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Biomedical subjects

L Kong

Publications and source records attributed to L Kong.

At least 73 records · Page 4Linked to original sources
PubMed1997

[Design and application of vacuum manifold for solid phase extraction].

Solid phase extraction (SPE) is now a common method for sample pretreatment. SPE vacuum manifold is a key instrument when SPE is used for sample pretreatment. It was developed by National Chromatographic R. & A. Center. Being connected with a vacuum pump, it can deal with 12 samples simultaneously. The manifold has a unique rotatable cover and can be conveniently rotated when switching between any two steps of SPE. An application of this SPE vacuum manifold in measuring bile acids in human serum was given.

Bile Acids and Salts↗
PubMed1997

Immobilized IDA-type Cu2+-chelating membrane affinity chromatography for purification of bovine liver catalase.

Cu2+-chelating membrane medium based on iminodiacetate-substituted modified short cotton cellulose was examined to purify bovine liver catalase (BLC) by affinity chromatography. The buffer pH notably affect BLC binding to the Cu2+-chelating membrane adsorbent. The results showed that under the chromatographic conditions chosen, 67.7% recovery of BLC attained with an overall 4.7-fold increase in specific activity in a single step. After purification of BLC, the chelating membrane adsorbent can easily be regenerated by imidazole or EDTA buffer. Different results were obtained by the two regeneration methods: the later is more effective than the former in reviving the purification characters of the chelating membrane adsorbent.

Animals↗
PubMed1996

Autoimmunity associated with TGF-beta1-deficiency in mice is dependent on MHC class II antigen expression.

The progressive inflammatory process found in transforming growth factor beta1 (TGF-beta1)-deficient mice is associated with several manifestations of autoimmunity, including circulating antibodies to nuclear antigens, immune complex deposition, and increased expression of both class I and class II major histocompatibility complex (MHC) antigens. The contribution of MHC class II antigens to the genesis of this phenotype has been determined by crossing the TGF-beta1-null [TGF-beta1(-/-)] genotype into the MHC class II-deficient [MHC-II(-/-)] background. Mice homozygous for both the TGF-beta1 null allele and the class II null allele [TGF-beta1(-/-);MHC-II(-/-)] are without evidence of inflammatory infiltrates, circulating autoantibodies, or glomerular immune complex deposits. Instead, these animals exhibit extensive extramedullary hematopoiesis with progressive splenomegaly and adenopathy, surviving only slightly longer than TGF-beta1(-/-);MHC-II(+/+) mice. The role of CD4+ T cells, which are also absent in MHC class II-deficient mice, is directly demonstrated through the administration of anti-CD4 monoclonal antibodies in class II-positive, TGF-beta1(-/-) mice. The observed reduction in inflammation and improved survival emphasize the significance of CD4+ cells in the pathogenesis of the autoimmune process and suggest that the additional absence of class II antigens in TGF-beta1(-/-);MHC-II(-/-) mice may contribute to their extreme myeloid metaplasia. Thus, MHC class II antigens are essential for the expression of autoimmunity in TGF-beta1-deficient mice, and normally may cooperate with TGF-beta1 to regulate hematopoiesis.

Animals↗
PubMed1996

Ha-ras oncogene expression abrogates a pH dependent endonuclease activity of apoptosis in normal rat kidney cells.

To investigate the role of oncogene expression in the resistance to tumor necrosis factor-alpha (TNF), we transfected the mutated T24-Ha-ras oncogene into the murine kidney cell line NRK and an alternative murine cell line C127 cells. The resulting transfectants, NRK-Ha and HC127, were assayed for TNF mediated cytotoxicity. Cellular cytotoxicity of 45% over 48 h occurred with the NRK cells. However, ras transfectant NRK-Ha cells demonstrated 0% cytotoxicity over the same period. Both C127 cells and the ras transfectant HC127 demonstrated 40% and 25% cytotoxicity, respectively, over 48 h when incubated with TNF. Furthermore, DNA isolated from NRK, C127, HC127, but not NRK-Ha cells revealed the presence of DNA fragmentation 'ladders' indicative of successful apoptosis when the cells were incubated with TNF. To determine the possible mechanism in which the ras oncogene may have protected the NRK-Ha cells from TNF mediated cytotoxicity and apoptosis, total nuclear endonucleases from the NRK cells and the ras transfectant NRK-Ha cells were isolated. We determined that the endonuclease activity in the NRK and the ras transfectant NRK-Ha cells was a pH dependent endonuclease. Significant degradation of the target DNA was observed only in pH 4-6 buffers containing the endonuclease. Furthermore, preliminary intracellular pH analysis suggested that while the NRK cells have an intracellular pH of 6.0, the ras transfectant NRK-Ha cells have an intracellular pH of 7.2 and may have abrogated its pH dependent endonuclease. Both the C127 cells and the ras transfectant HC127 cells did not express a pH dependent endonuclease but rather a Ca2+/Mg2+ dependent endonuclease. Furthermore, preliminary intracellular pH analysis suggested that both the C127 and HC127 cells have the same intracellular pH. Our results indicate that in normal rat kidney cells, ras oncogene transfection may cause a disruption in the endonuclease activation involved in apoptosis.

Animals↗
PubMed1996

Lymphocyte apoptosis and apoptosis-associated gene expression in Sjögren's syndrome.

OBJECTIVE: To study the mechanism and regulation of apoptosis in peripheral blood T and B lymphocytes from patients with Sjögren's syndrome (SS). METHODS: The mode of in vitro lymphocyte death in the peripheral blood of patients with SS was determined by fluorescence microscopic analysis, terminal deoxynucleotidyl transferase assay, and DNA fragmentation analysis. Apoptotic cell death of T and B cells was determined at 48 hours of culture by fluorescence-activated cell sorter analysis of propidium iodidestained cells. Messenger RNA (mRNA) expression of bcl-2, bcl-x, bax, and c-myc in T and B cells was determined by enzyme-linked immunosorbent assay-polymerase chain reaction (ELISA-PCR). Expression of bcl-xL and bcl-xS was determined by Southern blot analysis of PCR products. Gene expression was calculated as the ratio of each gene message to the message of the GAPDH gene. Bcl-2 protein levels in SS T cells were determined by ELISA. RESULTS: SS T cells showed increased in vitro apoptosis compared with normal T cells (mean +/- SD 12.3 +/- 4.5% versus 7.3 +/- 2.0%; P < 0.01). Freshly isolated SS T cells showed increased expression of bcl-2 mRNA compared with normal controls (mean +/- SD 1.50 +/- 0.65 versus 0.88 +/- 0.23; P < 0.05). There was no significant difference in levels of bax or c-myc mRNA in T cells and B cells between SS patients and normal controls. When SS T lymphocytes were cultured in vitro for 72 hours, Bcl-2 protein levels decreased with time. CONCLUSION: SS T cells showed accelerated apoptosis in vitro. Freshly isolated SS T cells had increased expression of bcl-2. An increase in death-promoter signals and decrease in death-suppressor signals in vitro may have been responsible, in part, for the apoptosis in SS T lymphocytes.

Adult↗
PubMed1996

Nitric oxide reduces tumor cell adhesion to isolated rat postcapillary venules.

Adhesion of circulating tumor cells to microvascular endothelium plays an important role in tumor metastasis to distant organs. The purpose of this study was to determine whether nitric oxide (NO) would attenuate tumor cell adhesion (TCA) to naive or lipopolysaccharide (LPS)-treated postcapillary venules. A melanoma cell line, RPMI 1846, was shown to be much more adhesive to postcapillary venules isolated from rat mesentery than to corresponding precapillary arterioles. Although venules exposed to LPS for 4 h demonstrated an increased adhesivity for the melanoma cells, TCA to LPS-treated arterioles was not altered. Isolated venules exposed to DETA/NO (1 mM), an NO donor, for 30 min prior to tumor cell perfusion prevented the increment in adhesion induced by LPS and attenuated TCA to naive postcapillary venules. While L-arginine (100 microM), an NO precursor, failed to decrease TCA to naive postcapillary venules, this treatment abolished LPS-stimulated TCA to postcapillary venules. The effect of L-arginine was reversed by administration of N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM), an NO synthase (NOS) inhibitor. These observations indicate that both exogenous and endogenous NO modulate TCA to postcapillary venules. To assess the role of NO-induced activation of cGMP in the reduction in TCA produced by DETA/NO, two additional series of experiments were conducted. In the first series, LY-83583 (10 microM), a guanylyl cyclase inhibitor, was shown to completely reverse the effect of DETA/NO on TCA to both naive and LPS-activated postcapillary venules. On the other hand, administration of 8-bromoguanosine 3',5'-cyclic monophosphate (8-B-cGMP) (1 mM), a cell permeant cGMP analog, mimicked the effect of DETA/NO and reduced TCA to LPS-stimulated postcapillary venules. These data suggest that (a) tumor cells are more likely to adhere to postcapillary venules than to corresponding precapillary arterioles, (b) LPS enhances TCA to postcapillary venules, (c) both exogenously applied (DETA/NO) and endogenously generated (L-arginine) NO attenuate the enhanced adhesion induced by LPS, but only DETA/NO reduced TCA to naive postcapillary venules, and (d) the NO-induced reduction in TCA to LPS-activated postcapillary venules occurs by a cGMP-dependent mechanism.

Aminoquinolines↗
PubMed1996

Production of premature delivery in pregnant rhesus monkeys by androstenedione infusion.

The endocrine mechanism involved in term and preterm delivery in primates, including pregnant women, are poorly understood. In the term monkey, fetal plasma androgen concentration rises to two hundred times the maternal concentration which remains unchanged. Placental conversion of androgen to estrogen results in increased maternal plasma estrogen concentration at term in both pregnant nonhuman primates and women. In the present study, continuous infusion of androstenedione to 0.8 gestation monkeys resulted in the premature occurrence of labor-type myometrial activity and increases in maternal plasma estrogen, oxytocin and amnion fibronectin concentrations similar to those measured at normal-term labor. Androstenedione induction of these normal-term biochemical and endocrine changes accompanied by fetal membrane rupture, cervical dilatation and live delivery provides a rich opportunity to study the molecular and physiological mechanisms of both term and preterm labor in primates.

Androstenedione↗
PubMed1996

PCR detection of colonization by Helicobacter pylori in conventional, euthymic mice based on the 16S ribosomal gene sequence.

Many animal models of Helicobacter infection have been described, including infection in rhesus monkeys, ferrets, gnotobiotic piglets, and mice. These animal models utilize a combination of detection methods, including culture, urease testing, and histopathology, all of which may be unreliable, insensitive, or labor-intensive. Development of new animal models of Helicobacter pylori requires new methods of detection with increased sensitivity and specificity. We have developed sensitive and specific PCR primers based on the 16S ribosomal gene sequence of H. pylori. The primers detected single-copy 16S DNA representing 0.2 cell of pure H. pylori (2 cells in the presence of mouse stomach mucosal DNA) and did not cross-react with closely related bacteria. We were able to detect colonization by H. pylori in conventional, euthymic, outbred mice up to 4 weeks postinoculation with a high percentage of isolates tested. One isolate of H. pylori was detected by PCR in 100% of the mice at 6 months and 60% of the mice 1 year after inoculation. Approximately 10(3) to 10(4) H. pylori cells per stomach were detected by utilizing this PCR methodology semiquantitatively. These primers and PCR methodology have facilitated detection of H. pylori colonization in conventional, euthymic mice, colonization which may not have been detectable by other methods.

Animals↗
PubMed1996

A sensitive and specific PCR method to detect Helicobacter felis in a conventional mouse model.

Although many detection methods have been used to determine Helicobacter colonization in small animal models, the sensitivity and specificity of these detection methods are limited. To improve the Helicobacter felis conventional mouse model for accurate evaluation of therapeutic regimens, we developed a PCR for detection of, and a competitive PCR for quantitation of, H. felis in viral antibody-free (VAF) mice. The PCR was based on the H. felis 16S rRNA gene. An internal control DNA was used for competitive quantitation of the PCR. VAF conventional Swiss-Webster mice were infected with an H. felis culture by oral gavage. At various times after H. felis challenge and therapy, stomach mucosa was collected and evaluated by PCR. PCR detected approximately 50 to 100 H. felis cells per mouse stomach and showed no cross-reaction with other bacteria commonly found in mouse stomachs. Colonization of H. felis in the mouse stomach was confirmed by culture isolation from germfree mice and histological examination of VAF mice. Response to therapy in this H. felis model correlated well with results seen in human clinical trials with H. pylori. A model utilizing PCR detection which may be useful for discovering new antibiotics and/or vaccines against Helicobacter ulcer disease has been developed.

Animals↗
PubMed1996

Tumor necrosis factor mediates apoptosis via Ca++/Mg++ dependent endonuclease with protein kinase C as a possible mechanism for cytokine resistance in human renal carcinoma cells.

PURPOSE: Because renal cell cancers have been found to be resistant to numerous chemotherapeutic agents, other agents including tumor necrosis factor are now being considered for clinical use. In this study, we used 2 renal cancer cell lines. SK-RC-42 and SK-RC-49, and determined the cytotoxic effects of tumor necrosis factor (TNF) and the possible mechanism of TNF resistance. MATERIALS AND METHODS: Cytotoxic assays, comparative reverse transcriptase-polymerase chain reaction (RT-PCR) and nuclease digestion analyses were used. RESULTS: Cytotoxic assays with SK-RC-42 demonstrated that TNF at 50 ng./ml. for 24 hours resulted in 19% cytotoxicity of the cells. Similar assay with SK-RC-49 only demonstrated less than 4% cytotoxicity. Based on these results, we defined our TNF-sensitive cells as SK-RC-42 and SK-RC-49 as our TNF-resistant cells. To determine whether protein kinase C (PKC), which is involved in the signal transduction pathway of a cell, could regulate endogenous basal TNF mRNA levels, comparative PCR analyses for TNF expression were used. The PCR results demonstrated that the TNF-resistant cell, SK-RC-49, had a higher basal expression of TNF mRNA than the TNF-sensitive cell SK-RC-42. With PMA, a PKC activator, for various time points, both cell lines demonstrated an induction of endogenous TNF mRNA. To further confirm our findings that PKC may regulate endogenous TNF expression, a PKC inhibitor, staurosporine, was used. When the cells were treated with staurosporine prior to PMA stimulation, no increase in TNF mRNA expression was seen. To determine whether PKC is involved in providing resistance against TNF, we incubated the SK-RC-42 and SK-RC-49 cells with staurosporine at 100 nM. and TNF at 50 ng./ml. for 24 hours. After factoring out the cytotoxicity of staurosporine, the TNF-mediated cytotoxicity increased to 39.3% and 28.7% for the SK-RC-42 and SK-RC-49 cells, respectively. Furthermore, by using a nuclease digestion assay, we demonstrated that TNF activated a Ca++/Mg++ dependent endonuclease responsible for programmed cell death or apoptosis. CONCLUSIONS: Our data suggest that protein kinase C may play a role in protecting renal cancer cells from undergoing cytokine-mediated apoptosis.

Apoptosis↗
PubMed1996

[Transgenic Brassica napus resistant to turnip mosaic virus].

A system for obtaining regenerated plantlets of "double low" Brassica napus by using cotyledonary petioles as material was established. The turnip mosaic virus coat protein (TuMV-CP) gene inserted in the binary vector pBTu in the Agrobacterium tumefaciens LBA 4404 was integrated into Brassica napus through co-culture of cotyledonary petioles with LBA4404, and the material in co-culture was selected under the stress of Kanamycin. Regenerated plantlets were obtained, and the specific TuMV-CP gene was proved to be integrated into the genomic DNA of the regenerated plants by the specific PCR amplification, dot hybridization and Southern blot. All these transgenic plants were proved to be resistant to virulent TuMV by virus challenge in different degree.

Brassica↗
PubMed1996

[Acute toxicity and anti-inflammatory effect of processed products of gamboge].

The acute toxicity and anti-inflammatory effect of processed and raw Gamboge were compared. The results showed that the acute toxicity of processed Gamboge was less than that of the raw one, while both had significant anti-inflammatory effect alike. Among the differently processed products, those boiled with lotus leaf juice and steamed by high pressure were better.

Animals↗
PubMed1996

[The effect of salmeterol on the activity of neutrophil chemotactic factor in patients with asthma].

OBJECTIVE: The activity of neutrophil chemotactic factor (NCF) in serum of 40 patients with asthma on their acute attacks was measured with membrane filter method. METHODS: Of the 26 patients, 13 were administed salmeterol 50 micrograms bid by inhaler and another 13 took procaterol 50 micrograms bid orally for four weeks. RESULTS: It was found that in patients with asthma NCF was 82 +/- 17 cell/10 HP, which was significantly higher than that in normal subjects (33 +/- 5 cell/10 HP, P < 0.01). After treatment with salmeterol the decrease in NCF activity was 26 cell/10 HP (P < 0.01), while the decrease in another group treated with procaterol was 8 cell/10 HP (P > 0.05). The difference between the two treated groups was significant (P < 0.01). In salmeterol treated group the decrease in NCF activity was more remarkable than the improvement in lung function. CONCLUSIONS: It was considered that the result may be related to the anti-inflammatory effect of salmeterol.

Adrenergic beta-Agonists↗
PubMed1996

[Effects of different processed products of radix Angelica sinensis on clearing out oxygen free radicals and anti-lipid peroxidation].

Different processed products of Radix Angelica Sinensis could clear out superoxide radical (O2.-) generated through hypoxanthinexanthine oxidase system and hydroxyl radical (.OH) generated through Fenton action, and inhibit lipid peroxidation of supernatant hepati homogenate in mice induced by free radical generation system. There exist significant differences among the different processed products.

Animals↗
PubMed1995

SLE-like autoantibodies and Sjögren's syndrome-like lymphoproliferation in TGF-beta knockout mice.

Mice bearing the TGF-beta 1 null mutation (-/-) develop lymphoid infiltrates in the heart, lungs, salivary glands, and other organs similar to those seen in the pseudolymphoma of Sjögren's Syndrome. We studied sera from -/- mice and found elevated Ab levels to dsDNA, ssDNA, and Sm ribonucleoprotein. No Abs to SSA/Ro or SSB/La and no IgM rheumatoid factor were found. Serum autoantibodies were predominately IgG and were specific as shown by ELISA inhibition studies. Antinuclear Ab patterns on Western blots varied from one mouse to the next, indicating a random process responsible for the diversity. Wild-type and heterozygote mice had no autoantibodies. Ig glomerular deposits were found in -/- mice, indicating that these autoantibodies may be pathogenic. Treatment of -/- mice with dexamethasone or TGF-beta 1 failed to suppress autoantibody production. These mice represent an overlap combining the autoimmune serology of SLE with the tissue infiltrates of SS. Our results support the concept that TGF-beta 1 is an important naturally occurring immunosuppressive cytokine whose absence can lead to a systemic autoimmune disease.

Animals↗