Biomedical subjects
K H Chen
Publications and source records attributed to K H Chen.
Gene suture--a novel method for intramuscular gene transfer and its application in hypertension therapy.
In this report, reporter gene beta-galactosidase (LacZ) was chosen to compare two different intramuscular gene transfer methods, direct injection and gene suture. Evidence showed that gene suture can produce a higher foreign gene express efficiency in skeletal muscle compared with the direct injection method. The highly efficient eukaryotic expressing vectors of human atrial natriuretic factor (ANF) were constructed (pcD2/pAdVAntage/hANF and pcDNA3/hANF), and in vivo ANF gene delivery was performed by intramuscular gene suture. The effects of ANF gene transfer on blood pressure and renal sodium and water excretion were studied in three models of hypertensive animals. Results showed that a marked decrease of mean arterial pressure (MAP) and a significant increase of urine volume and urinary sodium excretion was produced in rats receiving the hANF construct due to the local expression of ANF and its secretion into plasma. Taken together, these results indicate that gene suture may represent a novel gene delivery modality in gene therapy.
Mitochondrial glutathione modulates TNF-alpha-induced endothelial cell dysfunction.
The effect of glutathione (GSH) depletion by L-buthionine-[S,R]-sulphoximine (BSO) on tumor necrosis factor-alpha (TNF-alpha)-induced adhesion molecule expression and mononuclear leukocyte adhesion to human umbilical vein endothelial cells (HUVECs) was investigated. Cells with marked depletion of cytoplasmic GSH, but with an intact pool of mitochondrial GSH, only slightly enhanced TNF-alpha-induced E-selectin and vascular cell adhesion molecule-1 (VCAM-1) expression, compared with the control. However, TNF-a-induced expression of both molecules was markedly enhanced when the mitochondrial GSH pool was diminished to <15% of the control. In contrast, TNF-alpha-induced intercellular adhesion molecule-1 (ICAM-1) expression was not affected by the depletion of either cytoplasmic or mitochondrial GSH. Marked enhancement of TNF-alpha-induced adhesion molecule expression by the depletion of mitochondrial GSH resulted in increased in mononuclear leukocyte adhesion to treated HUVECs, compared with the control. These effects parallel reactive oxygen species (ROS) formation by the depletion of mitochondrial but not cytoplasmic GSH. Our findings demonstrate that depletion of mitochondrial GSH renders more ROS generation in HUVECs, and mitochondrial GSH modulates TNF-alpha-induced adhesion molecule expression and mononuclear leukocyte adhesion in HUVECs.
TGF-beta2 in aqueous humor suppresses S-phase entry in cultured corneal endothelial cells.
PURPOSE: Corneal endothelium in vivo is arrested in G1, the phase of the cell cycle that prepares cells for DNA synthesis. In many cell types, transforming factor (TGF)-beta inhibits proliferation by inducing G1-phase arrest. Evidence indicates that corneal endothelial cells synthesize mRNA for TGF-beta1 and are also bathed in aqueous humor that contains TGF-beta2 (mainly in a latent form). As such, this cytokine may maintain the corneal endothelium in a G1-phase-arrested state in vivo. The purpose of these studies was to determine the effect of exogenous TGF-beta2 and TGF-beta2 in aqueous humor on DNA synthesis in cultured corneal endothelial cells. METHODS: Rat corneal endothelial cells were grown in explant culture and identified by morphology and reverse transcription-polymerase chain reaction using primers for specific corneal cell markers. Subconfluent cells were synchronized in the G0 phase (quiescence) by serum starvation for 24 hours. Serum was then added to the cells in the presence or absence of exogenous TGF-beta2 or activated rat aqueous humor. [3H]Thymidine was added, and radioactivity was measured at various time points to detect DNA synthesis. Preincubation of exogenous TGF-beta2 or activated rat aqueous humor with neutralizing antibody was used to test for cytokine specificity. RESULTS: A linear increase in [3H]thymidine incorporation began approximately 16 hours after serum addition, and peak incorporation occurred at approximately 24 hours. Exposure of cells to serum plus TGF-beta2 suppressed [3H]thymidine incorporation in a dose-dependent manner at concentrations ranging from 5 pg/ml to 5 ng/ml. [3H]Thymidine incorporation was also suppressed in cells exposed to serum plus rat aqueous humor diluted 1:10. Neutralizing antibody reversed the effects of both exogenous TGF-beta2 and aqueous humor. CONCLUSIONS: Exogenous TGF-beta2 and TGF-beta2 in aqueous humor suppress S-phase entry of rat corneal endothelial cells. These results suggest that this cytokine in aqueous humor could help maintain the corneal endothelium in a G1-phase-arrested state in vivo.
Increased interleukin-6 in aqueous humor of neovascular glaucoma.
PURPOSE: To demonstrate the involvement of proinflammatory cytokines in intraocular neovascularization by detecting the presence of interleukin (IL)-6, IL-2, and tumor necrosis factor (TNF)-alpha in aqueous humor and serum of patients with neovascular glaucoma (NVG) secondary to central retinal vein occlusion (CRVO). METHODS: According to the grade of iris neovascularization (NVI), patients with CRVO were divided into three groups: CRVO without NVI, CRVO with NVI, and CRVO with regressed NVI. Healthy patients with cataract were enrolled as control subjects. Enzyme-linked immunosorbent assay was used to quantitate the concentrations of the cytokines IL-6, IL-2, and TNF-alpha in aqueous humor and serum from patients with NVG and control subjects. RESULTS: In serum, the levels of IL-6, IL-2, and TNF-alpha did not differ among groups. In aqueous humor, only IL-6 showed significant change among groups. IL-6 levels in aqueous humor of group 2, CRVO with NVI (1532.0+/-221.1 pg/ml; P < 0.001), and group 3, CRVO with regressed NVI (234+/-154.6 pg/ml; P < 0.001), were significantly higher. There was no significant difference in IL-6 levels between the control group (26.4+/-21.8 pg/ml) and group 1 (15.6+/-0.9 pg/ml). CONCLUSIONS: The inflammatory cytokine IL-6 in aqueous humor increased spatially and temporally correlated with the grade of NVI in patients of NVG secondary to CRVO. The aqueous IL-6 increased in NVI and decreased after vessels regressed. It is possible that the significantly higher level of IL-6 was due to intraocular synthesis because of the minimal change in serum. The increased level of IL-6 may have a putative role along with other angiogenic factors in angiogenesis of NVG as a possible predictor of NVI.
An SRRC elliptically polarizing undulator prototype to examine mechanical design feasibility and magnetic field performance.
In this work, a 1 m long Sasaki-type elliptically polarizing undulator (EPU) prototype with 5.6 cm period length is used to examine the mechanical design feasibility as well as magnetic field performance. The magnetic field characteristics of the EPU5.6 prototype at various phase shifts and gap motion are described. The field errors from mechanical tolerances, magnet block errors, end field effects and phase/gap motion effects are analysed. The procedures related to correcting the field with the block position tuning, iron shimming and the trim blocks at both ends are outlined.
Up-regulation of base excision repair correlates with enhanced protection against a DNA damaging agent in mouse cell lines.
DNA polymerase beta is required in mammalian cells for the predominant pathway of base excision repair involving single nucleotide gap filling DNA synthesis. Here we examine the relationship between oxidative stress, cellular levels of DNA polymerase beta and base excision repair capacity in vitro , using mouse monocytes and either wild-type mouse fibroblasts or those deleted of the DNA polymerase beta gene. Treatment with an oxidative stress-inducing agent such as hydrogen peroxide, 3-morpholinosydnonimine, xanthine/xanthine oxidase or lipopolysaccharide was found to increase the level of DNA polymerase beta in both monocytes and fibroblasts. Base excision repair capacity in vitro , as measured in crude cell extracts, was also increased by lipopolysaccharide treatment in both cell types. In monocytes lipopolysaccharide-mediated up-regulation of the base excision repair system correlated with increased resistance to the monofunctional DNA alkylating agent methyl methanesulfonate. By making use of a quantitative PCR assay to detect lesions in genomic DNA we show that lipopolysaccharide treatment of fibroblast cells reduces the incidence of spontaneous DNA lesions. This effect may be due to the enhanced DNA polymerase beta-dependent base excision repair capacity of the cells, because a similar decrease in DNA lesions was not observed in cells deficient in base excision repair by virtue of DNA polymerase beta gene deletion. Similarly, fibroblasts treated with lipopolysaccharide were more resistant to methyl methanesulfonate than untreated cells. This effect was not observed in cells deleted of the DNA polymerase beta gene. These results suggest that the DNA polymerase beta-dependent base excision repair pathway can be up-regulated by oxidative stress-inducing agents in mouse cell lines.
Induction of thymocyte apoptosis in mice by Yersinia enterocolitica products.
In-vivo administration of the culture supernates from Yersinia enterocolitica resulted in thymus atrophy in C3H/HeJ mice, known to be lipopolysaccharide (LPS)-nonresponders. The thymocytes underwent apoptosis as characterised by fragmented DNA ladders on agarose gel electrophoresis, a cell death detection ELISA and a morphological study by the TUNEL reaction. As a control, LPS treatment did not induce thymocyte apoptosis in C3H/HeJ mice. Flow cytometric analysis indicated that thymus atrophy was due predominantly to the deletion of CD4+ CD8+ T cells. When cells were undergoing apoptosis, an elevation in the percentage of T-cell receptor (TCR)-alphabeta(high) cells was observed at 24 h, which was correlated with the increase in the percentages of cells expressing high levels of the Vbeta6 and Vbeta8 TCR. Gel electrophoretic analysis demonstrated the presence of protein bands with mol.wts ranging from 17 to 65 kDa in Y. enterocolitica culture supernates.
Milk yield and composition of lactating cows fed steam-flaked sorghum and graded concentrations of ruminally degradable protein.
To determine the effect of various amounts of ruminally undegradable protein in the diets of lactating cows fed steam-flaked sorghum, 24 Holstein cows (90 +/- 50 d in milk) were assigned to three treatments: 0.8% urea, 6% soybean meal, or 5% fish meal. Respective percentages of ruminally undegradable protein in the diets (as a percentage of crude protein) were 30, 35, and 39%. All diets contained 37% alfalfa hay; 3 to 5% cottonseed hulls; 10 to 13% whole cottonseed; 39% steam-flaked sorghum (360 g/L); 5% of a molasses, mineral, and vitamin supplement; and the different protein supplements. Intake of dry matter was higher for cows fed urea than for cows fed soybean meal or fish meal diets. In cows that yielded more than 40 kg/d of milk (4 cows per treatment), the soybean meal and fish meal diets resulted in higher yields of milk and 3.5% fat-corrected milk and a greater efficiency of conversion of feed to milk than did the urea diet. Cows that yielded less than 40 kg/d of milk (4 cows per treatment) at the beginning of treatment tended to yield more milk when fed urea than when fed the protein supplements. Nutrient digestibilities were not greatly affected by source of N, suggesting a beneficial effect of urea supplementation on nutrient digestibilities because replacement of protein supplements with cottonseed products caused the neutral detergent fiber content of the urea diet to be about 7% higher than that of the other diets. These data show that response to ruminally undegradable protein in diets of lactating cows fed steam-flaked sorghum was related to milk yield.
Influence of sorghum grain processing on performance and nutrient digestibilities in dairy cows fed varying concentrations of fat.
Forty Holstein cows averaging 85 +/- 50 d in milk were assigned to five dietary treatments for 56 d to determine the effects of ruminal starch degradability and supplemental fat on milk yield and composition and nutrient digestibilities. Treatments were 1) dry-rolled sorghum, no added fat; 2) dry-rolled sorghum plus 2.5% prilled fatty acids; 3) steam-flaked sorghum, no added fat; 4) steam-flaked sorghum plus 2.5% prilled fatty acids; and 5) steam-flaked sorghum plus 5% prilled fatty acids. Fat supplementation at 2.5 and 5% increased the content of fatty acids in diets from 3.0 to 5.4 and 7.7%, respectively. Milk yield was increased 2.0 kg/d when steam-flaked sorghum plus 0 or 2.5% added fat was fed and was 2.4 kg/d higher when prilled fatty acids were fed at 2.5 versus 0%. Cows fed supplemental fat at 5% did not increase milk yield over that of cows fed no added fat. For cows fed 2.5% fat, steam-flaking decreased dry matter intake 12% and increased efficiency of conversion of feed to milk 11%. Milk composition was unaffected by treatments, except that lactose was depressed by fat supplementation. Milk protein yield and efficiency of conversion of dietary protein to milk protein were increased when steam-flaked sorghum was fed. Starch digestibilities were increased from 92.6 to 98.2% when sorghum was steam-flaked compared with dry-rolled and fat supplementation tended to decrease digestibility of neutral detergent fiber. Steam-flaking tended to improve, and the addition of 2.5% fat did improve, the lactational performances of the cows. These effects appeared additive, but 5% fat appeared to be excessive.
Cloning and expression of a novel partial cDNA related to hypertension.
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Use of transvaginal color Doppler ultrasound to diagnose vasa previa.
Vasa previa, though rare (1 in 3000 births), has a significantly high mortality rate. Diagnosis is usually difficult, especially during the antenatal period. However, the use of transvaginal color Doppler ultrasound has increased the recognition of this condition during the antenatal period. Transvaginal color Doppler ultrasound is a more accurate way to view the vulnerable vessels in the area of question. This diagnostic tool will assist in the management of the patient to prevent possible disaster. In this report, transvaginal color Doppler ultrasound helped to recognize and follow up a patient with vasa previa to ensure a successful outcome for both the mother and the fetus. As the antenatal diagnosis of the condition increases with the use of transvaginal color Doppler ultrasound, the high mortality rate should decrease. The importance of meticulous evaluation of the placenta during routine obstetric ultrasound scanning is emphasized.
Effects of supplemental fat and protein source on ruminal fermentation and nutrient flow to the duodenum in dairy cows.
Four lactating Holstein cows, fitted with ruminal and T-type duodenal cannulas, were used in a 4 x 4 Latin square design for four periods (14 d each). Treatments were 1) medium fat and low quality protein, 2) medium fat and high quality protein, 3) high fat and low quality protein, and 4) high fat and high quality protein. Fat was supplemented by addition of 2.5% prilled fatty acids to high fat diets. The low quality diets contained corn gluten meal and 0.47% Lys, and the high quality diets contained a mixture of fish, blood, and soybean meals and 0.77% Lys. The percentage of Met was similar for all diets (mean, 0.28%). Diets contained 35 to 38% steam-flaked sorghum and 32% chopped alfalfa hay. Dietary CP averaged 18%, and all diets were high in RUP (44% of CP). Yields of milk, milk protein, and SNF were increased by added fat and by high quality protein. Ruminal NH3 and butyrate were increased by supplemental fat. Cows fed high quality protein had increased ruminal VFA, propionate, butyrate, and valerate, but decreased ratios of acetate to propionate. Essential AA concentrations in coccygeal plasma and arteriovenous differences across the mammary gland were higher for Lys and lower for Leu in cows fed high quality protein than in those fed low quality protein.
Effects of ruminally inert fat and evaporative cooling on dairy cows in hot environmental temperatures.
Under hot summer conditions of Tucson, Arizona, 24 Holstein cows (mean = 80 d of lactation) were assigned for 56 d to four treatments in a randomized block design with a 2 x 2 factorial arrangement of treatments. Factors were 1) medium [4.6% of dry matter (DM)] versus high (7.4% of DM) amounts of dietary fat and 2) corral shade only versus shade equipped with evaporative cooling. The high fat diet contained 3% prilled fatty acids. The efficiency of the conversion of feed to milk tended to be better for cows fed prilled fat than for cows fed medium dietary fat, but other lactation measurements were unaffected. Cows with access to evaporative cooling had greater milk yields than did cows with access to shade only. Prilled fatty acids did not depress the percentage of milk protein, but reduced short- and medium-chain fatty acids (C6:0 to C14:0) in milk fat and increased palmitic acid. Digestibilities of DM, organic matter, crude protein, acid detergent fiber, neutral detergent fiber, and starch were unaffected by amount of fat or by cooling method, but prilled fatty acids tended to decrease apparent digestibility of fatty acids. No differences were observed among treatments in respiration rates or rectal temperatures. When rectal temperatures were determined, cows were crowded, which probably negated detection of an effect of evaporative cooling. Evaporative cooling increased milk yield of cows in hot weather, but the addition of 3% fatty acids did not increase yield, and no interactions were observed.
Comparison of barley and sorghum grain processed at different densities for lactating dairy cows.
To vary ruminally degradable starch, sorghum grain was dry-rolled or steam-flaked to different densities and compared with dry-rolled barley in total mixed diets fed to 40 lactating cows (111 d of lactation) assigned to five dietary treatments. Diets contained (percentage of dry matter) 35% alfalfa hay, 4.1% cottonseed hulls, 10% whole cottonseed, 2% fish meal, 4% soybean meal, and 4.9% of a molasses, mineral, and vitamin supplement. Treatments were 40% sorghum grain either dry-rolled or steam-processed at flake densities of 437, 360, and 283 g/L. A fifth diet containing 42% dry-rolled barley was fed. Cows were blocked according to pretreatment (14 d) milk yield and received experimental diets for 56 d. Increasing ruminal starch degradability by including steam-flaked sorghum grain or barley in the diet did not increase milk yield or milk protein percentage and yield, as was shown in eight previous studies. Steam-flaked sorghum or dry-rolled barley in the diet decreased dry matter intake, resulting in a 10 to 19% higher efficiency of conversion of feed dry matter to milk than that for dry-rolled sorghum. Milk urea N was decreased, and milk casein yield tended to be increased, by steam-flaking sorghum at the moderate density compared with dry-rolling or fine flaking. Dietary protein was more efficiently converted to milk protein and casein from flaked sorghum and dry-rolled barley than from dry-rolled sorghum. In this study, increasing ruminal starch degradability resulted in higher feed efficiency and lower feed intakes, bu optimal flake densities for steam-processed sorghum gain to maximize milk and milk protein yield were not clarified.
Influence of fat source and sorghum grain treatment on performance and digestibilities of high yielding dairy cows.
Forty-eight lactating Holstein cows averaging 81 d in milk were allotted to eight blocks based on milk yield during the 14-d pretreatment period and randomly assigned to six treatment groups in a 2 x 3 factorial arrangement of treatments for 64 d. Factors were type of sorghum grain processing [dry-rolled vs. steam-flaked; fed at 34% of dry matter (DM) in a total mixed ration (TMR) based on alfalfa] and type of supplemental fat (2.5% of DM as cottonseed oil, tallow, or prilled fatty acids). Compared with dry-rolled sorghum, steam-flaked sorghum did not affect milk yield, fat percentage, or fat yield but did increase milk protein percentage, body weight gains, and estimated net energy for lactation (22%). Fat source did not affect lactational response, but, compared with tallow, prilled fatty acids tended to decrease DM intake. Steam-flaked sorghum, compared with dry-rolled sorghum, increased digestibilities of DM, organic matter, crude protein, and starch, regardless of fat source. The TMR containing prilled fat had lower digestibilities of DM and organic matter than did TMR containing cottonseed oil or tallow; and TMR containing prilled fat had lower digestibilities of crude protein and total fatty acids than did TMR containing tallow. This study showed that steam-flaking of sorghum grain increased milk protein content, body weight gains, and estimated net energy for lactation, regardless of dietary fat source.
Effect of steam-flaked or steam-rolled corn with or without Aspergillus oryzae in the diet on performance of dairy cows fed during hot weather.
The objective of this study was to determine the effects of steam-rolled versus steam-flaked corn in the diet with or without the addition of a culture of Aspergillus oryzae on the performance of high producing dairy cows during hot summer weather. Thirty-two Holstein cows averaging 92 (+/- 60) d in milk were fed a pretreatment diet for 21 d followed by a 70-d experimental period in a completely randomized block design with a 2 x 2 factorial arrangement of treatments. Diets were 1) steam-flaked corn plus 3 g/d of A. oryzae, 2) steam-flaked corn, 3) steam-rolled corn plus 3 g/d of A. oryzae, and 4) steam-rolled corn. Intake was not affected significantly by grain processing or addition of A. oryzae. Compared with effects from steam-rolled corn in the diet, steam-flaked corn increased milk production; percentage of milk protein; yields of milk protein, lactose, and SNF; and the efficiency of conversion of dry matter to fat-corrected milk. Addition of A. oryzae tended to increase protein percentage and increased the percentage of SNF. Changes in body weight and body condition score tended to be higher, and somatic cell count tended to be lower, for cows fed the flaked corn than for cows fed the rolled corn. No interactions were significant. Treatments did not affect rectal temperatures or respiration rates; however, high mean values measured at 1400 h once weekly indicated thermal stress. These data show improved milk production from cows fed steam-flaked corn but not from those fed diets supplemented with A. oryzae.
Soluble tumor necrosis factor receptor in serum of patients with arthritis.
Tumor necrosis factor (TNF) induces the production of two forms of soluble receptor (p55 and p75) that are present in human serum at concentrations that increase greatly in inflammatory rheumatic disease, as well as varying among healthy individuals. The purpose of this study was to evaluate the usefulness of soluble TNF receptors in distinguishing different forms of arthritis. Serum from patients with gout, rheumatoid arthritis, and osteoarthritis, and normal control subjects was analyzed for p55, p75, and TNF-alpha by enzyme-linked immunosorbent assay. Patients with gout had the highest level of soluble TNF receptor p55, while there was no significant difference in the level of this receptor between rheumatoid arthritis patients and controls. Both rheumatoid arthritis and gout patients had higher soluble TNF receptor p75 levels than osteoarthritis patients and control subjects, but there was no difference in the p75 level between rheumatoid arthritis and gout patients. Osteoarthritis patients had higher levels of p55 and lower levels of p75 than control subjects. The level of TNF-alpha in rheumatoid arthritis patients was higher than in osteoarthritis patients, gout patients, and control subjects. Determination of soluble TNF receptor levels, especially p55, might enable differentiation of rheumatoid arthritis from osteoarthritis and gout. The level of p75 cannot be utilized to differentiate rheumatoid arthritis and gout, in contrast to the results of previous investigations.