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Biomedical subjects

J Fu

Publications and source records attributed to J Fu.

At least 199 records · Page 11Linked to original sources
PubMed1996

Use of green fluorescent protein variants to monitor gene transfer and expression in mammalian cells.

Two mutants of the green fluorescent protein (GFP), RSGFP4 and GFPS65T, have been recently created which differ from the wildtype GFP of A. victoria in their excitation maxima. Here we show that human fibroblasts transfected with either of the two mutant GFP genes emit a green fluorescence that is 18-fold brighter than the cells transfected with the wildtype GFP gene. Retroviral vectors expressing the improved GFP gene were also constructed to determine their suitability for stable gene transduction into mammalian cells. The inclusion of the RSGFP4 gene in a retroviral vector did not reduce the viral titer and resulted in a fluorescent signal in viable transduced cells detectable by both fluorescence microscopy and fluorescence-activated cell sorter (FACS) analysis. Therefore, the improved mutant GFP provides a vital marker for monitoring gene transfer and expression in mammalian cells.

Animals↗
PubMed1996

Respiratory toxicity of direct lytic factor in the venom of the southern Chinese cobra (N. naja atra) in dogs.

The effects of direct lytic factor (DLF) on respiratory ventilation, gas exchange as well as hemodynamics were studied in anesthetized dogs. After an intravenous DLF dose of 1 mg/kg, the initial manifestation of intoxication was observed as follows: (1) Increase in airway impedance characterized by slowed air flow rate and increased negative transpulmonary pressure. (2) Decrease in dynamic compliance. (3) Progressive increase in venoarterial shunt (Qs/Qt) and decrease in PaO2, (4) Elevation of pulmonary artery blood pressure and fall of mean systemic blood pressure and maximal left ventricular pressure. Above actions reached the peak values at 15 min and thereafter all respiratory functional parameters, except Qs/Qt and hypoxemia, returned gradually to approach the normal levels at 50 min. The tidal volume, PaCO2 and LVEDP remained unchanged until another DLF dose of 1.5 mg/kg was given. After a second dose of DLF (total 2.5 mg/kg), the respiratory functions and the cardiac performance deteriorated as follows: (1) Further increase in Qs/Qt and hypoxemia. (2) Appearance of hypercapnea and acidosis. (3) Fall of dP/dtmax and elevation of LVEDP, widening of QRS complex of ECG. (4) Blood pressure run a downhill course. From above experimental evidence, we came to the conclusion that as well as the basic cardiotoxicity, respiratory toxicity of DLF must be considered as the primary, because of broad spectrum of action of DLF and early effect on respiratory function.

Animals↗
PubMed1996

Abnormally expressed low-voltage-activated calcium channels in beta-cells from NOD mice and a related clonal cell line.

A macroscopic low-voltage-activated (LVA) inward current was found in pancreatic beta-cells isolated from NOD mice. However, this current was not present in nondiabetic prone mouse (e.g., Swiss-Webster) pancreatic beta-cells. We performed pharmacological analyses on this current in NOD insulinoma tumor cells (NIT-1). This cell line was developed from pancreatic beta-cells of a transgenic NOD mouse. The sodium-channel blocker, tetrodotoxin (TTX; 2 micromol/l) had no effect on this LVA current. The amplitudes of currents elicited by a -20 mV test pulse retained similarity when the extracellular sodium concentration was increased from 0 to 115 mmol/l; when the extracellular calcium concentration was decreased from 10 to 2 mmol/l, there was an approximate 50% reduction of this current elicited by a -30 mV test pulse. Neither the L-type calcium-channel blocker, nifedipine (3 micromol/l), nor the N-type calcium-channel blocker, omega-CgTx-GVIA (1 micromol/l), at -30 mV produced an appreciable effect. The T-type calcium-channel blockers, nickel (3 micromol/l) and amiloride (250 micromol/l), effectively reduced the peak of this current. In 2 mmol/l calcium external solution, the threshold of voltage-dependent activation of this calcium current was approximately -65 mV, and the peak current occurred at -20 mV. Half-maximum steady-state inactivation was around -43 mV. The mean time constant of slow deactivating tail currents generated by a preceding 20 mV pulse was 2.53 ms. The intracellular free calcium concentration was two- to threefold higher in NOD mouse pancreatic beta-cells compared with Swiss-Webster pancreatic beta-cells. We concluded that there are LVA calcium channels abnormally expressed in NOD mouse beta-cells. This LVA calcium channel may be factorial to the high cytosolic free calcium concentration observed in these cells, and thereby may contribute to the pathogenesis of NOD mouse beta-cells.

Amiloride↗
PubMed1996

A comparison of continuous versus intermittent light exposure on apoptosis.

PURPOSE: We recently found that continuous light exposure at a moderate intensity triggered apoptosis of photoreceptor cells. Since intermittent light exposure is known to cause more severe retinal damage than is continuous light exposure, we sought to determine if intermittent light exposure also triggered apoptosis of photoreceptor cells. METHODS: Lewis albino rats were reared, for 2 weeks, in cyclic light and dark adapted for 24 hr before light exposure. Rats were exposed to intermittent light or continuous light for 6 or 9 hr, respectively. Light-exposed rats were killed by lethal injection at three timepoints: immediately after light exposure, after 6 hr of dark recovery following light exposure and after 24 hr of dark recovery following light exposure. Retinal damage after light exposure was evaluated by morphology, morphometry, the terminal transferase-mediated biotin dUTP nick end labeling (TUNEL) technique for identification of nicked/cleaved nuclear DNA and agarose gel electrophoresis of retinal DNA. RESULTS: Evaluation of morphology confirmed that intermittent light exposure caused more photoreceptor cell damage than did continuous light exposure of the same duration and intensity. The TUNEL technique showed that photoreceptor nuclei contained nicked or cleaved DNA after either intermittent or continuous light exposure, although more TUNEL-positive nuclei were observed after intermittent exposure. Agarose gel electrophoresis of retinal DNA showed internucleosomal DNA fragmentation, which is associated with apoptosis in samples from intermittent light exposure. CONCLUSIONS: These data demonstrated that intermittent light exposure triggered apoptosis in more photoreceptor cells than did continuous light exposure of the same intensity and duration.

Animals↗
PubMed1996

Tissue transglutaminase in apoptosis of photoreceptor cells in rat retina.

PURPOSE: The possible involvement of tissue transglutaminase (tTG) in apoptosis during photoreceptor degeneration was examined in retinal photic injury in rats and in retinal dystrophy of Royal College of Surgeons (RCS) rats. METHODS: Retinal photic injury was induced in 48 male Lewis albino rats by exposure to green fluorescent light of 300 to 320 foot-candles. The retinal tTG was examined by enzyme assay, immunohistochemistry, and Western blot analysis after 9, 12, or 24 hours of exposure or at 6 or 24 hours of dark adaptation after 24 hours of light exposure. Retinas from RCS rats at various stages of degeneration also were examined with similar methods. RESULTS: There was a progressive increase in retinal tTG activity after 300 to 320 ft-c of light exposure, reaching a peak after 24 hours of light exposure. In the RCS rats, tTG activity increased with age. Western blot analysis revealed an immunoreactive band at 80 kDa, which increased in accordance with the transglutaminase activity in both models. In normal rat retinas, tTG immunolabeling was present only in the outer segments. There was an increased number of immunolabeled photoreceptor nuclei from 12 hours of light exposure to 24 hours of light exposure. In the RCS rat, increasing numbers of immunopositive photoreceptor nuclei from 20 to 50 days of age were noted. CONCLUSIONS: The data associated increased retinal tTG activity and enzyme levels with photoreceptor cells undergoing apoptosis. The tTG-dependent irreversible cross-linking of intracellular protein may play an important role in causing the structural changes in cells undergoing apoptosis in the retina.

Aging↗
PubMed1996

[Study of chromosome painting for one rare carrier with complex translocation].

This paper reports that one rare male carrier with complex translocation, being married for eight years, made his wife consecutive seven times abortions, stillbirth and teratogenesis of early death. Using human chromosome 7 and 8-specific probe pools constructed with the technique of chromosome microdissection and PCR, we carried out chromosome painting research of the patients, and identified the karyotype was: 46, XY, -7, -8, -9, +der (7)t(7; 9) (q2200; p24), +der(8)inv ins(8; 7) (q2100; q31.2q2200), +der (9)t(9; 7) (p24; q31.2).ish der(7)t(7; 9) (wcp7+), der(8)inv ins(8; 7)(wcp7+, wcp8+), der(9)t(9; 7) (wcp7+). The technique of chromosome painting provided us an effective technique of molecular cytogenetics for the study of chromosomal abnormalities. The formation mechanism of the complex translocation of the carrier is discussed.

Adult↗
PubMed1996

[Establishment of human amnion cell mutagenesis system by using a shuttle vector pS189].

Using transient shuttle vector pS189, we established a subclone human amnion cell (FL) mutagenesis detection system, and we detected the mutation specificity of N-methy-N'-nitro-N-nitrosoguanidine (MNNG) in this system. The spontaneous mutant frequency of target gene supF was 1.7 x 10(-5) and MNNG-induced mutant frequency was increased with dosage. The results from 0.8% agarose gel electrophoresis, PCR, PCR-single strand conformation polymorphism (PCR-SSCP) analysis showed that MNNG-induced 89% mutants were point mutants. These results indicated that this system can be used to detect and study the mutagenesis mechanism of potential mutagens.

Amnion↗
PubMed1996

[The role of p53 gene during the development of human oral malignant lesions: a comparative study of p53 gene mutation with P53 protein positive immunostaining].

In order to increase the sensitivity to immunostaining in formalinfixed, paraffin-embedded tissues, we developed an immunohistochemical method by microwave heating of tissue sections instead of trypsin digestion. The results of this study showed that there was no positive P53 protein reaction in normal and hyperplastic mucousa of human mouth, whereas 90% (27/30) of cases with dysplasia, 61% (30/49) of oral squamous cell carcinomas and 86% (13/15) of regional metastatic lymph nodes were positive. And all positive reactions were localized in nuclei. Comparison of these positive results of p53 gene mutation detected by silver staining method with the results by polymerase chain reaction-single strand conformation polymorphism analysis did not reveal matched results, especially during the precancerous period. The authors analysed the causes of difference not only by methodology, but also by cell groups which had different genetic changes in tissues of precancerous lesions.

Carcinoma, Squamous Cell↗
PubMed1996

[The role of P53 gene in the development of oral squamous cell carcinoma: study of P53 gene mutation by silver staining polymerase chain reaction-single strand conformation polymorphism].

It is widely accepted that the inactivation of P53 tumor suppressor gene is related to the tumorigenesis of many cancers. The study on P53 tumor suppressor gene has become a hotspot in molecular oncology. But until now, the reports on the research of the role of P53 gene in the tumorigenesis and prognosis of oral squamous cell carcinoma are scarce. So, two studies have been carried out. (1) A silver staining method of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis was established by detecting the mobility shifts of the DNA bands of the products of PCR of mutant P53 gene plasmid and the Sup F mutants which had been proved to have point mutation by DNA sequencing in shuttle vector plasmid pS 189 mutagenesised by N-methyl-N-nito-N-nitosoguanidine (MNNG). For the method is non-radiation with high sensitive, it has applicable value. (2) By silver staining PCR-SSCP system, tissues from normal oral mucoas membrane to metastatic oral squamous cell carcinoma (OSCC) were analyzed and the results showed that tissues from 53% oral precancerous lesions and 65% OSCC had abnormal SSCP bands and the abnormal frequency of metastatic cancers was higher than that of nonmetastatic ones. These suggest that P53 gene mutation plays some role on the process of initiation, development and metastasis of OSCC.

Adult↗
PubMed1995

Signal transduction by the receptors for thrombopoietin (c-mpL) and interleukin-3 in hematopoietic and nonhematopoietic cells.

Antisense oligonucleotide to the translation initiation sequence of human c-mpI reduced the proliferation of human CD34+ bone marrow cells in response to interleukin-3 (IL-3) alone or to the combination of IL-3 and thrombopoietin (TPO). To investigate the molecular basis for these cytokine interactions, we analyzed the relationship between the receptor subunits for IL-3 and TPO and determined whether both receptors activate identical signal transduction pathways. The function of the receptor subunits was characterized in transiently transfected hepatoma cells and fibroblasts by the activation of gene expression via specific regulatory elements and by the stimulation of DNA-binding activity of STAT proteins. Although c-mpl and IL-3 receptor (IL-3R) reconstituted a qualitatively comparable gene regulatory response, there was no detectable functional interaction between their respective receptor subunits. By comparing the receptor action in different cell lines, we observed that in human hepatoma cells the signaling of c-mpI was 100-fold less sensitive to TPO than in rat hepatoma cells. However, IL-3R signaling was comparable between the two cell types, suggesting that c-mpI and IL-3R do not use identical signal transducing mechanisms. The cytoplasmic domains necessary for c-mpI signaling were determined by testing deletion mutants. The membrane-proximal box 1 sequence motif was critical for gene regulation and for STAT protein activation that seemed to involve the Janus kinase 2 (JAK2). Because IL-3R was less dependent on JAK2 than c-mpI, different levels of JAK2 expression may account, in part, for the quantitative difference in IL-3 and TPO response among various cell lines.

Animals↗
PubMed1995

Systemic hypertension exaggerates retinal photic injury.

OBJECTIVE: To determine whether chronic systemic hypertension alters the response of photoreceptors to photic stress. METHODS: Spontaneously hypertensive rats and strain-matched, normotensive Wistar-Kyoto rats were exposed to green fluorescent light (490 to 580 nm, 180 to 200 foot-candles) for 24 hours. Retinal changes were evaluated by histopathologic examination, morphometry of the outer nuclear layer, and rhodopsin levels. RESULTS: Before light exposure, spontaneously hypertensive rats developed elevated systolic blood pressure and showed mild sclerosis of choroidal vasculature. After exposure, retinas of the spontaneously hypertensive rats revealed exaggerated light damage with increased loss of photoreceptor cells, more distortion, and shortening of the inner and outer segments relative to the normotensive Wistar-Kyoto rats. The outer nuclear layer thickness and rhodopsin level were significantly lower in spontaneously hypertensive rats than in the normotensive Wistar-Kyoto rats by day 14 after light exposure. CONCLUSION: Photic injury to photoreceptor cells was exaggerated in spontaneously hypertensive rats. This may have clinical relevance given the association of both systemic hypertension and light exposure in patients with age-related macular degeneration.

Animals↗
PubMed1995

The NSY mouse: a new animal model of spontaneous NIDDM with moderate obesity.

The NSY (Nagoya-Shibata-Yasuda) mouse was established as an inbred strain of mouse with spontaneous development of diabetes mellitus, by selective breeding for glucose intolerance from outbred Jcl:ICR mice. NSY mice spontaneously develop diabetes mellitus in an age-dependent manner. The cumulative incidence of diabetes is 98% in males and 31% in females at 48 weeks of age. Neither severe obesity nor extreme hyperinsulinaemia is observed at any age in these mice. Glucose-stimulated insulin secretion was markedly impaired in NSY mice after 24 weeks of age. In contrast, fasting plasma insulin level was higher in male NSY mice than that in male C3H/He mice (545 +/- 73 vs 350 +/- 40 pmol/l, p < 0.05, at 36 weeks of age). Pancreatic insulin content was higher in male NSY mice than that in male C3H/He mice (76 +/- 8 vs 52 +/- 5 ng/mg wet weight, p < 0.05, at 36 weeks of age). Morphologically, no abnormal findings, such as hypertrophy or inflammatory changes in the pancreatic islets, were observed in NSY mice at any age. These data suggest that functional changes of insulin secretion in response to glucose from pancreatic beta cells may contribute to the development of non-insulin-dependent diabetes mellitus (NIDDM) in the NSY mouse. Although insulin sensitivity was not measured, fasting hyperinsulinaemia in NSY mice suggests that insulin resistance may also contribute to the pathogenesis of NIDDM. Since these findings are similar to the pathophysiologic features of human NIDDM patients, the NSY mouse is considered to be useful for investigating the pathogenesis and genetic predisposition to NIDDM.

Aging↗
PubMed1995

Detection of MspI RFLP in human THY1 gene by the polymerase chain reaction.

THY1 gene encodes a cell surface glycoprotein predominantly expressed in brain and peripheral nerves. Human THY1 gene region on chromosome 11q23 has been implicated in susceptibility to type 1 diabetes (Wong et al., 1991). Two primers derived from the sequences flanking the polymorphic MspI site in intron 2 of the human THY1 gene (Gatti et al., 1988) were selected for RCP to amplify a 566 bp fragment that spans the MspI polymorphism. Polymorphism was detected by MspI digestion of the PCR product.

Base Sequence↗
PubMed1995

Recent advances in the large scale fermentation of Neisseria meningitidis group B for the production of an outer membrane protein complex.

The Outer Membrane Protein Complex (OMPC) of the bacterium Neisseria meningitidis group B has been used successfully as a protein carrier in a Haemophilus influenza type b (Hib) polysaccharide conjugate vaccine and a Streptococcus pneumoniae (Pn) polysaccharide conjugate vaccine to elicit antipolysaccharide immune responses in young infants. The OMPC carrier is derived by detergent extraction of whole cells and, thus, the consistent generation of suitable biomass is central to an effective production process. Therefore, we have developed a large-scale, high-cell density (5 g/L dry cell weight) fermentation process for the cultivation of N. meningitidis B11. Since current requirements for the production of human biologics mandate strict control of all aspects of the manufacturing process, several key features of the process, including a chemically defined medium and a rational event-based harvest criterion, support current good manufacturing practice (cGMP) and increased productivity.

Bacterial Outer Membrane Proteins↗
PubMed1995

Non-enzymatically glycated tau in Alzheimer's disease induces neuronal oxidant stress resulting in cytokine gene expression and release of amyloid beta-peptide.

Paired helical filament (PHF) tau is the principal component of neurofibrillary tangles, a characteristic feature of the neurodegenerative pathology in Alzheimer's disease (AD). Post-translational modification of tau, especially phosphorylation, has been considered a major factor in aggregation and diminished microtubule interactions of PHF-tau. Recently, it has been recognized that PHF-tau is also subject to non-enzymatic glycation, with formation of advanced glycation end products (AGEs). We now show that as a consequence of glycation, PHF-tau from AD and AGE-tau generate oxygen free radicals, thereby activating transcription via nuclear factor-kappa B, increasing amyloid beta-protein precursor and release of approximately 4 kD amyloid beta-peptides. These data provide insight into how PHF-tau disturbs neuronal function, and add to a growing body of evidence that oxidant stress contributes to the pathogenesis of AD.

Alzheimer Disease↗
PubMed1995

The effect of aurintricarboxylic acid, an endonuclease inhibitor, on ischemia/reperfusion damage in rat retina.

Apoptosis is a form of cell death distinct from necrosis showing distinctive morphologic features and may require energy. It is under various control mechanisms and may involve an endonuclease, which cleavages genomic DNA in the internucleosomal linker regions. Previously, we reported that ischemic/reperfusion injury to rat retina induced endonuclease mediated apoptosis of retinal neurons. In this study, we examined the effect of aurintricarboxylic acid (ATA), an endonuclease inhibitor, on ischemia/reperfusion damage in rat retina in our established rat model. A single intraperitoneal injection of ATA at 2 mg/kg given immediately after 60 minutes of ischemia to the retina showed no observable effect. At 10 mg/kg, there was notable beneficial effect morphologically but not morphometrically. ATA at 100 mg/kg showed significant effect both morphologically and morphometrically. This observation is consistent with the hypothesis that endonuclease mediated apoptosis may be involved in retinal cell loss after ischemia/reperfusion insult.

Animals↗