Allosteric modification of oxygen delivery by hemoglobin.

UNLABELLED: Hemoglobin affinity for oxygen is altered by pH, temperature, and high altitude, making oxygen more readily available to the tissues. RSR13 (Allos Therapeutics, Denver, CO), an analog of the drugs clofibrate and bezofibrate, causes a dose-dependent, rightward shift of the oxygen dissociation curve in animals and humans. We tested the safety, pharmacodynamic, and pharmacokinetics of RSR13, an allosteric modifier of hemoglobin, in patients having general surgery in a prospective, randomized, double-blinded, placebo-controlled, dose-escalation clinical trial. After the induction of general anesthesia with endotracheal intubation, 26 patients who consented were randomly assigned to receive an infusion of RSR13 or placebo (2:1) in an ascending dose scheme. Doses studied were 10, 20, 30, 40, 50, 60, 75, and 100 mg/kg infused for 30--60 minutes. Samples were taken for determination of RSR13 concentration in plasma, red blood cells, and urine, as well as for determination of the p50 in blood by using three-point tonometry at frequent intervals after the infusion of the study drug. The RSR13 administration resulted in a dose-dependent rightward shift of the oxygen dissociation curve, with the target p50 shift of 10 mm Hg achieved at the 75- and 100-mg/kg doses. No differences were seen between RSR13 and placebo groups in laboratory or hemodynamic findings, with the exception of a transient, limited increase in serum creatinine in 3 patients who received RSR13. These increases peaked at 48 h (2.2, 3.5, and 4.5 mg/dL respectively), were not associated with oliguria, did not require treatment, and did not prolong hospitalization in any patient. The reasons for the unexplained increases in serum creatinine were not evident, but potentially included surgery itself (nephrectomy), patient condition, or the concomitant administration of renally cleared medications or drugs that affect renal blood flow. IMPLICATIONS: We studied the safety and tolerance of an investigational drug, RSR13 (Allos Therapeutics, Denver, CO), in general surgery patients. This drug, which increases the amount of oxygen available to the body, was well tolerated by the 17 patients who received it. There were clinically relevant increases in serum creatinine in 3 patients, indicating a decrease in renal function, but these increases were short-lived and resolved without treatment.

Genomic and genetic dissection of an archaeal regulon.

The extremely halophilic archaeon Halobacterium sp. NRC-1 can grow phototrophically by means of light-driven proton pumping by bacteriorhodopsin in the purple membrane. Here, we show by genetic analysis of the wild type, and insertion and double-frame shift mutants of Bat that this transcriptional regulator coordinates synthesis of a structural protein and a chromophore for purple membrane biogenesis in response to both light and oxygen. Analysis of the complete Halobacterium sp. NRC-1 genome sequence showed that the regulatory site, upstream activator sequence (UAS), the putative binding site for Bat upstream of the bacterio-opsin gene (bop), is also present upstream to the other Bat-regulated genes. The transcription regulator Bat contains a photoresponsive cGMP-binding (GAF) domain, and a bacterial AraC type helix-turn-helix DNA binding motif. We also provide evidence for involvement of the PAS/PAC domain of Bat in redox-sensing activity by genetic analysis of a purple membrane overproducer. Five additional Bat-like putative regulatory genes were found, which together are likely to be responsible for orchestrating the complex response of this archaeon to light and oxygen. Similarities of the bop-like UAS and transcription factors in diverse organisms, including a plant and a gamma-proteobacterium, suggest an ancient origin for this regulon capable of coordinating light and oxygen responses in the three major branches of the evolutionary tree of life. Finally, sensitivity of four of five regulon genes to DNA supercoiling is demonstrated and correlated to presence of alternating purine-pyrimidine sequences (RY boxes) near the regulated promoters.

Genomic perspective on the photobiology of Halobacterium species NRC-1, a phototrophic, phototactic, and UV-tolerant haloarchaeon.

Halobacterium species display a variety of responses to light, including phototrophic growth, phototactic behavior, and photoprotective mechanisms. The complete genome sequence of Halobacterium species NRC-1 (Proc Natl Acad Sci USA 97: 12176-12181, 2000), coupled with the availability of a battery of methods for its analysis makes this an ideal model system for studying photobiology among the archaea. Here, we review: (1) the structure of the 2.57 Mbp Halobacterium NRC-1 genome, including a large chromosome, two minichromosomes, and 91 transposable IS elements; (2) the purple membrane regulon, which programs the accumulation of large quantities of the light-driven proton pump, bacteriorhodopsin, and allows for a period of phototrophic growth; (3) components of the sophisticated pathways for color-sensitive phototaxis; (4) the gas vesicle gene cluster, which codes for cell buoyancy organelles; (5) pathways for the production of carotenoid pigments and retinal, (6) processes for the repair of DNA damage; and (7) putative homologs of circadian rhythm regulators. We conclude with a discussion of the power of systems biology for comprehensive understanding of Halobacterium NRC-1 photobiology.

Genome sequence of Halobacterium species NRC-1.

We report the complete sequence of an extreme halophile, Halobacterium sp. NRC-1, harboring a dynamic 2,571,010-bp genome containing 91 insertion sequences representing 12 families and organized into a large chromosome and 2 related minichromosomes. The Halobacterium NRC-1 genome codes for 2,630 predicted proteins, 36% of which are unrelated to any previously reported. Analysis of the genome sequence shows the presence of pathways for uptake and utilization of amino acids, active sodium-proton antiporter and potassium uptake systems, sophisticated photosensory and signal transduction pathways, and DNA replication, transcription, and translation systems resembling more complex eukaryotic organisms. Whole proteome comparisons show the definite archaeal nature of this halophile with additional similarities to the Gram-positive Bacillus subtilis and other bacteria. The ease of culturing Halobacterium and the availability of methods for its genetic manipulation in the laboratory, including construction of gene knockouts and replacements, indicate this halophile can serve as an excellent model system among the archaea.

Saturation mutagenesis of the haloarchaeal bop gene promoter: identification of DNA supercoiling sensitivity sites and absence of TFB recognition element and UAS enhancer activity.

Transcription from the bop promoter in the haloarchaeon Halobacterium NRC-1, is highly induced under oxygen-limiting conditions. A DNA gyrase inhibitor, novobiocin, was previously shown to block bop gene induction and suggested that DNA supercoiling mediates transcriptional induction. A region of non-B structure was found 3' to the TATA box within an 11 bp alternating purine-pyrimidine sequence (RY box), which correlated to both increased DNA supercoiling and transcriptional induction. Here, saturation mutagenesis of the RY box region has been used to show that single-base substitutions of A(r)G either 23 or 19 bp 5' to the transcription start site temper the effect of DNA supercoiling based on novobiocin insensitivity of transcription. Mutagenesis of the region 5' to the TATA box showed its involvement in DNA supercoiling modulation of transcription, defined the 3' end of the upstream activator sequence (UAS) regulatory element, and ruled out the requirement for a TFB (TFIIB) Recognition Element. Spacing between the TATA box and UAS was found to be critical for promoter activity because insertion of partial or whole helical turns between the two elements completely inhibited transcription indicating that the UAS element does not function as a transcriptional enhancer. The results are discussed in the context of DNA melting and flexibility around the TATA box region and the involvement of multiple regulatory and transcription factors in bop promoter activity.

Improving the thromboresistivity of chemical sensors via nitric oxide release: fabrication and in vivo evaluation of NO-releasing oxygen-sensing catheters.

The development and in vivo analytical performance of a nitric oxide (NO)-releasing amperometric oxygen sensor with greatly enhanced thromboresistivity are reported. Gas permeable coatings formulated with cross-linked silicone rubber (SR) containing NO-generating compounds (diazeniumdiolates) are shown to release NO for extended periods of time (> 20 h) while reducing platelet adhesion and activation. Oxygen-sensing catheters prepared by dip-coating the NO-releasing films over the outer SR tubes of the implantable devices display similar analytical response properties in vitro (sensitivity, selectivity, response times) when compared to analogous sensors prepared without the NO release coatings. Superior analytical accuracy (relative to blood PO2 values measured in vitro) and greatly reduced thrombus formation on the outer surface of the sensors are observed in vivo (in canine model) with the NO release PO2 sensors compared to control sensors (without NO release) implanted simultaneously within the same animals. Based on these preliminary studies, the use of NO release polymers to fabricate catheter-style chemical sensors may be a potential solution to lingering biocompatibility and concomitant performance problems encountered when attempting to employ such devices for continuous intravascular measurements of blood gases and electrolytes.

Saturation mutagenesis of the TATA box and upstream activator sequence in the haloarchaeal bop gene promoter.

Degenerate oligonucleotides were used to randomize 21 bp of the 53-bp minimal bop promoter in three 7-bp segments, including the putative TATA box and the upstream activator sequence (UAS). The mutagenized bop promoter and the wild-type structural gene and transcriptional terminator were inserted into a shuttle plasmid capable of replication in the halophilic archaeon Halobacterium sp. strain S9. Active promoters were isolated by screening transformants of an orange (Pum- bop) Halobacterium mutant for purple (Pum+ bop+) colonies on agar plates and analyzed for bop mRNA and/or bacteriorhodopsin content. Sequence analysis yielded the consensus sequence 5'-tyT(T/a)Ta-3', corresponding to the promoter TATA box element 30 to 25 bp 5' of the transcription start site. A putative UAS, 5'-ACCcnactagTTnG-3', located 52 to 39 bp 5' of the transcription start site was found to be conserved in active promoters. This study provides direct evidence for the requirement of the TATA box and UAS for bop promoter activity.

Determination of low-molecular-weight heparins and their binding to protamine and a protamine analog using polyion-sensitive membrane electrodes.

A polycation-sensitive membrane electrode based on the ion-exchanger dinonylnaphthalene sulfonate has previously been developed and used as an end-point detector for the determination of unfractionated heparin in whole blood samples via simple potentiometric titration with protamine. Herein, we report the application of the same methodology for the quantitation of a commercial low-molecular-weight heparin (LMWH) preparation (Fragmin) in whole blood samples at concentrations up to 2 U/ml. Further, an analogous polyanion (heparin)-sensitive electrode is used to estimate the binding constants between protamine and various LMWH preparations. The equilibrium constants (Keq) and the number of binding sites per mole of heparin (n) are determined by recasting the data in the form of a Scatchard plot. Results show that the average molecular weight and molecular weight distribution of the LMWH preparation are important parameters affecting their binding with protamine. Comparable binding constants are obtained for the same LMWH preparations titrated with a synthetic protamine analog, [+18RGD] [acetyl-EA(R2A2R2A)4R2GRGDSPA-NH2].

Improved protamine-sensitive membrane electrode for monitoring heparin concentrations in whole blood via protamine titration.

An improved protamine-sensitive electrode based on a polymeric membrane doped with the charged ion exchanger dinonylnaphthalenesulfonate (DNNS) is used for monitoring heparin concentrations in whole blood. The electrode exhibits significant nonequilibrium potentiometric response to polycationic protamine over the concentration range of 0.5-20 mg/L in undiluted whole-blood samples. The sensor can serve as a simple end point detector for the determination of heparin via potentiometric titrations with protamine. Whole-blood heparin concentrations determined by the electrode method (n > or = 157) correlate well with other protamine titration-based methods, including the commercial Hepcon HMS assay (r = 0.934) and a previously reported potentiometric heparin sensor-based method (r = 0.973). Reasonable correlation was also found with a commercial chromogenic anti-Xa heparin assay (r = 0.891) with corresponding plasma samples and appropriate correction for whole-blood hematocrit levels. Whereas a significant positive bias (0.62 kU/L; P < 0.001) is observed between the anti-Xa assay and the protamine sensor methods, insignificant bias is observed between the protamine sensor and the Hepcon HMS tests (0.08 kU/L; P = 0.02). The possibility of fully automating these titrations offers a potentially simple, inexpensive, and accurate method for monitoring heparin concentrations in whole blood.