Rapid and sensitive NMR method for osmolyte determination

We propose a rapid and sensitive method for osmolyte determination, based on one-dimensional and two-dimensional 1H NMR spectroscopy applied directly on culture of haloalkalophilic Halomonas pantelleriensis and acidothermophilic archaeon Sulfolobus solfataricus, without any extraction procedure. The osmoprotectants hydroxyectoine, ectoine, glutamate, glycine-betaine and treahalose can easily be quantified by integrating the peak areas with respect to an internal standard, and the concentrations evaluated with this method are in excellent agreement with the values previously reported. Furthermore, trace amount of osmoprotectants, often undetectable after extraction procedures, can also be evaluated.     

Extension of the Messapia x dicoccoides linkage map of Triticum turgidum (L.) Thell

A set of recombinant inbred lines (RIL) derived from a cross between the cultivar Messapia of durum wheat (Triticum turgidum var. durum) and the accession MG4343 of T. turgidum var. dicoccoides was analysed to increase the number of assigned markers and the resolution of the previously constructed genetic linkage map. An updated map of the durum wheat genome consisting of 458 loci was constructed. These loci include 261 Restriction Fragment Length Polymorphisms (RFLPs), 91 microsatellites (Simple Sequence Repeats, SSRs), 87 Amplified Fragment Length Polymorphisms (AFLPs), two ribosomal genes, and nine biochemical (seven seed storage proteins and two isozymes) and eight morphological markers. The loci were mapped on all 14 chromosomes of the A and B genomes, and covered a total distance of 3038.4 cM with an average distance of 6.7 cM between adjacent markers. The molecular markers were evenly distributed between the A and the B genomes (240 and 218 markers, respectively). An additional forty loci (8.8%) could not be assigned to a specific linkage group. A fraction (16.4%) of the markers significantly deviated from the expected Mendelian ratios; clusters of loci showing distorted segregation were found on the 1B, 2A, 2B, 3A, 4A, 7A and 7B chromosomes. The genetic lengths of the chromosomes range from 148.8 cM (chromosome 6B) to 318.0 cM (chromosome 2B) and approximately concur with their physical lengths. Chromosome 2B has the largest number of markers (47), while the chromosomes with the fewest markers are 3A and 6B (23). There are two gaps larger than 40 cM on chromosomes 2A and 3B. The durum wheat map was compared with the published maps of bread and durum wheats; the order of most common RFLP and SSR markers on the 14 chromosomes of the A and B genomes were nearly identical. A core-map can be extracted from the high-density Messapia x dicoccoides map and a subset of uniformly distributed markers can be used to detect and map quantitative trait loci.     

Oral feeding with ethinyl estradiol suppresses and treats experimental autoimmune encephalomyelitis in SJL mice and inhibits the recruitment of inflammatory cells into the central nervous system

There is much interest in the possible ameliorating effects of estrogen on various autoimmune diseases. We previously established the protective effects of 17 beta-estradiol (E2) on experimental autoimmune encephalomyelitis (EAE). In the current study we investigated the effectiveness of oral treatment with ethinyl estradiol (EE) on EAE and the mechanisms involved. Ethinyl estradiol is a semisynthetic estrogen compound found in birth control pills, and its chemical structure allows this compound to retain activity when given orally. We found that oral EE, like E2, drastically suppressed EAE induced by proteolipid protein 139-151 peptide when given at initiation of EAE. However, unlike E2, EE reduced clinical severity when given after the onset of clinical signs. Treatment with EE significantly decreased the secretion of proinflammatory cytokines (IFN-gamma, TNF-alpha, and IL-6) by activated T cells as well as the expression of a key matrix metalloproteinase, disease-mediating chemokines/receptors, and IgG2a levels, but increased the expression of TGF-beta 3 in the CNS. The absence of infiltrating lymphocytes together with the suppression of cytokines, matrix metalloproteinase, and chemokines/receptors suggests that EE, like E2, protects mice from EAE by inhibiting the recruitment of T cells and macrophages into the CNS. These results suggest that oral ethinyl estradiol might be a successful candidate as therapy for multiple sclerosis.     

Nitric oxide-related toxicity in cultured astrocytes: effect of Bacopa monniera

There is growing evidence that high concentrations of nitric oxide (NO), generated by activated astrocytes, might be involved in a variety of neurodegenerative diseases, such as Alzheimer's disease, ischemia and epilepsy. It has recently been suggested that glial cells may produce NO under superoxide radical stimulation by enzyme-independent mechanism. This suggests that also natural antioxidants may have therapeutical relevance in neurodegenerative diseases. Studies of Bhattacharya et al. have evidenced that Bacopa monniera (BM) (family Scrophulariaceae), an Ayurvedic medicinal plant clinically used for memory enhancing, epilepsy, insomnia and as a mild sedative, is able to reduce the memory-dysfunction in rat models of Alzheimer's disease, but the molecular mechanisms of this action are yet to be determined. In the present study, we examined the effect of a methanolic extract of BM on toxicity induced by the nitric oxide donor, S-nitroso-N-acetyl-penicillamine (SNAP), in culture of purified rat astrocytes. Our results indicate that, after 18 h of treatment, SNAP induced an increase in the production of reactive species, but did not induce the rupture of cellular membrane. Conversely, this NO donor induced a fragmentation of genomic DNA compared to control astrocytes. The extract of BM inhibited the formation of reactive species and DNA damage in a dose dependent manner. This data supports the traditional use of BM and indicates that this medicinal plant has a therapeutic potential in treatment or prevention of neurological diseases.     

The 35 kDa acid metallophosphatase of the frog Rana esculenta liver: studies on its cellular localization and protein phosphatase activity

The cellular localization of the 35 kDa, low molecular mass acid metallophosphatase (LMW AcPase) from the frog (Rana esculenta) liver and its activity towards P-Ser and P-Tyr phosphorylated peptides were studied. This enzyme was localized to the cytoplasm of hepatocytes but did not appear in other cells of liver tissue (endothelium, macrophages, blood cells). This LMW AcPase does not display activity towards (32)P-phosphorylase a under conditions standard for the enzymes of PPP family. Proteins containing P-Ser: rabbit (32)P-phosphorylasea and phosvitin are hydrolysed only at acidic pH and are poor substrates for this enzyme. The frog AcPase is not inhibited by okadaic acid and F(-) ions, the Ser/Thr protein phosphatase inhibitors. Moreover, the frog enzyme does not cross-react with specific antisera directed against N-terminal fragment of human PP2A and C-terminal conserved fragment of the eukaryotic PP2A catalytic subunits. These results exclude LMW AcPase from belonging to Ser/Thr protein phosphatases: PP1c or PP2Ac. In addition to P-Tyr, this enzyme hydrolyses efficiently at acidic pH P-Tyr phosphorylated peptides (hirudin and gastrin fragments). K(m) value for the hirudin fragment (7.55 +/- 1.59 x 10(-6) M) is 2-3 orders of magnitude lower in comparison with other substrates tested. The enzyme is inhibited competitively by typical inhibitors of protein tyrosine phosphatases (PTPases): sodium orthovanadate, molybdate and tungstate. These results may suggest that the LMW AcPase of frog liver can act as PTPase in vivo. A different cellular localization and different response to inhibition by tetrahedral oxyanions (molybdate, vanadate and tungstate) provide further evidence that LMW AcPase of frog liver is distinct from the mammalian tartrate-resistant acid phosphatases.     

Dissecting planarian central nervous system regeneration by the expression of neural-specific genes

The planarian central nervous system (CNS) can be used as a model for studying neural regeneration in higher organisms. Despite its simple structure, recent studies have shown that the planarian CNS can be divided into several molecular and functional domains defined by the expression of different neural genes. Remarkably, a whole animal, including the molecularly complex CNS, can regenerate from a small piece of the planarian body. In this study, a collection of neural markers has been used to characterize at the molecular level how the planarian CNS is rebuilt. Planarian CNS is composed of an anterior brain and a pair of ventral nerve cords that are distinct and overlapping structures in the head region. During regeneration, 12 neural markers have been classified as early, mid-regeneration and late expression genes depending on when they are upregulated in the regenerative blastema. Interestingly, the results from this study show that the comparison of the expression patterns of different neural genes supports the view that at day one of regeneration, the new brain appears within the blastema, whereas the pre-existing ventral nerve cords remain in the old tissues. Three stages in planarian CNS regeneration are suggested.     

Synthesis,antiprotozoal and antibacterial activity of nitro- and halogeno-substituted benzimidazole derivatives

Two series of benzimidazole derivatives were sythesised. The first one was based on 5,6-dinitrobenzimidazole, the second one comprises 2-thioalkyl- and thioaryl-substituted modified benzimidazoles. Antibacterial and antiprotozoal activity of the newly obtained compounds was studied. Some thioalkyl derivatives showed remarkable activity against nosocomial strains of Stenotrophomonas malthophilia, and an activity comparable to that of metronidazole against gram-positive and gram-negative bacteria. Of the tested compounds, 5,6-dichloro-2-(4-nitrobenzylthio)-benzimidazole showed the most distinct antiprotozoal activity.     

Induction of light emission by luminescent bacteria treated with UV light and chemical mutagens

Intensity of light emission by luminescent bacteria in response to UV irradiation and chemical mutagens was tested. We demonstrated that luminescence of six strains of marine bacteria (belonging to four species: Photobacterium leiognathi, P. phosphoreum, Vibrio fischeri and V. harveyi) is significantly increased by UV irradiation relatively shortly after dilution of cultures. Such a stimulation of luminescence was abolished in cells treated with chloramphenicol 15 min before UV irradiation, indicating that effective gene expression is necessary for UV-mediated induction of light emission. These results suggest that stimulation of luminescence in UV-irradiated bacterial cells may operate independently of the quorum sensing regulation. A significant induction of luminescence was also observed upon treatment of diluted cultures of all investigated strains with chemical mutagens: sodium azide (SA), 2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine x 2HCl (ICR-191), 4-nitro-o-phenylenediamine (NPD), 4-nitroquinolone-N-oxide (NQNO), 2-aminofluorene (2-AF), and benzo[alpha]pyrene. These results support the proposal that genes involved in bioluminescence belong to the SOS regulon. The use of bacterial luminescence systems in assays for detection of mutagenic compounds is discussed in the light of this proposal.     

Adrenomedullin improves cardiac function and prevents renal damage in streptozotocin-induced diabetic rats

Adrenomedullin (AM) is a potent vasodilating peptide and is involved in cardiovascular and renal disease. In the present study, we investigated the role of AM in cardiac and renal function in streptozotocin (STZ)-induced diabetic rats. A single tail-vein injection of adenoviral vectors harboring the human AM gene (Ad.CMV-AM) was administered to the rats 1-wk post-STZ treatment (65 mg/kg iv). Immunoreactive human AM was detected in the plasma and urine of STZ-diabetic rats treated with Ad.CMV-AM. Morphological and chemical examination showed that AM gene delivery significantly reduced glycogen accumulation within the hearts of STZ-diabetic rats. AM gene delivery improved cardiac function compared with STZ-diabetic rats injected with control virus, as observed by decreased left ventricular end-diastolic pressure, increased cardiac output, cardiac index, and heart rate. AM gene transfer significantly increased left ventricular long axis (11.69 +/- 0.46 vs. 10.31 +/- 0.70 mm, n = 10, P < 0.05) and rate of pressure rise and fall (+6,090.1 +/- 597.3 vs. +4,648.5 +/- 807.1 mmHg/s), (-4,902.6 +/- 644.2 vs. -3,915.5 +/- 805.8 mmHg/s, n = 11, P < 0.05). AM also significantly attenuated renal glycogen accumulation and tubular damage in STZ-diabetic rats as well as increased urinary cAMP and cGMP levels, along with increased cardiac cAMP and Akt phosphorylation. We also observed that delivery of the AM gene caused an increase in body weight along with phospho-Akt and membrane-bound GLUT4 levels in skeletal muscle. These results suggest that AM plays a protective role in hyperglycemia-induced glycogen accumulation and cardiac and renal dysfunction via Akt signal transduction pathways.