Severe cefepime-induced status epilepticus treated with haemofiltration / Estatus epiléptico grave inducido por cefepime tratado con hemofiltración

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 (AU)

La neurotoxicidad por cefepime puede producirse principalmente en pacientes con insuficiencia renal. Presentamos un caso de status epiléptico no convulsivo producido por cefepime. Una mujer de 65 años con fracaso renal agudo en tratamiento con cefepime sufrió un episodio grave de status epiléptico no convulsivo que requirió inducción de coma barbitúrico con tiopental sódico. Tras el cambio de cefepime a meropenem se produjo también una interacción grave entre meropenem y ácido valproico. Se utilizó hemofiltración venovenosa continua para acelerar el aclaramiento de cefepime y la paciente recuperó progresivamente su situación neurológica previa. En conclusión, la dosis de cefepime debe ser ajustada a la función renal para evitar toxicidad en pacientes con insuficiencia renal. Debería considerarse la utilización del electroencefalograma en casos de estado confusional agudo en pacientes en tratamiento con cefepime para un diagnóstico precoz del status epiléptico no convulsivo. La terapia continua de reemplazo renal puede ser empleada en casos graves para acelerar la eliminación de cefepime. Además el meropenem no debe de utilizarse concomitantemente con el ácido valproico (AU)

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.

[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.

Variaciones hemodinámicas inducidas por la realización de unacurva presión–volumen y una maniobra de reclutamiento alveolaren pacientes con síndrome de distrés respiratorio agudo / Hemodynamic variability caused by pressurevolume plotting and alveolar recruitment maneuvers in patients with adult respiratory distress syndrome

OBJETIVOS: La realización de curvas presión-volumen(P-V) y maniobras de reclutamiento (MR) en pacientescon SDRA es una práctica extendida, si bien el mantenimientode una presión intratorácica elevada se asocia aefectos hemodinámicos potencialmente deletéreos. Nuestroobjetivo fue evaluar las alteraciones hemodinámicas y respiratoriasasociadas a la realización de ambas maniobrasy la eficacia a corto plazo de la MR.PACIENTES Y MÉTODOS: Pacientes con criterios de SDRAmonitorizados con un catéter PiCCOTM, en ritmo sinusal yadecuado relleno vascular. Se registraron los valores delíndice cardiaco (IC), presión arterial media (PAM), frecuenciacardiaca (FC), índice de volumen sistólico (IVS) ySpO2 durante la realización de la curva P-V (método quasi-estático) y MR (presión de 40 cmH2O mantenida). Seobtuvieron gasometrías previa a las técnicas y a los 15minutos de su finalización.RESULTADOS: En los 14 pacientes incluidos todos losparámetros estudiados disminuyeron significativamente.La máxima disminución del IC (26 ± 16%), PAM (6 ±6%), FC (4 ± 5%), IVS (21 ± 15%) y SpO2 (3 ± 3%) tuvolugar durante la MR, y se recuperó en menos de 2 minutosen todos los casos. El incremento de la PaO2 (7 ± 6%)y la PaO2/FiO2 (9 ± 8%) tras la MR fue significativo(p = 0,016 y 0,014 respectivamente), pero de escasa relevanciaclínica.CONCLUSIONES: Las alteraciones hemodinámicas asociadasy la escasa eficacia de una maniobra de reclutamientobasada en el mantenimiento de una presión teleinspiratoriaelevada hacen cuestionable su uso rutinario en pacientescon SDRA (AU)

OBJECTIVES: The plotting of pressure-volume curvesand the performance of alveolar recruitment maneuversare common practices in the care of patients with adultrespiratory distress syndrome (ARDS), even thoughpotentially harmful hemodynamic effects are associatedwith sustaining a high intrathoracic pressure. Our aimwas to analyze hemodynamic and ventilatory changesrelated to these 2 maneuvers and to assess the short-termeffectiveness of recruitment.PATIENTS AND METHODS: The patients had ARDS andwere being monitored with a catheter connected to aPiCCO system. All measurements were taken in sinusrhythm and with adequate vascular filling. Values recordedduring plotting of the quasistatic pressure-volume curveand the recruitment maneuver (sustained airway pressureof 40 cm H2O) were the cardiac index, mean arterialpressure, heart rate, systolic volume index, and oxygensaturation (SpO2). Blood gas measurements were recordedbefore the maneuvers and 15 minutes afterwards.RESULTS: All parameters decreased significantly in the14 patients studied. The mean (SD) maximum decreases,from which all patients recovered within 2 minutes, wereas follows: cardiac index, 26% (16%); mean arterialpressure, 6% (6%); heart rate, 4% (5%), systolic volumeindex, 21% (15%); and SpO2, 3% (3%). Significantincreases in PaO2 (7% [6%]) and the ratio of PaO2 to thefraction of inspired oxygen were recorded after therecruitment maneuver (P=.016 and P=.014, respectively),but the changes were not clinically significant.CONCLUSIONS: The hemodynamic disturbancesassociated with the alveolar recruitment maneuver basedon sustaining a high end-expiratory pressure and theminor improvement in oxygenation achieved as a resultsuggest that the routine use of that maneuver in ARDSpatients is of questionable value (AU)

[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.

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

OBJETIVO: El transporte de pacientes críticos supone un incremento de la morbi-mortalidad. Con frecuencia estos pacientes requieren sedación. La sedación insuficiente se acompaña de hipertensión, taquicardia y desadaptación a la ventilación mecánica. El objetivo de este estudio ha sido valorar el grado de sedación durante el transporte intrahospitalario de pacientes críticos monitorizando el índice biespectral (BIS). PACIENTES Y MÉTODO: Se estudiaron prospectivamente 30 pacientes críticos que requirieron transporte intrahospitalario a la Unidad de Críticos. Se recogieron el tiempo de transporte, el fármaco sedante elegido y la dosis empleada, valor BIS, la tensión arterial media (TAM) y la frecuencia cardiaca (FC) antes de iniciar el traslado y a su llegada. Los datos fueron recogidos por un observador no involucrado en el tratamiento de los enfermos. RESULTADOS: El tiempo medio de transporte fue 13,9 ± 4,2 minutos. Se empleó midazolam en 26 casos y en 4 casos propofol. En 10 pacientes se administró un bolo de cisatracurio antes de su traslado. Se observó un incremento significativo del BIS (de 47 a 78, p<0,001), de la TAM (de 73 a 91 mmHg, p<0,001) y de la FC a la llegada a la Unidad de Críticos (de 72 a 97, p<0,001). Las variaciones del BIS mostraron una correlación significativa con las variaciones de la FC (r=0,418, p=0,024), pero no con las de la TAM (r=0,249, p=0,19). CONCLUSIONES: La monitorización del BIS durante el transporte intrahospitalario de pacientes sedados con ventilación mecánica puede ser útil para detectar un grado de sedación superficial

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

Monitoring sedation in critically ill patients: bispectral index, Ramsay and observer scales.

BACKGROUND AND OBJECTIVE: Sedation is commonly required by critically ill patients and inadequate sedation may be hazardous. Traditionally, subjective scales have been used for monitoring sedation. Bispectral index has been proposed, although its utility in the intensive care unit is debated. Our aim was to evaluate the depth of sedation in intubated surgical critically ill patients by means of two sedation scales (Ramsay and Observer's Assessment of Alertness and Sedation) and bispectral index. METHODS: Sedation was assessed prospectively in 50 postoperative intubated patients requiring at least 24 h of sedation (35 propofol, 15 midazolam/fentanyl), every 8 h for a 24 -h period. The bispectral index value recorded was the mean value obtained during a 10-min observation period, whenever the quality signal index was above 75% and the electromyographic signal was below 25%. RESULTS: Most of the patients (78%) were oversedated (bispectral index < 60). The three sedation scores (global data) correlated significantly (P < 0.001). This correlation was lost in the midazolam group in which the patients were also significantly more sedated than the propofol group (P = 0.001). The correlation between the bispectral index and the scales in the midazolam group reappeared when the measurements with a Ramsay = 6 or an Observer's Assessment of Alertness and Sedation = 1 were excluded. CONCLUSIONS: Sedation should be monitored routinely in intensive care units. The Ramsay and the Observer's Assessment of Alertness and Sedation scales showed equal efficacy. Bispectral index might prove useful for discriminating between deeper levels of sedation.

Pressure-volume curve variations after a recruitment manoeuvre in acute lung injury/ARDS patients: implications for the understanding of the inflection points of the curve.

BACKGROUND AND OBJECTIVE: Although the pressure-volume (P-V) curve has been proposed in the management of mechanically ventilated patients, its interpretation remains unclear. Our aim has been to study the variations of the P-V curve after a recruitment manoeuvre (RM). Our hypothesis was that the lower inflection point (LIP) represents the presence of compressive atelectases, so it should not change after lung recruitment, while the upper inflection point (UIP) reflects reabsorptive atelectases, and an effective recruitment should result in changes at this level. METHODS: Two P-V curves (quasi-static method) separated by an RM (40 cmH2O, two consecutive manoeuvres) were plotted in 35 postoperative patients with criteria of acute lung injury/acute respiratory distress syndrome (ARDS). LIP, UIP and expiratory inflection point (EIP) were defined as the first point where the curve consistently starts to separate from the line. RESULTS: One to six measurements were obtained per patient (73 procedures). Neither the lower nor the EIPs varied significantly after the RM (P = 0.11 and 0.35, respectively). An UIP was observed in 18 curves (25%) before the RM and disappeared on nine occasions after the recruitment. Similar results were obtained when first measurements only were analysed, and when the cause (pulmonary vs. extrapulmonary), severity of lung injury or duration of mechanical ventilation at first measurement were studied. CONCLUSIONS: An RM does not modify the LIP significantly, but induces the disappearance of the UIP in 50% of the cases in which this point is found.

Adjusting positive end-expiratory pressure and tidal volume in acute respiratory distress syndrome according to the pressure-volume curve.

BACKGROUND: Management of acute respiratory distress syndrome (ARDS) patients implies the selection of the adequate ventilatory parameters, essentially PEEP and tidal volume (Vt), to prevent ventilator-induced lung injury. These parameters should be reset as the lung injury evolves. Among the different methods proposed for the adjustment of the ventilator, the measurement of the P-V curve has emerged as a useful, although debated, tool. Our aim has been to study the relationship between the different inflection points of the P-V curve in ARDS patients, and to assess the changes in the empiric PEEP and Vt (PEEP(emp), V(temp) following its use. METHODS: P-V curves were measured in 27 patients (lung injury score [LIS] >or= 2, 69 measurements) by means of the low-flow continuous inflation method. RESULTS: A lower inflection point (LIP) was found in all patients and, although it correlated with the PEEP(emp), there was only a fair concordance, so the PEEP was modified in 80% of the cases. The expiratory inflection point (EIP) was significantly lower than the LIP (6.3 +/- 1.7 vs. 8.1 +/- 3.2, P = 0.008). An upper inflection point was observed in 16 measurements (23%) and the Vt was reset in 20% of the cases. Both PEEP and Vt were readjusted on 10 occasions (14%). Only the EIP was significantly higher on the first 3 days of mechanical ventilation. The LIS was correlated with all the inflection points. There were no differences for any parameter independent of the cause of the ARDS (pulmonary/extrapulmonary). CONCLUSIONS: The quasi-static measurement of the P-V curve is a simple method, easy to interpret, for objective adjustment of the ventilatory parameters in ARDS patients as the lung injury evolves. The implementation of this strategy may vary the empiric clinical practice. The role of the EIP for the evaluation of the severity of lung injury deserves further investigation.

[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.

Hexokinase regulates kinetics of glucose transport and expression of genes encoding hexose transporters in Saccharomyces cerevisiae.

Glucose transport kinetics and mRNA levels of different glucose transporters were determined in Saccharomyces cerevisiae strains expressing different sugar kinases. During exponential growth on glucose, a hxk2 null strain exhibited high-affinity hexose transport associated with an elevated transcription of the genes HXT2 and HXT7, encoding high-affinity transporters, and a diminished expression of the HXT1 and HXT3 genes, encoding low-affinity transporters. Deletion of HXT7 revealed that the high-affinity component is mostly due to HXT7; however, a previously unidentified very-high-affinity component (K(m) = 0.19 mM) appeared to be due to other factors. Expression of genes encoding hexokinases from Schizosaccharomyces pombe or Yarrowia lipolytica in a hxk1 hxk2 glk1 strain prevented derepression of the high-affinity transport system at high concentrations of glucose.

Regulation of pyc1 encoding pyruvate carboxylase isozyme I by nitrogen sources in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the existence of PYC1 and PYC2 encoding cytosolic pyruvate carboxylase isoform I and II is rather puzzling, owing to the lack of potent differential gene regulation by the carbon sources. We report several findings indicating that these two genes are differentially regulated by the nature of the nitrogen source. In wild-type cells, the activity of pyruvate carboxylase, which is the sum of pyruvate carboxylase isoform I and II, was two- to fivefold lower in carbon medium containing aspartate, asparagine, glutamate or glutamine instead of ammonium as the nitrogen source, whereas it was 1.5- to threefold higher when the ammonium source was substituted by arginine, methionine, threonine or leucine. These enzymatic changes were independent of the nature of the carbon source and closely correlated to the changes in beta-galactosidase from PYC1-lacZ gene fusion and in PYC1 transcripts. Transfer of exponentially growing cells of the pyc2 mutant from an aspartate or a glutamate medium to an ammonium medium caused a fivefold increase in PYC1 mRNA in less than 30 min, whereas in the inverse experiment, PYC1 transcripts returned within 30 min to the low levels found in aspartate/glutamate medium. By contrast, these conditions affected neither the pyruvate carboxylase activity encoded by PYC2 nor PYC2 mRNA. Considering that changes in PYC1 expression inversely correlated with changes in alpha-ketoglutarate concentration or in alpha-ketoglutarate/glutamate ratio following the nitrogen shift experiments, and taking into account the pivotal role of this metabolite in ammonium assimilation, it is suggested that changes in alpha-ketoglutarate or in the alpha-ketoglutarate/glutamate ratio might be implicated in triggering the nitrogen effects on PYC1 expression. The physiological significance of the differential sensitivity of PYC1 and PYC2 genes with respect to the nitrogen source in the growth medium is also discussed.

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.

An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains.

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.

The alm1+ gene from Schizosaccharomyces pombe encodes a coiled-coil protein that associates with the medial region during mitosis.

We have isolated a cDNA that encodes a 142 kDa protein by immunoscreening of a Schizosaccharomyces pombe expression library with a new antibody, mAb8, that reveals spindle poles and equatorial ring-like structures in several organisms. This cDNA encodes a putative protein which we termed Alm (for abnormal long morphology). The protein is predicted to be a coiled-coil protein, containing a central alpha-helical domain flanked by non-helical terminal domains. Immunofluorescence analysis showed that Alm1 is localized in the medial region of the cell from anaphase to the end of cytokinesis. Cells carrying an alm1::ura4+ disruption are viable and exhibit an elongated morphology. Homozygous alm1::ura4+ diploids sporulated normally but the spores did not germinate. Spores that have inherited the disruption allele from a heterozygous alm1+/ alm1::ura4+ diploid germinated but generated smaller colonies. We propose that Alm1 participates in the structural organization of the medial region in S. pombe.

Mth1 receives the signal given by the glucose sensors Snf3 and Rgt2 in Saccharomyces cerevisiae.

We have determined that the mutant genes DGT1-1 and BPC1-1, which impair glucose transport and catabolite repression in Saccharomyces cerevisiae, are allelic forms of MTH1. Deletion of MTH1 had only slight effects on the expression of HXT1 or SNF3, but increased expression of HXT2 in the absence of glucose. A two-hybrid screen revealed that the Mth1 protein interacts with the cytoplasmic tails of the glucose sensors Snf3 and Rgt2. This interaction was affected by mutations in Mth1 and by the concentration of glucose in the medium. A double mutant, snf3 rgt2, recovered sensitivity to glucose when MTH1 was deleted, thus showing that glucose signalling may occur independently of Snf3 and Rgt2. A model for the possible mode of action of Snf3 and Rgt2 is presented.

Isolation of the MIG1 gene from Candida albicans and effects of its disruption on catabolite repression.

We have cloned a Candida albicans gene (CaMIG1) that encodes a protein homologous to the DNA-binding protein Mig1 from Saccharomyces cerevisiae (ScMig1). The C. albicans Mig1 protein (CaMig1) differs from ScMig1, in that, among other things, it lacks a putative phosphorylation site for Snf1 and presents several long stretches rich in glutamine or in asparagine, serine, and threonine and has the effector domain located at some distance (50 amino acids) from the carboxy terminus. Expression of CaMIG1 was low and was similar in glucose-, sucrose-, or ethanol-containing media. Disruption of the two CaMIG1 genomic copies had no effect in filamentation or infectivity. Levels of a glucose-repressible alpha-glucosidase, implicated in both sucrose and maltose utilization, were similar in wild-type or mig1/mig1 cells. Disruption of CaMIG1 had also no effect on the expression of the glucose-repressed gene CaGAL1. CaMIG1 was functional in S. cerevisiae, as judged by its ability to suppress the phenotypes produced by mig1 or tps1 mutations. In addition, CaMig1 formed specific complexes with the URS1 region of the S. cerevisiae FBP1 gene. The existence of a possible functional analogue of CaMIG1 in C. albicans was suggested by the results of band shift experiments.

Molecular cloning and characterization of the gene HXK1 encoding the hexokinase from Yarrowia lipolytica.

We have cloned the gene HXK1 from the dimorphic yeast Yarrowia lipolytica that encodes the unique hexokinase of this yeast. The gene has an intron located 39 base pairs after the A of the first ATG. The putative protein contains a sequence of 40 amino acids which is absent from other known hexokinase sequences. Y. lipolytica strains devoid of hexokinase grew in glucose slower than wild-type. This growth was due to the existence of a glucokinase. The hexokinase from Y. lipolytica substituted effectively for hexokinase II from S. cerevisiae in catabolite repression of invertase. The hexokinases from Schizosaccharomyces pombe or Kluyveromyces lactis were much less effective in this role. The K(m) for glucose and fructose of hexokinase was 0.38 mM and 3.56 mM, respectively. The K(m) of glucokinase for glucose was 0.17 mM. While the hexokinase was strongly inhibited by trehalose-6-phosphate (K(i)=3.6 microM), glucokinase was not affected by this compound.

Disruption and basic functional analysis of six novel ORFs of chromosome XV from Saccharomyces cerevisiae.

We report the disruption and functional analysis of six open reading frames (ORFs) from chromosome XV, namely YOL155c, YOL154w, YOL119c, YOL118c, YOR301w and YOR306c, in FY1679 and CEN.PK2 backgrounds. We constructed replacement cassettes and cloned each ORF into the pRS416 centromeric plasmid. No obvious phenotype was observed for the corresponding deleted strains with respect to growth, mating or sporulation. YOL155c encodes a protein with a secretion signal and putative GPI-anchor recognition site and is possibly a cell wall protein, although its deletion did not present morphogenetic defects under any of the conditions tested. Although YOL119c and YOR306c are members of the monocarboxylate permease family, the growth of the double disruptant in acetate, lactate and pyruvate was similar to that of the parental strains.

The lactate-proton symport of Saccharomyces cerevisiae is encoded by JEN1.

A mutant of Saccharomyces cerevisiae deficient in the lactate-proton symport was isolated. Transformation of the mutant with a yeast genomic library allowed the isolation of the gene JEN1 that restored lactate transport. Disruption of JEN1 abolished uptake of lactate. The results indicate that, under the experimental conditions tested, no other monocarboxylate permease is able to efficiently transport lactate in S. cerevisiae.