Bird feeding at urban wetlands: A comparison of demographics, attitudes and norms between feeders and non-feeders.

The feeding of wild birds is a popular but often controversial activity. This study explored differences in demographics, attitudes, and normative beliefs between waterbird feeders and non-feeders at an urban wetland residential estate in Melbourne, Australia. An online survey of nearby residents and visitors (n = 206) identified those who have fed waterbirds at least once in the past two years (feeders; 32.4 %) and those who have not (non-feeders). No differences were observed in demographic profile or connection to nature between feeders and non-feeders, but feeders were significantly more likely to believe that waterbird feeding is an acceptable activity. When compared with non-feeders, feeders exhibited different injunctive and descriptive norms relating to waterbird feeding; feeders believed that most people would be relatively happy with them feeding waterbirds in their community, while non-feeders thought that most people would be moderately unhappy. Feeders believed that more than half of the people in their community fed waterbirds (55.5 %), while non-feeders believed that less than half fed birds (36.7 %). These findings suggest that education or behaviour change programs with bird feeding objectives could be enhanced with information about the actual and perceived social norms for this common activity.

Ferroptosis is induced following siramesine and lapatinib treatment of breast cancer cells.

Ferroptosis is an iron-dependent, oxidative cell death, and is distinct from apoptosis, necrosis and autophagy. In this study, we demonstrated that lysosome disrupting agent, siramesine and a tyrosine kinase inhibitor, lapatinib synergistically induced cell death and reactive oxygen species (ROS) in MDA MB 231, MCF-7, ZR-75 and SKBr3 breast cancer cells over a 24 h time course. Furthermore, the iron chelator deferoxamine (DFO) significantly reduced cytosolic ROS and cell death following treatment with siramesine and lapatinib. Furthermore, we determined that FeCl3 levels were elevated in cells treated with siramesine and lapatinib indicating an iron-dependent cell death, ferroptosis. To confirm this, we treated cells with a potent inhibitor of ferroptosis, ferrastatin-1 that effectively inhibited cell death following siramesine and lapatinib treatment. The increase levels of iron could be due to changes in iron transport. We found that the expression of transferrin, which is responsible for the transport of iron into cells, is increased following treatment with lapatinib alone or in combination with siramesine. Knocking down of transferrin resulted in decreased cell death and ROS after treatment. In addition, ferroportin-1 (FPN) is an iron transport protein, responsible for removal of iron from cells. We found its expression is decreased after treatment with siramesine alone or in combination with lapatinib. Overexpression FPN resulted in decreased ROS and cell death whereas knockdown of FPN increased cell death after siramesine and lapatinib treatment. This indicates a novel induction of ferroptosis through altered iron regulation by treating breast cancer cells with a lysosome disruptor and a tyrosine kinase inhibitor.

BNIP3 acts as transcriptional repressor of death receptor-5 expression and prevents TRAIL-induced cell death in gliomas.

Glioblastoma multiforme (GBM) is the most common and malignant brain tumor, and current treatment modalities such as surgical resection, adjuvant radiotherapy and temozolomide (TMZ) chemotherapy are ineffective. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a novel cancer therapeutic agent for GBM because of its capability of inducing apoptosis in glioma cells. Unfortunately, the majority of glioma cells are resistant to TRAIL-induced apoptosis. The Bcl-2 nineteen kilodalton interacting protein (BNIP3) is a pro-cell death BH3-only member of the Bcl-2 family that is one of the highest expressed genes in hypoxic regions of GBM tumors. We previously found that BNIP3 is localized to the nucleus in GBM tumors and suppresses cell death in glioma cells. Herein, we have discovered when BNIP3 nuclear expression is knockdown in glioma cell lines and in normal mouse astrocytes, TRAIL and its death receptor, death receptor-5 (DR5) expression is increased. In addition, when nuclear BNIP3 expression is increased, the amount of TRAIL-induced apoptosis is reduced. Using a streptavidin pull-down assay, we found that BNIP3 binds to the DR5 promoter and nuclear BNIP3 binds to the DR5 promoter. Furthermore, nuclear BNIP3 expression in GBM tumors correlates with decreased DR5 expression. Taken together, we have discovered a novel transcriptional repression function for BNIP3 conferring a TRAIL resistance in glioma cells.

Epidermal growth factor regulates Mcl-1 expression through the MAPK-Elk-1 signalling pathway contributing to cell survival in breast cancer.

Myeloid cell leukaemia-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 family that is elevated in a variety of tumour types including breast cancer. In breast tumours, increased Mcl-1 expression correlates with high tumour grade and poor patient survival. We have previously demonstrated that Her-2 levels correspond to increased Mcl-1 expression in breast tumours. Epidermal growth factor (EGF) receptor signalling is frequently deregulated in breast cancer and leads to increased proliferation and survival. Herein, we determined the critical downstream signals responsible for the EGF mediated increase of Mcl-1 and their role in cell survival. We found that both Mcl-1 mRNA and protein levels are rapidly induced upon stimulation with EGF. Promoter analysis revealed that an Elk-1 transcription factor-binding site is critical for EGF activation of the Mcl-1 promoter. Furthermore, we found that knockdown of Elk-1 or inhibition of the Erk signalling pathway was sufficient to block EGF upregulation of Mcl-1 and EGF mediated cell survival. Using chromatin immunoprecipitation and biotin labelled probes of the Mcl-1 promoter, we found that Elk-1 and serum response factor are bound to the promoter after EGF stimulation. To determine whether Mcl-1 confers a survival advantage, we found that knockdown of Mcl-1 expression increased apoptosis whereas overexpression of Mcl-1 inhibited drug induced cell death. In human breast tumours, we found a correlation between phosphorylated Elk-1 and Mcl-1 protein levels. These results indicate that the EGF induced activation of Elk-1 is an important mediator of Mcl-1 expression and cell survival and therefore a potential therapeutic target in breast cancer.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) up-regulates death receptor 5 (DR5) mediated by NFkappaB activation in epithelial derived cell lines.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL/APO-2L) activates nuclear factor kappaB (NFkappaB). This activation is regulated by the recruitment of an adaptor protein Fas associating death domain (FADD) to TRAIL death receptors, death receptor 4 (DR4, TRAIL-R1) and death receptor 5 (DR5 TRAIL-R2). This leads to recruitment of caspase 8 and receptor interacting protein (RIP) to the receptor complex. Upon recruitment of caspase 8 and RIP, NFkappaB inducing kinase (NIK) becomes activated causing NFkappaB activation. The role of TRAIL induced NFkappaB activation in epithelial cells is unknown. Herein we demonstrate that TRAIL increases expression of DR5 in human embryonic kidney (HEK) 293, MCF-7 and MDA MB 231 epithelial cell lines while DR4 expression remains unchanged. Blockage of NFkappaB activation either by expression of dominant negative IkappaB or treatment with proteasome inhibitor lactacystin eliminates TRAIL induced DR5 expression. Expression of FADD dominant negative in HEK 293 cells that prevents the recruitment of caspase 8 and RIP to TRAIL death receptors also eliminates this increase. By over expression of the p65 subunit of NFkappaB that increases NFkappaB transcriptional activity, DR5 expression was increased compared to vector alone expressing cells. By blocking TRAIL induced NFkappaB activation, the sensitivity of cells to undergo TRAIL induced apoptosis was significantly decreased. Conversely, the amount of TRAIL induced apoptosis was increased in HEK 293 cells over expressing p65 subunit of NFkappaB. Finally blockage of NFkappaB activation eliminates the synergistic apoptotic response of TRAIL and etoposide. Thus, TRAIL mediated NFkappaB activation increases DR5 expression thereby amplifying the apoptotic response of TRAIL in epithelial derived cells.

Placental changes associated with fetal outcome in the Plasmodium coatneyi/rhesus monkey model of malaria in pregnancy.

Term placentas collected surgically from seven Plasmodium coatneyi-infected rhesus monkeys, one abortion, and five controls were evaluated histopathologically. The placentas from Plasmodium-infected dams had more significant pathologic changes than those from controls for six parameters (P < 0.05) and higher numbers of activated (LN5 + Zymed) macrophages in the intervillous space (IVS) (P = 0.0173). Total parasite load (TPL) was defined as the sum of all weekly peripheral infected red blood cell counts for each trimester and for the entire pregnancy. High first trimester PLs were more likely to result in fetal demise (P = 0.0476) or increased placental damage in surviving infants. As trimester 2-3 TPL increased, so did the number of activated macrophages (P < 0.05) and the total malaria pigment scores (P < 0.05). Low birth weight (LBW) and intrauterine growth retardation (IUGR) were associated with high pigment scores and high numbers of activated macrophages in the IVS. High placental damage scores were not associated with IUGR, LBW, or early infant mortality.

Defects in base excision repair combined with elevated intracellular dCTP levels dramatically reduce mutation induction in yeast by ethyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine.

Previously, we determined that elimination of deoxycytidylate (dCMP) deaminase (DCD1) in the yeast Saccharomyces cerevisiae increases the intracellular dCTP:dTTP ratio and reduces the induction of G x C --> A x T transitions in the SUP4-o gene by ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Simultaneously, the G x C --> C x G transversion frequency rises substantially. We attributed the first response to dCTP outcompeting dTTP for incorporation opposite O6-alkylguanine, and the second outcome to the increased dCTP pool causing error-prone repair of apurinic (AP) sites resulting from the removal or lability of N7-alkylguanine. To test the latter hypothesis, we used isogenic dcd1 strains deleted for either of two genes (MAG1: 3-methyladenine glycosylase; APN1: apurinic endonuclease) involved in the repair of N7-alkylguanine. In these backgrounds, EMS or MNNG induction of total SUP4-o mutations, G x C --> A x T transitions and G x C --> C x G transversions were reduced by >98%, >97%, and >80%, respectively. Mutation frequencies in the dcd1 apn1 strain were close to those for spontaneous mutagenesis in the wild-type parent. These findings argue that misincorporation of dCTP during repair of alkylation-induced AP sites is responsible for the increased G x C --> C x G transversion frequency in the dcd1 strain treated with EMS or MNNG. The data also demonstrate that defective repair of AP sites coupled with an elevated dCTP:dTTP ratio eliminates most EMS and MNNG mutagenesis. In addition, the results point to a role for AP sites in the production of some EMS- and MNNG-induced G x C --> A x T transitions as well as other substitutions in the dcd1 strain.

Plasmodium coatneyi in the rhesus monkey (Macaca mulatta) as a model of malaria in pregnancy.

Pregnant women with Plasmodium falciparum infection are at increased risk for complications such as anemia and cerebral malaria. In addition, the infants of these women suffer intrauterine growth retardation (IUGR), low birth weight (LBW), congenital infection, and high infant mortality. Although much has been learned from studies of malaria during human pregnancy, progress has been limited by the lack of a suitable animal model. Nonhuman primates are of particular interest because, other than the armadillo, they are the only animals with a discoidal, villous, hemochorial placenta like that of humans. We have established a model of malaria during human pregnancy by inoculating pregnant rhesus monkeys (Macaca mulatta) with Plasmodium coatneyi (a sequestering parasite) during the first trimester. In our initial experiment, four monkeys were inoculated with a fresh inoculum containing 10(8) viable parasites from an infected donor monkey. All four monkeys became parasitemic seven days postinoculation (PI) and three monkeys aborted 7-10 days PI coincident with high peak parasitemias (41,088-374,325 parasites/mm3). Although abortion is one of the outcomes observed in Plasmodium-infected women, the intent of this study was to examine the effects of Plasmodium infection throughout gestation. Since the rapid onset of high parasitemia may have been responsible for the abortions, a decision was made to reduce the size of the effective inoculum. Six additional pregnant monkeys were inoculated with a frozen isolate taken from the same donor containing 10(6) parasites. These six animals became parasitemic by 14 days PI and, along with monkey E412, carried their infants to term. These seven infants weighed significantly less at term than the infants of uninfected mothers (P = 0.0355). Symmetrical IUGR was detected by ultrasound in one fetus with an LBW of 334 g. Another LBW infant (300 g) had asymmetrical growth retardation, which has been associated with uteroplacental insufficiency and was consistent with the lower placental weights found in infected dams compared with controls (P = 0.0455). The infant with symmetric IUGR died at five days of age, while the other is alive but congenitally infected. The IUGR, LBW, congenital infection, postnatal infant mortality, and early abortions observed in these animals suggest that P. coatneyi in pregnant rhesus monkeys is a valid model of malaria in human pregnancy. This model should provide the opportunity to study questions about malaria in pregnancy that have been difficult to study in humans.

Position and velocity coupling of postural sway to somatosensory drive.

Light touch contact of a fingertip to a stationary surface provides orientation information that enhances control of upright stance. Slight changes in contact force at the fingertip lead to sensory cues about the direction of body sway, allowing attenuation of sway. In the present study, the coupling of postural sway to a moving contact surface was investigated in detail. Head, center of mass, and center of pressure displacement were measured as the contact surface moved rhythmically at 0.1, 0.2, 0.4, 0.6, and 0.8 Hz. Stimulus amplitude decreased with frequency to maintain peak velocity constant across frequency. Head and body sway were highly coherent with contact surface motion at all frequencies except 0.8 Hz, where a drop-off in coherence was observed. Mean frequency of head and body sway matched the driving frequency

Cost-effective poster and print production with digital camera and computer technology.

OBJECTIVE: The purpose of this report is to describe a cost-effective method for producing black-and-white prints and color posters within a radiology department. CONCLUSION: Using a high-resolution digital camera, personal computer, and color printer, the average cost of a 5 x 7 inch (12.5 x 17.5 cm) black-and-white print may be reduced from $8.50 to $1 each in our institution. The average cost for a color print (8.5 x 14 inch [21.3 x 35 cm]) varies from $2 to $3 per sheet depending on the selection of ribbons for a color-capable laser printer and the paper used. For a 30-panel, 4 x 8 foot (1.2 x 2.4 m) standard-sized poster, the cost for materials and construction is approximately $100.

Effect of naproxen on gastroesophageal reflux and esophageal function: a randomized, double-blind, placebo-controlled study.

OBJECTIVES: Gastrointestinal symptoms, particularly pyrosis, complicate nonsteroidal anti-inflammatory drug (NSAID) use. NSAIDs cause esophageal injury, and H2 blockers are often prescribed for, and successfully control, NSAID-related symptoms. To determine whether NSAIDs can induce gastroesophageal reflux, we studied the effect of a commonly used NSAID, naproxen, on reflux parameters and esophageal function. METHODS: Nine healthy volunteers (five males, four females, age 23-34 yr) were studied. After basal measurements were taken, the subjects randomly received naproxen 500 mg p.o. b.i.d. or placebo for 1 wk. On day 6, the subjects underwent esophageal manometry with a water-perfused system and Dent sleeve. Body pressures, contraction velocity, and duration of contraction were recorded in the distal 7 cm of the esophagus. The lower esophageal sphincter pressure (LESP) and number of transient relaxations (TLESRs) were monitored. This was followed by 24-h pH monitoring. The subjects then crossed over to the other drug after a minimum 14-day wash-out period. RESULTS: No subject experienced any GI symptoms during the study. One subject developed reflux-induced symptoms a few months after completing the study and was excluded from the analysis. The total fraction of time (pH < 4) was 4.9 +/- 1.0% in the basal state, 5.5 +/- 1.4% on placebo, and 5.4 +/- 1.5% on naproxen. These differences were not significant. The number of reflux episodes and the esophageal clearance time were not affected by naproxen. The LESP in the basal state was 32.1 +/- 5.6 mm Hg, 32.3 +/- 4.2 mm Hg on placebo, and 29.9 +/- 3.3 mm Hg on naproxen (p = NS). The number of TLESRs per 30 minutes in the basal state was 3.5 +/- 0.9, 4.6 +/- 1.2 on placebo, and 5.8 +/- 1.0 on naproxen (p = NS). The speed and duration of contractions were not affected by naproxen. The excluded subject had marked basal reflux (total fraction of time pH < 4 = 10.7%), low LESP (8 mm Hg), and a marked increase in reflux on naproxen (total fraction of time pH < 4 = 53%). CONCLUSIONS: Naproxen did not induce reflux in normal subjects, although reflux did increase in some subjects. Naproxen had no significant effect on motility parameters. Our data suggest that NSAIDs do not impair the anti-reflux barrier or induce reflux. Pyrosis experienced during NSAID use may not arise from the esophagus or may reflect altered esophageal sensitivity. A single subject with decreased LESP and asymptomatic increased acid exposure in the basal state had a marked increase in reflux on naproxen. This person subsequently developed symptomatic gastroesophageal reflux. The effect of NSAIDs on individuals with a propensity to reflux deserves further study.

Characterization of the Epstein-Barr virus Fp promoter.

Expression of the Epstein-Barr virus nuclear antigen-1 (EBNA-1) protein is mediated by the virus Fp promoter in Burkitt lymphoma and nasopharyngeal carcinoma. This promoter is silent in latently infected B lymphoblastoid and most Burkitt lymphoma-derived cell lines in vitro, which utilize separate promoters approximately 50 kb upstream of Fp to express EBNA proteins. Fp-mediated activation of EBNA-1 expression is also activated upon induction of the virus replication cycle. We previously demonstrated that activation of Fp in Burkitt cells requires cis-regulatory elements downstream of the site of transcription initiation. We have now mapped two positive regulatory elements within the Fp promoter. One element contains two potential binding sites for the cellular transcription factor LBP-1 between +138 and +150. A second regulatory element was mapped between +177 and +192 and can be specifically bound in vitro by protein from nuclear extracts of Burkitt cells. Although this element overlaps two partial E2F binding sites and Fp reporter plasmids could be activated in trans by the adenovirus E1A protein in cotransfection experiments, mutational analysis and DNA binding studies suggest that these are unlikely to be functional E2F response elements within Fp. We also demonstrate that Fp-directed transcription initiates at multiple sites within both the genome and the Fp reporter plasmids. However, the principal site of transcription initiation within the genome is not utilized within reporter plasmids, in which the majority of transcripts initiate at multiple sites between +150 and +200. This finding suggests that additional elements may be necessary for Fp to function normally in these assays or that the context of Fp within the viral genome is critical to its regulation.

Characterization of trimethoprim- and sulphisoxazole-resistant Chlamydia trachomatis.

Trimethoprim and sulphisoxazole were used as selective agents in culture to isolate, by a stepwise procedure, a series of Chlamydia trachomatis L2 populations resistant to the cytotoxic effects of the drugs. Two trimethoprim-resistant populations, L2TriR-60 and L2TriR-100, and one sulphonamide-resistant population, L2SulfR-100, were characterized in more detail. In addition to being resistant to trimethoprim, L2TriR-60 was cross-resistant to methotrexate, sensitive to sulphisoxazole and displayed a ribonucleotide auxotrophy similar to that of its parental wild type, C. trachomatis L2. Surprisingly, L2TriR-100 and L2SulfR-100 appeared phenotypically identical. Both mutants were highly resistant to trimethoprim, sulphisoxazole, and methotrexate. In contrast to wild-type C. trachomatis L2, these populations were sensitive to 5-fluorouracil. L2TriR-100 and L2SulfR-100 were incapable of taking pyrimidine ribonucleotides from the host cell and no longer synthesized thymidine nucleotides de novo. The pyrimidine requirement of these mutants was met by salvaging host-cell uracil and thymidine, a property which can account for their drug-resistant characteristics. L2TriR-100 and L2SulfR-100 could also salvage adenine and guanine. Using L2TriR-100 as a starting stock, a mutant population resistant to the cytotoxic effects of trimethoprim and 5-fluorouracil (L2Tri/5-FU) was selected. L2Tri/5-FU was resistant to 5-fluorouracil because it had regained the capacity to take pyrimidine ribonucleotides from the host cell.

Specificity of the mutator caused by deletion of the yeast structural gene (APN1) for the major apurinic endonuclease.

The loss of bases from cellular DNA occurs via both spontaneous and mutagen-induced reactions. The resulting apurinic/apyrimidinic (AP) sites are cytotoxic and mutagenic but are counteracted by repair initiated by AP endonucleases. Previously, in vitro and bacterial transfection studies suggested that AP sites often prompt insertion of dAMP residues during replication, the A-rule. Dissimilar results have been obtained by transfecting DNA into eukaryotic cells. It seemed possible that these differences might be due to idiosyncrasies of transfection or aberrant replication of the transecting DNA. The observation that AP endonuclease-deficient strains of the yeast Saccharomyces cerevisiae have elevated spontaneous mutation rates allowed us to determine the mutational specificity of endogenously generated AP sites in nuclear DNA. With the yeast SUP4-o gene as a mutational target, we found that a deficiency in the major yeast AP endonuclease, Apn1, provoked mainly single base-pair substitution; the rate of transposon Ty insertion was also enhanced. The rate of transversion to a G.C pair was increased 10-fold in Apn1-deficient yeast, including a 59-fold increase in the rate of A.T-->C.G events. In contrast, the rate of transversion to an A.T pair was increased by only 3-fold. A deficiency in N3-methyladenine glycosylase offset these substitution rate increases, indicating that they are due primarily to AP sites resulting from glycosylase action. Thus, the A-rule does not seem to apply to the mutagenic processing of endogenous abasic sites in S. cerevisiae. Other results presented here show that AP endonuclease-deficient Escherichia coli exhibit a mutator phenotype consistent with the A-rule.

Is the antioxidant, anti-inflammatory putative new vitamin, PQQ, involved with nitric oxide in bone metabolism?

Our laboratory recently isolated free PQQ (2,7,9-tricarboxy-pyrroloquinoline quinone, methoxatin), a bacterial redox cofactor, from red cells, neutrophils, serum and milk and found free PQQ in CSF, synovial fluid and bile. The metabolism and functions of PQQ and ascorbate may be coupled. Physiologically, free PQQ catalyzes dioxygen-superoxide interconversion, and participates in both superoxide generation (respiratory burst) and scavenging (cell protection). Using a labeled aromatic o-diamine, superoxide formation by activated neutrophils was inhibited and the labeled phenazine adduct of PQQ could be isolated from the inhibited cells (Karnovsky et al., 1992). PQQ may convert xanthine oxidase to xanthine dehydrogenase (XD) and could be the physiological coenzyme of XD. PQQ plus copper, form a potent amine-oxidizing system. Shah et al., 1992 found that PQQ-Cu2+ catalyzes the oxidation of epsilon-amino groups in collagen and elastin. Rucker's lab (Smidt et al., 1991) has found that PQQ may be a vitamin for mouse pups. Watanabe et al., 1988 and Nishigori et al., 1989, showed that injected PQQ protects animals against oxidative stress injury. PQQ's in vivo antioxidant action, spares reduced glutathione. PQQ, as an actively transported organic anion, concentrates in cells. In other experiments (Aizenman et al., 1992), PQQ protected neurons against the neurotoxin action of the glutamate-receptor against NMDA. We shall consider possible roles for PQQ in the biosynthesis of nitric oxide (NO, endothelium-derived relaxing factor, EDRF) from L-arginine and in NO removal by superoxide. NO has now been linked to the inhibition of osteoclastic bone resorption.

The Epstein-Barr virus nuclear protein 1 promoter active in type I latency is autoregulated.

The only member of the Epstein-Barr virus family of nuclear proteins (EBNAs) expressed during type I and type II latent infections is EBNA-1. This is in contrast to type III latency, during which all six nuclear proteins are expressed from a common transcription unit. The exclusive expression of EBNA-1 during type I and II latency is mediated through a recently identified promoter, Fp. The objective of this study was to characterize Fp in the Burkitt lymphoma cell background, where it is known to be differentially utilized. Using a short-term transfection assay and reporter gene plasmids containing Fp linked to the human growth hormone, we examined Fp activity in type I and type III latently infected and virus-negative Burkitt lymphoma cells. The data suggested that Fp is predominantly regulated through two distinct elements located between +24 and +270 relative to the transcription start site. One element positively mediates Fp activity, probably at the level of transcription, and acts in a virus-independent manner. The second element contains the EBNA-1 DNA binding domain III and negatively regulates Fp-directed gene expression in trans with EBNA-1 in type III as well as type I latency. Thus, we have identified a third function of EBNA-1, i.e., that of a repressor of gene expression, in addition to its known role in viral DNA replication and its ability to trans-activate gene expression. The overall activity of Fp in type I latently infected Burkitt cells was approximately sixfold lower than in virus-negative Burkitt cells, in which there is no autoregulation, suggesting that there is a fine balance between these two opposing regulatory elements during type I latency.

Acid-promoted tautomeric lactonization and oxidation-reduction of pyrroloquinoline quinone (PQQ).

Acid-treatment facilitates PQQ detection by electron ionization mass spectroscopy with a molecular ion at M/e 330 and a base ion formed by triple decarboxylation at M/e 198. Other ions found probably arise through acid-catalyzed tautomeric lactonization of PQQ to PQQ-lactone (PQQL) with subsequent oxidation of PQQL and reduction of PQQ. We propose that a carboxyl group, presumably the 9-carboxyl, attacks a double bond in PQQ, reversibly converting the 4,5-orthoquinone into an 4,5-enediol and forming an isomeric lactone, PQQL, of 330 daltons. The masking of carbonyls may explain the low reactivity of PQQ with carbonyl reagents in acid. Acid-promoted tautomeric lactonization with carbonyl-masking is known to occur with fluoresceins, phenolphthalein and other compounds, but has not been recognized before with PQQ. Acid-treated PQQ demonstrates molecular and other ions derived from reduced PQQ (PQQ(2H] or its lactone at M/e 332 with a base ion at M/e 200. There is compelling evidence for a dehydrogenated lactone, PQQ(-2H)L), at M/e 328 with a base ion at M/e 196. We suggest that PQQ, in tautomeric equilibrium with PQQL, oxidizes PQQL to PQQ(-2H)L (328 daltons), with its concurrent reduction to PQQ(2H) (332 daltons). With acidified D2O, PQQ shows deuterated products with ions at M/e values consistent with lactonization and oxidation-reduction. An analytically useful quinoxaline adduct, formed from PQQ and 2,3-diaminonaphthalene (PQQ-DAN) of 452 daltons, also undergoes acid-tautomerization-lactonization and oxidation-reduction similar to PQQ showing molecular ions at M/e 450, 452 and 454 and decarboxylation-derived strong (base) ions at M/e 318, 320 and 322.(ABSTRACT TRUNCATED AT 250 WORDS)

Separation and determination of alpha-amino acids by boroxazolidone formation.

Reaction of an alpha-amino acid (alpha-AA) with 1,1-diphenylborinic acid (DPBA) leads to the formation of a kinetically stable adduct at pH 2-5 in which both the alpha-amino and the alpha-carboxyl groups are bound to boron forming a cyclic mixed anhydride termed a boroxazolidone. In this adduct, the greater than N:B bond is coordinate, involving the free electron pair of nitrogen, thereby satisfying the octet rule for the second electron shell of boron (Group IIIA). Consequently, the alpha-amino function of the boroxazolidone can be primary, secondary, or tertiary, as demonstrated by boroxazolidone formation with glycine, N-methylglycine, and N,N-dimethylglycine. On reaction with DPBA, the alpha-AA moiety of N-terminal gamma-glutamyl peptides is also derivatized as demonstrated by the formation of a glutathione boroxazolidone. The 1,1-diphenylboroxazolidone adducts of alpha-AA may be separated by reversed-phase (RP)-HPLC (AA-DPBA/RP-HPLC) enabling the derivatization procedure to be used as a precolumn reaction for alpha-AA analysis. Under the conditions we describe here, DPBA is not stably reactive with the epsilon-amino group of lysine. Furthermore, it does not complex with amide bonds of the peptide backbone or to any side chains of the common amino acids. Reaction of an alpha-AA mixture with DPBA, followed by RP-HPLC (AA-DPBA/RP-HPLC) is then a simple method by which to analyze alpha-AA in a mixture with peptides and amines. Precolumn reaction with DPBA may be used to separate peptides from alpha-AA and from those peptides which contain an alpha-AA moiety. Unreacted peptides are bound only weakly to the HPLC column and thus are separated from reacted alpha-amino acids which are retained as 1,1-diphenylboroxazolidones until their selective elution. This method is particularly suited for the analysis of alpha-amino acids that are derived from post-translational modification of protein side chains.