Biomedical subjects
Publications and source records attributed to F Li.
The spectral and non-spectral inteferences of tantalum to the determination of Al, Ca, Cr, Cu, Fe, Mg, Mn, Mo, Nb, Ni, Pb, Sn and Ti by ICP-AES were studied. The non-spectral inteference can be eliminated by optimizing operating conditions. Three methods for correction of spectral inteferences were evaluated. The proposed method was used for the determination of impurities in synthetic samples with satisfactory results.
In this paper, the spectral interferences in ICP-AES were reviewed continuously.
PURPOSE: Exogenous basic fibroblast growth factor (bFGF) induces bFGF gene expression in cultured rat Müller cells. To elucidate the mechanism that links exogenous bFGF to transcriptional regulation of bFGF gene expression in these cells, the authors examined mRNA expression of the proto-oncogenes c-fos and c-jun in response to exogenous bFGF in cultured rat Müller cells. METHODS: Müller cells from 1- to 3-day-old Sprague-Dawley rats were isolated and cultured in essential modified Eagle's medium + 10% fetal calf serum. Cultured cells were identified by immunocytochemical analysis using antibodies against vimentin, carbonic anhydrase C, and glutamine synthetase. Cells of passages 1 through 4 were treated with bFGF (0.01, 0.1, 1, 10, and 100 ng/ml), either the protein kinase C (PKC) inhibitors H-7 (30 microM) and GF109203X (1 microM) or the PKC activator phorbol 12-myristate 13-acetate (PMA; 1, 10, 100, 500 nM), and either adenylate cyclase activator forskolin (5 microM) or adenylate cyclase inhibitor SQ22536 (100 microM). Northern blot analysis was performed to determine the mRNA expression of c-fos, c-jun, and bFGF. RESULTS: Addition of bFGF to culture medium induced c-fos and c-jun mRNA expression in a dose- and time-dependent manner. Induction of c-fos mRNA was observed as early as 10 minutes (9.6-fold) after exposure to bFGF at a dose of 10 ng/ml. It reached a maximum of 17.4-fold by 30 minutes. A rapid decline of c-fos mRNA level was observed after 45 minutes of bFGF treatment. The temporal pattern of c-jun gene expression was similar to that of c-fos, whereas a maximum induction of c-jun mRNA (8.2-fold) was seen after 45 minutes of treatment. Induction of c-fos and c-jun gene expression started at a bFGF concentration of 0.1 ng/ml. It reached peak levels of 15-fold for c-fos and 7.6-fold for c-jun mRNA at 10 ng/ml. A dose-dependent upregulation of c-fos and c-jun gene expression by the PKC activator PMA was also observed. A maximum induction was seen at 100 nM PMA. The induction of c-fos and c-jun gene expression by bFGF or by PMA was blocked by the PKC inhibitors H-7 (30 microM) or GF109203X (1 microM). SQ22536 (100 microM), an adenylate cyclase inhibitor, did not inhibit bFGF-induced c-fos and c-jun gene expression, whereas forskolin (5 microM), an adenylate cyclase activator, upregulated the expression. CONCLUSIONS: These results indicate that exogenous bFGF induces c-fos and c-jun gene expression in cultured rat Müller cells through PKC activation. The proto-oncogenes c-fos and c-jun may play a role in the regulation of bFGF gene expression in response to exogenous bFGF in retinal Müller cells. These findings provide further insight into the roles of Müller cells and exogenous bFGF in protecting against photoreceptor degeneration.
Human nerve growth factor (hNGF) was purified to near homogeneity on a large scale from human term placenta with an improved and inexpensive method. The purification procedure included tissue homogenization, ultrafiltration and single CM-cellulose column chromatography. The purified hNGF was a 14.4-kDa protein with an isoelectric point of approximately 9.3. The specific activity of the purified hNGF was approximately 38000 units/mg, and the activity was completely inhibited by the monoclonal antibody against recombinant hNGF (rhNGF). Western-blot analysis showed that the purified hNGF could interact with the monoclonal antibody against rhNGF.
Bcl-xL, an antiapoptotic member of the Bcl-2 family, inhibits programmed cell death in a broad variety of cell types. Recent reports have demonstrated that cytochrome c is released from mitochondria during apoptosis and have suggested that this release may be a critical step in the activation of proapoptotic caspases and subsequent cell death. Furthermore, it has been demonstrated that Bcl-2 can prevent the release of cytochrome c from mitochondria in cells triggered to undergo apoptosis. This has led to the hypothesis that the antiapoptotic effects of Bcl-2 family members are due specifically to their ability to prevent cytochrome c release thus preventing subsequent cytochrome c-dependent caspase activation. In the present report, we use microinjection techniques to investigate the relationship between cytochrome c release, induction of apoptosis, and Bcl-xL activity in intact cells. We demonstrate that microinjection of cytochrome c into the cytosol of human kidney 293 cells results in a dose-dependent induction of apoptosis. In contrast, MCF7 breast carcinoma cells (stably transfected to express the Fas antigen CD95, and denoted MCF7F) that lack detectable levels of caspase 3 (CPP32), are totally resistant to microinjection of cytochrome c. However, transfection of MCF7F cells with an expression plasmid coding for pro-caspase 3, but not other pro-caspases, restores cytochrome c sensitivity. Although MCF7F cells are insensitive to cytochrome c microinjection, they rapidly undergo apoptosis in a caspase-dependent manner in response to either tumor necrosis factor or anti-Fas plus cycloheximide, and these deaths are strongly inhibited by Bcl-xL expression. Furthermore, microinjection of cytochrome c does not overcome these antiapoptotic effects of Bcl-xL. Our results support the concept that the release of cytochrome c into the cytoplasm can promote the apoptotic process in cells expressing pro-caspase 3 but that cytochrome c release is not sufficient to induce death in all cells. Importantly, the ability of Bcl-xL to inhibit cell death in the cytochrome c-insensitive MCF7F cells cannot be due solely to inhibition of cytochrome c release from mitochondria.
Reverse transcriptase-polymerase chain reaction was used to study the biosynthesis of two different cholesteryl ester hydrolases by human and mouse macrophages. Oligonucleotide primers for bile salt-stimulated cholesterol esterase yielded positive reactions with RNA isolated from human peripheral blood monocytes, monocyte-derived macrophages, the human monocytic THP-1 cells, and phorbol ester-induced THP-1 macrophages. In contrast, oligonucleotide primers for hormone-sensitive lipase yielded positive reactions only with RNA isolated from non-differentiated human THP-1 monocytic cells and peripheral blood monocytes, but not those obtained from differentiated THP-1 macrophages or monocyte-derived macrophages. Thus, while human monocytes were capable of synthesizing both enzymes, human macrophages synthesized only bile salt-stimulated cholesterol esterase and not the hormone-sensitive lipase. The synthesis of bile salt-stimulated cholesterol esterase by human macrophages was confirmed by detection of bile salt-stimulated cholesteryl ester hydrolytic activity in conditioned media of differentiated THP-1 cells and human peripheral blood monocyte-derived macrophages. Moreover, incubating human macrophages with oxidized low density lipoprotein (LDL) or acetylated LDL increased bile salt-stimulated cholesterol esterase activity in the conditioned media of these cells. These results with human macrophages were contrasted with results of studies with mouse macrophages, which showed the presence of hormone-sensitive lipase mRNA but not the bile salt-stimulated cholesterol esterase mRNA. Taken together, these results demonstrated species-specific differences in expression of cholesteryl ester hydrolytic enzymes in macrophages. The expression of bile salt-stimulated cholesterol esterase by human macrophages, in a process inducible by modified LDL, suggests a role of this protein in atherogenesis.
Retinoic acid (RA) and interferons (IFNs) are negative regulators of cell proliferation. In vitro and in vivo, their combination leads to a more potent growth inhibition. However, the molecular mechanisms by which RA and IFNs potentiate each other are not fully understood. As some IFN-induced gene products regulate cell growth and/or antiviral activity, we analysed the effects of RA on their expressions. RA increases the level of 2'5'oligoadenylate synthetase, p68 kinase, the promyelocytic leukemia protein (PML) and Sp100 in both HL-60 and WISH cells. Moreover, RA and IFN act cooperatively to increase the expression of these proteins. RA also inhibits vesicular stomatitis virus replication and induces a higher antiviral state and growth inhibition when combined with IFN. RA stimulates the IFN regulatory factor 1 (IRF-1) gene expression directly through the GAS motif and causes the induction and secretion of IFNalpha. Additional mechanisms could be involved as RA increases the level of signal transducing activators of transcription (STAT) proteins, and enhances the IFN-induced STAT activation, suggesting that cooperative effects by RA and IFN are mediated through multiple pathways.
Little is known about the potential for engraftment of autologous hematopoietic stem cells in human adults not subjected to myeloablative conditioning regimens. Five adult patients with the p47(phox) deficiency form of chronic granulomatous disease received intravenous infusions of autologous CD34(+) peripheral blood stem cells (PBSCs) that had been transduced ex vivo with a recombinant retrovirus encoding normal p47(phox). Although marrow conditioning was not given, functionally corrected granulocytes were detectable in peripheral blood of all five patients. Peak correction occurred 3-6 weeks after infusion and ranged from 0.004 to 0.05% of total peripheral blood granulocytes. Corrected cells were detectable for as long as 6 months after infusion in some individuals. Thus, prolonged engraftment of autologous PBSCs and continued expression of the transduced gene can occur in adults without conditioning. This trial also piloted the use of animal protein-free medium and a blood-bank-compatible closed system of gas-permeable plastic containers for culture and transduction of the PBSCs. These features enhance the safety of PBSCs directed gene therapy.
Integrin-linked kinase (ILK) is a recently identified human protein kinase that has been implicated in integrin-mediated signal transduction and tumorigenesis. We have identified a mouse molecule that is highly homologous to human ILK. The mouse ILK homologue protein is readily recognized by antibodies raised against the human ILK protein, and the gene encoding the mouse ILK homologue is widely expressed in mouse tissues. The mouse ILK homologue gene has been mapped to chromosome 7E1 band. A second locus in the mouse chromosome 9E1-3 region has also been detected with a mouse ILK homologue cDNA probe by fluorescence in situ hybridization, suggesting the possible existence of an ILK pseudo-gene or a family of ILK genes in the mouse.
The p47phox-/- mouse exhibits a phenotype similar to that of human chronic granulomatous disease (CGD) and, thus, is an excellent model for the study of gene transfer technology. Using the Moloney murine leukemia virus-based retroviral vector MFG-S encoding the human form of p47phox, we performed ex vivo gene transfer into Sca-1+ p47phox-/- marrow progenitor cells without conditioning of donors with 5-fluorouracil. Transduced progenitors were transplanted into moderately irradiated (500 cGy), G-CSF preconditioned sibling p47phox-/- mice. Using the fluorescent probe dihydrorhodamine 123 (DHR), in vivo biochemical correction of the superoxide-generating NADPH oxidase system was detected by flow cytometry in 12.3% +/- 0.9% of phorbol myristate acetate-stimulated peripheral blood neutrophils at 4 weeks and 2.6% +/- 1.0% at 14 weeks after transplantation. Following gene therapy, mice were challenged with the CGD pathogen Burkholderia (formerly Pseudomonas) cepacia and bacteremia levels were assessed at 24 hours and 7 days after inoculation. At both time points, bacteremia levels in gene corrected p47phox-/- mice were significantly lower than untreated p47phox-/- mice (0.89 +/- 0.30 colonies v 237.7 +/- 83.6 colonies at 24 hours, P < .02; 4.0 +/- 2.0 colonies v 110.2 +/- 26.5 colonies at 7 days, P < .0014). More importantly, Kaplan-Meier survival analysis showed a significant survival advantage of gene corrected versus untreated p47phox-/- mice (P < .001). Thus, stem-cell-directed ex vivo gene therapy is capable of restoring phagocyte oxidant-dependent host-defense function in this mouse model of a human immune-system disorder.
Chronic granulomatous disease (CGD) can result from any of four single gene defects involving the components of the superoxide (O-2) generating phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. We show that transduction of peripheral blood CD34+ hematopoietic progenitors from a p67phox deficient CGD patient with replication defective amphotropic retrovirus encoding p67phox (MFGS-p67phox) significantly corrected the CGD functional defect in phagocyte oxidase activity in vitro. Using a chemiluminescence assay of oxidase activity, we showed that transduced patient CD34+ progenitors differentiating to myeloid cells in culture produced 25% of the total superoxide produced by normal CD34+ progenitors differentiating in culture. A flow cytometric assay of oxidase activity used to assess the oxidase function of individual cells in the cultures indicated that up to 32% of maturing granulocytes derived from transduced CD34+ progenitors from the p67phox CGD patient were oxidase positive with the average level of correction per granulocyte of 85% of that seen with granulocytes in similar cultures of CD34+ progenitors from normal volunteers. Nitroblue tetrazolium dye reduction assays of colonies of transduced progenitors in soft agar indicated that in some studies restoration of oxidase activity occurred in myeloid cells within 44% of granulocyte-erythrocyte-monocyte colonies, and within 28% of the combined group of granulocyte colonies/monocyte colonies/granulocyte monocyte colonies. These high correction rates were achieved without any selective regimen to enrich for transduced cells. This study provides a basis for development of gene therapy for the p67phox deficient form of CGD.
The origin of spongiform change seen in diffuse Lewy body disease (DLBD) cases was elucidated, as compared with Alzheimer-type dementia (ATD) cases. Spongiform change was composed of numerous vacuoles in the neuropil, predominantly in layers II-IIIab of the transentorhinal cortex and in the intermediate area of the accessory basal amygdaloid nucleus. The distribution of spongiform change was identical to that of ubiquitin-positive granular structures (UPG) in non-demented cases. The degree of spongiform change was correlated with loss of the large pyramidal neurons in layers IIIc and V of the transentorhinal cortex and with the disappearance of their ubiquitin-positive granular processes. With electron microscopy, the early vacuoles of spongiform change appeared to arise in the presynaptic terminals as well as in the postsynaptic terminals and dendritic processes. These findings indicate that the vacuoles derive from degeneration of terminal axons of the large pyramidal neurons. The occurrence of spongiform change characteristic to DLBD suggests that the large pyramidal neurons degenerate more rapidly in DLBD than in ATD.
We constructed a panel of overlapping and non-overlapping fragments of cDNA derived from open reading frame 2 (ORF2) of hepatitis E virus (HEV) and fused to the gene encoding glutathione S-transferase (GST), from which proteins were expressed in Escherichia coli. IgG-specific immunoreactivity against each protein was measured by Western immunoblotting using sera from experimentally infected Rhesus macaques (Macaca mulatta) or from HEV-infected patients. Under these conditions, full-length ORF2 protein (GST-ORF2) was strongly reactive with acute-phase sera from either macaques or patients, but was poorly reactive with convalescent sera. Recombinant protein GST-ORF2.3, representing amino acids 1-110 of the 660 encoded by ORF2, demonstrated a pattern of reactivity largely indistinguishable from the full-length protein. Conversely, GST-ORF2.1, representing amino acids 394-660 of the ORF2 protein was strongly reactive with both acute- and convalescent-phase sera. Extension of GST-ORF2.1 towards the N-terminus led to a progressive loss of convalescent-phase reactivity, apparent with as few as 20 additional HEV-specific amino acids. Deletion of 40 or more amino acids from the N-terminus of ORF2.1 also led to reduced convalescent-phase reactivity, however a protein representing this "reactive" region, containing amino acids 394-473, was poorly reactive, suggesting that the convalescent-reactive epitopes are conformational. Expression of full-length ORF2 protein in E. coli therefore masks the convalescent-reactive epitopes within the C-terminal part of the protein, without affecting N-terminal, acute-reactive epitopes.
Excess osteoclast activity is believed to be responsible for the early bone changes associated with Charcot neuroarthropathy in diabetes mellitus. Markers of osteoclast and osteoblast activity were measured in four groups of patients: 16 with an acute Charcot foot, 16 with a chronic Charcot foot, 10 diabetic controls, and 10 non-diabetic controls. Serum carboxyterminal telopeptide of type 1 collagen (1CTP), a marker of osteoclastic bone resorption, was significantly raised in the dorsal venous arch of the acute Charcot foot, 6.1 +/- 1.5 microg l(-1) (mean +/- SD) compared with the chronic Charcot foot 4.1 +/- 1.4, diabetic controls 3.3 +/- 1.4, and non-diabetic controls 2.8 +/- 1.4, p < 0.0001. This local increase in 1CTP was also reflected systemically in a study subgroup of 6 patients with acute Charcot neuroarthropathy, in whom peripheral antecubital vein 1CTP was 9.2 +/- 2.6 compared with 9.0 +/- 3.1 in the foot. In 6 chronic Charcot neuroarthropathy patients, foot (3.8 +/- 1.3) and systemic (4.0 +/- 1.5) 1CTP values were similar. Serum procollagen carboxyterminal propeptide (P1CP), an indicator of osteoblastic bone formation, was not significantly different between the feet of patients with acute Charcot neuroarthropathy 112 +/- 1.5 microg l(-1), patients with chronic Charcot neuroarthropathy 109 +/- 1.5 microg l(-1), diabetic controls 93.5 +/- 2.3 microg l(-1), and non-diabetic controls 90.1 +/- 1.5 microg l(-1). These results suggest that the acute Charcot foot demonstrates excess osteoclastic activity without concomitant increase in osteoblastic function. This may be important in its pathogenesis.
OBJECTIVE: Polymyalgia rheumatica (PMR) has an abrupt onset of inflammatory symptoms, making it a useful model for studying the effects of inflammation in bone. PMR requires corticosteroid treatment, which may itself have a detrimental effect on bone. This study used serially measured biochemical markers of bone turnover and bone density to address the relative contributions of systemic inflammation and corticosteroid therapy to bone loss. METHODS: Fifty untreated patients with PMR were randomized to receive oral prednisolone or intramuscular methylprednisolone. Biochemical bone markers (pyridinoline [PYR], deoxypyridinoline [DPYR], procollagen type I carboxy-terminal peptide [PICP]) and bone mineral density (BMD) were measured at baseline and at 6, 12, and 24 months. RESULTS: The median disease duration at presentation was 12 weeks (range 5-32 weeks). Levels of urinary crosslinks were increased in patients with untreated PMR compared with controls (PYR 74.9 +/- 30.0 nmoles/mmole creatinine, DPYR 14.6 +/- 6.4 nmoles/mmole creatinine [mean +/- SD]; P = 0.0001); the PICP level was normal (115.0 +/- 39.0 microg/liter). With treatment, the crosslinks levels fell and PICP levels rose within 6 months (P = 0.01). Bone resorption (PYR) correlated with untreated disease activity (erythrocyte sedimentation rate [ESR]) (r = 0.5, P = 0.003) and with interleukin-6 levels (r = 0.48, P = 0.05). There was a significant reduction in BMD of both the hip and the spine after 12 months of treatment (P = 0.0002), with no difference between treatment groups. As the steroid dosage was reduced, bone mass improved. Initial ESR influenced the percent change in BMD at 1 year (r = 0.35, P = 0.05), while cumulative steroid dose, mean ESR, and type of steroid used did not. CONCLUSION: Inflammation in PMR increases bone resorption and appears to have a more detrimental effect on bone than does low-dose corticosteroid. If corticosteroids can be tapered and discontinued, bone loss in PMR can be a transient phenomenon.
We characterized the survival-factor response of the normal mouse retina to mechanical injury by examining the expression of mRNAs for basic fibroblast growth factor (bFGF), ciliary neurotrophic factor (CNTF), and their receptors, FGF receptor-1 (FGFR-1) and CNTF receptor alpha (CNTFR-alpha). The retina was injured by making an incision through the choroid and retinal pigment epithelium that penetrated the subretinal space of each eye of an adult BALB/c mouse. Retinas were taken 6 hr, 12 hr, 1, 2, 4, 7, 10 and 16 days post-injury. Control animals were without injury. Northern blot analysis was performed to determine bFGF, CNTF and their receptor mRNA levels. A significant increase in bFGF and CNTF mRNAs was observed after injury, along with an increase in glial fibrillary acidic protein (GFAP) expression. More than 2-fold of upregulation of bFGF mRNA was seen as early as 6 hr after injury. This increase reached a maximum of more than 5-fold at day 2 post-injury and then declined slowly, and was still about 2.5-fold of the control level by day 16. Expression of CNTF showed a small increase of about 1.6-fold at 6 hr after injury. The upregulation reached a peak level of about 2.7-fold at day 4 after injury, then declined to control level by day 16. There was only a very small increase in FGFR-1 at 6, 12 and 24 hr after injury, and no significant increases in FGFR-1 at time points longer than 1 day post-injury. Expression of GFAP followed a time course similar to that of bFGF. We conclude that mechanical injury induces bFGF, CNTF, and GFAP expression in the mouse retina with time courses similar to the upregulation of these molecules in rat retina. Compared to the upregulation in rat retina, however, the injury-induced upregulation of bFGF and GFAP is much less in the mouse retina. In addition, there was only a very small induction of FGFR-1 expression in the mouse retina. These findings may explain, at least in part, the lack of injury-induced photoreceptor protection in the mouse retina.
Cardiac myocytes in rat hearts lose their ability to undergo cytokinesis between day 3 and day 4, resulting in the formation of binucleated myocytes. Failure in the formation of the actin-myosin contractile ring could cause cardiac myocytes to be defective in cytokinesis. Enzymatically isolated cardiac myocytes from 2- and 4-day-old rats were employed to investigate the organisation and distribution of actin, myomesin, and myosin by rhodamine phalloidin, anti-myomesin, and isoform-specific anti-myosin antibodies, respectively. Interestingly, the actin-myosin contractile ring was formed in mitotic myocytes from both 2- and 4-day-old animals. The changes in organisation and distribution of actin, myosin and myomesin in mitotic myocytes from 4-day-old rats were similar to those from 2-day-old rats, except that there were longitudinal actin filaments in the cytoplasm of mitotic myocytes from 4-day-old rats. In mitotic myocytes from both 2- and 4-day-old rats, actin disassembled in prometaphase, concentrated in the equator of the mitotic spindle in late anaphase, and formed a circumferential intensely staining band in early telophase. Cytoplasmic myosin was evenly distributed in the cytoplasm as small spots, and appeared to associate with the cell membrane from interphase to early anaphase. It became progressively more concentrated in association with the cortical membrane in the equator region in late anaphase, formed a ring-like structure in early telophase, and remained associated with adjacent membrane at the cleavage furrow until late telophase. Sarcomeric myosin and myomesin were only partially disassembled in mitotic myocytes from both 2- and 4-day-old animals. The present study showed that the actin-myosin contractile ring was actually formed during the binucleation process of cardiac myocytes. Molecules involved in the latter stages of cytokinesis may be responsible for incomplete cytokinesis during the binucleation process.
Neonatal cardiac myocytes continue to undergo nuclear division, but lose their ability to complete cell division between 3 and 4 days of age. To examine cytoskeletal organisation of cardiac myocytes during mitosis, freshly isolated cardiac myocytes from 2-, 4-, 6- and 8-day-old rats were fixed and labeled with anti-tubulin, vinculin, desmin and sarcomeric alpha-actinin antibodies. The central, nuclear region of cardiac myocytes is expanded to form a balloon-like structure when they entered prophase. The organisation of microtubules, vinculin and desmin in mitotic myocytes from 4-, 6- and 8-day-old rats was identical to that in dividing myocytes from 2-day-old animals. Microtubules emanating from the nuclear membrane mainly ran along the longitudinal axis of cardiac myocytes in interphase. Microtubules were disassembled and reorganised into the mitotic spindle during mitosis. Desmin was disassembled, either diffusely distributed in the cytoplasm or formed spotty cytoplasmic aggregates during mitosis. Vinculin was disassembled in prometaphase, diffusely distributed in the cytoplasm and associated with cell membranes. During telophase it concentrated in the equator of mitotic spindles. Sarcomeric alpha-actinin became dispersed in the cytoplasm of mitotic myocytes from 2-day-old rats in prometaphase. It remained diffusely distributed in the cytoplasm and associated with cell membranes until the completion of cytokinesis. However, sarcomeric alpha-actinin was only partially disassembled in 4-, 6- and 8-day-old myocytes. Striations of alpha-actinin with full sarcomere length were observed in the cytoplasm as well as in the region of furrow formation. Thus, incomplete disassembly and presence of myofibrils in the equator region where cleavage furrows from may physically impede the furrowing of sarcolemma driven by the contractile ring, resulting in the formation of binucleated cardiac myocytes.