Convenient colorimetric and fluorometric assays for S-nitrosothiols

S-nitrosothiols have been shown to affect a number of physiological functions. Several techniques have been used to detect these species in biological systems, primarily by methods utilizing chemiluminescence. Since the apparatus required for measurement of chemiluminescence are not readily available in most laboratories, methods employing more conventional techniques such as uv-vis and fluorescence spectroscopy may be of greater use. Herein, we report the development of colorimetric and fluorometric methods for the reliable quantitation of S-nitrosothiols. Solutions containing sulfanilamide/N-(1-naphthyl)- ethylenediamine dihydrochloride or 2,2'-azinobis (3-ethylbenzthiazoline-6-sulfonic acid), when exposed to S-nitrosoglutathione (GSNO), S-nitrosocysteine, or S-nitrosoacteylpenicillamine, resulted in no absorbance changes in the range of 400-800 nm. Exposure to HgCl2 or Cu(acetate)2 resulted in release of nitric oxide (NO) from the S-nitrosothiols. The liberated NO reacted subsequently with oxygen and formed a chemical species which reacted with either analysis solution, resulting in an increase in absorption between 400 and 800 nm. A plot of RSNO versus absorbance was linear for both mercury(II) and copper(II) ions where the slope in the presence of mercury ion was significantly greater than that for copper ion. The sensitivity was as low as 5 microM RSNO using HgCl2. The fluorometric method using 2, 3-diaminonaphthalene as the scavenger of the NOsolidusO2 products gave a sensitivity of 50 nM for GSNO. In addition, S-nitrosylated proteins were quantitated using the fluorometric technique. These methods provide accurate determination of low concentrations of S-nitrosothiols, utilizing conventional spectroscopic techniques available in most laboratories.     

Videographic and histologic comparison of Nd:YAG and diode laser contact transscleral cyclophotocoagulation

We performed contact transscleral cyclophotocoagulation in two human autopsy eyes with continuous-wave Nd:YAG and diode lasers. Duration of exposure was two seconds, and powers were 4 to 7.3 W with the Nd:YAG laser and 1.75 to 2.5 W with the diode laser. In both procedures, we used hand-held quartz fiberoptic contact probes for energy delivery. Tissue responses were viewed with high-magnification videographic recording technique to analyze the real-time laser effects. The treated tissues were then studied by light microscopy. We observed different tissue responses both videographically and histologically. Nd:YAG laser lesions were characterized by prominent tissue whitening and contraction of the ciliary epithelium, while the diode laser lesions had less whitening and the tissue contraction appeared to be deeper in the ciliary body. The histologic correlate was coagulation and disruption of the ciliary epithelium and little effect on the underlying ciliary muscle with the Nd:YAG laser, while the diode laser had less effect on the ciliary epithelium but caused a significant coagulative response in the ciliary muscle. Comparative trials are needed to establish the clinical significance of these videographic and histologic observations.     

Triggering the false suffocation alarm in panic disorder patients by using a voluntary breath-holding procedure

The authors compared the maximal duration of voluntary breath-holding in patients with panic disorder (N = 23), patients with generalized social phobia (N = 10), and healthy subjects (N = 26). Patients with panic disorder had significantly shorter breath-holding durations than either comparison group. Groups did not otherwise differ in physiologic response to the breath-holding. Implications for a false suffocation alarm in panic disorder are discussed.     

Lesion-to-background ratio in nonattenuation-corrected whole-body FDG PET images

The purpose of this study was to compare the diagnostic efficacy of attenuation-corrected and nonattenuation-corrected whole-body 18F-fluorodeoxyglucose (FDG) PET images to determine an adequate method that can semiquantitatively evaluate nonattenuation-corrected images. METHODS: Whole-body PET studies were performed in 24 fasting patients with various tumors (lung cancers, n = 18; mediastinal tumors, n = 4; breast cancers, n = 2) 30-40 min after a bolus injection of 18F-FDG. Transmission scans followed emission data acquisition. Reconstructed attenuation-corrected and uncorrected images were displayed simultaneously and the relative FDG uptake in lesions and corresponding background areas was evaluated by the region of interest method. Both types of images were also compared with X-CT scans and conventional nuclear medicine scans for diagnostic efficacy. RESULTS: Attenuation-corrected and uncorrected images were found to be equally sensitive for detecting lesions. There was a strong linear correlation between lesion-to-background (L/B) ratios calculated on attenuation-corrected and uncorrected images (r = 0.98; p < 0.001). Significant differences in L/B ratios between attenuation-corrected and uncorrected images were present in only 6 of 55 lesions (11%). Standardized uptake ratios (SURs) in attenuation-uncorrected images did not correlate with SURs in attenuation-corrected images nor with L/B ratios in uncorrected images. CONCLUSION: The efficacy of attenuation-uncorrected FDG PET images in evaluating tumors is similar to that using attenuation-corrected images. Uncorrected images provide not only clinically useful but also quantitative information equivalent to that provided by attenuation-corrected images. However the L/B ratio is the only available index that can be used for quantification of uncorrected images.     

Nephrectomy before interleukin-2 therapy for patients with metastatic renal cell carcinoma

PURPOSE: The management of metastatic renal cell carcinoma remains challenging and controversial. There is some evidence of improved response to interleukin-2 (IL-2) based immunotherapy in patients who undergo nephrectomy before systemic treatment. However, recent reports have suggested that surgery prior to immunotherapy may not be an efficient strategy, since many patients will not be able to receive systemic treatment after nephrectomy. We describe our criteria for determining which patients are candidates for nephrectomy before immunotherapy and present our series of patients treated with this approach. MATERIALS AND METHODS: Based on our initial experience with IL-2 based immunotherapy we developed certain inclusion criteria for treatment with initial nephrectomy followed by systemic immunotherapy, including greater than 75% debulking of tumor burden possible, no central nervous system, bone or liver metastases, adequate pulmonary and cardiac function, and Eastern Cooperative Oncology Group performance status of 0 or 1. In addition, patients in whom biopsies show other than predominantly clear cell type histology are excluded. From 1991 through 1996, 28 patients met these criteria and were treated with this approach. Patients were followed to determine the number receiving immunotherapy as well as overall response and survival rates. RESULTS: Radical nephrectomy was performed in all patients. One patient died of respiratory failure from disease progression 1 month after nephrectomy. Another patient had poor pulmonary function and, therefore, was treated with an alternative cytokine therapy. The remaining 26 patients (93%) received at least 1 course of IL-2. Median interval between nephrectomy and initiation of immunotherapy was 1.5 months (range 1 to 3). Overall response rate was 39% with 5 complete (18%) and 6 partial (21%) responses. Actuarial median survival of the entire group was 20.5 months (range 1 to 66) from the initiation of treatment. Currently 13 patients are alive, including 8 who are disease and/or progression-free. CONCLUSIONS: Using these strict criteria nephrectomy can be effectively performed before immunotherapy without compromising the likelihood that patients will receive systemic treatment. The activity of IL-2 in patients treated with this approach is encouraging and justifies its consideration in properly selected patients.     

Effect of calcium intake on nonheme-iron absorption from a complete diet

Recent studies based on radioiron measurements from single meals have suggested that calcium has a strongly inhibitory influence on nonheme-iron absorption. In view of evidence that the importance of various dietary enhancers and inhibitors of absorption is greatly diminished when assessed by labeling a complete diet, the present study evaluated the effect of variations in calcium intake on total dietary nonheme-iron absorption. Nonheme-iron absorption was measured in 14 healthy volunteers during three periods in which the diet was freely chosen or modified to decrease or increase dietary calcium intake maximally. The diet was labeled during each 5-d period by including with each of the two main meals of the day a small bread roll tagged extrinsically with radioiron. Carefully maintained dietary records indicated that 69-78% of the daily iron intake was labeled by this method. The basal calcium intake of 684 mg/d varied from 280 to 1281 mg/d when calcium intake was reduced or increased, respectively. Geometric mean iron-absorption values of 5.01%, 4.71%, and 5.83% for the three dietary periods were not significantly different from one another. No significant relation was observed between nonheme-iron absorption and dietary factors known to influence iron absorption. We conclude that calcium intake had no significant influence on nonheme-iron absorption from a varied diet.     

Use of chlorotoxin for targeting of primary brain tumors

Gliomas are primary brain tumors that arise from differentiated glial cells through a poorly understood malignant transformation. Although glioma cells retain some genetic and antigenic features common to glial cells, they show a remarkable degree of antigenic heterogeneity and variable mutations in their genome. Glioma cells have recently been shown to express a glioma-specific chloride ion channel (GCC) that is sensitive to chlorotoxin (CTX), a small peptide purified from Leiurus quinquestriatus scorpion venom [N. Ullrich et al, Neuroreport, 7: 1020-1024, 1996; and N. Ullrich and H. Sontheimer, Am. J. Physiol. (Cell Physiol.), 270: C1511-C1521, 1996]. Using native and recombinant 125I-labeled CTX, we show that toxin binding to glioma cells is specific and involves high affinity [dissociation constant (Kd)=4.2 nM] and low affinity (Kd=660 nml) binding sites. In radioreceptor assays, 125I-labeled CTX binds to a protein with Mr=72,000, presumably GCC or a receptor that modulates GCC activity. In vivo targeting and biodistribution experiments were obtained using 125I- and (131)I-labeled CTX injected into severe combined immunodeficient mice bearing xenografted gliomas. CTX selectively accumulated in the brain of tumor-bearing mice with calculated brain: muscle ratios of 36.4% of injected dose/g (ID/g), as compared to 12.4% ID/g in control animals. In the tumor-bearing severe combined immunodeficient mice, the vast majority of the brain-associated radioactivity was localized within the tumor (tumor:muscle ratio, 39.13% ID/g; contralateral brain:muscle ratio, 6.68%ID/g). Moreover, (131)I-labeled CTX distribution, visualized through in vivo imaging by gamma ray camera scans, demonstrates specific and persistent intratumoral localization of the radioactive ligand. Immunohistochemical studies using biotinylated and fluorescently tagged CTX show highly selective staining of glioma cells in vitro, in situ, and in sections of patient biopsies. Comparison tissues including normal human brain, kidney, and colon were consistently negative for CTX immunostaining. These data suggest that CTX and CTX-conjugated molecules may serve as glioma-specific markers with diagnostic and therapeutic potential.     

Efficient adaptive learning for classification tasks with binary units

This article presents a new incremental learning algorithm for classification tasks, called NetLines, which is well adapted for both binary and real-valued input patterns. It generates small, compact feedforward neural networks with one hidden layer of binary units and binary output units. A convergence theorem ensures that solutions with a finite number of hidden units exist for both binary and real-valued input patterns. An implementation for problems with more than two classes, valid for any binary classifier, is proposed. The generalization error and the size of the resulting networks are compared to the best published results on well-known classification benchmarks. Early stopping is shown to decrease overfitting, without improving the generalization performance.