Sericin enhances ammonia detoxification by promotes urea cycle enzyme genes and activates hepatic autophagy in relation to CARD-9/MAPK pathway.

Urea cycle is an important metabolic process that initiates in liver mitochondria and converts ammonia to urea. The impairment of ammonia detoxification, both primary and secondary causes, lead to hyperammonemia, a life-threatening condition affecting to the brain. Current treatments are not enough effective. In addition, our recent proteomics study in hypercholesterolemic rat model demonstrated that sericin enhances hepatic nitrogenous waste removal through carbamoyl-phosphate synthase 1 (CPS-1), aldehyde dehydrogenase-2 (ALDH-2), and uricase proteins. However, the underlining mechanisms regard to this property is not clarified yet. Therefore, the present study aims to examine the effect of sericin on urea cycle enzyme genes (CPS-1 and ornithine transcarbamylase; OTC) and proteins (mitogen-activated protein kinase; MAPK, caspase recruitment domain-containing protein 9; CARD-9, Microtubule-associated protein light chain 3; LC-3), which relate to urea production and liver homeostasis in hepatic cell line (HepG2) and hypercholesterolemic rat treated with or without sericin. qRT-PCR, immunohistochemistry, and electron microscopy techniques were performed. In vitro study determined that high dose of sericin at 1 mg/ml increased liver detoxification enzyme (Cytochrome P450 1A2; CYP1A2 and ALDH-2) and urea cycle enzyme (CPS-1 and OTC) genes. Both in HepG2 cell and rat liver mitochondria, sericin significantly downregulated CARD-9 (apoptotic protein) expression while upregulated MAPK (hepatic homeostasis protein) and LC-3 (autophagic protein) expressions. Hence, it might be concluded that sericin promotes ammonia detoxification by both increases urea cycle enzyme genes and enhances hepatic autophagy in associated with CARD-9/MAPK pathway (as shown by their own negative relationship). This study presents another beneficial property of sericin to develop an upcoming candidate for ammonia toxicity alleviation and liver function improvement.

Sericin-mediated improvement of dysmorphic cardiac mitochondria from hypercholesterolaemia is associated with maintaining mitochondrial dynamics, energy production, and mitochondrial structure.

CONTEXT: Sericin is a component protein in the silkworm cocoon [Bombyx mori Linnaeus (Bombycidae)] that improves dysmorphic cardiac mitochondria under hypercholesterolemic conditions. This is the first study to explore cardiac mitochondrial proteins associated with sericin treatment. OBJECTIVE: To investigate the mechanism of action of sericin in cardiac mitochondria under hypercholesterolaemia. MATERIALS AND METHODS: Hypercholesterolaemia was induced in Wistar rats by feeding them 6% cholesterol-containing chow for 6 weeks. The hypercholesterolemic rats were separated into 2 groups (n = 6 for each): the sericin-treated (1,000 mg/kg daily) and nontreated groups. The treatment conditions were maintained for 4 weeks prior to cardiac mitochondria isolation. The mitochondrial structure was evaluated by immunolabeling electron microscopy, and differential mitochondrial protein expression was determined and quantitated by two-dimensional gel electrophoresis coupled with mass spectrometry. RESULTS: A 32.22 ± 2.9% increase in the percent striated area of cardiac muscle was observed in sericin-treated hypercholesterolemic rats compared to the nontreatment group (4.18 ± 1.11%). Alterations in mitochondrial proteins, including upregulation of optic atrophy 1 (OPA1) and reduction of NADH-ubiquinone oxidoreductase 75 kDa subunit (NDUFS1) expression, are correlated with a reduction in mitochondrial apoptosis under sericin treatment. Differential proteomic observation also revealed that sericin may improve mitochondrial energy production by upregulating acetyl-CoA acetyltransferase (ACAT1) and NADH dehydrogenase 1α subcomplex subunit 10 (NDUFA10) expression. DISCUSSION AND CONCLUSIONS: Sericin treatment could improve the dysmorphic mitochondrial structure, metabolism, and energy production of cardiac mitochondria under hypercholesterolaemia. These results suggest that sericin may be an alternative treatment molecule that is related to cardiac mitochondrial abnormalities.

Cytoskeletal Alteration Is an Early Cellular Response in Pulmonary Epithelium Infected with Aspergillus fumigatus Rather than Scedosporium apiospermum.

Invasive aspergillosis and scedosporiosis are life-threatening fungal infections with similar clinical manifestations in immunocompromised patients. Contrarily, Scedosporium apiospermum is susceptible to some azole derivative but often resistant to amphotericin B. Histopathological examination alone cannot diagnose these two fungal species. Pathogenesis studies could contribute to explore candidate protein markers for new diagnosis and treatment methods leading to a decrease in mortality. In the present study, proteomics was conducted to identify significantly altered proteins in A549 cells infected with or without Aspergillus fumigatus and S. apiospermum as measured at initial invasion. Protein validation was performed with immunogold labelling alongside immunohistochemical techniques in infected A549 cells and lungs from murine models. Further, cytokine production was measured, using the Bio-Plex-Multiplex immunoassay. The cytoskeletal proteins HSPA9, PA2G4, VAT1, PSMA2, PEX1, PTGES3, KRT1, KRT9, CLIP1 and CLEC20A were mainly changed during A. fumigatus infection, while the immunologically activated proteins WNT7A, GAPDH and ANXA2 were principally altered during S. apiospermum infection. These proteins are involved in fungal internalisation and structural destruction leading to pulmonary disorders. Interleukin (IL)-21, IL-1α, IL-22, IL-2, IL-8, IL-12, IL-17A, interferon-γ and tumour necrosis factor-α were upregulated in both aspergillosis and scedosporiosis, although more predominately in the latter, in accordance with chitin synthase-1 and matrix metalloproteinase levels. Our results demonstrated that during invasion, A. fumigatus primarily altered host cellular integrity, whereas S. apiospermum chiefly induced and extensively modulated host immune responses.

Draft Genome Sequence of Diaporthe sp. Strain HANT25, an Endophytic Fungus Producing Mycoepoxydiene.

Diaporthe sp. strain HANT25 is an endophytic fungus that produces mycoepoxydiene, a rare bioactive natural compound. Here, we report the genome sequence of Diaporthe sp. HANT25, comprising 55.3 Mb in 80 scaffolds. The genome sequence should enhance understanding of the biology and bioactive compound production potential of the genus Diaporthe.

A combination extract of kaffir lime, galangal, and lemongrass maintains blood lipid profiles, hepatocytes, and liver mitochondria in rats with nonalcoholic steatohepatitis.

Kaffir lime leaves and the rhizomes of galangal and lemongrass are the main ingredients in several Thai foods with desirable medicinal effects. Based on their beneficial activities, this study aimed to indicate the chemical properties and in vivo efficacy of a combination of the herbs at a 1:2:1 ratio in a water extract form. Its volatile constituents were analyzed by gas chromatography coupled with mass spectrometer, which mainly consists of eucalyptol, citronellal, and citral. Clinicohistopathological and electron microscopic studies demonstrated that the extract corrected blood cholesterol, LDL, HDL, and triglyceride levels similarly as simvastatin treatment in association with its antioxidative properties, as indicated by the levels of superoxide dismutase and malondialdehyde in serum and the increment of nuclear factor erythroid 2-related factor 2 levels in hepatocytes. Hepatitis was significantly less severe in rats fed the extract for 14 days than in simvastatin-treated rats. Regarding its immunomodulatory properties, the extract also accelerated hepatic resolution from steatohepatitis during hypercholesterolemia as indicated by the upregulation of vimentin, cytokeratin, and CD206+. Interestingly, liver mitochondria were also preserved in hypercholesterolemic rats treated with the extract in relation to their architecture and the expression of haloacid dehalogenase-like hydrolase domain-containing protein 3 as well as metabolic and energy regulators. Therefore, the study concluded that the water extract of kaffir lime leaves and the rhizomes of galangal and lemongrass has beneficial effects on blood cholesterol, the severity of steatohepatitis, and the maintenance of mitochondrial architecture via its antioxidative and immunomodulatory activities.

Adaptive effect of sericin on hepatic mitochondrial conformation through its regulation of apoptosis, autophagy and energy maintenance: a proteomics approach.

We recently demonstrated that in addition to its protective effect on pancreatic and adrenal biosynthesis, antioxidant properties of sericin decrease blood cholesterol levels and improve the liver mitochondrial architecture. However, little is known about the detailed mechanisms underlying these effects. Using proteomics and electron microscopy, we identified mitochondrial proteins that play important roles in the preservation of the mitochondrial ultrastructure and cholesterol-lowering properties of sericin. Our results showed that sericin maintains the mitochondrial architecture during conditions of high blood cholesterol by regulating apoptotic (NADH-ubiquinone oxidoreductase 75 kDa subunit) and autophagic (mitochondrial elongation factor Tu and prohibitin-2) proteins as well as energy maintenance proteins [haloacid dehalogenase-like hydrolase domain-containing protein 3, succinate dehydrogenase (ubiquinone) flavoprotein subunit, ATP synthase-α subunit precursor, enoyl-CoA hydratase domain-containing protein 3 and electron transfer flavoprotein subunit-α]. Sericin also exerts anti-oxidative properties via aconitate hydratase and Chain A, crystal structure of rat carnitine palmitoyltrasferase 2 proteins. Together, these activities may reduce hepatocytic triglyceride deposition, thereby decreasing steatosis, as demonstrated by the modulatory effects on ornithine aminotransferase, mitochondrial aspartate aminotransferase, acyl-CoA synthase, hydroxyacyl-CoA dehydrogenase and D-beta-hydroxybutyrate dehydrogenase. Sericin activity further balanced nitrogenous waste detoxification, characterised by carbamoyl-phosphate synthase (ammonia), aldehyde dehydrogenase and uricase, or folate biosynthesis via sarcosine dehydrogenase and dimethyl glycine dehydrogenase. These results suggest that sericin maintains the hepatic mitochondrial architecture through apoptotic, autophagic, energy maintenance and anti-oxidative mitochondrial proteins for alleviating hepatic steatosis and promoting liver function under conditions of hypercholesterolaemia.

A kinetic spectrophotometric method for the determination of pyridoxal-5'-phosphate based on coenzyme activation of apo-d-phenylglycine aminotransferase.

A new PLP assay method based on the coenzyme activation of apo-d-phenylglycine aminotransferase (apo-d-PhgAT) is reported. The assay process is comprised of two steps. First, PLP present in plasma samples is allowed to reconstitute apo-d-PhgAT, forming active holo-d-PhgAT. In the second step, the enzymatic activity of reconstituted d-PhgAT is determined using d-4-OH-phenylglycine as the amino donor substrate with 4-OH-benzoylformate (OH-BZF) as the reaction product. OH-BZF absorbs UV light strongly at 334 nm (molar absorption coefficient = 25.4 × 103 M-1cm-1) and its rate of formation is monitored spectrophotometrically. The rate of the transamination reaction catalyzed by the reconstituted d-PhgAT is directly proportional to the amount of PLP in the sample. The method is applicable for determining PLP in the concentration range from 5.2 to 250 nM and requires 50 µL of plasma sample. The mean within- and between-run coefficient of variations (CVs) were 8.1% and 12.4%, respectively. Analytical recoveries ranged from 98 to 108%. The assay was specific and showed good correlation with the established method (CDC, Method No: 4002.05). The assay requires one reaction catalyzed by a single enzyme, does not require a radioactive substrate, and a derivatization reagent is not needed. This PLP determination process is relatively simple to perform and can be completed using common laboratory equipment.

Gene Expression and Molecular Characterization of a Xylanase from Chicken Cecum Metagenome.

A xylanase gene xynAMG1 with a 1,116-bp open reading frame, encoding an endo-ß-1,4-xylanase, was cloned from a chicken cecum metagenome. The translated XynAMG1 protein consisted of 372 amino acids including a putative signal peptide of 23 amino acids. The calculated molecular mass of the mature XynAMG1 was 40,013 Da, with a theoretical pI value of 5.76. The amino acid sequence of XynAMG1 showed 59% identity to endo-ß-1,4-xylanase from Prevotella bryantii and Prevotella ruminicola and 58% identity to that from Prevotella copri. XynAMG1 has two conserved motifs, DVVNE and TEXD, containing two active site glutamates and an invariant asparagine, characteristic of GH10 family xylanase. The xynAMG1 gene without signal peptide sequence was cloned and fused with thioredoxin protein (Trx.Tag) in pET-32a plasmid and overexpressed in Escherichia coli Tuner™(DE3)pLysS. The purified mature XynAMG1 was highly salt-tolerant and stable and displayed higher than 96% of its catalytic activity in the reaction containing 1 to 4 M NaCl. It was only slightly affected by common organic solvents added in aqueous solution to up to 5 M. This chicken cecum metagenome-derived xylanase has potential applications in animal feed additives and industrial enzymatic processes requiring exposure to high concentrations of salt and organic solvents.

Sericin improves heart and liver mitochondrial architecture in hypercholesterolaemic rats and maintains pancreatic and adrenal cell biosynthesis.

Hypercholesterolaemia is well known to be associated with mitochondrial dysfunction, subsequently leading to multiple organ failure. Similar to other natural products, sericin is a candidate for adjunctive therapy in hyperlipidaemic conditions. However, the cholesterol-lowering mechanisms of sericin are multifactorial and controversial. Here, a high-cholesterol-fed rat model with or without sericin treatment was established using a dosage of 1000mg/kg/day for 30 days. Blood lipid profiles, oxidative stress markers (superoxide dismutase, SOD; malondialdehyde, MDA; nuclear factor erythroid 2-related factor, Nrf-2), dysmorphic mitochondria in relation to fission (dynamin-related protein-1; Drp-1) and fusion (guanosine triphosphatase mutated in dominant optic atrophy; OPA-1) markers and biosynthetic markers (aquaporin, AQP-1; tubulin-4ß, Tb4B) in the pancreas and adrenal gland were evaluated. The results showed that sericin reduced blood cholesterol and increased high-density lipoprotein (HDL) by acting against oxidative stress. Hypocholesterolaemic and antioxidant conditions further preserved heart and liver mitochondrial architecture; however, this protection was not exhibited in the kidney, where a high level of renal mitophagy, indicating by LC-3 up-regulation, was presented. The steps of ultrastructural alteration of mitochondria from degenerative changes to necrosis were also demonstrated. Sericin also conserved AQP-1 and Tb4B levels in the exocrine pancreatic acinar cells and zona glomerulosa cells, which were positively correlated with serum lipase, HDL, antioxidative markers and mitochondrial integrity. The present study revealed that sericin not only has antioxidant capacity but also balances pancreatic and adrenal cell biosynthesis, especially lipase activity, which may have played an important role in improving lipid dysregulation in the hypercholesterolaemic rat model, leading to the reduction of dysmorphic mitochondria, particularly in the heart and liver.

Sericin ameliorated dysmorphic mitochondria in high-cholesterol diet/streptozotocin rat by antioxidative property.

Sericin has been implicated in lower cholesterolemic effect due to its properties with several mechanisms. Mitochondria are one of the most important targets to be affected in high blood cholesterol and glucose conditions. The protective role of sericin on mitochondria remains doubtful. To examine this role, electron microscopic, histopathologic, immunohistochemical, and biochemical studies were performed in a high-cholesterol diet/streptozotocin rat model. The results demonstrated that sericin reduced blood cholesterol without hypoglycemic effect. Sericin alleviated dysmorphic mitochondria in heart and liver but not in kidney and also decreased peculiar endoplasmic reticulum in the exocrine pancreas. In addition, sericin decreased hepatic steatosis and preserved zymogen granule referable to the decline of reactive oxygen species production in hepatic mitochondrial extraction and down-regulation of malondialdehyde expression in the liver and exocrine pancreas however irrelevant to lipase activity. This study suggests that sericin has antioxidative property to reduce blood cholesterol by means of diminishing fat deposit in hepatocyte and improves mitochondria and endoplasmic reticulum integrities. [Box: see text].

Mitochondrial and cytoskeletal alterations are involved in the pathogenesis of hydronephrosis in ICR/Mlac-hydro mice.

The pathogenesis of congenital hydronephrosis in laboratory animals has been studied for many years, yet little is known about the underlying mechanism of this disease. In this study, we investigated a MS-based comparative proteomics approach to characterize the differently expressed proteins between kidney tissue samples of ICR/Mlac-hydro and wild-type mice. Interestingly, proteomic results exhibited several mitochondrial protein alterations especially the up-regulation of 60 kDa heat shock protein (Hsp60), stress-70 protein (GRP75) dysfunction, and down-regulation of voltage-dependent anion-selective channel protein 1 (VDAC-1). The results demonstrated that mitochondrial alteration may lead to inadequate energy-supply to maintain normal water reabsorption from the renal tubule, causing hydronephrosis. Moreover, the alteration of cytoskeleton proteins in the renal tubule, in particular the up-regulation of tubulin beta-4B chain (Tb4B) and N-myc downstream-regulated gene 1 protein (Ndr-1) may also be related due to their fundamental roles in maintaining cell morphology and tissue stability. In addition, cytoskeletal alterations may consequence to the reduction of glyceraldehydes-3-phosphate dehydrogenase (GAPDH), cytoplasmic enzyme, which modulates the capacity of structural proteins. Our findings highlight a number of target proteins that may play a crucial role in congenital hydronephrosis and emphasize that the disorder of mitochondria and cytoskeleton proteins may be involved.

Optimization of zofimarin production by an endophytic fungus, Xylaria sp. Acra L38.

To optimize the medium for high zofimarin production, sucrose maltose, glucose, tryptone and peptone were used in an orthogonal array design experiment, where the highest value of zofimarin produced was 25.6 µg/mL. This value was about 3 times higher than that obtained with Czapek yeast extract (CzYE) culture medium. A study with Plackett-Burman design showed that sucrose, maltose, glucose and NaNO3 were significant factors in zofimarin production. Further studies using central composite design (CCD) showed the significance of glucose and the interactions of these critical components affecting zofimarin production. Multiple regression analysis of the data yielded a poor fit as shown by the mismatch of the model with these variable factors. When a polynomial equation was applied, the maximum zofimarin production was predicted to be 201.9 µg/mL. Experimental verification yielded a much lower amount of zofimarin, at around 70 µg/mL. Reconsideration of the CCD data and repetition of some runs with high zofimarin production resulted in reproducible zofimarin yield at 79.7 µg/mL. Even though the amount was lower than the predicted value, the medium optimization study was considered to be quite successful as the yield increased to around 8 times that obtained with the original CzYE culture medium.

Effects of halophilic peptide fusion on solubility, stability, and catalytic performance of D-phenylglycine aminotransferase.

D-Phenylglycine aminotransferase (D-PhgAT) from Pseudomonas stutzeri ST-201 is useful for enzymatic synthesis of enantiomerically pure D-phenylglycine. However, its low protein solubility prevents its application at high substrate concentration. With an aim to increase the protein solubility, the N-terminus of D-PhgAT was genetically fused with short peptides (A1 α- helix, A2 α-helix, and ALAL, which is a hybrid of A1 and A2) from a ferredoxin enzyme of a halophilic archaeon, Halobacterium salinarum. The fused enzymes A1-D-PhgAT, A2-D-PhgAT, and ALAL-D-PhgAT displayed a reduced pI and increased in solubility by 6.1-, 5.3-, and 8.1- fold in TEMP (pH 7.6) storage, respectively, and 5-, 4.5-, and 5.9-fold in CAPSO (pH 9.5) reaction buffers, respectively, compared with the wild-type enzyme (WT-D-PhgAT). In addition, all the fused D-PhgAT displayed higher enzymatic reaction rates than the WT-DPhgAT at all concentrations of L-glutamate monosodium salt used. The highest rate, 23.82 ± 1.47 mM/h, was that obtained from having ALAL-D-PhgAT reacted with 1,500 mM of the substrate. Moreover, the halophilic fusion significantly increased the tolerance of D-PhgAT in the presence of NaCl and KCl, being slightly in favor of KCl, where under the same condition at 3.5 M NaCl or KCl all halophilic-fused variants showed higher activity than WT-D-PhgAT.

Optimization of zofimarin production by an endophytic fungus, Xylaria sp. Acra L38

To optimize the medium for high zofimarin production, sucrose maltose, glucose, tryptone and peptone were used in an orthogonal array design experiment, where the highest value of zofimarin produced was 25.6 µg/mL. This value was about 3 times higher than that obtained with Czapek yeast extract (CzYE) culture medium. A study with Plackett-Burman design showed that sucrose, maltose, glucose and NaNO3 were significant factors in zofimarin production. Further studies using central composite design (CCD) showed the significance of glucose and the interactions of these critical components affecting zofimarin production. Multiple regression analysis of the data yielded a poor fit as shown by the mismatch of the model with these variable factors. When a polynomial equation was applied, the maximum zofimarin production was predicted to be 201.9 µg/mL. Experimental verification yielded a much lower amount of zofimarin, at around 70 µg/mL. Reconsideration of the CCD data and repetition of some runs with high zofimarin production resulted in reproducible zofimarin yield at 79.7 µ/mL. Even though the amount was lower than the predicted value, the medium optimization study was considered to be quite successful as the yield increased to around 8 times that obtained with the original CzYE culture medium.

Effective improvement of D-phenylglycine aminotransferase solubility by protein crystal contact engineering.

Structure-guided genetic engineering of D-phenylglycine aminotransferase (D-PhgAT) aimed at increasing protein solubility was attempted. In silico analyses predicted the Asn439 and Gln444 as highly solvent-exposed ß-turn residues involved with protein crystal contact (CC) potential candidates for solubility-improving mutations. They were replaced with Asp and Glu creating the N439D and Q444E single mutants, and N439D/Q444E double mutant with 2.5-, 3.3- and 5.9-fold increases in solubility, respectively. The protein CC prevention effect rather than the net charge effect accounted for the dramatically improved solubility since the N439D, Q444E and N439D/Q444E mutations altered the isoelectric point of D-PhgAT by only 0.1, 0.1 and 0.3 units, respectively. Examination of the D-PhgAT structural model revealed that the N439D mutation weakened the CC attraction force and the Q444E mutation created electrostatic repulsion at the CC point. Analysis of circular dichroism spectra, melting temperature, and D-PhgAT-specific activity showed that the mutations posed no unfavorable effect on the conformational stability and catalytic performance of the enzyme. The protein solubility-improving strategy employed on D-PhgAT in this study was successful with minimal protein structure modification required. It should be applicable with a high chance of success for other proteins, especially those with 3-D structural models available.

A Thermostable phytase from Neosartorya spinosa BCC 41923 and its expression in Pichia pastoris.

A phytase gene was cloned from Neosartorya spinosa BCC 41923. The gene was 1,455 bp in size, and the mature protein contained a polypeptide of 439 amino acids. The deduced amino acid sequence contains the consensus motif (RHGXRXP) which is conserved among phytases and acid phosphatases. Five possible disulfide bonds and seven potential N-glycosylation sites have been predicted. The gene was expressed in Pichia pastoris KM71 as an extracellular enzyme. The purified enzyme had specific activity of 30.95 U/mg at 37°C and 38.62 U/mg at 42°C. Molecular weight of the deglycosylated recombinant phytase, determined by SDS-PAGE, was approximately 52 kDa. The optimum pH and temperature for activity were pH 5.5 and 50°C. The residual phytase activity remained over 80% of initial activity after the enzyme was stored in pH 3.0 to 7.0 for 1 h, and at 60% of initial activity after heating at 90°C for 20 min. The enzyme exhibited broad substrate specificity, with phytic acid as the most preferred substrate. Its K (m) and V (max) for sodium phytate were 1.39 mM and 434.78 U/mg, respectively. The enzyme was highly resistant to most metal ions tested, including Fe(2+), Fe(3+), and Al(3+). When incubated with pepsin at a pepsin/phytase ratio of 0.02 (U/U) at 37°C for 2 h, 92% of its initial activity was retained. However, the enzyme was very sensitive to trypsin, as 5% of its initial activity was recovered after treating with trypsin at a trypsin/phytase ratio of 0.01 (U/U).

Characterization, gene cloning, and heterologous expression of ß-mannanase from a thermophilic Bacillus subtilis.

Bacillus subtilis BCC41051 producing a thermostable ß-mannanase was isolated from soybean meal-enriched soil and was unexpectedly found to be thermophilic in nature. The extracellular ß-mannanase (ManA) produced was hydrophilic, as it was not precipitated even with ammonium sulfate at 80% saturation. The estimated molecular weight of ManA was 38.0 kDa by SDS-PAGE with a pi value of 5.3. Optimal pH and temperature for mannan-hydrolyzing activity was 7.0 and 60°C, respectively. The enzyme was stable over a pH range of 5.0-11.5, and at temperatures of up to 60°C for 30 min, with more than 80% of its activity retained. ManA was strongly inhibited by Hg(2+) (1 mM), but was sensitive to other divalent ions to a lesser degree. The gene of ManA encoded a protein of 362 amino acid residues, with the first 26 residues identified as a signal peptide. High expression of recombinant ManA was achieved in both Escherichia coli BL21 (DE3) (415.18 U/ml) and B. megaterium UNcat (359 U/ml).

3-Nitropropionic acid (3-NPA), a potent antimycobacterial agent from endophytic fungi: is 3-NPA in some plants produced by endophytes?

3-Nitropropionic acid (3-NPA, 1) was found in extracts of several strains of endophytic fungi. 3-NPA (1) exhibited potent antimycobacterial activity with the minimum inhibition concentration of 3.3 microM, but was inactive against NCI-H187, BC, KB, and Vero cell lines. Endophytes were found to produce high levels of 3-NPA (1), and therefore 3-NPA (1) accumulated in certain plants may be produced by the associated endophytes. 3-NPA (1) may be used as a chemotaxonomic marker for endophytic fungi. The structure of 3-hydroxypropionic acid, a nematicidal agent, should be revised to 3-NPA (1).