Novel Antioxidants Isolated from Perilla frutescens Britton var. crispa (Thunb.)

Two novel antioxidants (vinyl caffeate and trans-p-menth-8-en-7-yl caffeate) and seven known antioxidants (3,4-dihydroxybenzaldehyde, methyl 3,4-dihydroxy-benzoate, methyl caffeate, 3′,4′,5,7-tetra-hydroxy-flavone, caffeic acid, 6,7-dihydroxycoumarin, and rosmarinic acid) were isolated from Perilla frutescens Britton var. crispa (Thunb.). The redox potentials of the novel isolated antioxidative compounds were comparable to those of known antioxidants. trans-p-menth-8-en-7-yl caffeate was effective to prevent the oxidative degradation of perillaldehyde in the essential oil of P. frutescens.     

Subunit structure and functional properties of the predominant globulin of perilla (Perilla frutescens var. frutescens) seeds

Approximately 40% of defatted perilla seeds consists of proteins which are primarily composed of globulin (84%). The amino acid profile of perilla proteins demonstrated balanced amounts of all essential amino acids, except for lysine. The molecular mass of the predominant globulin was estimated to be 340 kDa by gel filtration. This globulin was separated into three intermediary subunits (54, 57 and 59 kDa) by SDS-PAGE. It is suggested from these results that the globulin exists as a hexamer. A treatment with 50 mM dithiothreitol enabled the intermediary subunits to be separated into three acidic subunits (31-34 kDa) and four basic subunits (23-25 kDa). It is interesting that this subunit structure is the same as that of sesame α-globulin, despite them coming from different families. Compared to sesame α-globulin, the heat-induced gel of perilla globulin had better water-holding ability, despite it displaying the same degree of gel hardness.     

Hormonal Control of Morphogenesis in Leaf Explants of Perilla frutescens Britton var. crispa Decaisne f. viridi-crispa Makino

Leaf discs of Perilla frutescens var. crispa f. viridi-crispawere cultured on a defined medium to investigate factors influencingbud and root formation, callus induction, somatic embryogenesis,and floral bud formation. Addition of naphthalene-acetic acid(NAA) to the culture medium caused compact callus whereas 2,4-dichlorophenoxyacetic acid (2,4-D) promoted soft and friable callus formationon the surface of the explants. Benzyladenine, when appliedwith auxin, suppressed callus and root formation. Somatic embryogenesisoccurred, when the explants were first grown on nutrient mediumcontaining 2,4-D and organic elements, and then transferredto the 2,4-D free medium. Treatments with cytokinins, N-phenyl-N'-(4-pyridyl)urea and its derivatives induced bud formation. A low concentrationof NAA and naphthoxy-acetic acid promoted bud development. Occasionalfloral bud formation was observed depending on the originalleaf positions on mother plants from which the leaf discs wereexcised. A gradient of floral bud forming capacity along thestem was noted. Perilla frutescens, tissue culture, embryogenesis, morphogenesis, benzyl adenine, kinetin, naphthalene-acetic acid, naphthoxy-acetic acid, 2,4-dichlorophenoxy acetic acid, indol-3yl-acetic acid, cytokinins, auxins     

Analysis of expressed sequence tags from a normalized cDNA library of perilla (Perilla frutescens)

Perilla (Perilla frutescens), an herbal plant belonging to the Lamiaceae family, has long been cultivated in Asia. Perilla is notable as an aroma-rich leaf vegetable and as the oilseed crop richest in omega-3 fatty acids. However, molecular analysis of this herbal plant is lacking due to insufficient genetic resources. Here, we constructed a normalized cDNA library from whole young perilla plants and analyzed expressed sequence tags (ESTs). A total of 4,582 uniESTs were generated from analysis of 5,435 ESTs. Among these, 307 uniESTs (6.7%) were identified as unique in perilla and 3,625 uniESTs were assigned at least one GO term through similarity searches in public databases. The most frequent GO terms were related to abiotic and biotic stress responses. In addition, we identified 141 uniESTs involved in lipid metabolism, of which four genes encoded fatty acid desaturases. We found one new candidate omega-3 fatty acid desaturase gene, in addition to the four that were previously reported. This analysis of uniESTs from perilla provides a valuable genetic resource to elucidate the molecular underpinnings of lipid metabolism and for molecular breeding of perilla species.     

Isolation, identification, and biological evaluation of HIF-1-modulating compounds from Brazilian green propolis

The tumor microenvironment is characterized by hypoxia, low-nutrient levels, and acidosis. A natural product chemistry-based approach was used to discover small molecules that modulate adaptive responses to a hypoxic microenvironment through the hypoxia-inducible factor (HIF)-1 signaling pathways. Five compounds, such as baccharin (3), beturetol (4), kaempferide (5), isosakuranetin (6), and drupanin (9), that modulate HIF-1-dependent luciferase activity were identified from Brazilian green propolis using reporter assay. Compounds 3, 9 and 5 reduced HIF-1-dependent luciferase activity. The cinnamic acid derivatives 3 and 9 significantly inhibited expression of the HIF-1α protein and HIF-1 downstream target genes such as glucose transporter 1, hexokinase 2, and vascular endothelial growth factor A. They also exhibited significant anti-angiogenic effects in the chick chorioallantoic membrane (CAM) assay at doses of 300 ng/CAM. On the other hand, flavonoids 4 and 6 induced HIF-1-dependent luciferase activity and expression of HIF-1 target genes under hypoxia. The contents (g/100g extract) of the HIF-1-modulating compounds in whole propolis ethanol extracts were also determined based on liquid chromatography-electrospray ionization mass spectrometry as 1.6 (3), 14.2 (4), 4.0 (5), 0.7 (6), and 0.7 (9), respectively. These small molecules screened from Brazilian green propolis may be useful as lead compounds for the development of novel therapies against ischemic cardiovascular disease and cancer based on their ability to induce or inhibit HIF-1 activity, respectively.     

Monoterpene biosynthesis: specificity of the hydroxylations of (-)-limonene by enzyme preparations from peppermint (Mentha piperita), spearmint (Mentha spicata), and perilla (Perilla frutescens) leaves

Microsomal preparations from the epidermal oil glands of Mentha piperita, Mentha spicata, and Perilla frutescens leaves catalyze the NADPH- and O2-dependent allylic hydroxylation of the monoterpene olefin (-)-limonene at C-3, C-6, and C-7, respectively, to produce the corresponding alcohols, (-)-trans-isopiperitenol, (-)-trans-carveol, and (-)-perillyl alcohol. These transformations are the key steps in the biosynthesis of oxygenated monoterpenes in the respective species, and the responsible enzyme systems meet most of the established criteria for cytochrome P450-dependent mixed function oxygenases. The reactions catalyzed are completely regiospecific and, while exhibiting only a modest degree of enantioselectivity, are highly specific for limonene as substrate. Of numerous monoterpene olefins tested, including several positional isomers of limonene, only the 8,9-dihydro analog served as an alternate substrate for ring (C-3 and C-6) hydroxylation, but not side chain (C-7) hydroxylation. In addition to the regiospecificity of the allylic hydroxylation, these enzymes are also readily distinguishable based on differential inhibition by substituted imidazoles.     

Triterpene acids from the leaves of Perilla frutescens and their anti-inflammatory and antitumor-promoting effects

Nine triterpene acids, viz., six of the ursane type, ursolic acid (1), corosolic acid (2), 3-epicorosolic acid (3), pomolic acid (4), tormentic acid (5) and hyptadienic acid (6), and three of the oleanane type, oleanolic acid (7), augustic acid (8) and 3-epimaslinic acid (9), among which 1 constituted the most predominant triterpene acid, were isolated and identified from ethanol extracts of the leaves of red perilla [Perilla frutescens (L.) Britton var. acuta Kudo] and green perilla [P. frutescens (L.) Britton var. acuta Kudo forma viridis Makino]. These eight compounds, 1, 2, 4-9, were evaluated for their inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation (1 microg/ear) in mice. All the compounds tested showed a marked anti-inflammatory effect, with a 50% inhibitory dose (ID50) of 0.09-0.3 mg per ear. In addition, an evaluation against the Epstein-Barr virus early antigen (EBV-EA) activation induced by TPA showed five compounds, 1-3, 5 and 9, with a potent inhibitory effect on EBV-EA induction (91-93% inhibition at 1x10(3) mol ratio/TPA). Furthermore, compound 5 exhibited strong antitumor-promoting activity in an in vivo two-stage carcinogenesis test of mouse tumor by using 7,12-dimethylbenz(a)anthracene (DMBA) as an initiator and TPA as a promoter.     

First identification of a ranavirus from green pythons (Chondropython viridis)

Ten juvenile green pythons (Chondropython viridis) died or were euthanized shortly after having been illegally imported into Australia from Indonesia in 1998. Histologic examination of two of the three snakes that died revealed moderately severe chronic ulceration of the nasal mucosa and focal or periacinar degeneration and necrosis of the liver. In addition there was severe necrotizing inflammation of the pharyngeal submucosa accompanied by numerous macrophages, heterophils, and edema. An iridovirus was isolated in culture from several tissues and characterized by immunohistochemistry, electron microscopy, enzyme-linked immunosorbent Assay, polyacrylamide gel electrophoresis, polymerase chain reaction and sequence analysis, restriction endonuclease digestion, and DNA hybridization. This is the first report of a systemic ranavirus infection in any species of snake and is a new member of the genus, Ranavirus.     

Isolation and characterization of polymorphic microsatellites from Asian green mussel (Perna viridis)

Asian green mussels (Perna viridis) belonging to the family Mytilidae are native to the coastal and tropical marine waters of the Indo‐Pacific region. Ten polymorphic microsatellites were isolated and their polymorphisms were determined in 36 individuals. The average allele number of the 10 microsatellites was 11.7/locus with a range of two to 24 and the expected heterozygosity averaged at 0.69, ranging from 0.18 to 0.94. Eight out of 10 microsatellites agreed with the Hardy–Weinberg equilibrium and showed independent segregation. These microsatellites will facilitate the studying of the genetic diversity and population structure of the green mussel in and outside of the Indo‐Pacific region of Asia.     

Isolation of new microsatellite loci from the Green Lizard (Lacerta viridis viridis)

Twelve new microsatellite loci were isolated from the Green Lizard (Lacerta viridis viridis). Primers for 28 loci were designed and 18 of these loci amplified well for 10 individuals of four populations. Twelve of these loci were further characterized for a population in Hungary. The results document the suitability of these identified loci for the characterization of the genetic diversity of the endangered species L. viridis viridis.     

Isolation and biological activity of agrostophillinol from kaffir lime (Citrus hystrix) leaves

The leaves of the kaffir lime (Citrus hystrix) are commonly used in cuisine and folk medicine. The aim of this study was to isolate a bioactive compound in kaffir lime leaves and characterize its biological activity. The compound was isolated from a hexane fractional extract and identified as agrostophillinol. This is the first report of agrostophillinol isolated from kaffir lime leaves. In terms of cytotoxicity, agrostophillinol exhibited IC₅₀ values of 36.27 ± 7.30 and 53.44 ± 10.63 μg/mL against EoL-1 and HL60 cells, respectively. Agrostophillinol also exhibited potent anti-inflammatory activity, significantly inhibiting IL-6 secretion.     

Neuroprotective effect of polysaccharide separated from Perilla frutescens Britton var. acuta Kudo against H2O2-induced oxidative stress in HT22 hippocampus cells

This study was carried out to evaluate the neuroprotective activity of polysaccharide extracts isolated from Perilla frutescens (PEPF) in H₂O₂-treated HT22 hippocampus cells. The PEPF treatment was found to increase the anti-oxidant activities of HT22 hippocampus cells. PEPF treatment resulted in a significant protection of HT22 hippocampus cells against H₂O₂-induced neurotoxicity, this protection ultimately occurred through an inhibition of ROS-mediated intracellular Ca²⁺ levels leading to MAPKs and NF-κB, as well as the accumulation of PI3K/AKT and Nrf2-mediated HO-1/NQO1 pathways. Furthermore, PEPF not only decreased the expression of Bax, cytochrome c, and cleaved caspases-3, -8, and -9, but also increased the expression of PARP and Bcl-2 in the H₂O₂-treated HT22 hippocampus cells, which overall contributed to the neuroprotective action. PEPF retains its mitochondrial membrane potential and reduces the elevated levels of sub-G1 phase and apoptotic morphological features induced by H₂O₂. It also reduces the malondialdehyde levels and enhances the intracellular SOD activity.     

Perilla frutescens seed agar, a new medium for identification of the Cryptococcus species complex: evaluation for all major molecular types

Here we present a new and simple medium made by Perilla frutescens seed as a tool for identification of the Cryptococcus species complex, namely Cryptococcus neoformans and Cryptococcus gattii. Its usefulness was evaluated for all major molecular types within the Cryptococcus species complex. As a result, this medium is better for identification of the species complex compared with Guizotia abyssinica or Helianthus annuus seed medium.     

Occurrence of Amoebae on Oak Leaf Lettuce (Lactuca sativa var. crispa) and Boston Lettuce (L. sativa var. capitata)1

The colonization, distribution, population density, and species diversity of amoebae on leaves of Oak Leaf lettuce, Lactuca sativa var. crispa, and Boston lettuce, L. sativa var. capitata, were investigated. The role of soil in the colonization of Oak Leaf lettuce was determined by comparing numbers of amoebae present on basal leaves (those that pass through soil) with numbers on wrapped leaves (those that do not pass through soil). Amoebae were present in ten samples of basal leaves and ranged from 154–1510/g of leaf tissue. Wrapped leaves failed to yield amoebae in seven of ten trials and contained <4 amoebae/g of tissue. Mean values for the population density of amoebae on Oak Leaf basal leaves and Boston lettuce leaves were 484 ± 133 and 453 ± 93, respectively. The distribution of amoebae on green and white parts of leaves from both kinds of lettuce was studied. The occurrence of amoebae on rinsed, unrinsed, visibly clean, and visibly dirty samples of Boston lettuce leaves was established.     

鹅源鸭疫里默氏杆菌的分离、鉴定与生物学特性研究

【目的】从疑似鸭疫里默氏杆菌病的病死雏鹅分离病原菌进行鉴定。【方法】根据细菌培养特性、生化特性、动物试验、血清型鉴定及分子生物学特性对分离菌株进行鉴定。【结果】分离菌株为革兰氏阴性菌,不发酵糖类和醇类,尿素酶试验和氧化酶还原试验为阳性,致病,不同分离株的16S rRNA基因经多重序列比对分析,结果显示鹅源分离株与鸭源鸭疫里默氏杆菌处于同一进化支上,与鸡源鸭疫里默氏杆菌进化关系稍远,血清型鉴定为1型。【结论】分离菌株为血清1型的鸭疫里默氏杆菌,对鸭和鹅均有高致病性,自家疫苗能够较好地保护雏鹅。     

邻苯二甲酸二乙基己酯对翡翠贻贝(Perna viridis)生化指标的影响

实验条件下,研究邻苯二甲酸二乙基己酯(DEHP)5个浓度组(0、0.38、1.92、9.60和48.00mg.L-1)长时间胁迫下翡翠贻贝(Perna viridis)内脏团和外套膜中抗氧化酶(SOD和CAT)活性和丙二醛(MDA)含量的变化,以及胁迫解除后这些指标的恢复情况。结果表明:在胁迫过程中,翡翠贻贝内脏团SOD活性表现为先显著升高,随后受抑制而逐渐降低(P<0.05),CAT活性则表现为先被抑制后受诱导,15d后恢复到对照组水平,MDA含量呈显著增加的趋势(P<0.05);外套膜中的SOD活性在胁迫初期在低浓度组被抑制,而在高浓度组则被诱导(P<0.05),4d后SOD活性逐渐恢复到对照组水平,各浓度组MDA含量均出现明显的增加(P<0.05);净化阶段,低浓度组(0.38mg.L-1)内脏团SOD活性和CAT活性逐渐恢复到对照组水平,但MDA含量升高;净化7d后,除高浓度组(48.00mg.L-1)外,其余浓度组外套膜中SOD活性均已经恢复到对照组水平,MDA含量也没有出现明显升高的现象。研究表明,DEHP对翡翠贻贝内脏团和外套膜抗氧化防御系统酶具有明显的影响,DEHP诱导引起2种组织内脂质过氧化损伤,并且短期内这种损伤无法消除。