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Sex steroids are pervasive in mammals and evolutionarily conserved, but differences in the nuances of endocrine profiles characterize distinct species. Two sex steroids, testosterone and progesterone, feature prominently in the life history of mammalian taxa, but neither one has been analyzed from wild giraffes. Our study was designed to address this gap in knowledge by examining how these sex steroids are related to biological features and giraffe life history. We conducted the research at Rooipoort Nature Reserve a 44,000 ha private nature reserve in South Africa on a population of South African giraffes (Giraffa camelopardalis giraffa). Eleven adult giraffe cows and seven adult bulls were immobilized and various biological samples and morphological measurements were obtained. We analyzed both testosterone and progesterone using Ultra High Performance Liquid Chromotography - Tandem Mass Spectrometry (UHPLC-MS/MS). We found that non-pregnant females had lower progesterone concentrations than pregnant females and that those in the last trimester of pregnancy showed a slight drop in progesterone, as well as an increase in testosterone. Among males, chronological age was correlated with testicle size, testosterone concentrations, and ossicone volume. We propose that the progesterone decline functions partly to accelerate resumption of ovulation because giraffes become pregnant while lactating, and that the testosterone elevation provides an endocrine mileu for female defense of neonatal calves, given that lion predation is a major threat to calf survival. We suggest that male reproductive strategies are mediated by the age-related impacts of testosterone on growth in both body mass and ossicone volume as a consequence of sexual selection. We conclude by noting that the robust and solid ossicones of male giraffes function in mate competition as wedges that are used to topple opponents by raising their legs and placing them in an off-balanced position that can seriously injure them when falling to the ground.
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Girafas , Gravidez , Animais , Bovinos , Masculino , Feminino , África do Sul , Lactação , Progesterona , Seleção Sexual , Espectrometria de Massas em Tandem , Ruminantes , TestosteronaRESUMO
PURPOSE: Mebos, a traditional South Africa confection consisting of dried, pulped, and sugared apricots, is rich in fibre and vitamins, but also contains salicylic acid, flavonoids, and citric acid. We report a case of postoperative surgical site bleeding in a healthy patient who consumed approximately 2 kg of mebos per day prior to his elective spinal surgery. METHODS: The clinical course of a previously healthy 54-year-old male patient with cauda equina syndrome secondary to lumbar spinal stenosis who underwent surgical intervention with subsequent bleeding into the surgical site is discussed. The cause was investigated through biochemical analysis, thromboelastometry (ROTEM®) and mass and absorption spectrometry were applied to assess flavonoid, citric acid, and salicylic acid content. RESULTS: ROTEM® revealed an abnormal clotting profile with an increased clot forming time, suggesting intrinsic coagulopathy. Mass and absorption spectrometry revealed a high total flavonoid content as well as citric acid concentration in the mebos. Salicylic acid was at detection limits of the instrument. CONCLUSION: Results highlighted the effect of flavonoids and citric acid and therefore explain the abnormal clotting profile in this patient. Inhibition of coagulation prior to elective surgery is a known contraindication and may pose great risks in spinal surgery. In the present report, we demonstrated an association between inhibition of coagulation and an excess of the flavonoids content and citric acid concentration in mebos consumed in the days prior to elective spinal surgery.
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Vértebras Lombares , Humanos , Masculino , Pessoa de Meia-Idade , Vértebras Lombares/cirurgia , Frutas , Estenose Espinal/cirurgia , Hematoma Epidural Espinal/etiologia , Hematoma Epidural Espinal/cirurgia , Hematoma Epidural Espinal/diagnóstico por imagem , Síndrome da Cauda Equina/etiologia , Síndrome da Cauda Equina/cirurgiaRESUMO
South Africa has a rich history of medicinal plant species and their documented uses as traditional medicines, and is also home to three well-known, blue-flowered sage species of ethnobotanical importance. The Namaqualand bloublomsalie (Salvia dentata) has so far remained unstudied and apparently overlooked. Our study is the first to report on the essential oil chemistry of this medicinally relevant species and provide a comparison with the other two (well-studied) closely related Cape bloublomsalies (Salvia africana and S. chamelaeagnea). The data, generated from three geographically isolated populations comprised of 13 individual plants of S. dentata, revealed diagnostically high levels of camphor (14.37%), α-pinene (11.43%), camphene (10.18%), 1,8-cineole (eucalyptol) (9.42%) and bornyl acetate (8.56%) which provide a distinct chemical profile from the other two species.
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Óleos Voláteis , Plantas Medicinais , Salvia , Monoterpenos Bicíclicos , Cânfora , Eucaliptol , Óleos Voláteis/química , Salvia/química , África do SulRESUMO
The urease enzyme of Cryptococcus neoformans is linked to different metabolic pathways within the yeast cell, several of which are involved in polyamine metabolism. Cryptococcal biogenic amine production is, however, largely unexplored and is yet to be investigated in relation to urease. The aim of this study was therefore to explore and compare polyamine metabolism in wild-type, urease-negative and urease-reconstituted strains of C. neoformans. Mass spectrometry analysis showed that agmatine and spermidine were the major extra- and intracellular polyamines of C. neoformans and significant differences were observed between 26 and 37 °C. In addition, compared to the wild-type, the relative percentages of extracellular putrescine and spermidine were found to be lower and agmatine higher in cultures of the urease-deficient mutant. The inverse was true for intracellular spermidine and agmatine. Cyclohexylamine was a more potent polyamine inhibitor compared to DL-α-difluoromethylornithine and inhibitory effects were more pronounced at 37 °C than at 26 °C. At both temperatures, the urease-deficient mutant was less susceptible to cyclohexylamine treatment compared to the wild-type. For both inhibitors, growth inhibition was alleviated with polyamine supplementation. This study has provided novel insight into the polyamine metabolism of C. neoformans, highlighting the involvement of urease in biogenic amine production.
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Cryptococcus neoformans , Poliaminas/metabolismo , Urease/metabolismo , Putrescina , EspermidinaRESUMO
Wildly grown in most regions of the world, Carissa edulis is a highly underutilised fruit with significant antioxidant characteristics. The phyto and physicochemical properties of C. edulis berries at different stages of ripening are evaluated in this work. Total flavonoids (TF), total phenolic content (TPC) and antioxidant activity were determined spectrophotometrically, while concentration of polyphenols was determined using liquid chromatography coupled to diode array detection and electrospray ionization mass spectrometry. Results showed that antioxidant activity was lowest (18.36 ± 0.12 mg TE/g) in RS3 and decreased with TPC upon increased ripening. Conversely, TF increased with ripening progression with TF found to be highest in RS3 (5.92 ± 0.03 mg CE/g). Identified phenolic acids in C. edulis were quinic acid, protocatechuoyl-hexose, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and dicaffeoylquinic acid. Identified flavonoids included rutin, catechin, procyanidin dimer, procyanidin trimer, quercetin-3-O-glucosyl-xyloside, quercetin-3-O-robinobioside, quercetin-3-O-glucoside and quercetin-3-OH-3-methylglutaryl-glucoside. Physicochemical properties of C. edulis varied among samples with sugar/acid ratio of C. edulis ranging from 25.70 for RS1 to 50.36 for RS3. Ripening stage of C. edulis undoubtedly affects the phyto and physicochemical properties of C. edulis.
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Apocynaceae/química , Fenômenos Químicos , Frutas/química , Compostos Fitoquímicos/química , Polifenóis/química , Antioxidantes/química , Antioxidantes/farmacologia , Concentração de Íons de Hidrogênio , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Polifenóis/isolamento & purificação , Polifenóis/farmacologia , Solubilidade , Análise EspectralRESUMO
BACKGROUND: Preterm preeclampsia has a high rate of fetal death or disability. There is no treatment to slow the disease, except delivery. Preclinical studies have identified proton pump inhibitors as a possible treatment. OBJECTIVE: The purpose of this study was to examine whether esomeprazole could prolong pregnancy in women who have received a diagnosis of preterm preeclampsia. STUDY DESIGN: We performed a double-blind, randomized controlled trial at Tygerberg Hospital in South Africa. Women with preterm preeclampsia (gestational age 26 weeks+0 days to 31 weeks+6 days) were assigned randomly to 40-mg daily esomeprazole or placebo. The primary outcome was a prolongation of gestation of 5 days. Secondary outcomes were maternal and neonatal outcomes. We compared circulating markers of endothelial dysfunction that was associated with preeclampsia and performed pharmacokinetic studies. RESULTS: Between January 2016 and April 2017, we recruited 120 participants. One participant was excluded because of incorrect randomization, which left 59 participants in the esomeprazole and 60 participants in the placebo group. Median gestational age at enrolment was 29+4 weeks gestation. There were no between-group differences in median time from randomization to delivery: 11.4 days (interquartile range, 3.6-19.7 days) in the esomeprazole group and 8.3 days (interquartile range, 3.8-19.6 days) in the placebo group (3 days longer in the esomeprazole arm; 95% confidence interval, -2.9-8.8; P=.31). There were no placental abruptions in the esomeprazole group and 6 (10%) in the placebo group (P=.01, P=.14 adjusted). There were no differences in other maternal or neonatal outcomes or markers of endothelial dysfunction. Esomeprazole and its metabolites were detected in maternal blood among those treated with esomeprazole, but only trace amounts in the umbilical cord blood. CONCLUSION: Daily esomeprazole (40 mg) did not prolong gestation in pregnancies with preterm preeclampsia or decrease circulating soluble fms-like tyrosine kinase 1 concentrations. Higher levels in the maternal circulation may be needed for clinical effect.
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Esomeprazol/uso terapêutico , Pré-Eclâmpsia , Nascimento Prematuro/prevenção & controle , Cuidado Pré-Natal , Inibidores da Bomba de Prótons/uso terapêutico , Adulto , Esomeprazol/administração & dosagem , Feminino , Humanos , Gravidez , Terceiro Trimestre da Gravidez , Inibidores da Bomba de Prótons/administração & dosagem , África do Sul , Resultado do Tratamento , Adulto JovemRESUMO
RATIONALE: The speciation of the purely inorganic [PtCl6-n Brn](2-) (n = 0-6) anions and their corresponding mono-aquated [PtCl5-n Brn (H2O)](-) (n = 0-5) anions is of considerable importance to the precious metal refining and recycling industry, to ensure optimum recovery and separation efficiencies. Speciation of platinum complexes present in precursor solutions used for the preparation of precious metal nano-crystals of defined size and morphology appears also to be important. The various possible Pt(IV) complex anions in dilute aqueous can be characterized using ion-pairing reversed-phase high-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOFMS). METHODS: Ion-pairing reversed-phase ultra-high-performance LC separation of the Pt(IV) complex anions present in aqueous solutions prior to detection by means of high-resolution ESI-Q-TOFMS using a low ESI source cone voltage (5 V) allows for the clear identification of all the platinum complexes from the characteristic pattern of fragment ions (m/z), presumably generated by 'reductive conversion' in the ESI source of the mass spectrometer. Sufficient chromatographic resolution for the series of Pt(IV) complexes is achieved using the (n-butyl)3 NH(+) ion generated in a formic acid/water/methanol (pH ~3.5) mobile phase. This mobile phase composition facilitates a low-background for optimal ESI-Q-TOFMS detection with enhanced sensitivity. RESULTS: Direct-infusion mass spectrometry of the inorganic platinum complexes in aqueous solution is impractical due to their low volatility, but more importantly as a result of the very extensive series of fragment ions generated in the ESI source, which leads to virtually uninterpretable mass spectra. However, with prior separation, and by using low ESI cone voltages (5 V), the mass spectra of the separated analyte ions show simpler and systematic fragmentation patterns [Pt(IV) X5](-) â [Pt(III) X4 ](-) â [Pt(II) X3](-) â [Pt(I)X2 ](-) (X = Cl(-) and Br(-)), resulting in clear assignments. This methodology facilitates the characterization of the partially aquated [PtCl5-n Brn (H2O)](-) (n = 0-5) anions derived from the homo- and heteroleptic [PtCl6-n Brn](2-) (n = 0-6) anions, in equilibrated solutions at low concentrations. CONCLUSIONS: Speciation of homo- and heteroleptic [PtCl6-n Brn](2-) (n = 0-6) anions, together with some of their partially aquated [PtCl5-n Brn (H2O)](-) (n = 0-5) species in dilute solution, can successfully be carried out by means of prior ion-pairing reversed-phase LC separation coupled to high-resolution ESI-Q-TOFMS at low ESI cone-voltage settings.
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A group of non-ribosomally produced antimicrobial peptides, the tyrocidines from the tyrothricin complex, have potential as antimicrobial agents in both medicine and industry. Previous work by our group illustrated that the more polar tyrocidines rich in Trp residues in their structure were more active toward Gram-positive bacteria, while the more non-polar tyrocidines rich in Phe residues had greater activity toward Plasmodium falciparum, one of the major causative pathogens of malaria in humans. Our group also found that the tyrocidines have pronounced antifungal activity, dictated by the primary sequence of the tyrocidine. By simply manipulating the Phe or Trp concentration in the culture medium of the tyrothricin producer, Bacillus aneurinolyticus ATCC 10068, we were able to modulate the production of subsets of tyrocidines, thereby tailoring the tyrothricin complex to target specific pathogens. We optimized the tailored tyrothricin production using a novel, small-scale, high-throughput deep 96-well plate culturing method followed by analyses of the peptide mixtures using ultra-performance liquid chromatography linked to mass spectrometry. We were able to gradually shift the production profile of the tyrocidines and analogues, as well as the gramicidins between two extremes in terms of peptide subsets and peptide hydrophobicity. This study demonstrated that tyrothricin peptide subsets with targeted activity can be efficiently produced by simple manipulation of the aromatic amino acid profile of the culture medium.
Assuntos
Anti-Infecciosos/metabolismo , Bacillus/metabolismo , Tirotricina/metabolismo , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Cromatografia Líquida , Meios de Cultura/química , Fungos/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Espectrometria de Massas , Fenilalanina/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Triptofano/metabolismo , Tirotricina/química , Tirotricina/farmacologiaRESUMO
Secondary metabolic profiling, using UPLC-MSE and molecular networking, revealed the secondary metabolites produced by Serratia marcescens NP10. The NP10 strain co-produced cyclic and open-ring stephensiolides (i.e., fatty acyl chain linked to Thr-Ser-Ser-Ile/Leu-Ile/Leu/Val) and glucosamine derivatives (i.e., fatty acyl chain linked to Val-glucose-butyric/oxo-hexanoic acid), with the structures of sixteen new stephensiolides (L-Y) and three new glucosamine derivatives (L-N) proposed. Genome mining identified sphA (stephensiolides) and gcd (glucosamine derivatives) gene clusters within Serratia genomes available on NBCI using antiSMASH, revealing specificity scores of the adenylation-domains within each module that corroborates MSE data. Of the nine RP-HPLC fractions, two stephensiolides and two glucosamine derivatives exhibited activity against Staphylococcus aureus (IC50 of 25-79 µg/mL). 1H NMR analysis confirmed the structure of the four active compounds as stephensiolide K, a novel analogue stephensiolide U, and glucosamine derivatives A and C. Stephensiolides K and U were found to cause membrane depolarisation and affect the membrane permeability of S. aureus, while glucosamine derivatives A and C primarily caused membrane depolarisation. New members of the stephensiolide and glucosamine derivative families were thus identified, and results obtained shed light on their antibacterial properties and mode of membrane activity.
Assuntos
Serratia marcescens , Staphylococcus aureus , Humanos , Serratia marcescens/genética , Glucosamina/farmacologia , Cromatografia Líquida , Espectrometria de Massas em TandemRESUMO
Antibiotic production as a defence mechanism is a characteristic of a wide variety of organisms. In natural evolutionary adaptation, cellular events such as sporulation, biofilm formation and resistance to antibiotics enable some micro-organisms to survive environmental and antibiotic stress conditions. The two antimicrobial cyclic peptides in this study, gramicidin S (GS) from Aneurinibacillus migulanus and the lipopeptide surfactin (Srf) from Bacillus subtilis, have been shown to affect both membrane and intercellular components of target organisms. Many functions, other than that of antimicrobial activity, have been assigned to Srf. We present evidence that an additional function may exist for Srf, namely that of a detoxifying agent that protects its producer from the lytic activity of GS. We observed that Srf producers were more resistant to GS and could be co-cultured with the GS producer. Furthermore, exogenous Srf antagonized the activity of GS against both Srf-producing and non-producing bacterial strains. A molecular interaction between the anionic Srf and the cationic GS was observed with circular dichroism and electrospray MS. Our results indicate that the formation of an inactive complex between GS and Srf supports resistance towards GS, with the anionic Srf forming a chemical barrier to protect its producer. This direct detoxification combined with the induction of protective stress responses in B. subtilis by Srf confers resistance toward GS from A. migulanus and allows survival in mixed cultures.
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Antibacterianos/farmacologia , Antibacterianos/farmacocinética , Antibiose , Bacillales/efeitos dos fármacos , Gramicidina/farmacocinética , Inativação Metabólica , Lipopeptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Antibacterianos/metabolismo , Bacillales/metabolismo , Dicroísmo Circular , Farmacorresistência Bacteriana , Gramicidina/metabolismo , Lipopeptídeos/metabolismo , Peptídeos Cíclicos/metabolismo , Ligação Proteica , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The Sceletium genus has been of medicinal importance in southern Africa for millennia and Sceletium tortuosum (Aizoaceae), one of eight species in the genus has gained pharmaceutical importance as an anxiolytic and anti-depressant due to the presence of mesembrine alkaloids. S. tortuosum is used for the manufacture of herbal teas, dietary supplements and other phytopharmaceutical products. This study aimed to provide a metabolomic characterization of S. tortuosum and its sister species as these are not easy to distinguish using morphology alone. Plant samples were thus collected from various locations in the succulent Karoo (South Africa) and analyzed through liquid chromatography-mass spectrometry (LC-MS), using MSE fragmentation as a putative tool for chemical identities. Metabolomics-based analyses in combination with molecular networking were able to distinguish between the four species of Sceletium based on the presence of 4-(3,4-dimethyoxyphenyl)-4-[2-acetylmethlamino)ethyl]cyclohexanone (m/z 334.2020; RT 6.60 min), mesembrine (m/z 290.1757; RT 5.10 min) and 4'-O-demethylmesembrenol (m/z 276.1597; RT 4.17 min). Metabolomic profiles varied according to the different localities and metabolites occurred at variable quantitative levels in Sceletium ecotypes. Molecular networking provided the added advantage of being able to observe mesembrine alkaloid isomers and coeluting metabolites (from the joubertiamine group) that were difficult to discern without this application. By combining high-throughput metabolomics together with global and feature based-molecular networking, a powerful metabolite profiling platform that is able to discern chemical patterns within and between populations was established. These techniques were able to reveal chemotaxonomic relationships and allowed for the discovery of chemical markers that may be used as part of monitoring protocols during the manufacture of phytopharmaceutical and dietary products based on Sceletium.
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[This corrects the article DOI: 10.3389/fnut.2022.819753.].
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Species from the genus Xenorhabdus, endosymbiotic bacteria of Steinernema nematodes, produce several antibacterial and antifungal compounds, some of which are anti-parasitic. In this study, we report on the effect growth conditions have on the production of antimicrobial compounds produced by Xenorhabdus khoisanae J194. The strain was cultured in aerated and non-aerated broth, respectively, and on solid media. Production of antimicrobial compounds was detected after 24 h of growth in liquid media, with highest levels recorded after 96 h. Highest antimicrobial activity was obtained from cells cultured on solid media. By using ultraperformance liquid chromatography linked to mass spectrometry and HPLC, a plethora of known Xenorhabdus compounds were identified. These compounds are the PAX lipopeptides (PAX 1', PAX 3', PAX 5, and PAX 7E), xenocoumacins and xenoamicins. Differences observed in the MS-MS fractionation patterns collected in this study, when compared to previous studies indicated that this strain produces novel xenoamicins. Three novel antimicrobial compounds, khoicin, xenopep and rhabdin, were identified and structurally characterized based on MS-MS fractionation patterns, amino acid analysis and whole genome analysis. The various compounds produced under the three different conditions indicates that the secondary metabolism of X. khoisanae J194 may be regulated by oxygen, water activity or both. Based on these findings X. khoisanae J194 produce a variety of antimicrobial compounds that may have application in disease control.
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Tryptocidine C (TpcC, cyclo[D-Phe1-Pro2-Trp3-D-Trp4-Asn5-Gln6-Trp7-Val8-Orn9-Leu10]) is a broad-spectrum antimicrobial peptide in the tyrothricin complex produced by a soil bacterium, Brevibacillus parabrevis. Electrospray mass spectrometric studies reveal the oligomerisation of TpcC into dimers and higher oligomers, analogous to tyrocidine C (TrcC, Trp7 replaced by Tyr7). Ion mobility mass spectrometry (IMMS) further confirms the formation of stable peptide dimers and tetramers with diameters of 2.7 nm and 3.3 nm, respectively, calculated from collisional cross section (CCS). Molecular dynamic simulations and docking studies support the formation of amphipathic dimers, with a diameter of 2.5 ± 0.07 nm calculated from low energy model CCS. Circular dichroism and IMMS studies point towards dynamic hydrogen-bonded conformational changes up to 28-33 µM after which the structures become more static (or in equilibrium). Fluorescence studies indicate aromatic stacking of Trp residues with a CMC of 18 µM in aqueous solutions. The concentration and time dependent interaction of Trp in oligomers indicate cooperativity in the TpcC oligomerisation that leads to the formation of higher order microscopic structures. Scanning electron microscopy studies unequivocally shows that TpcC forms nanospheres with a mean diameter of 25 nm. Repeated smaller oligomeric units, possibly dimers and tetramers, self-assemble to form these nanospheres.
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Antibacterianos/química , Brevibacillus/química , Simulação de Dinâmica Molecular , Tirocidina/química , Dicroísmo Circular , Espectrometria de MassasRESUMO
Para-aminosalicylic acid (PAS), often the last drug remaining for treatment of drug-resistant tuberculosis, is notorious for causing gastrointestinal intolerance; however, the cause of PAS intolerance is uncertain. The objective of this study was to assess relationships between peak concentrations of PAS administered as a granular slow-release enteric coated formulation, and its metabolites acetyl-PAS and glycine-PAS, and intolerance. PAS and its metabolites were measured in 29 adult patients with drug-resistant tuberculosis at Brooklyn Hospital, Cape Town, randomized to receive granular slow-release enteric-coated PAS 4 g twice daily or 8 g once daily for 1 week, followed by the alternative regimen. Concentrations of PAS and its metabolites were determined by liquid chromatography and tandem mass spectrometry, and a visual analogue scale evaluated intolerance. Spearman's correlation test assessed the relationship between maximum plasma concentrations (Cmax ) and intolerance scores. A large interindividual variability was observed for the PAS Cmax (40.42-68.55 mg/L) following 4 g twice daily; (62.69-102.41 mg/L) for 8 g once daily and a similar wide Cmax range found for the metabolites acetyl-PAS and glycine-PAS. Twenty-six patients reported at least 1 intolerance episode, but most visual analogue scale scores clustered around 0. Significant inverse associations were found between acetyl-PAS Cmax and bloating (rho = -0.448; P = .025) and diarrhea (rho = -0.407; P = .044) for the twice-daily regimen and a similar inverse association found for glycine-PAS and diarrhea (rho = -0.412; P = .041). Plasma concentrations of the metabolites did not correlate with the occurrence of gastrointestinal symptoms, but higher metabolite concentrations correlated with lower intolerance scores; slow metabolism of PAS and its continued presence in the intestinal tract may be the main cause of intolerance.
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Ácido Aminossalicílico/efeitos adversos , Ácido Aminossalicílico/farmacocinética , Antituberculosos/efeitos adversos , Antituberculosos/farmacocinética , Adulto , Ácido Aminossalicílico/administração & dosagem , Ácido Aminossalicílico/sangue , Ácidos Aminossalicílicos/efeitos adversos , Ácidos Aminossalicílicos/sangue , Ácidos Aminossalicílicos/farmacocinética , Antituberculosos/administração & dosagem , Antituberculosos/sangue , Área Sob a Curva , Estudos Cross-Over , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/efeitos adversos , Preparações de Ação Retardada/farmacocinética , Esquema de Medicação , Resistência Microbiana a Medicamentos , Tolerância a Medicamentos , Feminino , Gastroenteropatias/sangue , Gastroenteropatias/induzido quimicamente , Humanos , Masculino , África do Sul , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológicoRESUMO
The interaction between a common soil yeast, Cryptococcus laurentii, and a slow-growing medicinal plant adapted to low-nutrient soils, Agathosma betulina (Berg.) Pillans, was studied. C. laurentii CAB 578 was isolated from the rhizosphere of wild A. betulina, and liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis revealed that the yeast was capable of producing polyamines, such as cadaverine and spermine, while growing in vitro in a chemically defined medium. Since the exogenous application of polyamines are known to impact on root growth, these findings supported the results obtained when axenic cultures of A. betulina seedlings were inoculated with C. laurentii CAB 578 and cultivated for 5 months under glasshouse conditions. The presence of the yeast increased root growth by 51%. Using soil dilution plates, it was demonstrated that yeast numbers were greater in the vicinity of the roots than in the bulk soil. In addition, fluoromicroscopy, in combination with the fluorescent probes Fungolight and Calcofluor white, revealed the presence of metabolic active yeast colonies on the rhizoplane 5 months after initiation of the experimentation. The study provided evidence for a symbiosis between C. laurentii and A. betulina.
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Cryptococcus/crescimento & desenvolvimento , Rutaceae/microbiologia , Microbiologia do Solo , Simbiose , Cryptococcus/genética , Cryptococcus/isolamento & purificação , Cryptococcus/metabolismo , DNA Fúngico/genética , DNA Espaçador Ribossômico , Raízes de Plantas/microbiologia , Poliaminas/metabolismoRESUMO
Tyrothricin, a complex mixture of antibiotic peptides from Bacillus brevis, was reported in 1944 to have antimalarial activity rivalling that of quinine in chickens infected with Plasmodium gallinaceum. We have isolated the major components of tyrothricin, cyclic decapeptides collectively known as the tyrocidines, and tested them against the human malaria parasite Plasmodium falciparum using standard in vitro assays. Although the tyrocidines differ from each other by conservative amino acid substitutions in only three positions, their observed 50% parasite inhibitory concentrations (IC(50)) spanned three orders of magnitude (0.58 to 360 nM). Activity correlated strictly with increased apparent hydrophobicity and reduced total side-chain surface area and the presence of ornithine and phenylalanine in key positions. In contrast, mammalian cell toxicity and haemolytic activities of the respective peptides were considerably less variable (2.6 to 28 microM). Gramicidin S, a structurally analogous antimicrobial peptide, was less active (IC(50)=1.3 microM) and selective than the tyrocidines. It exerted its parasite inhibition by rapid and selective lysis of infected erythrocytes as judged by fluorescence and light microscopy. The tyrocidines, however, did not cause an overt lysis of infected erythrocytes, but an inhibition of parasite development and life-cycle progression.
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Antibacterianos/farmacologia , Antiprotozoários/farmacologia , Bacillus/química , Estágios do Ciclo de Vida/efeitos dos fármacos , Plasmodium falciparum/crescimento & desenvolvimento , Tirocidina/farmacologia , Animais , Relação Dose-Resposta a Droga , Células HeLa , Hemólise/efeitos dos fármacos , Humanos , Microscopia de Fluorescência , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-Atividade , Testes de ToxicidadeRESUMO
It was previously observed that the lipopeptide surfactants in surfactin (Srf) have an antagonistic action towards the highly potent antimicrobial cyclodecapeptide, gramicidin S (GS). This study reports on some of the molecular aspects of the antagonism as investigated through complementary electrospray ionization mass spectrometry techniques. We were able to detect stable 1:1 and 2:1 hetero-oligomers in a mixture of surfactin and gramicidin S. The noncovalent interaction between GS and Srf, with the proposed equilibrium: GS~SrfâGS+Srf correlated to apparent K d values of 6-9 µM in gas-phase and 1 µM in aqueous solution. The apparent K d values decreased with a longer incubation time and indicated a slow oligomerization equilibrium. Furthermore, the low µM K dapp values of GS~SrfâGS+Srf fell within the biological concentration range and related to the 2- to 3-fold increase in [GS] needed for bacterial growth inhibition in the presence of Srf. Competition studies indicated that neither Na+ nor Ca2+ had a major effect on the stability of preformed heterodimers and that GS in fact out-competed Ca2+ and Na+ from Srf. Traveling wave ion mobility mass spectrometry revealed near symmetrical peaks of the heterodimers correlating to a compact dimer conformation that depend on specific interactions. Collision-induced dissociation studies indicated that the peptide interaction is most probably between one Orn residue in GS and the Asp residue, but not the Glu residue in Srf. We propose that flanking hydrophobic residues in both peptides stabilize the antagonistic and inactive peptide hetero-oligomers and shield the specific polar interactions in an aqueous environment. Graphical Abstract á .
Assuntos
Anti-Infecciosos/química , Gramicidina/química , Lipopeptídeos/química , Peptídeos Cíclicos/química , Sítios de Ligação , Dimerização , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
11-Oxygenated steroids such as 11-ketotestosterone and 11-ketodihydrotestosterone have recently been shown to play a putative role in the development and progression of castration resistant prostate cancer. In this study we report on the development of a high throughput ultra-performance convergence chromatography tandem mass spectrometry (UPC(2)-MS/MS) method for the analysis of thirteen 11-oxygenated and six canonical C19 steroids isolated from a cell culture matrix. Using an Acquity UPC(2) BEH 2-EP column we found that UPC(2) resulted in superior selectivity, increased chromatographic efficiency and a scattered elution order when compared to conventional reverse phase ultra-performance liquid chromatography (UPLC). Furthermore, there was a significant improvement in sensitivity (5-50 times). The lower limits of quantification ranged between 0.01-10ngmL(-1), while the upper limit of quantification was 100ngmL(-1) for all steroids. Accuracy, precision, intra-day variation, recovery, matrix effects and process efficiency were all evaluated and found to be within acceptable limits. Taken together we show that the increased power of UPC(2)-MS/MS allows the analyst to complete in vitro assays at biologically relevant concentrations for the first time and in so doing determine the routes of steroid metabolism which is vital for studies of androgen responsive cancers, such as prostate cancer, and could highlight new mechanisms of disease progression and new targets for cancer therapy.
Assuntos
Androgênios/análise , Cromatografia Líquida/métodos , Cromatografia com Fluido Supercrítico/métodos , Ensaios de Triagem em Larga Escala , Espectrometria de Massas em Tandem/métodos , Humanos , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
Sotolon has been reported to play an important role in the atypical ageing and aroma character of many wines. A number of analytical techniques for sotolon analysis in wine have been reported, but these often require extensive sample preparation. In this work we report a HPLC-UV method and a novel UPLC-MS method to determine sotolon concentrations in white wines with little sample preparation applied for the first time for the evaluation of sotolon levels in South African wines. The validation showed that the instrumental methods had good accuracy, repeatability and linearity, but the UPLC-MS method proved more sensitive. For both methods, quantification limits were lower than the sotolon odour threshold in wine (10µg/L), 0.86µg/L and 0.013µg/L, for HPLC-UV and UPLC-MS methods, respectively. Sotolon levels in 65 South African white wines were often found to be lower than the reported odour threshold, with the highest concentration being 9.11µg/L. However, for low levels (<1µg/L), unknown interferences in certain wines led to sotolon not being quantified with the HPLC-UV method, which made the UPLC-MS method more suitable.