Evolution of foot-and-mouth disease virus

Foot-and-mouth disease virus evolution is strongly influenced by high mutation rates and a quasispecies dynamics. Mutant swarms are subjected to positive selection, negative selection and random drift of genomes. Adaptation is the result of selective amplification of subpopulations of genomes. The extent of adaptation to a given environment is quantified by a relative fitness value. Fitness values depend on the virus and its physical and biological environment. Generally, infections involving large population passages result in fitness gain and population bottlenecks lead to fitness loss. Very different types of mutations tend to accumulate in the foot-and-mouth disease virus (FMDV) genome depending on the virus population size during replication. Quasispecies dynamics predict higher probability of success of antiviral strategies based on multivalent vaccines and combination therapy, and this has been supported by clinical and veterinary practice. Quasispecies suggest also new antiviral strategies based on virus entry into error catastrophe, and such procedures are under investigation. Studies with FMDV have contributed to the understanding of quasispecies dynamics and some of its biological implications.     

Determination of hepatitis C virus genotype by Pyrosequencing

A simple sequencing-based assay is described for genotyping of hepatitis C virus (HCV). RT-PCR was employed to amplify a 237-nucleotide-long fragment from the 5' untranslated region (UTR) of the genome using one biotinylated and one normal primer. Subsequent to capture of the PCR products on streptavidin-coated beads, single-stranded DNA separation, and hybridization of sequencing primer, Pyrosequencing was performed. The genotype of 98 samples out of which 77 samples were from American veterans and 21 samples were from Iran was determined. The samples from the American veterans contained six different subtypes, while five subtypes were found in Iranian samples. For rapid population-specific HCV subtyping, a multiplex assay was developed. This study demonstrates the suitability of this technology for low-cost, high throughput and accurate microbial genotyping.     

Potassium currents in primary cultured astrocytes from the rat corpus callosum

The corpus callosum (CC) is the main white matter tract in the brain and is involved in interhemispheric communication. Using the whole-cell voltage-clamp technique, a study was made of K⁺-currents in primary cultured astrocytes from the CC of newborn rats. These cells were positive to glial fibrillary acidic protein after culturing in Dulbecco’s Modified Eagle Medium (> 95% of cells) or in serum-free neurobasal medium with G5 supplement (> 99% of cells). Astrocytes cultured in either medium displayed similar voltage-activated ion currents. In 81% of astrocytes, the current had a transient component and a sustained component, which were blocked by 4-aminopyridine and tetraethylammonium, respectively; and both had a reversal potential of −66 mV, indicating that they were carried by K⁺ ions. Based on the Ba²⁺-sensitivity and activation kinetics of the K⁺-current, two groups of astrocytes were discerned. One group (55% of cells) displayed a strong Ba²⁺ blockade of the K⁺-current whose activation kinetics, time course of decay, and the current-voltage relationship were modified by Ba²⁺. This current was greatly blocked (52%) by Ba²⁺ in a voltage-dependent way. Another group (45% of cells) presented weak Ba²⁺-blockade, which was only blocked 24% by Ba²⁺. The activation kinetics and time course of decay of this current component were unaffected by Ba²⁺. These results may help to understand better the roles of voltage-activated K⁺-currents in astrocytes from the rat CC in particular and glial cells in general.     

Oral hairy leukoplakia. Histopathologic and cytopathologic features of a subclinical phase

Accurate diagnosis of oral hairy leukoplakia (OHL) is important because it may be an early indicator of undiagnosed HIV infection; moreover, it may be a prognostic indicator. Our purpose was to investigate the histopathologic features of subclinical OHL and to evaluate and support the rationale of detecting subclinical OHL with cytopathology. The Epstein-Barr virus (EBV) was detected by immunohistochemistry and in situ hybridization in 4 cases of macroscopically normal lateral borders of tongue mucosa from 8 AIDS necropsies and in none of 8 controls. The histopathologic features were specific when based on nuclear changes: Cowdry type A inclusion, ground glass, and nuclear beading. Smears were obtained from 50 patients with AIDS, without OHL, from the scraping of lateral borders of the tongue. Numerous clusters of the cells were associated with Candida organisms (30% of cases). Nuclear changes were observed in 12 patients (24%) on both sides of the tongue. We describe the histopathologic features of subclinical OHL, and our observations suggest that cytopathology can detect OHL in the subclinical phase.     

A novel fungal prenyl diphosphate synthase in the dimorphic zygomycete Mucor circinelloides

Two Mucor circinelloides structural genes involved in isoprenoid biosynthesis were isolated and characterised. The isoA gene encodes a typical eukaryotic farnesyl diphosphate synthase (EC 2.5.1.10), whereas the isoB gene deduced amino acid sequence shows similarity to fungal medium-chain prenyl diphosphate synthases. By functional complementation in Escherichia coli, the isoB gene product was shown to be a solanesyl diphosphate synthase (EC 2.5.1.11), which is the first fungal enzyme reported having this specificity. In addition, a M. circinelloides one-marker-per-chromosome map was completed by contour-clamped homogeneous electric field localisation of isoA, isoB and three other isoprenoid biosynthesis genes to individual chromosomes.Abbreviations FPP farnesyl diphosphate (or pyrophosphate) - GGPP geranylgeranyl diphosphate - PrenylPP prenyl diphosphate - DPP decaprenyl diphosphate - HPP hexaprenyl diphosphate - SPP solanesyl diphosphate     

Comparison of FDTD-calculated specific absorption rate in adults and children when using a mobile phone at 900 and 1800 MHz

In this paper, the specific absorption rate (SAR) in scaled human head models is analysed to study possible differences between SAR in the heads of adults and children and for assessment of compliance with the international safety guidelines, while using a mobile phone. The finite-difference time-domain method (FDTD) has been used for calculating SAR values for models of both children and adults, at 900 and 1800 MHz. Maximum 1 g averaged SAR (SAR1 g) and maximum 10 g averaged SAR (SAR10 g) have been calculated in adults and scaled head models for comparison and assessment of compliance with ANSI/IEEE and European guidelines. Results show that peak SAR1 g and peak SAR10 g all trend downwards with decreasing head size but as head size decreases, the percentage of energy absorbed in the brain increases. So, higher SAR in children's brains can be expected depending on whether the thickness of their skulls and surrounding tissues actually depends on age. The SAR in eyes of different sizes, as a critical organ, has also been studied and very similar distributions for the full size and the scaled models have been obtained. Standard limits can only be exceeded in the unpractical situation where the antenna is located at a very short distance in front of the eye.     

Genetic load caused by variation in the amount of rDNA in a wasp

Extensive variation in the size of the short (heterochromatic) arm of chromosome 14 was found in the wasp Trypoxylon (Trypargilum) albitarse. Ten different variants were differentiated by size and C-banding pattern. Fluorescent in-situ hybridization (FISH) revealed that ribosomal DNA in this species is clustered in the darkly C-banded parts of the heterochromatic short arm of chromosome 14. On this basis, we got an indirect estimate of the amount of rDNA from the area of these dark C-bands. The significant absence in males of the three chromosome variants with lower amounts of rDNA indicates that these three variants are lethal in this sex, and suggests the existence of a threshold marking the minimum amount of rDNA which is tolerable in haploidy. This implies about 4% genetic load in the population caused by variation in rDNA amount. This revised version was published online in July 2006 with corrections to the Cover Date.     

Longitudinal study of daily variation of rats' behavior in the elevated plus-maze

We conducted a longitudinal study about daily variation of Wistar male rats' behavior in the elevated plus-maze (EPM) evaluated in the 1st, 2nd, 3rd, 6th, 12th, and 18th months of life. Animals were submitted to the plus-maze in 12 sessions at 2-h intervals (n=72, 6 per time point). Spontaneous rest-activity rhythm of four animals was assessed by observation of 24-h videotape records. Time series were analyzed by Cosinor method. Behavioral rates on the six occasions and in light and dark phases were compared by means of two-way ANOVA with repeated measures. Exploratory behavior in EPM was smaller in the light phase and in older animals. Higher values of open and closed arms exploration were observed in the first and third months of the dark phase, and in the first month of the light phase. Adjustment to the 24-h period was significant at all stages for rest-activity data, number of entries in closed arms, and time on center, and for three to five stages for open-arm exploration. In general, 24 h variability was more pronounced in younger animals compared with older ones. The present study showed that: (1). a significant amount of total variability of the behavioral indexes analyzed could be attributed to 24 h variation, (2). light/dark phases differences in EPM exploration were present at all developmental stages, (3). older Wistar rats explored less the EPM and were less active in their home cage compared with younger ones, and (4). behavioral indexes (EPM) decrease was phase related and partially related to a reorganization of rest-activity rhythm.     

Interaction of Arc with CaM kinase II and stimulation of neurite extension by Arc in neuroblastoma cells expressing CaM kinase II

We investigated the relationship between Arc (activity-regulated cytoskeleton-associated protein) and Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). Arc and CaM kinase II were concentrated in the postsynaptic density. These proteins were accumulated after electroconvulsive treatment. Arc increased about 2.5-fold within 30 min and was maintained at this level for 8h after the stimulation. CaM kinase II also increased within 30 min and remained at this level for at least 24h. The interaction of Arc with CaM kinase II was demonstrated using GST-Arc fusion protein, and confirmed in neuroblastoma cells by immunoprecipitation. We examined the function of Arc by introducing Arc cDNA into neuroblastoma cells expressing CaM kinase II. The cells expressing both Arc and CaM kinase II had longer neurites than those expressing CaM kinase II alone. Arc itself did not promote neurite outgrowth. The growth of neurites by Arc was completely blocked by treatment with KN62, an inhibitor of CaM kinases. These results indicated that Arc potentiated the action of CaM kinase II for neurite extension.