Biochemical properties of the heme oxygenase inhibitor, Sn-protoporphyrin. Interactions with apomyoglobin and human serum albumin

Sn-protoporphyrin is a strong competitive inhibitor of heme oxygenase and a potential pharmacological agent for the treatment of neonatal hyperbilirubinemia. Little is otherwise known about the biochemistry of tin porphyrins. We have investigated aspects of the chemistry of tin-protoporphyrin in aqueous solution and of its interactions with heme-binding proteins other than heme oxygenase, specifically apomyoglobin and human serum albumin. In the pH region 7-10, Soret region absorption studies of unbound Sn-protoporphyrin demonstrate a pH-dependent monomer-dimer equilibrium (KD congruent to 10(6) M-1 at pH 7) with little higher aggregation. Dissociation of the dimer is relatively slow at neutral pH, permitting interaction of protein ligands with monomeric and dimeric species to be distinguished and providing insights into kinetic mechanisms of porphyrin binding by heme-binding proteins. In the present study, the kinetics of interaction of Sn-protoporphyrin with apomyoglobin are presented as novel evidence that this binding proceeds by an induced fit mechanism. Binding of Sn-protoporphyrin to both apomyoglobin and serum albumin is unexpectedly weak. Between pH 7 and 9, the apparent affinity of Sn-protoporphyrin for apomyoglobin is less than 1/200 that of heme and, at pH 9, is also significantly less than that of protoporphyrin. The apparent affinity of Sn-protoporphyrin for human serum albumin is less than 1/1000 that of heme and 1/30 to 1/100 that of protoporphyrin. Competition studies between heme and Sn-protoporphyrin and between bilirubin and Sn-protoporphyrin indicate that Sn-protoporphyrin distributes differently among porphyrin-binding sites on serum albumin than does heme and that it is also not an effective competitor with bilirubin for bilirubin-binding sites. These results argue that Sn-protoporphyrin should not significantly alter normal mechanisms for the binding and transport of heme or of preformed bilirubin by serum albumin. From a more general perspective, the results indicate potentially unusual binding site selectivity by tin chelates; possible origins of this selectivity are discussed.     

Immune stimulatory properties of metalloporphyrins

A possible approach to the immunotherapy of tumors is to stimulate either specific or nonspecific immune responses in vivo. We recently found that provision of a mitogenic signal to PBMC, by incubation with the oxidizing mitogens, enhanced the effect of IL-2 in generating cytolytic activity. We therefore searched for a mitogen that might safely be administered to patients. The present study is an investigation of the mitogenic properties of iron and tin (Sn)-protoporphyrin and their capacity to induce cytotoxicity in human PBMC. These agents have been administered to humans with little toxicity. Both iron- (hemin) and Sn-protoporphyrin induce mitogenicity in peripheral T cells. This effect is markedly enhanced by low concentrations of IL-2. Hemin and Sn-protoporphyrin, in combination with IL-2, increase IL-2R on PBMC. Hemin alone, and to a greater extent in combination with IL-2, induces cytotoxicity for NK-sensitive and NK-resistant cell lines. Sn-protoporphyrin, a more potent mitogen than hemin, fails to induce cytotoxicity, and has a marked inhibitory effect on cytotoxicity induced by IL-2. Hemin and Sn-protoporphyrin stimulate TNF-alpha and IFN-gamma production by PBMC. IL-2 is synergistic with the metalloporphyrins in eliciting this effect. Metalloporphyrin-induced mitogenesis has a stringent requirement for macrophages. Scavengers of oxygen-free radicals and inhibitors of peroxidase inhibit mitogenicity induced by hemin but not that induced by Sn-protoporphyrin. Hence, an oxidative event may mediate the mitogenic effect of hemin. Our results indicate that hemin is an immunostimulatory agent in vitro and the data warrant further evaluation of its in vivo immunostimulatory and antitumor effect.     

Protective effect of Sn-protoporphyrin against doxorubicin-induced perturbations of heme metabolism

The administration of doxorubicin, an anti-tumor antibiotic, to rodents resulted in an increase in heme oxygenase activity and a decrease in delta-aminolevulinate (ALA) synthase activity and in cellular heme and cytochrome P450 content in liver. Sn-protoporphyrin, a potent inhibitor of heme degradation both in vitro and in vivo, when administered to rodents prior to doxorubicin, mitigates the drug-induced toxic actions which are reflected by the drug-induced decreases of both cellular heme and cytochrome P450 content. Sn-protoporphyrin thus provides a pharmacological means of protecting against the toxic effects of doxorubicin and other drugs which enhance heme oxygenase activity and thus decrease cellular heme and cytochrome P450 content in vivo.     

Reduction of the C2 and C4 vinyl groups of Sn-protoporphyrin to form Sn-mesoporphyrin markedly enhances the ability of the metalloporphyrin to inhibit in vivo heme catabolism

Sn (tin)-mesoporphyrin (Sn-protoporphyrin in which the vinyl groups at C2 and C4 have been reduced to ethyl groups) when incubated with rat splenic microsomal heme oxygenase proved to be a potent competitive inhibitor of enzyme activity in vitro, with a Ki of 0.014 microM. Sn-mesoporphyrin (1 mumol/kg body wt) also inhibited hepatic, renal, and splenic heme oxygenase activity in vivo in adult animals for extended periods of time. Sn-mesoporphyrin (1 mumol/kg body wt) prevented the transient increase in serum bilirubin 24 h after birth in the rat neonate and substantially reduced the levels of serum bilirubin in ALA (delta-aminolevulinic acid) induced hyperbilirubinemia in the 7-day-old suckling neonate. Tissue heme oxygenase activity was decreased in both animal models of hyperbilirubinemia. Sn-mesoporphyrin administration led to a prolonged increase in the heme saturation of hepatic tryptophan pyrrolase indicating an increase in the "heme pool" related to tryptophan pyrrolase and the compound also suppressed chemically induced hepatic porphyria. The administration of Sn-mesoporphyrin to bile duct-cannulated rats was followed by a prompt and sustained decrease in bilirubin output in bile. In addition the excretion of heme in bile was enhanced in these animals. These studies indicate that Sn-mesoporphyrin, like Sn-protoporphyrin, decreases serum bilirubin by inhibiting the production of bilirubin in vivo and its mode of action is through a sustained competitive inhibition of heme oxygenase. However, when a direct comparison of Sn-protoporphyrin and Sn-mesoporphyrin was made, these studies clearly established that the reduction of the C2 and C4 vinyl groups of the porphyrin macrocycle to ethyl groups increases the effectiveness of the Sn-mesoporphyrin derivative 10-fold or more as compared with Sn-protoporphyrin in inhibiting heme catabolism in the animal model systems examined. Thus alterations in the side chain substituents as well as of the central metal atom can influence in a significant manner the potency of the resultant synthetic heme analog as an agent capable of inhibiting heme degradation in vivo.     

Detection of sulfa drugs and antibiotics in milk

A disc assay method for testing sulfa drugs and antibiotics in milk was developed wherein Bacillus megaterium ATCC 9855 was used as the test organism and Mueller-Hinton agar was used as the test substrate. Incubation was at 37 C for 4 to 5 hr. The test procedure is an improvement over the Bacillus subtilis-Antibiotic Medium No. 1 method, as described in Standard Methods for the Examination of Dairy Products, in that it is sensitive to eight sulfa drugs and to bacitracin without a significant change in sensitivity to eight other antibiotics commonly used for mastitis therapy.     

Sensitive fluorescence assays for urokinase using synthetic peptide 4-methoxy-beta-naphthylamide substrates

Two assays for the plasminogen activator urokinase using peptide fluorogenic substrates are described. N-carbobenzoxy-glycyl-glycyl-l-arginine-4-methoxy-β-naphthylamide (CBZ-Gly-Gly-Arg-4MβNA) can be used in a direct assay that is simple, rapid, and sensitive to as little as 0.5 IU/ml urokinase. Additional sensitivity, to 0.01 IU/ml urokinase, is obtained in a second method that uses plamsinogen as the primary substrate followed by a fluorogenic substrate assay employing N-carbobenzoxy-l-alanyl-l-alanyl-l-lysine-4-methoxy-β-naphthylamide (CBZ-Ala-Ala-Lys-4MβNA) as a specific substrate for the activated plasmin. These assays are as sensitive as the best assays presently in use and are simpler to perform. In addition, these assays can readily be used for kinetic analysis of the hydrolytic activity of urokinase or other plasminogen activators.     

Development and evaluation of a dot blot assay for rapid determination of invasion-associated gene ibeA directly in fresh bacteria cultures of E. coli

The ibeA gene, one of the important invasion-associated genes in neonatal meningitis Escherichia coli (NMEC), has been recently detected in avian pathogenic E. coli (APEC). Thus, it is necessary to close monitor the possible contamination of the poultry farms and its products to people. Here, a dot blot method for detecting the ibeA gene in E. coli was developed and validated. For the present study, probe sequence was designed and optimized for the specificity of dot blot. A 342-bp conserved fragment of ibeA gene was selected and labeled with digoxigenin (DIG)-dUTP according to the manufacturer??s guidelines, which indicated that this probe hybridizes with ibeA. In our established method, the bacteria culture samples were directly spotted on the membrane, following simple lyses on the membrane. Hence, the extraction of genomic DNA is not required, which reduces the workload and shortens the time. Furthermore, this assay was very sensitive, which could detect as few as 2.5?×?10³?CFU bacteria. The diagnostic reliability of this dot blot was evaluated on 467 APEC bacteria samples by using PCR analysis. Both methods showed that the result was in complete concordance. The dot blot assay was proved to be a simple, rapid, highly accurate, and cost-effective method to identify invasion-associated genes ibeA, which could be applied for initial screening of a large number of clinical samples or direct detection of bacteria culture.     

Enzyme-Linked Immunosorbent Assay for Specific Identification and Enumeration of Azospirillum brasilense Cd. in Cereal Roots

The enzyme-linked immunosorbent assay is suggested as a reliable, sensitive, and highly specific method for the identification and enumeration of Azospirillum brasilense Cd. As few as 10⁵ CFU/ml can be practically identified by this method. At higher bacterial numbers, sensitivity increased linearly up to 5 × 10⁸ CFU/ml, yielding useful standard curves. No cross-reaction was found either with different closely related Azospirillum strains or with other rhizosphere bacteria. The method allows for a specific identification of A. brasilense Cd. both in pure cultures and in mixtures with other bacterial species, even when the colony morphology is variable. The method was successfully applied to assess the degree of root colonization on various cereals by A. brasilense Cd.     

A sensitive colorimetric high-throughput screening method for lipase synthetic activity assay

A sensitive and practical high-throughput screening method for assaying lipase synthetic activity is described. Lipase-catalyzed transesterification between vinyl acetate and n-butanol in n-hexane was chosen as a model reaction. The released acetaldehyde was determined by the colorimetric method using 3-methyl-2-benzothialinone (MBTH) derivatization. In comparison with other methods, the major advantages of this process include high sensitivity, simple detection, inexpensive reagents, and low requirements for instruments.     

Comparison between radiometric and proportional methodologies in susceptibility testing of mycobacteria other than tubercle bacilli (MOT)

109 strains of mycobacteria other than tubercle bacilli (MOT) were tested against four primary antituberculous drugs; 27 strains of M. tuberculosis served as controls. While the latter species showed excellent overall agreement with the reference (proportion) method of Canetti (particularly for sensitive strains), MOTT gave such results only if there was a prevalence of sensitive or resistant strains in a species. Since this could not be predicted, the radiometric method cannot substitute for the proportion method for susceptibility testing of MOTT, in spite of early availability of results.     

Sn-protoporphyrin inhibits both heme degradation and hemozoin formation in Rhodnius prolixus midgut

Hematophagy is a feeding habit that involves the ingestion of huge amounts of heme. The hematophagous hemipteran Rhodnius prolixus evolved many genetic resources to protect cells against heme toxicity. The primary barrier against the deleterious effects of heme is the aggregation of heme into hemozoin in the midgut lumen. Hemozoin formation is followed by the enzymatic degradation of heme by means of a unique pathway whose end product is dicysteinyl-biliverdin IX-γ (Rhodnius prolixus biliverdin, RpBv). These mechanisms are complemented by a heme-binding protein (RHBP) in the hemolymph that attenuates the pro-oxidant effects of heme. In this work, we show that when insects are fed with blood enriched with a heme analog, Sn-protoporphyrin (SnPP-IX), both hemozoin synthesis and RpBv production are inhibited in a dose-dependent manner. These effects are accompanied by increased oxidative damage to the midgut epithelium and inhibition of oviposition, indicating that hemozoin formation and heme degradation are protective mechanisms that work together and contributed to the adaptation of this insect to successfully feed on vertebrate blood.     

Establishment and application of hyperbranched rolling circle amplification coupled with lateral flow dipstick for the sensitive detection of Karenia mikimotoi

The dinoflagellate Karenia mikimotoi is a noxious and harmful algal bloom (HAB)-forming microalga. Establishing a rapid, accurate, and sensitive method of detecting this harmful alga is necessary to provide warnings of imminent HABs through field monitoring. Here, an isothermal amplification technique combined with a rapid analytical method for nucleic acid-based amplified products, i.e., hyperbranched rolling circle amplification (HRCA) coupled with lateral flow dipstick (LFD), hereafter denoted as HRCA-LFD, was established to detect K. mikimotoi. The HRCA-LFD assay relied on a padlock probe (PLP) targeting DNA template and an LFD probe targeting PLP. The sequenced internal transcribed spacer of K. mikimotoi through molecular cloning was used as the target of PLP. The optimized HRCA conditions was determined to be as follows: PLP concentration, 20 pM; ligation temperature, 65 °C; ligation time, 10 min; amplification temperature, 61 °C; and amplification time, 30 min. The developed HRCA-LFD assay was specific for K. mikimotoi, displaying no cross-reactivity with other common microalgae. Sensitivity-comparison tests indicated that HRCA-LFD assay was 100-fold more sensitive than PCR, with a detection limit of 0.1 cell mL⁻¹ when used to analyze spiked field samples. The analysis with field samples also indicated that HRCA-LFD assay was suitable for samples with a target cell density range of 1–1000 cells mL⁻¹. All of these results suggested that HRCA-LFD assay is an alternative method for the sensitive and reliable detection of K. mikimotoi from marine water samples.     

Rapid Method for Enumeration of Viable Legionella pneumophila and Other Legionella spp. in Water

A sensitive and specific method has been developed to enumerate viable L. pneumophila and other Legionella spp. in water by epifluorescence microscopy in a short period of time (a few hours). This method allows the quantification of L. pneumophila or other Legionella spp. as well as the discrimination between viable and nonviable Legionella. It simultaneously combines the specific detection of Legionella cells using antibodies and a bacterial viability marker (ChemChrome V6), the enumeration being achieved by epifluorescence microscopy. The performance of this immunological double-staining (IDS) method was investigated in 38 natural filterable water samples from different aquatic sources, and the viable Legionella counts were compared with those obtained by the standard culture method. The recovery rate of the IDS method is similar to, or higher than, that of the conventional culture method. Under our experimental conditions, the limit of detection of the IDS method was <176 Legionella cells per liter. The examination of several samples in duplicates for the presence of L. pneumophila and other Legionella spp. indicated that the IDS method exhibits an excellent intralaboratory reproducibility, better than that of the standard culture method. This immunological approach allows rapid measurements in emergency situations, such as monitoring the efficacy of disinfection shock treatments. Although its field of application is as yet limited to filterable waters, the double-staining method may be an interesting alternative (not equivalent) to the conventional standard culture methods for enumerating viable Legionella when rapid detection is required.     

Molecular serogrouping of porcine enterotoxigenic Escherichia coli from Australia

Enterotoxigenic Escherichia coli (ETEC) is a common etiological agent of neonatal, pre and post weaning diarrhoea in piglets. One of the most important steps in the diagnosis and epidemiological understanding of this organism is accurate serogrouping. In many instances, however, conventional serogrouping fails to produce accurate identification of serogroups. In this communication we report a modified and simplified molecular serogrouping method (rfb-RFLP) for the accurate identification of the most common porcine ETEC strains that cause neonatal, pre and post weaning diarrhoea in Australia.     

Novel Indirect Calorimetry Technology to Analyze Metabolism in Individual Neonatal Rodent Pups

Background

The ability to characterize the development of metabolic function in neonatal rodents has been limited due to technological constraints. Low respiratory volumes and flows at rest pose unique problems, making it difficult to reliably measure O₂ consumption, CO₂ production, respiratory quotient (RQ), and energy expenditure (EE). Our aim was to develop and validate a commercial-grade indirect calorimetry system capable of characterizing the metabolic phenotype of individual neonatal rodents.

Methodology/Principal Findings

To address this research need, we developed a novel, highly sensitive open-circuit indirect calorimetry system capable of analyzing respiratory gas exchange in a single neonatal rodent pup. Additionally, we derived an equation from known metabolic relationships to estimate inlet flow rates, improving the efficiency of data collection. To validate the neonatal rodent indirect calorimetry system and evaluate the applicability of the derived equation for predicting appropriate flow rates, we conducted a series of experiments evaluating the impact of sex, litter size, time of day (during the light phase), and ambient temperature on neonatal rat metabolic parameters. Data revealed that the only metabolic parameter influenced by litter size is a neonatal rat''s RQ, with rat pups reared in a small litter (5 pups) having lower RQ''s than rat pups reared in either medium (8 pups) or large (11 pups) litters. Furthermore, data showed that ambient temperature affected all metabolic parameters measured, with colder temperatures being associated with higher CO₂ production, higher O₂ consumption, and higher energy expenditure.

Conclusion/Significance

The results of this study demonstrate that the modified Panlab Oxylet system reliably assesses early postnatal metabolism in individual neonatal rodents. This system will be of paramount importance to further our understanding of processes associated with the developmental origins of adult metabolic disease.     

DNA barcode ITS2 coupled with high resolution melting (HRM) analysis for taxonomic identification of Sideritis species growing in Greece

Identification of genotypes in Sideritis is complicated owing to the morphological similarity and common occurrence of natural hybridisation within Sideritis species. Species- and genotype-specific DNA markers are very useful for plant identification, breeding and preservation programs. Herein, a real-time polymerase chain reaction (PCR) of ITS2 barcode region coupled with high resolution melting-curve (HRM) analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on seven Sideritis species growing in Greece. The HRM assay developed in this study is a rapid and straightforward method for the identification and discrimination of the investigated Sideritis species. This assay is simple compared to other genotyping methods as it does not require DNA sequencing or post-PCR processing. Therefore, this method offers a new alternative for rapid detection of Sideritis species.     

Highly Sensitive Quantitative PCR for the Detection and Differentiation of Pseudogymnoascus destructans and Other Pseudogymnoascus Species

White-nose syndrome is a fungal disease that has decimated bat populations across eastern North America. Identification of the etiologic agent, Pseudogymnoascus destructans (formerly Geomyces destructans), in environmental samples is essential to proposed management plans. A major challenge is the presence of closely related species, which are ubiquitous in many soils and cave sediments and often present in high abundance. We present a dual-probe real-time quantitative PCR assay capable of detecting and differentiating P. destructans from closely related fungi in environmental samples from North America. The assay, based on a single nucleotide polymorphism (SNP) specific to P. destructans, is capable of rapid low-level detection from various sampling media, including sediment, fecal samples, wing biopsy specimens, and skin swabs. This method is a highly sensitive, high-throughput method for identifying P. destructans, other Pseudogymnoascus spp., and Geomyces spp. in the environment, providing a fundamental component of research and risk assessment for addressing this disease, as well as other ecological and mycological work on related fungi.     

Biochemical Measurement of Neonatal Hypoxia

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment ^1-3. Decreased adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy phosphate bonds are degraded ². This leads to increased levels of adenosine which is subsequently degraded to inosine, hypoxanthine, xanthine, and finally to uric acid. The final two steps in this degradation process are performed by xanthine oxidoreductase. This enzyme exists in the form of xanthine dehydrogenase under normoxic conditions but is converted to xanthine oxidase (XO) under hypoxia-reperfusion circumstances ^{4, 5}. Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation ^{4, 6}. This hydrogen peroxide in combination with other reactive oxygen species (ROS) produced during hypoxia, oxidizes uric acid to form allantoin and reacts with lipid membranes to generate malondialdehyde (MDA) ^7-9. Most mammals, humans exempted, possess the enzyme uricase, which converts uric acid to allantoin. In humans, however, allantoin can only be formed by ROS-mediated oxidation of uric acid. Because of this, allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have uricase.We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure biochemical markers of neonatal hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for uricase-generated allantoin. Purine metabolites were linked to hypoxia as early as 1963 and the reliability of hypoxanthine, xanthine, and uric acid as biochemical indicators of neonatal hypoxia was validated by several investigators ^10-13. The HPLC method used for the quantification of purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al⁷. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere ^{14, 15}. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. ^{16, 17}. Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin ¹⁸. This approach proved to be sufficiently sensitive and reproducible. Download video file.^{(77M, mov)}