Immunohistochemical analysis of Fas ligand expression in normal human tissues

Cross-linking of Fas and Fas ligand (FasL) induces apoptosis in Fas-bearing cells and regulates apoptosis. Fas is widely expressed in normal human tissues, but FasL expression has been considered to be restricted to lymphoid tissues. Recent studies have demonstrated that FasL is also expressed in some nonlymphoid tissues. To screen the in situ expression of FasL in normal human tissues, immunohistochemistry was performed using paraffin-embedded human tissues. FasL immunostaining was easily detected in testis, neurons, trophoblasts, tonsil, lymph node, Paneth cells, hepatocytes, renal tubular epithelium and bronchial epithelium, consistent with previous reports. Surprisingly, FasL was also expressed in many other cell types, including thymic medulla, skeletal muscle, cardiac muscle, pituitary gland, parathyroid gland, prostate glands, oocytes, epithelium of fallopian tube, endometrial glands, and gastric parietal cells. These findings demonstrate that FasL is widely expressed in human tissues and suggest that wide but cell-type specific expression of FasL may not only be implicated in the regulation of immune homeostasis but also in the regulation of cell death and life in many cell types in vivo.     

Internal derangements of the shoulder: decision tree and cost-effectiveness analysis of conventional arthrography, conventional MRI, and MR arthrography

Purpose. In a patient with internal derangement of the shoulder, the diagnostic method of choice is controversial. Conventional arthrography can diagnose most rotator cuff tears accurately; however, in many institutions MR arthrography is usually necessary to diagnose labral tears. We utilized decision tree methodology to compare the cost- effectiveness of conventional arthrography and conventional MRI with a hypothetical algorithm in which a patient underwent arthrography, performed with admixed gadolinium, which if negative, was followed by MRI. Design. The use of double-contrast arthrography alone, conventional MRI alone, and gadolinium-enhanced MRI used as an adjunct to conventional arthrography were modeled for the diagnosis of full-thickness rotator cuff tears (RCT), partial RCTs, labral tears, and the absence of cuff/labral tears using decision analysis methodology. English language medical publications were searched to determine the base probabilities for the accuracy of the diagnostic tests. The outcome utilities ranged from –1 to +1 to reflect the value of correct diagnostic evaluation. Charges for diagnostic tests and appropriate surgical treatments were based on 1997 Medicare reimbursement rates for professional fees and hospital charges in an outpatient setting. Sensitivity analyses were performed to evaluate the effects of uncertainty regarding the prevalence of each disease state and the accuracy of several diagnostic tests. Results. In the base-case analysis, the average effectiveness of double-contrast arthrography alone, MRI alone and arthrography selectively followed by MRI were 0.6610, 0.6715, and 0.7204, respectively. The average costs for each of these strategies were $1090, $2033, and $2339, respectively. Conclusion. Arthrography performed with admixed diluted gadolinium, which if negative is immediately followed by MRI, was somewhat more expensive than conventional MRI. However, because of much greater effectiveness, cost-effectiveness was significantly higher for our proposed algorithm. Conventional arthrography without gadolinium, although less expensive, had severely limited effectiveness. Received: 4 June 1999 Revision requested: 6 August 1999 Revision received: 30 August 1999 Accepted: 1 September 1999     

Effect of dopamine denervation and dopamine agonist administration on serine phosphorylation of striatal NMDA receptor subunits

Sensitization of striatal N-methyl-d-aspartate (NMDA) receptors has been implicated in the pathogenesis of the response alterations associated with dopaminomimetic treatment of parkinsonian animals and patients. To determine whether serine phosphorylation of NMDA receptor subunits by activation of Ca2+/calmodulin-dependent protein-kinase II (CaMKII) contributes to this process, we examined the effects of unilateral nigrostriatal ablation with 6-hydroxydopamine and subsequent treatment with levodopa, SKF 38393 (D1-preferring dopamine agonist), or quinpirole (D2-preferring agonist) on motor responses and phosphorylation states. Three weeks of twice-daily levodopa administration to rats shortened the duration of their rotational response to levodopa or SKF 38393 challenge, but prolonged the duration of quinpirole-induced rotation. At the same time, levodopa treatment elevated serine phosphorylation of striatal NR2A (p<0.02), but not that of NR2B subunits, without associated changes in subunit protein levels. Chronic treatment with SKF 38393 increased NR2A (p<0.0001) but decreased NR2B (p<0.004) serine phosphorylation. In contrast, chronic quinpirole treatment had no effect on NR2A but increased NR2B phosphorylation (p<0.0001). The acute intrastriatal injection of the CaMKII inhibitor KN93 (1.0 micrograms) not only normalized the levodopa-induced motor response alterations but also attenuated the D1 and D2 receptor-mediated serine phosphorylation of NR2A and NR2B subunits, respectively (p<0.02). These results suggest that a CaMKII-mediated rise in serine phosphorylation of NMDA receptor subunits induced by intermittent stimulation of D1 or D2 dopaminergic receptors contributes to the apparent enhancement in striatal NMDA receptor sensitivity and thus to the dopaminergic response plasticity in levodopa-treated parkinsonian rats.     

17 Beta-estradiol increases VEGF receptor-2 and promotes DNA synthesis in retinal microvascular endothelial cells.

PURPOSE: Estrogen is known to promote angiogenesis in gonads. The presence of estrogen receptors in the vascular endothelium of organs other than gonads has been reported. The goal of this study was to determine whether estrogen promotes the proliferation of retinal microvascular endothelial cells and to explore the mechanism of it. METHODS: DNA was quantitated using primary cultures of bovine retinal endothelial cells that were incubated with different doses of 17 beta-estradiol (E2), VEGF, or both. The changes in expression level of VEGF and VEGF receptor-2 (VEGFR2) were measured using northern blot analysis after treatment with E2. The presence of estrogen receptors in the endothelial cells was studied by immunohistochemistry and western blot analysis. RESULTS: 17 Beta-estradiol (E2) increased the DNA level in bovine retinal capillary endothelial cells (BRECs) by 177% at 1 nM (P < 0.05) and 150% at 10 nM (P < 0.05) by comparison with unstimulated BREC. One hundred nanomole tamoxifen completely blocked the E2-induced DNA synthesis in BRECs. Ten nanomole E2 augmented vascular endothelial growth factor (VEGF)-induced DNA synthesis in BRECs significantly (160%, P < 0.01). Ten nanomole E2 also increased VEGF mRNA expression, which peaked after 24 hours (6.7 times, P < 0.05), and VEGF receptor-2 (VEGFR2) mRNA expression, which peaked after 9 hours (2.4 times, P < 0.05). The mRNA expression level of VEGFR2 peaked with 10 nM E2 (P < 0.05) and that of VEGF reached maximum with 1 nM E2 (15 times, P < 0.001). VEGFR2 and VEGF proteins increased in parallel with their mRNA levels. Immunocytochemistry showed estrogen receptor expression in BRECs, and western blot analysis indicated the presence of a 67-kDa protein that was compatible with the estrogen receptor. CONCLUSIONS: These findings suggest that E2 may stimulate BREC growth by the receptor-mediated pathway and that E2 may augment the VEGF-dependent angiogenesis partly through the upregulation of VEGFR2.     

Synaptic connectivity of two types of recoverin-labeled cone bipolar cells and glutamic acid decarboxylase immunoreactive amacrine cells in the inner plexiform layer of the rat retina.

We investigated the synaptic connectivity of two populations of recoverin-labeled bipolar cells and GABAergic neurons in the inner plexiform layer (IPL) of the rat retina. Two types of cone bipolar cells, type 2 and type 8, were stained with anti-recoverin antibodies, and GABAergic neurons were stained with anti-glutamic acid decarboxylase (GAD) antibodies. Type 2 cone bipolar axons received synaptic input from amacrine cell processes in 177 cases; among these amacrine cell processes, 92 processes (52.0%) were GAD-like immunoreactive. A total of 159 amacrine cell processes, which are presynaptic to type 8 cone bipolar cells, were observed. Among these processes, 117 processes (73.6%) were GAD-like immunoreactive. The postsynaptic elements at the ribbon synapses of recoverin-labeled cone bipolar cells were observed in 482 processes. In both type 2 and type 8 cone bipolar cells, the major output was to amacrine cell processes. At the ribbon synapses of the type 2 cone bipolar cells, 224 of the postsynaptic profiles were amacrine cell processes, 97 processes (43.3%) were GAD-like immunoreactive. In type 8 cone bipolar cells, 45 processes (30.2%) of 149 amacrine cell processes were GAD-like immunoreactive. Our results provide morphological evidence that GABA is a major transmitter involved in the visual processing of type 2 and 8 cone bipolar cells and GABA may have a stronger influence on type 8 cone bipolar cells than type 2 cone bipolar cells in the IPL of the rat retina.     

CHO/hPEPT1 cells overexpressing the human peptide transporter (hPEPT1) as an alternative in vitro model for peptidomimetic drugs

The present study characterized Chinese hamster ovary cells overexpressing a human intestinal peptide transporter, CHO/hPEPT1 cells, as an in vitro model for peptidomimetic drugs. The kinetic parameters of Gly-Sar uptake were determined in three different cell culture systems such as untransfected CHO cells (CHO-K1), transfected CHO cells (CHO/hPEPT1) and Caco-2 cells. Vmax in CHO/hPEPT1 cells was approximately 3-fold higher than those in Caco-2 cells and CHO-K1 cells, while Km values were similar in all cases. The uptake of beta-lactam antibiotics in CHO/hPEPT1 cells was three to twelve fold higher than that in CHO-K1 cells, indicating that CHO/hPEPT1 cells significantly enhanced the peptide transport activity. However, amino acid drugs also exhibited high cellular uptake in both CHO-K1 and CHO/hPEPT1 cells due to the high background level of amino acid transporters. Thus, cellular uptake study in CHO/hPEPT1 cells is not sensitive enough to distinguish the peptidyl drugs from amino acid drugs. The potential of CHO/hPEPT1 cells as an in vitro model for peptidomimetic drugs was also examined through the inhibition study on Gly-Sar uptake. Peptidomimetic drugs such as beta-lactam antibiotics and enalapril significantly inhibited Gly-Sar uptake whereas the nonpeptidyl compounds, L-dopa and alpha-methyldopa, did not compete with Gly-Sar for cellular uptake within the therapeutic concentrations. In conclusion, the present study demonstrates the further characterization of CHO/hPEPT1 cells as an uptake model as well as inhibition study and suggests their utility as an alternative in vitro model for drug candidates targeting the hPEPT1 transporter.     

Acute and chronic effects of ginseng total saponin and amphetamine on fixed-interval performance in rats

The effect of ginseng total saponin (GTS) on amphetamine (AMPH)-induced disruption of fixed-interval (FI) responding in rats was examined. GTS (50 mg/kg) significantly improved the temporal responding impaired by 2 mg/kg of AMPH. A higher dose of 100 mg/kg GTS disrupted performance when given alone; this disruption was reversed by a low dose of AMPH (0.5 mg/kg) and tolerance developed to the effects of GTS with its repeated administration. Neurochemical analysis revealed that GTS (50 mg/kg) attenuated the increase in striatal dopamine caused by AMPH leading to the conclusion that brain dopamine may partially mediate the behavioral effects of GTS.