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The regular documentation of anthropometric data in an electronic medical record (EMR) is one tracking method used by primary care providers to follow the growth trajectory and development of children in their health care practices. EMR reminders have been proposed as a method to increase recording of pediatric height and weight by primary care providers, leading to potentially better detection and management of children classified as overweight or obese. The aim of this pre–post study was to improve a Family Health Team’s physician documentation of pediatric height and weight through the implementation of an EMR reminder alert tool. The documentation rate for children 4–7 years old in the 6 months before intervention was 36% of children seen. After implementation of EMR reminder alerts, primary care physicians’ documentation rate rose to 45% (9% increase; P

Glutamine synthetase (GS), which catalyzes the production of glutamine, plays essential roles in nitrogen metabolism. There are two main bacterial GS isoenzymes, GSI-α and GSI-β. GSI-α enzymes, which have not been structurally characterized, are uniquely feedback-inhibited by Gln. To gain insight into GSI-α function, we performed biochemical and cellular studies and obtained structures for all GSI-α catalytic and regulatory states. GSI-α forms a massive 600-kDa dodecameric machine. Unlike other characterized GS, the Bacillus subtilis enzyme undergoes dramatic intersubunit conformational alterations during formation of the transition state. Remarkably, these changes are required for active site construction. Feedback inhibition arises from a hydrogen bond network between Gln, the catalytic glutamate, and the GSI-α-specific residue, Arg(62), from an adjacent subunit. Notably, Arg(62) must be ejected for proper active site reorganization. Consistent with these findings, an R62A mutation abrogates Gln feedback inhibition but does not affect catalysis. Thus, these data reveal a heretofore unseen restructuring of an enzyme active site that is coupled with an isoenzyme-specific regulatory mechanism. This GSI-α-specific regulatory network could be exploited for inhibitor design against Gram-positive pathogens.

CodY is a global transcriptional regulator that is activated by branched-chain amino acids. A palindromic 15-bp sequence motif, AATTTTCNGAAAATT, is associated with CodY DNA binding. A gel mobility shift assay was used to examine the effect of pH on the binding of Bacillus subtilis CodY to the hutPp and ureAp 3 promoters. CodY at pH 6.0 has higher affinity for DNA, more enhanced activation by isoleucine, and a lower propensity for nonspecific DNA binding than CodY at pH 8.0. DNase I footprinting was used to identify the CodY-protected regions in the hutPp and ureAp 3 promoters. The CodY-protected sequences for both promoters were found to contain multiple copies of the 15-bp motif with 6-bp overlaps. Mutational analysis of the hutPp regulatory region revealed that two overlapping sequence motifs were required for CodY-mediated regulation. The presence of overlapping sequence motifs in the regulatory regions of many B. subtilis CodY-regulated genes suggests that CodY binds to native operators that contain overlapping binding sites.

The Bacillus subtilis GlnR transcription factor regulates gene expression in response to changes in nitrogen availability. Glutamine synthetase transmits the nitrogen regulatory signal to GlnR. The DNA-binding activity of GlnR is activated by a transient protein-protein interaction with feedback-inhibited glutamine synthetase that stabilizes GlnR-DNA complexes. This signal transduction mechanism was analysed by creating mutant GlnR proteins with partial or complete truncations of their C-terminal domains. The truncated GlnR proteins were found to constitutively repress gene expression in vivo. This constitutive repression did not require glutamine synthetase. Purified mutant GlnR proteins bound DNA in vitro more tightly than wild-type GlnR protein and this binding was not activated by feedback-inhibited glutamine synthetase. While full-length GlnR is monomeric, the truncated GlnR proteins contained significant levels of dimers. These results indicate that the C-terminal region of GlnR acts as an autoinhibitory domain that prevents GlnR dimerization and thus impedes DNA binding. The GlnR C-terminal domain is also required for the interaction between GlnR and feedback-inhibited glutamine synthetase. Compared with the full-length GlnR protein, the truncated GlnR proteins were defective in their interaction with feedback-inhibited glutamine synthetase in cross-linking experiments.

Two Bacillus subtilis transcriptional factors, TnrA and GlnR, regulate gene expression in response to changes in nitrogen availability. These two proteins have similar amino acid sequences in their DNA binding domains and bind to DNA sites (GlnR/TnrA sites) that have the same consensus sequence. Expression of the tnrA gene was found to be activated by TnrA and repressed by GlnR. Mutational analysis demonstrated that a GlnR/TnrA site which lies immediately upstream of the −35 region of the tnrA promoter is required for regulation of tnrA expression by both GlnR and TnrA. Expression of the glnRA operon, which contains two GlnR/TnrA binding sites ( glnRAo1 and glnRAo2 ) in its promoter region, is repressed by both GlnR and TnrA. The glnRAo2 site, which overlaps the −35 region of the glnRA promoter, was shown to be required for regulation by both GlnR and TnrA, while the glnRAo1 site which lies upstream of the −35 promoter region is only involved in GlnR-mediated regulation. Examination of TnrA binding to tnrA and glnRA promoter DNA in gel mobility shift experiments showed that TnrA bound with an equilibrium dissociation binding constant of 55 nM to the GlnR/TnrA site in the tnrA promoter region, while the affinities of TnrA for the two GlnR/TnrA sites in the glnRA promoter region were greater than 3 μM. These results demonstrate that GlnR and TnrA cross-regulate each other's expression and that there are differences in their DNA-binding specificities.

In Bacillus subtilis, the activity of the nitrogen regulatory factor TnrA is regulated through a protein- protein interaction with glutamine synthetase. During growth with excess nitrogen, the feedback-inhibited form of glutamine synthetase binds to TnrA and blocks DNA binding by TnrA. Missense mutations in glutamine synthetase that constitutively express the TnrA-regulated amtB gene were characterized. Four mutant proteins were purified and shown to be defective in their ability to inhibit the in vitro DNA-binding activity of TnrA. Two of the mutant proteins exhibited enzymatic properties similar to those of wild-type glutamine synthetase. A model of B. subtilis glutamine synthetase was derived from a crystal structure of the Salmonella typhimurium enzyme. Using this model, all the mutated amino acid residues were found to be located close to the glutamate entrance of the active site. These results are consistent with the glutamine synthetase protein playing a direct role in regulating TnrA activity.

Bacillus subtilis TnrA, a global regulator of transcription, responds to nitrogen availability, but the specific signal to which it responds has been elusive. Genetic studies indicate that glutamine synthetase is required for the regulation of TnrA activity in vivo. We report here that the feedback-inhibited form of glutamine synthetase directly interacts with TnrA and blocks the DNA binding activity of TnrA. Mutations in the tnrA gene ( tnrA C ) that allow constitutive high level expression of tnrA -activated genes were isolated and characterized. Feedback-inhibited glutamine synthetase had a significantly reduced ability to block the in vitro DNA binding by three of the TnrA C proteins. Thus, glutamine synthetase, an enzyme of central metabolism, directly interacts with and regulates the DNA binding activity of TnrA.

Ten saponins have been isolated from the deacylated saponin mixture of Bellis perennis L. The structures of these saponins can be divided into two groups that show differences both in the aglycone and the carbohydrate moieties bound at C-28. The first group consists of glycosides of polygalacic acid with tri- or tetrasaccharide moieties consisting mainly of deoxyhexoses and a second group of glycosides of bayogenin or asterogenic acid which possess glucose as major carbohydrate moiety and one xylose or rharnnose unit. From the most polar fraction of the alkaline hydrolysate obtained from the underground parts of Bellis perennis one saponin possessing only glucose residues has been obtained. On the basis of a sugar methylation analysis and NMR spectral data, the structure was established as 3-O-ß-D-glucopyranosyl-2ß,3ß,23-trihydroxyolean-12-en-28-oic acid 28-O-ß-D-glucoyranosyl-(1→2)-[ß-D-glucopyranosyl(1→6)]-ß-D-glucopyranoside.

Synthesis of glutamate, the cell's major donor of nitrogen groups and principal anion, occupies a significant fraction of bacterial metabolism. In Bacillus subtilis , the gltAB operon, encoding glutamate synthase, requires a specific positive regulator, GltC, for its expression. In addition, the gltAB operon was shown to be repressed by TnrA, a regulator of several other genes of nitrogen metabolism and active under conditions of ammonium (nitrogen) limitation. TnrA was found to bind directly to a site immediately downstream of the gltAB promoter. As is true for other genes, the activity of TnrA at the gltAB promoter was antagonized by glutamine synthetase under certain growth conditions.

The bacterium Microbacterium sp., isolated from the sponge Halichondria panicea, produced four unusual cell-associated glycoglycerolipids and one diphosphatidylglycerol when grown on marine broth and on artificial seawater media. The lipids were isolated by chromatography on silica columns and their structures elucidated using a combination of multidimensional NMR and MS techniques. The main compound was 1-O-acyl-3-[alpha-glucopyranosyl-(1-3)-(6-O-acyl-alpha-mannopyranosyl )]glycerol (GGL.2) with 14-methyl-hexadecanoic acid and 12-methyl-tetradecanoic acid positioned at C-6 of the mannose unit and at the glycerol moiety. Glycolipid production was correlated with growth and reached a maximum value of 200 mg/L when grown on artificial seawater medium with 20 g/L glucose. The main compound decreased the surface tension of water down to 33 mN/m and the interfacial tension of the water/n-hexadecane system down to 5 mN/m. In addition to this good surface-active behavior, the main glycoglycerolipid showed antitumor activities.

Bioassay-guided fractionation of an extract obtained from roots of Aglaia duperreana led to the isolation of 17 1H-cyclopenta[b]benzofurans of the rocaglamide type. Of the compounds isolated, four rocaglamide derivatives (2, 6, 11, and 16) were obtained as new natural products, and their structure elucidation was conducted by spectral methods. For bioassay-guided fractionation and determination of LC50 and EC50 values, neonate larvae of Spodoptera littoralis were employed. The results of chronic feeding assays have shown new aspects of the structure−activity relationship of rocaglamide derivatives. The substitution of a hydroxyl group at C-8b by a methoxyl substituent leads to a loss of insecticidal activity in a manner not previously documented in this compound class.

Two new saponins were isolated as the major components of the deacylated saponin extract from the underground parts of Celmisia spectabilis. Their structures were established by NMR and mass spectral data and derivative formation as 2 beta,3 beta,17,23- tetrahydroxy-28-norolean-12-en-16-one-3-O-alpha-L-arabinopyrano syl (1--2)-alpha-L-arabinopyranosyl(1--6)-beta-D-glucopyranoside and 2 beta,3 beta,17,23- tetrahydroxy-28-norolean-12-en-16-one-3-O-alpha-L-arabinopyrano syl (1--2)-alpha-L-arabinopyranosyl(1--6)-[alpha-L-rhamnopyranosyl (1--2)]-beta-D-glucopyranoside.

Transcription of the Bacillus subtilis nrgAB promoter is activated during nitrogen-limited growth by the TnrA protein. A common inverted repeat, TGTNAN 7 TNACA (TnrA site), is centered 49 to 51 bp upstream of the transcriptional start sites for the TnrA-regulated nrgAB , gabP P2, and nas promoters. Oligonucleotide-directed mutagenesis of the nrgAB promoter region showed that conserved nucleotides within the TnrA site, the A+T-rich region between the two TnrA half-sites, and an upstream A tract are all required for high-level activation of nrgAB expression. Mutations that alter the relative distance between the two half-sites of the nrgAB TnrA site abolish nitrogen regulation of nrgAB expression. Spacer mutations that change the relative distance between the TnrA site and −35 region of the nrgAB promoter reveal that activation of nrgAB expression occurs only when the TnrA site is located 49 to 51 bp upstream of the transcriptional start site. Mutational analysis of the conserved nucleotides in the gabP P2 TnrA site showed that this sequence is also required for nitrogen-regulated gabP P2 expression. The TnrA protein, expressed in an overproducing Escherichia coli strain, had a 625-fold-higher affinity for the wild-type nrgAB promoter DNA than for a mutated nrgAB promoter DNA fragment that is unable to activate nrgAB expression in vivo. These results indicate that the proposed TnrA site functions as the binding site for the TnrA protein. TnrA was found to activate nrgAB expression during late exponential growth in nutrient sporulation medium containing glucose, suggesting that cells become nitrogen limited during growth in this medium.

Two new saponins were isolated from the seeds of Agrostemma githago L. var. githago. On the basis of chemical and spectral evidence their structures were determined to be 3-O-beta-D-xylopyranosyl-(1-->3)-[beta-D-galactopyranosyl-(1-->2)]-beta- D- glucuronopyranosylgypsogenin-28-O-beta-D-xylopyranosyl-(1-->4)- [beta-D-glucopyranosyl-(1-->3)]-alpha-L-rhamnopyranosyl-(1-->2)-beta- D-4-O-acetylfucopyranoside and 3-O-beta-D-xylopyranosyl-(1-->3)- [beta-D-galactopyranosyl-(1-->2)]-beta-D-glucuronopyranosyl-gyp sogenin- 28-O-beta-D-xylopyranosyl-(1-->3)-beta-D-xylopyranosyl-(1-->4)- [beta-D-glucopyranosyl-(1-->3)]-alpha-L-rhamnopyranosyl-(1-->2)-beta-D- 4-O-acetylfucopyranoside.

The first enzyme of the Bacillus subtilis histidine-degradative (hut) pathway, histidase, was expressed at higher levels during the onset of the stationary growth phase in nutrient sporulation medium in early-blocked sporulation mutants (spo0A) than in wild-type strains. Histidase expression was also elevated in spo0A mutant cultures compared with wild-type cultures during the logarithmic growth phase in minimal medium containing slowly metabolized carbon sources. Histidase expression was not derepressed in spo0A abrB mutant cultures under these growth conditions, suggesting that the AbrB protein is responsible for the derepression of histidase synthesis seen in spo0A mutant cultures. spo0A mutants contain higher levels of the AbrB protein than do wild-type strains because the Spo0A protein represses AbrB expression. A direct correlation between the levels of abrB transcription and histidase expression was found in spo0A mutant cultures. The hutOCR2 operator, which is required for wild-type regulation of hut expression by catabolite repression, was also required for AbrB-dependent derepression of hut expression in spo0A mutants. Purified AbrB protein bound to the hutOCR2 operator in vitro, suggesting that AbrB protein alters hut expression by competing with the hut catabolite repressor protein for binding to the hutOCR2 site. During the logarithmic growth phase in media containing slowly metabolized carbon sources, the expression of several other enzymes subject to catabolite repression was elevated in spo0A mutants but not in spo0A abrB mutants. This suggests that the AbrB protein acts as a global modulator of catabolite repression during carbon-limited growth.

Pestalotiopsis is generally found as endophytes of tropical plants and as prolific producers of structurally unusal natural products [1]. Our previous chemical investigations on fungus species Pestalotiopsis JCM2A4 isolated from the Chinese Mangrove plant Rhizophora mucronata Lam. have led to the isolation of a series of natural products and yielded over twenty different compounds with seventeen of them being new natural products including chromones, cytosporones and coumarins [2–3]. Following cultivation of the fungus now yielded eight new pyrone derivatives (1) as well as a known compound. The structures of all compounds were unambiguously established from their spectroscopic data, that included HR-ESIMS and 1- and 2-dimensional NMR spectroscopy, and by comparison with the literature [4]. Our findings proved endophyte genus Pestalotiopsis to be particularly productive. Acknowledgement: Financial support by grants of Hainan University Youth Foundation (qnjj1005) and Dr. Research Starting Found (kyqd1066) are gratefully acknowledged. References: 1. Xu J et al. (2010) Fungal Divers 44:15–31. 2. Xu J et al. (2009)J Nat Prod 72: 662–665. 3. Xu J et al. (2009) Bioorg Med Chem 17: 7362–7367. 4. Xu J et al. (2011) Tetrahedron Lett 52: 21–25.

The Streptomyces coelicolor glnR gene positively regulates the transcription of the glutamine synthetase-encoding glnA gene. The nucleotide sequence of a 1682-bp DNA segment containing glnR was determined. The deduced amino acid sequence of the GlnR protein was found to be similar to the sequence of several bacterial response regulators that are known to function as transcriptional activators. Primer extension analysis of glnR mRNA identified three transcriptional tart points ( tsp ) upstream from the glnR coding sequence.

During growth of Bacillus subtilis in nutrient sporulation medium containing histidine (DSM-His medium), the expression of histidase, the first enzyme in the histidine-degradative pathway (hut), is derepressed 40- to 200-fold at the onset of stationary phase. To identify the gene products responsible for this regulation, histidase expression was examined in various hut regulatory mutants as well as in mutants defective in stationary-phase gene regulation. Histidase expression during growth in DSM-His medium was significantly altered only in a strain containing the hutC1 mutation. The hutC1 mutation allows the hut operon to be expressed in the absence of its inducer, histidine. During logarithmic growth in DSM-His medium, histidase levels were 25-fold higher in the HutC mutant than in wild-type cells. Moreover, histidase expression in the HutC mutant increased only four- to eightfold after the end of exponential growth in DSM-His medium. This suggests that histidine transport is reduced in wild-type cells during exponential growth in DSM-His medium and that this reduction is largely responsible for the repression of hut expression in cells growing logarithmically in this medium. Indeed, the rate of histidine uptake in DSM-His medium was fourfold lower in exponentially growing cells than in stationary-phase cells. The observation that the degradation of histidine is inhibited when B. subtilis is growing rapidly in medium containing a mixture of amino acids suggests that a hierarchy of amino acid utilization may be present in this bacterium.

Six Streptomyces coelicolor mutants that required glutamine for growth at the wild-type rate on all nitrogen sources (Gln-) were isolated. The phenotypes of all six mutants were similar. The glutamine synthetase (GS) levels were 20- to 100-fold lower in extracts of the Gln- mutants than in extracts of their parents. The reduced levels of GS activity in the Gln- mutants were not due to adenylylation of the GS protein, because GS activity in Gln- extracts did not increase after snake venom phosphodiesterase treatment. No transcripts of the GS structural gene (glnA) could be detected in RNA isolated from the Gln- mutants in primer extension experiments. All six gln mutations mapped adjacent to adeA. S. coelicolor chromosomal DNA complementing the Gln- mutants was isolated from a library of S. coelicolor chromosomal DNA constructed in the low-copy-number S. coelicolor plasmid pIJ922. Subcloning experiments showed that a 1.45-kb DNA fragment could complement all six Gln- mutants. This DNA fragment did not hybridize with either the cloned S. coelicolor glnA gene or the cloned S. viridochromogenes GSII gene in Southern blots. Since glnA transcription was restored in the Gln- mutants containing the complementing DNA, the gln mutations appear to lie in one or more closely linked genes that are required for glnA transcription in S. coelicolor.

Summary The anaerobic, thermophilic archaebacterium, Pyrobaculum organotrophum was examined for the presence of lipoquinones. Thin layer chromatographic, HPLC, UV, mass spectroscopic, 1 H- and 13 C-NMR analysis showed that menaquinones were present in the form of MK-6H 12 and a new lipoquinone with a partially cyclized side chain. The new compound was shown to correspond to 2-{14-[3-(1,5-dimethylhexyl)-cyclopentyl] -3,7,11-trimethyltetradecyl}-3-methyl-1,4-naphthoquinone.

The Bacillus subtilis GlnR transcription factor regulates gene expression in response to changes in nitrogen availability. Glutamine synthetase transmits the nitrogen regulatory signal to GlnR. The DNA-binding activity of GlnR is activated by a transient protein-protein interaction with feedback-inhibited glutamine synthetase that stabilizes GlnR-DNA complexes. This signal transduction mechanism was analysed by creating mutant GlnR proteins with partial or complete truncations of their C-terminal domains. The truncated GlnR proteins were found to constitutively repress gene expression in vivo. This constitutive repression did not require glutamine synthetase. Purified mutant GlnR proteins bound DNA in vitro more tightly than wild-type GlnR protein and this binding was not activated by feedback-inhibited glutamine synthetase. While full-length GlnR is monomeric, the truncated GlnR proteins contained significant levels of dimers. These results indicate that the C-terminal region of GlnR acts as an autoinhibitory domain that prevents GlnR dimerization and thus impedes DNA binding. The GlnR C-terminal domain is also required for the interaction between GlnR and feedback-inhibited glutamine synthetase. Compared with the full-length GlnR protein, the truncated GlnR proteins were defective in their interaction with feedback-inhibited glutamine synthetase in cross-linking experiments.

The Bacillus subtilis GlnR repressor controls gene expression in response to nitrogen availability. Because all GlnR-regulated genes are expressed constitutively in mutants lacking glutamine synthetase (GS), GS is required for repression by GlnR. Feedback-inhibited GS (FBI-GS) was shown to activate GlnR DNA binding with an in vitro electophoretic mobility shift assay (EMSA). The activation of GlnR DNA binding by GS in these experiments depended on the feedback inhibitor glutamine and did not occur with mutant GS proteins defective in regulating GlnR activity in vivo . Although stable GS–GlnR–DNA ternary complexes were not observed in the EMSA experiments, cross-linking experiments showed that a protein–protein interaction occurs between GlnR and FBI-GS. This interaction was reduced in the absence of the feedback inhibitor glutamine and with mutant GS proteins. Because FBI-GS significantly reduced the dissociation rate of the GlnR–DNA complexes, the stability of these complexes is enhanced by FBI-GS. These results argue that FBI-GS acts as a chaperone that activates GlnR DNA binding through a transient protein–protein interaction that stabilizes GlnR–DNA complexes. GS was shown to control the activity of the B. subtilis nitrogen transcription factor TnrA by forming a stable complex between FBI-GS and TnrA that inhibits TnrA DNA binding. Thus, B. subtilis GS is an enzyme with dual catalytic and regulatory functions that uses distinct mechanisms to control the activity of two different transcription factors.

The first enzymes of the histidine (hut) and proline degradative pathways, histidase and proline oxidase, could not be induced in Bacillus subtilis cells growing in glucose minimal medium containing a mixture of 16 amino acids. Addition of the 16-amino-acid mixture to induced wild-type cells growing in citrate minimal medium repressed histidase synthesis 25- to 250-fold and proline oxidase synthesis 16-fold. A strain containing a transcriptional fusion of the hut promoter to the beta-galactosidase gene was isolated from a library of Tn917-lacZ transpositions. Examination of histidase and beta-galactosidase expression in extracts of a hut-lacZ fusion strain grown in various media showed that induction, catabolite repression, and amino acid repression of the hut operon were mediated at the level of transcription. This result was confirmed by measurement of the steady-state level of hut RNA in cells grown in various media. Since amino acid repression was not defective in B. subtilis mutants deficient in nitrogen regulation of glutamine synthetase and catabolite repression, amino acid repression appears to be mediated by a system that functions independently of these regulatory systems.

A new acylated triterpenoid saponin was obtained from the leaves of Sanicula europaea. The structure has been elucidated from NMR and MS data, and by chemical derivatisation as 21-O-[2-methylbutanoyl]-3 beta, 15 alpha, 16 alpha, 21 beta, 22 alpha, 28-hexahydroxyolean-12-ene 3-O-[alpha-L-arabinopyranosyl (1-->3)] beta-D-glucopyranosyl(1-->2)-beta-D-glucuronopyranoside.

The Bacillus subtilis TnrA transcription factor belongs to the MerR family of proteins and regulates gene expression during nitrogen-limited growth. When B. subtilis cells are grown with excess nitrogen, feedback-inhibited glutamine synthetase forms a protein-protein complex with TnrA that prevents TnrA from binding to DNA. The C-terminal region of TnrA is required for the interaction with glutamine synthetase. Alanine scanning mutagenesis of the C-terminal region of TnrA identified three classes of mutants that altered the regulation by glutamine synthetase. While expression of the TnrA-regulated amtB gene was expressed constitutively in the class I (M96A, Q100A, and A103G) and class II (L97A, L101A, and F105A) mutants, the class II mutants were unable to grow on minimal medium unless a complex mixture of amino acids was present. The class III tnrA mutants (R93A, G99A, N102A, H104A, and Y107A mutants) were partially defective in the regulation of TnrA activity. In vitro experiments showed that feedback-inhibited glutamine synthetase had a significantly reduced ability to inhibit the DNA-binding activity of several class I and class II mutant TnrA proteins. A coiled-coil homology model of the C-terminal region of TnrA is used to explain the properties of the class I and II mutant proteins. The C-terminal region of TnrA corresponds to a dimerization domain in other MerR family proteins. Surprisingly, gel filtration and cross-linking analysis showed that a truncated TnrA protein which contained only the N-terminal DNA binding domain was dimeric. The implications of these results for the structure of TnrA are discussed.

The Bacillus subtilis ureABC operon encodes homologs of the three subunits of urease enzymes of the family Enterobacteriaceae. Disruption of ureC prevented utilization of urea as a nitrogen source and resulted in a partial growth defect in minimal medium containing limiting amounts of arginine or allantoin as the sole nitrogen source.

In view of the reports that polymorpho- nuclear leukocytes (PMN) of patients with localized aggressive periodontitis (LAP) exhibit hyper-re- sponsiveness to stimulation, it has been suggested that such abnormalities could lead to PMN-medi- ated tissue damage during inflammation. To deter- mine whether these abnormalities include signal transduction, we compared cytoplasmic calcium concentration ((Ca 2 )i) and cytoplasmic pH (pH i ) changes, early stimulus responses to chemo- tactic agents, of LAP versus control (C)-PMN and explored whether these could be modulated by sensitizing cytokines or calcium channel-blocking agents. PMN responses of LAP patients were compared with age- and gender-matched con- trols. (Ca 2 )i and pHi were measured fluori- metrically using 1H-indole-6-carboxylic acid, 2-(4-(bis(2-((acetyloxy)methoxy)-2-oxoethyl)- amino)-3-(2-(2-(bis(2-((acetyloxy)methoxy)-2- oxoethyl)amino)-5-methylphenoxy)ethoxy)phenyl)-1 and 2,7-bis-(carboxyethyl)-5(6)-carboxyfluores- cein as respective probes. Not only was the maxi- mal calcium response to chemoattractants higher in LAP-PMN, but also their subsequent intracellu- lar calcium redistribution was significantly slower. The slower calcium redistribution of LAP-PMN, but not their higher maximal calcium response, was successfully mimicked in C-PMN treated with Nifedipine™ or 1-(b-(3-(4-methoxyphenyl)propoxy)- 4-methoxyphenethyl)-1H-imidazole-HCl, both known to be inhibitors of membrane-associated calcium influx, but this redistribution was not af- fected when inhibitors of other calcium influx mechanisms, Diltiazem™ or Verapamil™, were used. Taken together, our findings indicate that certain early stimulus responses are aberrant in LAP-PMN, that internal redistribution of cytoplas- mic-free calcium is compromised, and, addition- ally, that a membrane-associated Ca 2 transport defect may be present. J. Leukoc. Biol. 78: 612-619; 2005.

Marine natural products with their unique structural features and pronounced biological activities continue to provide lead structures in the search for new drugs from nature. Invertebrates such as sponges, tunicates, mollusks and others that are either sessile or slow moving and mostly lack morphological defense structures have so far provided the largest number of marine-derived secondary constituents including some of the most interesting drug candidates. This review highlights recent research findings of our group related to natural products from marine invertebrates. Areas that are covered include ecological functions of secondary constituents from sponges against predatory fish, the search for new pharmacologically active constituents from sponges and tunicates, and sponge-associated fungi as an evolving source for new bioactive natural products.

Fractionation of the EtOAc extract of a static culture of Aspergillus niger isolated from the Mediterranean sponge Axinella damicornis yielded eight secondary metabolites, out of which seven compounds (2-8) proved to be new natural products, whereas one was identified as the known fungal pigment cycloleucomelone (1). The new compounds included the 3,3'-bicoumarin bicoumanigrin (2), the structurally unusual 4-benzyl-1H-pyridin-6-one derivatives aspernigrins A and B (3 and 4), and pyranonigrins A-D (5-8), the latter featuring a novel pyrano[3,2-b]pyrrole skeleton hitherto unprecedented in nature. All structures were elucidated on the basis of extensive one- and two-dimensional NMR spectroscopic studies ((1)H, (13)C, COSY, HMQC, HMBC, NOE difference spectra) and mass spectral analysis. For the two chiral molecules 4 and 5, the absolute configurations were established by quantum chemical calculations of their circular dichroism (CD) spectra. In each case, two independent methods, i.e., a molecular dynamics approach taking into consideration the molecular flexibility, and a conformational analysis followed by Boltzmann weighting of the single CD spectra calculated for the conformers thus obtained, led to identical results without the need of any empirical comparison of chiroptical data reported for reference compounds. Bicoumanigrin (2) showed moderate cytotoxicity against human cancer cell lines in vitro. In addition, aspernigrin B (4) was found to display a strong neuroprotective effect against glutamic acid.

Expression of the P3 promoter of the Bacillus subtilis ureABC operon is activated during nitrogen-limited growth by PucR, the transcriptional regulator of the purine-degradative genes. Addition of allantoic acid, a purine-degradative intermediate, to nitrogen-limited cells stimulated transcription of ure P3 twofold. Since urea is produced during purine degradation in B. subtilis , regulation of ureABC expression by PucR allows purines to be completely degraded to ammonia. The nitrogen transcription factor TnrA was found to indirectly regulate ure P3 expression by activating pucR expression. The two consensus GlnR/TnrA binding sites located in the ure P3 promoter region were shown to be required for negative regulation by GlnR. Mutational analysis indicates that a cooperative interaction occurs between GlnR dimers bound at these two sites. B. subtilis is the first example where urease expression is both nitrogen regulated and coordinately regulated with the enzymes involved in purine transport and degradation.

The ectomycorrhizal fungi Laccaria amethystina and Lactarius deterrimus grown in liquid culture were used to study the fate of added ferulic acid. Laccaria amethystina degraded ferulic acid to the major metabolite vanillic acid. The intermediate vanillin was not detected. Lactarius deterrimus showed a completely different detoxification pattern. Two dimers and one trimer of ferulic acid could be identified as polymerization products of this fungus. A bioassay of the possible biological activities of ferulic acid and vanillic acid on these fungi revealed that vanillic acid was less toxic than ferulic acid for Laccaria amethystina but that both phenolic acids were toxic for Lactarius deterrimus. The results are discussed with respect to ectomycorrhizal fungal growth in the organic layer of forest soils and between living root cells of ectomycorrhizas.

The Bacillus subtilis nitrogen transcriptional factor TnrA is inactive in cells grown with excess nitrogen, e.g., glutamine or glutamate plus ammonium, because feedback-inhibited glutamine synthetase (product of glnA ) binds to TnrA and blocks its DNA-binding activity. Two conditional mutations that allow TnrA-dependent gene expression in cells grown with glutamate plus ammonium, but not in glutamine-grown cells, were characterized. One mutant contained a mutation in the glnA ribosome binding site, while the other mutant synthesized a truncated GlnR protein that constitutively repressed glnRA expression. The levels of glutamine synthetase were reduced in both mutants. As a result, when these mutants are grown with excess nitrogen in the absence of glutamine, there is insufficient production of the feedback inhibitors necessary to convert glutamine synthetase into its feedback-inhibited form and TnrA-activated genes are expressed at high levels.

In Bacillus subtilis, the activity of the nitrogen regulatory factor TnrA is regulated through a protein- protein interaction with glutamine synthetase. During growth with excess nitrogen, the feedback-inhibited form of glutamine synthetase binds to TnrA and blocks DNA binding by TnrA. Missense mutations in glutamine synthetase that constitutively express the TnrA-regulated amtB gene were characterized. Four mutant proteins were purified and shown to be defective in their ability to inhibit the in vitro DNA-binding activity of TnrA. Two of the mutant proteins exhibited enzymatic properties similar to those of wild-type glutamine synthetase. A model of B. subtilis glutamine synthetase was derived from a crystal structure of the Salmonella typhimurium enzyme. Using this model, all the mutated amino acid residues were found to be located close to the glutamate entrance of the active site. These results are consistent with the glutamine synthetase protein playing a direct role in regulating TnrA activity.

Expression of the two Bacillus subtilis genes encoding l -asparaginase is controlled by independent regulatory factors. The ansZ gene (formerly yccC ) was shown by mutational analysis to encode a functional l -asparaginase, the expression of which is activated during nitrogen-limited growth by the TnrA transcription factor. Gel mobility shift and DNase I footprinting experiments indicate that TnrA regulates ansZ expression by binding to a DNA site located upstream of the ansZ promoter. The expression of the ansA gene, which encodes the second l -asparaginase, was found to be induced by asparagine. The ansA repressor, AnsR, was shown to negatively regulate its own expression.

Five cyanoglucosides have been isolated from extracts of the epidermal layer of barley and the structure of their trimethylsilyl derivatives have been elucidated spectroscopically (UV, NMR, MS). Three of these, namely 3-β- d -glucopyranosyloxy-3-methylbutyronitrile, 1-cyano-3-β- d -glucopyranosyloxy-2-methylpropene and 4-β- d -glucopyranosyloxy-3-hydroxy-3-hydroxymethyl-butyronitrile, are novel compounds.

Two new monodesmosidic saponins, herniaria saponins E and F, were isolated from the aerial parts of Herniaria hirsuta. On the basis of chemical and spectral evidence, their structures were established to be 2-O-acetyl medicagenic acid 28-O-beta-D-xylopyranosyl- (1--4)-alpha-L-rhamnopyranosyl(1--2)-[beta-D-glucopyranosyl(1--6)]-be ta- D-glucopyranoside (herniaria saponin E, 1) and medicagenic acid 28-O-beta-D-xylopyranosyl(1--4)-alpha-L- rhamnopyranosyl(1--2)-[alpha-L-rhamnopyranosyl-(1--4)-beta-D- glucopyranosyl-(1--6)]-beta-D-glucopyranoside (herniaria saponin F, compound 2).

The Bacillus subtilis nitrogen regulatory protein TnrA was purified and its interaction with the nrgAB regulatory region examined. The TnrA protein activates transcription from the nrgAB promoter in vitro . DNase I footprinting and methylation protection experiments demonstrated that TnrA binds to an inverted repeat, upstream of the −35 region of the nrgAB promoter. Gel mobility retardation assays were used to determine the affinity of TnrA for its DNA-binding site. The equilibrium dissociation binding constant for the interaction of TnrA with the nrgAB promoter fragment was 7.7 nM under the conditions used here. Mutations in the TnrA consensus sequence that reduce nrgAB expression in vivo were found to reduce significantly the in vitro affinity for TnrA. An A + T rich region located upstream of the TnrA-binding site was found to be necessary for optimal transcriptional activation. A mutant protein, TnrA HTH , was constructed in which the putative helix-turn-helix DNA-binding motif was altered by exchanging two arginine residues for alanine residues. The TnrA HTH protein was unable to activate the in vivo expression of nrgAB and had an in vitro affinity for the nrgAB promoter that was significantly lower than that of the wild-type protein.

Three major and two minor ferulic acid esters were identified in a hexane extract of Pavetta owariensis stembark. Their structures were elucidated using mass, 1 H and 13 C NMR spectroscopy.

In Bacillus subtilis , CcpA-dependent carbon catabolite repression (CCR) mediated at several cis -acting carbon repression elements ( cre ) requires the seryl-phosphorylated form of both the HPr ( ptsH ) and Crh ( crh ) proteins. During growth in minimal medium, the ptsH1 mutation, which prevents seryl phosphorylation of HPr, partially relieves CCR of several genes regulated by CCR. Examination of the CCR of the histidine utilization ( hut ) enzymes in cells grown in minimal medium showed that neither the ptsH1 nor the crh mutation individually had any affect on hut CCR but that hut CCR was abolished in a ptsH1 crh double mutant. In contrast, the ptsH1 mutation completely relieved hut CCR in cells grown in Luria-Bertani medium. The ptsH1 crh double mutant exhibited several growth defects in glucose minimal medium, including reduced rates of growth and growth inhibition by high levels of glycerol or histidine. CCR is partially relieved in B. subtilis mutants which synthesize low levels of active glutamine synthetase ( glnA ). In addition, these glnA mutants grow more slowly than wild-type cells in glucose minimal medium. The defects in growth and CCR seen in these mutants are suppressed by mutational inactivation of TnrA, a global nitrogen regulatory protein. The inappropriate expression of TnrA-regulated genes in this class of glnA mutants may deplete intracellular pools of carbon metabolites and thereby result in the reduction of the growth rate and partial relief of CCR.

Transgalactosylation of chitobiose and chitotriose employing beta-galactosidase from bovine testes yielded mixtures with beta1-3 linked galactose (type I) and beta1-4 linked galactose (type II) in a final ratio of 1:1 for the tri- and 1:1.4 for the tetrasaccharide. After 24 h incubations of the two purified oligosaccharide mixtures with large amounts (20-fold increase compared with standard conditions) of human alpha1,3/4-fucosyltransferase III (FucT III), the type I tri-/tetrasaccharides were completely converted to the Lewis(a) structure, whereas approximately 10% fucosylation of the type II isomers to the Lewis(x) oligosaccharides was observed in long-term incubations. Employing large amounts of human alpha1,3-fucosyltransferase VI (FucT VI), the type I trisaccharide substrate was exclusively fucosylated at the proximal O-4 substituted N-acetylglucosamine (GlcNAc) (20%) whereas almost all of the type II isomers was converted to the corresponding Lewis(x) product. 45% of the type I tetrasaccharide was fucosylated at the second GlcNAc solely by FucT VI. The type II isomer was almost completely alpha1-3 fucosylated to yield the Lewisx derivative with traces of a structure that contained an additional fucose at the reducing GlcNAc. The results obtained in the present study employing high amounts of enzyme confirmed our previous results that FucT III acts preponderantly as a beta1-4 fucosyltransferase onto GlcNAc in vitro. Human FucT VI attaches fucose exclusively in an alpha1-3 linkage to 4-substituted GlcNAc in vitro and does not modify any 3-substituted GlcNAc to yield Lewis(a) oligosaccharides. With 8-methoxycarbonyloctyl glycoside acceptors used under standard conditions, FucT III acts exclusively on the type I and FucT VI only on the type II derivative. With lacto-N-tetraose, lacto-N-fucopentraose I, or LS-tetrasaccharide as substrates, FucT III modified the 3-substituted GlcNAc and the reducing glucose; FucT VI recognized only lacto-N-neotetraose as a substrate.

In Bacillus subtilis , carbon catabolite repression (CCR) of many genes is mediated at cis -acting carbon repression elements ( cre ) by the catabolite repressor protein CcpA. Mutations in transcription-repair coupling factor ( mfd ) partially relieve CCR at cre sites located downstream of transcriptional start sites by abolishing the Mfd-mediated displacement of RNA polymerase stalled at cre sites which act as transcriptional roadblocks. Although the acsA cre is centered 44.5 bp downstream of the acsA transcriptional start site, CCR of acsA expression is not affected by an mfd mutation. When the acsA cre is centered 161.5 bp downstream of the transcriptional start site for the unregulated tms promoter, CCR is partially relieved by the mfd mutation. Since CCR mediated at an acsA cre centered 44.5 bp downstream of the tms start site is not affected by the mfd mutation, the inability of Mfd to modulate CCR of acsA expression most likely results from the location of the acsA cre . Higher levels of CCR were found to occur at cre sites flanked by A+T-rich sequences than at cre sites bordered by G and C nucleotides. This suggests that nucleotides adjacent to the proposed 14-bp cre consensus sequence participate in the formation of the CcpA catabolite repression complex at cre sites. Examination of CCR of acsA expression revealed that this regulation required the Crh and seryl-phosphorylated form of the HPr proteins but not glucose kinase.

A Bacillus subtilis mutant that partially relieves carbon catabolite repression (CCR) of the hut operon was isolated by transposon mutagenesis. Characterization of this mutant revealed that the transposon had inserted into the gene, mfd, that encodes transcription-repair coupling factor. The Mfd protein is known to promote strand-specific DNA repair by displacing RNA polymerase stalled at a nucleotide lesion and directing the (A)BC excinuclease to the DNA damage site. A set of transcriptional lacZ fusions was used to demonstrate that the mfd mutation relieves CCR of hut and gnt expression at the cis-acting cre sequences located downstream of the transcriptional start site but does not affect CCR at sites located at the promoters. CCR of the amyE and bglPH genes, which contain cre sequences that overlap their promoters, is not altered by the mfd mutation. These results support a model in which the Mfd protein displaces RNA polymerase stalled at downstream cre sites that function as transcriptional roadblocks and reveal a new role for Mfd in cellular physiology.

Expression of urease, which is encoded by the ureABC operon, is regulated in response to nitrogen availability in Bacillus subtilis. Three ureABC promoters were identified in primer extension experiments and by examination of beta-galactosidase expression from ure-lacZ fusions. P1, a low-level constitutive promoter, lies immediately upstream of ureA. The P2 promoter is transcribed by the E sigmaH form of RNA polymerase and initiates transcription 270 bp upstream of the ureA start codon. The transcriptional start site for the sigmaA-dependent P3 promoter is located 839 bp upstream of the ureA start codon. To identify transcription factors that control ureABC expression, regulation of the P2 and P3 promoters was examined in wild-type and mutant strains. During rapid growth in minimal medium containing glucose and amino acids, CodY represses expression of the P2 and P3 promoters 30- and 60-fold, respectively. TnrA activates expression of the P3 promoter 10-fold in nitrogen-limited cells, while GlnR represses transcription from the P3 promoter 55-fold during growth on excess nitrogen. Expression of the ureABC operon increases 10-fold at the end of exponential growth in nutrient sporulation medium. This elevation in expression results from the relief of CodY-mediated repression during exponential growth and increased sigmaH-dependent transcription during stationary phase.