[Genome composition analysis of the reassortant influenza viruses used in seasonal and pandemic live attenuated influenza vaccine].

The cold-adapted, temperature sensitive and attenuated influenza master donor viruses A/Leningrad/134/17/57 (H2N2) and B/USSR/ 60/69 were used to generate the vaccine viruses to be included in live attenuated influenza vaccine. These vaccine viruses typically are 6:2 reassortant viruses containing the surface antigens hemagglutinin and neuraminidase of current wild type influenza A and influenza B viruses with the gene segments encoding the internal viral proteins, and conferring the cold-adapted, temperature sensitive and attenuated phenotype, being inherited from the master donor viruses. The 6:2 reassortant viruses were selected from co-infections between master donor virus and wild type viruses that theoretically may yield as many as 256 combinations of gene segments and thus 256 genetically different viruses. As the time to generate and isolate vaccine viruses is limited and because only 6:2 reassortant viruses are allowed as vaccine viruses, screening needs to be both rapid and unambiguous. The screening of the reassortant viruses by RT-PCRs using master donor virus and wild type virus specific primer sets was described to select both influenza A and influenza B 6:2 reassortant viruses to be used in seasonal and pandemic live attenuated vaccine.

Three-dimensional quantification of regional left-ventricular dyssynchrony by magnetic resonance imaging.

Heart failure accounts for over five million patients in the United States alone. Many of them present dyssynchronous left ventricular (LV) contraction, whose treatment by cardiac resynchronization therapy (CRT) is until now guided by electrocardiographic analysis. One third of the selected patients, however, does not respond to the therapy. Aiming at improving the response rate, recent studies showed the importance of left bundle branch block (LBBB) configurations. Therefore, in order to detect motion patterns that relate to LBBB, this paper presents a novel method for three-dimensional quantification of regional LV mechanical dyssynchrony. LV wall-motion analysis is performed on magnetic resonance imaging (MRI) cines segmented by commercial software. Mutual delays between endocardial wall motion in different LV regions are estimated by cross correlation followed by phase difference analysis in frequency domain, achieving unlimited time resolution. Rather than focusing on the systolic phase, the full cardiac cycle is used to estimate the contraction timing. The method was successfully validated against MRI tagging in five dogs before and after LBBB induction. Preliminary validation in humans with 10 LBBB patients and 7 healthy subjects showed the method feasibility and reproducibility, with sensitivity and specificity in LBBB detection equal to 95.1% and 99.4%, respectively.

Intra-thoracic blood volume measurement by contrast magnetic resonance imaging.

The intra-thoracic blood volume (ITBV) is a cardiovascular parameter related to the cardiac preload and left ventricular function. Its assessment is, therefore, important for diagnosis and follow-up of several cardiovascular dysfunctions. Nowadays, the ITBV can be accurately measured only by invasive indicator dilution techniques, which require a double catheterization of the patient. In this study, a novel technique is presented for ITBV assessment by dynamic magnetic resonance imaging after intravenous injection of a small bolus of gadolinium chelate. The dose was chosen on the basis of in vitro calibration. The bolus first pass is detected from a simultaneous dynamic image series of the right and left ventricles. Two indicator dilution curves are derived and used to inspect the transpulmonary dilution system. Various mathematical models for the interpretation of the measured indicator dilution curves are compared. The ITBV is assessed as the product of the transpulmonary mean transit time of the indicator and the cardiac output, obtained by phase contrast magnetic resonance angiography. In vitro measurements showed a correlation coefficient larger than 0.99 and preliminary tests with volunteers proved the feasibility of the method, opening new possibilities for noninvasive quantitative cardiovascular diagnostics.

Towards patient-specific risk assessment of abdominal aortic aneurysm.

Diagnosis of vascular disease and selection and planning of therapy are to a large extent based on the geometry of the diseased vessel. Treatment of a particular vascular disease is usually considered if the geometrical parameter that characterizes the severity of the disease, e.g. % vessel narrowing, exceeds a threshold. The thresholds that are used in clinical practice are based on epidemiological knowledge, which has been obtained by clinical studies including large numbers of patients. They may apply "on average", but they can be sub-optimal for individual patients. To realize more patient-specific treatment decision criteria, more detailed knowledge may be required about the vascular hemodynamics, i.e. the blood flow and pressure in the diseased vessel and the biomechanical reaction of the vessel wall to this flow and pressure. Over the last decade, a substantial number of publications have appeared on hemodynamic modeling. Some studies have provided first evidence that this modeling may indeed be used to support therapeutic decisions. The goal of the research reported in this paper is to go one step further, namely to investigate the feasibility of a patient-specific hemodynamic modeling methodology that is not only effective (improves therapeutic decisions), but that is also efficient (easy to use, fast, as much as possible automatic) and robust (insensitive to variation in the quality of the input data, same outcome for different users). A review is presented of our research performed during the last 5 years and the results that were achieved. This research focused on the risk assessment for one particular disease, namely abdominal aortic aneurysm, a life-threatening dilatation of the abdominal aorta.

Subunit vaccines based on intimin and Efa-1 polypeptides induce humoral immunity in cattle but do not protect against intestinal colonisation by enterohaemorrhagic Escherichia coli O157:H7 or O26:H-.

Enterohaemorrhagic Escherichia coli (EHEC) infections in humans are an important public health concern and are commonly acquired via contact with ruminant faeces. Cattle are a key control point however cross-protective vaccines for the control of EHEC in the bovine reservoir do not yet exist. The EHEC serogroups that are predominantly associated with human infection in Europe and North America are O157 and O26. Intimin and EHEC factor for adherence (Efa-1) play important roles in intestinal colonisation of cattle by EHEC and are thus attractive candidates for the development of subunit vaccines. Immunisation of calves with the cell-binding domain of intimin subtypes beta or gamma via the intramuscular route induced antigen-specific serum IgG1 and, in some cases salivary IgA responses, but did not reduce the magnitude or duration of faecal excretion of EHEC O26:H- (Int(280)-beta) or EHEC O157:H7 (Int(280)-gamma) upon subsequent experimental challenge. Similarly, immunisation of calves via the intramuscular route with the truncated Efa-1 protein (Efa-1') from EHEC O157:H7 or a mixture of the amino-terminal and central thirds of the full-length protein (Efa-1-N and M) did not protect against intestinal colonisation by EHEC O157:H7 (Efa-1') or EHEC O26:H- (Efa-1-N and M) despite the induction of humoral immunity. A portion of the serum IgG1 elicited by the truncated recombinant antigens in calves was confirmed to recognise native protein exposed on the bacterial surface. Calves immunised with a mixture of Int(280)-gamma and Efa-1' or an EHEC O157:H7 bacterin via the intramuscular route then boosted via the intranasal route with the same antigens using cholera toxin B subunit as an adjuvant were also not protected against intestinal colonisation by EHEC O157:H7. These studies highlight the need for further studies to develop and test novel vaccines or treatments for control of this important foodborne pathogen.

Comparison of the efficacy of a subunit and a live streptomycin-dependent porcine pleuropneumonia vaccine.

OBJECTIVE: To evaluate the efficacy of two new-generation porcine pleuropneumonia vaccines when challenged with Australian isolates of Actinobacillus pleuropneumoniae of serovars 1 and 15. DESIGN: The Porcilis APP vaccine and an experimental streptomycin-dependent strain of A pleuropneumoniae were evaluated in a standardised pen trial. Each vaccine/challenge group consisted of 10 pigs. RESULTS: With the serovar 1 challenge, the Porcilis APP vaccine and the live vaccine, compared with the control group, gave significant protection in terms of clinical signs, lung lesions, re-isolation scores and average daily gain (ADG) postchallenge. Only the Porcilis APP vaccine provided significant protection against mortality. In the serovar 15 challenged pigs, the only significant difference detected was that the Porcilis APP vaccinated pigs had a better postchallenge ADG than the controls. None of the Porcilis APP vaccinated pigs showed signs of depression postvaccination and none were euthanased after challenge with either serovar 1 or 15. The pigs vaccinated with the live vaccine showed obvious depression after each vaccination and a total of 3 pigs were euthanased after challenge (one with serovar 1 and two with serovar 15). CONCLUSIONS: Both of the vaccines provided significant protection against a severe challenge with serovar 1 A pleuropneumoniae. Neither vaccine was effective against a serovar 15 A pleuropneumoniae challenge. There was evidence that the Porcilis APP vaccine did provide some protection against the serovar 15 challenge because the ADG, after challenge of pigs given this vaccine, was greater than the control pigs.

An evaluation of the role of antibodies to Actinobacillus pleuropneumoniae serovar 1 and 15 in the protection provided by sub-unit and live streptomycin-dependent pleuropneumonia vaccines.

OBJECTIVE: To evaluate the serological response of pigs receiving either the Porcilis APP vaccine or a modified live vaccine based on a streptomycin-dependent (SD) strain of Actinobacillus pleuropneumoniae, and then challenged with an Australian isolate of A. pleuropneumoniae of either serovar 1 or 15 as a means of understanding the protection provided by both vaccines against serovar 1 but not against serovar 15. DESIGN: The serological tests evaluated were serovar-specific polysaccharide ELISA tests (for serovar 1 and 15), ELISA tests for antibodies to three A. pleuropneumoniae toxins (ApxI, ApxII and ApxIII) as well as to a 42 kDa outer membrane protein (OMP), a haemolysin neutralisation (HN) assay and immunoblotting. The tests were used to detect antibodies in vaccinated pigs that had been shown to be protected against serovar 1 but not serovar 15. RESULTS: In the polysaccharide antigen ELISA assays, both vaccines resulted in a significant rise in the titre in the serovar 1 ELISA but not the serovar 15 ELISA. The Porcilis APP vaccinated pigs showed a significant response in the ApxI, ApxIII and 42 kDa OMP ELISA. In the ApxII ELISA, all pigs tested (the Porcilis APP vaccinates and the controls) were positive on entry to the trial. In the HN assay, the Porcilis APP vaccinated pigs showed a significant response after one dose while the SD vaccinated pigs required two doses of vaccine before a marked rise in titre was induced. Immunoblotting revealed that neither vaccine generated antibodies that recognised the ApxIII produced by serovar 15. CONCLUSIONS: The failure of these vaccines to provide protection against serovar 15 may be due to novel virulence factors possessed by serovar 15, significant differences between the ApxIII toxin of serovar 15 and those present in the Porcilis APP vaccine or failure by both vaccines to induce antibodies to the serovar 15 specific polysaccharide.

Cytosolic phospholipase A2 and lipoxygenase are involved in cell cycle progression in neuroblastoma cells.

Arachidonic acid has been implicated in regulating cellular proliferation, and is preferentially released by the 85-kDa cytosolic phospholipase A2 (cPLA2). Recently, we demonstrated that cPLA2 is activated at distinct periods during the ongoing cell cycle of neuroblastoma cells. The purpose of the present study was to establish the role of these cPLA2 activity peaks in cell cycle progression. Inhibition of cPLA2 activity with arachidonyl trifluoromethylketone (ATK) in early G1 phase reduced DNA synthesis markedly. A 24-h incubation with ATK revealed no significant difference in cell number compared to untreated cells, although cPLA2 activity was still inhibited. This suggests redundancy of different PLA2 enzymes. Lipoxygenase inhibition in early G1 resulted in G1 phase arrest, whereas inhibitors for cyclooxygenase had no effect. Furthermore, cells stopped progressing through S phase when lipoxygenase was inhibited in early S phase, demonstrating the requirement of lipoxygenase products for S phase progression.

Proposal of a new serovar of Actinobacillus pleuropneumoniae: serovar 15.

We report on the re-examination of nine Australian isolates of Actinobacillus pleuropneumoniae that have been previously assigned to serovar 12. In the ring precipitation test, none of the nine isolates reacted with antisera to serovars 1-14 of A. pleuropneumoniae. Antiserum prepared against one of the Australian isolates gave no reaction with any of the 14 recognised serovar reference strains, except serovar 7. This reaction of the HS143 antiserum with serovar 7 antigen could be removed by adsorption with serovar 7 antigen. The adsorbed antiserum remained reactive with HS143 and the other eight Australian isolates. The nine Australian isolates were all shown to express ApxII and ApxIII, found in serovars 2, 4, 6 and 8, as well as the 42kDa outer membrane protein found in all serovars of A. pleuropneumoniae. The nine Australian isolates were found to possess the following toxin associated genes apxIBD, apxIICA, apxIIICA, apxIIIBD and apxIVA. The toxin gene profile of the Australian isolates is typical of A. pleuropneumoniae serovars 2, 4, 6 and 8. On the basis of the serological characterisation results and the toxin gene profiles, we propose that these isolates represent a new serovar of A. pleuropneumoniae--serovar 15--with HS143 being the reference strain for the new serovar.

Cytosolic phospholipase A(2) activity during the ongoing cell cycle.

Cytosolic phospholipase A(2) (cPLA(2)) is of special interest because it selectively releases arachidonic acid from membrane phospholipids. Arachidonic acid has been implicated to play an important role in various cellular responses. Recently arachidonic acid release and prostaglandin synthesis have been shown to be cell cycle dependent and therefore the activity of cPLA(2) during the ongoing cell cycle was investigated, using the mitotic shake off method for cell synchronisation. cPLA(2) activity was high in mitotic cells and decreased rapidly in the early G1 phase. A strong increase in activity was measured following the G1/S transition in both neuroblastoma and Chinese hamster ovary cells. The changes in activity were not due to a difference in cPLA(2) expression but due to phosphorylation of cPLA(2). Phosphorylation of cPLA(2) occurs through MAPK since the use of a specific MAPK kinase inhibitor and serum depletion of synchronised cells inhibited cPLA(2) activity.

Phosphorylation of p42/44(MAPK) by various signal transduction pathways activates cytosolic phospholipase A(2) to variable degrees.

Arachidonic acid has been implicated to play a role in physiological and pathophysiological processes and is selectively released by the 85-kDa cytosolic phospholipase A(2) (cPLA(2)). The activity of cPLA(2) is regulated by calcium, translocating the enzyme to its substrate, and by phosphorylation by a mitogen-activated protein kinase (MAPK) family member and a MAPK-activated protein kinase. In this study, the signal transduction pathways in growth factor-induced phosphorylation of p42/44(MAPK) and cPLA(2) activation were investigated in Her14 fibroblasts. p42/44(MAPK) in response to epidermal growth factor was not only phosphorylated via the Raf-MEK pathway but mainly through protein kinase C (PKC) or a related or unrelated kinase in which the phosphorylated p42/44(MAPK) corresponded with cPLA(2) activity. Serum-induced phosphorylation of p42/44(MAPK) also corresponded with cPLA(2) activity but is predominantly mediated via Raf-MEK and partly through PKC or a related or unrelated kinase. In contrast, activation of PKC by phorbol ester did not result in increased cPLA(2) activity, while p42/44(MAPK) is phosphorylated, mainly via Raf-MEK and through MEK. Moreover, p42/44(MAPK) phosphorylation is present in quiescent and proliferating cells, and p42/44(MAPK) is entirely phosphorylated via Raf-MEK, but it only corresponds to cPLA(2) activity in the former cells. Collectively, these data show that p42/44(MAPK) in proliferating, quiescent, and stimulated cells is phosphorylated by various signal transduction pathways, suggesting the activation of different populations of p42/44(MAPK) and cPLA(2).

Group IIA and group V secretory phospholipase A(2): quantitative analysis of expression and secretion and determination of the localization and routing in rat mesangial cells.

Mesangial cells can be induced to express group IIA and group V secretory phospholipase A(2) (sPLA(2)) at the mRNA level and at the protein level. In this report we quantitatively analyze the expression of both proteins in stimulated cells by Western blot techniques. We found that 75-80% of the total amount of synthesized group IIA sPLA(2) was secreted. The synthesized group V sPLA(2), however, was present almost exclusively intracellularly. The amount of group V present in the cell was comparable to the intracellular amount of group IIA sPLA(2). We furthermore studied the localization and routing of both proteins. Using fusion proteins of the group IIA or group V pre-sPLA(2) with green fluorescent protein it was established that both presequences are able to direct the proteins to the Golgi system. In immunofluorescence studies group V sPLA(2) expressed by rat mesangial cells was located in a punctate pattern in the cytosol with an enrichment near the nucleus. Immunofluorescent confocal laser scanning microscopy revealed that the group V and IIA sPLA(2) show partial colocalization in a Golgi-like structure in the inner part in the cell, but no colocalization was seen in the vesicles in the cytoplasm. The images also showed that group IIA sPLA(2) was located throughout the cell while group V was mainly present in the inner part of the cell. After treatment of the cells with brefeldin A or monensin the group IIA enzyme could no longer be detected, while group V sPLA(2) was still present although its localization was somewhat dependent on the treatment. Collectively, these results indicate that the two enzymes differ in both localization and routing in the cell, which underscores the hypothesis that the enzymes might have different functions.

Cholesterol in the year 2000.

Cholesterol research was one of the key areas of scientific investigation in the 20th century. Little was known about the structure of cholesterol until the pioneering research of A. Windaus and H. Wieland in the first part of the century. The structure of cholesterol was completely elucidated in 1932. With the development of isotopic tracers in the 1930s studies on cholesterol biosynthesis were initiated. In 1942 K. Bloch and D. Rittenberg showed that deuterium-labeled acetate was incorporated into the ring structure and side chain of cholesterol. Another important discovery from Bloch's laboratory was that squalene was a precursor of cholesterol. In 1956, the main elements of the biosynthetic pathway became known when isopentenyl pyrophosphate was discovered as a precursor. In 1966, J. Cornforth and G. Popjak predicted that there were 16234 possible stereochemical pathways by which mevalonate could be converted into squalene. They subsequently showed which of these pathways was correct. In the 1970s and 1980s K. Bloch was able to provide intriguing evidence for an evolutionary advantage of cholesterol over lanosterol or some of the intermediates in the conversion of lanosterol to cholesterol. The last quarter of the 20th century was when M. Brown and J. Goldstein showed that the low density lipoprotein receptor was a key regulator of cholesterol homeostasis. They have also demonstrated that cholesterol balance in the cell is transcriptionally regulated via the sterol regulatory element binding protein. In the later part of the 20th century drugs were developed that effectively lower plasma cholesterol and lessen the risk of atherosclerosis and cardiovascular disease.

Sera of patients suffering from inflammatory diseases contain group IIA but not group V phospholipase A(2).

During recent years, the high phospholipase A(2) (PLA(2)) concentrations at sites of inflammation and in circulation in several life-threatening diseases, such as sepsis, multi-organ dysfunction and acute respiratory distress syndrome, has generally been ascribed to the non-pancreatic group IIA PLA(2). Recently the family of secreted low molecular mass PLA(2) enzymes has rapidly expanded. In some cases, a newly described enzyme appeared to be cross-reactive with antibodies against the group IIA enzyme. For this reason, reports describing the expression of group IIA PLA(2) during inflammatory conditions need to be reevaluated. Here we describe the identification of the PLA(2) activity in sera of acute chest syndrome patients and in sera of trauma victims. In both cases, the PLA(2) activity was identified as group IIA. This classification was based upon cross-reactivity with monoclonal antibodies against group IIA PLA(2) which do not recognize the recombinant human group V enzyme. Moreover, purification of the enzymatic activity from the two sera followed by N-terminal amino acid sequence analyses revealed only the presence of group IIA enzyme.

Regulation of cytosolic phospholipase A(2) in a new perspective: recruitment of active monomers from an inactive clustered pool.

cPLA(2) plays a key role in many signal transduction cascades by hydrolyzing arachidonic acid from membrane phospholipids. Tight control of cPLA(2) activity by a number of regulatory mechanisms is essential to its cellular function. We recently described the localization of cPLA(2) in clusters in fibroblasts and now propose that these clusters reflect a localized inactive pool from which active monomers can be recruited to keep cPLA(2) activity under control on the subcellular level. Using an electron microscopic in vitro approach, we show that cPLA(2) monomers, but not the clusters, bind to membranes in a Ca(2+)-dependent manner. This binding is accompanied by hydrolytic activity. The present data combined with our previous observation of a relative abundance of clusters over monomers in fixed fibroblasts [Bunt, G., de Wit, J., van den Bosch, H., Verkleij, A., and Boonstra, J. (1997) J. Cell Sci. 110, 2449-2459] gives rise to a concept of cPLA(2) regulation in which small amounts of active monomers are recruited to fulfill their function upon stimulation. This is in contrast to processes described for inflammatory cells, where a substantial part of the cytoplasmically localized cPLA(2) translocates to the perinuclear region upon stimulation to become active. Small-scale regulation of cPLA(2) by the proposed cluster-monomer cycle allows local and strictly confined control of cPLA(2) activity, apparently necessary for its cellular role in fibroblasts.

A functional cra gene is required for Salmonella enterica serovar typhimurium virulence in BALB/c mice.

A minitransposon mutant of Salmonella enterica serovar Typhimurium SR-11, SR-11 Fad(-), is unable to utilize gluconeogenic substrates as carbon sources and is avirulent and immunogenic when administered perorally to BALB/c mice (M. J. Utley et al., FEMS Microbiol. Lett., 163:129-134, 1998). Here, evidence is presented that the mutation in SR-11 Fad(-) that renders the strain avirulent is in the cra gene, which encodes the Cra protein, a regulator of central carbon metabolism.

Regulation of the expression of group IIA and group V secretory phospholipases A(2) in rat mesangial cells.

Rat mesangial cells synthesize and secrete a secretory phospholipase A(2) upon stimulation of the cells with cytokines, like IL-1beta and TNF and with cAMP elevating agents like forskolin. This enzyme was previously characterized to belong to group IIA sPLA(2). The discovery of several other low molecular weight phospholipases, like group IIC in murine testis and group V in human and rat heart, prompted investigations on the presence of group IIC and group V sPLA(2) in rat mesangial cells. This was done by isolating the RNA from stimulated cells and performing RT-PCR, using primers specific for group IIC and V sPLA(2). The results indicate that rat mesangial cells upon stimulation express next to group IIA also group V sPLA(2). No indications were obtained for the expression of group IIC sPLA(2). The regulation of the expression of group V sPLA(2) at the mRNA level was further investigated by examining the time-dependent expression, the influence of dexamethasone and the signaling route of the IL-1beta stimulation. The results show that the IL-1beta induced expression of group V sPLA(2) mRNA was time dependent and, similar to that of group IIA sPLA(2) mRNA, involves activation of NF-kappaB. However, in contrast to the group IIA sPLA(2), the expression of group V sPLA(2) was not influenced by the presence of dexamethasone. The expression of both phospholipases was also examined at the protein level in stimulated mesangial cells. Western blot analysis shows that stimulated mesangial cells synthesize both group IIA and group V sPLA(2) protein but the expression of group V is lower compared to that of group IIA sPLA(2). In addition, the extent of secretion into the medium appears to be considerably higher for group IIA than for group V sPLA(2).

Extracellular nucleotides activate the p38-stress-activated protein kinase cascade in glomerular mesangial cells.

1. Extracellular ATP and UTP have been reported to activate a nucleotide receptor (P2Y2-receptor) that mediates arachidonic acid release with subsequent prostaglandin formation, a reaction critically depending on the activity of a cytosolic phospholipase A2. In addition, extracellular nucleotides trigger activation of the classical mitogen-activated protein kinase (MAPK) cascade and cell proliferation as well as of the stress-activated protein kinase (SAPK) cascade. 2. In this study, we report that ATP and UTP are also able to activate the p38-MAPK pathway as measured by phosphorylation of the p38-MAPK and its upstream activators MKK3/6, as well as phosphorylation of the transcription factor ATF2 in a immunocomplex-kinase assay. 3. Time courses reveal that ATP and UTP induce a rapid and transient activation of the p38-MAPK activity with a maximal activation after 5 min of stimulation which declined to control levels over the next 20 min. 4. A series of ATP and UPT analogues were tested for their ability to stimulate p38-MAPK activity. UTP and ATP were very effective analogues to activate p38-MAPK, whereas ADP and gamma-thio-ATP had only moderate activating effects. 2-Methyl-thio-ATP, beta gamma-imido-ATP, AMP, adenosine and UDP had no significant effects of p38-MAPK activity. In addition, the extracellular nucleotide-mediated effect on p38-MAPK was almost completely blocked by 1 mM of suramin, a putative P2-purinoceptor antagonist. 5. In summary, these results demonstrate for the first time that extracellular nucleotides are able to activate the MKK3/6- p38-MAPK cascade most likely via the P2Y2-receptor. Moreover, this finding implies that all three MAPK subtypes are signalling candidates for extracellular nucleotide-stimulated cell responses.

Alkyl-dihydroxyacetonephosphate synthase. Presence and role of flavin adenine dinucleotide.

Alkyl-dihydroxyacetonephosphate synthase is a peroxisomal enzyme involved in ether lipid synthesis. It catalyzes the exchange of the acyl chain in acyl-dihydroxyacetonephosphate for a long chain fatty alcohol, yielding the first ether linked intermediate, i.e. alkyl-dihydroxyacetonephosphate, in the pathway of ether lipid biosynthesis. Although this reaction is not a net redox reaction, the amino acid sequence of the enzyme suggested the presence of a flavin adenine dinucleotide (FAD)-binding domain. In this study we show that alkyl-dihydroxyacetonephosphate synthase contains an essential FAD molecule as cofactor, which is evidenced by fluorescence properties, UV-visible absorption spectra and the observation that the enzyme activity is dependent on the presence of this cofactor in a coupled in vitro transcription/translation assay. Furthermore, we could demonstrate that the FAD cofactor directly participates in catalysis. Upon incubation of the enzyme with the substrate palmitoyl-dihydroxyacetonephosphate, the flavin moiety is reduced, indicating that in this initial step the substrate is oxidized. Stopped flow experiments show that the reduction of the flavin moiety is a monophasic process yielding a oxygen stable, reduced enzyme species. Upon addition of hexadecanol to the reduced enzyme species, the flavin moiety is efficiently reoxidized. A hypothetical reaction mechanism is proposed that is consistent with the data in this paper and with previous studies.