Cellular Activity Profile Research Articles

Simulation of the cellular anabolic activity within biofilms: Where a new immobilized cell will preferably be born?

A mathematical model, based on structured growth kinetics, is presented to simulate the profile of assimilated radioactive sulphur by a biofilm, after the release of calculated amounts of 35SO 4 2− in the biofilm environment. The radiolabelled sulphur ( 35S) stands as a biomass growth indicator, thus allowing the estimation of the cellular anabolic activity (growth) profile inside the biofilm. The model predicts that the maximum rate of biomass synthesis occurs at the external layer of the biofilm. A good correlation between the model predictions and previously published experimental data is reported, indicating the applicability of the structured model to describe microbial growth in immobilized cell systems.

The Role of Complement in Danger Sensing and Transmission

Self-non-self discrimination has long been considered the main function of the immune system. Increasing evidence supports the view of the immune system as a network of complex danger sensors and transmitters in which self-non-self discrimination is only one facet. To meet the challenge of danger sensing, the immune system carries a large stock of germline-encoded, highly conserved molecules that can recognize microbial as well as modified host structures. Among those are the Toll-like receptors (TLR), which comprise a dozen membrane-bound pattern-recognition receptors that directly link danger recognition to danger transmission through activation of several distinct cellular signaling pathways. Here, I discuss the function and biology of a complex, evolutionary ancient system, the complement system, which has long been considered critical to host defense. In contrast to TLRs, the complement system senses danger by a panel of soluble molecules that can directly bind to specific complement receptors and/or initiate a complex cascade of proteolytic events that lead to the generation of soluble complement fragments able to bind to another, distinct set of specific complement receptors. As I will outline in this review, complement- mediated danger sensing and the complex transition of this information into distinct cellular activation profiles is critical for tissue homeostasis under steady-state conditions and in response to infection and cell injury. Furthermore, I will discuss recent findings that support a concept of intense cross-talk between the complement system and TLRs, which defines the quality and the magnitude of immune responses in vivo.

An Integrated Approach to Analyzing Activation Profiles in Immune Cells by Combining Cytomic and Proteomic Techniques of Cell Analysis/Sorting and Protein Fractionation.

Understanding complex responses in biological systems is important to the development of new biomarkers, diagnostic assays, drugs and biologics required for early disease detection, prognosis, monitoring, and treatment. Although several cutting edge technologies have been introduced in the last decade, they have not significantly impacted the speed and economics of new drug applications and introduction of relevant diagnostic tests. In order to overcome this predicament, a Systems Biology approach is necessary to interrogate complex biological responses. This requires that assays that have so far been carried out in isolation, for e.g. gene expression, protein synthesis, phenotype and function, etc. need to be integrated and evaluated in a complementary fashion.In the current study, the authors have attempted such an evaluation by integrating well-known techniques of flow cytometry-based phenotypic analysis and cell sorting with protein fractionation and analysis to evaluate immune cellular activation profiles. To evaluate changes in protein profiles associated with the activation of an immune response, peripheral blood mononuclear cells (PBMCs) were stimulated with the superantigen, Staphylococcus enterotoxin B (SEB) and anti-CD28 or left un-treated for 24 hours. Lysates of PBMC populations from each culture were directly fractionated by two-dimensional gel-free liquid chromatography using isoelectric point (pI) and hydrophobicity. Alternatively, flow-based sorting techniques were 1st utilized to isolate T cells of interest from activated and control cultures and then fractionated as described above. Cell free supernatants from activated and non activated cultures were also fractionated and analyzed.The 1st dimension fractions obtained by separation based on pI alone did not reveal significant differences in activated and non activated lysates. The majority of the proteins in both the sorted and non-sorted lysates eluted in the acidic fractions of the pH gradient. However, using advanced differential display software, the hydrophobicity-dependent second dimension separations of the 1st dimension pI fractions did reveal significant quantitative and qualitative differences for several peaks in both the acidic and basic regions of the gradient in the activated and non activated fractions. Further combination of sorting of T cell populations with two-dimensional gel-free liquid chromatography permitted the characterization of an increased number of proteins that were up or down regulated by activation as compared to non sorted cell cultures. Thus the cumulative approach of sorting and 2-dimensional analysis was the most sensitive in discovering changes associated with activation. Such profiles could potentially be used as surrogate markers of efficacy in vaccine development and immunotoxicity in drug development. Further combinations of genomic, proteomic and cytomic profiling will further advance and ultimately simplify the discovery process and enable the mapping of biological pathways to accomplish a unified evaluation of relevant biomarkers in this response and other clinical models.

Cellular Profiling of Small‐Molecule Bioactivities: an Alternative Tool for Chemical Biology

Are they a good match? Target identification is critical to the success of chemical genetics projects that start with phenotypic screens. The method highlighted herein is a novel way to decrease the number of potential biological targets of compounds identified in phenotypic screens by comparison of their cellular activity profiles with those obtained with reference compounds. Activity profiles for hypothetical compounds A and B are shown.

Effect of inhibitors of histone deacetylase on the induction of cell differentiation in murine and human erythroleukemia cell lines

Histone deacetylase (HDAC) inhibitors are a novel class of promising anti-cancer agents. Little information is available on the capacity of structurally different HDAC inhibitors to induce terminal cell differentiation in different cell types in relation to enzyme inhibition and subtype selectivity. Consequently, the aim of this study was to provide a comprehensive comparison of these effects. New biarylalanine inhibitors of HDAC were synthesized and compared to a series of standard inhibitors from different laboratories. Chromatographically purified rat liver and immunoprecipitated FLAG-tagged recombinant human HDACs were used as sources of HDAC activity. Enzyme inhibition was studied using a fluorescent substrate and its conversion was monitored by high-performance liquid chromatography. The ability to induce cell differentiation was compared in murine (Friend DS-19) and human (K562) erythroleukemic cell lines, and was quantified by benzidine staining. Inhibition of cell proliferation was evaluated by cell counting. All HDAC inhibitors were identified as potent inhibitors of erythroleukemic cell proliferation. However, we observed a complex pattern of differentiation induction: structurally similar inhibitors resulted in disparate activity profiles, whereas similar profiles were detected within distinct structural classes. Among the newly synthesized biarylalanine compounds, a 3'-methoxy derivative was identified as a very effective inducer of terminal cell differentiation. We conclude that investigation of subtype selectivity of selected HDAC inhibitors does not provide a clear link between selectivity and the observed cellular activity profile. The predictive value of in vitro HDAC inhibition assays for identifying anti-proliferative compounds has been emphasized.

Discovery of a novel bicycloproline P2 bearing peptidyl α-ketoamide LY514962 as HCV protease inhibitor

We describe herein the design, syntheses and evaluation of a number of bicycloproline P2 bearing HCV protease inhibitors endowed with impressive enzyme potency, enzyme selectivity, cellular activity and favorable ADME profiles.

Improved P1/P1' substituents for cyclic urea based HIV-1 protease inhibitors: synthesis, structure-activity relationship, and X-ray crystal structure analysis.

We present several novel P1/P1' substituents that can replace the characteristic benzyl P1/P1' moiety of the cyclic urea based HIV protease inhibitor series. These substituents typically provide 5-10-fold improvements in binding affinity compared to the unsubstituted benzyl analogs. The best substituent was the 3,4-(ethylenedioxy)benzyl group. Proper balancing of the molecule's lipophilicity facilitated the transfer of this improved binding affinity into a superior cellular antiviral activity profile. Several analogs were evaluated further for protein binding and resistance liabilities. Compound 18 (IC90 = 8.7 nM) was chosen for oral bioavailability studies based on its log P and solubility profile. A 10 mg/kg dose in dogs provided modest bioavailability with Cmax = 0.22 microg/mL. X-ray crystallographic analysis of two analogs revealed several interesting features responsible for the 3,4-(ethylenedioxy)benzyl-substituted analog's potency: (1) Comparing the two complexes revealed two distinct binding modes for each P1/P1' substituent; (2) The ethylenedioxy moieties are within 3.6 A of Pro 81 providing additional van der Waals contacts missing from the parent structure; (3) The enzyme's Arg 8 side chain moves away from the P1 substituent to accommodate the increased steric volume while maintaining a favorable hydrogen bond distance between the para oxygen substituent and the guanidine NH.