Functional characterization of protease-treated Bacillus anthracis protective antigen.

Characterization of the functional domains of Bacillus anthracis protective antigen (PA, 83-kDa), the common cellular binding molecule for both anthrax edema toxin and anthrax lethal toxin, is important for understanding the mechanism of entry and action of the anthrax toxins. In this study, we generated both biologically active (facilitates killing of J774A.1 cells in combination with lethal factor, LF) and inactive preparations of PA by protease treatment. Limited proteolytic digestion of PA in vitro with trypsin generated a 20-kDa fragment and a biologically active 63-kDa fragment. In contrast, limited digestion of PA with chymotrypsin yielded a preparation containing 37- and 47-kDa fragments defective for biological activity. Treatment with both chymotrypsin and trypsin generated three major fragments, 20, "17," and 47 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This PA preparation was also biologically inactive. To investigate the nature of the defect resulting from chymotrypsin treatment, we assayed PA preparations for the ability to bind to the cellular receptor and to bind and internalize 125I-LF. All radiolabeled PA preparations bound with specificity to J774A.1 cells and exhibited affinities similar to native 83-kDa PA. Once bound to the cell surface receptor, both trypsin-treated PA and chymotrypsin/trypsin-treated PA specifically bound 125I-LF with high affinity. Finally, these PA preparations delivered 125I-LF to a Pronase-resistant cellular compartment in a time- and temperature-dependent fashion. Thus, the biological defect exhibited by chymotrypsin-treated PA is not at the level of cell binding or internalization but at a step later, such as toxin routing or processing by J774A.1 cells. These protease-treated preparations of PA should prove useful in both elucidating the intracellular processing of anthrax lethal toxin and determining the structure-function relationship of PA and LF.     

A highly conserved nuclear gene for low-level phylogenetics: elongation factor-1 alpha recovers morphology-based tree for heliothine moths

Molecular systematists need increased access to nuclear genes. Highly conserved, low copy number protein-encoding nuclear genes have attractive features for phylogenetic inference but have heretofore been applied mostly to very ancient divergences. By virtue of their synonymous substitutions, such genes should contain a wealth of information about lower-level taxonomic relationships as well, with the advantage that amino acid conservatism makes both alignment and primer definition straightforward. We tested this postulate for the elongation factor-1 alpha (EF-1 alpha) gene in the noctuid moth subfamily Heliothinae, which has probably diversified since the middle Tertiary. We sequenced 1,240 bp in 18 taxa representing heliothine groupings strongly supported by previous morphological and allozyme studies. The single most parsimonious gene tree and the neighbor-joining tree for all nucleotides show almost complete concordance with the morphological tree. Homoplasy and pairwise divergence levels are low, transition/transversion ratios are high, and phylogenetic information is spread evenly across gene regions. The EF-1 alpha gene and presumably other highly conserved genes hold much promise for phylogenetics of Tertiary age eukaryote groups.      

The discovery of a potent and selective lethal factor inhibitor for adjunct therapy of anthrax infection

A potent and selective anthrax LF inhibitor 40, (2R)-2-[(4-fluoro-3-methylphenyl)sulfonylamino]-N-hydroxy-2-(tetrahydro-2H-pyran-4-yl)acetamide, was identified through SAR study of a high throughput screen lead. It has an IC50 of 54 nM in the enzyme assay and an IC50 of 210 nM in the macrophage cytotoxicity assay. Compound 40 is also effective in vivo in several animal model studies.     

Astrocyte pVHL and HIF-α isoforms are required for embryonic-to-adult vascular transition in the eye

Successful transition from embryonic to adult circulation is critical for survival of mammalian organisms. This shift occurs in the central cardiovascular circulation and in the eye as oxygen tension increases. However, its regulation is not well understood. We have used combinatorial gene deletion and overexpression assays to assess the effect of astrocyte-targeted deletion of von Hippel-Lindau tumor suppressor (Vhl), hypoxia-inducible factor-αs (Hif-αs), and Vegf on the normal regression of the hyaloidal vessels, the fetal ocular circulation system. Astrocytic Vhl deletion induced accelerated hyaloidal regression and subsequent massive secondary outgrowth. Combinatorial gene deletion involving Vhl, Hif-αs, and Vegf genes revealed that HIF-2α/vascular endothelial growth factor signaling induces secondary outgrowth in Vhl mutants. Conversely, HIF-1α regulated macrophage migration inhibitory factor and promoted macrophage infiltration that accelerates hyaloidal vessel regression. The phenotype observed in Vhl mutants strongly resembles human persistent hyperplastic primary vitreous cases and may provide insights into vascular remodeling mechanisms in other systems.     

Stem cell biology in thyroid cancer: Insights for novel therapies

Currently, thyroid cancer is one of the most common endocrine cancer in the United States. A recent involvement of sub-population of stem cells, cancer stem cells, has been proposed in different histological types of thyroid cancer. Because of their ability of self-renewal and differentiation into various specialized cells in the body, these putative cells drive tumor genesis, metastatic activity and are responsible to provide chemo- and radioresistant nature to the cancer cells in the thyroid gland. Our Review was conducted from previously published literature to provide latest apprises to investigate the role of embryonic, somatic and cancer stem cells, and discusses the hypothesis of epithelial-mesenchymal transition. Different methods for their identification and isolation through stemness markers using various in vivo and in vitro methods such as flow cytometry, thyrosphere formation assay, aldehyde dehydrogenase activity and ATP-binding cassette sub-family G member 2 efflux-pump mediated Hoechst 33342 dye exclusion have been discussed. The review also outlines various setbacks that still remain to target these tumor initiating cells. Future perspectives of therapeutic strategies and their potential to treat advanced stages of thyroid cancer are also disclosed in this review.     

Harmonised Australian Reference Intervals for Serum PINP and CTX in Adults

Bone turnover markers (BTMs) are classified as either formation or resorption markers. Their concentrations in blood or urine of adults are considered to reflect the rate of bone remodelling and may be of use in the management of patients with bone disease. Major inter-method differences exist for BTMs, and harmonisation of methods is currently being pursued at an international level. Based on published data, this article describes age- and sex-specific Australian consensus reference intervals for adults for serum procollagen type I amino-terminal propeptide (s-PINP) and serum β-isomerised carboxy-terminal cross-linking telopeptide of type I collagen (s-CTX).     

Gracilaria and its epiphytes: 4. The response of two Gracilaria species to Ulva lactuca in a bacteria-limited environment

The responses of Gracilaria lemaneiformis, an easily epiphytized host,and the relatively resistant G. cornea mutant, to the green alga Ulva lactuca were studied using biculture experiments with and withoutantibiotics. Both Gracilaria species grown with and without U.lactuca showed different levels of growth rate, release of hydrogenperoxide and of halogenated hydrocarbons. These quantitative differencesled to a successful response against Ulva lactuca in the case of G.cornea mutant and to a failure in response in the case of G.lemaneiformis. The response of each Gracilaria species to U.lactuca was qualitatively similar to its response to bacteria. This suggeststhe involvement of oligosaccharide elicitors produced in the presence ofepiphytes and bacteria. A clear Gracilaria inhibition was demonstratedwith extracts of the culture medium. It appears that hydrogen peroxide,halogenated hydrocarbons and oligosaccharides may be components of theinhibitory activity of the extracts. The responses of Gracilaria speciesto the presence of U. lactuca suggest the characterization of adefence response.     

Phylogenetic studies of ribosomal RNA variation in higher moths and butterflies (Lepidoptera: Ditrysia).

The selection of exemplars has been shown both theoretically and empirically to affect tree topology, but the importance of the number and nature of taxa used to represent higher taxonomic lineages in molecular studies is rarely stressed. In our rRNA study of higher moths and butterflies (Lepidoptera: Ditrysia), the selection of different exemplars and outgroups caused major tree rearrangements. We also examined the effectiveness with which conserved rRNA regions track the diversification of Lepidoptera. Homoplasy is as prevalent at the few variable sites of conserved regions (18E, 18J, 28F) as at the many variable sites of a more rapidly evolving region (28B). Finally, 28B sequence variation differs qualitatively among lepidopteran superfamilies of presumed comparable age, the Papilionoidea (true butterflies) and Noctuoidea (cutworm moths and relatives).