Two Widely Accessible Media for Growth and Reproduction of Phytophthora and Pythium Species

Tomato agar and soybean agar were found to be comparable to or in some cases better than the popular V8 vegetable juice agar in supporting linear growth of Phytophthora cactorum, Phytophthora capsici, Phytophthora parasitica, Pythium aphanidermatum, and Pythium splendens; sporangium production of P. capsici, P. palmivora, and Pythium splendens; and oospore formation of P. cactorum, P. parasitica, Pythium aphanidermatum and Pythium splendens. These two media with readily accessible ingredients can be used to substitute for V8 juice agar in pathological and physiological studies of these two important groups of fungi in countries where V8 vegetable juice is not available or difficult to obtain.     

Two New Pyrrolosesquiterpenes Produced by a Streptomyces Species

Two new pyrrolosesquiterpenes, 1 and 2 , were isolated from cultures of the soil actinomycete Streptomyces sp. Hd7–21. The structures of these compounds were established as (2Z,4E,9E)‐6,7,11‐trihydroxy‐3,7,11‐trimethyl‐1‐(1H‐pyrrol‐2‐yl)dodeca‐2,4,9‐trien‐1‐one ( 1 ) and (2Z,4E)‐5‐{3‐[(2E)‐4‐hydroxy‐4‐methylpent‐2‐en‐1‐yl]‐3‐methyloxiran‐2‐yl}‐3‐methyl‐1‐(1H‐pyrrol‐2‐yl)penta‐2,4‐dien‐1‐one ( 2 ) by extensive spectroscopic analyses including MS, and 1D‐ and 2D‐NMR data. Their cytotoxic activities against a panel of human cancer cell lines were evaluated.     

Partial Purification and Characterization of a Bacteriocin Produced by Propionibacterium thoenii

A partially purified bacteriocin produced by Propionibacterium thoenii designated propionicin PLG-1 was found to be active against closely related species and exhibited a broad spectrum of activity against other microorganisms. Propionicin PLG-1 was found to be heat labile, sensitive to several proteolytic enzymes, and stable at pH 3 to 9. Propionicin PLG-1 was isolated from solid medium, partially purified by ammonium sulfate precipitation, and purified further by gel filtration. Gel filtration experiments revealed that bacteriocin PLG-1 was present as two different protein aggregates with apparent molecular weights of more than 150,000 and approximately 10,000. Resolution of these protein aggregates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a protein common to both with an apparent molecular weight of 10,000.     

Partial Characterization of Bacteriocins Produced by Two New Enterococcus faecium Isolated from Human Intestine

This study aimed at characterizing two novel bacteriocin-producing enterococcal strains isolated from human intestine. A total of 200 lactic acid bacteria were isolated from a woman stool sample. Two of them were selected for characterization due to their high antimicrobial activity against five strains of Listeria monocytogenes. The selected bacteria were identified as two different strains of Enterococcus faecium and designated MT 104 and MT 162. The bacteriocins produced by MT 104 and MT 162 were stable at different pH ranging from 2 to 11 and were active after different treatments such as heat, enzymes, detergents, and γ-irradiation. The two isolated strains exhibited some probiotic properties such as survival in simulated gastric fluid and intestinal fluid, lack of expression of bile salt hydrolase or hemolytic activity, adhesion to Caco-2 cells efficiently, and sensitivity to clinical antimicrobial agents. Thus, the two isolated strains of E. faecium could become new probiotic bacteria and their bacteriocins could be used for controlling L. monocytogenes in combination with irradiation for food preservation.     

一株曲霉的一种中温糖化酶的纯化及其部分酶学特性

本研究从广西花坪自然保护区采集的土壤中筛选获得了一株产糖化酶丝状真菌菌株57-45,通过形态学观察和真菌内转录间隔区(internal transcribed spacer,ITS)序列比对分析,将其初步鉴定为曲霉属(Aspergillus sp.)的一个种。纯化真菌57-45所产的一种胞外糖化酶经过硫酸铵分级沉淀、疏水层析和阴离子交换层析三步蛋白质纯化步骤,得到在SDS-PAGE上约60kD的单一蛋白质条带,薄层层析分析表明该纯化的蛋白质水解可溶性淀粉的产物只有葡萄糖,证明该纯化的蛋白质为糖化酶。纯化的糖化酶Km值为1.9mg/mL,Vmax为4148μmol/(min·mg),最适作用pH5.5,最适作用温度50℃,在同步糖化发酵中有应用的潜力。金属离子Fe3+、Zn2+、Cu2+对酶活有较强的抑制作用,EDTA对酶活有较强的促进作用。本文结果将为进一步研究曲霉糖化酶的酶学特性提供新的材料。     

Characterization of a Temperate Phage and Four Bacteriocins Produced by Nonpathogenic Clostridium Species

We previously reported the isolation of a temperate phage (named KT) and several bacteriocins (named clostocins) from strains of nonpathogenic Clostridium species. Later, the induction and some properties of the phage and four clostocins (A, B, C and D) were examined.

The phage was induced by UV light and mitomycin C. The phage had a polygonal head (about 85mμ in diameter) and a tail with contractile sheath (about 100mμ in length). Some other properties of the phage were also studied; plaque morphology, stability in salt solution, inactivation by UV light, pH stability, thermal inactivation, host-range and lysis of infected culture.

Clostocins A and D were partially induced by UV light and mitomycin C, whereas that of B and C were not. All clostocins failed to pass through a dialysis membrane, and were insensitive to UV light and to ribo- and deoxyribonuclease. They were destroyed by some proteolytic enzymes, but differences in degree of their susceptibility were observed among them. Clostocins A and D were very thermo-stable, whereas B and C were relatively thermo-labile. Clostocins A and D acted on some strains in the genus Clostridium, whereas B and C did on many strains in the family Bacillaceae.

There was no demonstrable serological relationship between phage KT and clostocin A, although they seemed to adsorb on the same bacterial receptor.     

Suppression of Pythium Root Rot of Cucumber by a Fluorescent Pseudomonad s Related to Reduced Root Colonization by Pythium Apbanidermatum

Root colonization of cucumber with P aphanidermatum OP4 was examined in the absence and in the presence of either fluorescent pseudomonad strain CH31 or strain CHI. Quantification was performed by antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA) using an antiserum obtained from a rabbit immunized with zoospore cysts: this antiserum did not cross-react with non-infested roots of cucumber, or with the fluorescent pseudomonads CH31 and CHI. Application of the previously developed biotest enabled us to assess the ability of the two strains of fluorescent pseudomonad to suppress root rot caused by P. aphanidermaium OP4.
At all bacterial densities tested, the fluorescent pseudomonad CH31 led to a significant reduction of both the root colonization of cucumber with P aphanidermatum OP4 and the severity of Pythium root rot. In contrast, the fluorescent pseudomonad CHI had no effect on either root colonization oe disease sevenity.     

Identification and Partial Characterization of Antilisterial Compounds Produced by Dairy Yeasts

Food-grade yeasts make significant contributions to flavor development in fermented foods. Some yeast species also inhibit undesirable bacteria, yeasts and molds, apparently by producing antimicrobial compounds called mycocins. The aim of this study was to evaluate the ability of wild yeasts, isolated from raw milk and cheese in the Quebec province area, to produce antilisterial compounds. Based on an agar-membrane screening test, 22 of 95 isolates, namely one Candida catenulata, one Candida parapsilosis, five Candida tropicalis, four Debaryomyces hansenii, one Geotrichum candidum, nine Pichia fermentans and one Pichia anomala, exhibited a significant inhibitory effect against Listeria ivanovii HPB28. Four in particular, namely C. tropicalis LMA-693, D. hansenii LMA-916, P. fermentans LMA-256 and P. anomala LMA-827, produced substances extractable from culture supernatant and capable of decreasing 18-h growth of L. ivanovii by, respectively, 97, 92, 84 and 78 %. Heating the extracted material (100 °C for 10 min) decreased these values to 72, 62, 58 and 31 %, respectively, while treatment with trypsin or pronase E decreased them to as little as 27 %. The extracts reduced the numbers of viable Listeria monocytogenes by as much as four log cycles within an hour. Transmission electron microscopy revealed a high proportion of lysis among the cells, apparently due to pore formation. This study clearly shows the potential of these four yeast isolates for use as bio-preservatives in a variety of dairy products.     

Partial Chemical and Physical Characterization of Two Extracellular Polysaccharides Produced by Marine, Periphytic Pseudomonas sp. Strain NCMB 2021

The marine bacterium Pseudomonas sp. strain NCMB 2021, which can attach to solid, and especially hydrophobic, surfaces, elaborates two different extracellular polysaccharides in batch cultures. One (polysaccharide A) was produced only during exponential growth and contained glucose, galactose, glucuronic acid, and galacturonic acid in a molar ratio of 1.00:0.81:0.42:0.32. It produced viscous solutions, formed gels at high concentrations, and precipitated with several multivalent cations. The other (polysaccharide B) was released at the end of the exponential phase and in the stationary phase. It contained equimolar amounts of N-acetylglucosamine, 2-keto-3-deoxyoctulosonic acid, an unidentified 6-deoxyhexose, and also O-acetyl groups. Despite its high molecular weight (10⁵ to 10⁶ as judged by gel filtration), the polysaccharide produced aqueous solutions with very low viscosities and was also soluble in 90% aqueous phenol, 80% methanol, and 80% ethanol.     

Isolation and Partial Characterization of a Tenuivirus from Wheat in Iran

A tenuivirus, tentatively designated Iranian wheat stripe virus (IWSV), was transmitted by the delphacid Unkanodes tanasijevici and induced dwarfing, striping and yellowing symptoms in wheat. It could also infect barley, oat, rice, rye, sorghum and a number of other gramineous species. The virus was purified from wheat by chloroform clarification and differential and density-gradient centrifugation. It formed several layers in density-gradient columns. Purified virus preparations had a UV absorption spectrum typical of nucleoproteins and contained flexuous supercoils of 8.7 nm and fine filaments of 4.3 nm width. The antiserum produced against the virus reacted with infected plant sap in agar-gel diffusion and enzyme-linked immunosorbent assay (ELISA). The virus could be readily detected in individual viruliferous planthoppers by ELISA. IWSV was serologically related to rice hoja blanca virus but not to maize stripe virus. The relationship of IWSV with other tenuiviruses is discussed.     

膜荚黄芪内生真菌次生代谢产物鉴定及抑菌活性

从膜荚黄芪(Astragalus membranaceus)叶中分离出一株内生真菌———瓶霉菌属(Phialophorasp.)。鉴定次生代谢产物中含有皂甙类、多糖类和黄酮类物质,并通过薄层层析证明内生真菌次生代谢产物的粗提物与黄芪植物水煎液的粗提物含有相同的成分。证明发酵液及菌丝体提取物对4种常见细菌具有不同程度的抑菌活性。     

Purification and Partial Characterization of a Murein Hydrolase, Millericin B, Produced by Streptococcus milleri NMSCC 061

Streptococcus milleri NMSCC 061 was screened for antimicrobial substances and shown to produce a bacteriolytic cell wall hydrolase, termed millericin B. The enzyme was purified to homogeneity by a four-step purification procedure that consisted of ammonium sulfate precipitation followed by gel filtration, ultrafiltration, and ion-exchange chromatography. The yield following ion-exchange chromatography was 6.4%, with a greater-than-2,000-fold increase in specific activity. The molecular weight of the enzyme was 28,924 as determined by electrospray mass spectrometry. The amino acid sequences of both the N terminus of the enzyme (NH₂ SENDFSLAMVSN) and an internal fragment which was generated by cyanogen bromide cleavage (NH₂ SIQTNAPWGL) were determined by automated Edman degradation. Millericin B displayed a broad spectrum of activity against gram-positive bacteria but was not active against Bacillus subtilis W23 or Escherichia coli ATCC 486 or against the producer strain itself. N-Dinitrophenyl derivatization and hydrazine hydrolysis of free amino and free carboxyl groups liberated from peptidoglycan digested with millericin B followed by thin-layer chromatography showed millericin B to be an endopeptidase with multiple activities. It cleaves the stem peptide at the N terminus of glutamic acid as well as the N terminus of the last residue in the interpeptide cross-link of susceptible strains.     

Earliest Symptoms of Coronary Heart Disease and their Recognition

We have reviewed the histories of 320 patients in whom a diagnosis of coronary heart disease was ultimately established and traced the symptoms back to their first appearance. In 51% the first symptom was effort angina. Difficulties in recognition arose when the symptom was localized to an unusual site, when its occurrence was dependent on a combination of exercise with cold or a recent meal, or when it was induced by excitement rather than by effort. In a quarter of the cases the onset of angina was abrupt, and in these there was usually evidence of acute infarction.In 43% of cases the first symptom was an attack of pain or discomfort in the torso occurring without any discernable precipitating factor. Again, diagnosis was difficult when the pain was in an atypical site and also when it was of brief duration associated with skeletal or abdominal disease which could cause pain at the same site, or if the patient was able to undertake strenuous exertion. In four patients cardiac pain was first experienced during a paroxysm of tachycardia. In 6% of cases the onset was marked by a symptom other than pain—most frequently dyspnoea, tiredness, faintness, or syncope.Clinical examination was of no direct value in diagnosis. Its importance lay in disclosing factors which had to be taken into account in interpreting the electrocardiogram. The electrocardiogram was invaluable, though by no means infallible. In over half of the patients the first tracing showed major abnormalities of coronary type, and nearly a quarter more showed minor S-T/T depression consistent with coronary disease. Ten per cent. showed miscellaneous abnormalities, such as left ventricular hypertrophy or bundle-branch block, and 15% no definite abnormality.There is as yet no completely reliable objective method of diagnosing early coronary heart disease, so that the recognition of symptoms remains of paramount importance.     

Isolation and Partial Characterization of Aluminium-Induced Cytoplasmic Polypeptides from Barley Root

Using preparative isoelectric focusing, fast performance liquid chromatography, and polyacrylamide gel electrophoresis accumulation of several Al-induced cytoplasmic proteins was described in barley roots. Two of them, 27 and 28 kDa polypeptides were isolated by continuous-elution electrophoresis system in sufficient quantities for their further characterisation.     

Isolation and Partial Characterization of Two Aeromonas hydrophila Bacteriophages

Two Aeromonas hydrophila bacteriophages, Aeh1 and Aeh2, were isolated from sewage. Both phages showed binal symmetry. The dimensions of A. hydrophila phages Aeh1 and Aeh2 differed from those of the other Aeromonas phages. Also, phage Aeh2 was the largest Aeromonas phage studied to date. Phage Aeh1 formed small, clear plaques, and phage Aeh2 formed turbid plaques with clear centers. Both phages were sensitive to chloroform treatment, being totally inactivated after treatment for 1 h at 60°C at pH 3 and 11. However, the infectivity of Aeh1 phage stocks increased by approximately fivefold after they were treated at pH 10 for 1 h at 22°C. Phages Aeh1 and Aeh2 were serologically unrelated and had latent periods of 39 and 52 min, respectively. The average burst sizes of phages Aeh1 and Aeh2 were 17 and 92 PFU per cell, respectively. Phage Aeh1 infected 13 of 22 A. hydrophila strains tested, whereas phage Aeh2 infected only its original host. Phage Aeh1 infected some A. hydrophila strains only at or below 37°C. Neither phage infected the two A. (Plesiomonas) shigelloides strains used in this study.     

Characterization of a Superantigen Produced by Yersinia pseudotuberculosis

Abstract

Yersinia species are Gram-negative coccobacilli consisting of three pathogenic species, Y. pestis, Y. pseudotuberculosis, Y. enterocolitica, and five nonpathogenic species, Y. kristensenii, Y. frederiksenii, Y. intermedia, Y. rohdei, and Y. aldovae. The former three species are primary pathogens of wild and domestic animals and birds. In the human, Y. pestis causes plague, or black death, while Y. pseudotuberculosis and Y. enterocolitica produce milder forms of disease varying from diarrhea and abdominal pain to more systemic symptoms such as fever, scarlatiniform skin rash, conjunctivitis, erythema nodosum, and lymphadenopathy (1–3). Complications of reactive arthritis, acute uveitis, coronary aneurysms, and acute renal failure are not infrequently reported after the latter two Yersinia infections (4–8). The mechanisms by which these organisms mediate these complicated symptoms are poorly understood. However, the preferential avidity for lymphoid tissues seen in these species and the characteristic histopathological finding of lymphoid hyperplasia mainly seen in mesenteric lymph nodes (9–10) suggest that the stimulation of a large proportion of T lymphocytes may be involved in the pathogenesis of this infection.     

Characterization and Identification of Asexual Strains of Pythium Associated with Root Rot of Rose in Japan

This study was conducted to survey the distribution of asexual isolates of Pythium in rose production and to characterize and identify them. Asexual isolates with proliferating globose sporangia belong to group P according to the key of van der Plaats‐Niterink (1981; Monograph of the genus Pythium. Studies in Mycology, Vol. 21, Centraalbueau Voor Schimmelcultures, Baarn, The Netherlands). Group P isolates were recovered from rotted roots of both cutting and miniature roses cultured in rock wool and ebb‐and‐flow culture systems, respectively, throughout the main rose production area of Japan. The typical feature of the P group isolates was that they could grow fast at high temperature, at least 30 mm per 24 h at 35°C. There was no difference between the P group isolates and P. helicoides in morphology and size of sporangia and sporangial germination mode. The symptoms caused by the group P isolates were root rot, followed by leaf blight and plant death in severe cases. In restriction fragment length polymorphism analysis of the rDNA‐ITS region, the banding patterns with five of six enzymes were identical between group P and P. helicoides, the only difference being seen with HhaI. In direct amplification analysis of minisatellite‐region DNA with M13 primer, group P and P. helicoides shared three of five distinct bands. In contrast, P. oedochilum and P. ostracodes showed different banding patterns except for each one band. The results suggest that the group P isolates obtained from rose root rot may be asexual strains of P. helicoides.     

Isolation and Partial Characterization of Antifungal Metabolites Produced by <Emphasis Type="Italic">Bacillus</Emphasis> sp. IBA 33

Antifungal proteins produced by Bacillus sp. IBA 33 were purified by ammonium sulfate precipitation and DEAE-Sephacel column chromatography. The two purified proteins inhibited the growth of Geotrichum candidum, the sour rot disease agent in lemon. The proteins were stable at 20 (3 months), 40, 60 and 100°C (30 min) and remained active after sterilization at 121°C for 15 min. Their hydrophobic nature was proved and when were developed with ninhydrin they did not show any free amino groups. The infrared spectrum showed vibrational modes corresponding to peptide, ester or ketone links and saturated CH links corresponding to long chain fatty acids. UV scan spectroscopy showed tyrosine and or tryptophan amino acids in their composition. The remarkable thermo-resistance of proteins may be a good feature to be used in the development of a new biocontrol method of Geotrichum candidum.