alternate structures in all) to test eight empirical energy models for their ability to discriminate alternate from native structures. Seven of the models fail to correctly identify at least some of the alternate structures as nonnative. An atomic solvation model is presented that succeeds in discriminating all 1200 alternate structures from native.
Amino Acid Sequence↗
Minimal deviation adenocarcinoma of endometrioid type may arise in the isthmus: clinicopathological and immunohistochemical study of two cases.
Clinicopathological and immunohistochemical features of minimal deviation adenocarcinoma (MDA) of endometrioid type which may have arisen in the isthmus are presented. Hysterectomy specimens from two women, one 39 years of age and the other 64 years of age, showed the presence of endometrioid-type adenocarcinoma with minimal atypia and without back-to-back arrangement of the glands, which was consistent with a histological diagnosis of endometrioid MDA. However, the tumorous glands were most abundant in the isthmus, and extensively infiltrated into the cervical stroma, vagina, parametrium, and corpus in one case, and the tumor was confined to the isthmus in the other. Immunohistochemically, the tumorous glands were CEA-positive and vimentin-negative in both cases. p53 protein expression was found in one case. Histogenetic origin of MDA of endometrioid type is discussed.
Adult↗
Phosphatidylcholine hydrolysis and DNA synthesis in cultured retinal capillary pericytes.
In order to verify the role of activation of phosphatidylcholine (PC) hydrolysis by phospholipase D (PLD) in the initiation of mitogenic process of retinal capillary pericytes, platelet-derived growth factor (PDGF), a known PC hydrolysis stimulator, and exogenous PLD have been used to stimulate pericytes. Exogenous PLD (Streptomyces chromofuscus PLD) or PDGF BB homodimer (PDGF) was added to a medium of quiescent pericytes prelabeled with [32P]orthophosphate. In the presence of ethanol (300 mM), phosphatidic acid (PA) and its stable transphosphatidylated product, phosphatidylethanol (PEt), were determined. In parallel, [3H]thymidine incorporation was measured. Downregulation of PKC was achieved by long-term treatment with a phorbol ester. The addition of exogenous PLD or PDGF stimulated both [3H]thymidine incorporation and [32P]PEt formation in a similar kinetic fashion, suggesting that PC hydrolysis is involved in PDGF-mitogenic signaling pathway. PDGF-stimulated [3H]PA formation was significantly higher in the presence than in the absence of PA phosphohydrolase (PAP) inhibitor, indicating the activation of PLD/PAP pathway. In the presence of ethanol, a substantial level of PA at the steady state can be abolished by an inhibitor of diacylglycerol (DAG) kinase. This phenomenon indicates the existence of PC-phospholipase C (PLC)/DAG kinase pathway in PC hydrolysis. Insulin potentiated both PLD- and PDGF-induced DNA synthesis. Though similarities occur in the induction of DNA synthesis and PC hydrolysis by exogenous PLD and PDGF, the maximum extent of DNA synthesis of exogenous PLD was only approximately 43% of that induced by PDGF. Moreover, exogenous PLD-induced DNA synthesis was not blunted, while PDGF-elicited DNA synthesis was markedly reduced, by PKC downregulation. In addition, PDGF-induced PC hydrolysis was attenuated by a tyrosine kinase inhibitor, whereas exogenous PLD-induced PC hydrolysis was unchanged. Taken together, exogenous PLD may mimic PDGF action and partially account for the efficacy on DNA synthesis elicited by PDGF. The signal transduction initiated by exogenous PLD is able to bypass the PKC- and PTK-dependent activation of endogenous PLD. These findings provide evidence for the importance of PLD-mediated PC hydrolysis in pericyte DNA synthesis stimulated by PDGF.
Animals↗
Expression of cytokine mRNA in human melanoma tissues.
We have reported that patients with metastatic melanoma treated with an autologous, dinitrophenol-modified vaccine develop inflammatory responses at tumor sites. Histologically, these inflamed lesions are characterized by T cell infiltration, which is sometimes associated with tumor cell destruction. We tested biopsy specimens of eight subcutaneous metastases that had developed inflammation following vaccine treatment for expression of mRNA for interferon gamma (IFN gamma), interleukin-4 (IL-4), tumor necrosis factor alpha (TNF alpha), and IL-10. Post-vaccine, inflamed biopsies contained mRNA for IFN gamma (5/8), IL-4 (4/8) or both (3/8), and for TNF alpha (4/7). In contrast, IFN gamma mRNA was detected in only 1/17 and TNF alpha mRNA in 2/16 control specimens (pre-treatment lymph node metastases or non-inflamed subcutaneous metastases). mRNA for IL-10, a cytokine with anti-inflammatory properties, was detected in 24/25 melanoma metastases and was independent of lymphoid content; in situ the reverse transcriptase/polymerase chain reaction confirmed that melanoma cells were the major source. These findings may provide a new parameter by which to measure the effects of cancer immunotherapy.
Humans↗
Induction of peroxisome proliferation and increase of catalase activity in yeast, Candida albicans, by cadmium.
The effects of cadmium on the growth rate, catalase activity, and peroxisome proliferation in yeast, Candida albicans, were evaluated. The yeast growth was markedly inhibited by 1 mM cadmium at the initial hours. The toxic effect of cadmium on the cell growth persisted. The catalase activity of the cells treated with 1 mM Cd2+ first decreased, and then rose at 24 h to about 2.6 times that of the controls. The average number of peroxisomes per cell in the yeast treated with 1 mM Cd2+ was about sixfold higher than the control groups. The proliferation of peroxisomes and the increase of catalase activity following cadmium toxicity gives credence to the hypothesis that cadmium toxicity is related to its potential to induce oxidative stress in cells.
Cadmium↗
Animal models of human-derived cancer vaccines.
Preclinical cancer vaccine studies must address vaccine safety, immunogenicity, and efficacy, as well as mechanism of vaccine action. Animal models of vaccines employing human tumor-associated antigen or epitopes (TAA, TAE) differ fundamentally from those employing tumor-specific antigens or epitopes (TSA, TSE). TSA and TSE vaccines will most likely demonstrate similar toxicity, immunogenicity, and efficacy in both tumor-bearing animals and patients. In contrast, TAA/TAE immunizations may have to overcome a host's immunological tolerance to TAA/TAE expressed not only on tumor, but also on normal tissues; immunity to TAA/TAE will potentially target normal tissues and thus may induce autoimmunity. Various experimental models for human-derived TAA/TAE vaccines have been developed. These models include transgenic mice, mice with severe combined immunodeficiency (SCID), and non-human primates. Recently, unique animal models of TAA/TAE cancer vaccines have been developed, taking advantage of the discovery of animal tissue antigens with significant sequence homologies to human TAA/TAE. These models mimic perhaps most closely the situation in cancer patients.
Animals↗
Improved definition of anomalous left coronary artery by magnetic resonance coronary angiography.
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Aged↗
Identification and molecular characterization of the murine cytomegalovirus homolog of the human cytomegalovirus UL100 gene.
The UL100 gene of human cytomegalovirus (HCMV) has been shown to encode an envelope glycoprotein that might play an important role in HCMV infection. Using the HCMV UL100 gene as a probe in low stringency hybridization studies, we were able to identify the putative UL100 homologous gene of murine cytomegalovirus (MCMV), strain Smith. The MCMV gene was sequenced and localized to the left end of the EcoRI fragment H on the MCMV physical map. This genomic location is similar to that found for the HCMV UL100 gene. The MCMV UL100 mRNA is 1.6 kb in size and is expressed exclusively in the late stages of infection. The 5' and 3' boundaries of the transcript were determined. The open reading frame (ORF) of the UL100 gene could encode a protein of 371 amino acid residues with a calculated molecular mass of 42 kDa. Computer analysis of the deduced amino acid sequence of this gene predicted the presence of eight transmembrane domains and four N-linked glycosylation sites in the protein. Sequence comparison revealed that this putative protein shares similarity with the predicted UL100 homologs of several other herpesviruses, and is most similar to the HCMV UL100 protein (47% identity).
3T3 Cells↗
Assessment of regional vascular distensibility in diseased iliofemoral arteries by intravascular ultrasound.
The influence of atherosclerosis on distensibility of iliac and superficial femoral arteries was assessed retrospectively in 28 patients using intravascular ultrasound. Distensibility was related to lesion morphology, lesion geometry, percentage area stenosis, effect of balloon angioplasty, hypertension and patient's age. In 10 patients, free lumen area remained unchanged during the cardiac cycle. From the remaining 18 patients, a total of 135 cross-sections underwent qualitative and quantitative analysis. Cross-sections without a lesion were compared with those showing; soft/hard and eccentric/concentric lesions. At normal sites, iliac arteries showed greater distensibility than femoral arteries (6.5 +/- 2.4% vs. 3.5 +/- 0.9%; p < or = 0.05). Hard lesions in iliac arteries were less distensible than lesions; in femoral arteries this difference was less pronounced. Lesion geometry did not influence arterial distensibility. Intravascular ultrasound revealed no difference in distensibility when normal cross-sections were compared with those having a < 50% or a 50% to 90% area stenosis. In contrast, a significant decrease in femoral artery distensibility was found in the presence of > 90% stenosis (0.4%). Comparison of cross-sections before and after balloon angioplasty revealed a marked increase in distensibility of iliac arteries following intervention; in the femoral artery, there was practically no change in distensibility. Hypertension and increasing age proved to have no significant influence on arterial distensibility. This study demonstrates that intravascular ultrasound is potentially a powerful tool to assess arterial distensibility and the influence of atherosclerosis on vascular dynamics.
Age Factors↗
C-terminals on motoneurons: electron microscope localization of cholinergic markers in adult rats and antibody-induced depletion in neonates.
C-terminals on motoneurons are defined as those accompanied by characteristic postsynaptic specializations termed subsurface cisterns. We have previously shown, by light microscope immunolabelling methods, that subsurface cisterns occur regularly beneath choline acetyltransferase- and acetylcholinesterase-containing boutons on motoneurons. In the present study, the cholinergic nature of C-terminals suggested by these results was further investigated by immunohistochemistry and electron microscopy in adult rats and in neonates treated with a murine monoclonal acetylcholinesterase antibody which was previously shown to cause immunological lesions of central cholinergic systems. In both the facial nucleus and lumbar segment of spinal cord of adult rats, C-terminals were seen intensely immunostained for the cholinergic markers choline acetyltransferase and acetylcholinesterase. Immunolabelled terminals made contact with either neuronal somata or large calibre dendrites, which were positive for the cholinergic markers, and exhibited club-shaped or thin elongated morphologies suggestive of terminal or en passant type synaptic interactions. The close relationship found between cholinergic markers and immunolabelled subsurface cisterns in adults was maintained on motoneurons of eight-day-old rats. While subcutaneous treatment of newborn rat with acetylcholinesterase antibody appeared to have no effect on the distribution of immunopositive subsurface cisterns in motoneurons when examined on postnatal day 8, the density of labelling for the two cholinergic markers around these neurons was reduced. Areas of neuropil immediately surrounding motoneurons in treated animals often showed signs of extensive swelling and deterioration indicative of a lesion event, and these motoneurons frequently displayed subsurface cisterns unapposed to C-terminals. These results support our earlier conclusion, based on light microscope investigation, that the majority if not all C-terminals are cholinergic in the areas investigated and demonstrate the potential utility of immunolesion methods in the study of C-terminal function.
Animals↗
Recognition of a 170 kD protein in mammalian Golgi complexes by an antibody against malarial intraerythrocytic lamellae.
Human erythrocytes infected with the malarial parasite Plasmodium falciparum contain flattened membrane lamellae. It has been suggested that the lamellae may be involved in the sorting of malarial proteins to the cytoplasm and the cell membrane of the host erythrocyte. We have previously shown that the lamellae accumulate sphingolipids by virtue of their lipid composition in a manner similar to the trans-Golgi and the trans-Golgi network in mammalian cells. In this paper, we show by immunofluorescence microscopy that a monoclonal antibody to the lamellae labeled a perinuclear organelle that colocalized with WGA and the mannose-6-phosphate receptor in cultured mammalian cells. Immunoelectron microscopy experiments revealed that LWLI labels cisternae of the trans-face and the trans-Golgi network. Western blot analysis of subcellular fractions using LWLI detected a 170 kD protein which is associated with the luminal side of Golgi membranes of rat liver and is conserved in all cell lines studied. Our results indicate that (i) the 170 kD protein is a novel marker of the mammalian trans-Golgi and the trans-Golgi network and (ii) in addition to similarities in their morphological and lipid characteristics, the lamellae induced by P. falciparum in erythrocytes share proteinaceous determinants with the Golgi apparatus of mammalian cells.
Animals↗
Intravascular ultrasound predictors of outcome after peripheral balloon angioplasty.
OBJECTIVE: This study investigates the potential role of intravascular ultrasound (IVUS) in the outcome in patients undergoing percutaneous transluminal angioplasty (PTA) of the superficial femoral artery. MATERIALS: Angiographic and the qualitative and quantitative IVUS data obtained at the narrowest site derived from 39 patients before and after PTA were analysed. RESULTS: Angiographically the diameter of the remaining stenosis seen after PTA was classified as < 50% in 31 patients (success); in eight patients a failure was encountered. Evaluating at 6 months the functional and anatomic results of the PTA in 31 patients, the intervention was a success in 14 patients (Group I) and a failure in 17 patients (Group II). The remaining eight patients defined as angiographic failure following PTA comprised Group III. Neither qualitative nor quantitative IVUS data obtained before PTA could predict outcome. Conversely, after PTA, the extent of dissection was significantly more severe in Groups II and III than in Group I. Similarly, significant differences were found between Groups I and II for mean free lumen area (13.2 vs. 9.7 mm2, respectively) and mean free lumen diameter (4.1 vs. 3.5 mm, respectively). Quantitative data obtained in Group II were similar to those in Group III. CONCLUSION: This preliminary study demonstrates that following PTA the extent of dissection, free lumen area and diameter seen with IVUS are predictive factors of patency. Future studies with more patients are mandatory to further highlight the sensitivity of these observations.
Adult↗
The olfactory system of migratory adult sea lamprey (Petromyzon marinus) is specifically and acutely sensitive to unique bile acids released by conspecific larvae.
Larval sea lamprey inhabit freshwater streams and migrate to oceans or lakes to feed after a radical metamorphosis; subsequently, mature adults return to streams to spawn. Previous observations suggested that lamprey utilize the odor of conspecific larvae to select streams for spawning. Here we report biochemical and electrophysiological evidence that this odor is comprised of two unique bile acids released by larvae. High performance liquid chromatography and mass spectrometry demonstrated that larval sea lamprey produce and release two unique bile acids, allocholic acid (ACA) and petromyzonol sulfate (PS). Electro-olfactogram (EOG) recording also demonstrated that the olfactory system of migratory adult sea lamprey is acutely and specifically sensitive to ACA and PS; detection thresholds for these compounds were approximately 10(-12) M. ACA and PS were the most potent of 38 bile acids tested and cross-adaptation experiments suggested that adult sea lamprey have specific olfactory receptor sites associated with independent signal transduction pathways for these bile acids. These receptor sites specifically recognize the key substituents of ACA and PS such as a 5 alpha-hydrogen, three axial hydroxyls, and a C-24 sulfate ester or carboxyl. In conclusion, the unique lamprey bile acids, ACA and PS, are potent and specific stimulants of the adult olfactory system, strongly supporting the hypothesis that these unique bile acids function as migratory pheromones in lamprey.
Animals↗
Cloned antigens and antiidiotypes.
Both monoclonal and polyclonal antiidiotypic antibodies mimicking the human colorectal carcinoma (CRC) associated antigen CO17-1A/GA733 have induced antigen-specific humoral and cellular immunity in CRC patients. The immune responses may underlie the clinical responses observed in some of the treated patients. Recently, the CO17-1A/GA733 antigen has been molecularly cloned and expressed in baculo-, adeno-, and vaccinia viruses. In preclinical studies, these recombinant antigen preparations elicited specific humoral immunity (cytotoxic antibodies) and cellular immunity (DTH-reactive and proliferative T cells). Antibody titers elicited in animals by recombinant antigen were significantly higher than those elicited by antiidiotypes. The recombinant antigen has a potential as a vaccine for CRC patients.
Animals↗