[Hemodynamic variability caused by pressure-volume plotting and alveolar recruitment maneuvers in patients with adult respiratory distress syndrome]. / Variaciones hemodinámicas inducidas por la realización de una curva presión-volumen y una maniobra de reclutamiento alveolar en pacientes con síndrome de distrés respiratorio agudo.

OBJECTIVES: The plotting of pressure-volume curves and the performance of alveolar recruitment maneuvers are common practices in the care of patients with adult respiratory distress syndrome (ARDS), even though potentially harmful hemodynamic effects are associated with sustaining a high intrathoracic pressure. Our aim was to analyze hemodynamic and ventilatory changes related to these 2 maneuvers and to assess the short-term effectiveness of recruitment. PATIENTS AND METHODS: The patients had ARDS and were being monitored with a catheter connected to a PiCCO system. All measurements were taken in sinus rhythm and with adequate vascular filling. Values recorded during plotting of the quasistatic pressure-volume curve and the recruitment maneuver (sustained airway pressure of 40 cm H2O) were the cardiac index, mean arterial pressure, heart rate, systolic volume index, and oxygen saturation (SpO2). Blood gas measurements were recorded before the maneuvers and 15 minutes afterwards. RESULTS: All parameters decreased significantly in the 14 patients studied. The mean (SD) maximum decreases, from which all patients recovered within 2 minutes, were as follows: cardiac index, 26% (16%); mean arterial pressure, 6% (6%); heart rate, 4% (5%), systolic volume index, 21% (15%); and SpO2, 3% (3%). Significant increases in PaO2 (7% [6%]) and the ratio of PaO2 to the fraction of inspired oxygen were recorded after the recruitment maneuver (P=.016 and P=.014, respectively), but the changes were not clinically significant. CONCLUSIONS: The hemodynamic disturbances associated with the alveolar recruitment maneuver based on sustaining a high end-expiratory pressure and the minor improvement in oxygenation achieved as a result suggest that the routine use of that maneuver in ARDS patients is of questionable value.

[Bispectral index monitoring during intrahospital transport]. / Monitorización del índice biespectral en el transporte intrahospitalario.

BACKGROUND AND OBJECTIVE: Risk of morbidity and mortality increases for critically ill patients during transfers within the hospital. Such patients often require sedation, and suboptimal sedation is associated with hypertension, tachycardia, and ventilator dyssynchrony. The aim of this study was to assess level of sedation as indicated by monitoring of the bispectral (BIS) index during intrahospital transport of critical patients. PATIENTS AND METHODS: Thirty patients who required transport to the critical care unit within the hospital were studied prospectively. We recorded time in transport, the agent used for sedation and the dosage, the BIS index, mean arterial pressure (MAP), and heart rate before starting transport and upon arrival at the critical care unit. The data were recorded by an observer who was not assigned to patient care. RESULTS: The mean (SD) transport time was 13.9 (4.2) minutes. Midazolam was used in 26 patients and propofol in 4. Ten patients were given a bolus dose of cisatracurium before transfer started. Significant increases were observed in the BIS index (from 47 to 78, (P < .001), MAP (from 73 to 91 mmHg, P < .001), and heart rate (from 72 to 97 beats/min, P < .001) between the moment of starting transport and arrival at the critical care unit. Changes in the BIS index correlated significantly with changes in heart rate (r = 0.418, P = .024) but not with changes in MAP (r = 0.249, P = .19). CONCLUSIONS: Monitoring the BIS index during intrahospital transport of sedated, mechanically ventilated patients may be useful for detecting inadequate sedation.

Carbohydrate and energy-yielding metabolism in non-conventional yeasts.

Sugars are excellent carbon sources for all yeasts. Since a vast amount of information is available on the components of the pathways of sugar utilization in Saccharomyces cerevisiae it has been tacitly assumed that other yeasts use sugars in the same way. However, although the pathways of sugar utilization follow the same theme in all yeasts, important biochemical and genetic variations on it exist. Basically, in most non-conventional yeasts, in contrast to S. cerevisiae, respiration in the presence of oxygen is prominent for the use of sugars. This review provides comparative information on the different steps of the fundamental pathways of sugar utilization in non-conventional yeasts: glycolysis, fermentation, tricarboxylic acid cycle, pentose phosphate pathway and respiration. We consider also gluconeogenesis and, briefly, catabolite repression. We have centered our attention in the genera Kluyveromyces, Candida, Pichia, Yarrowia and Schizosaccharomyces, although occasional reference to other genera is made. The review shows that basic knowledge is missing on many components of these pathways and also that studies on regulation of critical steps are scarce. Information on these points would be important to generate genetically engineered yeast strains for certain industrial uses.

Severe cefepime-induced status epilepticus treated with haemofiltration.

Neurotoxicity caused by cefepime may occur predominantly in patients with impaired renal function. A case of a cefepime-induced non-convulsive status epilepticus (NCSE) is presented. A 65-year-old woman suffered a severe NCSE due to cefepime in the presence of acute renal failure, requiring coma induction with sodium thiopental. A serious interaction between valproic acid (VPA) and meropenem was also produced after changing cefepime to meropenem. Continuous veno-venous haemofiltration was employed to improve cefepime clearance, and the patient progressively regained her previous mental condition. In conclusion, the cefepime dose must be adjusted according to renal function in order to avoid toxicity in patients with renal failure. Electroencephalogram should be considered in cases of acute confusional state in patients receiving cefepime, to achieve early detection of NCSE. Continuous renal replacement therapy may be successfully employed in severe cases in order to accelerate cefepime removal. Likewise, meropenem should not be used concomitantly with VPA.

Purification and properties of three intracellular proteinases from Candida albicans.

Three intracellular proteinases termed A, B and C were purified to homogeneity from the unicellular form of the yeast Candida albicans. Enzyme A is an aspartic proteinase that acts on a variety of proteins. Its optimal pH is around 5 and it is displaced to 6.5 by KSCN. It is not significantly inhibited by PMSF, TLCK (Tos-Lys-CHCl2) or soybean trypsin inhibitor but it is inhibited by pepstatin. Its molecular weight is 60 000. Enzyme B is a dipeptidase that acts on esters or on dipeptides without blocks in either the carboxyl or amino ends. Its pH optimum is around 7.5 and the molecular weight is 57 000. It is inhibited by PMSF, TLCK and DANME (N2Ac-Nle-OMe). Proteinase C is an aminopeptidase with an optimum pH around 8. Its molecular weight was 67 000 when determined by SDS gel electrophoresis and 243 000 when determined by gel weight was 67 000 when determined by SDS gel electrophoresis and 243 000 when determined by gel filtration. It is active towards dipeptides in which at least one amino acid is apolar and is not active when the N-terminal amino acid is blocked. It is inhibited by EDTA or o-phenanthroline and activated by several divalent cations.

Activation of yeast plasma membrane ATPase by acid pH during growth.

When yeast grows on media in which a final external pH lower than 4 is attained there is a 2-3-fold increase in plasma membrane ATPase activity. This acid-mediated activation produces a 2-fold increase in the ATPase affinity for ATP but does not modify its optimum pH. The acid-mediated activation is dependent on the stage of growth, only late logarithmic or stationary cells being capable of being activated by incubation in an acidic buffer. Only cells able to activate the ATPase maintain a constant internal pH when incubated in acidic buffers. It is concluded that acid-mediated activation of the plasma membrane ATPase is a mechanism of maintaining constant internal pH during growth of yeast on acid media.

Expression of PEP carboxylase from Escherichia coli complements the phenotypic effects of pyruvate carboxylase mutations in Saccharomyces cerevisiae.

We investigated the effects of the expression of the Escherichia coli ppc gene encoding PEP carboxylase in Saccharomyces cerevisiae mutants devoid of pyruvate carboxylase. Functional expression of the ppc gene restored the ability of the yeast mutants to grow in glucose-ammonium medium. Growth yield in this medium was the same in the transformed yeast than in the wild type although the growth rate of the transformed yeast was slower. Growth in pyruvate was slowed down in the transformed strain, likely due to a futile cycle produced by the simultaneous action of PEP carboxykinase and PEP carboxylase.

A series of vectors to construct lacZ fusions for the study of gene expression in Schizosaccharomyces pombe.

We have constructed a series of plasmids to facilitate the fusion of promoters with or without coding regions of genes of Schizosaccharomyces pombe to the lacZ gene of Escherichia coli. These vectors carry a multiple cloning region in which fission yeast DNA may be inserted in three different reading frames with respect to the coding region of lacZ. The plasmids were constructed with the ura4+ or the his3+ marker of S. pombe. Functionality of the plasmids was tested measuring in parallel the expression of fructose 1,6-bisphosphatase and beta-galactosidase under the control of the fbp1+ promoter in different conditions.

Identification of two forms of 6-phosphofructo-2-kinase in yeast.

Two forms of 6-phosphofructo-2-kinase have been identified in Saccharomyces cerevisiae by their different chromatographic behaviour on CM-Sephadex C-50. One of them was not adsorbed and represented approximately 30% of the eluted activity. The other one emerged at about 120 mM KCl. A molecular mass of 120 kDa was found for both of them. No differences in kinetic behaviour in susceptibility to activation by cAMP-dependent protein kinase were found between the two forms.

A mutation affecting carbon catabolite repression suppresses growth defects in pyruvate carboxylase mutants from Saccharomyces cerevisiae.

Yeasts with disruptions in the genes PYC1 and PYC2 encoding the isoenzymes of pyruvate carboxylase cannot grow in a glucose-ammonium medium (Stucka et al. (1991) Mol. Gen. Genet. 229, 307-315). We have isolated a dominant mutation, BPC1-1, that allows growth in this medium of yeasts with interrupted PYC1 and PYC2 genes. The BPC1-1 mutation abolishes catabolite repression of a series of genes and allows expression of the enzymes of the glyoxylate cycle during growth in glucose. A functional glyoxylate cycle is necessary for suppression as a disruption of gene ICL1 encoding isocitrate lyase abolished the phenotypic effect of BPC1-1 on growth in glucose-ammonium. Concurrent expression from constitutive promoters of genes ICL1 and MLS1 (encoding malate synthase) also suppressed the growth phenotype of pyc1 pyc2 mutants. The mutation BPC1-1 is either allelic or closely linked to the mutation DGT1-1.

Schizosaccharomyces pombe possesses an unusual and a conventional hexokinase: biochemical and molecular characterization of both hexokinases.

Two hexokinases were characterized in Schizosaccharomyces pombe: hexokinase 1, with a low phosphorylation coefficient on glucose (Km 8.5 mM) and hexokinase 2, a kinetically conventional hexokinase. Genes hxk1+ and hxk2+ encoding these enzymes were cloned and sequenced. Disruption of hxk1+ had no effect on growth but disruption of hxk2+ doubled the generation time in glucose. Spores carrying the double disruption hxk1+ hxk2+ did not grow on glucose or fructose after one week. Expression of hxk1+ increased strongly during growth in fructose or glycerol. Expression of hxk2+ was highest during growth in glycerol. A NADP-dependent glucose dehydrogenase was detected, but not a glucokinase.

Isolation and characterization of the gene encoding phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae.

The yeast PCK1 gene coding for phosphoenolpyruvate carboxykinase (PEPCK) was isolated by functional complementation of pck1 strains from S. cerevisiae. Only one copy of the gene was found per haploid yeast genome. An RNA of about 2 kb which hybridized with a DNA probe internal to the PCK1 gene was found only in cells growing in non-fermentable carbon sources. Yeast strains carrying multiple copies of the PCK1 gene showed normal catabolite repression of PEPCK except those carrying the shortest insertion complementing the mutation (2.2 kb) that presented an altered kinetics of derepression. Catabolite inactivation was decreased in strains transformed with multicopy plasmids carrying the PCK1 gene.

Trehalose-6-phosphate, a new regulator of yeast glycolysis that inhibits hexokinases.

Trehalose-6-phosphate (P) competitively inhibited the hexokinases from Saccharomyces cerevisiae. The strongest inhibition was observed upon hexokinase II, with a Ki of 40 microM, while in the case of hexokinase I the Ki was 200 microM. Glucokinase was not inhibited by trehalose-6-P up to 5 mM. This inhibition appears to have physiological significance, since the intracellular levels of trehalose-6-P were about 0.2 mM. Hexokinases from other organisms were also inhibited, while glucokinases were unaffected. The hexokinase from the yeast, Yarrowia lipolytica, was particularly sensitive to the inhibition by trehalose-6-P: when assayed with 2 mM fructose an apparent Ki of 5 microM was calculated. Two S. cerevisiae mutants with abnormal levels of trehalose-6-P exhibited defects in glucose metabolism. It is concluded that trehalose-6-P plays an important role in the regulation of the first steps of yeast glycolysis, mainly through the inhibition of hexokinase II.

Regulatory regions in the promoters of the Saccharomyces cerevisiae PYC1 and PYC2 genes encoding isoenzymes of pyruvate carboxylase.

We have identified regions in the promoters of the PYC1 and PYC2 genes from Saccharomyces cerevisiae involved in their regulation in different culture conditions. In the case of PYC1, a UAS in the region between -330/-297 and three repressing sequences with the common central core CCGCC at positions -457, -432 and -399 were identified. Specific binding of nuclear proteins to the -330/-214 DNA fragment was abolished in rtg mutants suggesting a role for the RTG genes in the control of PYC1 expression. In the case of the PYC2 promoter, elimination of a fragment from -417 to -291 brings about a two-fold decrease in the expression in repressed conditions and a similar increase in derepression.

Mapping of the PCK1 gene encoding phosphoenolpyruvate carboxykinase on chromosome XI of Saccharomyces cerevisiae.

The gene PCK1 encoding phosphoenolpyruvate carboxykinase of Saccharomyces cerevisiae has been mapped on the right arm of chromosome XI, 12.7 centimorgans proximal to MAL4 and 20.1 centimorgans distal to MET1. In order to map the gene, hybridization of PCK1 DNA with separated yeast chromosomes and tetrad analysis of diploids with adequate markers were carried out.

Characterization of mutations that overcome the toxic effect of glucose on phosphoglucose isomerase less strains of Saccharomyces cerevisiae.

Glucose inhibits growth of yeast phosphoglucose isomerase mutants in permissive media. Mutants insensitive to this effect were isolated by selection on media containing 2% fructose + 2% glucose. A nuclear, monogenic, recessive mutation named rgl was responsible for this phenotype. The mutants isolated belonged to two complementation groups and have been termed rgl1 and rgl2. When the double mutants were grown on fructose, fermentation of fructose or glucose was similar to that of the parental pgi strain but was not measurable when grown on fructose+glucose. Under these conditions, respiration of glucose and to a lesser extent of fructose was enhanced. The double mutants pgi rgl did not grow on fructose+glucose in the presence of antimycin A or ethidium bromide and their cytochrome oxidase was no longer sensitive to glucose repression. The results are interpreted as an indication that in the double mutants the glucose may be channeled through the pentose phosphate pathway to respiration.

Use of Yarrowia lipolytica hexokinase for the quantitative determination of trehalose 6-phosphate.

This paper describes a procedure for the quantitative determination of trehalose 6-phosphate (T6P) based on its ability to inhibit hexokinase from Yarrowia lipolytica. The assay is linear between 1 nmol and at least 8 nmol. The concentration of T6P in wild-type Saccharomyces cerevisiae (0.15 mM) and in ras2 mutants (0.25 mM) remained unchanged in the exponential or stationary phase of growth or after heat shock. A tps1 mutant affected in T6P synthase did not show detectable T6P. Heat shock increased the concentration of T6P in Schizosaccharomyces pombe from 0.43 to 0.75 mM.

Lack of lactate-proton symport activity in pck1 mutants of Saccharomyces cerevisiae.

Mutants of Saccharomyces cerevisiae without phosphoenolpyruvate carboxykinase activity showed no measurable lactate proton symport, while mutants without fructose-1,6-bisphosphatase had normal transport activity. Incubation of a pck1 mutant, under derepression conditions in the presence of glycerol, restored the activity of the lactate-proton symport, with identical kinetic characteristics to that in the wild-type. For efficient lactate-proton symport activity, not only is an external inducer such as lactic acid needed, but also a molecule derived from the acid metabolism may be necessary.

[Anesthesia in a case of Holt-Oram syndrome]. / Anestesia en un caso de síndrome de Holt-Oram.

A 19-year-old man with Holt-Oram syndrome (HOS) underwent emergency surgical treatment of an occipital abscess. He presented total aplasia of the radius and first and second finger of the left hand, asymmetric thorax and complex cyanotic cardiopathy with double output of the right ventricle that had been treated on several occasions, malpositioning of the large vessels and interventricular conduction. He had been treated with digoxin for episodes of supraventricular tachycardia. After premedication with 0.4 mg of atropine, balanced general anesthesia was induced with etomidate and remifentanil and maintained with O2/air/desflurane and infused remifentanil. The patient remained hemodynamically stable during surgery and tubes were removed in the operating room with no complications. HOS, a hereditary disease characterized by congenital malformations of the upper extremities and the heart, is often associated with rhythm disorders. Problems that may develop in such patients during anesthesia include difficulty catheterizing vessels, difficult orotracheal intubation and ventilation, hemodynamic instability, and the presentation of arrhythmias and cardiac arrest.