Effect of subinhibitory concentrations of tigecycline and ciprofloxacin on the expression of biofilm-associated genes and biofilm structure of Staphylococcus epidermidis.

Staphylococcus epidermidis is a leading cause of foreign body-associated infections. This is related to the bacterium's ability to form biofilms on synthetic materials. Bacteria within a biofilm may be exposed to subinhibitory concentrations (sub-MICs) of antibiotics because of an agent's limited penetration into the biofilm core. Here, we investigated the effect of sub-MICs of tigecycline and ciprofloxacin on the expression of biofilm-associated genes, i.e. icaA, altE and sigB, and the biofilm structure of five clinical isolates of S. epidermidis. For most tested isolates, the expression of these genes increased after exposure to 0.25 MIC and 0.5 MIC tigecycline. A slight decrease in icaAmRNA levels was observed only in two isolates in the presence of 0.25 MIC tigecycline. The effect of ciprofloxacin exposure was isolate-dependent. At 0.5 MIC, ciprofloxacin induced an increase of sigB and icaAmRNA levels in three of the five tested isolates. At the same time, expression of the altE gene increased in all isolates (from 1.3-fold to 42-fold, depending on the strain). Confocal laser scanning microscopy analysis indicated that sub-MIC ciprofloxacin decreased biofilm formation, whereas tigecycline stimulated this process. Our data suggest that sub-MIC tigecycline may have bearing on the outcome of infections.

Overexpression of endogenous 1-deoxy-d-xylulose 5-phosphate synthase (DXS) in cyanobacterium Synechocystis sp. PCC6803 accelerates protein aggregation.

1-Deoxy-d-xylulose 5-phosphate synthase (DXS) is a rate-limiting enzyme in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, which is responsible for the production of precursors of all isoprenoids. In a previous study, we had examined the overexpression of an endogenous DXS in a Synechocystis sp. PCC6803 mutant (DXS_ox), and found that the dxs mRNA level was 4-fold higher than that in the wild-type (WT) strain. However, the DXS protein level was only 1.5-fold higher, leading to the assumption that the level might be regulated by post-transcriptional events. In this study, we have additionally introduced an exogenous isoprene synthase (IspS; which can release MEP pathway products from the cell as gaseous isoprene) into the WT and DXS_ox strains (WT-isP and DXSox-isP strains, respectively), and their detailed DXS expression profiles were investigated from the induction phase through to the late-logarithmic phase. In the induction phase, the isoprene productivity of the DXSox-isP strain was slightly but significantly (1.4- to 1.8-fold) higher than that of the WT-isP strain, whereas the levels were comparable in the other phases. Interestingly, the ratios of soluble:insoluble DXS protein were remarkably low in the DXSox-isP strain during the induction phase to the early-logarithmic phase, resulting in a moderate level of soluble DXS. All our results suggested that the high translation rate of DXS disturbs the refolding process of DXS. To enhance the concentration of the active DXS in cyanobacteria, the enhancement of the DXS maturation system or the introduction of exogenous and robust DXS proteins might be necessary.

Crosstalk between the lipopolysaccharide and phospholipid pathways during outer membrane biogenesis in Escherichia coli.

The outer membrane of gram-negative bacteria is composed of phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet. LPS is an endotoxin that elicits a strong immune response from humans, and its biosynthesis is in part regulated via degradation of LpxC (EC 3.5.1.108) and WaaA (EC 2.4.99.12/13) enzymes by the protease FtsH (EC 3.4.24.-). Because the synthetic pathways for both molecules are complex, in addition to being produced in strict ratios, we developed a computational model to interrogate the regulatory mechanisms involved. Our model findings indicate that the catalytic activity of LpxK (EC 2.7.1.130) appears to be dependent on the concentration of unsaturated fatty acids. This is biologically important because it assists in maintaining LPS/phospholipids homeostasis. Further crosstalk between the phospholipid and LPS biosynthetic pathways was revealed by experimental observations that LpxC is additionally regulated by an unidentified protease whose activity is independent of lipid A disaccharide concentration (the feedback source for FtsH-mediated LpxC regulation) but could be induced in vitro by palmitic acid. Further experimental analysis provided evidence on the rationale for WaaA regulation. Overexpression of waaA resulted in increased levels of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) sugar in membrane extracts, whereas Kdo and heptose levels were not elevated in LPS. This implies that uncontrolled production of WaaA does not increase the LPS production rate but rather reglycosylates lipid A precursors. Overall, the findings of this work provide previously unidentified insights into the complex biogenesis of the Escherichia coli outer membrane.

Disposition of pharmacologically active dietary isoflavones in biological systems.

Dietary isoflavones, popularly known as phytoestrogens, represent one of the most biologically active classes of flavonoids. Numerous in vitro and in vivo studies provide convincing evidence regarding their beneficial effects on human health. These isoflavones are increasingly being investigated as potential alternate therapies for a range of hormone-dependent conditions, including cancer, menopausal symptoms, osteoporosis and cardiovascular diseases. However, they exhibit poor oral bioavailability which limits their clinical utility in humans. The reason being, they are substrates of a plethora of enzymes and transporters and undergo extensive conjugative metabolism which facilitates their rapid elimination from biological systems. In addition, a number of experimental studies have also revealed that these isoflavones are potent inhibitors of various cytochrome P450 isoforms and transporters which play an important role in the disposition of many commonly prescribed drugs. Thus, there arise chances of observing clinically relevant herb-drug interactions which could sometimes be life-threatening. This review gives a comprehensive understanding of these dietary phytoestrogens with regard to their absorption, biodistribution and the role of enzyme-transporter interplay affecting their disposition in biological systems. Further, the effects of these phytoestrogens on the activity and kinetics of drug metabolizing enzymes and various clinically relevant influx/efflux transporters and the resulting diet-drug interactions have also been discussed.

Significantly enhanced production of isoprene by ordered coexpression of genes dxs, dxr, and idi in Escherichia coli.

We constructed a biosynthetic pathway of isoprene production in Escherichia coli by introducing isoprene synthase (ispS) from Populus alba. 1-deoxy-D-xylulose 5-phosphate synthase (dxs), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (dxr) and isopentenyl diphosphate (IPP) isomerase (idi) were overexpressed to enhance the isoprene production. The isoprene production was improved 0.65, 0.16, and 1.22 fold over the recombinant BL21 (pET-30a-ispS), respectively, and idi was found to be a key regulating point for isoprene production. In order to optimize the production of isoprene in E. coli, we attempted to construct polycistronic operons based on pET-30a with genes dxs, dxr, and idi in various orders. The highest isoprene production yield of 2.727 mg g(-1) h(-1) (per dry weight) was achieved by E. coli transformed with pET-30a-dxs/dxr/idi. Interestingly, the gene order was found to be consistent with that of the metabolic pathway. This indicates that order of genes is a significant concern in metabolic engineering and a sequential expression pattern can be optimized according to the biosynthetic pathway for efficient product synthesis.

First ever isolation of bacterial prolipoprotein diacylglyceryl transferase in single step from Lactococcus lactis.

The unique bacterial enzyme phosphatidylglycerol: prolipoprotein diacylglyceryl transferase (Lgt) is the least studied enzyme of the ubiquitous bacterial lipoprotein synthetic pathway, mostly due to the low abundance of the enzyme. So far, Lgt has been studied to a limited extent in gram-negative bacteria, mainly in Escherichia coli. We, for the first time, report the isolation of an adequate amount of Lgt from the gram-positive lactic acid bacteria, Lactococcus lactis and compare this wild-type bacterial enzyme with the E. coli enzyme. The L. lactis Lgt, when purified by cationic-exchange chromatography, showed a 20-fold increase in the specific activity compared to that of the load, and 75% of the total Lgt activity loaded was recovered. Kinetically, L. lactis Lgt was found to be similar to the E. coli enzyme with matching K(m) and V(max), whereas the specific activity of the L. lactis enzyme was about 20 times less than that of the E. coli enzyme. Comparative bioinformatic analysis of L. lactis, E. coli and Staphylococcus aureus Lgt revealed that the conserved and catalytically important His-103 residue in E. coli Lgt, was altered to Tyr in L. lactis. Investigations showed that other bacteria where this alteration is visible, form a diversion within the gram-positive bacteria in evolution. Further analysis revealed Mycobacterium smegmatis to be the species which evolved with the alteration of His to Tyr.

Evaluation of CoA biosynthesis proteins of Mycobacterium tuberculosis as potential drug targets.

Coenzyme A biosynthesis pathway proteins are potential targets for developing inhibitors against bacteria including Mycobacterium tuberculosis. We have evaluated two enzymes in this pathway: phosphopantetheine adenylyltransferase (CoaD) and dephospho CoA kinase (CoaE) for essentiality and selectivity. Based on the previous transposon mutagenesis studies, coaD had been predicted to be a non-essential gene in M. tuberculosis. Our bioinformatics analysis showed that there is no other functional homolog of this enzyme in M. tuberculosis, which suggests that coaD should be an essential gene. In order to get an unambiguous answer on the essentiality of coaD, we attempted inactivation of coaD in wild type and merodiploid backgrounds. It was found that coaD could only be inactivated in the presence of an additional gene copy, confirming it to be an essential gene. Using a similar approach we found that CoaE was also essential for the survival of M. tuberculosis. RT-PCR analysis showed that both coaD and coaE were transcribed in M. tuberculosis. Amino acids alignment and phylogenetic analysis showed CoaD to be distantly related to the human counterpart while CoaE was found to be relatively similar to the human enzyme. Analysis of CoaD and CoaE structures at molecular level allowed us to identify unique residues in the Mtb proteins, thus providing a selectivity handle. The essentiality and selectivity analysis combined with the published biochemical characterization of CoaD and CoaE makes them suitable targets for developing inhibitors against M. tuberculosis.

The expression of chicken selenoprotein W, selenocysteine-synthase (SecS), and selenophosphate synthetase-1 (SPS-1) in CHO-K1 cells.

Selenoprotein W (SelW) has been found to be ubiquitously expressed in tissues in vivo and was purified more than 18 years ago. However, little in vitro research has been performed on SelW from birds. To detect the mRNA levels of chicken SelW in cultured cell lines, chicken SelW cDNA was cloned into an expression vector. The chicken SelW expression construct was then transfected into CHO-K1 cells. Using RT-PCR and real-time quantitative reverse transcription PCR, we detected the expression of the chicken SelW mRNA. Moreover, the selenocysteine-synthase (SecS) and selenophosphate synthetase-1 (SPS-1) mRNA levels were analyzed. The expression of SelW was detected in SelW-transfected cells; no expression was observed in control cells. Significant increases in the SelW mRNA levels were obtained in chicken SelW-transfected cells relative to control cells. SecS mRNA levels were significantly increased in chicken SelW transfected cells. No significant difference in the SPS-1 level was observed. Our findings show that chicken SelW could be studied in vitro and that SecS and SPS-1 may have potential roles in SelW biosynthesis.

Preparative scale cell-free production and quality optimization of MraY homologues in different expression modes.

MraY translocase catalyzes the first committed membrane-bound step of bacterial peptidoglycan synthesis leading to the formation of lipid I. The essential membrane protein therefore has a high potential as target for drug screening approaches to develop antibiotics against gram-positive as well as gram-negative bacteria. However, the production of large integral membrane proteins in conventional cellular expression systems is still very challenging. Cell-free expression technologies have been optimized in recent times for the production of membrane proteins in the presence of detergents (D-CF), lipids (L-CF), or as precipitates (P-CF). We report the development of preparative scale production protocols for the MraY homologues of Escherichia coli and Bacillus subtilis in all three cell-free expression modes followed by their subsequent quality evaluation. Although both proteins can be cell-free produced at comparable high levels, their requirements for optimal expression conditions differ markedly. B. subtilus MraY was stably folded in all three expression modes and showed highest translocase activities after P-CF production followed by defined treatment with detergents. In contrast, the E. coli MraY appears to be unstable after post- or cotranslational solubilization in detergent micelles. Expression kinetics and reducing conditions were identified as optimization parameters for the quality improvement of E. coli MraY. Most remarkably, in contrast to B. subtilis MraY the E. coli MraY has to be stabilized by lipids and only the production in the L-CF mode in the presence of preformed liposomes resulted in stable and translocase active protein samples.

Glycine cleavage enzyme complex: molecular cloning and expression of the H-protein cDNA from cultured human skin fibroblasts.

The human H-protein is one of four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex and its function is involved in the pathogenesis and diagnosis of glycine encephalopathy. A transcript corresponding to the glycine cleavage H-protein functional gene was isolated from cultured human skin fibroblasts along with a transcript for a putative processed pseudogene on chromosome 2q33.3. Sequence analysis of the fibroblast H-protein functional gene transcript showed complete identity to that reported from human liver. The H-protein cDNA was subsequently cloned with a hexahistidine affinity tag in the Pichia pastoris plasmid vector pPICZαA and recombined into the yeast genome downstream of the alcohol oxidase promoter for methanol-induced expression. The recombinant H-protein was secreted into the culture medium and purified to homogeneity using a one-step nickel-nitrilotriacetic acid resin column. Approximately 4 mg of homogeneous H-protein was obtained from 1 L of culture medium. Since the attachment of a lipoic acid prosthetic group is required for H-protein function, we have expressed and purified E. coli lipoate protein ligase and succeeded in lipoylating H-protein, converting the apo-H-protein to the functional holo-H-protein. A lipoamide dehydrogenase assay was performed to confirm that the apo-H-protein was inactive, whereas the holo-H-protein was approximately 2.3-fold more active than free lipoic acid as a hydrogen donor in driving the reaction. The availability of copious amounts of human recombinant H-protein by using Pichia pastoris expression and affinity purification will facilitate the elucidation of the structure and function of the H-protein and its relationship to the P-, T-, and L-proteins in the glycine cleavage enzyme complex. In view of the fact that there is no detectable glycine cleavage enzyme activity in human skin fibroblasts, we speculate that a plausible function of the H-protein is to interact with the L-protein, which is also part of the l-ketoglutarate dehydrogenase complex present in fibroblasts.

Enhancing isoprene production by genetic modification of the 1-deoxy-d-xylulose-5-phosphate pathway in Bacillus subtilis.

To enhance the production of isoprene, a volatile 5-carbon hydrocarbon, in the Gram-positive spore-forming rod-shaped bacterium Bacillus subtilis, 1-deoxy-d-xylulose-5-phosphate synthase (Dxs) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr) were overexpressed in B. subtilis DSM 10. For the strain that overexpresses Dxs, the yield of isoprene was increased 40% over that by the wild-type strain. In the Dxr overexpression strain, the level of isoprene production was unchanged. Overexpression of Dxr together with Dxs showed an isoprene production level similar to that of the Dxs overproduction strain. The effects of external factors, such as stress factors including heat (48°C), salt (0.3 M NaCl), ethanol (1%), and oxidative (0.005% H(2)O(2)) stress, on isoprene production were further examined. Heat, salt, and H(2)O(2) induced isoprene production; ethanol inhibited isoprene production. In addition, induction and repression effects are independent of SigB, which is the general stress-responsive alternative sigma factor of Gram-positive bacteria.

The expression of 1-deoxy-D-xylulose synthase and geraniol-10-hydroxylase or anthranilate synthase increases terpenoid indole alkaloid accumulation in Catharanthus roseus hairy roots.

The terpenoid indole alkaloid (TIA) pathway in Catharanthus roseus produces two important anticancer drugs, vinblastine and vincristine, in very low yields. This study focuses on overexpressing several key genes in the upper part of the TIA pathway in order to increase flux toward downstream metabolites within hairy root cultures. Specifically, we constructed hairy root lines with inducible overexpression of 1-deoxy-D-xylulose synthase (DXS) or geraniol-10-hydroxylase (G10H). We also constructed hairy root lines with inducible expression of DXS and anthranilate synthase α subunit (ASA) or DXS and G10H. DXS overexpression resulted in a significant increase in ajmalicine by 67%, serpentine by 26% and lochnericine by 49% and a significant decrease in tabersonine by 66% and hörhammericine by 54%. Co-overexpression of DXS and G10H caused a significant increase in ajmalicine by 16%, lochnericine by 31% and tabersonine by 13%. Likewise, DXS and ASA overexpression displayed a significant increase in hörhammericine by 30%, lochnericine by 27% and tabersonine by 34%. These results point to the need for overexpressing multiple genes within the pathway to increase the flux toward vinblastine and vincristine.

The regulator RamA influences cmytA transcription and cell morphology of Corynebacterium ammoniagenes.

RamA plays a regulatory role for acetate utilization and S-layer biosynthesis in Corynebacterium glutamicum. Looking for any additional role, the function of RamA was analyzed in Corynebacterium ammoniagenes, which is closely related to C. glutamicum. In this study, we showed that the DeltaramA mutant constructed by a markerless knockout strategy possessed increased cell surface hydrophobicity, leading to the formation of aggregated cell masses in liquid media. In addition, the mutant exhibited an elongated cell shape as observed by SEM, suggesting that cell wall-associated proteins might be influenced. Furthermore, cell surface proteome analysis revealed that the expression of cmytA gene encoding corynomycoloyl transferase required for cell wall biosynthesis was down-regulated in the mutant, supporting the regulatory role of RamA in cell wall assembly. These studies support a novel regulatory role of RamA in inducing the expression of proteins required for cell wall assembly.

Replacement of lipopolysaccharide with free lipid A molecules in Escherichia coli mutants lacking all core sugars.

Escherichia coli mutants deficient in 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo) biosynthesis are conditionally lethal, but their phenotypes are bypassed by certain suppressor mutations or by overexpression of MsbA, the inner membrane flippase for core-lipid A. These strains grow on broth with the tetraacylated precursor lipid IV(A) replacing lipopolysaccharide [Meredith, T. C., et al. (2006) ACS Chem. Biol. 1, 33-42]. Deletion of kdtA, which encodes the Kdo transferase, is possible under these conditions. We now show that lipid IV(A) reaches the outer surface of the outer membrane in these strains, as judged by its accessibility to the lipase PagL. On the assumption that MsbA is optimized to transport penta- or hexaacylated lipid A, we overexpressed the lauroyl- or the myristoyltransferase of lipid A biosynthesis, encoded by lpxL and lpxM, respectively, and demonstrated that kdtA deletion mutants were also viable in this setting. Although E. coli LpxL is stimulated by the presence of the Kdo disaccharide in its acceptor substrate, LpxL does slowly acylate lipid IV(A). Overexpression of LpxL from a plasmid suppressed the lethality of kdtA deletions on nutrient broth at 30 or 37 degrees C without the need for MsbA overproduction. These strains accumulated penta- and hexaacylated free lipid A containing a secondary laurate chain or a laurate and a myristate chain, respectively. Deletion of kdtA in strains overexpressing LpxM accumulated pentaacylated lipid A with a secondary myristate moiety. None of the strains lacking kdtA grew in the presence of bile salts at any temperature or on nutrient broth at 42 degrees C. Our findings show that the main function of Kdo is to provide the right substrates for the acyltransferases LpxL and LpxM, resulting in the synthesis of penta- and hexaacylated lipid A, which is optimal for the MsbA flippase.

Transcription profiles analysis of genes encoding 1-deoxy-D-xylulose 5-phosphate synthase and 2C-methyl-D-erythritol 4-phosphate synthase in plaunotol biosynthesis from Croton stellatopilosus.

Genes encoding 1-deoxy-D-xylulose 5-phosphate synthase (DXS; EC 2.2.1.7) and 2C-methyl-D-erythritol 4-phosphate synthase (MEPS; EC 1.1.1.267), the first two enzymes in the deoxyxylulose phosphate (DXP) pathway, were cloned from young leaves of Croton stellatopilosus, and designated as 1-deoxy-D-xylulose 5-phosphate synthase (CSDXS) and 2C-methyl-D-erythritol 4-phosphate synthase (CSMEPS), respectively. Analysis of deduced amino acid sequences of the CSDXS and the CSMEPS confirmed their nucleotide sequences as they shared high identities to other known DXSs and MEPSs in higher plants. Physiological roles of the CSDXS and the CSMEPS were determined for the mRNA expressions in leaves, twigs and roots. Transcription profiles analyses of the CSDXS and the CSMEPS genes were investigated using semi-quantitative RT-PCR technique. Relative intensities of the CSDXS and the CSMEPS expressions to house-keeping gene (18S rRNA) were calculated. The results indicated that the levels of mRNAs expressions of the CSDXS and the CSMEPS were high in leaves and twigs. This evidence was in line with the high content of plaunotol, accumulated in leaves and twigs. Neither the CSDXS nor the CSMEPS were expressed in roots, where plaunotol was not detected. From this study, it can be concluded that plaunotol is biosynthesized in the chloroplastic tissue and regulated by the CSDXS and the CSMEPS.

Biosynthesis of selenocysteine on its tRNA in eukaryotes.

Selenocysteine (Sec) is cotranslationally inserted into protein in response to UGA codons and is the 21st amino acid in the genetic code. However, the means by which Sec is synthesized in eukaryotes is not known. Herein, comparative genomics and experimental analyses revealed that the mammalian Sec synthase (SecS) is the previously identified pyridoxal phosphate-containing protein known as the soluble liver antigen. SecS required selenophosphate and O-phosphoseryl-tRNA([Ser]Sec) as substrates to generate selenocysteyl-tRNA([Ser]Sec). Moreover, it was found that Sec was synthesized on the tRNA scaffold from selenide, ATP, and serine using tRNA([Ser]Sec), seryl-tRNA synthetase, O-phosphoseryl-tRNA([Ser]Sec) kinase, selenophosphate synthetase, and SecS. By identifying the pathway of Sec biosynthesis in mammals, this study not only functionally characterized SecS but also assigned the function of the O-phosphoseryl-tRNA([Ser]Sec) kinase. In addition, we found that selenophosphate synthetase 2 could synthesize monoselenophosphate in vitro but selenophosphate synthetase 1 could not. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that synthesize selenoproteins.

Identification of a novel CoA synthase isoform, which is primarily expressed in the brain.

CoA and its derivatives Acetyl-CoA and Acyl-CoA are important players in cellular metabolism and signal transduction. CoA synthase is a bifunctional enzyme which mediates the final stages of CoA biosynthesis. In previous studies, we have reported molecular cloning, biochemical characterization, and subcellular localization of CoA synthase (CoASy). Here, we describe the existence of a novel CoA synthase isoform, which is the product of alternative splicing and possesses a 29aa extension at the N-terminus. We termed it CoASy beta and originally identified CoA synthase, CoASy alpha. The transcript specific for CoASy beta was identified by electronic screening and by RT-PCR analysis of various rat tissues. The existence of this novel isoform was further confirmed by immunoblot analysis with antibodies directed to the N-terminal peptide of CoASy beta. In contrast to CoASy alpha, which shows ubiquitous expression, CoASy beta is primarily expressed in the brain. Using confocal microscopy, we demonstrated that both isoforms are localized on mitochondria. The N-terminal extension does not affect the activity of CoA synthase, but possesses a proline-rich sequence which can bring the enzyme into complexes with signalling proteins containing SH3 or WW domains. The role of this novel isoform in CoA biosynthesis, especially in the brain, requires further elucidation.

Differential expression of three 1-deoxy-D: -xylulose-5-phosphate synthase genes in rice.

1-Deoxy-D-: xylulose-5-phosphate synthase (DXS) encoded by a multigene family in plants, catalyzes the first step in the methylerythritol 4-phosphate (MEP) pathway. Three rice DXS-related sequences (OsDXS) were identified from available rice databases. The open reading frame of three OsDXS genes (dxs1, dxs2, and dxs3) were amplified against cDNA template. Ratio of their transcript levels in etiolated rice leaf was 9:181:1. While the expression levels were not changed along the growth stages of etiolated culture, UV-irradiation of the etiolated rice induced the expression of dxs3 up to nine-fold compared with that of unirradiated control. In the case of light-illumination, the relative expression of dxs1 based on unilluminated control increased two-fold. The differential expression of three OsDXS genes suggested their distinct and complementary roles in the control of the first step of the MEP pathway in response to environmental stimuli.

Expression of hyaluronan synthases and hyaluronan in malignant mesothelioma cells.

BACKGROUND: Hyaluronan is one of the main components of the extracellular matrix. It is synthesized at the cell plasma membrane by specific hyaluronan synthases (HAS). Although a large number of studies have described hyaluronan in pleural effusion from malignant mesothelioma, the source of hyaluronan in malignant mesothelioma has been subject to controversy. MATERIALS AND METHODS: The mRNA expression of all three HAS in malignant mesothelioma cells was studied using RT-PCR. The hyaluronan production in culture medium of malignant mesothelioma cells was also examined using high-performance liquid chromatography (HPLC). RESULTS: We found that 9/10 malignant mesothelioma cell lines and one primary culture of malignant mesothelioma cells expressed HAS-1, while 10/10 malignant mesothelioma cell lines and one primary culture of malignant mesothelioma cells expressed HAS-2 and HAS-3. In addition, we demonstrated hyaluronan in the culture medium of 6 out of 10 malignant mesothelioma cell lines and one primary culture of malignant mesothelioma cells. CONCLUSION: Our results show that malignant mesothelioma cells express all three HAS and synthesize hyaluronan. The expression of HAS isoforms and hyaluronan in malignant mesothelioma cells in cultures and previous observations by other investigators indicate that these cells are, at least in part, responsible for hyaluronan synthesis in vivo.