Chromatin organisation of transgenes in Dictyostelium.

The introduction of transgenes in Dictyostelium discoideum typically results in the integration of the transformation vector into the genome at one or a few insertion sites as tandem arrays of approximately 100 copies. Exceptions are extrachromosomal vectors, which do not integrate into chromosomes, and vectors containing resistance markers such as blasticidin, which integrate as single copies at one or a few sites. Here we report that low copy number vector inserts display typical euchromatic features while high copy number insertions are enriched for modifications associate with heterochromatin. Interestingly, high copy number insertions also colocalise with heterochromatin, are enriched for the centromeric histone CenH3 and display centromere-like behaviour during mitosis. We also found that the chromatin organisation on extrachromosmal transgenes is different from those integrated into the chromosomes.

Studies of trypanocidal (inhibitory) power of naphthoquinones: evaluation of quantum chemical molecular descriptors for structure-activity relationships.

Electronic, lipophilic and steric descriptors included in QSAR-2D and -3D are analyzed for a set of ortho- and para-naphthoquinones that have proved to be powerful oxidative agents with potent trypanocidal activities specially against Leptomonas seymouri and Trypanosoma cruzi. Electronic properties are calculated by means of semiempirical (PM3), ab initio (HF/3-21G) and density functional theory (B3LYP/6-31+G*) methodologies. Three different electronic states, neutral quinones, hydroquinones and semiquinones, are studied to investigate if any one of them are statistically related with the biological activities. The best correlations were obtained at the B3LYP level of theory because it includes electronic correlation. The QSAR-2D indicates that the best trypanocidal growth inhibitors are molecules in the semiquinone electronic state, with the following properties: (a) high negative value of EHOMO, (b) high negative charge in the oxygen atoms of the carbonyl groups, (c) high positive charge in the carbon atom of one of carbonyl moieties and (d) high electronegativity (chi). In a complementary way, the QSAR-3D indicates that the electrostatic field correlates with trypanocidal activity and the presence of bulk moieties would increase activity. The idea of comparing the three electronic states may prove to be of most importance in the general strategy to the design of new trypanocidal drugs. In fact, the experimental results showed that semiquinone is the one really statistically relevant indicating a clear connection between biochemical and theoretical aspects. Finally, we demonstrated that to be a good anti-trypanosomatid compound, the molecule must be a good electron acceptor to reach easily the essential semiquinone state. We expect that the present results motivate new experimental as well as theoretical investigations that confirm our findings.

Anti-Trypanosoma cruzi effects of cyclosporin A derivatives: possible role of a P-glycoprotein and parasite cyclophilins.

Cyclophilins are target molecules for cyclosporin A (CsA), an immunosuppressive antimicrobial drug. We have previously reported the in vitro anti-Trypanosoma cruzi activity of H-7-94 and F-7-62 non-immunosuppressive CsA analogues. In this work, we continue the study of the parasiticidal effect of H-7-94 and F-7-62 CsA analogues in vitro and in vivo and we analyse 3 new CsA derivatives: MeIle-4-CsA (NIM 811), MeVal-4-CsA (MeVal-4) and D-MeAla-3-EtVal-4-CsA, (EtVal-4). The most efficient anti-T. cruzi effect was observed with H-7-94, F-7-62 and MeVal-4 CsA analogues evidenced as inhibition of epimastigote proliferation, trypomastigote penetration, intracellular amastigote development and in vivo T. cruzi infection. This trypanocidal activity could be due to inhibition of the peptidyl prolyl cis-trans isomerase activity on the T. cruzi recombinant cyclophilins tested. Furthermore, CsA and F-7-62 derivative inhibited the efflux of rhodamine 123 from T. cruzi epimastigotes, suggesting an interference with a P-glycoprotein activity. Moreover, H-7-94 and F-7-62 CsA analogues were not toxic as shown by cell viability and by aminopyrine-N-demethylase activity on mammalian cells. Our results show that H-7-94, F-7-62 and MeVal-4 CsA analogues expressed the highest inhibiting effects on T. cruzi, being promissory parasiticidal drugs worthy of further studies.

The chemotherapy of chagas' disease: an overview.

The review presents: a) a brief description of the disease; b) a summary of the most important metabolic targets so far identified in Trypanosome cruzi (T. cruzi) along with corresponding inhibitor compounds; c) the current state of knowledge on the trypanothione reductase system of trypanosomatids with reference to oxidative stress defenses; d) detailed discussions on T. cruzi trypanothione reductase inhibitors such as nitrofuranes, naphthoquinones and phenothiazines. As yet, the chemotherapy of Chagas' disease remains an unsolved problem. Further search for new drugs must continue by means of nucleating existing chemotherapy efforts.

Apoptogenic effect of the lipophilic o-naphthoquinone CG 10-248 on rat hepatocytes: light and electron microscopy studies

CG 10-248 (3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione; CG-NQ), a beta-lapachone analogue, modified the ultrastructure of rat hepatocytes, as demonstrated by light and electron microscopy. After 4 h incubation with 100 microM CG-NQ, the following effects were observed: (a) nuclear chromatin condensation; (b) chromatin fragmentation; (c) displacement of mitochondria, concentrated around the nucleus; (d) disruption or expansion of mitochondrial outer or inner membranes, respectively; (e) displacement and alteration of endoplasmic reticulum (rough and smooth); (f) decrease of microvilli; (g) blebbing of plasma membrane and production of apoptotic bodies formed by folding of plasma membrane fragments around mitochondria or peroxysomes; and (h) production of hydrogen peroxide. Expression of such effects varied according to hepatocyte samples and taken together strongly support an apoptotic action of CG-NQ dependent on reactive oxygen species.

Apoptogenic effect of the lipophilic o-naphthoquinone CG 10-248 on rat hepatocytes: light and electron microscopy studies

CG 10-248 (3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione; CG-NQ), a beta-lapachone analogue, modified the ultrastructure of rat hepatocytes, as demonstrated by light and electron microscopy. After 4 h incubation with 100 microM CG-NQ, the following effects were observed: (a) nuclear chromatin condensation; (b) chromatin fragmentation; (c) displacement of mitochondria, concentrated around the nucleus; (d) disruption or expansion of mitochondrial outer or inner membranes, respectively; (e) displacement and alteration of endoplasmic reticulum (rough and smooth); (f) decrease of microvilli; (g) blebbing of plasma membrane and production of apoptotic bodies formed by folding of plasma membrane fragments around mitochondria or peroxysomes; and (h) production of hydrogen peroxide. Expression of such effects varied according to hepatocyte samples and taken together strongly support an apoptotic action of CG-NQ dependent on reactive oxygen species. (AU)

Choline deficiency causes reversible hepatic abnormalities in patients receiving parenteral nutrition: proof of a human choline requirement: a placebo-controlled trial.

BACKGROUND: Previous studies have shown that plasma free choline concentrations are significantly decreased in many long-term home total parenteral nutrition (TPN) patients. Furthermore, low choline status has been associated with both hepatic morphologic and hepatic aminotransferase abnormalities. A preliminary pilot study suggested choline-supplemented TPN may be useful in reversal of these hepatic abnormalities. METHODS: Fifteen patients (10 M, 5 F) who had required TPN for > or =80% of their nutritional needs were randomized to receive their usual TPN (n = 8), or TPN to which 2 g choline chloride had been added (n = 7) for 24 weeks. Baseline demographic data were similar between groups. Patients had CT scans of the liver and spleen, and blood for plasma free and phospholipid-bound choline, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, gamma glutamyl transferase (GGT), bilirubin, serum lipids, complete blood count (CBC), and chemistry profile obtained at baseline, and weeks 2, 4, 6, 12, 16, 20, 24, and 34. CT scans were analyzed for Hounsfield unit (HU) densities. RESULTS: There were no significant differences in any measured parameters after 2 weeks. However, at 4 weeks, a significant difference in liver HU between groups was observed (13.3+/-5.0 HU [choline] vs 5.8+/-5.2 HU [placebo], p = .04). This significant trend continued through week 24. Recurrent hepatic steatosis and decreased HU were observed at week 34, 10 weeks after choline supplementation had been discontinued. A significant increase in the liver-spleen differential HU was also observed in the choline group (10.6+/-6.2 HU [choline] vs 1.3+/-3.3 HU [placebo], p = .01). Serum ALT decreased significantly (p = .01 to .05) in the choline group vs placebo at weeks 6,12, 20, and 24. Serum AST was significantly decreased in the choline group by week 24 (p = .02). The serum alkaline phosphatase was significantly reduced in the choline group at weeks 2, 12, 20, 24, and 34 (p = .02 to 0.07). Total bilirubin was normal in these patients and remained unchanged during the study. Serum GGT tended to decrease more in the choline group, but the greater decrease was not statistically significant. CONCLUSIONS: Choline deficiency is a significant contributor to the development of TPN-associated liver disease. The data suggest choline is a required nutrient for long-term home TPN patients.

[Cytotoxicity of beta-lapachone, an naphthoquinone with possible therapeutic use]. / Citotoxicidad de la beta-lapachona: una o-naftoquinona con posibles usos terapéuticos.

beta-lapachone (beta-lap) is a lipophilic o-naphthoquinone isolated from the bark of the lapacho tree. Initial observations proved its capability for inhibiting growth of Yoshida tumor and Walker 256 carcinosarcoma. beta-Lap redox-cycling in the presence of reductants and oxygen yields "reactive oxygen species" (ROS: O2-, OH and H2O2) which cytotoxicity led to assume its role in beta-lap activity in cells. beta-Lap inhibited DNA synthesis in Trypanosoma cruzi as well as topoisomerases I and II, poly(ADP-ribose) polymerase (PARP) in different cells. These enzymes are essential for maintaining DNA structure. beta-Lap inhibited growth of a large variety of tumor cells including epidermoid laringeal cancer, prostate, colon, ovary and breast cancer and also different types of leukemia cells. Advances in knowledge of apoptosis ("programmed cell death") and necrosis provided useful information for understanding the mechanism of beta-lap cytotoxicity. Thiol-dependent proteases (Calpaine), kinases (e.g. c-JUN NH2-terminal kinase), caspases and nucleases are involved in beta-lap cytotoxicity. These enzymes activity, as well as ROS production by beta-lap redox-cycling, would be essential for beta-lap cytotoxicity. Diaphorase and NAD(P)H-quinone reductase, which catalyse beta-lap redox-cycling and ROS production, seem to play an essential role in beta-lap activity. On these grounds, clinical applications of beta-lap have been suggested.

Hair bundle profiles along the chick basilar papilla.

Cochlear hair cells play a central role in the transduction of sound into neural output. Anatomical descriptions of these cells, and their protruding hair bundles, are of fundamental interest since hair cell transduction is dependent on hair bundle micromechanics and hair bundle micromechanics depends on hair bundle morphology. In this paper, we describe quantitatively changes in the staircase profile of the hair bundle along the apical portion of the chick's basilar papilla. Images of hair cells from 8 discretely dissected segments of the apical 3rd of the basilar papilla were archived, and the profile contour outlined by the tips of the stereocilia was digitised and curves were fitted by linear and power equations. The hair bundles of tall hair cells exhibited both linear and curvilinear profiles, which were equally distributed along the papilla. All short hair cells in our sample had straight contours. The differences in hair bundle shape among the tall hair cells may lead to differential susceptibility to injury and some variance in the current-displacement transduction curves due to differences in the translation of forces throughout the hair bundle.

Citotoxicidad de la beta-lapachona: una o-naftoquinona con posibles usos terapéuticos. / [Cytotoxicity of beta-lapachone, an naphthoquinone with possible therapeutic use]

beta-lapachone (beta-lap) is a lipophilic o-naphthoquinone isolated from the bark of the lapacho tree. Initial observations proved its capability for inhibiting growth of Yoshida tumor and Walker 256 carcinosarcoma. beta-Lap redox-cycling in the presence of reductants and oxygen yields [quot ]reactive oxygen species[quot ] (ROS: O2-, OH and H2O2) which cytotoxicity led to assume its role in beta-lap activity in cells. beta-Lap inhibited DNA synthesis in Trypanosoma cruzi as well as topoisomerases I and II, poly(ADP-ribose) polymerase (PARP) in different cells. These enzymes are essential for maintaining DNA structure. beta-Lap inhibited growth of a large variety of tumor cells including epidermoid laringeal cancer, prostate, colon, ovary and breast cancer and also different types of leukemia cells. Advances in knowledge of apoptosis ([quot ]programmed cell death[quot ]) and necrosis provided useful information for understanding the mechanism of beta-lap cytotoxicity. Thiol-dependent proteases (Calpaine), kinases (e.g. c-JUN NH2-terminal kinase), caspases and nucleases are involved in beta-lap cytotoxicity. These enzymes activity, as well as ROS production by beta-lap redox-cycling, would be essential for beta-lap cytotoxicity. Diaphorase and NAD(P)H-quinone reductase, which catalyse beta-lap redox-cycling and ROS production, seem to play an essential role in beta-lap activity. On these grounds, clinical applications of beta-lap have been suggested.

[Programmed cell death and apoptosis. The role of mitochondria]. / Muerte celular programada y apoptosis. Función de las mitocondrias.

Physiological cell death and apoptosis are natural processes genetically programmed, subjected to control by complex molecular mechanisms which elucidation is of particular interest for biology and medicine. Mitochondria play an essential role in physiological cell death and apoptosis. Apoptogenic effects develop in three phases, namely: (a) premitochondrial; (b) mitochondrial and (c) post-mitochondrial. During the first phase, apoptogenic signals (genotoxic agents, oxygen free radicals, corticoids, antibodies, etc.) interact with cell receptors activating specific mechanisms including thiol dependent proteases (caspases). As a consequence of those signals, mitochondrial damage results (membrane permeabilization, collapse of the membrane potential, swelling, membrane disruption, inhibition of electron transfer and oxidative phosphorylation). Other consequences of the mitochondrial disruption are the enhancement of free radical production and the exit of cytochrome c, caspases and endonucleases to the cytosol. During the third phase of apoptosis, free radicals and activated enzymes attack the cell protein structure and ADN, thus causing cell death. The mitochondrial regulation of apoptosis is controlled by the mitochondrial transitory permeability pore (MTPP) which is constituted by caspases, hexokinases, cytochrome c, ATP and ADP. MTPP is subjected to control by apoptogenic or antiapoptogenic agents which open or close it, according to their structure and the cell metabolic conditions. Uncontrolled opening of MTPP determines a massive exit of mitochondrial apoptogenic factors which in the cytosol and the nucleus exert their apoptogenic effects, thus producing cell death. MTPP can be modified by drugs with potential therapeutic actions thus opening interesting therapeutic possibilities. The role of apoptosis in pathologies such as degenerative diseases of the nervous system, autoimmunity diseases, SIDA and cancer is discussed.

Allograft dermal implant (AlloDerm) in a previously irradiated field.

OBJECTIVE: To evaluate the integration of AlloDerm (LifeCell Corp., The Woodlands, TX) in a field exposed to external-beam radiation (EBR) by analyzing graft thickness, fibroblast recellularization, and neovascularization. STUDY DESIGN: Randomized control. METHODS: Thirty-six male Sprague-Dawley rats (n = 36) were randomly assigned to four groups. One hind leg of each rat was exposed to 20 Gy of EBR; the other limb served as the nonirradiated control. Two weeks after irradiation, AlloDerm was implanted into both hind legs. Grafts were harvested at 3, 4, 6, and 14 weeks after implantation and underwent histological analyses. RESULTS: There was no statistically significant difference in graft thickness, fibroblast count, or neovascularization between the grafts placed in the irradiated bed and the controls (n = 33, P = .332, P = .336, and P = .057, respectively). However, at week 3, fibroblast counts in the graft placed in the field exposed to EBR were significantly lower than those of controls (P = .019), although at week 14 the counts in the experimental limb were higher than those of the controls (P = .002). Graft thickness (P = .001) and fibroblast count (P < .004) were lower at week 14 than at earlier time periods for both the experimental and control grafts. CONCLUSIONS: In the rat model, graft thickness and neovascularization of the AlloDerm dermal implant do not appear to be adversely affected by a field that has received EBR. Fibroblast ingrowth may be hindered in the early postimplantation period but appears to normalize in the long term. Furthermore, overall graft thickness and fibroblast counts decrease over time, regardless of irradiation status.

Prenatal diagnosis of a subamniotic hematoma.

A case of a subamniotic hematoma was diagnosed at 34 weeks of gestation. Pregnancy and delivery were uneventful. The ultrasound features of a subamniotic hematoma, and the differential diagnosis with lesions of less favorable outcome are described.

Muerte celular programada y apoptosis. Función de las mitocondrias. / [Programmed cell death and apoptosis. The role of mitochondria]

Physiological cell death and apoptosis are natural processes genetically programmed, subjected to control by complex molecular mechanisms which elucidation is of particular interest for biology and medicine. Mitochondria play an essential role in physiological cell death and apoptosis. Apoptogenic effects develop in three phases, namely: (a) premitochondrial; (b) mitochondrial and (c) post-mitochondrial. During the first phase, apoptogenic signals (genotoxic agents, oxygen free radicals, corticoids, antibodies, etc.) interact with cell receptors activating specific mechanisms including thiol dependent proteases (caspases). As a consequence of those signals, mitochondrial damage results (membrane permeabilization, collapse of the membrane potential, swelling, membrane disruption, inhibition of electron transfer and oxidative phosphorylation). Other consequences of the mitochondrial disruption are the enhancement of free radical production and the exit of cytochrome c, caspases and endonucleases to the cytosol. During the third phase of apoptosis, free radicals and activated enzymes attack the cell protein structure and ADN, thus causing cell death. The mitochondrial regulation of apoptosis is controlled by the mitochondrial transitory permeability pore (MTPP) which is constituted by caspases, hexokinases, cytochrome c, ATP and ADP. MTPP is subjected to control by apoptogenic or antiapoptogenic agents which open or close it, according to their structure and the cell metabolic conditions. Uncontrolled opening of MTPP determines a massive exit of mitochondrial apoptogenic factors which in the cytosol and the nucleus exert their apoptogenic effects, thus producing cell death. MTPP can be modified by drugs with potential therapeutic actions thus opening interesting therapeutic possibilities. The role of apoptosis in pathologies such as degenerative diseases of the nervous system, autoimmunity diseases, SIDA and cancer is discussed.

[Physiopathologic effects of nitric oxide and their relationship with oxidative stress]. / Efectos fisiopatológicos del óxido nítrico y su relación con el estrés oxidativo.

Nitric oxide (NO.) is produced from L-arginine, as result of a reaction catalyzed by the enzyme nitric oxide synthase (NOS). The reaction is the sole source of NO. in animal tissues. NO. can control physiological processes (or systems) such as (a) blood pressure; (b) relaxation of arterial smooth muscle; (c) platelet aggregation and adhesion; (d) neurotransmission; (e) neuroendocrine secretion. NO. contributes to the killing of pathogenic microorganisms and tumoral cells by phagocytes. NO. reacts with superoxide anion thus producing peroxynitrite, a cytotoxic ion capable of destroying many biological targets. The superoxide/peroxinitrite balance determines the ONOO- production and, accordingly, is essential for the development of hypertension, atherosclerosis, neurodegenerative diseases, viral infections, ischemia-reperfusion injury, and cancer.

Intracranial pressure waveform analysis: computation of pressure transmission and waveform shape indicators.

We studied transmission of arterial blood pressure to intracranial pressure by observing how the two pressure waveforms varied from baseline conditions to after postural change or jugular compression. Such experiments may lead to pressure waveform-based estimates of intracranial compliance. Using a single database of arterial blood pressure, central venous pressure, and intracranial pressure waveforms collected during baseline, jugular compresison, and head-elevated conditions from six Yucatan minipigs, we computed several numerical indicators of waveform shape to find an estimator of intracranial compliance. Of these indicators, two were based on the Fourier-decomposition of all three waveforms, and one was based on a new method for approximating the systolic slope of the intracranial pressure waveform. We computed amplitude transfer functions for the first six harmonics of the Fourier spectrum, treating intracranial pressure as system output and independently treating arterial blood pressure and central venous pressure as system inputs. Using these same inputs and outputs, we computed a single quotient based on the Fourier coefficients of the first six harmonics of the input and output waveforms. Finally, applying a Gaussian high-pass filter, we computed systolic slope approximations for all intracranial pressure wave cycles contained in a single respiratory cycle. Our third indicator was the mean-normalized variation of the slope approximations over a respiratory cycle. We studied how each composite at baseline varied with baseline mean intracranial pressure and how each composite changed from baseline as a result of a physical manipulation. Our analysis suggests that the composite based on respiratory variation of systolic slope approximations was positively correlated with mean intracranial pressure during baseline. The quotient based on Fourier coefficients with arterial blood pressure input seemed to increase from baseline to jugular compression. Composites that treated central venous pressure as input were both less correlated with mean intracranial pressure during baseline and exhibited less predictable changes from baseline to a physical manipulation than their counterparts that used arterial blood pressure as input. However, none of these apparent trends was statistically significant. The lack of statistically significant results may be due to the nature of the composites and/or the small sample size (n = 6). However, we hope this study stimulates further investigation of both central venous pressure-to-intracranial pressure (in addition to arterial blood pressure-to-intracranial pressure) transfer and automated computation of intracranial pressure waveform systolic slope. Such research may lead to noninvasively determined estimators of intracranial compliance.

Semiquinone production by lipophilic o-naphthoquinones.

Several ß-lapachone analogues, the o-naphthoquinones CG 10-248, CG 9-442 and CG 8-935, were reduced to their semiquinones by sodium borohydride, the liver NADPH-P450 reductase system and Crithidia fasciculata cells, in anaerobic solutions. ESR spectra of the radical anions showed hyperfine spin couplings located at protons of the naphthalene ring. Borohydride reduction of another o-naphthoquinone, mansonone E, yielded spin couplings located at the naphthalene and methyl groups protons. The symmetrical polarized carbonyl groups were essential for electron capture and semiquinone production. These observations support the idea that quinones are capable of redox-cycling and oxygen radical generation.

Improving the diagnostic accuracy of MR in the detection of infraspinatus tendon injuries.

PURPOSE: Our goal was to determine the accuracy of MRI in the diagnosis of infraspinatus tendon injury and more specifically to determine if the antero-posterior extent of a rotator cuff tear is predictive of infraspinatus tearing. METHOD: The MR images of 41 shoulders with surgically proven supraspinatus tears at surgery were retrospectively reviewed. The following were assessed for each of the 41 studies: the number of oblique coronal images on which a tendon defect could be seen, the angle subtended by the tear on axial images (the rotator cuff "axial angle"), and the extent of signal abnormality on sagittal images. RESULTS: The rotator cuff axial angle was 75.6 degrees in patients with infraspinatus tendon tears (ITTs) versus 40 degrees in those without ITTs, and this difference was significant (p < 0.001, t = 3.06). The mean number of oblique coronal images (obtained with a 4 mm slice and 1 mm gap) showing signal abnormality was 5.4 in the ITT group versus 2.9 in those without ITTs, and this difference was also significant (p < 0.001, t = 4.45). The mean sagittal extent of the tendon abnormality was 24.6 mm in the ITT group and 11.6 mm in those without ITTs, but the difference was not significant (p > 0.05, t = 1.1364). CONCLUSION: The axial angle and the number of oblique coronal images in which signal abnormality was present were significantly related to a higher incidence of infraspinatus tears.