Repeated introduction of genetically modified Pseudomonas putida WCS358r without intensified effects on the indigenous microflora of field-grown wheat.

To investigate the impact of genetically modified, antibiotic-producing rhizobacteria on the indigenous microbial community, Pseudomonas putida WCS358r and two transgenic derivatives were introduced as a seed coating into the rhizosphere of wheat in two consecutive years (1999 and 2000) in the same field plots. The two genetically modified microorganisms (GMMs), WCS358r::phz and WCS358r::phl, constitutively produced phenazine-1-carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (DAPG), respectively. The level of introduced bacteria in all treatments decreased from 10(7) CFU per g of roots soon after sowing to less than 10(2) CFU per g after harvest 132 days after sowing. The phz and phl genes remained stable in the chromosome of WCS358r. The amount of PCA produced in the wheat rhizosphere by WCS358r::phz was about 40 ng/g of roots after the first application in 1999. The DAPG-producing GMMs caused a transient shift in the indigenous bacterial and fungal microflora in 1999, as determined by amplified ribosomal DNA restriction analysis. However, after the second application of the GMMs in 2000, no shifts in the bacterial or fungal microflora were detected. To evaluate the importance of the effects induced by the GMMs, these effects were compared with those induced by crop rotation by planting wheat in 1999 followed by potatoes in 2000. No effect of rotation on the microbial community structure was detected. In 2000 all bacteria had a positive effect on plant growth, supposedly due to suppression of deleterious microorganisms. Our research suggests that the natural variability of microbial communities can surpass the effects of GMMs.

Effect of genetically modified Pseudomonas putida WCS358r on the fungal rhizosphere microflora of field-grown wheat.

We released genetically modified Pseudomonas putida WCS358r into the rhizospheres of wheat plants. The two genetically modified derivatives, genetically modified microorganism (GMM) 2 and GMM 8, carried the phz biosynthetic gene locus of strain P. fluorescens 2-79 and constitutively produced the antifungal compound phenazine-1-carboxylic acid (PCA). In the springs of 1997 and 1998 we sowed wheat seeds treated with either GMM 2, GMM 8, or WCS358r (approximately 10(7) CFU per seed), and measured the numbers, composition, and activities of the rhizosphere microbial populations. During both growing seasons, all three bacterial strains decreased from 10(7) CFU per g of rhizosphere sample to below the limit of detection (10(2) CFU per g) 1 month after harvest of the wheat plants. The phz genes were stably maintained, and PCA was detected in rhizosphere extracts of GMM-treated plants. In 1997, but not in 1998, fungal numbers in the rhizosphere, quantified on 2% malt extract agar (total filamentous fungi) and on Komada's medium (mainly Fusarium spp.), were transiently suppressed in GMM 8-treated plants. We also analyzed the effects of the GMMs on the rhizosphere fungi by using amplified ribosomal DNA restriction analysis. Introduction of any of the three bacterial strains transiently changed the composition of the rhizosphere fungal microflora. However, in both 1997 and 1998, GMM-induced effects were distinct from those of WCS358r and lasted for 40 days in 1997 and for 89 days after sowing in 1998, whereas effects induced by WCS358r were detectable for 12 (1997) or 40 (1998) days. None of the strains affected the metabolic activity of the soil microbial population (substrate-induced respiration), soil nitrification potential, cellulose decomposition, plant height, or plant yield. The results indicate that application of GMMs engineered to have improved antifungal activity can exert nontarget effects on the natural fungal microflora.

Diversity and seasonal fluctuations of the dominant members of the bacterial soil community in a wheat field as determined by cultivation and molecular methods.

There is a paucity of knowledge on microbial community diversity and naturally occurring seasonal variations in agricultural soil. For this purpose the soil microbial community of a wheat field on an experimental farm in The Netherlands was studied by using both cultivation-based and molecule-based methods. Samples were taken in the different seasons over a 1-year period. Fatty acid-based typing of bacterial isolates obtained via plating revealed a diverse community of mainly gram-positive bacteria, and only a few isolates appeared to belong to the Proteobacteria and green sulfur bacteria. Some genera, such as Micrococcus, Arthrobacter, and Corynebacterium were detected throughout the year, while Bacillus was found only in July. Isolate diversity was lowest in July, and the most abundant species, Arthrobacter oxydans, and members of the genus Pseudomonas were found in reduced numbers in July. Analysis by molecular techniques showed that diversity of cloned 16S ribosomal DNA (rDNA) sequences was greater than the diversity among cultured isolates. Moreover, based on analysis of 16S rDNA sequences, there was a more even distribution among five main divisions, Acidobacterium, Proteobacteria, Nitrospira, cyanobacteria, and green sulfur bacteria. No clones were found belonging to the gram-positive bacteria, which dominated the cultured isolates. Seasonal fluctuations were assessed by denaturing gradient gel electrophoresis. Statistical analysis of the banding patterns revealed significant differences between samples taken in different seasons. Cluster analysis of the patterns revealed that the bacterial community in July clearly differed from those in the other months. Although the molecule- and cultivation-based methods allowed the detection of different parts of the bacterial community, results from both methods indicated that the community present in July showed the largest difference from the communities of the other months. Efforts were made to use the sequence data for providing insight into more general ecological relationships. Based on the distribution of 16S rDNA sequences among the bacterial divisions found in this work and in literature, it is suggested that the ratio between the number of Proteobacteria and Acidobacterium organisms might be indicative of the trophic level of the soil.

Analysis of fungal diversity in the wheat rhizosphere by sequencing of cloned PCR-amplified genes encoding 18S rRNA and temperature gradient gel electrophoresis.

Like bacteria, fungi play an important role in the soil ecosystem. As only a small fraction of the fungi present in soil can be cultured, conventional microbiological techniques yield only limited information on the composition and dynamics of fungal communities in soil. DNA-based methods do not depend on the culturability of microorganisms, and therefore they offer an attractive alternative for the study of complex fungal community structures. For this purpose, we designed various PCR primers that allow the specific amplification of fungal 18S-ribosomal-DNA (rDNA) sequences, even in the presence of nonfungal 18S rDNA. DNA was extracted from the wheat rhizosphere, and 18S rDNA gene banks were constructed in Escherichia coli by cloning PCR products generated with primer pairs EF4-EF3 (1. 4 kb) and EF4-fung5 (0.5 kb). Fragments of 0.5 kb from the cloned inserts were sequenced and compared to known rDNA sequences. Sequences from all major fungal taxa were amplified by using both primer pairs. As predicted by computer analysis, primer pair EF4-EF3 appeared slightly biased to amplify Basidiomycota and Zygomycota, whereas EF4-fung5 amplified mainly Ascomycota. The 61 clones that were sequenced matched the sequences of 24 different species in the Ribosomal Database Project (RDP) database. Similarity values ranged from 0.676 to 1. Temperature gradient gel electrophoresis (TGGE) analysis of the fungal community in the wheat rhizosphere of a microcosm experiment was carried out after amplification of total DNA with both primer pairs. This resulted in reproducible, distinctive fingerprints, confirming the difference in amplification specificity. Clear banding patterns were obtained with soil and rhizosphere samples by using both primer sets in combination. By comparing the electrophoretic mobility of community fingerprint bands to that of the bands obtained with separate clones, some could be tentatively identified. While 18S-rDNA sequences do not always provide the taxonomic resolution to identify fungal species and strains, they do provide information on the diversity and dynamics of groups of related species in environmental samples with sufficient resolution to produce discrete bands which can be separated by TGGE. This combination of 18S-rDNA PCR amplification and TGGE community analysis should allow study of the diversity, composition, and dynamics of the fungal community in bulk soil and in the rhizosphere.

Some observations on ethnoveterinary medicine in northern Nigeria.

The Fulani are herdsmen of Northern Nigeria. For generations they have been moving their cattle in an annual migration pattern in search of water. The tracks are chosen carefully, however: tsetse-fly-infected areas are avoided. In his ten years of experience in Nigeria's veterinary sector--part of this time as special officer in charge of eradicating rinderpest--the author saw numerous examples of indigenous knowledge and practices that will be useful for the future development of animal health and production.

Both the V2 and V3 regions of the human immunodeficiency virus type 1 surface glycoprotein functionally interact with other envelope regions in syncytium formation.

To map the regions of the external envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) involved in the process of membrane fusion, we determined the syncytium-inducing capacity of a panel of transiently expressed chimeric envelope genes. This panel was generated by exchanging gene fragments between four previously studied envelope genes that exhibited a high degree of sequence homology yet displayed marked differences in syncytium-inducing capacity when expressed by recombinant vaccinia virus. The results demonstrate that multiple regions of the HIV-1 envelope glycoproteins are involved in syncytium formation. Some fragments, most notably those containing the V2 or V3 region, can transfer syncytium-inducing capacity to envelope proteins previously not capable of inducing syncytia. Moreover, it is shown that such regions functionally interact with other envelope regions, especially one encompassing the V4 and V5 regions of gp120 or a region encompassing part of gp41, to exert their function in membrane fusion.

Genetic and functional analysis of a set of HIV-1 envelope genes obtained from biological clones with varying syncytium-inducing capacities.

To study HIV-1 envelope-mediated syncytium formation we have amplified, cloned, expressed, and sequenced individual envelope genes from a set of eight biological HIV-1 clones. These clones were obtained from two patients and display either a syncytium-inducing (SI) or nonsyncytium-inducing (NSI) phenotype. Upon expression through recombinant vaccinia virus, individual envelope gene products display heterogeneous syncytium-inducing capacities which reflect the phenotype of the parental biological HIV-1 clones in all cases. For the eight biological HIV-1 clones presented here, variation of the envelope gene alone is sufficient to explain the observed variable syncytium-inducing capacity of the respective parental viruses. In addition we determined the complete nucleotide sequence of these envelope genes. The predicted amino acid sequence revealed a considerable amount of variation located mainly in the previously denominated variable regions. In various regions of envelope genes obtained from the same patient, phenotype associated amino acid variation was found. This phenotype associated amino acid variation however, is not conserved between the two sets of envelope genes derived from different patients. Four envelope sequences derived from clones obtained from one patient showed phenotype-associated amino acid variation in the fusion domain. Sequencing of 12 additional fusion domains revealed that this same variation is found in four additional clones. However, a functional test performed on recombinant vaccinia expressing mutant envelope genes showed that this observed fusion domain variation does not contribute to the variation in syncytium-inducing capacity of the envelope gene product.

Interactions of methotrexate and cyclophosphamide with the pharmacokinetics of 5-fluorouracil in an animal model.

The interaction of methotrexate and/or cyclophosphamide with the pharmacokinetics of 5-fluorouracil (5-FU) was studied in tumor-bearing WAG/Rij rats. Four groups were formed including treatment with single-agent 5-FU (eight rats); 5-FU plus methotrexate (11 rats); 5-FU plus cyclophosphamide (12 rats); and 5-FU, cyclophosphamide, and methotrexate (13 rats). The area-under-the-plasma-concentration/time curve, total-body clearance, elimination half-life, mean residence time, and steady-state volume of distribution were computed and compared. The mean residence time and elimination half-life of 5-FU increased when methotrexate was included in the combination. The increase was significant (P less than 0.05) for 5-FU, cyclophosphamide, and methotrexate versus 5-FU and cyclophosphamide.

Variations in exposure to mitomycin C in an in vitro colony-forming assay.

The effect of mitomycin C on two human ovarian cancer cell lines was measured during several exposure times and concentrations using the Human Tumour Colony-forming Assay (HTCA). Changes in exposure time and concentration resulted in considerable differences in tumour cell survival. It is concluded that several exposure times and concentrations are necessary for in vitro sensitivity testing. We suggest alternative criteria derived from pharmacokinetic data in patients instead of one-tenth of the peak plasma level which is the usual practice.

Pharmacokinetic interactions of cyclophosphamide and 5-fluorouracil with methotrexate in an animal model.

In tumor-bearing WAG/Rij rats the interaction of cyclophosphamide and/or 5-fluorouracil (5-FU) with methotrexate as manifested at the pharmacokinetic level was studied. Four groups were formed of at least ten animals. The control group, which received single-agent methotrexate, was compared with groups that received methotrexate plus cyclophosphamide, methotrexate plus 5-FU, and methotrexate plus cyclophosphamide plus 5-FU. There appeared to be an increase of 40% in the clearance of methotrexate by the triple combination. Cyclophosphamide especially diminished the terminal part of the concentration-time curve of methotrexate.

Determination of the underivatized antineoplastic drugs cyclophosphamide and 5-fluorouracil and some of their metabolites by capillary gas chromatography combined with electron-capture and nitrogen-phosphorus selective detection.

A rapid and sensitive method for the determination of cyclophosphamide (CP) and 5-fluorouracil (5-FU) and some of their metabolites in one analysis has been developed. Surface-coated open-tubular OV-275 columns were combined with electron-capture detection and nitrogen-phosphorus selective detection. The influence of the column diameter on the separation is shown. Extraction with 2-propanol-diethyl ether (22:77) allows the isolation of CP, 5-FU and their analogues in one extraction step. The assay was applied to some pharmacokinetic experiments with chemotherapeutically treated patients and with a WAG/Rij rat.

Induction of chromosomal alterations as an assay for cytostatic drug activity in plasma.

Information about the extent and persistence of cytostatic activity in blood plasma after administration of a cytostatic drug into the body is needed for a better evaluation of the inter-individual variations in drug metabolism and disposition. As an assay for cytotoxic activity, a test system was chosen in which Chinese hamster ovary cells (CHO) were incubated with plasma containing active metabolites of cyclophosphamide (from human patients or rats), after which the frequencies of induced sister-chromatid exchanges per cell were determined. The treatment with plasma increased the frequencies of SCEs very effectively at concentrations of metabolites that were negative in the Salmonella typhimurium back-mutation test with strain TA100. The results obtained indicate that the SCE test system offers the possibility to follow the cytotoxic activity of plasma at various time intervals after administration of cyclophosphamide.

Tick-borne diseases of domestic animals in northern Nigeria. II. Research summary, 1966 to 1976.

During the first 10 years of its existence, the Department of Parasitology and Entomology of the Faculty of Veterinary Medicine, Ahmadu Bello University in Zaria, Nigeria, confirmed or described for the first time the occurrence of 25 different tick-borne parasites of domestic animals in the northern part of the country. Most of these organisms occur as inapparent infections but may serve as complicating factors in any adverse host condition. The transmission of Babesia bigemina by Boophilus decoloratus, Cowdria ruminantium by Amblyomma variegatum, and Borrelia anserina and Aegyptianella pullorum by Argas persicus was confirmed under Nigerian conditions.

Tick-borne diseases of domestic animals in northern Nigeria. I. Historical review, 1923-1969.

Information on the occurrence of tick-borne diseases of domestic animals in northern Nigeria for the period 1923 to 1966 is summarised. Due to lack of obvious outbreaks and diverse interest of the veterinary staff, the published information is very scanty. Significant for the period under review was the complicating factor of Babesia bigemina infections in cattle undergoing rinderpest immunisation procedures or suffering from trypanosomiasis. Tick-borne diseases were also considered important in imported breeds of livestock and in those kept under intensive systems of management.