Co-occurrence of mycotoxins, aflatoxin biosynthetic precursors, and Aspergillus metabolites in garlic (Allium sativum L) marketed in Zaria, Nigeria.

Multi-mycotoxin analysis of 72 samples of garlic bulbs sold in Zaria markets was carried out using a liquid chromatography-mass spectrometry (LC-MS/MS) method. The results indicated the presence of seven major mycotoxins, including aflatoxin B1 (AFB1), ochratoxin A (OTA), and the fumonisins B1, B2, B3, B4, and B6, at different levels of contamination. AFB1 and OTA were detected in 1 of the 72 samples (1.4%) with median concentrations of 5.48 and 12.3 µg/kg, respectively. FB1 and FB2 were detected in 77% and 100% of the analysed samples, with median concentrations of 401 µg/kg and 491 µg/kg, respectively. The observed levels of AFB1, OTA, FB1, and FB2 were above the EU maximum limit in herbal products. Sterigmatocystin (STC), an AFB1 biosynthetic precursor, was present in all tested samples. The contamination level of mycotoxins and Aspergillus metabolites of marketed garlic in the study area is of public health concern.

Brain-specific expression of a novel human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc-T9).

We isolated cDNA coding for the ninth of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-T9) from human brain by the polymerase chain reaction. The polypeptide encoded by GalNAc-T9 contained the structural features characteristic of GalNAc transferases, such as a GT1 motif, a Gal/GalNAc transferase motif, (QXW)(3) repeats, and conserved His, Cys, and acidic amino acid residues. Northern blot analysis revealed the mRNA expression of the enzyme to be confined to the brain. The brain-specific expression of GalNAc-T9 suggested that this isozyme catalyzes O-glycosylation in the brain.

Selective targeting of lysosomal cysteine proteases with radiolabeled electrophilic substrate analogs.

BACKGROUND: The lysosomal cysteine proteases of the papain family are some of the best studied proteolytic enzymes. Small-molecule inhibitors and fluorogenic substrate mimics have been used to probe the physiological roles of these proteases. A high degree of homology between family members and overlap in substrate specificity have made elucidating individual protease function, expression and activity difficult. RESULTS: Using peptide vinyl sulfones and epoxide as templates, we have generated probes that can be tagged with radioactive iodine. The resulting compounds covalently label various cathepsins and several unidentified polypeptides likely to be proteases. MB-074 was found to be a highly selective probe of cathepsin B activity. Probes that labeled several cathepsins were used to examine the specificity and cell permeability of the CA-074 family of inhibitors. Although CA-074 reportedly acts in vivo, we find it is unable to penetrate cells. Esterifying CA-074 resulted in a cell-permeable inhibitor with dramatically reduced activity and specificity for cathepsin B. The probes were also used to monitor protease activity in primary human tumor tissue and cells derived from human placenta. CONCLUSIONS: We have generated a highly selective cathepsin B probe and several less specific reagents for the study of cathepsin biology. The reagents have several advantages over commonly used fluorogenic substrates, allowing inhibitor targets to be identified in a pool of total cellular enzymes. We have used the probes to show that cathepsin activity is regulated in tumor tissues and during differentiation of placental-derived cytotrophoblasts to invasive cells required for establishing blood circulation in a developing embryo.

Binding preferences of hydroxamate inhibitors of the matrix metalloproteinase human fibroblast collagenase.

In this paper we report molecular dynamics (MD) and free energy perturbation (FEP) studies carried out on enzyme-inhibitor (two hydroxamates that only differ by a carbon-carbon double bond) complexes of human fibroblast collagenase to obtain insights into the structural and energetic preferences of these inhibitors. We have developed a bonded model for the catalytic and structural zinc centers (Hoops, S. C.; et al. J. Am. Chem. Soc. 1991, 113, 8262-8270) where the electrostatic representation for this model was derived using a novel quantum-mechanical/molecular-mechanical (QM/MM) minimization procedure followed by electrostatic potential fitting. The resulting bonded model for the zinc ions was then used to generate MD trajectories for structural analysis and FEP studies. This model has satisfactorily reproduced the structural features of the active site, and furthermore, the FEP simulations gave relative free energies of binding in good agreement with experimental results. MD simulations in conjunction with the FEP are able to provide a structural explanation regarding why one hydroxamate inhibitor is favored over the other, and we are also able to make predictions about changes in the inhibitor that would enhance protein-inhibitor interactions.

An O-acetylated sialyl-Tn is involved in ovarian cancer-associated antigenicity.

We reported previously that a monoclonal antibody, 6G9, raised against bovine submaxillary mucin (BSM) reacted with mucinous ovarian cancer and recognized tumor-associated sialylated carbohydrate antigens. To obtain structural information on the carbohydrate antigens recognized by 6G9, the reactivity of several mucins and carbohydrates with the antibody was determined by ELISA. Exoglycosidase digestion of BSM showed that 6G9 recognized Sia as a nonreducing monosaccharide but neither Gal nor GlcNAc. Reactivity of BSM with 6G9 decreased markedly on de-O-acetylation of BSM in mild alkali, and O-acetyl Sia obtained from BSM reacted with the antibody, indicating the presence of O-acetyl groups on Sia in the epitope. A sialyl-Tn structure located in the epitope was also demonstrated by the findings that de-O-acetylated BSM retained weak but significant reactivity with 6G9 and that ovine submaxillary mucin, major sugar chains of which are sialyl-Tn, reacted with 6G9 stronger than de-O-acetylated BSM. Furthermore, weak reactivity of NeuAcalpha2 --> 6GalNAc prepared from BSM demonstrated that 6G9 recognized the sialyl-Tn structure, but the modification of Sia with O-acetyl groups was essential for the recognition. The failure of 9-O-acetyl NeuAc, synthesized chemically, to react with the antibody implied that 6G9 recognized sialyl-Tn with O-acetylation on Sia distinct from C-9 O-acetylation.

New synthetic absorbable polymers as BMP carriers: plastic properties of poly-D,L-lactic acid-polyethylene glycol block copolymers.

Bone morphogenetic proteins (BMPs) are biologically active molecules capable of eliciting new bone formation. In combination with biomaterials, these proteins can be used in a clinical setting as bone-graft substitutes to promote bone repair. To find new synthetic absorbable polymers with plastic nature that can be used as BMP-carrier materials, six types of poly-D,L-lactic acid-polyethylene glycol block copolymer (PLA-PEG) with various molecular weights of PLA and PEG were synthesized. These were PLA6, 500-PEG3,000 (P-1), PLA11,500-PEG3,000 (P-2), PLA17,500-PEG3,000 (P-3), PLA6,500-PEG1,000 (P-4), PLA15,000-PEG8,000 (P-5), and PLA8, 500-PEG1,000 (P-6). Fifty milligrams of these polymers was mixed with 0 microg (control) or 5, 10, or 20 microg of recombinant human BMP-2 (rhBMP-2). These pellets were implanted into the dorsal muscle pouches of 144 mice (six pellets consisting of the same polymer and dose of rhBMP-2 for a specific group). Three weeks after surgery, the pellets were harvested and examined by radiographic and histological methods. All P-1 pellets with 10 or 20 microg of rhBMP-2 showed bone formation with hematopoietic marrow and bony trabeculae, as did one third of those with 5 microg of rhBMP-2. The incidence of new bone formation with P-2 pellets or that of P-5 pellets was lower than that of P-1 pellets. No bone was formed in any other type of pellet. These results indicated that the PLA6, 500-PEG3,000 polymer with plastic properties was found to work well as a BMP carrier.

Design, docking, and evaluation of multiple libraries against multiple targets.

We present a general approach to the design, docking, and virtual screening of multiple combinatorial libraries against a family of proteins. The method consists of three main stages: docking the scaffold, selecting the best substituents at each site of diversity, and comparing the resultant molecules within and between the libraries. The core "divide-and-conquer" algorithm for side-chain selection, developed from an earlier version (Sun et al., J Comp Aided Mol Design 1998;12:597-604), provides a way to explore large lists of substituents with linear rather than combinatorial time dependence. We have applied our method to three combinatorial libraries and three serine proteases: trypsin, chymotrypsin, and elastase. We show that the scaffold docking procedure, in conjunction with a novel vector-based orientation filter, reproduces crystallographic binding modes. In addition, the free-energy-based scoring procedure (Zou et al., J Am Chem Soc 1999;121:8033-8043) is able to reproduce experimental binding data for P1 mutants of macromolecular protease inhibitors. Finally, we show that our method discriminates between a peptide library and virtual libraries built on benzodiazepine and tetrahydroisoquinolinone scaffolds. Implications of the docking results for library design are explored.