Chymotrypsin and trypsin inhibitor isolated from potato tubers

Potato Kunitz-type chymotrypsin inhibitor (PKCI-23) was isolated from potato tubers (Solanum tuberosum L., cv. Zhukov’s Jubilee) and purified to a homogenous state. The protein was purified by gel-filtration chromatography and ion-exchange chromatography using Sephadex G-75 and CM-sepharose CL-6B, respectively. PKCI-23 protein has been shown to inhibit both chymotrypsin and trypsin with equal efficacy, forming equimolar complexes with these enzymes. However, much weaker inhibitory effect of PKCI-23 has been observed for subtilisin Carlsberg. The N-terminal 20 amino acid sequence of PKCI-23 has been sequenced. PKCI-23 has been shown to suppress, with different efficacy, the growth and development of pathogenic microorganisms Fusarium culmorum (Wm. G. Sm.) Sacc. and Phytophtora infestans (Mont.) de Bary that infect potato.     

Uncoupling activities of chalcones and dihydrochalcones on isolated mitochondria from potato tubers and mung bean hypocotyls

The uncoupling properties of 23 chalcones and dihydrochalcones were studied. Twelve compounds completely uncouple oxidative phosphorylation in mung bean hypocotyl and potato tuber mitochondria, four are weak uncouplers and seven are without effect. Usually, mung bean mitochondria are more sensitive to uncoupling agents than potato mitochondria. The uncoupling activity of chalcones and dihydrochalcones appears to be connected with the presence of hydrogen or hydroxyl groups in the 2′-position and hydrogen, hydroxyl or nitrate groups in the 4′-position. The α-β-unsaturated carbonyl system is not essential for activity. For the compounds which are not very lipophilic, substituents on the B-ring are without effect on the uncoupling properties. Phloretin appears to be an active uncoupler; its 6′-glucoside is without effect.     

Biological activities of novel derivatives of DIF-1 isolated from Dictyostelium

The differentiation-inducing factor-1 (DIF-1) is a lipophilic signal molecule (chlorinated alkylphenone) that induces stalk cell differentiation in the cellular slime mold Dictyostelium discoideum. In addition, DIF-1 and its derivatives have been shown to possess anti-leukemic activity and glucose consumption-promoting activity in vitro in mammalian cells. In this study, to assess the chemical structure-effect relationship of DIF-1, we synthesized eight derivatives of DIF-1 and investigated their stalk cell-inducing activity in Dictyostelium cells and pharmacological activities in mammalian cells. Of the derivatives, two amide derivatives of DIF-1, whose hydrophobic indexes are close to that of DIF-1, induced stalk cell differentiation as strongly as DIF-1 in Dictyostelium cells. It was also found that some derivatives suppressed cell growth in human K562 leukemia cells and promoted glucose consumption in mouse 3T3-L1 cells. These results give us valuable information as to the chemical structure-effect relationship of DIF-1.     

Synthesis,antibacterial and antifungal activities of some carbazole derivatives

A series of N-substituted carbazole derivatives were synthesized and evaluated for antibacterial and antifungal activities against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Bacillus proteus, Candida albicans and Aspergillus fumigatus by two fold serial dilution technique. Some of the synthesized compounds displayed comparable or even better antibacterial and antifungal activities than reference drugs fluconazole, chloramphenicol and norfloxacin against tested strains.     

Cold-lability of phosphofructokinase from potato tubers

The aim of this work was to study the cold-lability of phosphofructokinase from tubers of Solanum tuberosum cv Record, a variety that exhibits low temperature sweetening. The enzyme was purified by affinity chromatography and samples were examined by differential scanning calorimetry. Power-time curves were recorded for cooling and warming between 293 and 265 K. This revealed an exothermic dissociation, centred on 286 K, as the temperature was lowered. The latter temperature is close to that at which the tubers start to sweeten. It is suggested that hydrophobic interactions that contribute to the stability of the active configuration of the oligomeric enzyme are weakened at low temperatures, and that this causes spontaneous dissociation and consequent loss of activity of the enzyme. The results are discussed in relation to low temperature sweetening of potatoes.     

Taxonomic study on nonpathogenic streptomycetes isolated from common scab lesions on potato tubers

Numerical analysis was carried out to compare sixteen nonpathogenic actinomycetes isolated from common scab lesions on potato tubers with Streptomyces scabiei type strain as well as with other streptomycete groups. These isolates were divided into two classes according to their level of similarity with S. scabiei. Isolates resembling S. scabiei were associated with S. griseoruber or with S. violaceusniger while isolates exhibiting less than 61% of similarity with S. scabiei were phenotypically related to S. albidoflavus or to S. atroolivaceus. Sequence of the 16S rRNA gene of each isolate was obtained and compared against the GenBank nucleotide database. No significant match could be established between the sequences of two potato isolates and the ones available in the GenBank database. The other isolates were closely related with S. setonii (S. griseus), S. mirabilis, S. fimbriatus, S. violaceoruber, S. melanosporofaciens and S. thermocarboxydus.     

Biotransformation of potato stress metabolites rishitin,lubimin, and 15-dihydro lubimin by potato and soybean cell cultures

Potato callus and cell suspensions of potato and soybean were exogenously supplied with potato phytoalexin rishitin, much of which was converted by both species to an unknown tenatively identified as glutinosone. Exogenous lubimin was unaffected by the potato cell culture, but was transformed to 15-dihydro lubimin by the soybean cell suspensions. The stability of the exogenous lubimin may be ascribed to a second block in the rishitin pathway of the potato cell culture.Abbreviations MS Murashige and Skoog (1962) - NAA 1-naphthaleneacetic acid - TMV 2,4-dichlorophenoxyacetic acid, 2,4-D; Tobacco Mosaic Virus - TLC thin layer chromatography - GC gas chromatography - GC/MS gas chromatography/mass spectrometry - NMR nuclear magnetic resonance     

Detection of phlD gene in some fluorescent pseudomonads isolated from Iran and its relative with antifungal activities

Fluorescent pseudomonads that produce antibiotic 2,4-diacetylphloroglocinol (2,4-DAPG) are important group of PGRP that inhibit a broad spectrum of plant pathogenic fungi. Studying on genetic diversity of 2,4-diacetylphloroglucinol-producing fluorescent pseudomonads has been shown with special importance. The first step to investigate the genetic diversity of these bacteria is detecting of the genes required for the biosynthesis of this antibiotic. The objectives of the current study were detection of phlD gene within fluorescent pseudomonads by a PCR-based assay, and comparison of phenotypic and genotypic characteristics of fluorescent pseudomonads with proven biocontrol potential against some soil-borne phytopathogenic fungi. We used a collection of 47 fluorescent Pseudomonas spp. some with known biological control activity against Macrophomina phaseolina, Rhizoctonia solani, Phytophthora nicotianae var. parasitica, Pythium sp. and Fusarium sp. in vitro and the potential to produce known secondary metabolites such as, siderophore, HCN and protease. The results indicated that 66, 40.42, 63.82,48.94 and 27.65% of strains revealed antagonistic activity against R. solani, M. phaseolina, Pythium sp., P. nicotianae and Fusarium sp., respectively. Rhizoctonia solani recognized as the most vulnerable fungus. Among 47 strains, 76.59, 97.87 and 17% of strains produced protease, siderophore and HCN, respectively. We could detect phlD gene in strains P-5, P-32, P-47. Strain CHA0 was used as positive control for the detection this gene. Overall, there was no obvious link between the existence of phlD gene and inhibition of fungal growth or production of the antifungal metabolites in vitro. But in some strains such as CHA0 and P-5, we saw a link between the existence of phlD and antifungal activities. Studying on detection and diversity of phlD provides a fundamental knowledge for developing a rapid genetic screening system to identify a potential biocontrol strains.     

Isopestacin,an isobenzofuranone from Pestalotiopsis microspora,possessing antifungal and antioxidant activities

Isopestacin is an isobenzofuranone obtained from the endophytic fungus Pestalotiopsis microspora. While a few other isobenzofuranones are known from natural sources, isopestacin is the only one having a substituted benzene ring attached at the C-3 position of the furanone ring. The compound was isolated from culture broths of the fungus and crystallized and its structure was determined by X-ray crystallography. Both proton and carbon NMR spectral assignments are also reported for isopestacin. This compound possesses antifungal activity and, as measured by electron spin resonance specroscopy, it also behaves as an antioxidant scavenging both superoxide and hydroxy free radicals.     

Enzymatic activities of starch synthesis in potato tubers of different sizes

The objective of this study was to determine the relationship between tuber weight and enzymatic activities involved in tuber starch synthesis. As tuber weight increased, the activities of sucrose synthetase, UDPG pyrophosphorylase, and granular starch synthetase escalated, whereas the activities of soluble starch synthetase and ADPG pyrophosphorylase stayed constant and that of phosphorylase declined. This suggests that when samples are taken to determine specific enzymatic activities, the sampling procedure should ensure that results do not vary because of differences in the tuber weight or size distribution.     

Water permeability of periderm membranes isolated enzymatically from potato tubers (Solanum tuberosum L.)

The fine structure and water permeability of potato tuber periderm have been studied. Periderm membranes (PM) were isolated enzymatically using pectinase and cellulase. They were composed of, about six layers of phellem cells arranged in radial rows. The walls of phellem cells consist of cellulosic primary and tertiary walls and suberized secondary walls which are lamellated. Middle lamellae and primary walls contain lignin. Since the PM did not disintegrate during enzymatic isolation it appears that lignin also extends into the secondary suberized walls. The water permeability of PM was low, ranging from 1–3·10^-10 m s^-1. This low water permeability developed only during storage of tubers in air. Periderm membranes from freshly harvested tubers had a relatively high permeability. The low permeability of PM from stored tubers is attributed to soluble lipids associated with suberin since: (1) extraction of soluble lipids from PM increased permeability by more than 100-fold, (2) a phase transition of soluble lipids was observed between 46 and 51° C, and (3) only the permeability of PM decreased during storage while the permeability of extracted PM remained unchanged. Evidence is presented that two pathways for water movement exist in parallel. Pathway 1 is represented by middle lamellae and primary walls extending in radial direction across the membranes. This pathway has a relatively high specific permeability. Pathway 2 is represented by a polylaminated structure made up of tangential walls of phellem cells which are orientated normal to the direction of water flow. This pathway has a low specific permeability because of the properties of secondary walls incrusted with soluble lipids. It is calculated that about 10% of the water flows across pathway 1 and 90% across pathway 2 which has a volume fraction of 0.995.     

Purification and multiple forms of phosphoglucomutase from potato tubers

Two fractions (Peaks I and II) having phosphoglucomutase activitywere separated by DEAE-cellulose chromatography of the crudeenzyme preparation from potato tubers. Upon separate rechromatographyunder the same conditions, they were respectively eluted atthe same positions as initially eluted. Incubation of PeaksI and II with glucose 1,6-bisphosphate which should cause conversionof the dephosphorylated form to the phosphorylated form causedno change in their eluting positions in rechromatography. Theratio of their contents in the crude extracts were fairly constantamong different samples of potato tubers. Peak I was furtherseparated into three fractions on isoelectric focusing. Theresulting main fraction (Peak I_a) was purified 1,800-fold overthe crude extract with a 9% yield. It was homogeneous as judgedby polyacrylamide gel electrophoreses in the absence and presenceof sodium dodecyl sulfate. Crude extracts from peas and broadbeans each contain a single species of phosphoglucomutase whichcorresponds on the chromatograph to the Peak II enzyme frompotato tubers. It is suggested that phosphoglucomutase frompotato tubers contains at least two, possibly four, differentprotein species. (Received December 22, 1977; )     

Biological activities of extracts from endophytic fungi isolated from Garcinia plants

Sixty-five crude extracts from 51 selected endophytic fungi isolated from Garcinia species were tested for various bioactivities. Eighty per cent of the fungal extracts from fermentation broths and mycelia displayed bioactivities: antimycobacterial (76.9%), antimalarial (14.1%), antiviral (16.7%), antioxidant (22.2%), antiproliferation (11.1% against NCI-H187 and 12.7% against KB cells), and cytotoxicity to Vero cells (40.0%). Based on internal transcribed spacer rRNA sequence analysis, 15 bioactive isolates were identified as Aspergillus, Botryosphaeria, Curvularia, Fusicoccum, Guignardia, Muscodor, Penicillium, Pestalotiopsis, and Phomopsis spp. One isolate (N24) was matched with an unidentified fungal endophyte. These results indicate that endophytic fungi isolated from Garcinia plants in Thailand are potential sources of various bioactive natural products.     

小麦内生细菌及其对根茎部主要病原真菌的抑制作用

对小麦植株不同生育期、不同器官的内生细菌进行了分离和数量变化分析.结果表明,根、茎、叶及未成熟籽粒等器官中存在大量的内生细菌,鲜组织中平均约含内生细菌5.0×10⁵ CFU·g^-1,其中根系中内生细菌数量达7.8×10⁵ CFU·g^-1,而茎秆、叶片和未成熟籽粒中内生细菌数量分别为4.8×10⁵、3.2×10⁵和2.8×10⁵ CFU·g^-1.内生细菌数量在不同生育期也存在差异,幼苗期平均约为3.1×10⁵ CFU·g^-1、拔节期和灌浆期分别为5.7×10⁵和7.0×10⁵ CFU·g^-1.不同小麦田块之间存在明显差异,长武县一田块植物鲜组织中内生细菌的数量为6.1×10⁵ CFU·g^-1,而大荔县一田块约为3.9×10⁵ CFU·g^-1.试验结果发现,对小麦全蚀病菌具有拮抗作用的内生细菌有51株、对小麦纹枯病菌具有抑制作用的内生细菌有45株.用平板对峙法测定,有71株对两种病原真菌均有拮抗作用,对小麦全蚀病菌抑菌圈直径大于10 mm的有23株,其中来源于根系、茎秆、叶片和籽粒的分别为6株、7株、9株和1株;对小麦纹枯病菌抑菌圈超过10 mm的有20株,其中来源于根系、茎秆、叶片和籽粒的分别为7株、5株、7株和1株,说明从小麦叶片诱捕分离的内生细菌中,对小麦全蚀病菌和纹枯病菌抑菌作用较强的分离株比率最高,其次为茎秆,而根部和未成熟籽粒中比例明显较低.     

Novel derivatives of chitosan and their antifungal activities in vitro

Three kinds of Schiff bases of carboxymethyl chitosan (CMCTS) were prepared, and their antifungal activities were assessed according to Jasso de Rodríguez's method. The results indicated that 2-(2-hydroxybenzylideneamino)-6-carboxymethylchitosan (HNCMCTS) and 2-(5-chloro-2-hydroxybenzylideneamino)-6-carboxymethylchitosan (HCCMCTS) had better inhibitory effects than those of chitosan or CMCTS against Fusarium oxysporium f. sp. vasinfectum, Alternaria solani, and Valsa mali.