Environmental Assessment of Enzyme Production and Purification.

The importance of bioprocesses has increased in recent decades, as they are considered to be more sustainable than chemical processes in many cases. E factors can be used to assess the sustainability of processes. However, it is noticeable that the contribution of enzyme synthesis and purification is mostly neglected. We, therefore, determined the E factors for the production and purification of 10 g enzymes. The calculated complete E factor including required waste and water is 37,835 gwaste·genzyme-1. This result demonstrates that the contribution of enzyme production and purification should not be neglected for sustainability assessment of bioprocesses.

The cGAS-STING signaling pathway contributes to the inflammatory response and autophagy in Aspergillus fumigatus keratitis.

Fungal keratitis is a serious corneal infection, which can lead to significant visual impairment and blindness. The cGAS-STING signaling pathway has emerged as a key player in innate immunity by sensing of invading pathogens. However, the role of the cGAS-STING pathway in Aspergillus fumigatus (A. fumigatus) keratitis is still unknown. In this study, we showed that the cGAS-STING signaling pathway was activated in human corneal epithelial cells (HCECs) and in mouse corneas infected with A. fumigatus. Knockdown of cGAS reduced A. fumigatus-induced production of pro-inflammatory cytokines, including TNF-α, IL-1ß, IL-6, and IFN-ß. However, reconstruction of cGAS activity restored the inflammatory response in HCECs infected with A. fumigatus. A specific cGAS inhibitor, RU.521, could also significantly inhibit A. fumigatus-induced inflammatory cytokine expression. In addition, we found that cGAS was indispensable for the autophagy flux evoked by A. fumigatus infection. Moreover, inhibition of cGAS using siRNA or RU.521 alleviated the severity of A. fumigatus keratitis in the mouse cornea. Therefore, the cGAS-STING signaling pathway contributes to the progression of A. fumigatus keratitis and targeting this pathway may provide therapeutic potential.

NCTD elicits proapoptotic and antiglycolytic effects on colorectal cancer cells via modulation of Fam46c expression and inhibition of ERK1/2 signaling.

Colorectal cancer is a digestive tract malignancy and the third leading cause of cancer­related mortality worldwide. Norcantharidin (NCTD), the demethylated form of cantharidin, has been reported to possess anticancer properties. Family­with­sequence­similarity­46c (Fam46c), a non­canonical poly(A) polymerase, has been reported to be critical in NCTD­mediated effects in numerous types of cancer, including hepatoma. In the current study, it was found that Fam46c expression was reduced in colorectal cancer tissues and cells. Treatment with NCTD was observed to significantly enhance apoptosis and inhibit glycolysis in colorectal cancer cells. In addition, Fam46c and cleaved caspase 3 expression levels were found to be increased in response to NCTD treatment, in contrast to tumor­specific pyruvate kinase M2 and phosphorylated ERK expression, which was reduced. Importantly, overexpression of Fam46c exerted similar effects as NCTD treatment on the apoptosis and glycolysis of colorectal cancer cells, whereas Fam46c knockdown strongly attenuated the effect of NCTD. Moreover, epidermal growth factor, which acts as an agonist of ERK1/2 signaling, weakened the effects of NCTD on colorectal cancer cells. Taken together, the results indicated that NCTD promotes apoptosis and suppresses glycolysis in colorectal cancer cells by possibly targeting Fam46c and inhibiting ERK1/2 signaling, hence suggesting that Fam46c may act as a tumor suppressor in colorectal cancer. Thus, the present study identified a novel therapeutic target of NCTD in the clinical treatment of colorectal cancer.

Increased expression of the bacterial glycolipid MPIase is required for efficient protein translocation across membranes in cold conditions.

Protein integration into and translocation across biological membranes are vital events for organismal survival and are fundamentally conserved among many organisms. Membrane protein integrase (MPIase) is a glycolipid that drives membrane protein integration into the cytoplasmic membrane in Escherichia coli MPIase also stimulates protein translocation across the membrane, but how its expression is regulated is incompletely understood. In this study, we found that the expression level of MPIase significantly increases in the cold (<25 °C), whereas that of the SecYEG translocon does not. Using previously created gene-knockout E. coli strains, we also found that either the cdsA or ynbB gene, both encoding rate-limiting enzymes for MPIase biosynthesis, is responsible for the increase in the MPIase expression. Furthermore, using pulse-chase experiments and protein integration assays, we demonstrated that the increase in MPIase levels is important for efficient protein translocation, but not for protein integration. We conclude that MPIase expression is required to stimulate protein translocation in cold conditions and is controlled by cdsA and ynbB gene expression.

Bacterial Production, Characterization and Protein Modeling of a Novel Monofuctional Isoform of FAD Synthase in Humans: An Emergency Protein?

FAD synthase (FADS, EC 2.7.7.2) is the last essential enzyme involved in the pathway of biosynthesis of Flavin cofactors starting from Riboflavin (Rf). Alternative splicing of the human FLAD1 gene generates different isoforms of the enzyme FAD synthase. Besides the well characterized isoform 1 and 2, other FADS isoforms with different catalytic domains have been detected, which are splice variants. We report the characterization of one of these novel isoforms, a 320 amino acid protein, consisting of the sole C-terminal 3'-phosphoadenosine 5'-phosphosulfate (PAPS) reductase domain (named FADS6). This isoform has been previously detected in Riboflavin-Responsive (RR-MADD) and Non-responsive Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) patients with frameshift mutations of FLAD1 gene. To functionally characterize the hFADS6, it has been over-expressed in Escherichia coli and purified with a yield of 25 mg·L-1 of cell culture. The protein has a monomeric form, it binds FAD and is able to catalyze FAD synthesis (kcat about 2.8 min-1), as well as FAD pyrophosphorolysis in a strictly Mg2+-dependent manner. The synthesis of FAD is inhibited by HgCl2. The enzyme lacks the ability to hydrolyze FAD. It behaves similarly to PAPS. Combining threading and ab-initio strategy a 3D structural model for such isoform has been built. The relevance to human physio-pathology of this FADS isoform is discussed.

Expression of Cyclic GMP-AMP Synthase in Patients With Systemic Lupus Erythematosus.

OBJECTIVE: Type I interferon (IFN) is implicated in the pathogenesis of systemic lupus erythematosus (SLE) and interferonopathies such as Aicardi-Goutières syndrome. A recently discovered DNA-activated type I IFN pathway, cyclic GMP-AMP synthase (cGAS), has been linked to Aicardi-Goutières syndrome and mouse models of lupus. The aim of this study was to determine whether the cGAS pathway contributes to type I IFN production in patients with SLE. METHODS: SLE disease activity was measured by the Safety of Estrogens in Lupus Erythematosus National Assessment version of the Systemic Lupus Erythematosus Disease Activity Index. Expression of messenger RNA for cGAS and IFN-stimulated genes (ISGs) was determined by quantitative polymerase chain reaction analysis. Cyclic GMP-AMP (cGAMP) levels were examined by multiple reaction monitoring with ultra-performance liquid chromatography tandem mass spectrometry. RESULTS: Expression of cGAS in peripheral blood mononuclear cells (PBMCs) was significantly higher in SLE patients than in normal controls (n = 51 and n = 20 respectively; P < 0.01). There was a positive correlation between cGAS expression and the IFN score (P < 0.001). The expression of cGAS in PBMCs showed a dose response to type I IFN stimulation in vitro, consistent with it being an ISG. Targeted measurement of cGAMP by tandem mass spectrometry detected cGAMP in 15% of the SLE patients (7 of 48) but none of the normal (0 of 19) or rheumatoid arthritis (0 of 22) controls. Disease activity was higher in SLE patients with cGAMP versus those without cGAMP. CONCLUSION: Increased cGAS expression and cGAMP in a proportion of SLE patients indicates that the cGAS pathway should be considered as a contributor to type I IFN production. Whereas higher cGAS expression may be a consequence of exposure to type I IFN, detection of cGAMP in patients with increased disease activity indicates potential involvement of this pathway in disease expression.

Characterization of the transcriptional regulation of the tarIJKL locus involved in ribitol-containing wall teichoic acid biosynthesis in Lactobacillus plantarum.

Lactobacillus plantarum strains produce either glycerol (Gro)- or ribitol (Rbo)-backbone wall teichoic acid (WTA) (Gro-WTA and Rbo-WTA, respectively). The strain WCFS1 has been shown to be able to activate the tarIJKL locus involved in Rbo-WTA synthesis when the tagD1F1F2 locus for Gro-WTA synthesis was mutated, resulting in switching of the native Gro-WTA into Rbo-WTA. Here, we identify a regulator involved in the WTA backbone alditol switching and activation of the tarIJKL locus. Promoter reporter assays of the tarI promoter (Ptar) demonstrated its activity in the Rbo-WTA-producing mutant derivative (ΔtagF1-2) but not in the parental strain WCFS1. An electrophoresis mobility shift assay using a Ptar nucleotide fragment showed that this fragment bound to Ptar-binding protein(s) in a cell-free extract of WCFS1. Three proteins were subsequently isolated using Ptar bound to magnetic beads. These proteins were isolated efficiently from the lysate of WCFS1 but not from the lysate of its ΔtagF1-2 derivative, and were identified as redox-sensitive transcription regulator (Lp_0725), catabolite control protein A (Lp_2256) and TetR family transcriptional regulator (Lp_1153). The role of these proteins in Ptar regulation was investigated by knockout mutagenesis, showing that the Δlp_1153 mutant expressed the tarI gene at a significantly higher level, supporting its role as a repressor of the tarIJKL locus. Notably, the Δlp_1153 mutation also led to reduced expression of the tagF1 gene. These results show that Lp_1153 is a regulatory factor that plays a role in WTA alditol switching in Lb. plantarum WCFS1 and we propose to rename this gene/protein wasR/WasR, for WTA alditol switch regulator.

The transcription factor TFII-I promotes DNA translesion synthesis and genomic stability.

Translesion synthesis (TLS) enables DNA replication through damaged bases, increases cellular DNA damage tolerance, and maintains genomic stability. The sliding clamp PCNA and the adaptor polymerase Rev1 coordinate polymerase switching during TLS. The polymerases Pol η, ι, and κ insert nucleotides opposite damaged bases. Pol ζ, consisting of the catalytic subunit Rev3 and the regulatory subunit Rev7, then extends DNA synthesis past the lesion. Here, we show that Rev7 binds to the transcription factor TFII-I in human cells. TFII-I is required for TLS and DNA damage tolerance. The TLS function of TFII-I appears to be independent of its role in transcription, but requires homodimerization and binding to PCNA. We propose that TFII-I bridges PCNA and Pol ζ to promote TLS. Our findings extend the general principle of component sharing among divergent nuclear processes and implicate TLS deficiency as a possible contributing factor in Williams-Beuren syndrome.

Jasmonic acid-amino acid conjugation enzyme assays.

Jasmonic acid (JA) is activated for signaling by its conjugation to isoleucine (Ile) through an amide linkage. The Arabidopsis thaliana JASMONIC ACID RESISTANT1 (JAR1) enzyme carries out this Mg-ATP-dependent reaction in two steps, adenylation of the free carboxyl of JA, followed by condensation of the activated group to Ile. This chapter details the protocols used to detect and quantify the enzymatic activity obtained from a glutathione-S-transferase:JAR1 fusion protein produced in Escherichia coli, including an isotope exchange assay for the adenylation step and assays for the complete reaction that involve the high-performance liquid chromatography quantitation of adenosine monophosphate, a stoichiometric by-product of the reaction, and detection of the conjugation product by thin-layer chromatography or gas -chromatography/mass spectrometry.

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.

MiR-96 downregulates REV1 and RAD51 to promote cellular sensitivity to cisplatin and PARP inhibition.

Cell survival after DNA damage relies on DNA repair, the abrogation of which causes genomic instability. The DNA repair protein RAD51 and the trans-lesion synthesis DNA polymerase REV1 are required for resistance to DNA interstrand cross-linking agents such as cisplatin. In this study, we show that overexpression of miR-96 in human cancer cells reduces the levels of RAD51 and REV1 and impacts the cellular response to agents that cause DNA damage. MiR-96 directly targeted the coding region of RAD51 and the 3'-untranslated region of REV1. Overexpression of miR-96 decreased the efficiency of homologous recombination and enhanced sensitivity to the PARP inhibitor AZD2281 in vitro and to cisplatin both in vitro and in vivo. Taken together, our findings indicate that miR-96 regulates DNA repair and chemosensitivity by repressing RAD51 and REV1. As a therapeutic candidate, miR-96 may improve chemotherapeutic efficacy by increasing the sensitivity of cancer cells to DNA damage.

Evolution of the biosynthesis of di-myo-inositol phosphate, a marker of adaptation to hot marine environments.

The synthesis of di-myo-inositol phosphate (DIP), a common compatible solute in hyperthermophiles, involves the consecutive actions of inositol-1-phosphate cytidylyltransferase (IPCT) and di-myo-inositol phosphate phosphate synthase (DIPPS). In most cases, both activities are present in a single gene product, but separate genes are also found in a few organisms. Genes for IPCT and DIPPS were found in the genomes of 33 organisms, all with thermophilic/hyperthermophilic lifestyles. Phylogeny of IPCT/DIPPS revealed an incongruent topology with 16S RNA phylogeny, thus suggesting horizontal gene transfer. The phylogenetic tree of the DIPPS domain was rooted by using phosphatidylinositol phosphate synthase sequences as out-group. The root locates at the separation of genomes with fused and split genes. We propose that the gene encoding DIPPS was recruited from the biosynthesis of phosphatidylinositol. The last DIP-synthesizing ancestor harboured separated genes for IPCT and DIPPS and this architecture was maintained in a crenarchaeal lineage, and transferred by horizontal gene transfer to hyperthermophilic marine Thermotoga species. It is plausible that the driving force for the assembly of those two genes in the early ancestor is related to the acquired advantage of DIP producers to cope with high temperature. This work corroborates the view that Archaea were the first hyperthermophilic organisms.

A common structural blueprint for plant UDP-sugar-producing pyrophosphorylases.

Plant pyrophosphorylases that are capable of producing UDP-sugars, key precursors for glycosylation reactions, include UDP-glucose pyrophosphorylases (A- and B-type), UDP-sugar pyrophosphorylase and UDP-N-acetylglucosamine pyrophosphorylase. Although not sharing significant homology at the amino acid sequence level, the proteins share a common structural blueprint. Their structures are characterized by the presence of the Rossmann fold in the central (catalytic) domain linked to enzyme-specific N-terminal and C-terminal domains, which may play regulatory functions. Molecular mobility between these domains plays an important role in substrate binding and catalysis. Evolutionary relationships and the role of (de)oligomerization as a regulatory mechanism are discussed.

Infusion of Lin- bone marrow cells results in multilineage macrochimerism and skin allograft tolerance in minimally conditioned recipient mice.

Donor-specific immunological tolerance using high doses of donor bone marrow cells (BMC) has been demonstrated in mixed chimerism-based tolerance induction protocols; however, the development of graft versus host disease (GVHD) remains a risk. In the present study, we demonstrate that the infusion of low numbers of donor Lin(-) bone marrow cells (Lin(-) BMC) 7 days post allograft transplantation facilitates high level macrochimerism induction and graft tolerance. Full-thickness BALB/c skin allografts were transplanted onto C57BL/6 mice. Mice were treated with anti-CD4 and anti-CD8 mAbs on day 0, +2, +5, +7 and +14 along with low dose busulfan on day +5. A low dose of highly purified Lin(-) BMC from BALB/c donor mice was infused on day +7. Chimerism and clonal cell deletion were evaluated using flow cytometry. Donor-specific tolerance was tested by donor and third-party skin grafting and mixed leukocyte reaction (MLR). Lin(-) BMC infusion with minimal immunosuppression led to stable, mixed, multilineage macrochimerism and long-term allograft survival (>300 days). Mixed donor-recipient macrochimerism was observed. Donor-reactive T cells were clonally deleted and a 130% increase in CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) was observed in the spleen. Tolerant mice subsequently accepted second donor, but not third-party (C3H), skin grafts and recipient splenocytes failed to react with allogeneic donor cells indicating donor-specific immunological tolerance was achieved. We conclude that the infusion of donor Lin(-) BMC without cytoreductive recipient conditioning can induce indefinite survival of skin allografts via mechanisms involving the establishment of a multilineage macrochimeric state principally through clonal deletion of alloreactive T cells and peripherally induced CD4(+)Foxp3(+) Tregs.

[Multifunctional regulatory mutation in Bacillus subtilis flavinogenesis system].

Among Bacillus subtilis riboflavin-resistant mutants we identified one, which differed from other regulatory mutants by overproduction of riboflavin and simultaneous upregulation of the rib C gene encoding flavokinase/FAD-synthase. Genetic and biochemical analysis showed that the ribU1 mutation determines a trans-acting factor that simultaneously regulates activity of riboflavin and truB-ribC-rpsO operons. Regulatory activity of the ribU1 mutation comprises about 10% of Rfn element activity on interaction with flavins. The ribUl mutation can be presumably ascribed to a gene of the transcriptional regulators family.

Lin-Sca-1+CD49fhigh stem/progenitors are tumor-initiating cells in the Pten-null prostate cancer model.

We have shown previously that Pten deletion leads to the expansion of subset of prostate cancer cells positive for CK5 and p63. Although this subpopulation may be involved in tumor initiation or progression, studies to date have not functionally validated this hypothesis. Using in vitro sphere-forming assay and in vivo prostate reconstitution assay, we show here the presence of a tumor-initiating subpopulation in the Pten prostate cancer mouse model. Specifically, we show that the Lin(-)Sca-1(+)CD49f(high) (LSC) subpopulation overlaps with CK5(+);p63(+) cells and is significantly increased during prostate cancer initiation and progression and after castration. Mutant spheres mimic the structural organization of the epithelial compartment in the Pten-null primary tumor. Sorted LSC cells from either Pten-null spheres or primary tumors are able to regenerate prostate epithelial structure with cancerous morphology, closely mimicking that of primary cancers. Therefore, the LSC subpopulation is capable of initiating a cancerous phenotype that recapitulates the pathology seen in the primary lesions of the Pten mutant prostate model.

Biochemical characterization of the GTP:adenosylcobinamide-phosphate guanylyltransferase (CobY) enzyme of the hyperthermophilic archaeon Methanocaldococcus jannaschii.

The archaeal cobY gene encodes the nonorthologous replacement of the bacterial NTP:AdoCbi kinase (EC 2.7.7.62)/GTP:AdoCbi-P guanylyltransferase (EC 3.1.3.73) and is required for de novo synthesis of AdoCbl (coenzyme B(12)). Here we show that ORF MJ1117 of the hyperthermophilic, methanogenic archaeon Methanocaldococcus jannaschii encodes a CobY protein (Mj CobY) that transfers the GMP moiety of GTP to AdoCbi-P to form AdoCbi-GDP. Results from isothermal titration calorimetry (ITC) experiments show that MjCobY binds GTP (K(d) = 5 muM), but it does not bind the GTP analogues GMP-PNP (guanosine 5'-(beta,gamma)-imidotriphosphate) or GMP-PCP (guanylyl 5'-(beta,gamma)-methylenediphosphonate) nor GDP. Results from ITC experiments indicate that MjCobY binds one GTP per dimer. Results from in vivo studies support the conclusion that the 5'-deoxyadenosyl upper ligand of AdoCbi-P is required for MjCobY function. Consistent with these findings, MjCobY displayed high affinity for AdoCbi-P (K(d) = 0.76 muM) but did not bind nonadenosylated Cbi-P. Kinetic parameters for theMj CobY reaction were determined. Results from circular dichroism studies indicate that, in isolation, MjCobY denatures at 80 degrees C with a concomitant loss of activity. We propose that ORF MJ1117 of M. jannaschii be annotated as cobY to reflect its involvement in AdoCbl biosynthesis.

Beta-lactam and aminoglycoside resistance rates and mechanisms among Pseudomonas aeruginosa in French general practice (community and private healthcare centres).

OBJECTIVES: The aim of this study was to assess antibiotic resistance rates and mechanisms of beta-lactam and aminoglycoside resistance among isolates of Pseudomonas aeruginosa isolated in the extra-hospital setting (community and private healthcare centres). PATIENTS AND METHODS: During a 4 month period, 226 non-repetitive strains of P. aeruginosa were collected from patients residing in private healthcare centres (73.5%) or at home (26.5%). Resistance rates were evaluated by MIC determination, and beta-lactam and aminoglycoside resistance was analysed by phenotypic tests, PCR amplification, cloning and sequencing. RESULTS: Among the ticarcillin-resistant strains (38.1%), 33.7% overexpressed their chromosomal cephalosporinase, 27.9% produced acquired penicillinases (21 PSE-1, 2 OXA-21 and 1 TEM-2), 4.7% produced extended-spectrum beta-lactamases (ESBLs) (3 TEM-21 and 1 SHV-2a) and 45.3% possessed a non-enzymatic resistance (NER). Thus, 88.4% had a single mechanism of resistance, whereas 11.6% cumulated several mechanisms. No carbapenemases were detected among the 6.6% imipenem-resistant strains. With regard to aminoglycosides, 23.0% of the strains exhibited an acquired resistance to gentamicin (GEN), tobramycin (TOB), amikacin (AMK) or netilmicin (NET). Enzymatic resistance was more frequent (71.2%) than NER (34.6%). Various aminoglycoside modifying enzymes were associated with overlapping phenotypes: 36.5% strains produced AAC(6')-I with either a serine (GEN-TOB-NET) or a leucine (TOB-NET-AMK) at position 119, or both variants (GEN-TOB-NET-AMK); 21.2% expressed ANT(2'')-I (GEN-TOB), 7.7% AAC(3)-II (GEN-TOB-NET), 5.8% AAC(3)-I (GEN) and 1.9% AAC(6')-II (GEN-TOB-NET-AMK) or AACA7 (TOB-NET-AMK). CONCLUSIONS: Antibiotic resistance rates in P. aeruginosa were globally similar in general practice as in French hospitals. This first analysis of resistance mechanisms showed an unexpectedly high frequency of ESBLs and an unusual distribution of aminoglycoside modifying enzymes.

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.

The synthesis of UDP-N-acetylglucosamine is essential for bloodstream form trypanosoma brucei in vitro and in vivo and UDP-N-acetylglucosamine starvation reveals a hierarchy in parasite protein glycosylation.

A gene encoding Trypanosoma brucei UDP-N-acetylglucosamine pyrophosphorylase was identified, and the recombinant protein was shown to have enzymatic activity. The parasite enzyme is unusual in having a strict substrate specificity for N-acetylglucosamine 1-phosphate and in being located inside a peroxisome-like microbody, the glycosome. A bloodstream form T. brucei conditional null mutant was constructed and shown to be unable to sustain growth in vitro or in vivo under nonpermissive conditions, demonstrating that there are no alternative metabolic or nutritional routes to UDP-N-acetylglucosamine and providing a genetic validation for the enzyme as a potential drug target. The conditional null mutant was also used to investigate the effects of N-acetylglucosamine starvation in the parasite. After 48 h under nonpermissive conditions, about 24 h before cell lysis, the status of parasite glycoprotein glycosylation was assessed. Under these conditions, UDP-N-acetylglucosamine levels were less than 5% of wild type. Lectin blotting and fluorescence microscopy with tomato lectin revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite. The principal parasite surface coat component, the variant surface glycoprotein, was also analyzed. Endoglycosidase digestions and mass spectrometry showed that, under UDP-N-acetylglucosamine starvation, the variant surface glycoprotein was specifically underglycosylated at its C-terminal Asn-428 N-glycosylation site. The significance of this finding, with respect to the hierarchy of site-specific N-glycosylation in T. brucei, is discussed.