Aryl-indolyl maleimides as inhibitors of CaMKIIdelta. Part 2: SAR of the amine tether

A family of aryl-substituted maleimides was prepared and studied for their activity against calmodulin-dependant kinase. Inhibitory activities against the enzyme ranged from 34nM to >20microM and were dependant upon both the nature of the aryl group and the tether joining the basic amine to the indolyl maleimide core. Key interactions with the kinase ATP site and hinge region, predicted by homology modeling, were confirmed.     

Mobilization of rubisco and stroma-localized fluorescent proteins of chloroplasts to the vacuole by an ATG gene-dependent autophagic process

During senescence and at times of stress, plants can mobilize needed nitrogen from chloroplasts in leaves to other organs. Much of the total leaf nitrogen is allocated to the most abundant plant protein, Rubisco. While bulk degradation of the cytosol and organelles in plants occurs by autophagy, the role of autophagy in the degradation of chloroplast proteins is still unclear. We have visualized the fate of Rubisco, stroma-targeted green fluorescent protein (GFP) and DsRed, and GFP-labeled Rubisco in order to investigate the involvement of autophagy in the mobilization of stromal proteins to the vacuole. Using immunoelectron microscopy, we previously demonstrated that Rubisco is released from the chloroplast into Rubisco-containing bodies (RCBs) in naturally senescent leaves. When leaves of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing stroma-targeted fluorescent proteins were incubated with concanamycin A to inhibit vacuolar H(+)-ATPase activity, spherical bodies exhibiting GFP or DsRed fluorescence without chlorophyll fluorescence were observed in the vacuolar lumen. Double-labeled immunoelectron microscopy with anti-Rubisco and anti-GFP antibodies confirmed that the fluorescent bodies correspond to RCBs. RCBs could also be visualized using GFP-labeled Rubisco directly. RCBs were not observed in leaves of a T-DNA insertion mutant in ATG5, one of the essential genes for autophagy. Stroma-targeted DsRed and GFP-ATG8 fusion proteins were observed together in autophagic bodies in the vacuole. We conclude that Rubisco and stroma-targeted fluorescent proteins can be mobilized to the vacuole through an ATG gene-dependent autophagic process without prior chloroplast destruction.     

Functional impact of high protein intake on healthy elderly people

Decline in muscle mass, protein synthesis, and mitochondrial function occurs with age, and amino acids are reported to enhance both muscle protein synthesis and mitochondrial function. It is unclear whether increasing dietary protein intake corrects postabsorptive muscle changes in aging. We determined whether a 10-day diet of high [HP; 3.0 g protein x kg fat-free mass (FFM)(-1) x day(-1)] vs. usual protein intake (UP; 1.5 g protein x kg FFM(-1) x day(-1)) favorably affects mitochondrial function, protein metabolism, and nitrogen balance or adversely affects insulin sensitivity and glomerular filtration rate (GFR) in 10 healthy younger (24+/-1 yr) and 9 older (70+/-2 yr) participants in a randomized crossover study. Net daily nitrogen balance increased equally in young and older participants, but postabsorptive catabolic state also increased, as indicated by higher whole body protein turnover and leucine oxidation with no change in protein synthesis. Maximal muscle mitochondrial ATP production rate was lower in older people, with no change occurring in diet. GFR was lower in older people, and response to HP was significantly different between the two groups, with a significant increase occurring only in younger people, thus widening the differences in GFR between the young and older participants. In conclusion, a short-term high-protein diet increased net daily nitrogen balance but increased the postabsorptive use of protein as a fuel. HP did not enhance protein synthesis or muscle mitochondrial function in either young or older participants. Additionally, widening differences in GFR between young and older patients is a potential cause of concern in using HP diet in older people.     

COCCOID EUKARYOTIC MARINE ULTRAPLANKTERS WITH FOUR DIFFERENT HPLC PIGMENT SIGNATURES1

Pigment compositions of 16 coccoid eukaryotic ultraplanktonic clones isolated from coastal and oceanic waters were investigated by high-performance liquid chromatography (HPLC). Four distinct pigment signatures were observed, and clones were classified into subgroups based on the presence or absence and relative abundances of selected chlorphylls and carotenoids. The first subgroup (5 clones) was pigmented like chlorophyll b-containing higher plants and resembled true chlorophycean algae. The second subgroup (3 clones) contained chlorophyll b and relatively high levels of prasinoxanthin, a carotenoid characteristic of certain members of the Prasinophyceae (sometimes grouped as the Micromonadophyceae). The third subgroup (5 clones) was pigmented in a similar fashion but had a twofold lower prasinoxanthin-to-chlorophyll a ratio and an unidentified carotenoid. The fourth subgroup (3 clones) lacked chlorophyll b and was pigmented like certain members of the Chrysophyceae (e. g. 19′-butanoyloxyfucoxanthin-containing Pelagococcus subviridis Norris) Online diode array spectral analysis of selected clonal extracts revealed the presence of Mg 2,4-divinylphaeoporphyrin a₅ monomethyl ester-like and chlorophyll c-like pigments in representatives of the prasinophyte-like and chrysophyte like clones, respectively. These findings plus the occurrence of chlorophyll b, prasinoxanthin and 19′-butanoyloxyfucoxanthin in the North Atlantic Ocean suggest that chrysophyte- and prasinophyte-like organisms can be important biomass components of marine phytoplankton.     

Sunny-side up: flower heliotropism as a source of parental environmental effects on pollen quality and performance in the snow buttercup, Ranunculus adoneus (Ranunculaceae)

Floral traits affect mating success via their influence on the microenvironment in which sexual reproduction occurs as well as their impact on pollinator attraction. Here we investigate the importance of flower heliotropism as a source of parental environmental effects on pollen quality and performance. Flowers of the snow buttercup, Ranunculus adoneus, closely track the sun's rays. We experimentally restrained flowers to test for effects of heliotropism on pollen quality and performance after pollination. When equivalent amounts of pollen were transferred to recipient pistils, pollen from solar-tracking donor flowers exhibited a 32% advantage in germination compared to pollen from stationary (tethered) donor flowers. By the end of anthesis, pistils of tracking flowers contained 40% more germinating pollen grains and 44% more pollen tubes midway down the style than pistils of stationary ones. Solar tracking had no direct effect on pollen tube growth. The greater amount of germinating pollen in tracking flowers accounted for the treatment effect on pollen tube density. A survey of pollen receipt and pollen germination in naturally tracking flowers indicated that solar tracking primarily affects pollen tube density by promoting pollen germination rather than pollen deposition. We conclude that flower heliotropism, by enhancing the paternal environment for pollen development and the maternal environment for pollen germination, represents a source of positive parental environmental effects on pollen performance in snow buttercups.     

The mannose receptor fails to enhance processing and presentation of a glycoprotein antigen in transfected fibroblasts

One function proposed for the mannose receptor found on dendritic cells as well as on macrophages and hepatic endothelial cells is in enhancing uptake and processing of glycoprotein antigens for presentation by major histocompatibility complex (MHC) class II molecules. In this study, a direct assessment of the possible role of the mannose receptor in this process was made in the absence of other endocytic receptors that can internalize glycoproteins. Presentation of RNase A and B peptides was compared in transfected fibroblasts coexpressing the mannose receptor and MHC class II molecules. RNase B bears a high-mannose oligosaccharide and is a ligand for the mannose receptor, whereas RNase A is not glycosylated and is taken up by pinocytosis. Incubation of RNase A or B with the transfected cells resulted in identical stimulation of ribonuclease-specific T cells, indicating that endocytosis of the glycosylated protein by the mannose receptor does not enhance presentation of this antigen. The postulated role of the mannose receptor in presentation of glycoprotein-derived antigen is reevaluated in light of these results.     

Monoclonal antibodies to adhesive cell coat glycoproteins secreted by zoospores of the green alga Enteromorpha

Zoospores of Enteromorpha compressa (L.) Grev. secrete an adhesive cell coat which is involved in their attachment to various substrata. Two monoclonal antibodies (mAbs), designated Ent 1 and Ent 6, were raised against settled zoospores displaying secreted adhesive. Both antibodies labelled specifically the anterior region of the cell containing putative adhesive vesicles. During settlement the antigens recognised by both mAbs were secreted but whereas Ent 6 recognised a fibrillar material released within a few minutes of settlement, Ent 1 recognised components which were associated predominantly with the developing cell wall at later time points. Both mAbs also labelled a Golgi-rich region of settled spores, suggesting that these antigens are also synthesised after settlement. Both mAbs labelled the cell walls of vegetative tissue. Competitive enzyme-linked immunosorbent assay indicated that the two antibodies recognise separate, but overlapping epitopes. In spore settlement assays the Ent 6 immunoglobulin strongly reduced initial adhesion at low concentration whereas the inhibitory effects of Ent 1 occurred at later time points. On analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (SDS-PAGE) both MAbs recognised a major buffer- and SDS-soluble, polydisperse 110-kDa antigen. The 110-kDa component was present in extracts of zoospores and sporulating tissue, but absent, in soluble form, from vegetative tissue. Deglycosylation of zoospore extract with anhydrous HF and peptide N-glycosidase digestion, showed that the major 110-kDa antigen is an N-linked glycan, and that the epitope is borne by the protein component. Time-course experiments showed that the Ent 6 antigen became progressively insoluble after zoospore attachment. Taken together, the data indicate that the two antibodies recognise separate but closely related antigens which have distinctive roles in adhesion and cell wall development. Received: 8 February 1999 / Accepted: 26 July 1999     

Kinetic analysis of the interactions between troponin C (TnC) and troponin I (TnI) binding peptides: evidence for separate binding sites for the 'structural' N-terminus and the 'regulatory' C-terminus of TnI on TnC

The Ca(2+)/Mg(2+)-dependent interactions between TnC and TnI play a critical role in regulating the 'on' and 'off' states of muscle contraction as well as maintaining the structural integrity of the troponin complex in the off state. In the present study, we have investigated the binding interactions between the N-terminus of TnI (residues 1-40 of skeletal TnI) and skeletal TnC in the presence of Ca(2+) ions, Mg(2+) ions and in the presence of the C-terminal regulatory region peptides: TnI(96-115), TnI(96-131) and TnI(96-139). Our results show the N-terminus of TnI can bind to TnC with high affinity in the presence of Ca(2+) or Mg(2+) ions with apparent equilibrium dissociation constants of K(d(Ca(2+) ) ) = 48 nM and K(d(Mg(2+) ) ) = 29 nM. The apparent association and dissociation rate constants for the interactions were, k(on) = 4.8 x 10(5) M (-1) s(-1), 3.4 x 10(5) M (-1) s(-1) and k(off) = 2.3 x 10(-2) s(-1), 1.0 x 10(-2) s(-1) for TnC(Ca(2+)) and TnC(Mg(2+)) states, respectively. Competition studies between each of the TnI regions and TnC showed that both TnI regions can bind simultaneously to TnC while native gel electrophoresis and SEC confirmed the formation of stable ternary complexes between TnI(96-139) (or TnI(96-131)) and TnC-TnI(1-40). Further analysis of the binding interactions in the ternary complex showed the binding of the TnI regulatory region to TnC was critically dependent upon the presence of both TnC binding sites (i.e. TnI(96-115) and TnI(116-131)) and the presence of Ca(2+). Furthermore, the presence of TnI(1-40) slightly weakened the affinity of the regulatory peptides for TnC. Taken together, these results support the model for TnI-TnC interaction where the N-terminus of TnI remains bound to the C-domain of TnC in the presence of high and low Ca(2+) levels while the TnI regulatory region (residues 96-139) switches in its binding interactions between the actin-tropomyosin thin filament and its own sites on the N- and C-domain of TnC at high Ca(2+) levels, thus regulating muscle contraction.     

Indole-based heterocyclic inhibitors of p38alpha MAP kinase: designing a conformationally restricted analogue

p38alpha Mitogen Activated Protein Kinase (MAP kinase) is an intracellular soluble serine threonine kinase. p38alpha kinase is activated in response to cellular stresses, growth factors and cytokines such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha). The central role of p38alpha activation in settings of both chronic and acute inflammation has led efforts to find inhibitors of this enzyme as possible therapies for diseases such as rheumatoid arthritis, where p38alpha activation is thought to play a causal role. Herein, we report structure-activity relationship studies on a series of indole-based heterocyclic inhibitors that led to the design and identification of a new class of p38alpha inhibitors.