Differential localization of leu-enkephalin and hyperglycemic hormone in axon terminals of the sinus gland of the crabsCarcinus maenas,eriocheir sinensis,andUca pugilator

Summary Leu-enkephalin containing secretory granules were demonstrated in axon terminals of immunogoldlabeled electron-microscopic sections of the sinus gland of three brachyuran crustaceans. These granules have a diameter of 120+-15 nm and differ in electron density from those located in adjacent terminals containing hyperglycemic or molt-inhibiting hormone. These neurohormones do not show co-localization with leu-enkephalin. The cross-reactivity of leu-enkephalin antiserum with met-enkephalin is less than 1%. The sinus glands of the three species examined show no immunoreactivity for FMRF-amide. A modulatory activity of endogenous enkephalin by paracrine mechanisms is suggested.     

Modification of tyrosine hydroxylase activity by chloral derived beta-carbolines in vitro

Beta-carbolines have been suggested to be involved in the pathogenesis of Parkinson's disease as a result of their structural similarity to the neurotoxin N -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The chloral-derived beta-carboline derivative 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) causes cell loss in neuronal and glial cell cultures and induces a slowly developing neurodegenerative process in rats. In our experiments, effects of TaClo and its derivatives 2-methyl-TaClo (2-Me-TaClo), and 1-dichloromethylene-1,2,3,4-tetrahydro-beta-carboline (1-CCl(2) -THbetaC) on tyrosine hydroxylase (TH) activity were investigated in TH assays using homogenate preparations of the rat nucleus accumbens and recombinant human TH (hTH1). TH activity was determined in vitro by measuring l-DOPA production with HPLC-ECD. Using homogenate preparations, TaClo, 2-Me-TaClo, and 1-CCl(2) -THbetaC inhibited TH in concentrations of 0.1 mm, while 1-CCl(2) -THbetaC in low concentrations enhanced TH activity. When TH was activated by PACAP-27, TaClo, 2-Me-TaClo, or 1-CCl(2) -THbetaC also inhibited activated enzyme activity in high concentrations. However, in the case of 2-Me-TaClo and 1-CCl(2) -THbetaC a biphasic effect was observed with a marked increase of TH activity in the nanomolar range. In our experiments using recombinant hTH1, TaClo, 2-Me-TaClo, or 1-CCl(2) -THbetaC did not modify enzyme activity. After activation of hTH1 by PKA all the tetrahydro-beta-carbolines investigated in this study decreased l-DOPA formation. We suggest that these beta-carbolines modulate dopamine synthesis by interacting with a protein kinase TH-activating system.     

The genetic basis of fluconazole resistance development in Candida albicans

Infections by the opportunistic fungal pathogen Candida albicans are widely treated with the antifungal agent fluconazole that inhibits the biosynthesis of ergosterol, the major sterol in the fungal plasma membrane. The emergence of fluconazole-resistant C. albicans strains is a significant problem after long-term treatment of recurrent oropharyngeal candidiasis (OPC) in acquired immunodeficiency syndrome (AIDS) patients. Resistance can be caused by alterations in sterol biosynthesis, by mutations in the drug target enzyme, sterol 14alpha-demethylase (14DM), which lower its affinity for fluconazole, by increased expression of the ERG11 gene encoding 14DM, or by overexpression of genes coding for membrane transport proteins of the ABC transporter (CDR1/CDR2) or the major facilitator (MDR1) superfamilies. Different mechanisms are frequently combined to result in a stepwise development of fluconazole resistance over time. The MDR1 gene is not or barely transcribed during growth in vitro in fluconazole-susceptible C. albicans strains, but overexpressed in many fluconazole-resistant clinical isolates, resulting in reduced intracellular fluconazole accumulation. The activation of the gene in resistant isolates is caused by mutations in as yet unknown trans-regulatory factors, and the resulting constitutive high level of MDR1 expression causes resistance to other toxic compounds in addition to fluconazole. Disruption of both alleles of the MDR1 gene in resistant C. albicans isolates abolishes their resistance to these drugs, providing genetic evidence that MDR1 mediates multidrug resistance in C. albicans.     

TNF-α -308 genotypes are associated with TNF-α and TGF-β₁ mRNA expression in blood leucocytes of humans

AimTumor necrosis factor α (TNF-α) influences the pathogenesis of lung-fibrosis and carcinogenesis in normal cells. Polymorphisms of this gene are suggested to be associated with susceptibility to lung-diseases. Additionally TNF-α is postulated to play a significant role in regulating. Transforming growth factor (TGF-β₁) expression Therefore we investigated if the TNF-α or TGF-β₁ gene expression level is different within the ?308 TNF-α genotypes.MethodsQuantitative Real-time PCR of TNF-α and TGF-β₁ was performed in 178 Germans. Calculations of expression were made with the 2^?ΔΔCT method. Detection of the ?308 promoter polymorphism of the TNF-α gene was performed by rapid capillary PCR with melting curve analysis.ResultsThe relative TNF-α mRNA expression revealed significant differences between the TNF-α ?308 homozygote wild-type G/G (0.00079 ± 0.00011; n = 113) and the heterozygote genotype G/A (0.0005 ± 0.00008; n = 52; p = 0.030) as well as between homozygote wild-type G/G and the homozygote mutant A/A (0.00029 ± 0.00009; n = 5; p = 0.004). The relative TGF-β mRNA expression showed, similar to TNF-α, the highest mRNA expression was seen within the TNF-α ?308 homozygote wild-types, while the lowest mRNA expression lay within the homozygote mutant-types.ConclusionOur findings suggest that the G-allele of TNF-α ?308 is associated with a significantly higher TNF-α mRNA expression compared to the A-allele and that this also reflects in TGF-β expression. Therefore we support the thesis that TGF-β is regulated by TNF-α.     

The COMPASS family of H3K4 methylases in Drosophila

Methylation of histone H3 lysine 4 (H3K4) in Saccharomyces cerevisiae is implemented by Set1/COMPASS, which was originally purified based on the similarity of yeast Set1 to human MLL1 and Drosophila melanogaster Trithorax (Trx). While humans have six COMPASS family members, Drosophila possesses a representative of the three subclasses within COMPASS-like complexes: dSet1 (human SET1A/SET1B), Trx (human MLL1/2), and Trr (human MLL3/4). Here, we report the biochemical purification and molecular characterization of the Drosophila COMPASS family. We observed a one-to-one similarity in subunit composition with their mammalian counterparts, with the exception of LPT (lost plant homeodomains [PHDs] of Trr), which copurifies with the Trr complex. LPT is a previously uncharacterized protein that is homologous to the multiple PHD fingers found in the N-terminal regions of mammalian MLL3/4 but not Drosophila Trr, indicating that Trr and LPT constitute a split gene of an MLL3/4 ancestor. Our study demonstrates that all three complexes in Drosophila are H3K4 methyltransferases; however, dSet1/COMPASS is the major monoubiquitination-dependent H3K4 di- and trimethylase in Drosophila. Taken together, this study provides a springboard for the functional dissection of the COMPASS family members and their role in the regulation of histone H3K4 methylation throughout development in Drosophila.     

Selective inhibition of dipeptidyl peptidase 4 by targeting a substrate-specific secondary binding site

Dipeptidyl peptidase 4/CD26 (DP4) is a multifunctional serine protease liberating dipeptide from the N-terminus of (oligo)peptides which can modulate the activity of these peptides. The enzyme is involved in physiological processes such as blood glucose homeostasis and immune response. DP4 substrate specificity is characterized in detail using synthetic dipeptide derivatives. The specificity constant k(cat)/K(m) strongly depends on the amino acid in P?-position for proline, alanine, glycine and serine with 5.0 x 10? M?1 s?1, 1.8 x 10? M?1 s?1, 3.6 x 102 M?1 s?1, 1.1 x 102 M?1 s?1, respectively. By contrast, kinetic investigation of larger peptide substrates yields a different pattern. The specific activity of DP4 for neuropeptide Y (NPY) cleavage comprising a proline in P?-position is the same range as the k(cat)/K(m) values of NPY derivatives containing alanine or serine in P?-position with 4 x 10? M?1 s?1, 9.5 x 10? M?1 s?1 and 2.1 x 10? M?1 s?1, respectively. The proposed existence of an additional binding region outside the catalytic center is supported by measurements of peptide substrates with extended chain length. This 'secondary' binding site interaction depends on the amino acid sequence in P?'-P?'-position. Interactions with this binding site could be specifically blocked for substrates of the GRF/glucagon peptide family. By contrast, substrates not belonging to this peptide family and dipeptide derivative substrates that only bind to the catalytic center of DP4 were not inhibited. This more selective inhibition approach allows, for the first time, to distinguish between substrate families by substrate-discriminating inhibitors.     

Positive Feedback Mechanisms among Local Ca Releases,NCX, and ICaL Ignite Pacemaker Action Potentials

Recent data suggest that cardiac pacemaker cell function is determined by numerous time-, voltage-, and Ca-dependent interactions of cell membrane electrogenic proteins (M-clock) and intracellular Ca cycling proteins (Ca-clock), forming a coupled-clock system. Many aspects of the coupled-clock system, however, remain underexplored. The key players of the system are Ca release channels (ryanodine receptors), generating local Ca releases (LCRs) from sarcoplasmic reticulum, electrogenic Na/Ca exchanger (NCX) current, and L-type Ca current (I_CaL). We combined numerical model simulations with experimental simultaneous recordings of action potentials (APs) and Ca to gain further insight into the complex interactions within the system. Our simulations revealed a positive feedback mechanism, dubbed AP ignition, which accelerates the diastolic depolarization (DD) to reach AP threshold. The ignition phase begins when LCRs begin to occur and the magnitude of inward NCX current begins to increase. The NCX current, together with funny current and T-type Ca current accelerates DD, bringing the membrane potential to I_CaL activation threshold. During the ignition phase, I_CaL-mediated Ca influx generates more LCRs via Ca-induced Ca release that further activates inward NCX current, creating a positive feedback. Simultaneous recordings of membrane potential and confocal Ca images support the model prediction of the positive feedback among LCRs and I_CaL, as diastolic LCRs begin to occur below and continue within the voltage range of I_CaL activation. The ignition phase onset (identified within the fine DD structure) begins when DD starts to notably accelerate (～0.15 V/s) above the recording noise. Moreover, the timing of the ignition onset closely predicted the duration of each AP cycle in the basal state, in the presence of autonomic receptor stimulation, and in response to specific inhibition of either the M-clock or Ca-clock, thus indicating general importance of the new coupling mechanism for regulation of the pacemaker cell cycle duration, and ultimately the heart rate.     

Phylogeny of spiny frogs Nanorana (Anura: Dicroglossidae) supports a Tibetan origin of a Himalayan species group

Recent advances in the understanding of the evolution of the Asian continent challenge the long‐held belief of a faunal immigration into the Himalaya. Spiny frogs of the genus Nanorana are a characteristic faunal group of the Himalaya–Tibet orogen (HTO). We examine the phylogeny of these frogs to explore alternative biogeographic scenarios for their origin in the Greater Himalaya, namely, immigration, South Tibetan origin, strict vicariance. We sequenced 150 Nanorana samples from 62 localities for three mitochondrial (1,524 bp) and three nuclear markers (2,043 bp) and complemented the data with sequence data available from GenBank. We reconstructed a gene tree, phylogenetic networks, and ancestral areas. Based on the nuDNA, we also generated a time‐calibrated species tree. The results revealed two major clades (Nanorana and Quasipaa), which originated in the Lower Miocene from eastern China and subsequently spread into the HTO (Nanorana). Five well‐supported subclades are found within Nanorana: from the East, Central, and Northwest Himalaya, the Tibetan Plateau, and the southeastern Plateau margin. The latter subclade represents the most basal group (subgenus Chaparana), the Plateau group (Nanorana) represents the sister clade to all species of the Greater Himalaya (Paa). We found no evidence for an east–west range expansion of Paa along the Himalaya, nor clear support for a strict vicariance model. Diversification in each of the three Himalayan subclades has probably occurred in distinct areas. Specimens from the NW Himalaya are placed basally relative to the highly diverse Central Himalayan group, while the lineage from the Tibetan Plateau is placed within a more terminal clade. Our data indicate a Tibetan origin of Himalayan Nanorana and support a previous hypothesis, which implies that a significant part of the Himalayan biodiversity results from primary diversification of the species groups in South Tibet before this part of the HTO was uplifted to its recent heights.