Green synthesized Ag nanoparticles stimulate gene expression and paclitaxel production in Corylus avellana cells.

Synthesis of nanoparticles (NPs) through plant extracts has been suggested as an effective and nature-friendly method. Paclitaxel is one of the most valuable secondary metabolites with therapeutic uses, and hazelnut has been suggested as one of the sustainable resources for producing this metabolite. In the present study, we synthesized Ag NPs using the ethanolic extract of C. avellana leaves and were characterized using UV-visible, FTIR, XRD, EDX, DLS, SEM, and TEM analyses. In addition, we investigated the effect of green synthesized Ag (GS Ag) NPs (5 and 10 mg/L), para-aminobenzoic acid (PABA) (20 mg/L), and AgNO3 (10 mg/L) on cell viability, physiological characteristics, gene expression, and biosynthesis of secondary metabolites in hazelnut cell cultures. The results showed that 10 mg/L Ag NPs and AgNO3 significantly affected the cell viability, the content of ROS, peroxidation of lipids, antioxidant capacity, secondary metabolite production, and expression pattern of the genes involved in the taxanes biosynthesis pathway in the hazelnut cells. The cytotoxicity increased by increasing the GS Ag NPs concentration from 5 to 10 mg/L, which was associated with reduced membrane integrity and cell viability. Elicitation of the cells with 10 mg/L Ag NPs combined with 20 mg/L PABA (as a precursor) remarkably excited the expression of TAT and GGPPS genes and the production of secondary metabolites as well as paclitaxel. So that the highest expression of TAT and GGPPS genes (3.71 and 3.69) and the highest amount of taxol (230.21 µg g-1 FW) and baccatin (1025.8 µg g-1 FW) were observed in this treatment. KEY POINTS: • For the first time, we assessed and reported the molecular and physiological responses of C. avellana cells to GS Ag NPs, AgNO3, and PABA. • In hazel cells, GS Ag NPs stimulate several physiological and molecular responses. • In addition to increasing antioxidant activity, GS Ag NPs significantly increased the expression of genes involved in the paclitaxel biosynthesis pathway and the production of secondary metabolites.

Para-Aminobenzoic Acid, Calcium, and c-di-GMP Induce Formation of Cohesive, Syp-Polysaccharide-Dependent Biofilms in Vibrio fischeri.

The marine bacterium Vibrio fischeri efficiently colonizes its symbiotic squid host, Euprymna scolopes, by producing a transient biofilm dependent on the symbiosis polysaccharide (SYP). In vitro, however, wild-type strain ES114 fails to form SYP-dependent biofilms. Instead, genetically engineered strains, such as those lacking the negative regulator BinK, have been developed to study this phenomenon. Historically, V. fischeri has been grown using LBS, a complex medium containing tryptone and yeast extract; supplementation with calcium is required to induce biofilm formation by a binK mutant. Here, through our discovery that yeast extract inhibits biofilm formation, we uncover signals and underlying mechanisms that control V. fischeri biofilm formation. In contrast to its inability to form a biofilm on unsupplemented LBS, a binK mutant formed cohesive, SYP-dependent colony biofilms on tTBS, modified LBS that lacks yeast extract. Moreover, wild-type strain ES114 became proficient to form cohesive, SYP-dependent biofilms when grown in tTBS supplemented with both calcium and the vitamin para-aminobenzoic acid (pABA); neither molecule alone was sufficient, indicating that this phenotype relies on coordinating two cues. pABA/calcium supplementation also inhibited bacterial motility. Consistent with these phenotypes, cells grown in tTBS with pABA/calcium were enriched in transcripts for biofilm-related genes and predicted diguanylate cyclases, which produce the second messenger cyclic-di-GMP (c-di-GMP). They also exhibited elevated levels of c-di-GMP, which was required for the observed phenotypes, as phosphodiesterase overproduction abrogated biofilm formation and partially rescued motility. This work thus provides insight into conditions, signals, and processes that promote biofilm formation by V. fischeri. IMPORTANCE Bacteria integrate environmental signals to regulate gene expression and protein production to adapt to their surroundings. One such behavioral adaptation is the formation of a biofilm, which can promote adherence and colonization and provide protection against antimicrobials. Identifying signals that trigger biofilm formation and the underlying mechanism(s) of action remain important and challenging areas of investigation. Here, we determined that yeast extract, commonly used for growth of bacteria in laboratory culture, inhibits biofilm formation by Vibrio fischeri, a model bacterium used for investigating host-relevant biofilm formation. Omitting yeast extract from the growth medium led to the identification of an unusual signal, the vitamin para-aminobenzoic acid (pABA), that when added together with calcium could induce biofilm formation. pABA increased the concentrations of the second messenger, c-di-GMP, which was necessary but not sufficient to induce biofilm formation. This work thus advances our understanding of signals and signal integration controlling bacterial biofilm formation.

Folate Acts in E. coli to Accelerate C. elegans Aging Independently of Bacterial Biosynthesis.

Folates are cofactors for biosynthetic enzymes in all eukaryotic and prokaryotic cells. Animals cannot synthesize folate and must acquire it from their diet or microbiota. Previously, we showed that inhibiting E. coli folate synthesis increases C. elegans lifespan. Here, we show that restriction or supplementation of C. elegans folate does not influence lifespan. Thus, folate is required in E. coli to shorten worm lifespan. Bacterial proliferation in the intestine has been proposed as a mechanism for the life-shortening influence of E. coli. However, we found no correlation between C. elegans survival and bacterial growth in a screen of 1,000+ E. coli deletion mutants. Nine mutants increased worm lifespan robustly, suggesting specific gene regulation is required for the life-shortening activity of E. coli. Disrupting the biosynthetic folate cycle did not increase lifespan. Thus, folate acts through a growth-independent route in E. coli to accelerate animal aging.

Importance of bacterial replication and alveolar macrophage-independent clearance mechanisms during early lung infection with Streptococcus pneumoniae.

Although the importance of alveolar macrophages for host immunity during early Streptococcus pneumoniae lung infection is well established, the contribution and relative importance of other innate immunity mechanisms and of bacterial factors are less clear. We have used a murine model of S. pneumoniae early lung infection with wild-type, unencapsulated, and para-amino benzoic acid auxotroph mutant TIGR4 strains to assess the effects of inoculum size, bacterial replication, capsule, and alveolar macrophage-dependent and -independent clearance mechanisms on bacterial persistence within the lungs. Alveolar macrophage-dependent and -independent (calculated indirectly) clearance half-lives and bacterial replication doubling times were estimated using a mathematical model. In this model, after infection with a high-dose inoculum of encapsulated S. pneumoniae, alveolar macrophage-independent clearance mechanisms were dominant, with a clearance half-life of 24 min compared to 135 min for alveolar macrophage-dependent clearance. In addition, after a high-dose inoculum, successful lung infection required rapid bacterial replication, with an estimated S. pneumoniae doubling time of 16 min. The capsule had wide effects on early lung clearance mechanisms, with reduced half-lives of 14 min for alveolar macrophage-independent and 31 min for alveolar macrophage-dependent clearance of unencapsulated bacteria. In contrast, with a lower-dose inoculum, the bacterial doubling time increased to 56 min and the S. pneumoniae alveolar macrophage-dependent clearance half-life improved to 42 min and was largely unaffected by the capsule. These data demonstrate the large effects of bacterial factors (inoculum size, the capsule, and rapid replication) and alveolar macrophage-independent clearance mechanisms during early lung infection with S. pneumoniae.

Folate production by probiotic bacteria.

Probiotic bacteria, mostly belonging to the genera Lactobacillus and Bifidobacterium, confer a number of health benefits to the host, including vitamin production. With the aim to produce folate-enriched fermented products and/or develop probiotic supplements that accomplish folate biosynthesis in vivo within the colon, bifidobacteria and lactobacilli have been extensively studied for their capability to produce this vitamin. On the basis of physiological studies and genome analysis, wild-type lactobacilli cannot synthesize folate, generally require it for growth, and provide a negative contribution to folate levels in fermented dairy products. Lactobacillus plantarum constitutes an exception among lactobacilli, since it is capable of folate production in presence of para-aminobenzoic acid (pABA) and deserves to be used in animal trials to validate its ability to produce the vitamin in vivo. On the other hand, several folate-producing strains have been selected within the genus Bifidobacterium, with a great variability in the extent of vitamin released in the medium. Most of them belong to the species B. adolescentis and B. pseudocatenulatum, but few folate producing strains are found in the other species as well. Rats fed a probiotic formulation of folate-producing bifidobacteria exhibited increased plasma folate level, confirming that the vitamin is produced in vivo and absorbed. In a human trial, the same supplement raised folate concentration in feces. The use of folate-producing probiotic strains can be regarded as a new perspective in the specific use of probiotics. They could more efficiently confer protection against inflammation and cancer, both exerting the beneficial effects of probiotics and preventing the folate deficiency that is associated with premalignant changes in the colonic epithelia.

para-Aminobenzoic acid is a precursor in coenzyme Q6 biosynthesis in Saccharomyces cerevisiae.

Coenzyme Q (ubiquinone or Q) is a crucial mitochondrial lipid required for respiratory electron transport in eukaryotes. 4-Hydroxybenozoate (4HB) is an aromatic ring precursor that forms the benzoquinone ring of Q and is used extensively to examine Q biosynthesis. However, the direct precursor compounds and enzymatic steps for synthesis of 4HB in yeast are unknown. Here we show that para-aminobenzoic acid (pABA), a well known precursor of folate, also functions as a precursor for Q biosynthesis. A hexaprenylated form of pABA (prenyl-pABA) is normally present in wild-type yeast crude lipid extracts but is absent in yeast abz1 mutants starved for pABA. A stable (13)C(6)-isotope of pABA (p- amino[aromatic-(13)C(6)]benzoic acid ([(13)C(6)]pABA)), is prenylated in either wild-type or abz1 mutant yeast to form prenyl-[(13)C(6)]pABA. We demonstrate by HPLC and mass spectrometry that yeast incubated with either [(13)C(6)]pABA or [(13)C(6)]4HB generate both (13)C(6)-demethoxy-Q (DMQ), a late stage Q biosynthetic intermediate, as well as the final product (13)C(6)-coenzyme Q. Pulse-labeling analyses show that formation of prenyl-pABA occurs within minutes and precedes the synthesis of Q. Yeast utilizing pABA as a ring precursor produce another nitrogen containing intermediate, 4-imino-DMQ(6). This intermediate is produced in small quantities in wild-type yeast cultured in standard media and in abz1 mutants supplemented with pABA. We suggest a mechanism where Schiff base-mediated deimination forms DMQ(6) quinone, thereby eliminating the nitrogen contributed by pABA. This scheme results in the convergence of the 4HB and pABA pathways in eukaryotic Q biosynthesis and has implications regarding the action of pABA-based antifolates.

4-Aminobiphenyl downregulation of NAT2 acetylator genotype-dependent N- and O-acetylation of aromatic and heterocyclic amine carcinogens in primary mammary epithelial cell cultures from rapid and slow acetylator rats.

Aromatic and heterocyclic amine carcinogens present in the diet and in cigarette smoke induce breast tumors in rats. N-acetyltransferase 1 (NAT1) and N-acetyltransferase 2 (NAT2) enzymes have important roles in their metabolic activation and deactivation. Human epidemiological studies suggest that genetic polymorphisms in NAT1 and/or NAT2 modify breast cancer risk in women exposed to these carcinogens. p-Aminobenzoic acid (selective for rat NAT2) and sulfamethazine (SMZ; selective for rat NAT1) N-acetyltransferase catalytic activities were both expressed in primary cultures of rat mammary epithelial cells. PABA, 2-aminofluorene, and 4-aminobiphenyl N-acetyltransferase and N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine and N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline O-acetyltransferase activities were two- to threefold higher in mammary epithelial cell cultures from rapid than slow acetylator rats. In contrast, SMZ (a rat NAT1-selective substrate) N-acetyltransferase activity did not differ between rapid and slow acetylators. Rat mammary cells cultured in the medium supplemented 24 h with 10muM ABP showed downregulation in the N-and O-acetylation of all substrates tested except for the NAT1-selective substrate SMZ. This downregulation was comparable in rapid and slow NAT2 acetylators. These studies clearly show NAT2 acetylator genotype-dependent N- and O-acetylation of aromatic and heterocyclic amine carcinogens in rat mammary epithelial cell cultures to be subject to downregulation by the arylamine carcinogen ABP.

Folate fortification of rice by metabolic engineering.

Rice, the world's major staple crop, is a poor source of essential micronutrients, including folates (vitamin B9). We report folate biofortification of rice seeds achieved by overexpressing two Arabidopsis thaliana genes of the pterin and para-aminobenzoate branches of the folate biosynthetic pathway from a single locus. We obtained a maximal enhancement as high as 100 times above wild type, with 100 g of polished raw grains containing up to four times the adult daily folate requirement.

Resistance to sulphadrug-based antifolate therapy in malaria: are we looking in the right place?

Sulphadrug treatment failure in malaria therapy cannot solely be ascribed to the build-up of genetic resistance within the parasitic genome. Although numerous in vitro studies have tried to determine the exact genetic markers that could predict treatment outcome in patients, this research has not been conclusive. Sulphadrugs work by competitive inhibition with pABA at one point of the pathway to de novo folate synthesis. However, evidence suggests that the malaria parasite is capable of overcoming this competitive inhibition by switching over to other metabolic pathways, like direct folate salvage from a person's bloodstream. In other words, increased folic acid administration, via diet or supplementation, may have reduced the effectiveness of sulphadrugs more than genetic mutations. Although in vitro studies are valuable for understanding disease mechanisms, we should not forget that the human being is infinitely more complex than any laboratory model.

Bacterially synthesized folate and supplemental folic acid are absorbed across the large intestine of piglets.

A large pool of folate exists in the large intestine of humans. Preliminary evidence, primarily in vitro, suggests that this folate may be bioavailable. The purpose of this study was to test the hypothesis that supplemental folic acid and bacterially synthesized folate are absorbed across the large intestine of piglets. The pig was used as an animal model because it resembles the human in terms of folate absorption, at least in the small intestine. A tracer of [3H]-folic acid or [3H]-para-aminobenzoic acid ([3H]-PABA), a precursor of bacterially synthesized folate, was injected into the cecum of 11-day-old piglets. Feces and urine were collected for 3 days. Thereafter, piglets were killed, and livers and kidneys harvested. [3H]-Folate was isolated from biological samples by affinity chromatography using immobilized milk folate binding proteins and counted using a scintillation counter. In piglets injected with [3H]-folic acid, the feces, liver, urine and kidneys accounted for 82.1%, 12.3%, 3.9% and 1.7% of recovered [3H]-folate, respectively. In piglets injected with [3H]-PABA, the amount of recovered bacterially synthesized folate in the feces, liver and urine was 85.1%, 0.4% and 14.6%, respectively. Twenty-three percent and 13% of tritium were recovered in samples examined (liver, kidney, fecal and urine) from piglets injected with [3H]-folic acid and [3H]-PABA, respectively. Using our estimates of [3H]-folic acid absorption and the total and percent monoglutamyl folate content of piglet feces, we predict that at least 18% of the dietary folate requirement for the piglet could be met by folate absorption across the large intestine.

Candicidin biosynthesis in Streptomyces griseus.

The biosynthesis of the aromatic polyene macrolide antibiotic candicidin, produced by Streptomyces griseus IMRU 3570, begins with a p-aminobenzoic acid (PABA) molecule which is activated to PABA-CoA and used as starter for the head-to-tail condensation of four propionate and 14 acetate units to produce a polyketide molecule to which the deoxysugar mycosamine is attached. Using the gene coding for the PABA synthase ( pabAB) from S. griseusIMRU 3570 as the probe, a 205-kb region of continuous DNA from the S. griseus chromosome was isolated and partially sequenced. Some of the genes possibly involved in the biosynthesis of candicidin were identified including part of the modular polyketide synthase (PKS), genes for thioesterase, deoxysugar biosynthesis, modification, transport, and regulatory proteins. The regulatory mechanisms involved in the production of candicidin, such as phosphate regulation, were studied using internal probes for some of the genes involved in the biosynthesis of the three moieties of candicidin (PKS, aromatic moiety and amino sugar). mRNAs specific for these genes were detected only in the production medium (SPG) but not in the SPG medium supplemented with phosphate or in the inoculum medium, indicating that phosphate represses the expression of genes involved in candicidin biosynthesis. The modular architecture of the candicidin PKS and the availability of the PKSs involved in the biosynthesis of three polyene antibiotics (pimaricin, nystatin, and amphotericin B) shall make possible the creation of new, less toxic and more active polyene antibiotics through combinatorial biosynthesis and targeted mutagenesis.

Kinetics of folate turnover in pregnant women (second trimester) and nonpregnant controls during folic acid supplementation: stable-isotopic labeling of plasma folate, urinary folate and folate catabolites shows subtle effects of pregnancy on turnover of folate pools.

To investigate the effects of pregnancy on folate metabolism, we conducted an 84-d study in second-trimester (gestational wk 14-25) pregnant women (n = 6) and nonpregnant controls (n = 6) with stable-isotopic tracer methods. All subjects were fed a diet containing approximately 272 nmol/d (120 microg/d) folate from food, along with supplemental folic acid that contained 15% [3',5'-(2)H(2)] folic acid ([(2)H(2)]folic acid) during d 1--41 and that was unlabeled during d 42--84 to yield a constant total folate intake of 1.02 or 1.93 micromol/d (450 or 850 microg/d). Isotopic enrichment of plasma folate, urinary folate and the urinary folate catabolites para-aminobenzoylglutamate (pABG) and para-acetamidobenzoylglutamate (ApABG) was determined at intervals throughout the study. The labeling of pABG and ApABG reflected that of tissue folate pools from which the catabolites originate. After the intake of labeled folic acid was terminated on d 41, labeling of urinary folate exhibited a biphasic exponential decline with distinct fast and slow components. In contrast, during d 42--84, the enrichment of urinary pABG and ApABG exhibited primarily monophasic exponential decline, and plasma folate underwent little decline of labeling during this period. Pregnant women and controls did not differ in estimates of body folate pool size and most aspects of the excretion of labeled urinary folate and catabolites, rates of decline of excretion, and areas under the curves for folate and catabolite excretion. Pregnant women, however, tended to have a slower rate of decline of pABG than ApABG and higher enrichment at d 42 of ApABG and pABG. These data support and extend our previous findings indicating that pregnancy (gestational wk 14--26) causes subtle changes in folate metabolism but does not elicit substantial increases in the rate or extent of folate turnover at these moderately high folate intakes.

Inhibitory actions of luteolin on the growth and arylamine N-acetyltransferase activity in strains of Helicobacter pylori from ulcer patients.

Helicobacter pylori is now recognized as an important cause of type B gastritis, which is strongly associated with gastric and duodenal ulcer disease. H. pylori may be a causative factor in patients with gastric cancer. The growth inhibition and N-acetylation of 2-Aminofluorene (AF) or P-aminobenzoic acid (PABA) by arylamine N-acetyltransferase (NAT) in H. pylori were inhibited by luteolin, a component in herbal medicine. The growth inhibition was based on the changes of optical density (OD) by using a spectrophotometer. The N-acetylation of AF or PABA by NAT from H. pylori were assayed by the amounts of acetylated and non-acetylated AF or PABA in cytosols and intact bacteria of H. pylori by using HPLC. An inhibition of growth on H. pylori demonstrated that luteolin elicited a dose-dependent growth inhibition in the H. pylori cultures. Cytosols and suspensions of H. pylori with or without specific concentrations of luteolin co-treatment showed different percentages of AF or PABA acetylation. The data indicated that there was decreased NAT activity associated with increased levels of luteolin in H. pylori cytosols and suspensions. Using standard steady-state kinetic analysis, it was demonstrated that luteolin was a possible uncompetitive inhibitor to NAT enzyme in H. pylori. This report is the first demonstration to show that luteolin can inhibit H. pylori growth and NAT activity.

Effect of norcantharidin on N-acetyltransferase activity in HepG2 cells.

The inhibition ofarylamine N-acetyltransferase (NAT) activity by norcantharidin (NCTD), the demethylated form of cantharidin, in human hepatocellular carcinoma HepG2 cells was investigated. By using high performance liquid chromatography, NAT activity on acetylation of 2-aminofluorene (AF) and p-aminobenzoic acid (PABA) were examined. Two assay systems were performed, one with cellular cytosols, the other with intact HepG2 cell suspensions. The NAT activity in HepG2 cell line was inhibited by norcantharidin in a dose-dependent manner in both types of examined systems: i.e. the greater the concentration of norcantharidin in the reaction, the greater the inhibition of NAT activities. This report is the first to show that norcantharidin has an inhibitory effect on NAT activity in HepG2 cell.

Substrate-dependent regulation of human arylamine N-acetyltransferase-1 in cultured cells.

Arylamine N-acetyltransferase-1 (NAT1) is a polymorphically expressed enzyme that is widely distributed throughout the body. In the present study, we provide evidence for substrate-dependent regulation of this enzyme. Human peripheral blood mononuclear cells cultured in medium supplemented with p-aminobenzoic acid (PABA; 6 microM) for 24 h showed a significant decrease (50-80%) in NAT1 activity. The loss of activity was concentration-dependent (EC(50) approximately 2 microM) and selective because PABA had no effect on the activity of constitutively expressed lactate dehydrogenase or aspartate aminotransferase. PABA also induced down-regulation of NAT1 activity in several human cell lines grown at confluence. Substrate-dependent down-regulation was not restricted to PABA. Addition of other NAT1 substrates, such as p-aminosalicylic acid, ethyl-p-aminobenzoate, or p-aminophenol to peripheral blood mononuclear cells in culture also resulted in significant (P <.05) decreases in NAT1 activity. However, addition of the NAT2-selective substrates sulfamethazine, dapsone, or procainamide did not alter NAT1 activity. Western blot analysis using a NAT1-specific antibody showed that the loss of NAT1 activity was associated with a parallel reduction in the amount of NAT1 protein (r(2) = 0.95). Arylamines that did not decrease NAT1 activity did not alter NAT1 protein levels. Semiquantitative reverse transcriptase polymerase chain reaction of mRNA isolated from treated and untreated cells revealed no effect of PABA on NAT1 mRNA levels. We conclude that NAT1 can be down-regulated by arylamines that are themselves NAT1 substrates. Because NAT1 is involved in the detoxification/activation of various drugs and carcinogens, substrate-dependent regulation may have important consequences with regard to drug toxicity and cancer risk.

Effects of the garlic components diallyl sulfide and diallyl disulfide on arylamine N-acetyltransferase activity in human colon tumour cells.

Diallyl sulfide (DAS) and diallyl disulfide (DADS), major components of garlic, were used to determine inhibition of arylamine N-acetyltransferase (NAT) activity in a human colon tumour (adenocarcinoma) cell line. Two assay systems were performed, one with cellular cytosols (9000g supernatant), the other with intact bacterial cell suspensions. The NAT activity in a human colon tumour cell line was inhibited by DAS and DADS in a dose-dependent manner in both system: that is, the greater the concentration of DAS and DADS in the reaction, the greater the inhibition of NAT activities in both systems. The data also indicated that DAS and DADS decrease the apparent values of Km and Vmax of NAT enzymes from human colon tumour cells in both systems examined. This is the first report to demonstrate that garlic components do affect human colon tumour cell NAT activity.

Comparative effects of enteric-coated pancreatin microsphere therapy after conventional and pylorus-preserving pancreatoduodenectomy.

BACKGROUND: A comparative study was performed between patients with exocrine pancreatic insufficiency after conventional pancreatoduodenectomy (Whipple's procedure) and pylorus-preserving pancreatoduodenectomy (PPPD). In these patients the pharmacodynamics of 2-mm enteric-coated pancreatin microspheres (ECPMs) and their gastric transit time in relation to that of a solid meal were investigated. The efficacy of ECPM preparations may differ after Whipple's procedure compared with PPPD, because the latter procedure does not include gastrectomy. METHODS: Gastric transit was assessed by double-isotope scintigraphy. A pancake meal was labelled with 99mTc. ECPMs were cold-labelled with 170Er and neutron activated shortly before ingestion to enable imaging with a gamma camera. Intraluminal pancreatic enzyme activity was assessed during a 6-h period with two indirect tests: the cholesteryl [14C]octanoate breath test and the N-benzoyl-L-tyrosyl-p-aminobenzoic acid-p-aminosalicylic acid (NBT-PABA-PAS) test. RESULTS: In patients who had Whipple's procedure, the gastric transit time of ECPMs and of the pancake meal was not significantly different. The outcome of the indirect pancreatic function tests during enzyme supplementation was comparable, and not significantly different, from that in healthy volunteers. In patients who had PPPD, however, the gastric transit time of microspheres was greatly delayed compared with that of the pancake meal (P < 0.05). Improvement in the outcome of the indirect pancreatic function tests during enzyme supplementation was much less and remained well below that of healthy volunteers (P < 0.05). CONCLUSION: In cases of exocrine pancreatic insufficiency after Whipple's procedure, 2-mm ECPM treatment adequately restores pancreatic enzyme activity. Following PPPD, however, ECPM treatment is often ineffective because the microspheres are retained in the stomach. In these patients, use of conventional powdered pancreatin enzyme preparations may improve the efficacy of treatment.

Virulence studies of Aspergillus nidulans mutants requiring lysine or p-aminobenzoic acid in invasive pulmonary aspergillosis.

To identify steps in fungal intermediary metabolism required by Aspergillus spp. during invasive pulmonary aspergillosis, we have developed murine models involving Aspergillus nidulans as the inoculum. The advantages of using A. nidulans over Aspergillus fumigatus or Aspergillus flavus, which are the most common agents of clinical disease, are the well-understood genetics of A. nidulans and a large range of mutants of this species which are affected in a variety of metabolic pathways. Comparison of the virulence of A. nidulans strains carrying mutations which block the biosynthesis of lysine (lysA2) and p-aminobenzoic acid (pabaA1) shows that lysA2 strains have reduced virulence while pabaA1 strains are entirely nonpathogenic. The pathogenicity of pabaA1 strains can be restored by supplementing the drinking water of animals with p-aminobenzoic acid. The results indicate that the availability of lysine in the lung is limited, and p-aminobenzoic acid is probably not available at all. Thus, models of invasive pulmonary aspergillosis involving A. nidulans can be used to identify metabolic pathways that may be essential for the pathogenicity of A. fumigatus, the predominant pathogenic species, suggesting potential new targets for antifungal therapy.

Identification of a substance, previously shown to enhance mitogenesis of human lymphocytes, as the acetamide of p-aminobenzoic acid.

We characterize here an arachidonic acid (AA)-derived metabolite previously found to have an adjuvant effect in phytohemagglutinin-induced mitogenesis of lymphocytes from mothers of newborn babies and from immunodeficient infants. We named the metabolite 'compound 4' due to its position in a thin-layer chromatography system developed for isolation of eicosanoids. The compound was originally found to be produced by peripheral blood mononuclear leukocytes and the T cell leukemia line Jurcat after long-term (18-24 h) incubation with [1-14C]AA. Compound 4 is also produced by lymphocytes, monocytes, platelets, thrombocytes, cultured fibroblasts and various types of malignant cell lines. We purified this metabolite by means of high pressure liquid chromatography with synchronous detection of radioactivity and measurement of ultraviolet-light absorption at 278 nm. Proton nuclear magnetic resonance spectroscopy and mass spectrometry with electron impact techniques demonstrated that compound 4 is not an eicosanoid, but is identical to p-acetamidobenzoic acid (PACBA). The cells synthesize PACBA from p-aminobenzoic acid and a two-carbon residue from AA.