Potamogeton×exilis (P. alpinus×P. natans), a new hybrid pondweed from Finland

A Potamogeton hybrid recently discovered in Finland was proven to be a new taxon, which has a different parentage than the most similar P.×vepsicus (=P. natans×praelongus) described from Russia. Based on molecular and morphological investigation, the new hybrid was identified as P. alpinus×natans and is here described as P.×exilis nothosp. nov. The hybrid is known only from several nearby localities in a single river system. All discovered colonies almost certainly originate from a single hybridization event that occurred in the upper part of the river system. Other localities downstream have been established through fragmentation and dispersal of stems and rhizomes of this sterile hybrid. A detailed morphological comparison of the new hybrid with similar hybrids and species is provided.     

Synthesis, characterization and assessment of the cytotoxic properties of cis and trans-[Pd(L)(2)Cl(2)] complexes involving 6-benzylamino-9-isopropylpurine derivatives

A series of square-planar Pd(II) complexes of the composition cis-[Pd(L(n))(2)Cl(2)] {L(1)=2-chloro-6-benzylamino-9-isopropylpurine (1), L(2)=2-chloro-6-[(4-methoxybenzyl)amino]-9-isopropylpurine (2), L(3)=2-chloro-6-[(2-methoxybenzyl)amino]-9-isopropylpurine (3) and 2-[(chloropropyl)amino]-6-benzylamino-9-isopropylpurine (6)} has been synthesized by the reaction of PdCl(2) with L(n) in a 1:2 molar ratio. In contrast, the same reaction followed by recrystallization of the product from N,N'-dimethylformamide (DMF) leads to trans-[Pd(L(n))(2)Cl(2)] x nDMF {L(3), n=0 (4), n=1(4( *)DMF); L(4)=2-chloro-6-[(2,3-dimethoxybenzyl)-amino]-9-isopropylpurine, n=0 (5), n=1.5 (5( *)DMF). The compounds have been characterized by elemental analyses, conductivity measurements, electrospray mass spectra in the positive ion mode (ES+MS), FTIR, (1)H and (13)C NMR spectra, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). Moreover, the complexes 2 and 6 have been also investigated by (15)N NMR spectroscopy. The molecular structures of L(5), {(H(2+)L(5))(Cl(-))(2)} x H(2)O, i.e. the protonated form of L(5), trans-[Pd(L(3))(2)Cl(2)] (4) and trans-[Pd(L(4))(2)Cl(2)] (5) have been determined by single crystal X-ray analysis. NMR data and X-ray structures revealed that the organic molecules are coordinated to Pd via N7 atom of a purine moiety. All the complexes and the corresponding ligands have been tested in vitro for their cytotoxicity against four human cancer cell lines: breast adenocarcinoma (MCF7), malignant melanoma (G361), chronic myelogenous leukaemia (K562) and osteogenic sarcoma (HOS). Promising in vitro cytotoxic effect has been found for cis-[Pd(L(2))(2)Cl(2)] (2), having the IC(50) values of 12, 10, 25, and 14 microM against MCF7, G361, K562, and HOS, respectively, and for trans-[Pd(L(3))(2)Cl(2)].DMF (4) with the IC(50) value of 15 microM against G361.     

Rapid detection of CAA/CAG repeat polymorphism in the AIB1 gene using DHPLC

Denaturing high-performance liquid chromatography (DHPLC) has been used for rapid and accurate DNA mutation analysis; to extend the DNA fragment lengths analysis. Recently, polymorphism in polyglutamine-coding region of Amplified In Breast cancer gene 1 (AIB1) was analyzed as an independent genetic risk factor influencing breast cancer onset in carriers of mutation in breast cancer predisposing gene 1 (BRCA1). We have implemented efficient, cost-effective and rapid method for analysis of the AIB1 polyglutamine repeat polymorphism based on DHPLC analysis (WAVE system) of unlabeled PCR products. This strategy can be useful for genotyping of other trinucleotide repeat polymorphisms using DHPLC in medium/high throughput settings.     

An analysis of spontaneous behavior following acute MDMA treatment in male and female rats

3,4-methylenedioxymethamphetamine (MDMA, ecstasy) is a widely abused drug that impairs behavioral, emotional and cognitive functions in humans and animals. The aim of this study was to evaluate MDMA effects on the spontaneous behavioral repertoire of rats with a focus on the gender differences. MDMA was given subcutaneously in a single dose of 2.5, 5 and 10 mg/kg and the spontaneous behavior of male and female rats was studied using the open field test. Behavioral patterns (locomotion, rearing, floor-sniffing, air-sniffing, grooming, immobility and stereotypy) were registered in two sessions - 30 and 60 min following MDMA administration; each session lasting 5 min. We found that MDMA totally disrupted the structure and timing of spontaneous behavioral patterns in both genders; no evident differences were measured between either of the sessions. MDMA irrespective of the dosage produced hyperlocomotion, excessive floor-sniffing and almost absolute suppression of grooming and immobility. A biphasic effect of MDMA was found in rearing. Gender differences were present namely in rearing and sniffing stereotypy. This study also confirms that behavioral experiments should focus on more behavioral elements than only on e.g. locomotion and that the observer-based approach still gives the most reliable results.     

Stepwise reduction of functional spinal structures increase range of motion and change lordosis angle

Many investigators have performed studies on specific defect situations or determined the contribution on isolated structures. Investigating the contribution of functional structures requires obtaining the kinematic response directly on spinal segments. The purpose of this study was to quantify the function of anatomical components on lumbar segments for different loading magnitudes. Eight spinal segments (L4-5) with a median age of 52 years (ranging from 38 to 59 years) and a low degree of disc degeneration were utilized for the in vitro testing. Specimens were mounted in a custom-built spine tester and loaded with pure moments (1-10 N m) to move within three anatomical planes at a loading rate of 1.0 degrees /s. Anatomy was successively reduced by: ligaments, facet capsules, joints and nucleus. Data were evaluated for range of motion, neutral zone and lordosis angle. Transection of posterior ligaments predominantly increased specimen flexion for all bending moments applied. Supraspinous ligament also indicated to resist in extension slightly, whereas the facet capsules did not. Facet joints contributed to axial rotation, but not in lateral bending. The anterior longitudinal ligament was found to slightly resist in axial rotation, but strongly in extension. Nucleotomy caused largest increase of all movements. The unloaded posture of the specimens changed after ligament dissection, indicating ligament pretension. The region of lumbar spine is interesting for finite element (FE) simulation due to the high evidence of disc degeneration and injuries. This study may help to understand the function of specific anatomical structures and assists in FE model calibration. We suggest to start a calibration procedure for such models with the smallest functional structure (annulus) and to cumulatively add further structures.     

IrAE: an asparaginyl endopeptidase (legumain) in the gut of the hard tick Ixodes ricinus

Ticks are ectoparasitic blood-feeders and important vectors for pathogens including arboviruses, rickettsiae, spirochetes and protozoa. As obligate blood-feeders, one possible strategy to retard disease transmission is disruption of the parasite's ability to digest host proteins. However, the constituent peptidases in the parasite gut and their potential interplay in the digestion of the blood meal are poorly understood. We have characterised a novel asparaginyl endopeptidase (legumain) from the hard tick Ixodes ricinus (termed IrAE), which we believe is the first such characterisation of a clan CD family C13 cysteine peptidase (protease) in arthropods. By RT-PCR of different tissues, IrAE mRNA was only expressed in the tick gut. Indirect immunofluorescence and EM localised IrAE in the digestive vesicles of gut cells and within the peritrophic matrix. IrAE was functionally expressed in Pichia pastoris and reacted with a specific peptidyl fluorogenic substrate, and acyloxymethyl ketone and aza-asparagine Michael acceptor inhibitors. IrAE activity was unstable at pH > or = 6.0 and was shown to have a strict specificity for asparagine at P1 using a positional scanning synthetic combinatorial library. The enzyme hydrolyzed protein substrates with a pH optimum of 4.5, consistent with the pH of gut cell digestive vesicles. Thus, IrAE cleaved the major protein of the blood meal, hemoglobin, to a predominant peptide of 4kDa. Also, IrAE trans-processed and activated the zymogen form of Schistosoma mansoni cathepsin B1 -- an enzyme contributing to hemoglobin digestion in the gut of that bloodfluke. The possible functions of IrAE in the gut digestive processes of I. ricinus are compared with those suggested for other hematophagous parasites.     

Glycosidic juvenogens: derivatives bearing alpha,beta-unsaturated ester functionalities

A series of the protected alkyl glycosides 5a/5b-12a/12b was synthesized from the parent isomeric alcohols (insect juvenile hormone bioanalogs; juvenoids), 4-[4'-(2'-hydroxycyclohexyl)methylphenoxy]-3-methyl-but-2-enoic acid ethyl ester (1a/1b-4a/4b; racemic structures) and (1a-4a; enantiopure structures). Cadmium carbonate was used as a promoter of this Koenigs-Knorr reaction, and the products were obtained in 82-92% yields. Deprotection of the carbohydrate functionality of 5a/5b-12a/12b was carefully performed using ethanolysis in the presence of zinc acetate, due to the presence of another ester functionality in the aglycone part of the molecule of protected alkyl glycosides. Resulting alkyl glycosides 13a/13b-20a/20b (diastereoisomeric mixtures) and 13a-20a (enantiopure compounds), biochemically activated hormonogenic compounds (juvenogens), were obtained in 82-93% yields. Finally, chiral HPLC separation of the diastereoisomeric mixtures of alkyl glycosides was applied to get sufficient quantities of the respective enantiomers 13b-20b of the alkyl glycosides for their structure elucidation and (13)C chemical shift assignment by (1)H and (13)C NMR spectroscopy. Partial introductory entomological screening tests of the target alkyl glycosides 13a/13b-20a/20b were performed on the red firebug (Pyrrhocoris apterus). The results of this biological testing clearly demonstrated the time-extended effect of several juvenogens on P. apterus due to their biochemical activation, i.e., hydrolysis of the juvenogen molecule, which results in liberation of the biologically active juvenoid in the insect organism.     

Increased level of cytokines and matrix metalloproteinases in osteoarthritic subchondral bone

OBJECTIVE: The aim of this study was to investigate the expression of several cytokines, matrix metalloproteinases (MMPs), and tissue inhibitor of matrix metalloproteinases (TIMP)-1 in osteoarthritis (OA) and control sera and different joint tissues. METHODS: Serum, synovial fluid, cartilage, synovial and subchondral bone tissues were examined in OA and control subjects. The protein level of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1alpha, IL-8, IL-10 and MMP-2, MMP-3, MMP-9, and TIMP-1 were measured by immunoanalysis. RESULTS: Serum levels of TNF-alpha, MMP-3 and -9 were significantly higher in OA patients than in controls. Conversely, serum IL-10 was decreased in OA patients. CRP was elevated when compared to healthy controls and decreased significantly 6 months after the surgery. In contrast to control samples, OA cartilage and synovium revealed significantly higher MMP-2, -3, -9 and IL-10. IL-1alpha was significantly higher in OA cartilage and IL-8 in OA synovium. Interestingly, MMP-3, -9, TIMP-1 and all tested cytokines were up-regulated in OA subchondral bone. DISCUSSION: This study demonstrates pro-inflammatory condition of OA pathology and supports the idea that vascularized subchondral region may increase the synthesis of cytokines and MMPs leading to degradation of adjacent cartilage.     

Acetylcholinesterases--the structural similarities and differences

Acetylcholinesterase (AChE) is a widely spread enzyme playing a very important role in nerve signal transmission. As AChE controls key processes, its inhibition leads to the very fast death of an organism, including humans. However, when this feature is to be used for killing of unwanted organisms (i.e. mosquitoes), one is faced with the question - how much do AChEs differ between species and what are the differences? Here, a theoretical point of view was utilized to identify the structural basis for such differences. The various primary and tertiary alignments show that AChEs are very evolutionary conserved enzymes and this fact could lead to difficulties, for example, in the search for inhibitors specific for a particular species.