Peptides

Successful use of liposomes as immunological adjuvants in vaccines requires simple, easy to scale up technology capable of high-yield antigen entrapment. Recent work from this laboratory has led to the development of techniques that can generate liposomes of various sizes containing soluble antigens such as proteins or particulate antigens such as whole, live, or attenuated bacteria or viruses. Entrapment of proteins is carried out by the dehydration-rehydration procedure, which entails freeze-drying of a mixture of "empty" small unilamellar vesicles and free antigens. Upon rehydration, the large multilamellar vesicles that are formed incorporate up to 80% of the antigen used. When such liposomes are microfluidized in the presence of nonentrapped material, their size is reduced to about 100 nm in diameter, with much of the originally entrapped antigen still associated with the vesicles. A similar technique applied to the entrapment of particulate antigens (e.g., Bacillus s...

Fluorescent fusion proteins are an important tool for the study of vesicle trafficking and exocytosis, especially when combined with newer types of microscopy. We previously reported that the design of a vesicle-targeted fluorescent fusion construct strongly influences the kinetics of fluorescence change at exocytosis. In the present study we demonstrate that the cell in which a construct is expressed also affects the kinetics of fluorescence change at exocytosis. We fused enhanced green fluorescent protein to the carboxy terminus of the vesicular cargo protein rodent islet amyloid polypeptide. The two proteins were separated by a “linker” sequence of 18 amino acids. We then compared kinetics of fluorescence change at exocytosis for this fluorescent cargo protein expressed in three different types of peptidergic endocrine cell: pancreatic alpha cell, pancreatic beta cell, and adrenal chromaffin cell. In resting cells of all three types, fluorescent spots of similar size and membrane-proximal density appeared near the plasma membrane as expected if the probe is stored in large dense-core secretory vesicles. Upon stimulation, the fluorescent spots displayed sudden changes in fluorescence intensity that were consistent with exocytosis. In beta and alpha cells the fluorescent spots consistently brightened and persisted, whereas in chromaffin cells the fluorescent spots always dispersed rapidly. Thus, for fluorescent cargo proteins in peptidergic endocrine cells, cell type influences the kinetics of fluorescence change at exocytosis. Together with our previous findings, this observation strongly highlights the fact that the behavior of vesicle-targeted fluorescent cargo may be unrelated to that of native cargo, and it emphasizes the need for caution in interpreting fluorescence kinetics in terms of an exocytosis mechanism.

Abstract— Irradiation of 1,3–dimethyluracil (DMU) in 1–aminopropane (AP) with u.v. light resulted in the formation of reduced and dimeric forms of DMU and adducts between DMU and AP. The formation of these compounds may be rationalized from the activation of either DMU or AP. Irradiation of DMU and poly-l-lysine in water with u.v. light resulted in the extensive binding of DMU to poly-l-lysine. The pyrimidine is probably attached to the ε-carbon of the polypeptide at the 6–position. The biological implications of these studies are discussed.

We propose that deletion of pro-melanin-concentrating hormone (pMCH) would increase energy expenditure and further improve glucose tolerance in mice lacking stearoyl-coA desaturase-1 (SCD1). To test our hypothesis, we bred and metabolically challenged Pmch-/-; Scd1-/- double-knockout mice, with comparison to Pmch-/- mice; Scd1-/- mice and C57Bl/6J controls. Deletion of both Pmch and Scd1 increased both food intake and energy expenditure relative to control mice. Pmch-/-; Scd1-/- double-knockout mice had improved glucose tolerance relative to control mice. The majority of the metabolic effects were contributed by inactivation of the Scd1 gene. We conclude that the increased food intake and increased energy expenditure of Scd1-/- mice are independent of the neuropeptide melanin-concentrating hormone.

Although several strains ofB. subtiliswith antifungal activity have been isolated worldwide, to date there are no published reports regarding the isolation of a nativeB. subtilisstrain from strawberry plants in Mexico. A native bacterium (Bacillus subtilis21) demonstratedin vitroantagonistic activity against different plant pathogenic fungi. Under greenhouse conditions, it was shown that plants infected withRhizoctonia solaniandFusarium verticillioidesand treated withB. subtilis21 produced augment in the number of leaves per plant and an increment in the length of healthy leaves in comparison with untreated plants. In addition,B. subtilis21 showed activity against pathogenic bacteria. Secreted proteins byB. subtilis21 were studied, detecting the presence of proteases and bacteriocin-like inhibitor substances that could be implicated in its antagonistic activity. Chitinases and zwittermicin production could not be detected. Then,B. subtilis21 could potentially be used to control phyt...

The large surface area, good vascularization, immense capacity for solute exchange and ultra-thinness of the alveolar epithelium are unique features of the lung that can facilitate systemic delivery via pulmonary administration of peptides and proteins. Physical and biochemical barriers, lack of optimal dosage forms and delivery devices limit the systemic delivery of biotherapeutic agents by inhalation. Current efforts to overcome these difficulties in order to deliver metabolic hormones (insulin, calcitonin, thyroid-stimulating hormone [TSH], follicle-stimulating hormone [FSH] and growth hormones) systemically, to induce systemic responses (immunoglobulins, cyclosporin A [CsA], recombinant-methionyl human granulocyte colony-stimulating factor [r-huG-CSF], pancreatic islet autoantigen) and to modulate other biological processes via the lung are reviewed. Safety aspects of pulmonary peptide and protein administration are also discussed.

The drugs against tropical neglected diseases, especially Chagas’ Disease, were launched more than 30 years ago, and the development of resistance requires the discovery of new and more effective chemotherapeutic agents. Trypanosoma cruzi has a redox enzyme called trypanothione reductase which was successfully inhibited for peptide derivatives (McKie et al., Amino Acids, 2001, 20: 145). This work aims at studying the mechanism of inhibition of this enzyme through molecular dynamics simulations and evaluating the behavior of some derivatives when inhibiting this protein. We should affirm that any particular molecular dynamics analysis tools (Hbond pattern, 3-D root-mean-square deviation, solvent accessible surface area, etc.) cannot be used apart from the others to justify completely these peptides inhibitory patterns. Based on our results, we reproduced the experimental data and, moreover, we discriminated against a new site in enzyme aperture, which can assist the development of powerful inhibitors against trypanothione reductase enzyme.