Rapid detection of Escherichia coli O157:H7 in ground beef using a fiber-optic biosensor.

A portable fiber-optic biosensor was used to detect Escherichia coli O157:H7 in seeded ground beef samples. The principle of the system is a sandwich immunoassay using cyanine 5 dye-labeled polyclonal anti-E. coli O157:H7 antibodies for generation of a specific fluorescent signal. Signal acquisition is effected by launching light from a 635-nm diode laser into a dual tapered 600-microm silica fiber. Fluorescent molecules within approximately 100 nm of the fiber surface are excited by the evanescent field, and a portion of the emission recouples into the fiber. A photodiode allows for quantitation of the collected emission light at wavelengths of 670 to 710 nm. Biotin-avidin interactions are used to attach polyclonal antibodies specific for E. coli O157:H7 to the final 7.5 cm of the fiber probe. The biosensor was able to detect E. coli O157:H7 to 3 to 30 CFU/ml in seeded ground beef samples. The reaction was highly specific. Signals with Listeria monocytogenes, Salmonella Typhimurium, or E. coli nonO157:H7 were 2 to 3% of those observed with a similar concentration of E. coli O157:H7. Assays were conducted at or near real-time with results obtained within 20 min of sampling.

In vivo sequence diversity of the protease of human immunodeficiency virus type 1: presence of protease inhibitor-resistant variants in untreated subjects.

We have evaluated the sequence diversity of the protease human immunodeficiency virus type 1 in vivo. Our analysis of 246 protease coding domain sequences obtained from 12 subjects indicates that amino acid substitutions predicted to give rise to protease inhibitor resistance may be present in patients who have not received protease inhibitors. In addition, we demonstrated that amino acid residues directly involved in enzyme-substrate interactions may be varied in infected individuals. Several of these substitutions occurred in combination either more or less frequently than would be expected if their appearance was independent, suggesting that one substitution may compensate for the effects of another. Taken together, our analysis indicates that the human immunodeficiency virus type 1 protease has flexibility sufficient to vary critical subsites in vivo, thereby retaining enzyme function and viral pathogenicity.

Role of ECMO in the treatment of respiratory syncytial virus bronchiolitis: a collaborative report.

AIM: To report the collaborative experience of extracorporeal membrane oxygenation (ECMO) in the treatment of respiratory syncytial virus (RSV) bronchiolitis between April 1989 and January 1995. METHODS: The medical records of patients with confirmed RSV bronchiolitis referred to three centres (Leicester, Glasgow, and Great Ormond Street) were reviewed. RESULTS: Twenty four infants were identified. Seventeen had been born prematurely (gestational range 23-40 weeks, median 30 weeks). Thirteen infants had been mechanically ventilated after birth and seven of these had evidence of bronchopulmonary dysplasia (BPD). The age of onset of RSV infection varied from three to 64 weeks (mean 17.4 weeks, median 12 weeks). Ventilation before ECMO ranged from one to 16 days and oxygenation indices at the time of referral ranged from 21-73 (mean 39). Ribavirin was used in eight of the 24 patients. Sixteen patients received venoarterial and eight veno-venous ECMO. ECMO hours ranged from 32-647 (median 196 hours). One infant died (survival rate 96%). Cranial ultrasound abnormalities were detected in three patients. However, at follow up only one of the 23 survivors had evidence of developmental delay. CONCLUSION: A group of paediatric patients in whom ECMO can be of benefit has been identified. The use of ECMO should be considered when other means of support prove unsuccessful.

Development of a medical fiber-optic pH sensor based on optical absorption.

A new fiber-optic pH sensor system has been developed. The sensor uses an absorbtive indicator compound with a long wave-length absorption peak near 625 nm; change in absorption over the pH range 6.8 to 7.8 is reasonably linear. The sensor is interrogated by a pulsed, red LED. Return light signal is split into short and long wave-length components with a dichroic mirror; the respective signals are detected by photodiodes, and their photocurrents are used to form a ratiometric output signal. In laboratory tests, the sensor system provided resolution of 0.01 pH, accuracy of +/- 0.01 pH, and response time of 30-40 s. Following gamma sterilization, laboratory sensor testing with heparinized human blood yielded excellent agreement (e.g., r = 0.992 for n = 42) with a clinical blood gas analyzer. Excellent sensor performance and low cost, solid-state instrumentation are hallmarks of this sensor-system design.

Development of a medical fiber-optic oxygen sensor based on optical absorption change.

A new fiber-optic oxygen sensor has been developed for use in medical applications. The sensor's viologen indicator becomes strongly absorbent after brief UV stimulation, and then returns to the transparent state. The rate of indicator return to transparency is proportional to the local oxygen concentration. Indicator absorbance is monitored with a red LED and receiving photodiode, and absorbance data are processed by a dedicated cpu. The solid-state sensor system has performance comparable to existing oxygen measurement techniques, and may be applicable for both in vitro and in vivo oxygen measurements.

Molecular characterization of the sex steroid binding protein (SBP) of plasma. Re-examination of rabbit SBP and comparison with the human, macaque and baboon proteins.

Physico-chemical characterization of the sex steroid-binding protein, SBP, of rabbit plasma reveals that it is a dimer of mol. wt 85,800 composed of similar subunits of mol. wt 43,000. These data confirm our original proposal for a dimeric structure. The protein contains 9% carbohydrate, comprised of mannose, galactose, N-acetylglucosamine and sialic acid. It is devoid of N-acetylgalactosamine and fucose. The protein binds one molecule of 5 alpha-dihydrotestosterone per dimer with a Kd of 0.89 nM (12 degrees C). Comparison with the human, monkey and baboon SBPs indicates that all these proteins have the same dimeric molecular organization and exhibit microheterogeneity in SDS-PAGE and isoelectricfocusing. Rabbit SBP, however, contains less carbohydrate and has a higher polypeptide molecular weight than all the other SBPs. Spectrophotometric data also indicate that some tryptophan residues are in a different chemical environment than those in other SBPs. The observed microheterogeneity in all four SBP species is due for the most part to variable glycosylation of the subunit and variability at the amino-terminal region of the subunit. Combination of these and other phenomena will generate a significant number of isomeric forms of the SBP subunit which will then interact stoichiometrically to yield active dimeric SBP molecules. These differ slightly from each other depending upon the charge and size of the subunit comprising the dimeric structure, and will result in the observed microheterogeneity of pure SBP preparations. Based on these results along with more recent amino acid sequence data, we conclude that all four SBPs are dimers composed of identical polypeptide chains.

Omega chain methylated analogs of PGF2 alpha and PGE2.

Our previously published prostaglandin (PG) synthesis route, in which the omega-chain is added in the penultimate step, provides facile access to a wide variety of omega-chain variant PG analogs. Each series requires only the synthesis of the appropriate methylated acylphosphonate for the Emmons' condensation. The syntheses of analogs bearing the following methylation patterns are detailed: 15-Me; 17,17-(Me) 2; 17, 17, 20-(Me) 3; 18, 18, 20-(Me) 3; 15, 18, 18, 20-(Me) 4; and 15-OMe-18, 18, 20- (Me) 3. The well-known 16., 16-dimethyl prostaglandins have also been prepared by this sequence. The synthesis of 16, 16-tetramethylene-PG analogs is also described.

Methyl ethers of prostaglandins F2 alpha and I2.

Regiospecific monomethyl prostaglandin f2 alpha ethers (at 0-9, 0-11, and 0-15) have been prepared by total synthesis. The 9, 15-bis-ether was also prepared. The 11- and 15-monoethers have been converted to the corresponding prostacyclins. Nuclear Magnetic Resonance (NMR) spectral comparisons indicate conformational changes associated with ether formation; nonetheless, the PGF2 alpha monoethers all retain significant biological activity: 3-420% of natural PGF2 alpha. The 9- and 15- menthyl ethers show increased selectivity for luteolytic activity as measured in the hamster antifertility (HAF) assay. In contrast the prostacyclin ethers are essentially devoid of agonist activity on both the platelet and vasculature. Prostacyclin diastereomers [5a] also lack activity and it appears that any modification at or of the C-11 or C-15 functions reduces receptor binding by at least a factor of 100.