Biochemical and immunological characterization of pea nuclear intermediate filament proteins

In immunoblot assays, at least three putative nuclear intermediate filament (NIF) proteins were detected in nuclear envelope-matrix (NEM) and lamin (L1) fractions of nuclei from plumules of dark-grown pea (Pisum sativum L.) seedlings. These NIF proteins had apparent molecular masses of ca. 65, 60, and 54 kDa (also referred to as p65, p60, and p54), and appeared as multiple isoelectric forms, with pIs ranging from ca. 4.8 to 6.0. Polyclonal and monoclonal antibodies were raised to the 65-kDa NIF protein bands excised from gels after electrophoresis. These anti-pea antibodies were specifically cross-reactive with the pea nuclear p65, p60, and p54 proteins and also with chicken lamins. Sequence alignment of peptide fragments obtained from the 65- and 60-kDa pea NIF proteins showed similarity with animal intermediate filament proteins such as lamins and keratins and with certain plant proteins predicted to have long coiled-coil domains. These pea NIF proteins were further purified and enriched from the NEM fraction using methods similar to those used for isolating animal lamins. When negatively stained and viewed by transmission electron microscopy, the filaments in the pea lamin (L1) fraction appeared to be 6–12 nm in diameter. As assayed by immunofluorescence cytochemistry using a confocal laser-scanning microscope, fixed pea plumule cells displayed uniform as opposed to peripheral nuclear staining by several of the antibody preparations, both polyclonal and monoclonal. This report describes the biochemical and immunological properties of these pea NIF proteins.Abbreviations IF Intermediate filament - L Lamin fraction - LM Lamina-matrix fraction - MAb JLA20 Anti-chicken actin monoclonal antibody - MAb LN43 Anti-human lamin B2 monoclonal antibody - MAb PL19 Anti-pea lamin #19 monoclonal antibody - MAb TIB 131 Anti-intermediate filament monoclonal antibody - N Nuclei fraction - NEM Nuclear envelope-matrix fraction - NIF Nuclear intermediate filament - PAb PL3 Anti-pea lamin #3 polyclonal antibody     

Cartilage interstitial fluid load support in unconfined compression following enzymatic digestion

Interstitial fluid pressurization plays an important role in cartilage biomechanics and is believed to be a primary mechanism of load support in synovial joints. The objective of this study was to investigate the effects of enzymatic degradation on the interstitial fluid load support mechanism of articular cartilage in unconfined compression. Thirty-seven immature bovine cartilage plugs were tested in unconfined compression before and after enzymatic digestion. The peak fluid load support decreased significantly (p < 0.0001) from 84 +/- 10% to 53 +/- 19% and from 80 +/- 10% to 46 +/- 21% after 18-hours digestion with 1.0 u/mg-wet-weight and 0.7 u/mg-wet-weight of collagenase, respectively. Treatment with 0.1 u/ml of chondroitinase ABC for 24 hours also significantly reduced the peak fluid load support from 83 +/- 12% to 48 +/- 16% (p < 0.0001). The drop in interstitial fluid load support following enzymatic treatment is believed to result from a decrease in the ratio of tensile to compressive moduli of the solid matrix.     

Synthesis of chiral phosphoantigens and their activity in gamma delta T cell stimulation

Gammadelta T cells expressing Vgamma2Vdelta2 T cell receptors are activated by a broad range of phosphorus-containing small molecules, termed phosphoantigens, and are of interest in the context of the chemotherapy of B cell malignancies. Here, we report the synthesis of four pairs of chiral phosphoantigens: the bromohydrins of isopentenyl diphosphate (Phosphostim), the epoxides of isopentenyl diphosphate (EIPP); and the corresponding bromohydrin and epoxide analogs of but-3-enyl diphosphate. The ability of each compound to stimulate human Vgamma2Vdelta2 T cells was determined by TNF-alpha release and cell proliferation. In these assays, the (R)-bromohydrin diphosphates were, on average, about twice as active as the (S)-bromohydrin diphosphates. In contrast, the (S)-form of EIPP was about twice as active as (R)-EIPP. The activities of the epoxy but-3-enyl diphosphates were both very low. These results suggest that chiral phosphoantigens, as opposed to racemic mixtures, may have utility in immunotherapy.     

N-Benzoyl amino acids as ICAM/LFA-1 inhibitors. Part 2: structure-activity relationship of the benzoyl moiety

o-Bromobenzoyl l-tryptophan 1 inhibits the association of LFA-1 with ICAM-1 with an IC(50) of 1.7microM. Evaluation of the structure-activity relationship of the benzoyl moiety shows that 2,6-di-substitutions greatly enhance potency of this class of inhibitors. Electronegative substitutions that favor a 90 degrees angle between the benzoyl ring and the amide bond yield the most potent compounds. There is a strong correlation between the potency of the compounds and the difference between the ab initio energy at 90 degrees and the global minima energy for given compounds. Combining the favored benzoyl substitutions with l-histidine and l-asparagine resulted in a 15-fold increase in potency over compound 1.     

Discovery of N-propylurea 3-benzylpiperidines as selective CC chemokine receptor-3 (CCR3) antagonists

The discovery of novel and selective small molecule antagonists of the CC Chemokine Receptor-3 (CCR3) is presented. Simple conversion from a 4- to 3-benzylpiperidine gave improved selectivity for CCR3 over the serotonin 5HT(2A) receptor. Chiral resolution and exploration of mono- and disubstitution of the N-propylurea resulted in several 3-benzylpiperidine N-propylureas with CCR3 binding IC(50)s under 5 nM. Data from in vitro calcium mobilization and chemotaxis assays for these compounds ranged from high picomolar to low nanomolar EC(50)s and correlated well with antagonist binding IC(50)s.     

Aminoimidazo[1,2-a]pyridines as a new structural class of cyclin-dependent kinase inhibitors. Part 1: Design, synthesis, and biological evaluation

We have identified a novel structural class of protein serine/threonine kinase inhibitors comprised of an aminoimidazo[1,2-a]pyridine nucleus. Compounds from this family are shown to potently inhibit cyclin-dependent kinases by competing with ATP for binding to a catalytic subunit of the protein. Structure-based design approach was used to direct this chemical scaffold toward generating potent and selective CDK2 inhibitors. The discovery of this new class of ATP-site directed protein kinase inhibitors, aminoimidazo[1,2-a]pyridines, provides the basis of new medicinal chemistry tool in search for an effective treatment of cancer and other diseases that involve protein kinase signaling pathways.     

The development of new bicyclic pyrazole-based cytokine synthesis inhibitors

4-Aryl-5-pyrimidyl-based cytokine synthesis inhibitors of TNF-alpha production, which contain a novel bicyclic pyrazole heterocyclic core, are described. Many of these inhibitors showed low nanomolar activity against LPS-induced TNF-alpha production in a THP-1 cell-based assay and against human p38 alpha MAP kinase in an isolated enzyme assay. The X-ray crystal structure of a bicyclic pyrazole inhibitor co-crystallized with mutated p38 (mp38) is presented.     

Nanos and Pumilio are essential for dendrite morphogenesis in Drosophila peripheral neurons

Much attention has focused on dendritic translational regulation of neuronal signaling and plasticity. For example, long-term memory in adult Drosophila requires Pumilio (Pum), an RNA binding protein that interacts with the RNA binding protein Nanos (Nos) to form a localized translation repression complex essential for anterior-posterior body patterning in early embryogenesis. Whether dendrite morphogenesis requires similar translational regulation is unknown. Here we report that nos and pum control the elaboration of high-order dendritic branches of class III and IV, but not class I and II, dendritic arborization (da) neurons. Analogous to their function in body patterning, nos and pum require each other to control dendrite morphogenesis, a process likely to involve translational regulation of nos itself. The control of dendrite morphogenesis by Nos/Pum, however, does not require hunchback, which is essential for body patterning. Interestingly, Nos protein is localized to RNA granules in the dendrites of da neurons, raising the possibility that the Nos/Pum translation repression complex operates in dendrites. This work serves as an entry point for future studies of dendritic translational control of dendrite morphogenesis.     

Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization

BACKGROUND: 14-3-3 proteins are abundant and conserved polypeptides that mediate the cellular effects of basophilic protein kinases through their ability to bind specific peptide motifs phosphorylated on serine or threonine. RESULTS: We have used mass spectrometry to analyze proteins that associate with 14-3-3 isoforms in HEK293 cells. This identified 170 unique 14-3-3-associated proteins, which show only modest overlap with previous 14-3-3 binding partners isolated by affinity chromatography. To explore this large set of proteins, we developed a domain-based hierarchical clustering technique that distinguishes structurally and functionally related subsets of 14-3-3 target proteins. This analysis revealed a large group of 14-3-3 binding partners that regulate cytoskeletal architecture. Inhibition of 14-3-3 phosphoprotein recognition in vivo indicates the general importance of such interactions in cellular morphology and membrane dynamics. Using tandem proteomic and biochemical approaches, we identify a phospho-dependent 14-3-3 binding site on the A kinase anchoring protein (AKAP)-Lbc, a guanine nucleotide exchange factor (GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo. CONCLUSION: 14-3-3 proteins can potentially engage around 0.6% of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been validated by the broad inhibition of 14-3-3 phosphorylation-dependent binding in vivo and by the specific analysis of AKAP-Lbc, a RhoGEF that is controlled by its interaction with 14-3-3.