Apoptotic rate: a new indicator for the quantification of the incidence of apoptosis in cell cultures

BACKGROUND: Late apoptotic cells divide into apoptotic bodies and are missed by current detection methods. This results in an artificially low apoptotic index (AI). METHODS: This study proposes a flow cytometry-based ratiometric method that uses an internal reference standard of microbeads combined with fluorescein-annexin V binding and 7-aminoactinomycin D to enumerate viable, necrotic, and early and late apoptotic cells within specific subsets of a heterogeneous culture. RESULTS: In the absence of cell growth, the number of apoptotic cells that undergo fragmentation into apoptotic bodies in culture can also be determined accurately by this method. This information can then be used to obtain the apoptotic rate (AR), a new indicator of apoptosis that calculates the proportion of cells that have undergone apoptosis with respect to the total number of seeded cells. The main limitation of the method is that the AR is only suitable for the study of apoptosis in noncycling cells. CONCLUSIONS: This study reveals the superiority of the proposed method over the widely used Nicoletti method and current annexin-V binding methods. The AI did not reflect the true incidence of lymphocyte apoptosis, neither in response to lectins or phorbol esters, nor to serum deprivation. AR was more sensitive than AI, detecting apoptosis at lower concentrations of cell death inducers in all the subsets studied.     

Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators

Hepatocyte gap junction proteins, connexins (Cxs) 26 and 32, are downregulated during obstructive cholestasis (OC) and lipopolysaccharide hepatocellular cholestasis (LPS-HC). We investigated rat hepatic Cxs during ethynylestradiol hepatocellular cholestasis (EE-HC) and choledochocaval fistula (CCF) and compared them with OC and LPS-HC. Levels (immunoblotting) and cellular distribution (immunofluorescence) of Cx26, -32, and -43, as well as macrophage infiltration, were studied in livers of rats under each condition. Cx26 and -32 were reduced in LPS-HC, OC, and CCF. However, in EE-HC, Cx26 did not change and Cx32 was increased. Prominent inflammation occurred in LPS-HC, OC, and CCF, which was associated with increased levels of Cx43 in LPS-HC and OC but not CCF. No inflammation nor changes in Cx43 levels occurred during EE-HC. In cultured hepatocytes, dye coupling was reduced by tumor necrosis factor-alpha and interleukins-1beta and -6, whereas reduction induced by LPS required coculture with Kupffer cells. Thus hepatocyte gap junctions are downregulated in forms of cholestasis associated with inflammation, and reduced intercellular communication might be induced in part by proinflammatory mediators.     

Distribution of intrachromosomal telomeric sequences (ITS) on Macaca fascicularis (Primates) chromosomes and their implication for chromosome evolution

The intrachromosomal location of the telomeric sequence in the crab-eating macaque, Macaca fascicularis (F. Cercopithecidae, Catarrhini) has been analysed by fluorescent in situ hybridisation with a long synthetic (TTAGGG)(n) probe. A total of 237 metaphases was analysed. As expected, all telomeres hybridised with the probe and 90 intrachromosomal loci with different hybridisation frequencies were also detected. The chromosomal location of interstitial telomeric sequences in M. fascicularis and in Homo sapiens was then compared, 37 sites (41.11%) being found to be conserved. Some of these sequences can be derived from rearrangements, such as inversions (MFA13q23) or fusions (MFA2p13 and MFA13p12), that have taken place during karyotype evolution.     

Characterization of salt stress-enhanced phosphoenolpyruvate carboxylase kinase activity in leaves of <Emphasis Type="Italic">Sorghum</Emphasis><Emphasis Type="Italic">vulgare</Emphasis>: independence from osmotic stress,involvement of ion toxicity and significance of dark phosphorylation

C(4) phosphoenolpyruvate carboxylase (PEPCase: EC 4.1.1.31) is subjected to in vivo regulatory phosphorylation by a light up-regulated, calcium-independent protein kinase. Salt stress greatly enhanced phosphoenolpyruvate carboxylase-kinase (PEPCase-k) activity in leaves of Sorghum. The increase in PEPCase-k anticipated the time course of proline accumulation thereby suggesting that water stress was not involved in the kinase response to salt. Moreover, osmotic stress seemed not to be the main factor implicated, as demonstrated by the lack of effect when water availability was restricted by mannitol. In contrast, LiCl (at a concentration of 10 mM in short-term treatment of both excised leaves and whole plants) mimicked the effects of 172 mM NaCl salt-acclimation, indicating that the rise in PEPCase-k activity resulted primarily from the ionic stress. Both NaCl and LiCl treatments increased the activity of a Ca(2+)-independent, 35 kDa kinase, as demonstrated by an in-gel phosphorylation experiment. Short-term treatment of excised leaves with NaCl or LiCl partially reproduces the effects of whole plant treatments. Finally, salinization also increased PEPCase-k activity and the phosphorylation state of PEPCase in darkened Sorghum leaves. This fact, together with increased malate production during the dark period, suggests a shift towards mixed C(4) and crassulacean acid metabolism types of photosynthesis in response to salt stress.     

Temporal localization of porcine reproductive and respiratory syndrome virus in reproductive tissues of experimentally infected boars

Porcine reproductive and respiratory syndrome virus (PRRSV) has been reported to be shed in the semen of infected boars. To determine whether the reproductive tissues could be a persistent source of virus and the possible origin of PRRSV found in semen of infected boars, 20 PRRSV-seronegative boars were intranasally inoculated with 5 x 10(6) median tissue culture infective doses (TCID50) of PRRSV and necropsied at different times post-inoculation (p.i.) from Day 2 to Day 37 p.i. Blood samples were collected before experimental inoculation, at necropsy and at different times p.i. At necropsy, epididymal semen and reproductive tissues were collected and the presence of the virus determined by virus isolation. The infection of the boars was demonstrated by the isolation of the virus from the sera of all inoculated boars and by seroconversion. PRRSV was detected in serum samples from Day 2 to Day 23 p.i., although the viremic period was largely dependent on the individual response to infection. Viral replication was proven within different reproductive tissues from Day 2 to Day 23 p.i., being most consistently found in the epididymus. In addition, PRRSV was isolated in semen from Day 4 to Day 10 p.i. The correlation of a diminished viremia and the inability to isolate PRRSV from semen or reproductive tissues may be due to one of two possibilities. First, viremia is responsible for most of the virus isolated from reproductive tissues due to the movement of PRRSV-infected cells out of the blood and into the tissues. Second, viremia may initially seed the reproductive tissues with PRRSV, and then the virus is produced into the reproductive tract and shed into semen at low levels.     

Oral hydratation with a low osmolality solution in dehydrated children with diarrheic diseases: controlled clinical trial

A clinical trial was conducted to compare the efficacy of a low-osmolarity solution (245 mOsm/L), and a standard oral rehydration solution (ORS) recommended by WHO for children dehydrated by diarrhea. Group 1 (69 children) received WHO/ORS (311 mOsml/L) and group 2 (71 children) received a low-osmolarity solution (245 mOsm/L). Rehydration was successful in 88.4% in group 1 and 92.9% in group 2 (p = 0.35). Rehydration was completed in 5.2 h (SD +/- 1.8) in group 1 and 5.5 (SD +/- 1.7) in group 2 (p = 0.31). Stool output was 6.3 g/kg/h (SD +/- 5.0) in group 1 and 5.6 g/kg/h (SD +/- 5.1) in group 2 (p = 0.94). Sodium at rehydration-completion was 139.3 mEq/L (SD +/- 7.1) in group 1 and 136.7 mEq/L (SD +/- 4.3) in group 2 (p = 0.014). Group 1 was under observation for 21 hours (SD +/- 5.7) and group 2, for 22 hours (SD +/- 5.6). Stool output in group 1 was 5.2 g/kg/h (SD 4.1) and 4.2 gr./kg/h (SD +/- 4.1) in group 2 (p = 0.16). In group 1, 23.1% required intravenous solutions and 9.8% in group 2 (p = 0.03). In treating dehydrated children, the low-osmolarity solution diminished the need for intravenous solutions, corrected most plasmatic sodium disorders, and produced no-risk of developing hyponatremia.     

Neurological effects of American trypanosomyiasis: clinical aspects

Trypanosoma cruzi, causative agent of Chagas disease, affects not only cardiac and intestinal structures but also neurological structures. A high prevalence of T. cruzi infection occurs in Colombia, prompting the present study. First, a qualitative metaanalysis was undertaken using the PubMed database, the electronic internet engine Altavista, Colombian journals indexed by Colciencias, and three relevant textbooks. The following key words were used: Trypanosoma, Chagas disease, nervous system, spinal cord, central nervous system, peripheral nervous system, neuromuscular junction, autonomic nervous system, muscle, muscle disorders, neuromuscular disease, neuromuscular disorders, synapticopathies and dysautonomia. The documents analyzed numbered 116 and included original papers, reviews, case reports, editorials, brief communications, conferences and book chapters. At minimum, each document included data involving ELISA testing, indirect immunofluorescense, or parasitemia levels in the clinical, serological or histopathological studies. Polymerase chain reaction (PCR) studies were not included because of the recent introduction of PCR as a confirmatory technique for Chagas disease in Colombia. Chagas disease affects the central, the peripheral and the autonomic nervous system in humans, although its effects on the antonomic system is most commonly investigated in Colombia. Neurological lesions must be evaluated carefully, because patients may be misdiagnosed and treated as carriers of 'idiopathic' diseases. Neurological pathologies poses a serious threat in Colombia due to the prevalence of Chagas disease.     

Oxidative DNA damage of mixed copper(II) complexes with sulfonamides and 1,10-phenanthroline. Crystal structure of [Cu(N-quinolin-8-yl-p-toluenesulfonamidate)2(1,10-phenanthroline)

Mixed coordination compounds of Cu(II) with sulfonamides and 1,10-phenanthroline as ligands have been prepared and characterised. Single crystal structural determination of the complex [Cu(N-quinolin-8-yl-p-toluenesulfonamidate)(2)(phen)] shows Cu(II) ions are located in a highly distorted octahedral environment, probably as a consequence of the Jahn-Teller effect. The FT-IR and electronic paramagnetic resonance (EPR) spectra are also discussed. The mixed complexes prepared undergo an extensive DNA cleavage in the presence of ascorbate and hydrogen peroxide. Two of the complexes have higher nucleolytic efficiency than the bis(o-phenanthroline)copper(II) complex.     

Nucleation of protein crystals

This paper introduces nucleation theory applied to crystallizing protein solutions. It is shown that the classical approach explains the available nucleation data under most conditions used for growing protein crystals for structural studies and for industrial crystallization. However, it fails to explain most experimental data on the structure of the critical clusters. It is also shown that for open systems working out of equilibrium, such as hanging-drop and counterdiffusion techniques, the geometry of the Ostwald-Myers protein solubility diagram and the number, size, and quality of the forming crystals depend not only on supersaturation but also on the rate of development of supersaturation.