Regulatory volume decrease in human esophageal epithelial cells

In vivo human esophageal epithelial cells are regularly exposed to hyposmolal stress. This stress, however, only becomes destructive when the surface epithelial cell (barrier) layers are breached and there is contact of the hyposmolal solution with the basolateral cell membranes. The present investigation was designed to examine the effects of hyposmolal stress in the latter circumstance using as a model for human esophageal epithelial cells the noncancer-derived HET-1A cell line. Cell volume and the response to hyposmolal stress in suspensions of HET-1A cells were determined by cell passage through a Coulter Counter Multisizer II. HET-1A cells behaved as osmometers over the range of 280 to 118 mosmol/kg H(2)O with rapid increases in cell volume < or = 15-20% above baseline. Following swelling, the cells exhibited regulatory volume decrease (RVD), restoring baseline volume within 30 min, despite continued hyposmolal stress. With the use of pharmacologic agents and ion substitutions, RVD appeared to result from rapid activation of parallel K(+) and Cl(-) conductance pathways and this was subsequently joined by activation of a KCl cotransporter. Exposure to hyposmolal stress in an acidic environment, pH 6.6, inhibited, but did not abolish, RVD. These data indicate that human esophageal epithelial cells can protect against hyposmolal stress by RVD and that the redundancy in mechanisms may, to some extent, serve as added protection in patients with reflux disease when hyposmolal stress may occur in an acidic environment.     

Wavelet analysis of instantaneous heart rate: a study of autonomic control during thrombolysis

Myocardial infarction (MI) is known to elicit activation of the autonomic nervous system. Reperfusion, induced by thrombolysis, is thus expected to bring about a shift in the balance between the sympathetic and vagal systems, according to the infarct location. In this study, we explored the correlation between reperfusion and the spectral components of heart rate (HR) variability (HRV), which are associated with autonomic cardiac control. We analyzed the HR of patients during thrombolysis: nine anterior wall MI (AW-MI) and eight inferoposterior wall MI (IW-MI). Reperfusion was determined from changes in ST levels and reported pain. Reocclusion was detected in four patients. HRV was analyzed using a modified continuous wavelet transform, which provided time-dependent versions of the typically used low-frequency (LF) and high-frequency (HF) peaks and of their ratio, LF/HF. Marked alterations in at least one of the HRV parameters was found in all 18 reperfusion events. Patterns of HRV, compatible with a shift toward relative sympathetic enhancement, were found in all of the nine reperfusion events in IW-MI patients and in three AW-MI patients. Patterns of HRV compatible with relative vagal enhancement were found in six AW-MI patients (P < 0.001). Significant changes in HRV parameters were also found after reocclusion. Time-dependent spectral analysis of HRV using the wavelet transform was found to be valuable for explaining the patterns of cardiac rate control during reperfusion. In addition, examination of the entire record revealed epochs of markedly diminished HRV in two patients, which we attribute to vagal saturation.     

A 2S albumin-homologous protein from passion fruit seeds inhibits the fungal growth and acidification of the medium by Fusarium oxysporum

Antimicrobial proteins have been isolated from a wide range of plant species. More recently, it has become increasingly clear that these types of proteins play an important role in the protection of plants. In this study, we investigate the presence of defense-related proteins from passion fruit (Passiflora edulis f. flavicarpa) seeds. Initially, seed flour was extracted for 2h (at 4 degrees C) with phosphate buffer, pH 5.5. The precipitate obtained between 0 and 70% relative ammonium sulfate saturation was re-dissolved in distilled water and heated at 80 degrees C for 15 min. The resulting suspension was clarified by centrifugation and the supernatant (F/0-70) was extensively dialyzed. A Sephadex G-50 size exclusion column was employed for further separation of proteins. The fraction with antifungal activity was pooled and submitted to CM-Sepharose cation exchange. Two proteins, named Pf1 and Pf2, were eluted in 0.1 and 0.2M of salt, respectively, and submitted to reverse-phase chromatography in HPLC. This fraction inhibited the growth, in an in vitro assay, of the phytopathogenic fungi Fusarium oxysporum and colletotrichum lindemuthianum and the yeast Saccharomyces cerevisiae and strongly inhibited glucose-stimulated acidification of the medium by F. oxysporum in a dose-dependent manner. The molecular masses of these proteins, referred to now as Pf1-RP and Pf2-RP, were obtained by MALDI-TOF spectrometry and corresponded to 12,088 Da for Pf1-RP and 11,930 Da for Pf2-RP. These proteins were also subjected to automated N-terminal amino acid sequencing. Sequence comparisons for the heavy subunit of Pf2-RP showed the presence of a protein with a high degree of homology to storage 2S albumins.     

Characterization of novel Rab6-interacting proteins involved in endosome-to-TGN transport

Rab6 GTPase regulates intracellular transport at the level of the Golgi complex. Using the yeast two-hybrid screen, we have isolated two clones that specifically interact with the three isoforms of Rab6 present in mammalian cells (Rab6A, A' and B). The cDNAs encode two proteins of 976 and 1120 amino acids (calculated molecular mass of 112 and 128 kDa, respectively) that we named Rab6IP2A and Rab6IP2B (for Rab6 Interacting Protein 2). The two proteins likely correspond to spliced variants of the same gene. Rab6IP2s have no significant homology with other known proteins, including Rab effectors or partners. They are ubiquitously expressed, mostly cytosolic and found in high molecular mass complexes in brain cytosol. We show that Rab6IP2s can be recruited on Golgi membranes in a Rab6:GTP-dependent manner. The overexpression of any form of Rab6IP2 has no detectable effect on the secretory pathway. In contrast, the retrograde transport of the Shiga toxin B subunit between the plasma membrane and the Golgi complex is partly inhibited in cells overexpressing the Rab6-binding domain of Rab6IP2. Our data suggest that Rab6IP2s is involved in the pathway regulated by Rab6A'.     

Selection of cryoprotectants based on their toxic effects on oyster gametes and embryos

Cryopreservation is a valuable tool for aquaculture by providing continuous seed production, regardless of the spawning seasons. This study aimed to select the least toxic among the cryoprotectants dimethyl sulfoxide (Me2SO), propylene glycol (PG), and methanol (MET) based on their toxicological effects on Crassostrea rhizophorae gametes and trochophores. They were exposed for 10, 20, and 30 min to a range of concentrations of those cryoprotectants. The endpoint was EC15-24 h (effective concentration which causes abnormalities in 15% of the population exposed to the cryoprotectants for 24 h), recently determined as the chronic value (the concentration at which chronic effects are first observed) for C. rhizophorae embryonic phases. There were no significant differences (p>0.05) among the exposure times in Me2SO toxic effects to either gametes or trochophores. For MET, the increase in exposure time resulted in higher toxicity for gametes, but not for trochophores, while for PG there was a significant (p>0.05) increase in toxicity with the increase of exposure for trochophores and spermatozoa, but not for oocytes. For gametes, MET was the most toxic among the cryoprotectants, while PG was the most toxic for trochophores.     

Viremic blood donor found by a rapid screening method in a season of high human parvovirus B19 activity in Niterói, Rio de Janeiro, Brazil

Erythrovirus B19 infection is usually benign but may have serious consequences in patients with hemolytic anemia (transient aplastic crisis), immunodeficiency (in whom persistent infection can lead to chronic bone marrow failure with anemia), or who are in the first or second trimester of gestation (spontaneous abortion, hydrops fetalis, and fetal death). Being non-enveloped, B19 resists most inactivation methods and can be transmitted by transfusion. B19 is difficult to cultivate and native virus is usually obtained from viremic blood. As specific antibodies may be absent, and there is no reliable immunological method for antigen detection, hybridization or polymerase chain reaction are needed for detecting viremia. A rapid method, gel hemagglutination (Diamed ID-Parvovirus B19 Antigen Test), can disclose highly viremic donations, whose elimination lessens the viral burden in pooled blood products and may even render them non-infectious. In order to obtain native antigen and to determine the frequency of viremic donors, we applied this test to blood donors in a period of high viral activity in our community. Positive or indeterminate results were re-tested by dot-blot hybridization. We tested 472 donors in 1998 and 831 ones in 1999. One viremic donor was found in 1999. We suggest that in periods of high community viral activity the gel hemagglutination test may be useful in avoiding highly viremic blood being added to plasma pools or directly transfused to patients under risk.     

Synthesis and antitrypanosomal profile of new functionalized 1,3,4-thiadiazole-2-arylhydrazone derivatives, designed as non-mutagenic megazol analogues

In this work we reported the synthesis and the trypanocidal profile of new 1,3,4-thiadiazole-2-arylhydrazone derivatives of nitroimidazole series (4) or phenyl series (5), designed by exploring the molecular hybridization approach between megazol (2) and guanyl hydrazone derivative (3). The evaluation of the activity against bloodstream trypomastigote forms of Trypanosoma cruzi forms lead us to identify a new potent trypamomicide prototype, that is, brazilizone A (4k), which present an IC₅₀/24 h = 5.3 μM.