Retention ofB. burgdorferi pathogenicity and infectivity after multiple passages in a co-culture system

In vitro cultivation ofB. burgdorferi in BSK medium results in the loss of infectivity and pathogenicity after repeated passages. To prevent this loss, a feeder layer of tibio-tarsal joint tissue derived from newborn LEW/N rats was grown on Cytodex 3 microcarriers in ESG (formerly BSKE), a novel medium developed to support the growth of both the feeder layer andB. burgdorferi. A new pathogenic isolate (FNJ) and a high passage, non-pathogenic strain (TNJ) grew well in this co-culture system with high yields of viable organism. FNJ caused no growth inhibition or visible damage to the cells in the feeder layer. FNJ remained arthritogenic for newborn LEW/N rats after 22 passages in the co-culture system, but lost its arthritogenicity after 7 passages when cultured in BSK medium. This borrelia-mammalian tissue co-culture technique presents an experimental system to study the long term interactions ofB. burgdorferi with the infected host tissues in vitro, as well as facilitate diagnostic tests and vaccine development.This article forms New Jersey Agricultural Experiment Station paper no. D8420-19-92.     

Retention of<Emphasis Type="Italic">B. burgdorferi</Emphasis> pathogenicity and infectivity after multiple passages in a co-culture system

In vitro cultivation ofB. burgdorferi in BSK medium results in the loss of infectivity and pathogenicity after repeated passages. To prevent this loss, a feeder layer of tibio-tarsal joint tissue derived from newborn LEW/N rats was grown on Cytodex 3 microcarriers in ESG (formerly BSKE), a novel medium developed to support the growth of both the feeder layer andB. burgdorferi. A new pathogenic isolate (FNJ) and a high passage, non-pathogenic strain (TNJ) grew well in this co-culture system with high yields of viable organism. FNJ caused no growth inhibition or visible damage to the cells in the feeder layer. FNJ remained arthritogenic for newborn LEW/N rats after 22 passages in the co-culture system, but lost its arthritogenicity after 7 passages when cultured in BSK medium. This borrelia-mammalian tissue co-culture technique presents an experimental system to study the long term interactions ofB. burgdorferi with the infected host tissues in vitro, as well as facilitate diagnostic tests and vaccine development.     

Développement du virus de la maladie des noyaux denses deGalleria mellonella en culture de tissus de Lépidoptères

Summary In tissue cultures of ovarian tubes ofGalleria mellonella andBombyx mori (Lepidoptera) infected by the virus of the disease of dense nuclei ofG. mellonella, fibroblasts show changes characteristic of the disease. These changes consist of the formation of intranuclear, Feulgen-positive, basophilic masses and are followed by lysis of the cells.     

Shoot regeneration fromAgrobacterium tumefaciens-induced tumors of a tropical timber tree,Fagraea fragrans

Summary Agrobacterium tumefaciens A208 with nopaline plasmid pTiT37 was used to obtain stem tumors on plantlets ofFagraea fragrans grown in vitro. Bacterial elimination and tissue proliferation were simultaneously achieved by growing tumors on cefatoxime medium. After some tissue growth the shoots regenerated. An examination of these showed the presence of nopaline, indicating genetic transformation by T-DNA.     

The use of 5-fluorocytosine and ketoconazole in the culture of the erythrocytic stages ofPlasmodium falciparum and some tumor cell lines

Summary In vitro culture systems are often contaminated by bacteria and fungi. It is therefore often necessary to supplement culture media with agents such as penicillin/streptomycin, gentamycin or amphotericin B. The latter cannot be used in the in vitro culture of erythrocytic stages ofP. falciparum, and thus anti-fungal agents have not been regularly used in this system. We describe the prophylactic use of 5-fluorocytosine (5-FC) and ketoconazole (KTZ) in tissue cultures at concentrations up to 300 and 10 g/ml respectively which have no effect on the growth ofP. falciparum (FCR-3 strain). A melanoma cell line (C32) and a line of uterine carcinoma (C41) were also unaffected by similar concentrations of 5-FC and KTZ. When dissolved in complete culture medium (RPMI 1640) with 10% human plasma, the minimum inhibitory concentration of 5-FC for a susceptible strain ofCandida remained below 2 g/ml. These experiments suggest that 5-FC (at 50 g/ml) alone or in combination with KTZ (at 1 g/ml) is a useful addition to the armamentarium of antimicrobials available to the tissue culture biologist for a variety of cell culture systems.     

Aplysianin-A,an antibacterial and antineoplastic glycoprotein in the albumen gland of a sea hare,Aplysia kurodai

Summary Aplysianin-A, an antibacterial and antineoplastic factor in the albumen gland of the sea hareAplysia kurodai, was isolated. It had a molecular weight of approximately 320 kD and consisted of subunits with a molecular weight of 85 kD. It contained 9.8% neutral sugar. Aplysianin A showed 50% inhibition ofBacillus subtilis growth at a concentration of 4 g protein/ml and 50% lysis of murine MM46 tumor cells at 14 ng protein/ml. A partial identity of antigenic specificity of the purified specimen with an antineoplastic factor fromAplysia eggs was observed in immunodiffusion tests.Acknowledgment. We are indebted to the staff of Fisheries Research Laboratory, Faculty of Agriculture, University of Tokyo, Maisaka, for the collection of sea hares.     

Oxidative stress in the moderately halophilic bacteriumDeleya halophila: Effect of NaCl concentration

The sensitivity ofDeleya halophila to oxidative stress caused by hydrogen peroxide (H₂O₂) was found to vary, depending on the NaCl concentration of the growth medium. Pretreatment of the bacteria at a low concentration of H₂O₂ (50 M) protected the cells against the lethal effects of higher levels (1–2 mM) of H₂O₂. Exposure ofD. halophila cells to 50 M H₂O₂ resulted in the induction of several proteins (hydrogen peroxide-inducible proteins, hips). However, the kinetics of induction, the extent of induction and the number of hips appear to be influenced by the salt concentration of the growth medium. Five of the hips exhibited apparent molecular masses identical to those of five heat shock proteins (hsps).     

New phytotoxic butenolides produced bySeiridium cardinale,the pathogen of cypress canker disease

Summary Two new butenolides, seiridin andiso-seiridin, were isolated from culture filtrates ofSeiridium cardinale, the pathogen of cypress canker, a destructive disease ofCupressus and relatedConiferae These metabolites were characterized as 3-methyl-4-(2-hydroxyheptyl)-2(5H)-furanone and its 4-(3-hydroxyheptyl) isomer, respectively. Chlorotic, and necrotic symptoms were produced on leaves of either host or non-host test plants by absorption of 0.3 mg/ml solutions of either compound. These also showed antibacterial activity.This study was supported in part by the National Research Council, Rome (special ad hoc project Chimica fine e secondaria) and in part by the Ministry of Education, Rome.     

Effect of actinomycin D onTrypanosoma cruzi

Summary Viable metacyclic forms ofT. cruzi, Y strain, treated with an adequate dose of actinomycin D (50 g Act-D/ml/10⁷ parasites/ml for 72 h at 28° C) showed the following properties: 1) they lost their ability to replicate in culture medium, in blood and in tissues of normal mice and were no longer able to incorporate tritiated thymidine; 2) they could no penetrate into Vero cells and could not replicate inside normal macrophages; 3) they retained their immunogenicity and the ability to protect mice against a virulent infection; 4) they did not induce histological lesions as described in chronic experimental Chagas' disease.     

Detection of toxic metabolites in the hemolymph ofBeauveria bassiana infectedSpodoptera exigua larvae

Highly active metabolites have been detected in the hemolymph of the lepidopteranSpodoptera exigua infected with the mycopathogen,Beauveria bassiana. A combination of phenyl sepharose and CM ion exchange chromatography was utilized to extract the active metabolites from infected hemolymph samples. The active in vivo metabolites, having a molecular mass greater than 10 KDa, were thermolabile and were inactivated by proteinase K. These metabolites were characterized by their ability to disrupt metamorphosis, killing treated larvae at the wandering or pupal stage. Additionally, injection ofS. exigua larvae with active samples caused a reduction in the number of filopodial-producing hemocytes. The biological activities and biochemical properties suggest that novel compounds are produced duringB. bassiana mycosis.     

Slide test for detection of slime formation in vibrios

Summary and conclusion Slime production was detected by a simple slide test on a glass slide with distilled water and 0.5% sodium taurocholate solution in all the 64 strains of vibrios consisting of classicalV. cholerae, El Tor and NAG vibrios, isolated from different sources, 2 strains ofPs. aeruginosa and 2 of the 8 strains of Enterobacteriaceae, when the organisms were grown in a special phosphate agar medium. Phosphate was essential for SM in many strains of El Tor and NAG vibrios. Although detectable SM was observed in nutrient agar, presence of phosphate definitely augmented its production by classicalV. cholerae. Observation of such characteristic features in vibrios justified the consideration that the viscous material was extracellular slime of the organism. This also suggests that the simple slide test as employed here can be utilized for detection of SM of intestinal bacteria after growing in medium containing suitable concentration of phosphate.

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Zusammenfassung Nachweis der Schleimbildung von Vibrionen in Enterobakterien.

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The authors wish to convey their gratefulness to Prof. J. B.Chatterjea, School of Tropical Medicine, Calcutta, for his kind permission to publish this paper.     

Effect of tingenone,a quinonoid triterpene,on growth and macromolecule biosynthesis inTrypanosoma cruzi

Summary Tingenone and horminone, two natural quinonoid substances, inhibited the in vitro growth ofTrypanosoma cruzi, 30 M drug concentration producing total inhibition of growth. Tingenone inhibited total uptake and incorporation of [³H]thymidine, [³H]uridine, L-[³H]leucine into parasite macromolecules. Other quinonoids assayed were either less effective (abruquinone A) or even quite inactive (visminone B and ferruginin B). Investigation of several mechanisms for the cytotoxic action of tingenone pointed to the interaction with DNA as the most likely factor involved. Tingenone also inhibited the growth ofCrithidia fasciculata, but the drug was significantly less active on this organism than onT. cruzi.This work was supported by grants of UNDP/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases, Organization of American States (Multinational Programme of Biochemistry) and Programa Nacional de Enfermedades Endémicas (SECYT), República Argentina. A preliminary account was given at the Workshop on Oxidative Damage and Related Enzymes, Frascatti (Italy), 1983.     

Molecular characterization of the p83/100 proteins of variousBorrelia burgdorferi sensu lato strains

Conclusions As in OspA-serotyping experiments, theB garinii group (OspA-sterotype 3–7) showed highest diversity within this internal fragment of p83/100, whereas theB. afzelii group (OspA-type 2) and theB. burgdorferi sensu stricto group (OspA-type 1) were nearly identical. Determination of the size of the PCR products as well as restriction fragment length polymorphism analysis (DraI) can be used for classification into the three species ofB. burgdorferi sensu lato. Since p83/100 is chromosomally encoded, this protein might be a more stable marker for classification than the plasmid-encoded OspA. In contrast to the flagellin gene a subclassification of theB. garinii group is possible due to the diversity of the p83/100 internal fragment.     

Transgenic and mutant animal models to study mechanism of protection of red cell genetic defects against malaria

Malaria, caused by members of the genusPlasmodia, is still the most prevalent parasitic disease in the world. In an attempt to understand genetic factors conferring resistance to malaria, mouse models of thalassemia, sickle trait, and ankyrin and spectrin deficiency were studied during infection with species of malaria infectious to rodents. Although growth ofP. falciparum is not inhibited in thalassemic erythrocytes in culture, mice carrying a -thalassemia mutation were protected fromPlasmodium chabaudi adami, supporting epidemiologic findings. Transgenic mice expressing ^s hemoglobin were also significantly protected from two species of rodent malaria. Importantly, a significant role for the spleen in protection in the ^s transgenic mice was found. Finally, mice deficient in spectrin and ankyrin were studied with respect to their ability to support the growth of malaria. It was found that spectrin deficient mice were almost completely refractory toP. chabaudi adami andP. berghei. These models will allow further study of host factors in resistance to malaria.     

Autoinhibition of juvenile hormone production. The case of the cockroachBlattella germanica (L.)

In 6-day-old females ofBlattella germanica, the activity of corpora allata (CA) was inhibited in vitro by juvenile hormone III (JH III). Effective doses (281.5 and 375.4 M in the medium) were somewhat higher than (although of the same order of magnitude as) the estimated intraglandular concentration of JH III at this age, and they induced about 45% inhibition of hormonal release and a significant intraglandular accumulation of JH III and methyl farnesoate. The results suggest that autoinhibitory mechanisms operate in the CA to constrain the upper limit of JH III production at the end of the gonadotrophic cycle.     

Unusual antimicrobial compounds fromAeollanthus buchnerianus

Using bioassay guided isolation, three novel 12 carbon polyoxygenated fatty acids and a novel abietane diterpene have been isolated from the the chloroform extract of aerial parts ofAeollanthus buchnerianus (Lamiaceae). Rigorous spectroscopic methods were used for compound identification. (Z,Z)-8-acetoxy-5-hydroxydodeca-2,6-dienoic acid and (Z,Z)-5,8-dihydroxydodeca-2,6-dienoic acid inhibited the spore germination ofCladosporium cucumerinum (both with Minimum Inhibitory Dose (MID) values of 1 g) andAspergillus niger (MID 5 and 25 g respectively). Further, they also reduced the hyphal growth ofPythium ultimum. (Z)-5-hydroxy-6,7,8-triacetoxydodeca-2-dienoic acid exhibited short term inhibition of the growth ofCladosporium cucumerinum. The novel abietane diterpenoid, (rel)-14-acetoxyabiet-7-en-18-oic acid inhibited the growth of the gram positive bacteriaBacillus subtilis, staphylococcus aureus andStreptomyces scabies (MIC values 80, 20 and 20 g ml^–1 respectively).     

ZNS-aktive Phenylpropylamine vonCatha edulis FORSK. (Celastraceae) kenyanischer Herkunft

Summary The phenylpropylamine fraction ofCatha edulis FORSK. grown in Kenya has been investigated. The major components have been found to be (S)-(–)--aminopropiophenone (1), (+)-norpseudoephedrine (2) and(–)-norephedrine (3). Quantitative ratios of1, 2, 3 in tissues of different age are discussed.

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ZNS-aktive Phenylpropylamine vonCatha edulis FORSK. (Celastraceae) kenyanischer Herkunft

     

Effects of diapause and host species on some morphometric characters inTrichogramma (Hym.: Trichogrammatidae)

Adult morphology ofTrichogramma is modified by environmental conditions during the preimaginal development. For instance, the low temperatures inducing diapause cause a decrease of the ratio length of longest seta on male flagellum/width of flagellum inT. evanescens. The species of the host also influences numerous body ratios. So, in females, the development of tibiae with regard to wings is more important withScotia ipsilon than withGalleria mellonella. These results confirm the necessity of performing comparative morphological studies on individuals reared in the same conditions.     

Occurrence of thiaminase II inSaccharomyces cerevisiae

Summary It was found that cell-free extracts ofSaccharomyces cerevisiae contain thiaminase II which hydrolyzes thiamine and thiamine analogs. The possible involvement of this enzyme and thiamine-synthesizing enzymes in thiamine production from thiamine antagonists is discussed.30 September 1986Acknowledgments. We thank the late Dr S. Yurugi, Takeda Pharmaceutical Industries, Osaka, for his generous gifts of dimethialium, 2-northiamine, -hydroxyethylthiamine, hydroxymethylpyrimidine, 2-norhydroxymethylpyrimidine and hydroxyethylthiazole. We also thank Prof. H. Nakayama, Yamaguchi Women's College, for his kind supply ofEscherichia coli 70–17 and 26–43.     

Protective effect of vitamins against trichothecene toxicity towardsSaccharomyces cerevisiae

Summary Several trichothecene mycotoxins were shown to inhibit the growth ofSaccharomyces cerevisiae. This effect was most pronounced with the macrocyclic trichothecenes, especially verrucarin A. Much less growth inhibition was observed with T-2 toxin. Verrucarol, diacetoxyscirpenol, acetyl T-2 toxin, HT-2 toxin, T-2 tetraol and neosolaniol were inactive at a concentration of 75 g of toxin per disc. Incubation ofS. cerevisiae with verrucarin A together with vitamins resulted in a decrease in toxicity. Pyridoxine-HCl, Ca-pantothenate, thiamine-HCl and -tocopheryl acetate were amongst the most potent of the vitamins tested which reversed growth inhibition, overcoming the inhibitory potential of the toxins.9 December 1986The authors thank Dr J. Behrend, Makor Company, Israel, for a generous gift of verrucarin A and roridin A.