Deep subsurface sulfate reduction and methanogenesis in the Iberian Pyrite Belt revealed through geochemistry and molecular biomarkers.

The Iberian Pyrite Belt (IPB, southwest of Spain), the largest known massive sulfide deposit, fuels a rich chemolithotrophic microbial community in the Río Tinto area. However, the geomicrobiology of its deep subsurface is still unexplored. Herein, we report on the geochemistry and prokaryotic diversity in the subsurface (down to a depth of 166 m) of the Iberian Pyritic belt using an array of geochemical and complementary molecular ecology techniques. Using an antibody microarray, we detected polymeric biomarkers (lipoteichoic acids and peptidoglycan) from Gram-positive bacteria throughout the borehole. DNA microarray hybridization confirmed the presence of members of methane oxidizers, sulfate-reducers, metal and sulfur oxidizers, and methanogenic Euryarchaeota. DNA sequences from denitrifying and hydrogenotrophic bacteria were also identified. FISH hybridization revealed live bacterial clusters associated with microniches on mineral surfaces. These results, together with measures of the geochemical parameters in the borehole, allowed us to create a preliminary scheme of the biogeochemical processes that could be operating in the deep subsurface of the Iberian Pyrite Belt, including microbial metabolisms such as sulfate reduction, methanogenesis and anaerobic methane oxidation.

Functional analysis of the Streptomyces lividans type I signal peptidases.

Type I signal peptidases are responsible for the proteolytic cleavage of the signal peptide of secreted proteins. In the gram-positive bacterium Streptomyces lividans, four adjacent genes (sipW, sipX, sipY and sipZ) were isolated encoding putative type I signal peptidases. In this work, the different sip genes were cloned and expressed. Subsequently, the Sip proteins were purified to raise antibodies. Although the four Sip proteins share a low degree of sequence similarity and differ significantly in size and pI, anti-Sip antibodies cross-reacted intensively. Functional signal peptidase processing activity for each of these Sip proteins was shown both in vitro and in vivo. The different Sip proteins did not exhibit the same cleavage efficiency on the Bacillus subtilis pre-chitosanase.

Direct versus indirect signs of traumatic aortic injury revealed by helical CT: performance characteristics and interobserver agreement.

OBJECTIVE: The purpose of this study was to evaluate the relative value of and interobserver agreement on direct versus indirect (hematoma) signs of traumatic aortic injury using helical CT. MATERIALS AND METHODS: From April 1994 through January 1997, 40 patients who were suspected to have traumatic aortic injury and who underwent contrast-enhanced helical CT had subsequent proof or exclusion of aortic injury. All available CT scans of these patients were combined with CT scans of 13 randomly chosen patients that had been initially interpreted as negative, and clinical follow-up showed no evidence of aortic injury. Two emergency radiologists and a nonemergency radiologist who were unaware of clinical outcome performed independent review of these cases to evaluate for mediastinal hematoma, periaortic hematoma, and direct signs of aortic injury. RESULTS: Direct signs of injury were seen on helical CT by both emergency radiologists in all 17 cases of aortic injury with no false-positive interpretations. The nonemergency radiologist failed to observe subtle direct signs in two cases of aortic injury, but patient management would not have been adversely affected. All observers had more false-negative interpretations for both mediastinal hematoma and periaortic hematoma than for direct signs. Interobserver agreement was higher for direct signs (kappa = .93) than for either mediastinal hematoma (kappa = .65) or periaortic hematoma (kappa = .71). CONCLUSION: In this study, helical CT revealed direct signs of traumatic aortic injury that were more accurate and more often observed than were indirect signs. Emphasis on direct signs should improve confidence in using helical CT to evaluate traumatic aortic injury.

Cervical spine trauma: how much more do we learn by routinely using helical CT?

Assessment of suspected cervical spine injuries remains a major debate in trauma care. It is generally accepted that many fractures are missed or incompletely shown at radiography, mainly because of suboptimal studies obtained in obtunded, uncooperative trauma victims. In a series of 88 severely traumatized patients, the authors retrospectively determined the type, distribution, and significance of such missed lesions. This assessment was made by comparing radiographs and helical computed tomographic (CT) scans of the cervical spine and reviewing medical records in these cases. Of the 88 patients, 32 patients had cervical spine fractures (n = 50) that were not revealed or were incompletely demonstrated at radiography. Most missed fractures occurred at the C-1 to C-2 and C-6 to C-7 levels, and most involved the transverse processes and the posterolateral elements of the vertebrae. One-third of the patients with missed fractures had either clinically significant or unstable injuries, as determined on the basis of mechanistic or imaging criteria. Helical CT can depict significant fractures not shown by plain radiography and should be added routinely to the initial screening for cervical spine fractures in polytrauma victims.

Erythropoietin gene expression in human, monkey and murine brain.

The haematopoietic growth factor erythropoietin is the primary regulator of mammalian erythropoiesis and is produced by the kidney and the liver in an oxygen-dependent manner. We and others have recently demonstrated erythropoietin gene expression in the rodent brain. In this work, we show that cerebral erythropoietin gene expression is not restricted to rodents but occurs also in the primate brain. Erythropoietin mRNA was detected in biopsies from the human hippocampus, amygdala and temporal cortex and in various brain areas of the monkey Macaca mulatta. Exposure to a low level of oxygen led to elevated erythropoietin mRNA levels in the monkey brain, as did anaemia in the mouse brain. In addition, erythropoietin receptor mRNA was detected in all brain biopsies tested from man, monkey and mouse. Analysis of primary cerebral cells isolated from newborn mice revealed that astrocytes, but not microglia cells, expressed erythropoietin. When incubated at 1% oxygen, astrocytes showed >100-fold time-dependent erythropoietin mRNA accumulation, as measured with the quantitative reverse transcription-polymerase chain reaction. The specificity of hypoxic gene induction in these cells was confirmed by quantitative Northern blot analysis showing hypoxic up-regulation of mRNA encoding the vascular endothelial growth factor, but not of other genes. These findings demonstrate that erythropoietin and its receptor are expressed in the brain of primates as they are in rodents, and that, at least in mice, primary astrocytes are a source of cerebral erythropoietin expression which can be up-regulated by reduced oxygenation.

Localization of specific erythropoietin binding sites in defined areas of the mouse brain.

The main physiological regulator of erythropoiesis is the hematopoietic growth factor erythropoietin (EPO), which is induced in response to hypoxia. Binding of EPO to the EPO receptor (EPO-R), a member of the cytokine receptor superfamily, controls the terminal maturation of red blood cells. So far, EPO has been reported to act mainly on erythroid precursor cells. However, we have detected mRNA encoding both EPO and EPO-R in mouse brain by reverse transcription-PCR. Exposure to 0.1% carbon monoxide, a procedure that causes functional anemia, resulted in a 20-fold increase of EPO mRNA in mouse brain as quantified by competitive reverse transcription-PCR, whereas the EPO-R mRNA level was not influenced by hypoxia. Binding studies on mouse brain sections revealed defined binding sites for radioiodinated EPO in distinct brain areas. The specificity of EPO binding was assessed by homologous competition with an excess of unlabeled EPO and by using two monoclonal antibodies against human EPO, one inhibitory and the other noninhibitory for binding of EPO to EPO-R. Major EPO binding sites were observed in the hippocampus, capsula interna, cortex, and midbrain areas. Functional expression of the EPO-R and hypoxic upregulation of EPO suggest a role of EPO in the brain.

Synthesis of beta-glucans in Prototheca zopfii. Evidence for the existence of a glycoprotein primer.

Membrane preparations from the non-photosynthetic alga Prototheca zopfii incorporate glucose from UDP-[3H]glucose into the trichloroacetic-acid-insoluble fraction and the polysaccharides insoluble in hot alkali. Time course and pulse-chase experiments indicate that the acid-insoluble fraction was a precursor of the alkali-insoluble fraction. Isolation of 3H-labeled membrane or soluble fraction showed that only membrane fractions were able to transfer radioactivity into polysaccharides. Treatment of glucosylated membranes with trypsin or cellulase only partially affect their transfer ability, indicating that the precursor was internalized in vesicles. Analysis of the in vitro synthesized polysaccharides by enzymatic and acid hydrolysis showed that glucose and cellobiose were present as radioactive sugars. Permethylation of the polysaccharide indicates that 80% of the glucose was beta-1,4-bonded with 20% in beta-1,3-linkages. This polysaccharide was found to be identical with the cell-wall beta-glucan obtained in vivo [Rivas, L.A. & Pont Lezica, R. (1978) Planta (Berl.) 165, 348-353].

Synthesis of beta-glucans in Prototheca zopfii. Isolation and characterization of the glycoprotein primer.

When Prototheca zopfii cells were pulse-labeled with 14C-containing amino acids and homogenized, 14C-labeled membranes were obtained. In vitro incubations with the previously labeled membranes and UDP-[3H]Glc produced a trichloroacetic-acid-insoluble fraction having both isotopes. A double-labeled glucoprotein was isolated and characterized. It has a relative molecular mass of 28,000-30,000 and a carbohydrate content of 10%. The oligosaccharide chain is linked to the protein through an O-glycosidic bond between hydroxyproline and glucose. The oligosaccharide has a polymerization degree ranging over 10-20 hexose units. Glucose is the only monosaccharide found; most of the glucose residues are beta-1,4-linked (90%) but some are beta-1,3-linked (10%).

ß-Glucan biosynthesis in synchronous cells of Prototheca zopfii.

Synchronous cultures of the colorless chlorophyte Prototheca zopfii Kruger were obtained by a shift-down of the basal medium plus addition of 1.5 mM ethylenediaminetetracetic acid for a 10-14-h period, followed by periodic dilutions with the basal medium. The cell cycle has a duration of 10-12 h at 25°C and an average of eight autospores are produced at the end of each cycle. The incorporation of [(14)C]glucose into ß-glucans was determined in vivo. Very high incorporation occurs between hours 3 and 4 of culture, coincident with maximal cell expansion. The incorporation of glucose from uridine 5'diphospho-[(14)C]glucose into lipid-linked sugars and glycoproteins, as well as the activity of guanosine 5'-diphosphoglucose:ß-glucan synthase found in cell-free preparations are in good agreement with the in-vivo incorporation of glucose into ß-glucans.

Estudio cruzado y evaluación ecocardiográfica de prazosin, propranolol e hidroclorotiazida en hipertensión arterial leve y moderada / Cross study and ecocardiographia evaluation of prazosin, propranolol and hydroclorothiazide in mild and moderate arterial hypertension

Veinte pacientes con hipertensión arterial esencial leve a moderada concluyeron un estudio cruzado distribuidos al azar en dos grupos: Grupo A: 10 pacientes que recibieron primero propranolol y posteriormente prazosin. Grupo B: 10 pacientes que recibieron inicialmente hidroclorotiazida y después prazosin. La reducción de la presión arterial en posición supina y de pie fue estadísticamente significativa y comparable con las tres drogas. Los efectos colaterales fueron menores con el prazosin. El análisis ecocardiográfico modo "M" en los pacientes con hipertrofia ventricular izquierda mostró reducción de la masa ventricular en 5 que recibieron propranolol y en 4 tratados con hidroclorotiazida. No encontramos reducción ulterior con el prazosin. La fracción de eyección y el acortamiento circunferencial de la fibra mejoraron en forma significativa con el prazosin (16 pacientes), no así con el propranolol ni con la hidroclorotiazida. Se concluye que el prazosin es una droga que puede ser utilizada como terapia inicial con pocos efectos secundarios a dosis bajas. Se propone el tratamiento secuencial de la hipertensión arterial leve a moderada en vez del esquema tradicional

Inhibition of beta-1,4-Glucan Biosynthesis by Deoxyglucose : The Effect on the Glucosylation of Lipid Intermediates.

GDP- and UDP-deoxyglucose inhibit the incorporation of glucose from UDP-glucose into dolichyl phosphate glucose and dolichyl pyrophosphate oligosaccharides. GDP-deoxyglucose inhibits by competing with the physiological nucleotide sugars for dolichyl phosphate, and dolichyl phosphate deoxyglucose is formed. This inhibition is reversed by excess of dolichyl phosphate. UDP-deoxyglucose does not give rise to a lipid-linked derivative, and inhibition by this analog is not reversed by dolichyl phosphate. The UDP- and GDP-derivatives of deoxyglucose inhibit the incorporation of glucose into glucose-containing glycoproteins. This effect seems to be the result of the inhibition of lipid intermediates glucosylation and is comparable to the effect produced by coumarin. Cellulose synthetase activity is not affected by UDP- or GDP-deoxyglucose. On the other hand, deoxyglucose inhibits the formation of beta-1,4-glucans in vivo.