PoInTree： A Polar and Interactive Phylogenetic Tree

PoInTree （Polar and Interactive Tree） is an application that allows to build, visualize, and customize phylogenetic trees in a polar, interactive, and highly flexible view. It takes as input a FASTA file or multiple alignment formats. Phylogenetic tree calculation is based on a sequence distance method and utilizes the Neighbor Joining （N J） algorithm. It also allows displaying precalculated trees of the major protein families based on Pfam classification. In PoInTree, nodes can be dynamically opened and closed and distances between genes are graphically represented. Tree root can be centered on a selected leaf. Text search mechanism, color-coding and labeling display are integrated. The visualizer can be connected to an Oracle database containing information on sequences and other biological data, helping to guide their interpretation within a given protein family across multiple species. The application is written in Borland Delphi and based on VCL Teechart Pro 6 graphical component （Steema software）.     

Filter binding assay for the geldanamycin-heat shock protein 90 interaction

A filter binding assay to measure affinity of [3H-allyl]17-allylamino geldanamycin ([3H]AAG) for the ATP binding site of the N-terminal domain of human Hsp90alpha (hHsp90alpha9-236) was developed. Diethylaminoethyl cellulose or glass fiber filters impregnated with polyethyleneimine were used to capture the [3H]AAG-Hsp90 complex, and conditions which washed >98% of free [3H]AAG from the filters were developed. The complex formed at a rapid rate (k(on)=2.5 x 10(7)Lmol(-1) x s(-1)) and dissociated with a half-life of 2.3 min (k(off)=5 x 10(-3) x s(-1)). hHsp90alpha9-236 bound to [3H]AAG with a K(d) value of 0.4+/-0.1 microM. [3H]AAG had similar affinities for full-length hHsp90alpha and for hHsp90alpha9-236 variants containing biotinylated N-terminal biotinylation signal sequences and N- or C-terminal His(6) tags. Geldanamycin, ADP, ATP, and radicicol-all known to bind to the ATP domain of Hsp90-competed with [3H]AAG for binding to hHsp90alpha9-236, showing K(d) values in good agreement with reported values.     

Both aromatic and cationic residues contribute to the membrane-lytic and bactericidal activity of eosinophil cationic protein

Eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are proteins of the ribonuclease A (RNase A) superfamily that have developed biological properties related to the function of eosinophils. ECP is a potent cytotoxic molecule, and although the mechanism is still unknown this cytotoxic activity has been associated with its highly cationic character. Using liposome vesicles as a model, we have demonstrated that ECP tends to disrupt preferentially acidic membranes. On the basis of structure analysis, ECP variants modified at basic and hydrophobic residues have been constructed. Changes in the leakage of liposome vesicles by these ECP variants have indicated the role of both aromatic and basic specific amino acids in cellular membrane disruption. This is the case with the two tryptophans at positions 10 and 35, but not phenylalanine 76, and the two arginines 101 and 104. The bactericidal activity of both native ECP and point-mutated variants, tested against Escherichia coli and Staphylococcus aureus, suggests that basic amino acids play, in addition to the effect on the disruption of the cellular membrane, other roles such as specific binding on the surface of the bacteria cell.     

Comparative findings of lymphocytic thyroiditis and thyroid lymphoma

OBJECTIVE: To compare the cytologic features of histologically proven lymphocytic (Hashimoto's) thyroiditis (Hashimoto's thyroitidis) and primary thyroid lymphomas (TL). STUDY DESIGN: Clinical histories, smears (stained with Diff-Quik, Papanicolaou stain or hematoxylin and eosin [HE]) and surgical specimens (HE slides) were reviewed in 25 cases of lymphocytic thyroiditis and 12 of thyroid lymphomas. RESULTS: Surgical specimens of thyroiditis were obtained for other medical reasons: goiter and compressive symptomatology in 21 cases and neoplasms in 4 (2 papillary carcinomas, 1 follicular carcinoma and 1 oncocytic adenoma). Seven cases were primary lymphomas, and 5 were secondary. Histologically there were 6 large B-cell lymphomas, 2 mantle cell lymphomas, 1 Burkitt lymphoma, 2 mucosal-associated lymphoid tissue lymphomas in blastic transformation and 1 of unknown type. Sensitivity for the diagnosis was 67.5% for HT and 92.3% for lymphoma. CONCLUSION: A heterogeneous population of small and large lymphocytes was the most frequent pattern in both diseases. The presence of a monotonous population of large lymphocytes or, more rarely, of small cells indicates a probable TL. Plasma cells favor HT. Other techniques are mandatory for the differentiation of cases with inconclusive diagnoses.     

Protective effect of folic acid against oxidative stress produced in 21-day postpartum rats by maternal-ethanol chronic consumption during pregnancy and lactation period

In this paper we show the protective effect of folic acid on oxidative stress in offspring caused by chronic maternal ethanol consumption during pregnancy and the lactation period. Glutathione reductase (GR) specific activity was assayed in liver and pancreas of offspring and mothers. In the offspring, these tissues were also assayed for markers of oxidative damage to lipids and proteins. The results show that ethanol exposure during pregnancy and lactation increased the specific activity of GR in tissues of the mothers (32-34% increase) as well as in the liver of their progeny (24%). Thiobarbituric acid reactive substances (TBARS) were also increased in the liver and pancreas of 21-day-old rats (37- and 54%, respectively). Alcohol also increased the amount of carbonyl groups in proteins in both tissues. These measures of ethanol-mediated oxidative stress were mitigated when pregnant rats were treated with folic acid concomitantly to ethanol administration. The antioxidant capacity of folic acid seems to be involved in its protective effect. The results obtained in the present work suggest that folic acid may be useful in the prevention of damage and promotion of health of the progeny of ethanol-treated rats.     

Peroxynitrite-mediated mitochondrial dysfunction

Peroxynitrite anion (ONOO(-)) is a potent biological oxidant produced by the near diffusion-limited reaction of superoxide and nitric oxide. Peroxynitrite has been implicated in diverse forms of free radical-induced tissue injury. Experimental evidence showed that exogenous and endogenous peroxynitrite causes alterations of the structure and function of mitochondrial proteins, leading to mitochondrial dysfunction and cellular or organ injury. These data are discussed along with its physiopathological implications.     

Cellular and subcellular localization of endogenous nitric oxide in young and senescent pea plants

The cellular and subcellular localization of endogenous nitric oxide (NO.) in leaves from young and senescent pea (Pisum sativum) plants was studied. Confocal laser scanning microscopy analysis of pea leaf sections with the fluorescent probe 4,5-diaminofluorescein diacetate revealed that endogenous NO. was mainly present in vascular tissues (xylem and phloem). Green fluorescence spots were also detected in the epidermal cells, palisade and spongy mesophyll cells, and guard cells. In senescent leaves, NO. generation was clearly reduced in the vascular tissues. At the subcellular level, by electron paramagnetic resonance spectroscopy with the spin trap Fe(MGD)(2) and fluorometric analysis with 4,5-diaminofluorescein diacetate, NO. was found to be an endogenous metabolite of peroxisomes. The characteristic three-line electron paramagnetic resonance spectrum of NO., with g = 2.05 and a(N) = 12.8 G, was detected in peroxisomes. By fluorometry, NO. was also found in these organelles, and the level measured of NO. was linearly dependent on the amount of peroxisomal protein. The enzymatic production of NO. from l-Arg (nitric oxide synthase [NOS]-like activity) was measured by ozone chemiluminiscence. The specific activity of peroxisomal NOS was 4.9 nmol NO. mg(-1) protein min(-1); was strictly dependent on NADPH, calmodulin, and BH(4); and required calcium. In senescent pea leaves, the NOS-like activity of peroxisomes was down-regulated by 72%. It is proposed that peroxisomal NO. could be involved in the process of senescence of pea leaves.