Identification of Acetylcholinesterase Receptors in Rotifera

We have identified acetylcholinesterase (AChE) receptors in six freshwater rotifers. Using β-bungarotoxin labelled with fluoresceinisothiocyanate (FITC), muscarinic and nicotinic receptors were found in Brachionus quadridentatus (females and males), Lecane luna, Lecane quadridentata, Plationus patulus, and Rotaria neptunia. Using α-bungarotoxin-FITC, nicotinic receptors were identified in B. quadridentatus, Lecane bulla, L. luna, L. quadridentata, P. patulus and R. neptunia. Concentrations as low as 1.5 nM of β-bungarotoxin, and 5 nM of α-bungarotoxin identified receptors in the digestive tract. Higher concentrations of both toxins identified additional receptors associated with the lorica. A preliminary analysis of fluorescence intensity in L. quadridentata showed that response to α-bungarotoxin increases with age from newborn to 48-h old, but not in older individuals, thus suggesting an increase in binding sites, and possibly in number of nicotinic receptors, during the first 48-h of life. Our study extends the number of rotifer species in which AChE receptors have been reported.     

Design of novel α7-subtype-preferring nicotinic acetylcholine receptor agonists: Application of docking and MM-PBSA computational approaches, synthetic and pharmacological studies

In the search for nicotinic acetylcholine receptor (nAChRs) agonists with a selective affinity for the homomeric α7 channels, we carried out the virtual screening of a test set of potential nicotinic ligands, and adopted a simplified MM-PBSA approach to estimate their relative binding free energy values. By means of this procedure, previously validated by a training set of compounds, we reached a realistic compromise between computational accuracy and calculation rate, and singled out a small group of novel structurally related derivatives characterized by a promising theoretical affinity for the α7 subtype. Among them, five new compounds were synthesized and assayed in binding experiments at neuronal α7 as well as α4β2 nAChRs.     

Structure,Function, and Regulation of the Three β-Adrenergic Receptors

Three β-adrenergic receptor subtypes are now known to be functionally expressed in mammals. All three belong to the R₇G family of receptors coupled to G-proteins, and characterized by an extracellular glycosylated N-terminal and an intracellular C-terminal region and seven transmembrane domains, linked by three exta- and three intracellular loops. The catecholamine ligand binding domain, studied using affinity-labeling and site-directed mutagenesis, is a pocket lined by residues belonging to the transmembrane domains. The region responsible for the interaction with the Gs protein which, when activated, stimulates adenylyl cyclase, is composed of residues belonging to the parts most proximal to the membrane of intracellular loop i₃ and the C-terminal region. The pharmacology of the three subtypes is quite distinct: in fact most of the potent β₁/β₂ antagonists (the well known β blockers) act as agonists on β₃. The subtype is resistant to short-term desensitization mediated by phosphorylation through PKA or βARK, in stark contrast to the β₁ or β₂ subtypes. Various compounds (dexamethasone, butyrate, insulin) up regulate β₁ or β₁ subtypes while down-regulating β₃ whose expression strictly correlates with differentiation of 3T3-F442A fibroblasts into adipocytes, thus confirming that the expression of the three subtypes may each be regulated independently to exert a specific physiologic role in different tissues or at different stages of development.     

Induction of mouse β integrin expression following transfection with human α4 chain

We report here an analysis of the expression and function of the α chain of human VLA-4 in stable mouse L cell transfectants and the requirement for the β chain in these processes. L cells were transfected with human α4 cDNA or α4 and human β1 cDNA. Unexpectedly, human α4 cDNA, when transfected alone, could induce de novo surface expression of host β7 and increased expression of host β1. Induction of mouse β7 and β1 surface expression was not due to de novo gene activation, but instead represented α4/β intracellular subunit association and transport to the cell surface. Transfection with human β1 prevented surface expression of mouse β integrins. Whereas human α4 and human β1 subunits associated very tightly in anti-α4 immunoprecipitates, human α4 and mouse β subunits were only partially associated. Furthermore, binding of human/mouse chimeric receptors to recombinant VCAM, a major ligand for α4β7 and α4β1, was very poor, whereas human α4/human β1 receptors bound strongly to VCAM. One α4 transfectant, which exhibited a tight human α4/mouse β1 association, could be induced, but only after PMA activation, to bind strongly to VCAM. These results indicate that α4 subunits have specific affinity for β7 and β1 integrins and require β subunits for surface expression as well as high affinity ligand binding activity. Our results indicate that a tight association between the α4 and β subunit appears to be critical for ligand binding, consistent with a direct as well as regulatory role for the β subunit in ligand binding. Furthermore, these studies demonstrate that expression of foreign recombinant proteins can alter host cell protein expression resulting in de novo surface protein expression. © 1996 Wiley-Liss, Inc.     

A novel approach to predicting protein structural classes in a (20–1)-D amino acid composition space

The development of prediction methods based on statistical theory generally consists of two parts: one is focused on the exploration of new algorithms, and the other on the improvement of a training database. The current study is devoted to improving the prediction of protein structural classes from both of the two aspects. To explore a new algorithm, a method has been developed that makes allowance for taking into account the coupling effect among different amino acid components of a protein by a covariance matrix. To improve the training database, the selection of proteins is carried out so that they have (1) as many non-homologous structures as possible, and (2) a good quality of structure. Thus, 129 representative proteins are selected. They are classified into 30 α, 30 β, 30 α + β, 30 α/β, and 9 ζ (irregular) proteins according to a new criterion that better reflects the feature of the structural classes concerned. The average accuracy of prediction by the current method for the 4 × 30 regular proteins is 99.2%, and that for 64 independent testing proteins not included in the training database is 95.3%. To further validate its efficiency, a jackknife analysis has been performed for the current method as well as the previous ones, and the results are also much in favor of the current method. To complete the mathematical basis, a theorem is presented and proved in Appendix A that is instructive for understanding the novel method at a deeper level. © 1995 Wiley-Liss, Inc.     

Different conversion metabolic rates of testosterone are associated to hormone-sensitive status and -response of human prostate cancer cells

The main goal of the present work was to compare the ability of human prostate cancer (PCa) cells to metabolize testosterone (T) in living conditions. To this end we studied three different human PCa cell lines (LNCaP, DU145 and PC3) having different hormone-sensitive status and capability of response to androgens. We used an original approach which allows the evaluation of conversion metabolic rates in growing cells after administration of labeled steroid precursor (presently T), at physiological concentrations (1–10 nM). Analysis of both precursor degradation and formation of several products was carried out using reverse phase-high performance liquid chromatography (RP-HPLC) and “on line” radioactive detection. Comparison of the three human PCa cells revealed that their metabolic aptitude differed in many respects: (i) rates of precursor degradation, (ii) different products' formation, and (iii) extent of conjugate production. In detail, PC3 cells quickly degraded T and exhibited high formation rates of androstenedione (A-4-ene-Ad); both DU145 and LNCaP cells mostly retained high levels of unconverted T, with a limited production of A-4-ene-Ad and its 17-keto derivatives (if any). Either LNCaP or DU145 cells generated a relatively high amount of dihydrotestosterone (DHT). In contrast, neither DHT nor its main metabolites were detected in PC3 cells at both short and longer incubation times. As expected, T degradation and A-4-ene-Ad production were highly correlated (r = 0.97; P < 0.03); similarly, A-4-ene-Ad and DHT formation showed a negative, significant correlation. Negligible production of conjugates was noted in both PC3 and DU145 cells, whilst it was remarkable in LNCaP cells (ranging from 43 to 57%). Overall, our data indicate that human PCa cells degrade T quite differently, favoring alternatively reductive or oxidative patterns of androgen metabolism.     

Folding type-specific secondary structure propensities of amino acids,derived from α-helical, β-sheet, α/β, and α+β proteins of known structures

Folding type-specific secondary structure propensities of 20 naturally occurring amino acids have been derived from α-helical, β-sheet, α/β, and α+β proteins of known structures. These data show that each residue type of amino acids has intrinsic propensities in different regions of secondary structures for different folding types of proteins. Each of the folding types shows markedly different rank ordering, indicating folding type-specific effects on the secondary structure propensities of amino acids. Rigorous statistical tests have been made to validate the folding type-specific effects. It should be noted that α and β proteins have relatively small α-helices and β-strands forming propensities respectively compared with those of α+β and α/β proteins. This may suggest that, with more complex architectures than α and β proteins, α+β and α/β proteins require larger propensities to distinguish from interacting α-helices and β-strands. Our finding of folding type-specific secondary structure propensities suggests that sequence space accessible to each folding type may have differing features. Differing sequence space features might be constrained by topological requirement for each of the folding types. Almost all strong β-sheet forming residues are hydrophobic in character regardless of folding types, thus suggesting the hydrophobicities of side chains as a key determinant of β-sheet structures. In contrast, conformational entropy of side chains is a major determinant of the helical propensities of amino acids, although other interactions such as hydrophobicities and charged interactions cannot be neglected. These results will be helpful to protein design, class-based secondary structure prediction, and protein folding. © 1998 John Wiley & Sons, Inc. Biopoly 45: 35–49, 1998     

Integrin αvβ3 enhances the suppressive effect of interferon‐γ on hematopoietic stem cells

Hematopoietic homeostasis depends on the maintenance of hematopoietic stem cells (HSCs), which are regulated within a specialized bone marrow (BM) niche. When HSC sense external stimuli, their adhesion status may be critical for determining HSC cell fate. The cell surface molecule, integrin αvβ3, is activated through HSC adhesion to extracellular matrix and niche cells. Integrin β3 signaling maintains HSCs within the niche. Here, we showed the synergistic negative regulation of the pro‐inflammatory cytokine interferon‐γ (IFNγ) and β3 integrin signaling in murine HSC function by a novel definitive phenotyping of HSCs. Integrin αvβ3 suppressed HSC function in the presence of IFNγ and impaired integrin β3 signaling mitigated IFNγ‐dependent negative action on HSCs. During IFNγ stimulation, integrin β3 signaling enhanced STAT1‐mediated gene expression via serine phosphorylation. These findings show that integrin β3 signaling intensifies the suppressive effect of IFNγ on HSCs, which indicates that cell adhesion via integrin αvβ3 within the BM niche acts as a context‐dependent signal modulator to regulate the HSC function under both steady‐state and inflammatory conditions.     

Persistence and effects of α-methyl-substituted amino acids on 5-hydroxytryptamine and dopamine concentrations in cockroach (Periplaneta americana) nervous tissue

The α-methylated derivatives of tryptophan, tyrosine, and dihydroxyphenylalanine were injected into cockroaches (Periplaneta americana). The levels of these compounds and those of dopamine, 5-hydroxytryptamine, tyrosine, and tryptophan in the nervous tissue, hemolymph, and fat body were measured at various times after drug administration. Levels of 5-hydroxytryptamine and tryptophan in the nervous tissue are significantly reduced by α-methyltryptophan administration. Concentrations of dopamine in nervous tissue are reduced by α-methyltyrosine administration. This effect also persists for several weeks, and α-methyltyrosine is observed in the nervous tissue 3 weeks after injection. Levels of dopamine and 5-hydroxytryptamine in the nervous tissue are unaffected by α-methyldihydroxyphenylalanine, and this compound is less persistent in nervous tissue than α-methyltyrosine or α-methyltryptophan demonstrates that these compounds can be absorbed and affect amine levels in the nervous tissue when included in the diet. Inhibition of tryptophan hydroxylation by crude enzyme preparations of cockroach nervous tissue was demonstrated with both α-methyltryptophan and α-methyltyrosine, with α-methyltryptophan being the more effective inhibitor. Aromatic amino acid decarboxylase activity toward dihydroxyphenylalanine in crude enzyme preparations of cockroach nervous tissue was strongly inhibited by α-methyldihydroxyphenylalanine and monofluoromethyldihydroxyphenylalanine, slightly inhibited by α-methyltyrosine and unaffected by α-methyltryptophan at concentrations up to 10^?3 M. The results indicate that α-methyltyrosine and α-methyltryptophan, but not α-methyldihydroxyphenylalanine, can selectively alter amine concentrations in insect nervous tissue and that insects are only poorly able to metabolize or excrete these compounds. The selective and long-lasting depletion of dopamine or 5-hydroxytryptamine by some of these compounds suggest that they may be useful in behavioral studies designed to elucidate the roles of these amines in insects.