Recombination and deletion of sequences in shuttle vector plasmids in mammalian cells.

Shuttle vector plasmids were constructed with directly repeated sequences flanking a marker gene. African green monkey kidney (AGMK) cells were infected with the constructions, and after a period of replication, the progeny plasmids were recovered and introduced into bacteria. Those colonies with plasmids that had lost the marker gene were identified, and the individual plasmids were purified and characterized by restriction enzyme digestion. Recombination between the repeated elements generated a plasmid with a precise deletion and a characteristic restriction pattern, which distinguished the recombined molecules from those with other defects in the marker gene. Recombination among the following different sequences was measured in this assay: (i) the simian virus 40 origin and enhancer region, (ii) the AGMK Alu sequence, and (iii) a sequence from plasmid pBR322. Similar frequencies of recombination among these sequences were found. Recombination occurred more frequently in Cos1 cells than in CV1 cells. In these experiments, the plasmid population with defective marker genes consisted of the recombined molecules and of the spontaneous deletion-insertion mutants described earlier. The frequency of the latter class was unaffected by the presence of the option for recombination represented by the direct repeats. Both recombination and deletion-insertion mutagenesis were stimulated by double-strand cleavage between the repeated sequences and adjacent to the marker, and the frequency of the deletion-insertion mutants in this experiment was again independent of the presence of the direct repeats. We concluded that although recombination and deletion-insertion mutagenesis were both stimulated by double-strand cleavage, the molecules which underwent the two types of change were drawn from separate pools.     

Association Between the 4 bp Proinsulin Gene Insertion Polymorphism (IVS‐69) and Body Composition in Black South African Women

The objective of the study was to examine the association between a functional 4 bp proinsulin gene insertion polymorphism (IVS‐69), fasting insulin concentrations, and body composition in black South African women. Body composition, body fat distribution, fasting glucose and insulin concentrations, and IVS‐69 genotype were measured in 115 normal‐weight (BMI <25 kg/m²) and 138 obese (BMI ≥30 kg/m²) premenopausal women. The frequency of the insertion allele was significantly higher in the class 2 obese (BMI ≥35kg/m²) compared with the normal‐weight group (P = 0.029). Obese subjects with the insertion allele had greater fat mass (42.3 ± 0.9 vs. 38.9 ± 0.9 kg, P = 0.034) and fat‐free soft tissue mass (47.4 ± 0.6 vs. 45.1 ± 0.6 kg, P = 0.014), and more abdominal subcutaneous adipose tissue (SAT, 595 ± 17 vs. 531 ± 17 cm², P = 0.025) but not visceral fat (P = 0.739), than obese homozygotes for the wild‐type allele. Only SAT was greater in normal‐weight subjects with the insertion allele (P = 0.048). There were no differences in fasting insulin or glucose levels between subjects with the insertion allele or homozygotes for the wild‐type allele in the normal‐weight or obese groups. In conclusion, the 4 bp proinsulin gene insertion allele is associated with extreme obesity, reflected by greater fat‐free soft tissue mass and fat mass, particularly SAT, in obese black South African women.     

Relative accuracy of spatial predictive models for lynx Lynx canadensis derived using logistic regression-AIC,multiple criteria evaluation and Bayesian approaches

We compared probability surfaces derived using one set of environmental variables in three Geographic Information Systems (GIS) -based approaches: logistic regression and Akaike's Information Criterion (AIC),Multiple Criteria Evaluation (MCE),and Bayesian Analysis (specifically Dempster-Shafer theory). We used lynx Lynx canadensis as our focal species,and developed our environment relationship model using track data collected in Banff National Park,Alberta,Canada,during winters from 1997 to 2000. The accuracy of the three spatial models were compared using a contingency table method. We determined the percentage of cases in which both presence and absence points were correctly classified (overall accuracy),the failure to predict a species where it occurred (omission error) and the prediction of presence where there was absence (commission error). Our overall accuracy showed the logistic regression approach was the most accurate (74.51% ). The multiple criteria evaluation was intermediate (39.22%),while the Dempster-Shafer (D-S) theory model was the poorest (29.90%). However,omission and commission error tell us a different story: logistic regression had the lowest commission error,while D-S theory produced the lowest omission error. Our results provide evidence that habitat modellers should evaluate all three error measures when ascribing confidence in their model. We suggest that for our study area at least,the logistic regression model is optimal. However,where sample size is small or the species is very rare,it may also be useful to explore and/or use a more ecologically cautious modelling approach (e.g. Dempster-Shafer) that would over-predict,protect more sites,and thereby minimize the risk of missing critical habitat in conservation plans.     

RalA mediates v-Src, v-Ras, and v-Raf regulation of CD44 and fibronectin expression in NIH3T3 fibroblasts

Oncogenic transformation of fibroblasts by v-Src and v-Ras is often associated with downregulation of fibronectin (FN) and increased expression of CD44, a receptor for hyaluronan. Both v-Src and v-Ras as well as v-Raf activate phospholipase D through the small GTPase, RalA, an important mediator of transformation and tumorigenesis in vivo. We have therefore investigated whether RalA is involved in the downregulation of FN and overproduction of CD44 upon oncogenic transformation. We report here that compared to untransfected cells NIH3T3 cells transformed by v-Src, v-Ras, or v-Raf have reduced levels of FN and increased levels of CD44. Moreover, the ability to form extracellular FN fibrils was significantly reduced in the oncogene-transformed cells compared to parental controls. Coexpression of the dominant negative S28N-RalA mutant restored the levels of CD44 and FN and the capacity of v-Src-, v-Ras-, and v-Raf-expressing cells to form extracellular FN fibrils, to those observed in NIH3T3 cells. The data presented here show a novel regulatory role for RalA, which is required for tumor formation in transformed NIH3T3 cells, in mediating the signal transduction pathway activated by v-Src, v-Ras, and v-Raf, that leads to FN downregulation and CD44 overexpression.     

Effects of disease modifying agents and dietary intervention on insulin resistance and dyslipidemia in inflammatory arthritis: a pilot study

Patients with rheumatoid arthritis (RA) experience excess cardiovascular disease (CVD). We investigated the effects of disease-modifying antirheumatic drugs (DMARD) and dietary intervention on CVD risk in inflammatory arthritis. Twenty-two patients (17 women; 15 with RA and seven with spondyloarthropathy) who were insulin resistant (n = 20), as determined by the Homeostasis Model Assessment, and/or were dyslipidemic (n = 11) were identified. During the third month after initiation of DMARD therapy, body weight, C-reactive protein (CRP), insulin resistance, and lipids were re-evaluated. Results are expressed as median (interquartile range). DMARD therapy together with dietary intervention was associated with weight loss of 4 kg (0–6.5 kg), a decrease in CRP of 14% (6–36%; P < 0.006), and a reduction in insulin resistance of 36% (26–61%; P < 0.006). Diet compliers (n = 15) experienced decreases of 10% (0–20%) and 3% (0–9%) in total and low-density lipoprotein cholesterol, respectively, as compared with increases of 9% (6–20%; P < 0.05) and 3% (0–9%; P < 0.05) in diet noncompliers. Patients on methotrexate (n = 14) experienced a reduction in CRP of 27 mg/l (6–83 mg/l), as compared with a decrease of 10 mg/l (3.4–13 mg/l; P = 0.04) in patients not on methotrexate. Improved cardiovascular risk with DMARD therapy includes a reduction in insulin resistance. Methotrexate use in RA may improve CVD risk through a marked suppression of the acute phase response. Dietary intervention prevented the increase in total and low-density lipoprotein cholesterol upon acute phase response suppression.     

A new variant of the neodermatan-type spermiogenesis in a parasitic 'turbellarian', Notentera ivanovi (Platyhelminthes) and the origin of the Neodermata

Spermiogenesis is characterized by fully incorporated (in the testes of mature worms) or partially free (submature worms) axonemes in spermatids. Formation of free flagella correlates with tight arrangement of cells in the testes and small size of the zone of differentiation and vice versa . In both cases the axonemes elongate within the growing shaft, so that the organization of the resulting spermatids is different only with regard to the distal end. In late spermatids, the nucleus occupies the proximal half, the two mitochondria and the axonemes directed distally lie in the distal half. After detachment of the spermatid, a migration of the nucleus takes place. In the resulting mature sperm, the proximal (anterior) half is occupied by the mitochondria and axonemes the basal bodies of which lie at the anterior end of the spermatozoon; the nucleus occupies the distal (posterior) half. Because of the distal orientation of the axonemes and a peculiar mode of the migration of the nucleus, the spermiogenesis of Notentera should be classified as a new variant of the type characteristic of the Neodermata (parasitic Platyhelminthes). Based on the analysis of the available morphological and other relevant data it is argued (i) that a high-ranked taxon, the Fecampiida, should be established within the Neoophora to include Notentera and the closely related Fecampiidae and (ii) that all the Platyhelminthes with neodermatan-type spermiogenesis form a monophyletic taxon, the Revertospermata, which includes the sister groups Fecampiida and Mediofusata.     

Activation of morphogenesis and proliferation by low specificity chemical effectors

Activation of highly specific biochemical processes by simple chemical agents is demonstrated for morphogenesis (anlage and development of female gametophyte in cereal) and mitosis (in cell cultures and animal and plant tissues). The effects of these agents are tissue-specific. Structure--activity relationship is analyzed in this group of compounds. Thus, the phenomenon reveals the exact pathways of the influence of allelopathic and anthropogenic chemical agents on evolution of plant biocenoses.     

On the phylogeny of families and genera within the Temnocephalida

Temnocephalida includes species demonstrating many intermediate steps presenting the transition from commensalism to parasitism. Dramatic morphological changes also occurred within this group but the number of supraspecific taxa is small, making the Temnocephalida an excellent model for evolutionary studies. Having summarised original and relevant published morphological data, we suggest a cladogram (phylogenetic tree) which nearly fully resolves the order of branching of families and main genera within the Temnocephalida. We also introduce a new family, the Diceratocephalidae nov. fam. This revised version was published online in July 2006 with corrections to the Cover Date.     

Fusarium javanicum Koorders in Israel

Fusarium javanicum Kds. has been isolated from wilted vegetable marrows and melons and from the soil of a citrus grove in Israel. The morphology of the species is described, and measurements and drawings of macroconidia are presented.In vitro tests showed thatF. javanicum failed to develop at either 12 or 40° C, and developed better at 24° C than 18 or 30° C. Mycelial growth on potato dextrose agar was always better at pH 7 than at pH 4.2. Darkness favored such growth to a marked extent only at 18° C and pH 4.2. In glasshouse inoculation tests one isolate ofF. javanicum from vegetable marrow proved clearly pathogenic to cucumber, melon, watermelon and marrow. Measurements of macroconidia have been made, for comparative purposes, on isolates ofF. javanicum var.ensiforme and ofF. solani f.cucurbitae furnished from abroad, and on isolates ofF. solani from cucurbits in Israel. Comparing the data for 3-septate macroconidia with those given in literature forF. solani and forF. javanicum and its var.radicicola and var.ensiforme, it is concluded that two groups can be distinguished: One group comprises, with one exception, all isolates ofF. javanicum and its varieties as well asF. solani f.cucurbitae, and is characterized by slender macroconidia. The other group is represented byF. solani with its typically sausage shaped conidia. It is concluded thatF. javanicum should be maintained as a species distinct fromF. solani. It is further suggested that what has been calledF. solani f.cucurbitae may have closer affinity toF. javanicum than toF. solani.

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Zusammenfassung Fusarium javanicum Koorders wurde in Israel von verwelkten Cucurbitaceen(Cucurbita pepo Kussa andCucumis melo) sowie von dem Boden einer Citruspflanzung isoliert. Die Morphologie des Pilzes wird hier beschreiben. Inin vitro Versuchen entwickelte sichF. javanicum auf Kartoffel-Dextrose Agar weder bei 12 noch bei 40° C, und wuchs besser bei 24 als bei 18 oder 30° C. Das Wachstum des Myzeliums war stets besser bei pH 7 als bei pH 4.2. Dunkelheit förderte dieses Wachstum erheblich nur bei 18° C und pH 4.2. In Impfungsversuchen im Glasshaus wirkte ein Isolat desF. javanicum vonC. pepo stark pathogenisch auf Gurken, Melonen, Wassermelonen undC. pepo. Zwecks taxonomischer Vergleiche wurden Makrokonidien aus dem Ausland erhaltener Isolate vonF. javanicum var.ensiforme undF. solani f.cucurbitae gemessen, sowie solche desF. solani von Cucurbitaceen in Israel. Die Resultate dieser Messungen wurden mit den in der Literatur angegebenen Werten fürF. solani, F. javanicum und dessen var.radicicola und var.ensiforme verglichen. Dieser Vergleich zeigte 2 klar getrennte Gruppen: Die erste enthält, mit einer Ausnahme, alle Isolate desF. javanicum und desF. solani f.cucurbitae, und besitzt typisch schlanke Makrokonidien. Die zweite Gruppe besteht ausF. solani mit seinen typisch dickeren, Würstchenartigen Sporen. Dies führt zu dem Schluß daßF. javanicum weiterhin als eine vonF. solani getrennte Spezies zu betrachten ist.

     

The mycoflora of fresh and stored groundnut kernels in Israel

The mycoflora of groundnut kernels has been studied in Israel over a 5 year period. A total of 132 samples of fresh, and 419 samples of stored kernels was examined.A list is presented of 173 species isolated from these kernels, comprising 109 from fresh, and 153 from stored kernels.In 3 of the 4 years in which a comparative study was made of fresh and stored kernels, the number of fungal colonies from the former exceeded that from the latter by about 25 %. Aspergillus flavus was usually found in small quantities only.A. niger was the species almost invariably found in greatest numbers; on stored kernels it was even more predominant than on fresh ones.Penicillium funiculosum andP. rubrum were also prevalent.

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Zusammenfassung Die Mykoflora der Grundnüsse sind in Israel in einer Periode von über fünf Jahren untersucht worden. Insgesamt sind 132 Proben von frischen, und 419 Proben von eingelagerten Grundnüssen untersucht worden. Die Liste enthält 173 Arten wovon 109 von frischen und 153 von eingelagerten Grundnüssen isoliert worden sind. In drei von den vier Jahren, in welchen vergleichende Untersuchungen von frischen und eingelagerten Grundnüssen durchgeführt worden sind, überstieg die Anzahl der Pilzkolonien der ersten gegenüber der letzteren um 25 %.Aspergillus flavus war nur in geringer Menge gefunden worden.A. niger war fast immer in der größten Anzahl gefunden worden, an eingelagerten Grundnüssen war er noch vorherrschender als an den frischen.Penicillium funiculosum undP. rubrum waren auch vorherrschend.

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This research is supported by grant number FG-161 of the United States Department of Agriculture, to whom the author is indebted.