Alteration of glucose uptake in cultured human corneal endothelial cells grown in high glucose media via cAMP-dependent pathway

In this study, cultured human corneal endothelial cells were incubated in media containing various concentrations of glucose at 5 mM, 10 mM, and 25 mM for 2 days. Then, the cellular 2-deoxyglucose uptake and cAMP concentration of cultured human corneal endothelial cells were measured. The results indicated that the activity of cellular glucose uptake of nmole/min/mg protein was decreased gradually from 0.18 (5 mM), 0.10 (10 mM), 0.07 (20 mM) to 0.06 (25 mM) after 2 days incubation with a high concentration of glucose. The glucose uptake in insulin-treated human corneal endothelial cells also exhibited a similar declining effect in high glucose media from 0.30 (5 mM), 0.11 (10 mM), 0.08 (20 mM) to 0.05 (25 mM). The cAMP concentration in human corneal endothelial cells was measured in the presence of high glucose media. It was indicated that the cAMP concentrations of pmole/well in both insulin-treated and non-insulin treated cells were also decreased after increasing the glucose concentration in the media from 73 (5 mM) to 20 (25 mM) and 101 (5 mM) respectively. The cAMP concentration in insulin-treated cells was less than in non-insulin treated cells. This decreasing effect was significantly reversed by the addition of 1 mM dibutyryl-cAMP to the cells for 1 hour in both groups. These results suggest that the diabetic state may decrease the 2-deoxyglucose uptake in human corneal endothelial cells via cAMP-dependent pathway.     

Presynaptic modulation by L-glutamate and GABA of sympathetic co-transmission in rat isolated vas deferens

1. The modulatory effects of L-glutamate and its structural analogues, and of gamma-aminobutyric acid (GABA), on sympathetic co-transmission were studied in the rat isolated vas deferens exposed to electrical field stimulation (EFS). 2. Application of exogenous L-glutamate caused a concentration-dependent (1 microM-3 mM) inhibition of the rapid twitch component of the biphasic EFS contraction. However, L-glutamate (1 microM-3 mM) had a minimal effect on the phasic contraction induced by exogenous adenosine 5'-triphosphate (ATP, 150 microM) and noradrenaline (50 microM). Unlike L-glutamate, D-glutamate had no effect on the EFS contraction. 3. The L-glutamate-induced inhibition of the EFS contractions was significantly attenuated by the glutamate decarboxylase (GAD) inhibitor 3-mercapto-propionic acid (150 microM) and was abolished in the presence of the GABA transaminase (GABA-T) inhibitor, 2-aminoethyl hydrogen sulphate (500 microM). 4. The L-glutamate-induced inhibition of the electrically evoked contraction was not affected by the adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX)(30 nM), reactive blue 2 (30 microM) or the GABAA receptor antagonist bicuculline (50 microM). However, the GABAB receptor antagonist 2-hydroxysaclofen (50 microM) significantly inhibited the L-glutamate effect. 5. Similar to L-glutamate, GABA also caused a concentration-dependent (0.1-100 microM) inhibition of the EFS contractions. This GABA-induced inhibition was not affected by either the GABAA receptor antagonist bicuculline (50 microM) or reactive blue 2 (30 microM). However, a significant attenuation of the GABA-mediated effect was recorded with the GABAB receptor antagonist 2-hydroxysaclofen (50 microM). Contractions of the vas deferens induced by exogenous ATP and noradrenaline were not affected by GABA (0.1-100 microM). 6. The L-glutamate analogues, N-methyl-D-aspartate (NMDA) (1 microM-1 mM) and quisqualate (Quis 0.1 microM-0.3 mM) had no effect, whilst kainate (Kain, 1 microM-1 mM) caused an inhibition of the EFS-induced contractions. Effects of Kain could be abolished by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dioxine (CNQX, 10 microM). NMDA, Quis and Kain had no effect on the exogenous ATP- or noradrenaline-induced contractions. 7. It is concluded that the excitatory amino acid L-glutamate modulates the electrically evoked vas deferens contraction through conversion to the inhibitory amino acid GABA by a specific GABA transaminase. The GABA formed may then act on GABAB receptors and cause inhibition of the contraction through a presynaptic mechanism.     

Diagnostic thoracic-computed tomography in radiotherapy for loco-regional recurrent breast carcinoma

OBJECTIVE: To define the composition of cytoplasmic inclusions forming stacks and concentric whorls in histiocytes and mesothelial cells of serous fluids, imparting to them a resemblance to Gaucher cells, and to draw conclusions on the mechanism of their formation. STUDY DESIGN: Three serous fluids (one pleural and two pericardial) containing a fair number of the cells referred to were progressively subjected to the following studies: (1) cytochemistry for mucopolysaccharides, proteins, phospholipids and hemoglobin; (2) immunocytochemistry for immunoglobulins IgA, IgG, IgM and lysozyme; (3) transmission electron microscopy (TEM), and (4) scanning electron microscopy-based energy dispersive X-ray microanalysis (SEM-EDAX). RESULTS: All three specimens were blood stained and contained large numbers of histiocytes and mesothelial cells, arranged singly and in groups, with abundant cytoplasmic inclusions. The inclusions stained strongly positive for phospholipids, weakly positive for hemoglobin and negative for all other substances examined by cytochemistry and immunocytochemistry. By TEM the inclusions had a concentric lamellar membranous structure, reminiscent of myelinosomes or lamellar bodies of lipid-forming or -storing cells. There was also phagocytosis by histiocytes and mesothelial cells of red blood cells, which were mostly in a degenerated state. SEM-EDAX of inclusion-bearing cells showed a modest peak for phosphorus and a variable but small peak for iron, which corroborated the cytochemical and TEM findings. CONCLUSION: Since there was not metabolic or other systemic disease in the patients to account for these cells, we posit that phospholipids derived from cell membranes of phagocytized cells, especially red blood cells, provide the building blocks for the formation of such inclusions as they enter the metabolic pathway of phagocytic cells (mesothelial cells and histiocytes) and appear in their lysosomal structures. It is advantageous for cytologists to be familiar with significance of such changes and not to mistake them for metabolic or other systemic disease.     

Dexon and nylon-sutured wound reaction in conjunctival flap after trabeculectomy combined with or without topical application of mitomycin-C

In this study, rabbits were used to evaluate the sutured wound reaction with Dexon or nylon in the conjunctival flap 1, 4, 7, 14 and 28 days after trabeculectomy surgery with or without the use of mitomycin-C. Four major treated groups were used to compare their wound healing reaction; group 1--nylon-suture and non-mitomycin treatment; group 2--nylon-suture and mitomycin treatment; group 3--Dexon-suture and non-mitomycin treatment; group 4--Dexon-suture and mitomycin treatment. One day after surgery, the number of polymorphs was the greatest most in the nylon-sutured and non-mitomycin treated tissues (86 +/- 2). Four days after surgery, the number of polymorphs was the greatest most in Dexon-sutured and non-mitomycin treated tissues (109 +/- 87). The number of fibroblasts was the greatest most in nylon-sutured and non-mitomycin treated tissues (111 +/- 23). Seven days after surgery, the number of polymorphs was the greatest most in Dexon-sutured and mitomycin treated tissues (32 +/- 12). The number of fibroblasts was the greatest most in nylon-sutured and non-mitomycin treated tissues (126 +/- 15). Fourteen days after surgery, the number of fibroblasts was the greatest most in Dexon-sutured and non-mitomycin tissues (43 +/- 10). The number of goblet cells was the greatest most in nylon-sutured and non-mitomycin treated tissues (4 +/- 2). Twenty-eight days after surgery, the number of fibroblasts was the greatest most in Dexon-sutured and mitomycin treated tissues (40 +/- 15). The number of goblet cells was the greatest most in nylon-sutured and non-mitomycin treated tissues (4 +/- 2). Our conclusions are as follows: 1). The concentration of mitomycin in conjunctival wound edge should be maintained at as low a level as possible because the mitomycin will delay the wound healing process; 2). Nylon material is better than Dexon for conjunctival wound suture because nylon could induce a great quantity of fibroblasts before Dexon did.     

A rapid, sensitive and automated method for detection of Salmonella species in foods using AG-9600 AmpliSensor Analyzer

The AG-9600 AmpliSensor Analyzer is an automated fluorescence-based system for detection of polymerase chain reaction (PCR) products. The principle of the AmpliSensor PCR assay involves amplification-mediated disruption of a fluorogenic DNA signal duplex (AmpliSensor) that is homologous to a target sequence within a 284-bp amplified fragment of the Salmonella invA gene. Since the assay is homogenous, the data can be obtained by direct measurement of fluorescence of the amplification mixture. The accumulation of the amplified product, reflected by the fluorescence index, is monitored cycle by cycle by the AG-9600 Analyzer. The detection limit of the assay was less than 2 colony-forming units (cfu) per PCR reaction using a pure culture of Salmonella typhimurium. In post-spiking experiments in which Salmonella was added to the overnight pre-enriched samples (chicken carcass rinses, ground beef, ground pork and raw milk), the detection limit of the assay was 2-6 cfu per PCR reaction. In pre-spiking experiments in which Salmonella was added to the samples prior to overnight pre-enrichment, the detection limit was less than 3 cfu per 25 g or 25 ml of food. The assay was up to 2 orders of magnitude more sensitive than detection by ethidium bromide-stained agarose gel electrophoresis. To further evaluate assay performance, 54 naturally contaminated chicken carcass rinses, 65 raw milk and six ground pork samples were tested in the study. Thirty-eight Salmonella-positive samples confirmed by the Modified Semi-solid Rappaport-Vassiliadis (MSRV) culture assay were found positive using the AmpliSensor assay. Two chicken carcass rinses found positive using the assay were MSRV-negative. In addition, relative quantification using the AmpliSensor assay was linear up to 3 logs of initial target concentration in artificially contaminated food samples.     

CGG repeat interruptions in the FMR1 gene in patients with infantile autism

We determined the CGG repeat length and AGG interruptions in the FMR1 gene in normal Chinese subjects and patients with infantile autism and mild mental retardation. Genomic DNA was investigated by PCR and Southern hybridisation for CGG repeat number and PCR with Mnl I restriction analysis for AGG interruption. Both the normal subjects and the patients with autism have 53 CGG repeats in FMR1, and the majority have two interspersed AGG. Our normal Chinese subjects have a similar number of interspersed AGG as other populations. When compared with the normal subjects, the autism patients have less AGG interruptions and a different pattern of AGG distribution. There was a significant difference in the CGG configurations between normal subjects and patients with autism. The latter had less interspersed AGG, as in fragile X patients, but they did not have fragile X. A study on mentally retarded patients with no infantile autism should also be carried out to ascertain whether mental retardation alone may have contributed to such AGG pattern.     

Perturbation of fuel homeostasis caused by overexpression of the glucose-6-phosphatase catalytic subunit in liver of normal rats

The terminal step in hepatic gluconeogenesis is catalyzed by glucose-6-phosphatase, an enzyme activity residing in the endoplasmic reticulum and consisting of a catalytic subunit (glucose-6-phosphatase (G6Pase)) and putative accessory transport proteins. We show that Zucker diabetic fatty rats (fa/fa), which are known to exhibit impaired suppression of hepatic glucose output, have 2.4-fold more glucose-6-phosphatase activity in liver than lean controls. To define the potential contribution of increased hepatic G6Pase to development of diabetes, we infused recombinant adenoviruses containing the G6Pase cDNA (AdCMV-G6Pase) or the beta-galactosidase gene into normal rats. Animals were studied by one of three protocols as follows: protocol 1, fed ad libitum for 7 days; protocol 2, fed ad libitum for 5 days, fasted overnight, and subjected to an oral glucose tolerance test; protocol 3, fed ad libitum for 4 days, fasted for 48 h, subjected to oral glucose tolerance test, and then allowed to refeed overnight. Hepatic glucose-6-phosphatase enzymatic activity was increased by 1.6-3-fold in microsomes isolated from AdCMV-G6Pase-treated animals in all three protocols, and the resultant metabolic profile was similar in each case. AdCMV-G6Pase-treated animals exhibited several of the abnormalities associated with early stage non-insulin-dependent diabetes mellitus, including glucose intolerance, hyperinsulinemia, decreased hepatic glycogen content, and increased peripheral (muscle) triglyceride stores. These animals also exhibited significant decreases in circulating free fatty acids and triglycerides, changes not normally associated with the disease. Our studies show that overexpression of G6Pase in liver is sufficient to perturb whole animal glucose and lipid homeostasis, possibly contributing to the development of metabolic abnormalities associated with diabetes.     

Fused aminotetralins: novel antagonists with high selectivity for the dopamine D3 receptor

Starting from a series of 2-aminotetralins 1, a novel series of N-[4-(4-phenylbenzoylamino)butyl]-octahydrobenzoquinolines and hexahydrobenzoindoles with high potency and selectivity for the dopamine D3 receptor has been designed. The effect of ligand chirality on binding affinity has been established. Selected derivatives (e.g. 2o, 2p) show high functional selectivity and enhanced in vivo properties compared to 1.