Yarrowia lipolytica and its multiple applications in the biotechnological industry.

Yarrowia lipolytica is a nonpathogenic dimorphic aerobic yeast that stands out due to its ability to grow in hydrophobic environments. This property allowed this yeast to develop an ability to metabolize triglycerides and fatty acids as carbon sources. This feature enables using this species in the bioremediation of environments contaminated with oil spill. In addition, Y. lipolytica has been calling the interest of researchers due to its huge biotechnological potential, associated with the production of several types of metabolites, such as bio-surfactants, γ-decalactone, citric acid, and intracellular lipids and lipase. The production of a metabolite rather than another is influenced by the growing conditions to which Y. lipolytica is subjected. The choice of carbon and nitrogen sources to be used, as well as their concentrations in the growth medium, and the careful determination of fermentation parameters, pH, temperature, and agitation (oxygenation), are essential for efficient metabolites production. This review discusses the biotechnological potential of Y. lipolytica and the best growing conditions for production of some metabolites of biotechnological interest.

Screening filamentous tropical fungi for their nutritional potential as sources of crude proteins, lipids and minerals.

The use of fungal and yeast biomass in foodstuff, either as supplements or as major ingredients in formulations is an area of growing interest for the modern food industry. The aim of this study was to evaluate the nutritional potential of biomasses obtained from filamentous fungi Penicillium sclerotiorum, Penicillium janthinellum, Rhizopus stolonifer and Syncephalastrum racemosum. Biomasses presented 26-37% of total proteins, 1.7-3.5% of lipids and 4.6-9.1% of ashes. The humidity level reached 75-83%. Ashes were screened for minerals contents with a special outcome for S. racemosum biomass that presented 3438 mg/100 g (dw) of magnesium. Fatty acids present in the biomasses were screened and the palmitic (C16:0), estearic (C18:0), elaidic (18:1n9-t), oleic (18:1n9-c), linolelaidic (C18:2n6-t), linoleic (C18:2n6-c) and γ-linolenic (C18:3n6) acids were found to be the most abundant, from a total of 88-90% of identified fatty acids. Overall data indicate that the filamentous fungi studied have good nutritional properties, possessing a combination of good level of proteins, low level of fat, and presence of essential fatty acids, including omega-3 derivatives, along with the presence of Ca, Mg, Zn and Fe.

Novel agents for enzymatic and fungal hydrolysis of stevioside.

A comparative study on the potential of some biological agents to perform the hydrolysis of stevioside was carried out, aiming at establishing an alternative methodology to achieve the aglycon steviol or its rearranged derivative isosteviol, in high yields to be used in the preparation of novel bioactive compounds. Hydrolysis reactions were performed by using filamentous fungi (Aspergillus niger, Rhizopus stolonifer and Rhizopus arrhizus), a yeast (Saccharomyces cerevisiae) and enzymes (pancreatin and lipases PL250 and VFL 8000). Pancreatin showed the best hydrolytic activity, furnishing isosteviol at 93.9% of yield, at pH 4.0, using toluene as a co-solvent. Steviol was produced using both pancreatin at pH 7.0 (20.2% yield) and A. niger at pH 7 (20.8% yield).

Synthesis of a new allelopathic agent from the biotransformation of ent-15α-hydroxy-16-kauren-19-oic acid with Fusarium proliferatum.

The use of kaurane diterpenes as substrates in fungal biotransformation to achieve bioactive compounds has been widely reported. In this work, the natural product kaurenoic acid, a diterpene widely distributed in the plant Kingdom, was chemically converted into ent-15α-hydroxy-kaur-16-en-19-oic acid (1). Substrate 1 was subjected to biotransformation by the fungus Fusarium proliferatum, furnishing a new derivative, ent-2α,15α-dihydroxy-kaur-16-en-19-oic acid (2). The structure of metabolite 2 was deduced on the basis of spectroscopy and MS data. Derivative 2 showed allelopathic activity on germination and growth of root and stem of lettuce (Lactuca sativa), inhibiting 100% of germination and growth of roots and stem, at higher concentration assayed (10-4 mol/L).

Constituents from stem barks of Luehea ochrophylla Mart and evaluation of their antiparasitic, antimicrobial, and antioxidant activities.

Luehea species are found in almost all Central and South American countries. The present work describes the phytochemical study, isolation, and structural characterisation of friedelin, ß-friedelinol, lupeol, pseudotaraxasterol, ß-sitosterol, betulinic acid, taraxasterol, (-)-epicatechin, ß-sitosterol-3-O-ß-d-glucopyranoside, and (+)-epicatechin-(4ß→8)-epicatechin from stem barks of Luehea ochrophylla Mart. The structural identification of the isolated compounds was mainly performed by NMR analyses and comparison with the data from literature. These compounds were isolated for the first time in the genus Luehea, except ß-sitosterol glucopyranoside, (-)-epicatechin, and lupeol. Hexane extract (HE) and dichloromethane (DF) and ethyl acetate (AF) fractions exhibited antiparasitic activity against amastigote (intracellular) and trypomastigote culture forms of Trypanosoma cruzi. The ethanol extract (EE), DF, and ethanol fraction (EF) exhibited considerable antifungal activity against Candida albicans. Moreover, extracts and fractions exhibited significant percentage of capture free radicals of 2,2-diphenyl-picrylhydrazyl (DPPH) when compared to the standard of ascorbic acid.

New jacaranone glucoside from Jacaranda oxyphylla leaves.

Jacaranda oxyphylla Cham. is popularly known as 'caroba-de-São-Paulo' and it is used in traditional medicine for microbial infections. A new phytoquinoid (α/ß-glucoside-4-phenylacetate-6-(1-hydroxy-4-oxo-2,5-cyclohexadiene-1-acetate) (1) was isolated from J. oxyphylla leaves, together with three known compounds: quercetin-3-O-ß-d-galactoside (2), verbascoside (3) and polystyrene (4). Their chemical structures were elucidated using spectroscopic techniques and by comparison with the related known compounds. In addition, it was found a pronounced acetylcholinesterase inhibitory activity for the quinoid 1 (100.0 ± 0.8%) and phenolic compounds 2 and 3 (99.9 ± 0.7 and 99.3 ± 0.5%, respectively), if compared to the standard eserine (92.7 ± 0.4%), that was analysed by a microplate spectrophotometer.

Antioedematogenic activity, acetylcholinesterase inhibition and antimicrobial properties of Jacaranda oxyphylla.

Jacaranda oxyphylla Cham. (Bignoniaceae) is a shrub found in the Brazilian cerrado and used in folk medicine to treat microbial infections. The aim of this study was to carry out a phytochemical screening and evaluate antioedematogenic, antimicrobial and antiacetylcholinesterase properties of J. oxyphylla crude extracts. All extracts analysed showed presence of terpenoids, which are potentially active chemical substances. A high AChE inhibitory activity for hexane extract from leaves and for the extracts from twigs was found. Ethanol extract from leaves of J. oxyphylla showed activity against Gram-positive (Staphylococcus aureus and Bacillus cereus) and Gram-negative (Escherichia coli) bacteria. This extract was also effective in inhibiting the stages of inflammation evaluated. Biological investigation and phytochemical screening of J. oxyphylla extracts provided additional evidence of its traditional medicinal value.

Pomegranate extract inhibits Staphylococcus aureus growth and subsequent enterotoxin production.

In Brazil, pomegranate (Punica granatum L. (Punicaceae)) is widely used as a phytotherapeutic agent. This study evaluates the effect of pomegranate extract on Staphylococcus aureus FRI 722 growth and subsequent enterotoxin production. Bacterial susceptibility was determined by tube dilution method and production of enterotoxin was assessed using membrane-over-agar (MOA) plates. At a low extract concentration (0.01% v/v) bacterial growth was delayed, while a higher concentration (1% v/v) eliminated bacterial growth. Most interestingly, a 0.05% (v/v) concentration of extract was found to inhibit Staphylococcal enterotoxin (SE) A production. These data further implicate pomegranate extracts as potential antibacterial therapeutics with the added ability to inhibit enterotoxin production.

Inhibition of cell growth and tumorigenesis of human glioblastoma cells by a neutralizing antibody against human basic fibroblast growth factor.

We report here that a neutralizing mouse monoclonal antibody against basic FGF inhibited both anchorage-dependent and anchorage-independent growth of U-87MG and T98G human glioblastoma cells and HeLa cells, all of which express both the basic FGF and the FGF receptor genes. In addition, the subcutaneous administration of this antibody significantly suppressed the tumor development of these tumor cells in nude mice. Therefore, basic FGF plays an important role in neoplastic growth of these cells. The neutralization of basic FGF will be effective in controlling the growth of tumors, such as glioblastoma and other cancer cells which bear basic FGF and FGF receptors.

Tubular aggregates in the skeletal muscle of the senescence-accelerated mouse; SAM.

We investigated the skeletal muscles of nine strains of senescence accelerated mouse (SAM), DDD, AKR/J, C57BL/6J, A/J and BALB/c mice. We found that male SAMP8, SAMP7, C57BL/6J, A/J and BALB/c mice expressed tubular aggregates (TAs) in their skeletal muscle. Among these strains, the SAMP8 strain, which exhibits a short life span and various age-associated neurodegenerative disorders plus mitochondrial dysfunction, showed TAs more markedly than the others. Thus, we compared SAMP8 mice against SAMR1 mice, an accelerated senescence-resistant strain. Light- and electron micrographs showed that male SAMP8 mice exhibited an age-dependent aggravation of TA accumulation. There were no significant differences in the serum lactate/pyruvate levels between the SAMP8 and SAMR1 mice. However, the serum creatine kinase (CK) levels of the 3 and 6-month-old SAMP8 mice were higher than that of the corresponding SAMR1 mice. Considering the serum CK levels and the mitochondrial dysfunction of SAMP8 mice, we conclude that the TAs may be involved in the homeostasis of energy metabolism that is not appropriately regulated in the SAMP8 mouse mitochondrion.

Adenovirus-mediated gene transfer of basic fibroblast growth factor induces in vitro angiogenesis.

To investigate the possibility of adenovirus-mediated gene transfer in the treatment of vascular occlusive diseases, we constructed a replication-deficient recombinant adenovirus vector coding for human basic fibroblast growth factor (bFGF) and examined its effect on the proliferation and differentiation of vascular endothelial cells in vitro. Human umbilical vein endothelial cells (HUVECs) were successfully infected with high efficiency and expressed 18 kD protein which is immunoreactive to anti-bFGF monoclonal antibody. This protein was accumulated mainly in the nuclei of the cells, but was also detected in the culture medium although the complimentary DNA (cDNA) did not contain the classical secreting signal sequence. The proliferation assay of HUVECs infected with bFGF-expressing adenovirus revealed a significant increase in cell number over control. Infection with this virus also enhanced tubular formation of HUVECs on reconstituted basement membrane. Neovascularization and the formation of collateral vessels play important roles in minimizing tissue damage in ischemic disorders. These results imply that the use of bFGF-expressing recombinant adenovirus may be a suitable in vivo gene therapy for ischemic diseases.

Immunohistochemical study for basic fibroblast growth factor and fibroblast growth factor receptor I in pituitary adenomas.

Immunohistochemistry and RT-PCR of basic fibroblast growth factor (bFGF) and one of its receptors (FGFR-I) were performed in pituitary adenomas. Sixty percent of pituitary adenomas showed strong or moderate immunoreactivity to bFGF. The immunoreactivity for FGFR-I in tumor tissues showed positive correlation to that for bFGF (X2 = 6.176, P = 0.0456). Basic FGF-positive cells consisted of pituitary adenoma cells as well as folliculostellate cells and, their distribution was heterogeneous. Expressions of bFGF and FGFR-I were not related to cell proliferation of pituitary adenomas or hormones produced, suggesting that bFGF plays some role other than progression of pituitary adenomas.

An early stage mechanism of the age-associated mitochondrial dysfunction in the brain of SAMP8 mice; an age-associated neurodegeneration animal model.

In order to characterize the early stage of mitochondrial dysfunction, we investigated the redox state and oxidative phosphorylation of the brain mitochondria from 2-month-old Senescence-accelerated mouse (SAM)P8 and SAMR1 mice; SAMP8 mice exhibit various signs of age-associated neurodegeneration and rapid mitochondrial dysfunction, although SAMR1 mice do not. The redox state was estimated as the reduction rate of Cu-pyruvaldehyde-bis (N4-methylthiosemicarbazone) (Cu-PTSM), the reduction of which is closely related to the electron leakage from the mitochondrial electron transport system in the brain, using electron spin resonance spectrometry (ESRS). The oxidative phosphorylation was measured polarographically. The SAMP8 mouse brain mitochondria demonstrated higher redox state and a higher activity of mitochondrial respiration with lower respiration control ratio than the mitochondria of SAMR1 mouse brains. This indicates that an inefficient hyperactive state can exist in the mitochondrial electron transport system before the age-associated mitochondrial dysfunction develops.

The bio-transformation of ent-19-hydroxykaur-16-en-15-one by Cephalosporium aphidicola.

ent-19-Hydroxykaur-16-en-15-one is transformed to ent-3 beta,16 beta,19-trihydroxykauran-15-one by Cephalosporium aphidicola. The structure of this metabolite was established by X-ray crystallography.

The microbiological transformation of two ent-16 beta,17-epoxykaurane derivatives by Gibberella fujikuroi.

The biotransformation of ent-16beta-17-epoxy-7 alpha-hydroxykaurane by Gibberella fujikuroi affords ent-7 alpha,11 alpha,16 beta,17-tetrahydroxykaurane and ent-7 alpha, 9 alpha, 16 beta, 17-tetrahydroxykaurane. These results indicated that the presence of the 16 alpha, 17-diol group, into which the 16 alpha, 17-epoxy is transformed in the medium, inhibits oxidation at C-19 and favours hydroxylation at C-11(beta). Incubation of ent-16 beta, 17-epoxykauran-19-oic acid, via the 16 alpha, 17-diol, gave the 7-aldehyde of 16 alpha, 17-dihydroxy-GA12.

The biotransformation of ent-kaur-16-en-19-oic acid by Rhizopus stolonifer.

Microbial transformation of ent-kaur-16-en-19-oic acid was carried out with R. stolonifer. After seven days of incubation, two metabolites, ent-7 alpha-hydroxy-kaur-16-en-19-oic acid and ent-12 beta-hydroxy-kaur-9(11),16-dien-19-oic acid, were isolated as a result of hydroxylation and hydroxylation/dehydrogenation, respectively. Incubation for 15 days also afforded ent-16 beta,17-dihydroxy-kauran-19-oic acid. The metabolites were identified by spectroscopic methods.

Immunohistochemical reactions for fibroblast growth factor receptor in arteries of patients with moyamoya disease.

The cause of moyamoya disease remains unknown, and pathophysiological mechanisms remain uncertain. Basic fibroblast growth factor (FGF) is a pluripotent polypeptide that has been shown to play roles in angiogenesis, tumorigenesis and many other processes. In a previous study, we demonstrated immunohistochemically that the amount of basic FGF was increased above normal in the superficial temporal artery (STA) of patients with moyamoya disease. To clarify the function of basic FGF in moyamoya disease, we have performed an immunohistochemical study of the STA using a polyclonal antihuman FGF receptor antibody and also have tested immunohistochemical reactions for basic FGF. Twelve surgical specimens of the STA from patients with moyamoya disease were studied. Twelve specimens of the STA from skin flaps of patients with other neurological diseases were also investigated for comparison. The sections of the STA from patients with moyamoya disease showed dense and strong FGF receptor and basic FGF immunoreactivity in endothelial cells, in cells scattered in the thickened intima, and in smooth muscle cells in the media. In contrast, the sections of the STA of control patients showed faint basic FGF immunoreactivity. The statistical analysis revealed a significant difference of basic FGF immunoreactivity between moyamoya disease and other neurological diseases (chi 2 = 23; P = 0.0001). Moderately intense FGF receptor immunoreactivity was observed in most control patients. However, the statistical analysis revealed a significant difference of FGF receptor immunoreactivity between moyamoya disease and other neurological diseases (chi 2 = 13.382; P = 0.0012).(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of fibroblast growth factor receptor-1 in human glioma and meningioma tissues.

We examined the expression of fibroblast growth factor receptor-1 (FGFR-1), namely FLG, in tissues of 18 human gliomas, 10 human meningiomas, 3 human metastatic brain tumors, and 2 normal human brains by means of immunohistochemistry. All tissues were positively stained for FGFR-1. Primary brain tumors were more abundantly immunoreactive than normal brain tissues (Mann-Whitney U test, P < 0.05). There was significant correlation between the expression level of basic fibroblast growth factor (basic FGF) and that of FGFR-1 in tissues of human glioma (Spearman's test, P < 0.05). The expression level of FGFR-1 of tumor cells increased in correlation with that of endothelial cells in glioma tissues (Spearman's test, P < 0.001). We previously reported that basic FGF is produced in more than 90% of human glioma and meningioma tissues. Together with these data, it is suggested that basic FGF is involved in autonomous cell growth and tumorigenesis of gliomas and meningiomas as an autocrine growth factor in vivo.

Possible roles of basic fibroblast growth factor in the pathogenesis of moyamoya disease: an immunohistochemical study.

Prominent features of moyamoya disease are fibrocellular thickening of the intima and enhanced angiogenesis. The pathogenesis of moyamoya disease is, however, unknown. Basic fibroblast growth factor (FGF) is an angiogenic factor as well as a potent mitogen for a number of cell types including vascular endothelial and smooth-muscle cells. In order to test the possibility that basic FGF takes part in the pathogenesis of moyamoya disease, the authors tested for the presence of this factor using a mouse monoclonal antibody against human recombinant basic FGF. The surgical specimens studied included two sections of the superficial temporal artery (STA) and four samples of dura mater from four patients with moyamoya disease. Surgical specimens were obtained from three patients with other diseases as control tissue. Sections of the STA obtained from the patients with moyamoya disease showed strong basic FGF immunoreactivity in endothelial and smooth-muscle cells, while control sections had only faint and scattered immunoreactivity. All sections of the dura mater obtained from the patients with moyamoya disease also revealed more intense immunohistochemical staining of basic FGF in meningeal and vascular cells than did control sections. These observations indicate that the amount of basic FGF is increased in the tissues of patients with moyamoya disease; thus, basic FGF may play an important role in the pathogenesis of moyamoya disease.

Correlation of basic fibroblast growth factor expression levels with the degree of malignancy and vascularity in human gliomas.

Basic fibroblast growth factor (FGF) is a mitogen, a differentiation factor for neuroectoderm-derived cells, and a potent angiogenic factor. The authors have previously demonstrated that the messenger ribonucleic acid of basic FGF is expressed in more than 90% of human gliomas. In the present study, they examined the expression of basic FGF in human glioma tissues using immunohistochemical techniques with a mouse monoclonal antibody against human basic FGF. They also correlated the basic FGF level with the histological grades of malignancy assessed by the number of nucleolar organizer regions (NOR's). Basic FGF was detected in 18 of 19 gliomas, whereas it was undetectable in two normal brains. The expression level of basic FGF peptide increased proportionally with the degree of malignancy. There was also a tendency for the number of NOR's in glioma cells to increase in glioma samples with a high level of basic FGF expression. Furthermore, most of the cases with increased vascularity demonstrated on cerebral angiograms showed a relatively high level of basic FGF expression of tumor cells and a large number of NOR's in endothelial cells in tumor tissues. These results suggest that basic FGF is actually produced in most gliomas and is involved in tumorigenesis and malignant progression as an autocrine growth factor. Moreover, basic FGF may play an important role in tumor neovascularization as a paracrine angiogenic factor.