Identification and characterization of 3-substituted pyrazolyl esters as alternate substrates for cathepsin B: the confounding effects of DTT and cysteine in biological assays

Substituted pyrazole esters were identified as hits in a high throughput screen (HTS) of the NIH Molecular Libraries Small Molecule Repository (MLSMR) to identify inhibitors of the enzyme cathepsin B. Members of this class, along with functional group analogs, were synthesized in an effort to define the structural requirements for activity. Analog characterization was hampered by the need to include a reducing agent such as dithiothreitol (DTT) or cysteine in the assay, highlighting the caution required in interpreting biological data gathered in the presence of such nucleophiles. Despite the confounding effects of DTT and cysteine, our studies demonstrate that the pyrazole 1 acts as alternate substrate for cathepsin B, rather than as an inhibitor.     

Deconvolving single-molecule intensity distributions for quantitative microscopy measurements

In fluorescence microscopy, images often contain puncta in which the fluorescent molecules are spatially clustered. This article describes a method that uses single-molecule intensity distributions to deconvolve the number of fluorophores present in fluorescent puncta as a way to "count" protein number. This method requires a determination of the correct statistical relationship between the single-molecule and single-puncta intensity distributions. Once the correct relationship has been determined, basis histograms can be generated from the single-molecule intensity distribution to fit the puncta distribution. Simulated data were used to demonstrate procedures to determine this relationship, and to test the methodology. This method has the advantages of single-molecule measurements, providing both the mean and variation in molecules per puncta. This methodology has been tested with the avidin-biocytin binding system for which the best-fit distribution of biocytins in the sample puncta was in good agreement with a bulk determination of the avidin-biocytin binding ratio.     

Phylogenetic analysis of <Emphasis Type="Italic">Antrodia</Emphasis> species and <Emphasis Type="Italic">Antrodia camphorata</Emphasis> inferred from internal transcribed spacer region

The species of Antrodia are one of the difficult-to-classify and obscure groups of poroid Aphyllophorales based on morphological appearance. However, it is becoming increasingly important to reliably identify the entire suite of Antrodia camphorata strains and Antrodia species due to the potential pharmaceutical value of their biologically active ingredients. In this study, the internal transcribed spacer (ITS) region of the ribosomal RNA gene (rDNA) was sequenced and phylogenetically analyzed in a number of Antrodia fungal species and strains. ITS amplicons from the Antrodia species tested ranged in size from 543 to 610 bp; the size of the ITS of A. camphorata strains ranged from 592 to 596 bp. The overall sizes of ITS2 and 5.8S ribosomal RNA gene of all A. camphorata strains tested in this study were shown to be 217 and 158 bp, respectively. A phylogenetic analysis of ITS data generated, which included sequences of 11 A. camphorata strains and nine other Antrodia species, showed three clearly distinct groups. Group 1 includes A. camphorata, Antrodia salmonea, and Antrodia carbinca strains. Within Group 2, Antrodia sinuosa and Antrodia xantha were clustered together. Group 3 contained Antrodia albida, A. heteromorpha, A. serialis, and A. malicola. The observed sequence diversity among ITS alleles provided an effective tool for differentiating strains of A. camphorata, A. salmonea, A. xantha, A. sinuosa, or A. serialis. Polymorphisms arising within the ITS1-5.8S-ITS2 region can provide practical markers for establishing a foundation for the further expansion of an ITS sequence database of medically important fungi.     

Characterization of murine interferon-alpha 12 (MuIFN-alpha12): biological activities and gene expression

Interferon alpha (IFN-alpha) belongs to the type I interferon family and consists of multiple subtypes in many species. In the mouse, there are at least 14 IFN-alpha genes and 3 IFN-alpha pseudogenes, the most recently identified of which are murine interferon-alpha 12 (MuIFN-alpha12), MuIFN-alpha13 and MuIFN-alpha14. To further study the biological activities of MuIFN-alpha12, we have produced a recombinant MuIFN-alpha12 (rMuIFN-alpha12) protein using COS-1 cells. rMuIFN-alpha12 was found to inhibit the growth of murine myeloid leukemia JCS cells. Flow cytofluorometric analysis with propidium iodide staining showed that the growth inhibitory activity of rMuIFN-alpha12 may be caused by the induction of apoptosis. Flow cytofluorometric analysis also revealed that rMuIFN-alpha12 was able to up-regulate the expression of MHC-I on both JCS cells and primary macrophages. Functional studies indicated that a MuIFN-alpha12 transgene could induce an anti-viral state in L929 cells against Influenza A virus. Moreover, expression of MuIFN-alpha12 was not detectable by RT-PCR in untreated, Influenza A virus infected, polyI:polyC induced L929 cells, or in a wide range of normal murine tissues. Taken together, this data shows that MuIFN-alpha12 is a protein with all the biological traits of a type I IFN.     

Effective binding to protein antigens by antibodies from antibody libraries designed with enhanced protein recognition propensities

Antibodies provide immune protection by recognizing antigens of diverse chemical properties, but elucidating the amino acid sequence-function relationships underlying the specificity and affinity of antibody-antigen interactions remains challenging. We designed and constructed phage-displayed synthetic antibody libraries with enriched protein antigen-recognition propensities calculated with machine learning predictors, which indicated that the designed single-chain variable fragment variants were encoded with enhanced distributions of complementarity-determining region (CDR) hot spot residues with high protein antigen recognition propensities in comparison with those in the human antibody germline sequences. Antibodies derived directly from the synthetic antibody libraries, without affinity maturation cycles comparable to those in in vivo immune systems, bound to the corresponding protein antigen through diverse conformational or linear epitopes with specificity and affinity comparable to those of the affinity-matured antibodies from in vivo immune systems. The results indicated that more densely populated CDR hot spot residues were sustainable by the antibody structural frameworks and could be accompanied by enhanced functionalities in recognizing protein antigens. Our study results suggest that synthetic antibody libraries, which are not limited by the sequences found in antibodies in nature, could be designed with the guidance of the computational machine learning algorithms that are programmed to predict interaction propensities to molecules of diverse chemical properties, leading to antibodies with optimal characteristics pertinent to their medical applications.