Poly(A) tailing of ancient DNA: a method for reproducible microsatellite genotyping

Microsatellites could be of great potential use in the analysis of ancient remains, but so far such analyses have failed to be reproducible mainly because of the high degree of ancient DNA (aDNA) degradation. During PCR, annealing of the primers to the complementary sequences of microsatellites occurs together with cross-annealing of partially degraded repeated sequences. This could create chimeric alleles that do not correspond to the authentic ones. Here we report a simple method for processing aDNA fragments prior to PCR that greatly reduces the production of chimeric alleles. This approach eliminates aDNA molecules broken within the repeats as targets for Taq polymerase by adding poly(A) tails at the 3(') ends of the DNA fragments, which disrupts the homology in the region and thus prevents annealing out of register. We have analyzed one dinucleotide- (D6S337) and two trinucleotide-containing loci (IT15 and SCA1) using poly(A)-tailed and the same untreated aDNA as template. aDNAs were isolated from 28 human remains, 600 and 7000 years of age. In repeated experiments with untreated aDNAs we obtained three to five times more alleles compared to poly(A)-tailed aDNAs. According to our results, modification of aDNA by poly(A) tailing is an efficient pretreatment for accurate genotyping.     

Class A scavenger receptors mediate cell adhesion via activation of G(i/o) and formation of focal adhesion complexes

Class A macrophage scavenger receptors (SR-A) are multifunctional receptors with roles in modified lipoprotein uptake, innate immunity, and macrophage adhesion. Our previous studies conducted in mouse peritoneal macrophages demonstrated that pertussis toxin (PTX) mediated inhibition of G(i/o) attenuated SR-A-dependent uptake of modified lipoprotein. The finding that SR-A-mediated lipoprotein internalization was PTX-sensitive led us to hypothesize that SR-A-mediated cell adhesion might be similarly regulated by G(i/o)-dependent signaling pathways. To test this hypothesis, SR-A was expressed in HEK cells under inducible control. Relative to HEK cells that lack SR-A, SR-A expressing cells displayed enhanced adhesion to tissue culture dishes. SR-A-mediated adhesion was significantly reduced following PTX treatment and was insensitive to chelating divalent cations with EDTA. SR-A-expressing cells exhibited a distinct cell morphology characterized by fine filopodia-like projections. Both polymerized actin and vinculin were codistributed with SR-A in the filopodia-like projections indicating the formation of focal adhesion complexes. Overall, our results indicate that the ability of SR-A to enhance cell adhesion involves G(i/o) activation and formation of focal adhesion complexes.     

‘Therapeutic window’ for multiple drug treatment of experimental cerebral ischemia in gerbils

The effects of the following drugs: nimodipine (1 mg/kg b. w., i. p.), 2-amino-5-phosphonovaleric acid (4mg/kg b.w., i.p.) and propentofylline (25mg/kg b.w., i.p.), administered (alone or in combination) at the end of 15 min bilateral ischemia in gerbils were evaluated on mitochondrial superoxide dismutase (SOD), glutathione reductase (GR), glucose-6 phosphate dehydrogenase (G6PD), monoamine oxidase (MAO) activities, and thiobarbituric acid reactive material (TBARM), and brain water content at 1 hour of reperfusion. The combined treatment virtually abolished early postischemic brain edema (4.1% v.s. 0.6%) and efficiently counteracted ischemia-induced changes [decreased SOD (79% v.s. 98%), GR (52% v.s. 105%) and MAO (25% v.s. 79%), and increased TBARM (198% v.s. 108%)]. The same combination of drugs administered 15 min before ischemia had a similar effect (e.g., reduced brain swelling and lipid peroxidation) as when given at the end of ischemia, whereas a limited or absent impact was seen when the drugs were given 15 min or 1 hour after ischemia, respectively. The data suggest that (post)ischemic brain swelling and mitochondrial dysfunction can be reduced by drugs which synchronously prevent processes induced in the early stages of reperfusion.     

Sublethal Effects of Tebufenpyrad on the Eggs and Immatures of Two-Spotted Spider Mite, <Emphasis Type="Italic">Tetranychus urticae</Emphasis>

Sublethal effects of the mitochondrial electron transport inhibitor (METI) tebufenpyrad on Tetranychus urticae Koch females surviving treatment as eggs or immatures (≥90% mortality) were investigated in life-table assay. The developmental time of females that had survived treatment as eggs (2 mg/l) or larvae (2.5 mg/l) was 1 day longer, while that of protonymphs (2.5 mg/l) or deutonymphs (4 mg/l) was 2 days longer, in addition to reduced longevity and fertility, compared to control. The treatment significantly reduced the intrinsic rate of increase in female survivors: corresponding values were 0.258, 0.278, 0.207 and 0.209, respectively (0.307 in untreated females). The offspring age distribution was significantly affected by the treatment. Sublethal effect of tebufenpyrad and its impact on T. urticae management are discussed.     

On invariants of a 2-D proteome map derived from neighborhood graphs

We consider the problem of the construction of invariants for characterization of 2-D maps, such as 2-D proteome maps, 2-D NMR spectral maps, etc., that in addition to facilitating cataloguing such maps, can be used for comparison of maps and numerical evaluation of their degree of similarity. A novel approach, based on the concept that the nearest neighborhood of points (spots) on a map are sufficiently flexible to allow one not only to vary the number of points used for characterization of the map but also the density of information on their relative positions, is put forward. The method is illustrated with the Coomassie brilliant blue stained 2-D gel electrophoresis patterns of the proteomes from liver cells of healthy male Fisher F344 rats and the rats treated with four peroxisome proliferators.     

The J-protein family: modulating protein assembly, disassembly and translocation

DnaJ is a molecular chaperone and the prototypical member of the J-protein family. J proteins are defined by the presence of a J domain that can regulate the activity of 70-kDa heat-shock proteins. Sequence analysis on the genome of Saccharomyces cerevisiae has revealed 22 proteins that establish four distinguishing structural features of the J domain: predicted helicity in segments I-IV, precisely placed interhelical contact residues, a lysine-rich surface on helix II and placement of the diagnostic sequence HPD between the predicted helices II and III. We suggest that this definition of the J-protein family could be used for other genome-wide studies. In addition, three J-like proteins were identified in yeast that contain regions closely resembling a J domain, but in which the HPD motif is non-conservatively replaced. We suggest that J-like proteins might function to regulate the activity of bona fide J proteins during protein translocation, assembly and disassembly.     

Time-dependent increase of succinate dehydrogenase activity in low-frequency stimulated rabbit muscle: a comparison between microphotometric and biochemical methods

 We present an improved method for microphotometric measurement of enzyme activity in muscle fibres by determining maximum reaction rates using computer-assisted image analysis. The method was used to determine absolute and relative activities of succinate dehydrogenase (SDH) in 4801 whole-fibre cross-sections of rabbit tibialis anterior muscles stimulated at low frequency (10 Hz) for different time periods of up to 50 days. Measurements were performed on cross-sections of composite blocks from stimulated and contralateral control muscles. The validity of the method was checked by determining SDH activity in homogenates of the same muscles using a standard photometric assay. Both methods yielded similar results for the time-dependent increases of SDH activity in response to chronic low-frequency stimulation. Significant increases in catalytic activity were detected by the two methods only in muscles stimulated for longer than 8 days. According to homogenate measurements, overall SDH activity was 7.4-fold elevated in 50-day-stimulated total muscles. Depending on whether or not measurements were corrected for the so-called nothing-dehydrogenase activity, the average increase in microphotometrically determined SDH activity amounted to approximately 8-fold or 10-fold, respectively. Microphotometry revealed pronounced scattering of SDH activities within the fibre populations of both normal and stimulated muscles. The heterogeneity of the fibre population with regard to SDH activity increased in long-term stimulated muscles ranging between 5-fold and 15-fold elevations. Accepted: 2 September 1996     

Essential Oil from Blackcurrant Buds as Chemotaxonomy Marker and Antimicrobial Agent

Dormant buds are recognized as valuable side product of the blackcurrant cultivation. Four blackcurrant varieties cultivated in Serbia, i.e., Ben Sarek, Ometa, Ben Lomond, and Ben Nevis, were evaluated for the content, chemical composition, and antimicrobial activity of their bud essential oils. The oil yields of buds harvested during two different growth periods ranged from 1.2–2.0%, and the variety Ometa had the highest yield among the tested varieties. GC‐FID and GC/MS analysis of the oils allowed the identification of eight main components, i.e., α‐pinene (1.6–5.4%), sabinene (1.9–38.4%), δ‐car‐3‐ene (13.0–50.7%), β‐phellandrene (2.9–18.0%), terpinolene (6.6–11.9%), terpinen‐4‐ol (0.9–6.6%), β‐caryophyllene (3.8–10.4%), and α‐humulene (0.2–4.1%). In addition, the similarity degree of the essential‐oil compositions of buds harvested from the upper and lower parts of the shrubs was investigated by hierarchical clustering. All essential oils originating from the same genotype were grouped in the same cluster, indicating the reliability of essential oils as chemotaxonomic markers. For more detailed chemotaxonomic investigations, the three compounds with the greatest variance were chosen, i.e., sabinene, δ‐car‐3‐ene, and β‐phellandrene, which proved to be efficient for the variety distinction. Factor analysis showed that the essential‐oil composition as chemotaxonomic marker in blackcurrants was more reliable for variety Ben Sarek than for variety Ben Nevis. Moreover, it was demonstrated that the essential oils had very strong inhibitory activity against all tested microorganisms. Fungi were more sensitive than bacteria; indeed their growth was completely inhibited at much lower concentrations. In comparison to commercial antibiotics, significantly lower concentrations of the oils were necessary for the complete inhibition of fungal growth.     

Adaptive phenotypic plasticity of gypsy moth digestive enzymes

The adaptiveness of plasticity of digestive enzyme responses to allelochemical stress was tested on 32 full-sib families of gypsy moth larvae from an oak forest population (the Quercus population) and 26 families from a locust-tree forest (the Robinia population), reared either on control diet, or on tannin-supplemented diet. Using the duration of larval development as an indirect measure of fitness, phenotypic selection analyses revealed that lower specific activities of total proteases and trypsin, and higher specific activity of leucine aminopeptidase were adaptive for both populations in the control environment. Plasticity was only shown to be costly for total proteases and trypsin activity in Quercus larvae. In a stressful environment, the most apparent adaptive response was a significant increase in lipase activity. There was no plasticity cost for lipase activity. The two populations differed in the direction of selection acting on α-glucosidase activity, which favoured decreased activity in Quercus larvae and increased activity in Robinia larvae in the control environment. α-glucosidase activity in Quercus larvae is characterized by cost of homeostasis, while cost of plasticity was shown for Robinia larvae. The results obtained on the plasticity of digestive enzyme activity indicate how this generalist species copes with variation in plant allelochemicals.     

Variation in the Fatty‐Acid Content in Seeds of Various Black,Red, and White Currant Varieties

Currant seeds, a by‐product of juice production, are recognized as a valuable source of oil rich in polyunsaturated fatty acids. We have evaluated 28 currant varieties for their oil content and fatty‐acid composition. The oil content in the seeds ranged from 18.2–27.7%, and no statistical difference between varieties of different fruit color were recorded. Furthermore, the estimated oil yields in the field production ranged from 26.4–212.4 kg/ha. The GC and GC/MS chemical profiles of the seed oils extracted from all examined varieties were common for currants. Linoleic acid (LA) was the major component, with contents ranging from 32.7–46.9% of total fatty acids, followed by α‐linolenic acid (ALA; 2.9–32.0 %), oleic acid (OA; 9.8–19.9%), γ‐linolenic acid (GLA; 3.3–18.5%), palmitic acid (PA; 4.4–8.1%), stearidonic acid (SDA; 2.2–4.7%), and stearic acid (SA; 1.2–2.4%). Quantitative differences in the fatty‐acid profiles between varieties of different fruit color were observed. Blackcurrant varieties showed significantly higher contents of LA, GLA, and PA than red and white currant varieties, whereas significantly higher amounts of ALA and OL were detected in the red and white varieties. Cluster analysis based on the chemical oil profiles joined the blackcurrants in one group, while most of the red and white cultivars joined in a second group at the same linkage distance.