Selective regulation of neurosteroid biosynthesis in human neuroblastoma cells under hydrogen peroxide-induced oxidative stress condition.

Neurosteroid biosynthesis is demonstrated in many species but key factors interacting with neurosteroidogenesis under pathophysiological conditions are unknown. Hydrogen peroxide (H(2)O(2))-induced oxidative stress is an etiological factor involved in several disorders. We hypothesized that, if neurosteroidogenesis is a pivotal mechanism for nerve cell protection or viability, it might be selectively regulated under oxidative stress condition. To check our hypothesis, we investigated H(2)O(2) effects on neurosteroidogenesis in human neuroblastoma SH-SY5Y cells. Pulse-chase, high performance liquid chromatography and flow-scintillation analyses showed that, along neurosteroidogenic pathways converting pregnenolone into various neurosteroids, only estradiol synthesis selectively decreased in SH-SY5Y cells after H(2)O(2)-treatment. Testosterone conversion into estradiol was also inhibited by H(2)O(2). Real-time reverse transcription-polymerase chain reaction revealed aromatase gene repression in SH-SY5Y cells 12 h after the oxidative stress onset. Consistently, viability assays showed that chronic inhibition of aromatase activity by letrozole killed neuroblastoma cells. A 12-h pretreatment of SH-SY5Y cells with estradiol was protective against H(2)O(2)-induced death. In addition, estradiol was also capable of rescuing markedly neuroblastoma cells from letrozole-evoked death. Altogether, these results suggest that endogenous estradiol formation is pivotal for SH-SY5Y cell viability. Serum deprivation-evoked stress, which also killed SH-SY5Y cells, unaffected neurosteroidogenesis, indicating that inhibitory effect on neuroprotective-neurosteroid estradiol biosynthesis is specific for H(2)O(2)-induced stress. Selective targeting of neurosteroidogenic pathways may therefore constitute an interesting strategy against H(2)O(2)-evoked neurodegenerative processes.

Endogenous steroid production in the spinal cord and potential involvement in neuropathic pain modulation.

It has recently been demonstrated that the spinal cord (SC) is an active production center of neuroactive steroids including pregnenolone, dehydroepiandrosterone, progesterone and allopregnanolone. Indeed, anatomical, cellular and biochemical investigations have shown that the SC dorsal horn (DH), a pivotal structure in nociception, contains various active steroidogenic enzymes such as cytochrome P450side-chain-cleavage, cytochrome P450c17, 3beta-hydroxysteroid dehydrogenase, 5alpha-reductase and 3alpha-hydroxysteroid oxido-reductase. Reviewed here are several data obtained with in vitro and vivo experiments showing that endogenous steroids synthesized in the SC are involved in the modulation of nociceptive mechanisms. Various approaches were used as the real-time polymerase chain reaction after reverse transcription to determine the effects of neuropathic pain on the expression of genes encoding steroidogenic enzymes in the DH. Combination of the pulse-chase technique with high performance liquid chromatography and continuous flow scintillation detection allowed investigations of the impact of noxious signals on the activity of steroid-producing enzymes in the SC in vitro. Radioimmunological analyses of spinal tissue extracts contributed to determine the link between the painful state and endogenous steroid secretion in the SC in vivo. Finally, the physiological relevance of the modification of endogenous steroid formation in the SC during painful situation was discussed.

[Missed opportunities of in utero transfer of very preterm births (24-32 weeks gestation) in Aquitaine, 2003-2005]. / Opportunités manquées de transfert in utero des grands prématurés (24-32 semaines d'aménorrhée) en Aquitaine, 2003-2005.

OBJECTIVE: Describe and define the factors associated with missed opportunities of an in utero transfer (IUT), defined by by an absence of IUT where there was no counter-indication for a transfer. MATERIALS AND METHODS: Multicentric and retrospective cohort study within the Aquitaine perinatal healthcare network from 1st January 2003 to 30th June 2005 on deliveries between 24 and 32 weeks gestation, depending on whether the woman initially followed care in level I or II facilities benefited from an IUT at a level III facility or not. associated with missed opportunities of IUT were analysed by a logistic regression. RESULTS: Five hundred and twelve deliveries, eligible to deliver in level III facilities, were included in the study: 273 after an IUT and 239 in a level I or II maternity hospitals out of which 18% are defined as a missed opportunity of an in utero transfer. The multivariate analysis did not show a link between missed opportunities of an in utero transfer and the characteristics of maternities: status, size, level of care and distance from a level III facility. Only the delivery term appears to be linked to a missed opportunity of an in utero transfer (p=0.01): 32 weeks gestation versus 26-29 weeks gestation (RC=6.53; IC(95%): 2.00-21.25). While managing the delivery after 31 weeks gestation is considered suitable in a level IIb facility, the delivery term is no longer statistically related to a missed opportunity. CONCLUSION: This study serves as a first analysis of the Aquitaine perinatal healthcare network. It shows that missed opportunities of IUT does not seem to be linked to characteristics of maternities but seems to be linked to deliveries after 31 weeks gestation in level IIb facilities.

cdk1- and cdk2-mediated phosphorylation of MyoD Ser200 in growing C2 myoblasts: role in modulating MyoD half-life and myogenic activity.

We have examined the role of protein phosphorylation in the modulation of the key muscle-specific transcription factor MyoD. We show that MyoD is highly phosphorylated in growing myoblasts and undergoes substantial dephosphorylation during differentiation. MyoD can be efficiently phosphorylated in vitro by either purified cdk1-cyclin B or cdk1 and cdk2 immunoprecipitated from proliferative myoblasts. Comparative two-dimensional tryptic phosphopeptide mapping combined with site-directed mutagenesis revealed that cdk1 and cdk2 phosphorylate MyoD on serine 200 in proliferative myoblasts. In addition, when the seven proline-directed sites in MyoD were individually mutated, only substitution of serine 200 to a nonphosphorylatable alanine (MyoD-Ala200) abolished the slower-migrating hyperphosphorylated form of MyoD, seen either in vitro after phosphorylation by cdk1-cyclin B or in vivo following overexpression in 10T1/2 cells. The MyoD-Ala200 mutant displayed activity threefold higher than that of wild-type MyoD in transactivation of an E-box-dependent reporter gene and promoted markedly enhanced myogenic conversion and fusion of 10T1/2 fibroblasts into muscle cells. In addition, the half-life of MyoD-Ala200 protein was longer than that of wild-type MyoD, substantiating a role of Ser200 phosphorylation in regulating MyoD turnover in proliferative myoblasts. Taken together, our data show that direct phosphorylation of MyoD Ser200 by cdk1 and cdk2 plays an integral role in compromising MyoD activity during myoblast proliferation.

[Perinatal prognosis of pregnancies complicated by placental chronic intervillitis]. / Pronostic périnatal des grossesses compliquées d'intervillites chroniques placentaires.

UNLABELLED: SUBJECT. Massive Chronic Intervillositis is an infrequent inflammation lesion of the placenta, characterized by lymphohistiocytic intervillous infiltration, associated with fibrinoid deposition. The purpose of this study was to evaluate the perinatal outcome of pregnancies complicated by such lesions. MATERIAL AND METHODS: We conducted a descriptive retrospective multicentric analysis of a series of pregnancies for which placenta or products of abortion were analyzed between January 1995 and September 2005, at the University Hospital of Bordeaux. After re-examining the histology slides, we performed a semi-quantitative graduation of the cell infiltration and fibrinoid deposition. RESULTS: Twenty-five women were included (one twin-pregnancy and two histologic recurrences). We found three spontaneous abortions before 22 weeks, four intrauterine fetal deaths and three neonatals deaths. Seven of eight elective inductions pregnancies, were performed for intrauterine growth restriction less than 2.5 percentile. The rate of pregnancy loss was 55% and the perinatal mortality was 29%. 77% of fetuses are small for gestational age. Three mothers were pre-eclamptic. 21% of the fetuses had a congenital malformation. Only 32% of the fetuses were alive one week after birth. Histologically, 25% were associated with lesions of Villitis of Unknown Etiology. 77% of the cell infiltration was grade 3 and seemed to be correlated with severe growth restriction. We describe 3 cases of antenatal diagnosis of Chronic Intervillositis, realised after immunofixation on chorionic villous sampling. CONCLUSION: Massive Chronic Intervillositis is a recurrent lesion with a poor prognosis complicated by spontaneous abortion, intrauterine growth restriction and perinatal fetal death. Currently, there is no treatment. Chorionic villous sampling in severe growth restriction might be useful in order to obtain at the same time the fetal karyotype and an histological probe of the placenta.

A novel HLA-B*39 allele (HLA-B*3916) due to a rare mutation causing cryptic splice site activation.

A novel HLA-B*39 variant, found in an African patient with sickle cell anemia undergoing bone marrow transplantation is described. Initially suspected by inconsistent serological typing (B-blank, Bw6), then recognized by PCR-SSP, and finally characterized by nucleotide sequencing, this novel allele is designated HLA-B*3916. It differs from HLA-B*3910 by a point mutation (G to C) at position 17 of exon 3 causing glutamine to histidine change at codon 96 of alpha(2) domain, a conserved position among HLA class I alleles. cDNA sequence analysis further revealed the presence of both normally and abnormally spliced mRNA species in established cell lines. The abnormal species correspond to partial truncation of exon 3 presumably due to the nucleotide change in exon 3, which constitutes a new consensus acceptor splice site within this exon. We postulate that the observed blank is essentially the consequence of qualitative change in a critical region of this novel antigen as abnormal mRNA species are relatively less abundant than normal species. Because the residue 96 of the HLA class I heavy chain is directly involved in interaction with alpha(2)m, another interesting possibility is that an aminoacid change in this position would perturb such interaction and consequently could affect the serological specificity of B*3916, or its expression or both.

Analysis of monobutyryl and dibutyryl derivatives of adenosine 3',5'-monophosphate in biological samples using isocratic ion pair high-performance liquid chromatography.

Adenosine 3',5'-monophosphate (cyclic AMP), its dibutyryl and monobutyryl derivatives, and a number of other naturally occurring adenine-containing compounds were separated by isocratic ion pair high-performance liquid chromatography. A mobile phase consisting of 30% methanol in 0.1 M KH2PO4 (pH 4.0) containing 1 mM tetramethylammonium hydroxide as the counterion was used to separate the butyryl derivatives. To sufficiently separate cyclic AMP from other adenine-containing compounds, a mobile phase containing 6% methanol in the same aqueous buffer plus counterion was used. Extraction of these cyclic nucleotides from deproteinized biological samples using disposable reverse-phase extraction columns is described. This not only eliminated lipophilic contaminants, but also served to concentrate the samples. The outlined procedures were used to determine the concentrations of the butyryl derivatives in lung tissue and perfusate following a 35-min lung perfusion with 100 microM N6-O2'-dibutyryl cyclic AMP. The role of this technique in the analysis of cyclic nucleotide derivatives as compared with conventional assay procedures is discussed.

A strategy for the evaluation of sensory and pulmonary irritation due to chemical emissions from indoor sources.

Sensory and pulmonary irritation are physiological responses to chemical exposure which result in characteristic, measurable changes in respiratory activity in mice. A standard method has been applied to the estimation of sensory irritation associated with a specific chemical exposure. This method has been correlated with human responses to these chemicals. Symptoms associated with chemical irritants are consistent with complaints due to problems with indoor air quality, which may include eye and upper respiratory tract irritation, headaches, and nausea. A stepwise strategy for assessing the contribution of indoor products to sensory and pulmonary irritation is discussed in the current paper. The strategy includes product emissions testing using dynamic environmental chambers, the selection of suspected irritants for respiratory irritation testing, respiratory irritation testing of individual compounds are representative mixtures using synthesized atmospheres, and the evaluation of test data to determine those compounds which may contribute to sensory and pulmonary irritation in humans. The current strategy is being applied to evaluate carpet system materials and their constituent chemicals.

[Atypical hyperplasia of endometrium and hysteroscopy]. / Place de l'hystéroscopie dans le diagnostic et la prise en charge des hyperplasies atypiques de l'endomètre.

OBJECTIVE: To evaluate the risk of discovering an endometrial cancer when atypical hyperplasia was diagnosed by either endometrial samples using the pipelle device or hysteroscopic resection products. PATIENTS AND METHODS: A retrospective monocentric study from january 1990 to july 2000. Twenty-three patients with atypical hyperplasia were included. Initial endometrial status was provided by endometrial biopsyduring diagnosis hysteroscopy (12 cases) or by operative hysteroscopic resection products (11 cases). For 23 patients, operative hysteroscopy and analyse of products resected were performed. For all patients, there was no hysteroscopical aspect evocative of adenocarcinoma. For 23 patients, histopathological analysis of the hysterectomy piece precised the final diagnosis. RESULTS: Among the 23 hysterectomy pieces, 7 adenocarcinomas were diagnosed (30.4%). Risk for discovering adenocarcinoma when atypical hyperplasia was diagnosed by means of the pipelle biopsy device was 50% (6/12). Risk for discovering adenocarcinoma when atypical hyperplasia was diagnosed by means of operative hysteroscopy resection products was 5.9 % (1/17). DISCUSSION AND CONCLUSION: Atypical endometrial hyperplasia evidenced by pipelle biopsy device is often associated with adenocarcinoma. Diagnosis hysteroscopy however does not show evident pathological aspect of adenocarcinoma in such cases. Operative hysteroscopy allows in most cases correction of endometrial status. Risk of omitting adenocarcinoma when atypical hyperplasia is discovered on hysteroscopic resection pieces is low.

Toxic epidermal necrolysis.

Toxic epidermal necrolysis is a potentially fatal dermatological disease. Large bullae covering extensive areas of the body cause continuous exfoliation of skin, which requires immediate medical attention. Intraoral manifestations may precede cutaneous lesions. Two cases with different treatment protocols are presented.

Mechanism of transport for toxic cysteine conjugates in rat kidney cortex membrane vesicles.

Cysteine conjugate transport plays a key role in the interorgan transport of xenobitoic metabolites which are formed via the mercapturic acid pathway. In the rat, transport of cysteine conjugates could be an important factor in the selective nephrotoxicity of some toxic cysteine conjugates. However, little information is available on the molecular mechanism(s) of cysteine conjugate transport in the rat kidney. Therefore, we have investigated the polarity and the molecular driving forces for the transport of S-(1,2-dichlorovinyl)-L-cysteine (DCVC) in isolated membrane vesicles from rat kidney cortex. Our data suggest that Na+-dependent transport on the lumenal side is responsible for the uptake of cysteine conjugates across the apical membrane. No Na+-stimulated transport was found on the basolateral side and uptake of DCVC in basolateral membrane vesicles was not saturable. Na+-dependent transport in brush border membrane vesicles was inhibited by a variety of neutral amino acids and cysteine conjugates, but not by polar amino acids. Therefore, the transporter is similar to the Na+-dependent neutral amino acid transporter of rat kidney brush border membranes. The system L-specific substrate, 2-amino-2-norbornane carboxylic acid, was not inhibitory. The Km for the Na+-stimulated transport system in brush border membrane vesicles was 225 microM and the Vmax was 782 pmol/15 sec/mg of protein. We propose that the driving force for the apical transport of cysteine conjugates may be the coupling of the lumenal transport to the Na+-gradient. The data are discussed with regard to a transepithelial transport model for cysteine conjugates and the role transport plays in the molecular mechanism of cysteine conjugate toxicity.

The transport of S-cysteine conjugates in LLC-PK1 cells and its role in toxicity.

The transport of S-cysteine conjugates was studied in the kidney cell line, LLC-PK1, using the nephrotoxin, S-(1,2-dichlorovinyl)-L-cysteine (L-DCVC), as the model compound. The saturable uptake of this conjugate did not require sodium and was selectively inhibited by the amino acid transport system L-specific substrate, 2-amino-2-norbornane carboxylic acid, as well as a variety of other S-cysteine conjugates and neutral amino acids with large, nonpolar side chains. Kinetic studies suggested the existence of both low and high affinity transport systems with Km values that differed by 25-fold. Although these uptake systems showed no discernible differences in substrate specificity, the low affinity transport was more sensitive to trans-stimulation. L-DCVC uptake in subconfluent cultures was about 3-fold that of confluent cells, suggesting either adaptive regulation to cell growth or polarization of transport to the basolateral membrane. L-DCVC toxicity in LLC-PK1 cells was inhibited in the presence of nontoxic transport substrates but was potentiated when cells were preloaded with many of the same compounds, indicating that transport may be a rate-limiting factor in L-DCVC-induced toxicity under certain circumstances. The possible role of this system L-like uptake in the transport of S-cysteine conjugates in vivo is discussed.

High bicoid levels render the terminal system dispensable for Drosophila head development.

In Drosophila, the gradient of the Bicoid (Bcd) morphogen organizes the anteroposterior axis while the ends of the embryo are patterned by the maternal terminal system. At the posterior pole, expression of terminal gap genes is mediated by the local activation of the Torso receptor tyrosine kinase (Tor). At the anterior, terminal gap genes are also activated by the Tor pathway but Bcd contributes to their activation. Here we present evidence that Tor and Bcd act independently on common target genes in an additive manner. Furthermore, we show that the terminal maternal system is not required for proper head development, since high levels of Bcd activity can functionally rescue the lack of terminal system activity at the anterior pole. This observation is consistent with a recent evolution of an anterior morphogenetic center consisting of Bcd and anterior Tor function.

Immunohistochemical localization of glutamine transaminase K, a rat kidney cysteine conjugate beta-lyase, and the relationship to the segment specificity of cysteine conjugate nephrotoxicity.

Rat kidney glutamine transaminase K is a major rat kidney cysteine conjugate beta-lyase and is a key enzyme in the nephrotoxicity of some cysteine conjugates. However, it has not been demonstrated that the beta-lyase is present in the target cells. Furthermore, although all segments of the proximal tubule are affected by high doses of nephrotoxic cysteine conjugates, the S3 segment is the most sensitive. Because heterogeneous distribution of the beta-lyase could account for the enhanced sensitivity, antibody raised against rat kidney cysteine conjugate beta-lyase has been prepared and used to investigate the distribution of the enzyme in kidney and other tissues. The data show that the enzyme is highest in rat kidney, consistent with enzyme activity data. By immunohistochemical staining, no enzyme is present in the glomeruli or distal tubular elements of the kidney. The enzyme is present only in the target cells, the renal proximal tubular epithelium. However, the distribution of the beta-lyase within the proximal tubule is not consistent with the hypothesis that a higher concentration of the enzyme in the S3 segment accounts for the greater sensitivity of S3 to nephrotoxic cysteine conjugates compared to S1 and S2. Several alternative hypotheses are discussed.

Determination of DRB alleles using PCR amplification and allele-specific primers.

HLA class-II allelic diversity is commonly defined using polymerase chain reaction (PCR) in combination with sequence-specific oligotyping (PCR-SSO) or the combination of PCR and restriction fragment length polymorphism methods (PCR-RFLP). Nevertheless, the identification of the DRB polymorphism by PCR-SSO is a time-consuming procedure and the PCR-RFLP is cumbersome. A rapid technique which allows a precise and extensive HLA-DRB typing is required, particularly in order to study the role of class-II matching in organ transplantation. A DRB typing method based on the detection and length of PCR products amplified using combination of allele specific primers has been developed. Thirty-four DRB alleles (29 DRB1, 4DRB3, 1DRB4) can be detected using 29 primers distributed into 19 amplification mixtures.

Co-amoxiclav pharmacokinetics during posttraumatic hemorrhagic shock.

OBJECTIVE: To determine the effects of severe trauma with hemorrhagic shock on amoxicillin and clavulanate concentrations in plasma and their pharmacokinetics. DESIGN: A prospective, open, descriptive study. SETTING: A 12-bed, adult surgical intensive care unit in a university-affiliated hospital in France. SUBJECTS: Subjects were 12 patients (10 men, 2 women) with severe trauma: median (range) Injury Severity Score, 38 (17-48); Acute Physiology and Chronic Health Evaluation II, 16 (7-38); Simplified Acute Physiology Score II, 41 (23-77). Also enrolled were 12 healthy volunteers who were matched on age (+/-5 yrs), gender, and body-surface area (+/-20 cm2). All the trauma patients suffered hemorrhagic shock defined as the association of at least one episode of systolic blood pressure <90 mm Hg and an intravascular volume expansion >2000 mL between trauma and surgery. INTERVENTION: Prophylactic perioperative administration of 2 g of amoxicillin and 0.2 g of clavulanate in combination during the first 12 hrs posttrauma in patients, and at the start of the pharmacokinetic study in volunteers. MEASUREMENTS AND MAIN RESULTS: Serial plasma samples (n = 13) were obtained after the first antibiotic administration to measure antibiotic levels by using high-performance liquid chromatography assays. Compared with volunteers, trauma patients had higher plasma amoxicillin and clavulanate concentrations, attributed to a reduction of the volume of distribution (p =.001 and p =.06, respectively) and, to a lesser extent, of the total body clearance (p =.09 and p =.20, respectively). Consequently, amoxicillin and clavulanate elimination half-lives were similar for the two groups of subjects. The interindividual variabilities for all the amoxicillin pharmacokinetic parameters were higher in patients. CONCLUSIONS: In trauma patients with hemorrhagic shock requiring surgery, the administration of 2 g of amoxicillin and 0.2 g of clavulanate seems adequate, according to the antibiotic concentrations observed in plasma for both drugs. However, further studies exploring antibiotic concentrations in tissues are warranted.

Two distinct domains of Bicoid mediate its transcriptional downregulation by the Torso pathway.

The transcriptional activity of the Bicoid morphogen is directly downregulated by the Torso signal transduction cascade at the anterior pole of the Drosophila embryo. This regulation does not involve the homeodomain or direct phosphorylation of Bicoid. We analyse the transcriptional regulation of Bicoid in response to the Torso pathway, using Bicoid variants and fusion proteins between the Bicoid domains and the Gal4 DNA-binding domain. We show that Bicoid possesses three autonomous activation domains. Two of these domains, the serine/threonine-rich and the acidic domains, are downregulated by Torso, whereas the third activation domain, which is rich in glutamine, is not. The alanine-rich domain, previously described as an activation domain in vitro, has a repressive activity that is independent of Torso. Thus, Bicoid downregulation by Torso results from a competition between the glutamine-rich domain that is insensitive to Torso and the serine/threonine-rich and acidic activation domains downregulated by Torso. The alanine-rich domain contributes to this process indirectly by reducing the global activity of the protein and in particular the activity of the glutamine-rich domain that might otherwise prevent downregulation by Torso.

Bicoid functions without its TATA-binding protein-associated factor interaction domains.

Four maternal systems are known to pattern the early Drosophila embryo. The key component of the anterior system is the homeodomain protein Bicoid (Bcd). Bcd needs the contribution of another anterior morphogen, Hunchback (Hb), to function properly: Bcd and Hb synergize to organize anterior development. A molecular mechanism for this synergy has been proposed to involve specific interactions of Bcd and Hb with TATA-binding protein-associated factors (TAFIIs) that are components of the general transcription machinery. Bcd contains three putative activation domains: a glutamine-rich region, which interacts in vitro with TAFII110; an alanine-rich domain, which targets TAFII60; and a C-terminal acidic region, which has an unknown role. We have generated flies carrying bcd transgenes lacking one or several of these domains to test their function in vivo. Surprisingly, a bcd transgene that lacks all three putative activation domains is able to rescue the bcdE1 null phenotype to viability. Moreover, the development of these embryos is not affected by the presence of dominant negative mutations in TAFII110 or TAFII60. This means that the interactions observed in vitro between Bcd and TAFII60 or TAFII110 aid transcriptional activation but are dispensable for normal development.

Malolactic fermentation by engineered Saccharomyces cerevisiae as compared with engineered Schizosaccharomyces pombe.

The ability of yeast strains to perform both alcoholic and malolactic fermentation in winemaking was studied with a view to achieving a better control of malolactic fermentation in enology. The malolactic gene of Lactococcus lactis (mleS) was expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe. The heterologous protein is expressed at a high level in cell extracts of a S. cerevisiae strain expressing the gene mleS under the control of the alcohol dehydrogenase (ADH1) promoter on a multicopy plasmid. Malolactic enzyme specific activity is three times higher than in L. lactis extracts. Saccharomyces cerevisiae expressing the malolactic enzyme produces significant amounts of L-lactate during fermentation on glucose-rich medium in the presence of malic acid. Isotopic filiation was used to demonstrate that 75% of the L-lactate produced originates from endogenous L-malate and 25% from exogenous L-malate. Moreover, although a small amount of exogenous L-malate was degraded by S. cerevisiae transformed or not by mleS, all the exogenous degraded L-malate was converted into L-lactate via a malolactic reaction in the recombinant strain, providing evidence for very efficient competition of malolactic enzyme with the endogenous malic acid pathways. These results indicate that the sole limiting step for S. cerevisiae in achieving malolactic fermentation is in malate transport. This was confirmed using a different model, S. pombe, which efficiently degrades L-malate. Total malolactic fermentation was obtained in this strain, with most of the L-malate converted into L-lactate and CO2. Moreover, L-malate was used preferentially by the malolactic enzyme in this strain also.