Dual-function reporter protein for analysis of gene expression in living cells

The firefly luciferase (Luc) protein and the jellyfish green fluorescent protein (GFP) are two commonly used molecular reporters that can be detected noninvasively in living cells. The properties that make GFP or Luc useful for a particular experimental application are quite distinct. A recombinant protein with both fluorescent and bioluminescent characteristics might take advantage of the strengths of both reporters. An expression vector encoding a chimeric protein in which GFP was tethered to Luc through a 19-amino acid linker was prepared and characterized. Western blotting with antibodies specific for either GFP or Luc showed that a protein of appropriate size was expressed in transfected cells. Fluorescence microscopy revealed bright green fluorescence from transfected cells, indicating proper formation of the GFP chromophore. Luc enzymatic activity in protein extracts from transfected cells showed that Luc was fully functional. The treatment of living cell cultures stably expressing the GFP-Luc fusion protein with the protein translation-inhibitor cycloheximide (Chx) was used to show that the half-life for Luc protein activity was approximately 2 h at 37 degrees C. The utility of this dual-function reporter protein was shown by the identification of single living cells expressing the chimeric protein within a population by fluorescence microscopy, followed by quantification of Luc activity from the same living cells.     

International surveillance of bloodstream infections due to Candida species: frequency of occurrence and antifungal susceptibilities of isolates collected in 1997 in the United States, Canada, and South America for the SENTRY Program. The SENTRY Participant Group

An international program of surveillance of bloodstream infections (BSIs) in the United States, Canada, and South America between January and December 1997 detected 306 episodes of candidemia in 34 medical centers (22 in the United States, 6 in Canada, and 6 in South America). Eighty percent of the BSIs were nosocomial and 50% occurred in patients hospitalized in an intensive care unit. Overall, 53.3% of the BSIs were due to Candida albicans, 15.7% were due to C. parapsilosis, 15.0% were due to C. glabrata, 7.8% were due to C. tropicalis, 2.0% were due to C. krusei, 0.7% were due to C. guilliermondii, and 5.8% were due to Candida spp. However, the distribution of species varied markedly by country. In the United States, 43.8% of BSIs were due to non-C. albicans species. C. glabrata was the most common non-C. albicans species in the United States. The proportion of non-C. albicans BSIs was slightly higher in Canada (47.5%), where C. parapsilosis, not C. glabrata, was the most common non-C. albicans species. C. albicans accounted for 40.5% of all BSIs in South America, followed by C. parapsilosis (38.1%) and C. tropicalis (11.9%). Only one BSI due to C. glabrata was observed in South American hospitals. Among the different species of Candida, resistance to fluconazole (MIC, > or = 64 microg/ml) and itraconazole (MIC, > or = 1.0 microg/ml) was observed with C. glabrata and C. krusei and was observed more rarely among other species. Isolates of C. albicans, C. parapsilosis, C. tropicalis, and C. guilliermondii were all highly susceptible to both fluconazole (99.4 to 100% susceptibility) and itraconazole (95.8 to 100% susceptibility). In contrast, 8.7% of C. glabrata isolates (MIC at which 90% of isolates are inhibited [MIC90], 32 microg/ml) and 100% of C. krusei isolates were resistant to fluconazole, and 36.9% of C. glabrata isolates (MIC90, 2.0 microg/ml) and 66.6% of C. krusei isolates were resistant to itraconazole. Within each species there were no geographic differences in susceptibility to fluconazole or itraconazole.     

Neural network analysis of combined conventional and experimental prognostic markers in prostate cancer: a pilot study

Radiolabeling permits the detection of trace amounts of zwitterionic detergent remaining in extracted hydrophobic or membrane proteins. To develop a sensitive and specific assay for its presence, the commonly used zwitterionic membrane protein detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) was synthesized in a tritiated form. Synthesis via 7-ketodeoxycholic acid gave [7-3H]Chaps in 53% yield with a specific activity of 0.85 mCi/mmol. A novel solvent extraction system for cholic acid obviated the need for chromatographic isolation of this intermediate. The protocol can be readily modified to yield [7-3H]Chaps of higher specific activity. [7-3H]Chaps was used to monitor the efficiency of various strategies for detergent removal from concentrated bacterial culture supernatants containing 0.2% (w/v) Chaps. Dialysis removed 95% of Chaps and the addition of detergent-affinity beads to the dialysis buffer resulted in 97% removal of Chaps. Gel-filtration chromatography removed 99.9% of Chaps, while a detergent-affinity bead chromatography column removed 99.99%. Overall, gel-filtration chromatography was the most convenient and economical method for the one-step removal of the zwittergent from complex biological mixtures.     

Potential application of p53 as an intermediate biomarker in Barrett's esophagus

Diagnosis of the neoplastic progression in Barrett's esophagus using the histologic classification of dysplasia is frequently difficult. The tumor suppressor protein p53, when mutated, confers a promoter effect on cell growth. The purpose of this study was to evaluate the applicability of p53 as an intermediate biomarker of malignancy in Barrett's esophagus. Archival analysis of 100 biopsy specimens of Barrett's esophagus and 10 esophageal adenocarcinomas were compared with 35 chronic esophagitis biopsy specimens. Immunocytochemistry using an anti-p53 monoclonal antibody was performed and elevated immunoreactivity quantitated microscopically. Data were analyzed using a logistic regression model. Significant p53 immunoreactivity occurred as follows: chronic esophagitis (0%), Barrett's esophagus without dysplasia (10%), with low-grade dysplasia (60%), with high-grade dysplasia (100%), and adenocarcinoma (70%). All cases of Barrett's esophagus were significantly immunoreactive when compared with the chronic esophagitis cases (p = 0.001). There was an increase in p53 immunoreactivity as the histologic classification progressed toward adenocarcinoma (p = 0.001). Progression to high-grade dysplasia may be predicted based on p53 immunoreactivity. These findings suggest a role for p53 as an intermediate biomarker in Barrett's esophagus.     

Use of X-ray fluorescence for sorting vinyl from other packaging materials in municipal solid waste

X-ray fluorescence of the chlorine atom is a suitable method for identifying vinyl in a mixed plastics stream in municipal solid waste. The chlorine X-ray is weak and does not penetrate paper labels. There is also a rapid decrease in measured chlorine X-ray intensity as the sample is moved away from the X-ray source and detector which could be a potential problem for bottles of uneven shape.     

Electrolysis: observations from 13 years and 140,000 hours of experience

Electrolysis has been performed since 1875. Electrolysis satisfactorily removes hair from women with static hair growth, but women with hirsutism often require concomitant management of their hormonal problems. We have found the blend method to be the most effective modality for permanent hair removal. Attention must be given to proper electrolysis technique, including accurate needle insertion and appropriate intensities and duration. Scarring does not occur with properly performed electrolysis. Hair is not an electrical conductor and electronic tweezers do not result in permanent hair removal. Shaving 1 to 5 days before electrolysis greatly increases efficacy because it ensures that only growing anagen hairs are epilated. The recent availability of EMLA (eutectic mixture of local anesthetics) has been beneficial in reducing the sensations of electrolysis. The availability of prepackaged, presterilized, individual electrolysis needles has greatly reduced the need for more complicated sterilization procedures.     

The fibrinolytic effects of intermittent pneumatic compression: mechanism of enhanced fibrinolysis

BACKGROUND AND OBJECTIVES: Intermittent pneumatic compression (IPC) is an effective form of deep vein thrombosis prophylaxis for general surgery patients. The antithrombotic effect of IPC is thought to be the result of increased venous velocity and stimulation of endogenous fibrinolysis. However, the mechanism of enhanced fibrinolytic activity and the relative effects on normal and postthrombotic veins have not been defined. The purposes of this study are 1) to quantify changes in fibrinolytic activity with IPC; 2) to study the mechanism of fibrinolytic enhancement with IPC; and 3) to evaluate whether postthrombotic patients have the same capacity for fibrinolytic enhancement with IPC as do normal subjects. METHODS: Twelve volunteers (6 normal and 6 postthrombotic) had 5 IPC devices applied for 120 minutes in random fashion, 1 per week x 5 weeks. The devices included single-chamber, sequential, foot, calf, and long-leg compression. Subjects had an indwelling antecubital venous cannula placed for blood drawn at baseline, 60, 120, and 180 minutes after IPC devices were applied. Global fibrinolytic activity (euglobulin fraction, fibrin plate assay), tissue plasminogen activator (tPA) antigen (Ag) and activity (Act), plasminogen activator inhibitor-1 (PAI-1) Ag and Act, alpha-2-antiplasmin-plasmin complexes, and von Willebrand factor (vWF) antigen were assayed. RESULTS: A striking elevation in fibrinolytic activity was noted at 180 minutes with all devices in normal subjects and postthrombotic patients (p = 0.01-0.0001); however, baseline and stimulated fibrinolytic activity was attenuated in postthrombotic patients (<0.03). The tPA-Act increased only in normal subjects (3.8 +/- 1.9%) (p = 0.057), despite a decrease in plasma tPA-Ag, which was observed in both normal subjects (-12.4 +/- 3.8%) (p = 0.009) and patients (-17.2 +/- 3.1%) (p = 0.001). PAI-1-Ag decreased in both normal subjects (-13.4 +/- 3.8%) (p = 0.007) and patients (-12.0 +/- 3.1%) (p = 0.013) with a marked reduction in PAI-1-Act in both normal subjects (p = 0.003) and patients (p = 0.004). There were no changes in vWF, and alpha-2-antiplasmin-plasmin complexes increased only in postthrombotic patients (p = 0.021). CONCLUSIONS: Stimulation of endogenous fibrinolytic activity occurs after IPC, both in normal subjects and postthrombotic patients; however, baseline and overall fibrinolytic response in postthrombotic patients is reduced. The mechanism of increased fibrinolytic activity is likely because of a reduction in PAI-1, with a resulting increase of tPA activity.