Mohammed A. Meetani

Orthologs of magainin, PGLa, procaerulein-derived, and proxenopsin-derived peptides from skin secretions of the octoploid frog Xenopus amieti (Pipidae).

The Volcano clawed frog Xenopus amieti Kobel, du Pasquier, Fischberg, and Gloor, 1980, with a chromosome number of 2n=72, is believed to have undergone two rounds of genome duplication since evolving from a diploid ancestor. Nine peptides with differential antimicrobial activity against Escherichia coli and Staphylococcus aureus were isolated from norepinephrine-stimulated skin secretions of X. amieti that showed structural similarity to peptides previously isolated from the tetraploid frog X. laevis (2n=36) and the diploid frog Silurana (formerly Xenopus) tropicalis (2n=20). Two peptides (magainin-AM1 and -AM2) are othologous to the magainins, two peptides (PGLa-AM1 and -AM2) orthologous to peptide glycine-leucine-amide, four peptides (CPF-AM1, -AM2, -AM3, -AM4) orthologous to caerulein-precursor fragments, and one peptide (XPF-AM1) structurally similar to xenopsin-precursor fragments were characterized. CFP-AM1 (GLGSVLGKALKIGANLL.NH(2)) was the most potent peptide present in the secretions and magainin-AM2 (GVSKILHSAGKFGKAFLGEIMKS) was the most abundant. The data indicate that nonfunctionalization has been the most common fate of duplicated antimicrobial peptide genes following the polyploidization events in the X. amieti lineage. However, the very low antimicrobial activity of the magainin-AM1 and PGLa-AM2 paralogs suggests the possibility that certain peptides may have evolved toward a new, as yet undetermined, function (neofunctionalization).

Effect of cucurbit[n]urils on tropicamide and potential application in ocular drug delivery

The supramolecular interactions of the ocular drug tropicamide (TR) with cucurbit[7]uril (CB7) and cucurbit[8]uril (CB8) were investigated in aqueous solutions by using 1H NMR, ESI-MS and UV–vis spectroscopic techniques. The results indicate a 1:1 binding stoichiometry of TR with CB7 and CB8. The binding constants of TR in its protonated form were higher (e.g. K = 4 × 106 M− 1 with CB8) than in its neutral form (e.g. K = 1.4 × 104 M− 1 with CB8), which led to a complexation-induced increase in its pK a value of ca. 0.5 and 2 units with CB7 and CB8, respectively. In the presence of about 1% (w/v) CB8, the ionisation degree of 0.1% (w/v) TR was increased from 2% to 62% at neutral pH. The increase in the pK a value and thus stabilisation of the protonated TR species at neutral pH is discussed in the context of supramolecular drug delivery of ophthalmologic drugs.

Mechanistic studies of photoinduced degradation of Orange G using LC/MS

Photocatalytic degradation of Orange G dye was carried out with UV light in the presence of TiO2 mixed with 20% chromium oxide as a catalyst. The conditions for maximum dye degradation were optimized in terms of catalyst amount, dye concentration, pH and time. The dye degradation followed pseudo first-order kinetics. LC-MS/MS was used to track the numerous intermediate products formed during the course of dye degradation. Major intermediate products confirmed were substituted phenols, aromatic hydroxyl amine, nitroso hydroxyl aromatic amines and dicarboxyl aromatic compounds. These intermediates along with some minor products were correlated to suggest a possible degradation pathway of Orange G. The dye degradation occurred mainly by three pathways, namely the hydroxylation of the aromatic ring, desulfonation and oxidative cleavage of the azo bond.

Peptidomic analysis of skin secretions from the bullfrog Lithobates catesbeianus (Ranidae) identifies multiple peptides with potent insulin-releasing activity

Using a combination of reversed-phase HPLC and electrospray mass spectrometry, peptidomic analysis of norepinephrine-stimulated skin secretions of the American bullfrog Lithobates catesbeianus Shaw, 1802 led to the identification and characterization of five newly described peptides (ranatuerin-1CBb, ranatuerin-2CBc, and -CBd, palustrin-2CBa, and temporin-CBf) together with seven peptides previously isolated on the basis of their antimicrobial activity (ranatuerin-1CBa, ranatuerin-2CBa, brevinin-1CBa, and -1CBb, temporin-CBa, -CBb, and -CBd). The abilities of the most abundant of the purified peptides to stimulate the release of insulin from the rat BRIN-BD11 clonal β cell line were evaluated. Ranatuerin-2CBd (GFLDIIKNLGKTFAGHMLDKIRCTIGTCPPSP) was the most potent peptide producing a significant stimulation of insulin release (119% of basal rate, P<0.01) from BRIN-BD11 cells at a concentration of 30nM, with a maximum response (236% of basal rate, P<0.001) at a concentration of 3μM. Ranatuerin-2CBd did not stimulate release of the cytosolic enzyme, lactate dehydrogenase at concentrations up to 3μM, indicating that the integrity of the plasma membrane had been preserved. Brevinin-1CBb (FLPFIARLAAKVFPSIICSVTKKC) produced the maximum stimulation of insulin release (285% of basal rate, P<0.001 at 3μM) but the peptide was cytotoxic at this concentration.

Liquid chromatography tandem mass spectrometry analysis of photodegradation of a diazo compound: A mechanistic study

The photolytic degradation of the diazo dye, Amido Black, using UV/H(2)O(2) has been carried out experimentally and parameters for most efficient dye degradation have been determined. The degradation of the dye was followed by UV-Vis spectroscopy, HPLC, and LC-MS and is proposed to be initiated by ()OH radicals formed by the photolysis of H(2)O(2). A detailed study was also carried out using LC-MS and LC-MS/MS to determine the degradation pathway of the dye as well as to identify some of the intermediate products formed. Our results suggest that Amido Black degradation occurs preferentially by ()OH radical attack at the more electron rich diazo functionality of the molecule. Furthermore, evidence is presented that subsequent steps in this diazo dye degradation pathway include radical denitration, radical desulfonation and radical diazotization. This report is one of the very few studies that have proposed possible mechanistic pathways for the degradation pathways of a diazo compound.

Mechanistic study of a diazo dye degradation by Soybean Peroxidase

BackgroundEnzyme based remediation of wastewater is emerging as a novel, efficient and environmentally-friendlier approach. However, studies showing detailed mechanisms of enzyme mediated degradation of organic pollutants are not widely published.ResultsThe present report describes a detailed study on the use of Soybean Peroxidase to efficiently degrade Trypan Blue, a diazo dye. In addition to examining various parameters that can affect the dye degradation ability of the enzyme, such as enzyme and H2O2 concentration, reaction pH and temperature, we carried out a detailed mechanistic study of Trypan Blue degradation. HPLC-DAD and LC-MS/MS studies were carried out to confirm dye degradation and analyze the intermediate metabolites and develop a detailed mechanistic dye degradation pathway.ConclusionWe report that Soybean peroxidase causes Trypan Blue degradation via symmetrical azo bond cleavage and subsequent radical-initiated ring opening of the metabolites. Interestingly, our results also show that no high molecular weight polymers were produced during the peroxidase-H2O2 mediated degradation of the phenolic Trypan Blue.

Caerulein-and xenopsin-related peptides with insulin-releasing activities from skin secretions of the clawed frogs, Xenopus borealis and Xenopus amieti (Pipidae).

Caerulein-related peptides were identified in norepinephrine-stimulated skin secretions of the tetraploid frog Xenopus borealis and the octoploid frog Xenopus amieti using negative ion electrospray mass spectrometry and their primary structures determined by positive ion tandem (MS/MS) mass spectrometry. X. borealis caerulein-B1 (pGlu-Gln-Asp-Tyr(SO(3))-Gly-Thr-Gly-Trp-Met-Asp-Phe.NH2) contains an additional Gly(5) residue compared with X. laevis caerulein and caerulein-B2 (pGlu-Asp-Tyr(SO(3))-Thr-Gly-Trp-Met-Asp-Phe.NH2) contains a Gln(2) deletion. X. amieti caerulein was identical to the X. laevis peptide. In addition, xenopsin, identical to the peptide from X. laevis, together with xenopsin-AM2 (pGlu-Gly-Arg-Arg-Pro-Trp-Ile- Leu) that contains the substitution Lys(3)→Arg were isolated from X. amieti secretions. X. borealis caerulein-B1, and X. amieti xenopsin and xenopsin-AM2 produced significant (P<0.05) and concentration-dependent stimulations of insulin release from the rat BRIN-BD11 clonal β cell line at concentrations ⩾30nM. The peptides did not stimulate the release of lactate dehydrogenase at concentrations up to 3μM demonstrating that the integrity of the plasma membrane had been preserved. While their precise biological role is unclear, the caerulein- and xenopsin-related peptides may constitute a component of the animals chemical defenses against predators.

Antimicrobial peptides from the skin secretions of the New World frogs Lithobates capito and Lithobates warszewitschii (Ranidae)

Taxonomic revisions within the anuran family Ranidae have established the genus Lithobates that currently comprises 49 species of frogs from the New World. Peptidomic analysis, using reversed-phase HPLC with on-line detection by electrospray mass spectrometry, has led to the identification of multiple antimicrobial peptides in norepinephrine-stimulated skin secretions of the North American frog Lithobates capito and the Central American frog Lithobates warszewitschii. Structural characterization of the peptides demonstrated that the L. capito secretions contained brevinin-1 (1), esculentin-1 (1), esculentin-2 (1), ranatuerin-2 (3), and temporin (2) peptides. L. warszewitschii secretions contained brevinin-1 (1), esculentin-2 (1), ranatuerin-2 (2), and temporin (1) peptides. Values in parentheses indicate number of peptides in each family. Temporin-CPa from L. capito, with the atypical structure IPPFIKKVLTTVF.NH(2), also showed atypical growth-inhibitory activity having greater potency against Escherichia coli (MIC=25 microM) and Candida albicans (MIC=25 microM) than against Staphylococcus aureus (MIC=50 microM). Phylogenetic analysis based upon the amino acid sequences of 37 ranatuerin-2 peptides from 17 species belonging to the genus Lithobates provides support for currently accepted taxonomic relationships. L. capito is sister-group to Lithobates sevosus in a clade that also contains Lithobates areolatus, and Lithobates palustris. L. warszewitschii is most closely related to the Central American species Lithobates tarahumarae and Lithobates vaillanti.

Hybridization between the African clawed frogs Xenopus laevis and Xenopus muelleri (Pipidae) increases the multiplicity of antimicrobial peptides in skin secretions of female offspring.

Peptidomic analysis was used to compare the distribution of host-defense peptides in norepinephrine-stimulated skin secretions from laboratory-generated female F1 hybrids of the common clawed frog Xenopus laevis (Daudin, 1802) and Muellers clawed frog Xenopus muelleri (Peters, 1844) with the corresponding distribution in skin secretions from the parent species. A total of 18 peptides were identified in secretions from the hybrid frogs. Eleven peptides (magainin-1, magainin-2, CPF-1, CPF-3, CPF-4, CPF-5, CPF-6, CPF-7, XPF-1, XPF-2, and PGLa) were identified in secretions of both the hybrids and X. laevis. Four peptides (magainin-M1, XPF-M1, CPF-M1, and tigerinin-M1) were previously found in skin secretions of X. muelleri but magainin-M2 and CPF-M2 from X. muelleri were not detected. Three previously undescribed peptides (magainin-LM1, PGLa-LM1, and CPF-LM1) were purified from the secretions of the hybrid frogs that were not detected in secretions from either X. laevis or X. muelleri. Magainin-LM1 differs from magainin-2 from X. laevis by a single amino acid substitution (Gly(13)→Ala) but PGLa-LM1 and CPF-LM1 differ appreciably in structure from orthologs in the parent species. CPF-LM1 shows potent, broad-spectrum antimicrobial activity and is hemolytic. The data indicate that hybridization increases the multiplicity of skin host-defense peptides in skin secretions. As the female F1 hybrids are fertile, hybridization may represent an adaptive strategy among Xenopus species to increase protection against pathogenic microorganisms in the environment.

Intermolecular interactions between cucurbit[7]uril and pilocarpine.

The interactions between cucurbit[7]uril (CB7) macrocycles and pilocarpine (PIL) were investigated in aqueous solution by using (1)H NMR and circular dichroism (CD) spectroscopic techniques. The characterizations of the freeze-drying solid complex were conducted by electrospray ionization mass spectroscopy (ESI-MS), Fourier transform-infrared spectroscopy (FT-IR), thermogravimetry, and differential scanning calorimetry (DSC) techniques. The DSC and thermogravimetry confirmed the production of a thermally stable solid complex. The NMR, CD and ESI-MS measurements confirmed asymmetric induction during the complexation reaction, in which the γ-lactone ring of PIL (not the imidazole nucleus) has been fully encapsulated within the cavity of CB7. The stability of the drug has significantly enhanced as evidenced by the high-performance liquid chromatographic (HPLC) method. The results are discussed in the context of utilizing non-conventional supramolecular host-guest approaches to enhance the chemical stability in aqueous media of hydrophilic PIL drugs as model compounds. The non-classical stereospecific interactions between CB7 and PIL drugs are also highlighted.