Azithromycin Fails to Prevent Accelerated Airway Obliteration in T-bet-/- Mouse Lung Allograft Recipients.

BACKGROUND: Cellular and molecular mechanisms of acute and chronic lung allograft rejection have yet to be clearly defined, and obliterative bronchiolitis (OB) remains the primary limitation to survival in lung transplant recipients (LTRs). We have previously shown that T-bet-deficient recipients of full major histocompatibility complex (MHC)-mismatched, orthotopic left lung transplants develop accelerated obliterative airway disease (OAD) in the setting of acute cellular rejection characterized by robust alloimmune CD8+ interleukin (IL)-17 and interferon (IFN)-γ responses that are attenuated with neutralization of IL-17. Azithromycin has been shown to be beneficial in some LTRs with bronchiolitis obliterans syndrome/OB. Here, we evaluated the effects of azithromycin on rejection pathology and T-cell effector responses in T-bet-/- recipients of lung transplants. METHODS: Orthotopic left lung transplantation was performed in BALB/c → B6 wild type or BALB/c → B6 T-bet-/- strain combinations as previously described. Mice treated with azithromycin received 10 mg/kg or 50 mg/kg subcutaneously daily. Lung allograft histopathology was analyzed at day 10 or day 21 post-transplantation, and neutrophil staining for quantification was performed using anti-myeloperoxidase. Allograft mononuclear cells were isolated at day 10 for T-cell effector cytokine response assessment using flow cytometry. RESULTS: We show that while azithromycin significantly decreases lung allograft neutrophilia and CXCL1 levels and attenuates allospecific CD8+ IL-17 responses early post-transplantation, OAD persists in T-bet-deficient mice. CONCLUSIONS: Our results indicate that lung allograft neutrophilia is not essential for the development of OAD in this model and suggest allospecific T-cell responses that remain despite marked attenuation of CD8+ IL-17 are sufficient for obliterative airway inflammation and fibrosis.

Proteasome Inhibitor Carfilzomib-Based Therapy for Antibody-Mediated Rejection of the Pulmonary Allograft: Use and Short-Term Findings.

We present this observational study of lung transplant recipients (LTR) treated with carfilzomib (CFZ)-based therapy for antibody-mediated rejection (AMR) of the lung. Patients were considered responders to CFZ if complement-1q (C1q)-fixing ability of their immunodominant (ID) donor-specific anti-human leukocyte antibody (DSA) was suppressed after treatment. Treatment consisted of CFZ plus plasma exchange and immunoglobulins. Fourteen LTRs underwent CFZ for 20 ID DSA AMR. Ten (71.4%) of LTRs responded to CFZ. DSA IgG mean fluorescence intensity (MFI) fell from 7664 (IQR 3230-11 874) to 1878 (653-7791) after therapy (p = 0.001) and to 1400 (850-8287) 2 weeks later (p = 0.001). DSA C1q MFI fell from 3596 (IQR 714-14 405) to <30 after therapy (p = 0.01) and <30 2 weeks later (p = 0.02). Forced expiratory volume in 1s ( FEV1 ) fell from mean 2.11 L pre-AMR to 1.92 L at AMR (p = 0.04). FEV1 was unchanged after CFZ (1.91 L) and subsequently rose to a maximum of 2.13 L (p = 0.01). Mean forced expiratory flow during mid forced vital capacity (25-75) (FEF25-75 ) fell from mean 2.5 L pre-AMR to 1.95 L at AMR (p = 0.01). FEF25-75 rose after CFZ to 2.54 L and reached a maximum of 2.91 L (p = 0.01). Responders had less chronic lung allograft dysfunction or progression versus nonresponders (25% vs. 83%, p = 0.04). No deaths occurred within 120 days and 7 patients died post CFZ therapy of allograft failure. Larger prospective interventional studies are needed to further describe the benefit of CFZ-based therapy for pulmonary AMR.

Bioluminescence inhibition assay for the detection of hydroxylated polychlorinated biphenyls.

Hydroxylated polychlorinated biphenyls (OH-PCBs) are an important class of contaminants that mainly originate from polychlorinated biphenyl metabolism. They may conceivably be as dangerous and persistent as the parent compounds; most prominently, OH-PCBs are endocrine disruptors. Due to increasing evidence of the presence of OH-PCBs in the environment and in living organisms, including humans, and of their toxicity, methods of detection for OH-PCBs are needed in the environmental and medical fields. Herein, we describe the development and optimization of a protein-based inhibition assay for the quantification of OH-PCBs. Specifically, the photoprotein aequorin was utilized for the detection of OH-PCBs. We hypothesized that OH-PCBs interact with aequorin, and we established that OH-PCBs actually inhibit the bioluminescence of aequorin in a dose-dependent manner. We took advantage of this phenomenon to develop an assay that is capable of detecting a wide variety of OH-PCBs with a range of detection limits, the best detection limit being 11 nM for the compound 2-hydroxy-2',3,4',5',6-pentachorobiphenyl. The viability of this system for the screening of OH-PCBs in spiked biological and environmental samples was also established. We envision the implementation of this novel bioluminescence inhibition assay as a rapid, sensitive, and cost-effective method for monitoring OH-PCBs. Furthermore, to the best of our knowledge, this is the first time aequorin has been employed to detect an analyte by the inhibition of its bioluminescence reaction. Hence, this strategy may prove to be a general approach for the development of a new generation of protein-based inhibition assays.

Cyclic AMP receptor protein-aequorin molecular switch for cyclic AMP.

Molecular switches are designer molecules that combine the functionality of two individual proteins into one, capable of manifesting an "on/off" signal in response to a stimulus. These switches have unique properties and functionalities and thus, can be employed as nanosensors in a variety of applications. To that end, we have developed a bioluminescent molecular switch for cyclic AMP. Bioluminescence offers many advantages over fluorescence and other detection methods including the fact that there is essentially zero background signal in physiological fluids, allowing for more sensitive detection and monitoring. The switch was created by combining the properties of the cyclic AMP receptor protein (CRP), a transcriptional regulatory protein from E. Coli that binds selectively to cAMP with those of aequorin, a bioluminescent photoprotein native of the jellyfish Aequorea victoria . Genetic manipulation to split the genetic coding sequence of aequorin in two and genetically attach the fragments to the N and C termini of CRP resulted in a hybrid protein molecular switch. The conformational change experienced by CRP upon the binding of cyclic AMP is suspected to result in the observed loss of the bioluminescent signal from aequorin. The "on/off" bioluminescence can be modulated by cyclic AMP over a range of several orders of magnitude in a linear fashion in addition to the capacity to detect changes in cellular cyclic AMP of intact cells exposed to different external stimuli without the need to lyse the cells. We envision that the molecular switch could find applications in vitro as well as In Vivo cyclic AMP detection and/or imaging.

Paper strip whole cell biosensors: a portable test for the semiquantitative detection of bacterial quorum signaling molecules.

Herein, we report the development of a novel, inexpensive, and portable filter-paper-based strip biosensor for the detection of bacterial quorum sensing signaling molecules, N-acylhomoserine lactones (AHLs). AHLs are generally employed by Gram-negative bacteria for their cell-cell communication to control expression of specialized genes, such as those involved in biofilm formation and production of virulence factors, in a population-density-dependent manner. First, a bacterial cell-based sensing system employing components of AHL-mediated QS regulatory system as recognition elements and beta-galactosidase as the reporter protein was designed and developed. The bacterial-sensing cells were then liquid-dried on strips of filter paper. beta-Galactosidase as the reporter allows for the visual monitoring of the analyte-induced signal when a colorimetric method of detection is applied. The paper strip biosensor was able to detect low AHL concentrations down to 1 x 10(-8) M. Furthermore, it was successfully applied to the detection of AHLs in physiological samples, such as saliva. The filter-paper-based sensing strips could provide reproducible results upon storage at 4 degrees C for at least 3 months. In conclusion, a filter-paper-based strip biosensor was developed that allows for visual, fast, and convenient detection of AHLs in a dose-dependent manner in a test sample. In addition, it does not require expensive equipment or trained personnel and allows ease of transportation and storage. Therefore, we envision that this biosensor will serve as a simple and economical portable field kit for on-site monitoring of AHL in a variety of clinical and environmental samples.

Genetically modified semisynthetic bioluminescent photoprotein variants: simultaneous dual-analyte assay in a single well employing time resolution of decay kinetics.

Progress in the miniaturization and automation of complex analytical processes depends largely on increasing the sensitivity, diversity, and robustness of current labels. Because of their ubiquity and ease of use, fluorescent, enzymatic, and bioluminescent labels are often employed in such miniaturized and multiplexed formats, with each type of label having its own unique advantages and drawbacks. The ultrasensitive detection limits of bioluminescent reporters are especially advantageous when dealing with very small sample volumes and biological fluids. However, bioluminescent reporters currently do not have the multiplexing capability that fluorescent labels do. In an effort to address this limitation, we have developed a method of discriminating two semisynthetic aequorin variants from one another using time resolution. In this work we paired two aequorin conjugates with different coelenterazine analogues and then resolved the two signals from one another using the difference in decay kinetics and half-life times. Utilizing this time-resolution, we then developed a simultaneous, dual-analyte, single well assay for 6-keto-prostaglandin-FI-alpha and angiotensin II, two important cardiovascular molecules.

Spectral tuning of photoproteins by partnering site-directed mutagenesis strategies with the incorporation of chromophore analogs.

Aequorin and obelin are photoproteins whose calcium controlled bioluminescent light emission is used for labeling in assays, for the determination of calcium concentrations in vivo, and as a reporter in cellular imaging. Both of these photoproteins emit blue light from a 2-hydroperoxycoelenterazine chromophore, which is non-covalently bound in the hydrophobic core of the proteins. In an effort to produce aequorin and obelin variants with improved analytical properties, such as alternative emission colors and altered decay kinetics, seven mutants of aequorin and obelin were prepared and combined with 10 different coelenterazine analogs. These semi-synthetic photoprotein mutants exhibited shifts in bioluminescent properties when compared with wild-type proteins. The bioluminescent parameters determined for these semi-synthetic photoprotein mutants included specific activity, emission spectra and decay half-life time. This spectral tuning strategy resulted in semi-synthetic photoprotein mutants that had significantly altered bioluminescent properties. The largest emission maxima shift obtained was 44 nm, and the largest decay half-life difference was 23.91 s.

Pegylated arginine deiminase lowers hepatitis C viral titers and inhibits nitric oxide synthesis.

BACKGROUND: The arginine-degrading enzyme, arginine deiminase conjugated to polyethylene glycol (ADI-SS PEG 20,000 mw), reduces extracellular arginine, has minimal toxicity, decreases tumor burden and improves liver function in patients with chronic hepatitis C virus infection (HCV) and inoperable hepatocellular carcinoma (HCC). Reduced extracellular arginine inhibits viral replication through unknown mechanisms. It is hypothesized that ADI-SS PEG 20,000 mw reduces HCV viral titers through nitric oxide (NO)-dependent effects. METHODS: The effects of ADI-SS PEG 20,000 mw (dose, 160 IU/m2; three cycles of four once-weekly i.m. injections) on HCV titers, serum NO and plasma arginine, were evaluated using archived plasma from patients with HCC and HCV and in vitro cell model measurements of HCV replication. RESULTS: ADI-SS PEG 20,000 mw selectively inhibited HCV replication in vitro (IC50 = 0.027 IU/mL). Fifteen HCC/HCV patients completed treatment. The HCV titers were reduced by up to 99% in five out of 10 (50%) HCV-serotype 1b patients (P = 0.0093). These patients also experienced significant improvements in liver function (P = 0.0091). There were concomitant reductions of plasma arginine and serum NO levels. The HCV titer was not reduced in HCV-type 2c patients. CONCLUSION: Reduction of extracellular arginine by ADI-SS PEG 20,000 mw in HCC patients reduces HCV viral titers and improves liver function, possibly through suppression of NO.

Pegylated arginine deiminase treatment of patients with metastatic melanoma: results from phase I and II studies.

PURPOSE: Individuals with metastatic melanoma have a poor prognosis. Many human melanomas are auxotrophic for arginine, and arginine is not an essential amino acid in humans. We hypothesized that this auxotrophy may be therapeutically exploited. A novel amino acid-degrading enzyme (arginine deiminase) conjugated to polyethylene glycol (ADI-SS PEG 20,000 mw) was used to lower plasma arginine in individuals with metastatic melanoma. PATIENTS AND METHODS: Two cohort dose-escalation studies were performed. A phase I study in the United States enrolled 15 patients, and a phase I to II study in Italy enrolled 24 patients. The Italian patients also received two subsequent cycles of treatment, each consisting of four once-weekly injections of 160 U/m2. The goals of these studies were to determine pharmacokinetics (PK), pharmacodynamics (PD), safety, and the antitumor activity of ADI-SS PEG 20,000 mw. RESULTS: PK and PD studies indicated that a dose of 160 U/m2 lowered plasma arginine from a resting level of approximately 130 micromol/L to less than 2 micromol/L for at least 7 days; nitric oxide levels also were lowered. There were no grade 3 or 4 toxicities directly attributable to the drug. Six of 24 phase I to II patients responded to treatment (five partial responses and one complete response; 25% response rate) and also had prolonged survival. CONCLUSION Elimination of all detectable plasma arginine in patients with metastatic melanoma was well tolerated and may be effective in the treatment of this cancer. Further testing of ADI-SS PEG 20,000 mw in a larger population of individuals with metastatic melanoma is warranted.

Pegylated arginine deiminase treatment of patients with unresectable hepatocellular carcinoma: results from phase I/II studies.

PURPOSE: Recently, we reported that a large number of human hepatocellular cancer (HCC) cell lines were auxotrophic for arginine. Here we report the results obtained with the amino acid-degrading enzyme arginine deiminase (ADI) conjugated to polyethylene glycol (ADI-SS PEG 20,000 mw) as a means of lowering plasma arginine to treat HCC. The study was a cohort dose-escalation phase I/II study. PATIENTS AND METHODS: Pharmacodynamic studies indicated an ADI-SS PEG 20,000 mw dose level of 160 U/m(2) was sufficient to lower plasma arginine from a resting level of approximately 130 micromol/L to below the level of detection (< 2 micromol/L) for more than 7 days, a dose later defined as the optimal biologic dose. All patients were to receive three cycles at the optimum biologic dose. RESULTS: This therapy was well tolerated, even in patients who had no detectable plasma arginine for 3 continuous months of therapy. Of the 19 patients enrolled, two had a complete response, seven had a partial response, seven had stable disease, and three had progressive disease. The median survival for the 19 patients enrolled on this study was 410 days, with four patients still alive at present (> 680 days). CONCLUSION: Elimination of all detectable plasma arginine in patients with HCC was well tolerated and seemed to be effective in the treatment of some patients with HCC. Further testing of ADI-SS PEG 20,000 mw in a larger population of individuals with HCC as well as other human tumors auxotrophic for arginine is warranted.

Incidence and distribution of argininosuccinate synthetase deficiency in human cancers: a method for identifying cancers sensitive to arginine deprivation.

BACKGROUND: Argininosuccinate synthetase (ASS) was the first of two enzymes to convert citrulline to arginine. This pathway allowed cells to synthesize arginine from citrulline, making this amino acid nonessential for the growth of most mammalian cells. Previous studies demonstrated that several human tumor cell lines were auxotrophic for arginine due to an inability to express ASS. Selective elimination of arginine from the circulation of animals with these tumors is a potentially effective anticancer treatment. The purpose of these experiments was to determine the frequency of ASS deficiency and arginine auxotrophy in a variety of human malignant tumors. METHODS: The authors analyzed the expression of ASS by immunohistochemistry with a monoclonal antibody in a variety of human tumor biopsies. They found that the incidence of ASS deficiency varied greatly with the tumor type and tissue of origin. RESULTS: Melanoma, hepatocellular carcinoma, and prostate carcinoma were most frequently deficient in ASS. Some human cancers were almost always positive for ASS (e.g., lung and colon carcinomas). However, other human cancers, including sarcomas, invasive breast carcinoma, and renal cell carcinoma, also were sometimes ASS deficient. CONCLUSIONS: These data indicated that immunohistochemical detection of ASS may prove an effective means for determining ASS deficiency in malignant human tumors and for identifying patients most likely to respond to arginine deprivation therapy. Based on these results, human clinical trials using arginine-degrading enzyme therapy to treat patients with advanced melanoma or hepatocellular carcinoma have been initiated.

Regression of hepatocellular cancer in a patient treated with arginine deiminase.

We report the first pharmacokinetic and clinical response data from a patient with unresectable hepatocellular cancer treated with a new drug, ADI PEG20,000 mw (arginine deiminase-polyethylene glycol 20,000 molecular weight). A single patient with idiopathic cirrhosis and unresectable hepatocellular cancer was treated with escalating dosages of ADI-PEG20,000 mw. Human hepatocellular cancer has been found to be arginine-dependent for growth because of loss of expression or arginosuccinate synthetase, the rate-limiting enzyme in the conversion of citrulline to arginine. Thus, an arginine-degrading enzyme like ADI-PEG20,000 mw should produce cell death in hepatocellular cancer cells without significantly affecting normal cells. There was a dose-dependent reduction of plasma arginine levels after weekly intramuscular administration of ADI-PEG20,000 mw. Successive treatment cycles at the optimal biologic dose of 160 IU/m2 led to reduction in tumor size and serum alpha-fetoprotein levels. Sufficient tumor cytoreduction was achieved with ADI-PEG20,000 mw treatment to permit surgical treatment. The patient developed no toxicities or side effects related to ADI-PEG20,000 mw treatment. The results in a single patient with unresectable hepatocellular cancer treated with ADI-PEG20,000 mw suggests this may be a promising, low-toxicity treatment. Full-scale clinical trials have been initiated.

Uricase formulated with polyethylene glycol (uricase-PEG 20): biochemical rationale and preclinical studies.

OBJECTIVE: Humans have a non-sense codon inserted into the 5 prime end of the open reading frame of urate oxidase, and thus express an enzymatically inactive fragment of this enzyme; and consequently are unable to metabolize uric acid into allantoin and are prone to develop hyperuricemia and gout. Various urate oxidases (uricase) from mammals and microorganisms have been administered to humans with hyperuricemia and gout. Although successful in lowering plasma uric acid, these therapies have had limited application due to undesirable biochemical properties of the enzymes used, the short circulating half-life, and inherent antigenicity of these preparations. METHODS: We compared urate oxidase from a variety of sources for specific enzyme activity, pH optimum, affinity, and retention of enzyme activity under physiological conditions. A variety of polyethylene glycols (PEG) were tested to formulate uricase. RESULTS: Urate oxidase from Candida utilis had more favorable enzymatic properties and PEG of 20,000 MW (termed uricase-PEG 20) had greatly reduced antigenicity and increased circulating half-life as compared to those previously described. CONCLUSION: It is anticipated that uricase-PEG 20 may have utility as a treatment for hyperuricemia and gout.

Rationally designed fluorescently labeled sulfate-binding protein mutants: evaluation in the development of a sensing system for sulfate.

Periplasmic binding proteins from E. coli undergo large conformational changes upon binding their respective ligands. By attaching a fluorescent probe at rationally selected unique sites on the protein, these conformational changes in the protein can be monitored by measuring the changes in fluorescence intensity of the probe which allow the development of reagentless sensing systems for their corresponding ligands. In this work, we evaluated several sites on bacterial periplasmic sulfate-binding protein (SBP) for attachment of a fluorescent probe and rationally designed a reagentless sensing system for sulfate. Eight different mutants of SBP were prepared by employing the polymerase chain reaction (PCR) to introduce a unique cysteine residue at a specific location on the protein. The sites Gly55, Ser90, Ser129, Ala140, Leu145, Ser171, Val181, and Gly186 were chosen for mutagenesis by studying the three-dimensional X-ray crystal structure of SBP. An environment-sensitive fluorescent probe (MDCC) was then attached site-specifically to the protein through the sulfhydryl group of the unique cysteine residue introduced. Each fluorescent probe-conjugated SBP mutant was characterized in terms of its fluorescence properties and Ser171 was determined to be the best site for the attachment of the fluorescent probe that would allow for the development of a reagentless sensing system for sulfate. Three different environment-sensitive fluorescent probes (1,5-IAEDANS, MDCC, and acylodan) were studied with the SBP171 mutant protein. A calibration curve for sulfate was constructed using the labeled protein and relating the change in the fluorescence intensity with the amount of sulfate present in the sample. The detection limit for sulfate was found to be in the submicromolar range using this system. The selectivity of the sensing system was demonstrated by evaluating its response to other anions. A fast and selective sensing system with detection limits for sulfate in the submicromolar range was developed.

Biochemical characterization of the arginine degrading enzymes arginase and arginine deiminase and their effect on nitric oxide production.

BACKGROUND: Nitric oxide (NO) is a biomediator believed to be synthesized primarily from extracellular arginine. Various methodologies have been used to inhibit NO synthesis so as to elucidate its physiological and pathophysiological functions. Several investigators have utilized various argin ine degrading enzymes as a means of lowering extracellular arginine. Arginase, most commonly derived from mammalian sources, has been most often used. However, arginase has failed to inhibit NO synthesis. Therefore, a systematic biochemical characterization of arginase and arginine deiminase (ADI) derived from M. Hominus was undertaken. MATERIAL/METHODS: The murine macrophage cell line N-9 was treated with either arginase or arginine deiminase to determine the effect on intracellular and extracellular arginine and nitric oxide production. RESULTS: Arginase was found to have an alkaline pH optima(approximately 9.5) with little enzyme activity at physiological pH. In contrast, the pH optima of ADI was approximately 6.5, retaining >70% of its activity at physiological pH. ADI had more than 1000 fold higher affinity for arginine (Km approximately 30 KM for ADI vs approximately 45 mM for arginase), and was able to lower arginine levels to a much greater extent than arginase. ADI, unlike arginase, was effective in lowering extracellular arginine in tissue culture media and inhibit NO production by the murine macrophage cell line N-9 in response to gamma interferon and LPS stimulation. CONCLUSIONS: These data suggest that ADI may be useful for delineating the role of NO in a variety of biological systems as well as determining the role of extracellular arginine in its synthesis.

Enzymic degradation of plasma arginine using arginine deiminase inhibits nitric oxide production and protects mice from the lethal effects of tumour necrosis factor alpha and endotoxin.

Septic shock is mediated in part by nitric oxide (NO) and tumour necrosis factor alpha (TNFalpha). NO is synthesized primarily from extracellular arginine. We tested the ability of an arginine-degrading enzyme to inhibit NO production in mice and to protect mice from the hypotension and lethality that occur after the administration of TNFalpha or endotoxin. Treatment of BALB/c mice with arginine deiminase (ADI) formulated with succinimidyl succinimide polyethylene glycol of M(r) 20000 (ADI-SS PEG(20000)) eliminated all measurable plasma arginine (from normal levels of approximately 155 microM arginine to 2 microM). In addition, ADI-SS PEG(20000) also inhibited the production of NO, as quantified by plasma nitrate+nitrite. Treatment of mice with TNFalpha or endotoxin resulted in a dose-dependent increase in NO production and lethality. Pretreatment of mice with ADI-SS PEG(20000) resulted in increased resistance to the lethal effects of TNFalpha and endotoxin. These observations are consistent with NO production resulting, to some extent, from the metabolism of extracellular arginine. The toxic effects of TNFalpha and endotoxin may be partially inhibited by enzymic degradation of plasma arginine by ADI-SS PEG(20000). Interestingly, pretreatment with ADI-SS PEG(20000) did not inhibit the anti-tumour activity of TNFalpha in vitro or in vivo. This treatment may allow greater amounts of TNFalpha, as well as other cytokines, to be administered while abrogating side effects such as hypotension and death.

A fluorescence-based sensing system for the environmental monitoring of nickel using the nickel binding protein from Escherichia coli.

A sensing system for nickel based on the nickel binding protein (NBP) from Escherichia coli is shown to be feasible. The versatility of NBP was demonstrated by its use in three different assay formats. When the NBP binds nickel, it undergoes a conformational change that can be used as the basis for an optical sensing system for nickel. The NBP gene was overexpressed in E. coli and the protein purified in a single step using perfusion anion-exchange chromatography. A unique cysteine residue at position 15 in the NBP was labeled with the fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC). In a spectrofluorimetric assay, there was a maximum of 65% quenching of the fluorescence signal produced by NBP-MDCC in the presence of nickel. A response curve for nickel using NBP-MDCC revealed a detection limit of 8 x 10(-8) mol L(-1). NBP-MDCC was also used to develop assays in microtiter plate and fiber optic bundle formats. Detection limits for nickel using these formats were also in the submicromolar range. Selectivity studies conducted with other divalent metals, including copper, cobalt, iron, cadmium, and manganese, showed that fluorescence quenching for cobalt was similar in magnitude but with a detection limit more than 10-fold higher than for nickel. The quenching responses were lower for the other metals, with detection limits at least 10 to 100 times higher than for nickel. These results suggest that fluorescently labeled NBP is potentially useful in the development of a sensing system for nickel.

The psychological health of "joiners".

The current study examined the relationship between scores on social complexity and psychological health. A sample of 202 college students yielded positive correlations for propensity to join and maintain memberships in diverse groups with psychological health, thereby replicating past research.

A novel reagentless sensing system for measuring glucose based on the galactose/glucose-binding protein.

The galactose/glucose-binding protein (GBP) is synthesized in the cytoplasm of Escherichia coli in a precursor form and exported into the periplasmic space upon cleavage of a 23-amino-acid leader sequence. GBP binds galactose and glucose in a highly specific manner. The ligand induces a hinge motion in GBP and the resultant protein conformational change constitutes the basis of the sensing system. The mglB gene, which codes for GBP, was isolated from the chromosome of E. coli using the polymerase chain reaction (PCR). Since wild-type GBP lacks cysteines in its structure, introducing this amino acid by site-directed mutagenesis ensures single-label attachment at specific sites with a sulfhydro-specific fluorescent probe. Site-directed mutagenesis by overlap extension PCR was performed to prepare three different mutants to introduce a single cysteine residue at positions 148, 152, and 182. Since these residues are not involved in ligand binding and since they are located at the edge of the binding cleft, they experience a significant change in environment upon binding of galactose or glucose. The sensing system strategy is based on the fluorescence changes of the probe as the protein undergoes a structural change on binding. In this work a reagentless sensing system has been rationally designed that can detect submicromolar concentrations of glucose. The calibration plots have a linear working range of three orders of magnitude. Although the system can sense galactose as well, this epimer is not a potential interfering substance since its concentration in blood is negligible.