Endothelial Cell-Derived Nitric Oxide Enhances Aerobic Glycolysis in Astrocytes via HIF-1α-Mediated Target Gene Activation.

Astrocytes exhibit a prominent glycolytic activity, but whether such a metabolic profile is influenced by intercellular communication is unknown. Treatment of primary cultures of mouse cortical astrocytes with the nitric oxide (NO) donor DetaNONOate induced a time-dependent enhancement in the expression of genes encoding various glycolytic enzymes as well as transporters for glucose and lactate. Such an effect was shown to be dependent on the hypoxia-inducible factor HIF-1α, which is stabilized and translocated to the nucleus to exert its transcriptional regulation. NO action was dependent on both the PI3K/Akt/mTOR and MEK signaling pathways and required the activation of COX, but was independent of the soluble guanylate cyclase pathway. Furthermore, as a consequence of NO treatment, an enhanced lactate production and release by astrocytes was evidenced, which was prevented by downregulating HIF-1α. Several brain cell types represent possible sources of NO. It was found that endothelial cells, which express the endothelial NO synthase (eNOS) isoform, constitutively produced the largest amount of NO in culture. When astrocytes were cocultured with primary cultures of brain vascular endothelial cells, stabilization of HIF-1α and an enhancement in glucose transporter-1, hexokinase-2, and monocarboxylate transporter-4 expression as well as increased lactate production was found in astrocytes. This effect was inhibited by the NOS inhibitor l-NAME and was not seen when astrocytes were cocultured with primary cultures of cortical neurons. Our findings suggest that endothelial cell-derived NO participates to the maintenance of a high glycolytic activity in astrocytes mediated by astrocytic HIF-1α activation.

Prevalence and levels of IgG and IgM antibodies against Plasmodium falciparum and P. vivax in blood donors from Rondônia, Brazilian Amazon

Antibodies of IgG and IgM isotopes reacting with Plasmodium falciparum and P. vivax thick-smear antigens were searched for by the indirect fluorescent antibody test (IFAT) in a random sample of 230 blood donors at the transfusion centre of Porto Velho (HEMERON), Rondônia State, western Brazilian Amazon. A high prevalence of IgG seropositivity (32% against P. falciparum, 24% against P. vivax and 37% against either P. falciparum or P. vivax antigens) was detected among them, despite the fact that candidates reporting recent (<12 months) malaria attacks were not elegible. Only a small proportion of them had also detectable IgM antibodies to these antigens. These data suggest an intense, relatively recent exposure to malaria in such an urban population sample. However, parasitaemia (as detected by microscopical examination of Giemsa-stained thick smears) was patent in only one prospective donor. The antibody profile of blood donors was compared with that of healthy subjects of all age groups, living in a close endemic area (Candeias village, 21 km east of Porto Velho). The villagers were classified into two groups according to their history of a recent (<12 months) or a remote (>12 months) past malaria attack due to either P. falciparum or P. vivax. Extensive overlapping was observed when the distribution of antibody titres of healthy subjects from Candeias village with a recent and remote malaria history was compared. In conclusion, subjects with a recent or a remote malaria history could not be distinguished by sorological criteria alone.

Potassium restores vasorelaxation of resistance arterioles in non-hypertensive DOCA/salt fed mice.

BACKGROUND: Potassium-enriched diets exert renal and cardiovascular protective effects, but the underlying mechanisms are largely unknown. METHODS: Using the dorsal skinfold chamber model for intravital microscopy, we examined endothelium-dependent vasorelaxation of precapillary resistance arterioles in response to acetylcholine or the NO donor SNAP in awake mice. Experiments were performed in uni-nephrectomized one renin gene (Ren-1c) C57BL/6 mice (control group) and in mice having received a continuous administration of deoxycorticosterone acetate and a dietary supplementation of 1% sodium chloride for 8weeks (DOCA/salt group). An additional group of DOCA/salt treated animals received a dietary supplement of 0.4% KCl for 3weeks prior to the experiments (DOCA/salt + potassium group). RESULTS: DOCA/salt treatment for 8weeks resulted in hypokalemia, but blood pressure remained unchanged. In DOCA/salt mice, relaxation of resistance arterioles was blunted in response to acetylcholine, and to a lesser extent to SNAP, suggesting endothelial dysfunction. Endothelium-dependent vasorelaxation was restored by the potassium-enriched diet. CONCLUSION: This study is the first to demonstrate a protective effect of potassium on endothelium-dependent vasorelaxation in the absence of confounding anti-hypertensive effects, as observed in most animal models and the clinical situation. We propose that the known cardio- and nephro-protective effects of potassium might - at least in part - be mediated by the salutary effects on endothelium-dependent arteriolar relaxation.

Rekonstruktion von Oberkieferdefekten mit einem freien Skapula-angle-Lappen [Reconstruction of maxillary defects using a free scapular angle flap].

BACKGROUND: In addition to prosthetic rehabilitation, maxillary defects can also be surgically reconstructed. Soft-tissue reconstruction employs a radial forearm or latissimus dorsi muscle flap, while bony reconstruction can be achieved using a fibula, iliac crest, or scapular flap. Reconstruction using a scapular flap is further divided into two subgroups: the traditional scapular flap with the circumflex scapular artery as the donor vessel and the scapular angle flap with the angular artery originating from the thoracodorsal artery as the donor vessel. MATERIALS AND METHODS: We report on four patients who underwent successful reconstruction with a free scapular angle flap between 2009 and 2011, following maxillary resection due to malignancy. RESULTS: Vertical positioning of the scapular angle flap enables reconstruction of the facial contour, whereas its horizontal alignment and microvascular anastomosis makes a bony reconstruction of the hard palate possible. CONCLUSIONS: The versatility, low rate of donor site morbidity and shape of the scapular angle flap--which resembles that of the hard palate--render it ideal for plastic reconstruction. The suitability of bone quality for dental rehabilitation with implants is a topic of controversial discussion. The scapular angle flap represents an alternative to obturator prosthesis for the reconstruction of maxillary defects ≥ grade I according to Okay et al.

Monoamine oxidase A down-regulation contributes to high metanephrine concentration in pheochromocytoma.

CONTEXT: The high diagnostic performance of plasma-free metanephrines (metanephrine and normetanephrine) (MN) for pheochromocytoma (PHEO) results from the tumoral expression of catechol-O-methyltransferase (COMT), the enzyme involved in O-methylation of catecholamines (CAT). Intriguingly, metanephrine, in contrast to epinephrine, is not remarkably secreted during a stress in hypertensive or normotensive subjects, whereas in PHEO patients CAT and MN are both raised to high levels. Because epinephrine and metanephrine are almost exclusively produced by the adrenal medulla, this suggests distinct CAT metabolism in chromaffin cells and pheochromocytes. OBJECTIVE: The objective of the study was to compare CAT metabolism between adrenal medulla and PHEO tissue regarding related enzyme expression including monoamine oxidases (MAO) and COMT. DESIGN: A multicenter comparative study was conducted. STUDY PARTICIPANTS: The study included 21 patients with a histologically confirmed PHEO and eight adrenal glands as control. MAIN OUTCOME MEASURES: CAT, dihydroxyphenol-glycol, 3,4-dihydroxyphenylacetic acid, and MN were measured in adrenal medulla and PHEO tissue. Western blot, quantitative RT-PCR and immunofluorescence studies for MAOA, MAOB, tyrosine hydroxylase, dopamine β-hydroxylase, L-amino acid decarboxylase, and COMT were applied on tissue homogenates and cell preparations. RESULTS: At both the protein and mRNA levels, MAOA and COMT are detected less often in PHEO compared with adrenal medulla, conversely to tyrosine hydroxylase, L-amino acid decarboxylase, and dopamine β-hydroxylase, much more expressed in tumor tissue. MAOB protein is detected less often in tumor but not differently expressed at the mRNA level. Dihydroxyphenol-glycol is virtually absent from tumor, whereas MN, produced by COMT, rises to 4.6-fold compared with adrenal medulla tissue. MAOA down-regulation was observed in 100% of tumors studied, irrespectively of genetic alteration identified; on the other hand, MAOA was strongly expressed in all adrenal medulla collected independently of age, gender, or late sympathetic activation of the deceased donor. CONCLUSION: High concentrations of MN in tumor do not only arise from CAT overproduction but also from low MAOA expression, resulting in higher substrate availability for COMT.

Anàlisi genètica i molecular de les malalties Niemann-Pick tipus A/B i tipus C. Estratègies terapèutiques.

Aquest treball es basa en l’estudi de dues malalties lisosòmiques: la malaltia de Niemann-Pick A/B (NPAB) i la malaltia de Niemann-Pick tipus C (NPC). En relació a la malaltia de NPAB, s’ha realitzat l’expressió in vitro d’algunes de les mutacions de canvi d’aminoàcid trobades en pacients espanyols per tal de detectar les activitats enzimàtiques residuals. Totes les mutacions presenten una activitat molt baixa, gairebé nul•la, excepte la p.L225P i la R608del que tenen un 11% i 20% d’activitat respectivament. Els resultats obtinguts són coherents amb la severitat del fenotip que presenten els pacients. D’altra banda, s’ha caracteritzat un al•lel amb una mutació que afecta a una posició poc conservada d’un donador de splicing i que produeix la generació de trànscrits aberrants corresponents a trànscrits minoritaris de SMPD1, prèviament descrits, que no codifiquen per proteïna funcional. Respecte a malaltia de NPC, s’ha realitzat una anàlisi molecular de pacients espanyols prèviament estudiats identificant, en la majoria dels casos, la segona mutació responsable de la patologia. S’ha descrit per primer cop per aquesta malaltia una gran deleció que inclou el gen NPC1 i altres gens flanquejants i s’ha estudiat l’efecte que tenen les mutacions de splicing trobades a nivell de RNA. Per una d’aquestes mutacions, c.1554-1009G&A, s’ha assajat amb èxit una estratègia terapèutica basada en la utilització d’oligonuclèotids antisentit. D’altra banda, s’està desenvolupant un model cel•lular neuronal de la malaltia de Niemann-Pick tipus C, basat en la utilització de RNAs d’interferència, sobre el qual es podran assajar possibles estratègies terapèutiques en un futur.

CHO expression of a novel human recombinant IgG1 anti-RhD antibody isolated by phage display.

Replacement of the hyperimmune anti-Rhesus (Rh) D immunoglobulin, currently used to prevent haemolytic disease of the newborn, by fully recombinant human anti-RhD antibodies would solve the current logistic problems associated with supply and demand. The combination of phage display repertoire cloning with precise selection procedures enables isolation of specific genes that can then be inserted into mammalian expression systems allowing production of large quantities of recombinant human proteins. With the aim of selecting high-affinity anti-RhD antibodies, two human Fab libraries were constructed from a hyperimmune donor. Use of a new phage panning procedure involving bromelin-treated red blood cells enabled the isolation of two high-affinity Fab-expressing phage clones. LD-6-3 and LD-6-33, specific for RhD. These showed a novel reaction pattern by recognizing the D variants D(III), D(IVa), D(IVb), D(Va), D(VI) types I and II. D(VII), Rh33 and DFR. Full-length immunoglobulin molecules were constructed by cloning the variable regions into expression vectors containing genomic DNA encoding the immunoglobulin constant regions. We describe the first, stable, suspension growth-adapted Chinese hamster ovary (CHO) cell line producing a high affinity recombinant human IgG1 anti-RhD antibody adapted to pilot-scale production. Evaluation of the Fc region of this recombinant antibody by either chemiluminescence or antibody-dependent cell cytotoxicity (ADCC) assays demonstrated macrophage activation and lysis of red blood cells by human lymphocytes. A consistent source of recombinant human anti-RhD immunoglobulin produced by CHO cells is expected to meet the stringent safety and regulatory requirements for prophylactic application.

Desarrollo de una colección de Líneas Casi Isogénicas (NIL) para el estudio de caracteres de interés agronómico en especies del género Fragaria

Fragaria vesca posee varias características que la hacen interesante como especie modelo en la familia Rosaceae. Además, su naturaleza diploide permite sortear la complejidad genética de la fresa cultivada. Considerando que los genomas de las especies diploides y octoploides de Fragaria mantienen una relación de sintenia y colinearidad, estamos desarrollando una colección de Líneas Casi Isogénicas (NIL) en Fragaria diploide, usando Fragaria bucharica, una variedad asiática, como donante de introgresiones y Fragaria vesca var. Reine des Vallées, una variedad francesa comúnmente cultivada en España para usos industriales, como parental recurrente. Para obtener introgresiones de F. bucharica en un fondo genético homogéneo de F. vesca, se desarrolló un retrocruzamiento y se obtuvo una población BC1 que fue analizada para la presencia de alelos de F. bucharica a lo largo de los 7 grupos de ligamiento (LG) del mapa de referencia de Fragaria. Los individuos con bajo número de introgresiones de gran tamaño, que en conjunto abarcaron todo el genoma de F. bucharica, fueron seleccionados y retrocruzados con el parental recurrente. La descendencia fue analizada para los loci segregantes y los individuos BC2 con 1 ó 2 introgresiones fueron seleccionados como líneas donadoras de introgresiones de las NIL. Hasta el momento se han descrito tres fenotipos dominantes diferentes bajo condiciones de invernadero en varias NIL heterozigóticas: Las plantas con introgresiones en LG2 presentan estolones. Las plantas con introgresiones en LG1 y LG3 presentan floración temprana. Las plantas con introgresiones en LG6 presentan floración estacional. Actualmente se está trabajando en la selección y caracterización de introgresiones de pequeño tamaño mediante la autofecundación de las líneas seleccionadas. La caracterización fenotípica de los recombinantes seleccionados permitirá localizar y estimar los patrones de herencia de los genes implicados en los caracteres descritos, además de la identificación de nuevos caracteres recesivos que aparecerán en homozigosis.

MHC-II-independent CD4+ T cells induce colitis in immunodeficient RAG-/- hosts.

CD4(+) alpha beta T cells from either normal C57BL/6 (B6) or MHC-II-deficient (A alpha(-/-) or A beta(-/-)) B6 donor mice engrafted into congenic immunodeficient RAG1(-/-) B6 hosts induced an aggressive inflammatory bowel disease (IBD). Furthermore, CD4(+) T cells from CD1d(-/-) knockout (KO) B6 donor mice but not those from MHC-I(-/-) (homozygous transgenic mice deficient for beta(2)-microglobulin) KO B6 mice induced a colitis in RAG(-/-) hosts. Abundant numbers of in vivo activated (CD69(high)CD44(high)CD28(high)) NK1(+) and NK1(-) CD4(+) T cells were isolated from the inflamed colonic lamina propria (cLP) of transplanted mice with IBD that produced large amounts of TNF-alpha and IFN-gamma but low amounts of IL-4 and IL-10. IBD-associated cLP Th1 CD4(+) T cell populations were polyclonal and MHC-II-restricted when derived from normal B6 donor mice, but oligoclonal and apparently MHC-I-restricted when derived from MHC-II-deficient (A alpha(-/-) or A beta(-/-)) B6 donor mice. cLP CD4(+) T cell populations from homozygous transgenic mice deficient for beta(2)-microglobulin KO B6 donor mice engrafted into RAG(-/-) hosts were Th2 and MHC-II restricted. These data indicate that MHC-II-dependent as well as MHC-II-independent CD4(+) T cells can induce a severe and lethal IBD in congenic, immunodeficient hosts, but that the former need the latter to express its IBD-inducing potential.

A study of the aging of fingerprints by GC/MS analysis.

Determining the time since deposition of fingermarks may prove necessary in order to assess their relevance to criminal investigations. The crucial factor is the initial composition of fingermarks because it represents the starting point of any ageing model. This study mainly aimed to characterize the initial composition of fingerprints, which show a high variability between donors (inter-variability), but also to investigate the variations among fingerprints from the same donor (intra-variability). Solutions to reduce this initial variability using squalene and cholesterol as target compounds are proposed and should be further investigated. The influence of substrates was also evaluated and the initial composition was observed to be larger on porous surface than non-porous surfaces. Preliminary aging of fingerprints over 30 days was finally studied on a porous and a non-porous substrate to evaluate the potential for dating of fingermarks. Squalene was observed to decrease in a faster rate on a non-porous substrate.

Ganciclovir Exposure under a 450 mg Daily Dosage of Valganciclovir for the Prevention of Cytomegalovirus Disease in Kidney Transplant Recipients.

Background: It is suggested that a low dose of valganciclovir can be equally effective than a standard dose for cytomegalovirus (CMV) prophylaxis after kidney transplantation. The aim of our study was to determine the ganciclovir exposure observed under a routine daily dosage of 450 mg valganciclovir in kidney transplant recipients with a wide range of renal function. Methods: In this prospective study, kidney transplant recipients with a GFR MDRD above 25 mL/min at risk for CMV (donor or recipient seropositive for CMV) received a dose of valganciclovir (450 mg daily) prophylaxis for 3 months. Ganciclovir levels at trough (Ctrough) and at peak (C3h) were measured monthly. Ganciclovir exposure (AUC0-24) was estimated using Bayesian non-linear mixed-effect modelling (NONMEM) and compared between 3 groups of patients according to their kidney function: GFRMDRD 26-39 mL/min (Group 1), GFRMDRD 40-59 mL/min (Group 2) and GFRMDRD 60-90 mL/min (Group 3). CMV DNAemia was assessed during and after prophylaxis using PCR. Results: Thirty-six patients received 450 mg daily of valganciclovir for 3 months. Median ganciclovir C3h was 3.9 mg/L (range: 1.3-7.1) and Ctrough was 0.4 mg/L (range 0.1-2.7). Median (range) AUC0-24 of ganciclovir was 59.3 mg.h/L (39.0-85.3) in Group 1 patients, 35.8 mg.h/L (24.9-55.8) in Group 2 patients and 29.6 mg.h/L (22.0- 43.2) in Group 3 patients (p<0.001). Anemia was more common in Group 1 patients compared to patients on the other groups (p=0.01). No differences in other adverse events according to ganciclovir exposure were observed. CMV DNAemia was not detected during prophylaxis. After discontinuing prophylaxis, CMV DNAemia was seen in 8/34 patients (23.5%) and 4/36 patients (11%) developed CMV disease. Conclusion: A routine dosage of valganciclovir achieved plasma levels of ganciclovir in patients with GFR>60 mL/min similar to those previously reported using oral ganciclovir. A daily dose of 450 mg valganciclovir appears to be acceptable for CMV prophylaxis in most kidney transplant recipients.

Conjugative Transfer of Chromosomal Genes between Fluorescent Pseudomonads in the Rhizosphere of Wheat.

Bacteria released in large numbers for biocontrol or bioremediation purposes might exchange genes with other microorganisms. Two model systems were designed to investigate the likelihood of such an exchange and some factors which govern the conjugative exchange of chromosomal genes between root-colonizing pseudomonads in the rhizosphere of wheat. The first model consisted of the biocontrol strain CHA0 of Pseudomonas fluorescens and transposon-facilitated recombination (Tfr). A conjugative IncP plasmid loaded with transposon Tn5, in a CHA0 derivative carrying a chromosomal Tn5 insertion, promoted chromosome transfer to auxotrophic CHA0 recipients in vitro. A chromosomal marker (pro) was transferred at a frequency of about 10(sup-6) per donor on wheat roots under gnotobiotic conditions, provided that the Tfr donor and recipient populations each contained 10(sup6) to 10(sup7) CFU per g of root. In contrast, no conjugative gene transfer was detected in soil, illustrating that the root surface stimulates conjugation. The second model system was based on the genetically well-characterized strain PAO of Pseudomonas aeruginosa and the chromosome mobilizing IncP plasmid R68.45. Although originally isolated from a human wound, strain PAO1 was found to be an excellent root colonizer, even under natural, nonsterile conditions. Matings between an auxotrophic R68.45 donor and auxotrophic recipients produced prototrophic chromosomal recombinants at 10(sup-4) to 10(sup-5) per donor on wheat roots in artificial soil under gnotobiotic conditions and at about 10(sup-6) per donor on wheat roots in natural, nonsterile soil microcosms after 2 weeks of incubation. The frequencies of chromosomal recombinants were as high as or higher than the frequencies of R68.45 transconjugants, reflecting mainly the selective growth advantage of the prototrophic recombinants over the auxotrophic parental strains in the rhizosphere. Although under field conditions the formation of chromosomal recombinants is expected to be reduced by several factors, we conclude that chromosomal genes, whether present naturally or introduced by genetic modification, may be transmissible between rhizosphere bacteria.

Update of the BIPM comparison BIPM.RI(II)-K1.Cs-134 of activity measurements of the radionuclide 134Cs to include the 2008 results of the BEV (Austria), the 2009 result of the IRA (Switzerland) and the 2010 results of the NMISA (South Africa)

Purine nucleotide pyrophosphotransferase was purified to apparent homogeneity from a culture filtrate of Streptomyces morookaensis. It is a monomeric protein with a molecular weight of 24 000-25 000, and its isoelectric point is 6.9. The enzyme synthesizes purine nucleoside 5'-phosphate (mono, di, or tri) 3'-diphosphates such as pppApp, ppApp, pApp, pppGpp, ppGpp and pppIpp by transferring a pyrophosphoryl group from the 5'-position of ATP, dATP and ppApp to the 3'-position of purine nucleotides. The purified enzyme catalysed the formation of 435 mumol of pppApp and 620 mumol of pppGpp from ATP and GTP per min mg protein under the standard conditions. The enzyme requires absolutely a divalent cation for activity, and optimum pH for the enzyme activity lay above 10 for Mg2+, for Co2+ and Zn2+ from 9 to 9.5, and for Fe2+ from 7.5 to 8. The following Michaelis constants were determined: AMP, 2.78 mM; ADP, 3.23 mM; GMP, 0.89 mM; GDP, 0.46 mM and GTP, 1.54 mM, in the case of ATP donor. The enzyme is inhibited by guanine, guanosine, dGDP, dGTP, N-bromosuccinimide, iodacetate, sodium borate and mercuric acetate.

Value of allogeneic versus autologous stem cell transplantation and chemotherapy in patients with myelodysplastic syndromes and secondary acute myeloid leukemia. Final results of a prospective randomized European Intergroup Trial.

BACKGROUND: Allogeneic stem cell transplantation is usually considered the only curative treatment option for patients with advanced or transformed myelodysplastic syndromes in complete remission, but post-remission chemotherapy and autologous stem cell transplantation are potential alternatives, especially in patients over 45 years old. DESIGN AND METHODS: We evaluated, after intensive anti-leukemic remission-induction chemotherapy, the impact of the availability of an HLA-identical sibling donor on an intention-to treat basis. Additionally, all patients without a sibling donor in complete remission after the first consolidation course were randomized to either autologous peripheral blood stem cell transplantation or a second consolidation course consisting of high-dose cytarabine. RESULTS: The 4-year survival of the 341 evaluable patients was 28%. After achieving complete remission, the 4-year survival rates of patients under 55 years old with or without a donor were 54% and 41%, respectively, with an adjusted hazard ratio of 0.81 (95% confidence interval [95% CI], 0.49-1.35) for survival and of 0.67 (95% CI, 0.42-1.06) for disease-free survival. In patients with intermediate/high risk cytogenetic abnormalities the hazard ratio in multivariate analysis was 0.58 (99% CI, 0.22-1.50) (P=0.14) for survival and 0.46 (99% CI, 0.22-1.50) for disease-free survival (P=0.03). In contrast, in patients with low risk cytogenetic characteristics the hazard ratio for survival was 1.17 (99% CI, 0.40-3.42) and that for disease-free survival was 1.02 (99% CI, 0.40-2.56). The 4-year survival of the 65 patients randomized to autologous peripheral blood stem cell transplantation or a second consolidation course of high-dose cytarabine was 37% and 27%, respectively. The hazard ratio in multivariate analysis was 1.22 (95% CI, 0.65-2.27) for survival and 1.02 (95% CI, 0.56-1.85) for disease-free survival. CONCLUSIONS: Patients with a donor and candidates for allogeneic stem cell transplantation in first complete remission may have a better disease-free survival than those without a donor in case of myelodysplastic syndromes with intermediate/high-risk cytogenetics. Autologous peripheral blood stem cell transplantation does not provide longer survival than intensive chemotherapy.