Powder properties and their influence on dry powder inhaler delivery of an antitubercular drug

The purpose of this study was to determine if aerosol delivery of drug loaded microparticles to lungs infected withMycobacterium tuberculosis may be achieved by predicting dispersion of dry powders through knowledge of particle surface properties. Particle sizes of rifampicin-loaded poly(lactide-co-glycolide) microparticles (R-PLGA), rifampicin alone, and lactose and maltodextrin carrier particles (bulk and 75-125-μm sieved fractions) were determined by electron microscopy for the projected area diameter (D_p) and laser diffraction for the volume diameter (D_v). Surface energies (Y) of R-PLGA, rifampicin alone, lactose, and maltodextrin were obtained by inverse phase gas chromatography, surface areas (S_a) by N₂ adsorption, and cohesive energy densities by calculation. Particle dispersion was evaluated (Andersen nonviable impactor) for 10% blends of R-PLGA and rifampicin alone with bulk and sieved fractions of the carriers. D_p for R-PLGA and rifampicin alone was 3.02 and 2.83 μm, respectively. D_v was 13±1 and 2±1 μm for R-PLGA and rifampicin alone, respectively, indicating that R-PLGA was more aggregated. This was evident in Y of 35±1 and 19±6 mJ/m² for R-PLGA and rifampicin alone. D_p for lactose and maltodextrin (sieved and bulk) was approximately 40 mm. Bulk maltodextrin (D_v=119±6 mm) was more aggregated than bulk lactose (D_v=54±2 mm). This was a result of the higher S_a for maltodextrin (0.54 m²/g) than for lactose (0.21 m²/g). The Y of bulk lactose and maltodextrin was 40±4 and 60±6 mJ/m² and of sieved lactose and maltodextrin was 39±1 and 50±1 mJ/m². Impaction studies yielded higher fine particle fractions of R-PLGA from sieved lactose, 13%±3%, than from sieved maltodextrin, 7%±1%, at 90 L/min. An expression, based on these data, is proposed as a predictor of drug dispersion from carrier particles. Delivery of dry powder formulations can be achieved by characterizing particle surfaces and predicting impact on dispersion.     

Redox proteins in mammalian cell death: an evolutionarily conserved function in mitochondria and prokaryotes

Mammalian cell mitochondria are believed to have prokaryotic ancestry. Mitochondria are not only the powerhouse of energy generation within the eukaryotic cell but they also play a major role in inducing apoptotic cell death through release of redox proteins such as cytochrome c and the apoptosis-inducing factor (AIF), a flavoprotein with NADH oxidase activity. Recent evidence indicates that some present day prokaryotes release redox proteins that induce apoptosis in mammalian cells through stabilization of the tumour suppressor protein p53. p53 interacts with mitochondria either directly or through activation of the genes for pro-apoptotic proteins such as Bax or NOXA or genes that encode redox enzymes responsible for the production of reactive oxygen species (ROS). The analogy between the ancient ancestors of present day bacteria, the mitochondria, and the present day bacteria with regard to their ability to release redox proteins for triggering mammalian cell death is an interesting example of functional conservation during the hundreds of millions of years of evolution. It is possible that the ancestors of the present day prokaryotes released redox proteins to kill the ancestors of the eukaryotes. During evolution of the mitochondria from prokaryotes as obligate endosymbionts, the mitochondria maintained the same functions to programme their own host cell death.     

Enzymatic synthesis of 2'-deoxynucleoside diphosphates and triphosphates

2'-Deoxynucleoside diphosphates and triphosphates were synthesized from 2'-deoxy-nucleoside monophosphates by using nucleoside monophosphate kinase and nucleoside diphosphate kinase enzymes. This biocatalytic procedure is superior to chemical methods for obtaining these compounds and can be extended to other applications. © Rapid Science Ltd. 1998     

Dual role for Zn2+ in maintaining structural integrity and inducing DNA sequence specificity in a promiscuous endonuclease

We describe two uncommon roles for Zn2+ in enzyme KpnI restriction endonuclease (REase). Among all of the REases studied, KpnI REase is unique in its DNA binding and cleavage characteristics. The enzyme is a poor discriminator of DNA sequences, cleaving DNA in a promiscuous manner in the presence of Mg2+. Unlike most Type II REases, the active site of the enzyme comprises an HNH motif, which can accommodate Mg2+, Mn2+, or Ca2+. Among these metal ions, Mg2+ and Mn2+ induce promiscuous cleavage by the enzyme, whereas Ca2+-bound enzyme exhibits site-specific cleavage. Examination of the sequence of the protein revealed the presence of a zinc finger CCCH motif rarely found in proteins of prokaryotic origin. The zinc binding motif tightly coordinates zinc to provide a rigid structural framework for the enzyme needed for its function. In addition to this structural scaffold, another atom of zinc binds to the active site to induce high fidelity cleavage and suppress the Mg2+- and Mn2+-mediated promiscuous behavior of the enzyme. This is the first demonstration of distinct structural and catalytic roles for zinc in an enzyme, suggesting the distinct origin of KpnI REase.     

R.KpnI, an HNH superfamily REase, exhibits differential discrimination at non-canonical sequences in the presence of Ca2+ and Mg2+

KpnI REase recognizes palindromic sequence, GGTAC↓C, and forms complex in the absence of divalent metal ions, but requires the ions for DNA cleavage. Unlike most other REases, R.KpnI shows promiscuous DNA cleavage in the presence of Mg²⁺. Surprisingly, Ca²⁺ suppresses the Mg²⁺-mediated promiscuous activity and induces high fidelity cleavage. To further analyze these unique features of the enzyme, we have carried out DNA binding and kinetic analysis. The metal ions which exhibit disparate pattern of DNA cleavage have no role in DNA recognition. The enzyme binds to both canonical and non-canonical DNA with comparable affinity irrespective of the metal ions used. Further, Ca²⁺-imparted exquisite specificity of the enzyme is at the level of DNA cleavage and not at the binding step. With the canonical oligonucleotides, the cleavage rate of the enzyme was comparable for both Mg²⁺- and Mn²⁺-mediated reactions and was about three times slower with Ca²⁺. The enzyme discriminates non-canonical sequences poorly from the canonical sequence in Mg²⁺-mediated reactions unlike any other Type II REases, accounting for the promiscuous behavior. R.KpnI, thus displays properties akin to that of typical Type II REases and also endonucleases with degenerate specificity in its DNA recognition and cleavage properties.     

IL-1β promotes TGF-β1 and IL-2 dependent Foxp3 expression in regulatory T cells

Earlier, we have shown that GM-CSF-exposed CD8α- DCs that express low levels of pro-inflammatory cytokines IL-12 and IL-1β can induce Foxp3+ Tregs leading to suppression of autoimmunity. Here, we examined the differential effects of IL-12 and IL-1β on Foxp3 expression in T cells when activated in the presence and absence of DCs. Exogenous IL-12 abolished, but IL-1β enhanced, the ability of GM-CSF-exposed tolerogenic DCs to promote Foxp3 expression. Pre-exposure of DCs to IL-1β and IL-12 had only a modest effect on Foxp3- expressing T cells; however, T cells activated in the absence of DCs but in the presence of IL-1β or IL-12 showed highly significant increase and decrease in Foxp3+ T cell frequencies respectively suggesting direct effects of these cytokines on T cells and a role for IL-1β in promoting Foxp3 expression. Importantly, purified CD4+CD25+ cells showed a significantly higher ability to maintain Foxp3 expression when activated in the presence of IL-1β. Further analyses showed that the ability of IL-1β to maintain Foxp3 expression in CD25+ T cells was dependent on TGF-β1 and IL-2 expression in Foxp3+Tregs and CD25- effectors T cells respectively. Exposure of CD4+CD25+ T cells to IL-1β enhanced their ability to suppress effector T cell response in vitro and ongoing experimental autoimmune thyroidits in vivo. These results show that IL-1β can help enhance/maintain Tregs, which may play an important role in maintaining peripheral tolerance during inflammation to prevent and/or suppress autoimmunity.     

Ring-expanded analogues of natural oxetanocin: (+) and (-) hydroxymethyl isodideoxyadenosine

New enantiomeric isonucleoside analogues related to natural oxetanocin have been synthesized from D-glucosamine and D-glucose. The structures of the target compounds were confirmed by NMR, HRMS, UV, single crystal X-ray, and optical rotation data. Stability studies with respect to purine nucleoside phosphorylase and adenosine deaminase show that these compounds are not substrates. Antiviral results are discussed.     

Inhibitors of inosine monophosphate dehydrogenase: probes for antiviral drug discovery

The role of inosine monophosphate dehydrogenase (IMPDH) at the metabolic branch point of de novo purine nucleotide biosynthesis makes this enzyme an attractive probe for the discovery of antiviral compounds. Introduction of unsaturation at the 2-position of IMP, the natural substrate for IMPDH, produces Michael acceptors at that position, which results in these compounds being inhibitors of IMPDH. Consistent with this mechanism-based molecular design, some of the parent nucleosides exhibited antiviral activity.     

Cloning, expression, and characterization of an oligoxyloglucan reducing end-specific xyloglucanobiohydrolase from Aspergillus nidulans

An oligoxyloglucan reducing end-specific xyloglucanobiohydrolase from the filamentous fungus Aspergillus nidulans was cloned and expressed in Pichia pastoris as a secreted histidine-tagged protein and purified by affinity chromatography. The enzyme acts on xyloglucan oligomers and releases the first two glycosyl residue segments from the reducing end, provided that neither the first glucose nor the xylose attached to the third glucose residue from the reducing end is not further substituted. The enzyme has a specific activity of 7 U/mg at the pH optimum of 3 and at the temperature optimum of 42 degrees C.