Identification of hepatotoxin-producing cyanobacteria by DNA-chip

We developed a new tool to detect and identify hepatotoxin-producing cyanobacteria of the genera Anabaena , Microcystis , Planktothrix , Nostoc and Nodularia . Genus-specific probe pairs were designed for the detection of the microcystin ( mcyE ) and nodularin synthetase genes ( ndaF ) of these five genera to be used with a DNA-chip. The method couples a ligation detection reaction, in which the polymerase chain reaction (PCR)-amplified mcyE / ndaF genes are recognized by the probe pairs, with a hybridization on a universal microarray. All the probe pairs specifically detected the corresponding mcyE / ndaF gene sequences when DNA from the microcystin- or nodularin-producing cyanobacterial strains were used as template in the PCR. Furthermore, the strict specificity of detection enabled identification of the potential hepatotoxin producers. Detection of the genes was very sensitive; only 1–5 fmol of the PCR product were needed to produce signal intensities that exceeded the set background threshold level. The genus-specific probe pairs also reliably detected potential microcystin producers in DNA extracted from six lake and four brackish water samples. In lake samples, the same microcystin producers were identified with quantitative real-time PCR analysis. The specificity, sensitivity and ability of the DNA-chip in simultaneously detecting all the main hepatotoxin producers make this method suitable for high-throughput analysis and monitoring of environmental samples.     

Complete mitochondrial genome sequence of the Tyrolean Iceman

The Tyrolean Iceman was a witness to the Neolithic-Copper Age transition in Central Europe 5350-5100 years ago, and his mummified corpse was recovered from an Alpine glacier on the Austro-Italian border in 1991 [1]. Using a mixed sequencing procedure based on PCR amplification and 454 sequencing of pooled amplification products, we have retrieved the first complete mitochondrial-genome sequence of a prehistoric European. We have then compared it with 115 related extant lineages from mitochondrial haplogroup K. We found that the Iceman belonged to a branch of mitochondrial haplogroup K1 that has not yet been identified in modern European populations. This is the oldest complete Homo sapiens mtDNA genome generated to date. The results point to the potential significance of complete-ancient-mtDNA studies in addressing questions concerning the genetic history of human populations that the phylogeography of modern lineages is unable to tackle.     

Further studies on the pharmacological features of the nociceptin/orphanin FQ receptor ligand ZP120

ZP120 is a nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) ligand. In previous studies, the effects of ZP120 were found to be sensitive to J-113397 in mouse tissues while resistant to UFP-101 in rat tissues. The aim of this study was to further investigate the ZP120 pharmacological profile using mouse and rat preparations, J-113397 and UFP-101, as well as NOP receptor knockout (NOP(-/-)) mice. Electrically stimulated mouse and rat vas deferens were used to characterize the pharmacology of ZP120 in vitro. For in vivo studies the tail-withdrawal assay was performed in wild type (NOP(+/+)) and NOP knockout (NOP(-/-)) mice. In the mouse and rat vas deferens ZP120 mimicked the effects of N/OFQ showing higher potency but lower maximal effects. In both preparations, J-113397 antagonized N/OFQ and ZP120 effects showing similar pK(B) values ( approximately 7.8). UFP-101 antagonized the actions of N/OFQ (pK(B) values approximately 7.3) but did not modify the effects of ZP120. The inhibitory effects of N/OFQ and ZP120 were no longer evident in vas deferens tissues taken from NOP(-/-) mice. In NOP(+/+) mice subjected to the tail-withdrawal assay, ZP120 (1 nmol) mimicked the pronociceptive action of N/OFQ (10 nmol), producing longer lasting effects. The effects of both peptides were absent in NOP(-/-) animals. The NOP receptor ligand ZP120 is a high potency NOP selective partial agonist able to evoke long-lasting effects; its diverse antagonist sensitivity in comparison with N/OFQ may derive from different modality of binding to the NOP receptor.     

EPR studies of the Mo-enzyme aldehyde oxidoreductase from Desulfovibrio gigas: An application of the Bloch-Wangsness-Redfield theory to a system containing weakly-coupled paramagnetic redox centers with different relaxation rates

Electron transfer proteins and redox enzymes containing paramagnetic redox centers with different relaxation rates are widespread in nature. Despite both the long distances and chemical paths connecting these centers, they can present weak magnetic couplings produced by spin-spin interactions such as dipolar and isotropic exchange. We present here a theoretical model based on the Bloch-Wangsness-Redfield theory to analyze the dependence with temperature of EPR spectra of interacting pairs of spin 1/2 centers having different relaxation rates, as is the case of the molybdenum-containing enzyme aldehyde oxidoreductase from Desulfovibrio gigas. We analyze the changes of the EPR spectra of the slow relaxing center (Mo(V)) induced by the faster relaxing center (FeS center). At high temperatures, when the relaxation time T₁ of the fast relaxing center is very short, the magnetic coupling between centers is averaged to zero. Conversely, at low temperatures when T₁ is longer, no modulation of the coupling between metal centers can be detected.     

Antioxidant treatment reduces matrix metalloproteinase-2-induced vascular changes in renovascular hypertension

Mounting evidence indicates that structural and functional vascular changes associated with two-kidney, one-clip (2K-1C) hypertension result, at least in part, from altered activity of matrix metalloproteinases (MMPs). Because MMPs are upregulated by increased formation of reactive oxygen species (ROS), we hypothesized that antioxidant approaches could attenuate the increases in MMP-2 expression/activity and the vascular dysfunction and remodeling associated with 2K-1C hypertension. Sham-operated or 2K-1C hypertensive rats were treated with tempol 18 mg/kg/day or apocyanin 25 mg/kg/day (or vehicle). Systolic blood pressure was monitored weekly. After 8 weeks of treatment, aortic rings were isolated to assess endothelium-dependent and -independent relaxation. Quantitative morphometry of structural changes in the aortic wall was studied in hematoxylin/eosin sections. Aortic and systemic ROS levels were measured using dihydroethidine and thiobarbituric acid-reactive substances, respectively. Aortic MMP-2 levels and activity were determined by gelatin and in situ zymography, fluorimetry, and immunohistochemistry. Tempol and apocyanin attenuated 2K-1C hypertension (181 ± 20.8 and 192 ± 17.6 mm Hg, respectively, versus 213 ± 18 mm Hg in hypertensive controls; both p < 0.05) and prevented the reduction in endothelium-dependent vasorelaxation found in 2K-1C rats. Tempol, but not apocyanin (p > 0.05), prevented the vascular remodeling found in 2K-1C rats (all p < 0.01). Tempol was more effective than apocyanin in attenuating hypertension-induced increases in oxidative stress (both p < 0.05), MMP-2 levels, and MMP-2 activity in hypertensive rats (all p < 0.05). Our results suggest that antioxidant approaches decrease MMP-2 upregulation and attenuate the vascular dysfunction and remodeling during 2K-1C hypertension.     

Spindle assembly checkpoint genes reveal distinct as well as overlapping expression that implicates MDF-2/Mad2 in postembryonic seam cell proliferation in Caenorhabditis elegans

Background

The spindle assembly checkpoint (SAC) delays anaphase onset by inhibiting the activity of the anaphase promoting complex/cyclosome (APC/C) until all of the kinetochores have properly attached to the spindle. The importance of SAC genes for genome stability is well established; however, the roles these genes play, during postembryonic development of a multicellular organism, remain largely unexplored.

Results

We have used GFP fusions of 5' upstream intergenic regulatory sequences to assay spatiotemporal expression patterns of eight conserved genes implicated in the spindle assembly checkpoint function in Caenorhabditis elegans. We have shown that regulatory sequences for all of the SAC genes drive ubiquitous GFP expression during early embryonic development. However, postembryonic spatial analysis revealed distinct, tissue-specific expression of SAC genes with striking co-expression in seam cells, as well as in the gut. Additionally, we show that the absence of MDF-2/Mad2 (one of the checkpoint genes) leads to aberrant number and alignment of seam cell nuclei, defects mainly attributed to abnormal postembryonic cell proliferation. Furthermore, we show that these defects are completely rescued by fzy-1(h1983)/CDC20, suggesting that regulation of the APC/C^CDC20 by the SAC component MDF-2 is important for proper postembryonic cell proliferation.

Conclusion

Our results indicate that SAC genes display different tissue-specific expression patterns during postembryonic development in C. elegans with significant co-expression in hypodermal seam cells and gut cells, suggesting that these genes have distinct as well as overlapping roles in postembryonic development that may or may not be related to their established roles in mitosis. Furthermore, we provide evidence, by monitoring seam cell lineage, that one of the checkpoint genes is required for proper postembryonic cell proliferation. Importantly, our research provides the first evidence that postembryonic cell division is more sensitive to SAC loss, in particular MDF-2 loss, than embryonic cell division.     

Synthesis and anticonvulsant activity of a class of 2-amino 3-hydroxypropanamide and 2-aminoacetamide derivatives

Several studies have demonstrated that N-substituted amino acid derivatives exhibit weak anticonvulsant activities in vivo. In the present study, a series of amides of aminoacids structurally related to aminoacetamide have been synthesised and investigated for anticonvulsant activity. Among the molecules investigated, those containing a bicyclic (tetralinyl, indanyl) group linked to the aminoacetamide chain (40, 47 and 59) were among the most active as anticonvulsants (ED50 > 10, <100 mg/kg after oral administration) against tonic seizures in the mouse maximal electroshock, bicuculline and picrotoxin tests at doses devoid of neurotoxic activity. Altogether, these results suggest the described compounds as a class of orally available anticonvulsants. The ability of these compounds to partially block veratridine-induced aspartate efflux from rat cortical synaptosomes suggests that their anticonvulsant activity may be only partly the consequence of an interaction with neuronal voltage-dependent sodium channels. Some of the most potent compounds appear worthy of a further investigation aimed at assessing their anticonvulsant activity in other models and at elucidating the underlying mechanism of action.     

Structure-activity studies on neuropeptide S: identification of the amino acid residues crucial for receptor activation

Neuropeptide S (NPS) has been recently recognized as the endogenous ligand for the previous orphan G-protein-coupled receptor GPR154, now referred to as the NPS receptor (NPSR). The NPS-NPSR receptor system regulates important biological functions such as sleeping/wakening, locomotion, anxiety, and food intake. To collect information on the mechanisms of interaction between NPS and its receptor, a classical structure-activity relationship study was performed. Human (h) NPS derivatives obtained by Ala and d-scan and N- and C-terminal truncation were assessed for their ability to stimulate calcium release in HEK293 cells expressing the human recombinant NPSR. The results of this study indicate that (i) the effect of hNPS is mimicked by the fragment hNPS-(1-10); (ii) Phe(2), Arg(3), and Asn(4) are crucial for biological activity; (iii) the sequence Thr(8)-Gly(9)-Met(10) is important for receptor activation, although with non-stringent chemical requirements; and (iv) the sequence Val(6)-Gly(7) acts as a hinge region between the two above-mentioned domains. However, the stimulatory effect of hNPS given intracerebroventricularly on mouse locomotor activity was not fully mimicked by hNPS-(1-10), suggesting that the C-terminal region of the peptide maintains importance for in vivo activity. In conclusion, this study identified the amino acid residues of this peptide most important for receptor activation.     

Hierarchical clustering using the arithmetic-harmonic cut: complexity and experiments

Clustering, particularly hierarchical clustering, is an important method for understanding and analysing data across a wide variety of knowledge domains with notable utility in systems where the data can be classified in an evolutionary context. This paper introduces a new hierarchical clustering problem defined by a novel objective function we call the arithmetic-harmonic cut. We show that the problem of finding such a cut is NP-hard and APX-hard but is fixed-parameter tractable, which indicates that although the problem is unlikely to have a polynomial time algorithm (even for approximation), exact parameterized and local search based techniques may produce workable algorithms. To this end, we implement a memetic algorithm for the problem and demonstrate the effectiveness of the arithmetic-harmonic cut on a number of datasets including a cancer type dataset and a corona virus dataset. We show favorable performance compared to currently used hierarchical clustering techniques such as k-Means, Graclus and Normalized-Cut. The arithmetic-harmonic cut metric overcoming difficulties other hierarchical methods have in representing both intercluster differences and intracluster similarities.