Neuronal responses to purinoceptor agonists in the leech central nervous system

Exracellular nucleotides like ATP and its derivatives are possible chemical messengers in vertebrate nervous systems. In invertebrate nervous systems, however, little is known about their role in neurotransmission. We have studied the reponse of identified neurones of the leech Hirudo medicinalis to the purinoceptor agonist ATP, ADP, AMP, and adenosine using conventional intracellular microelectrodes and whole-cell patch-clamp recording. Bath application of the agoinsts depolarized the different neurons, but not neuropil glial cells. The most effective responses (up to 10 mV) were observed with ATP (100 μM) or ADP (100 μM) in the noxious and touch cells. In most neurons the nonhydrolyzable ATP derivative ATP-γ-S (5 μM) induced larger depolarizations that 100 μM ATP, indicating that most of the potency of ATP is lost presumably due to its degradation by ectonucleotidases. In medial noxios cells, ATP (100 μM) induced an inward current of 1.7 ± 1.1 nA at a holding potential of ?60 mV. The ATP-induced current-voltage relationship showed an inward rectification and a reversal potential close to 0 m V. In a Na⁺-free extracellular solution, the ATP-induced inward current decreased and in a Na⁺- and Ca²⁺-free saline only a small residual current persisted. The possible P₂ purinoceptor antagonist suramin did not antagonize the ATP-induced current, but itself evoked an inward current and a conductance increase. We conclude that ATP activates nonselective cation channels in medial noxious cells of the leech with the order of potency of purinoceptor agonists ATP ≥ ADP > AMP. The results suggest that these cells express purinoceptors of the P₂ type. 1994 John Wiley & Sons, Inc.     

Two-trait-locus linkage analysis: a powerful strategy for mapping complex genetic traits.

Recent advances in molecular biology have provided geneticists with ever-increasing numbers of highly polymorphic genetic markers that have made possible linkage mapping of loci responsible for many human diseases. However, nearly all diseases mapped to date follow clear Mendelian, single-locus segregation patterns. In contrast, many common familial diseases such as diabetes, psoriasis, several forms of cancer, and schizophrenia are familial and appear to have a genetic component but do not exhibit simple Mendelian transmission. More complex models are required to explain the genetics of these important diseases. In this paper, we explore two-trait-locus, two-marker-locus linkage analysis in which two trait loci are mapped simultaneously to separate genetic markers. We compare the utility of this approach to standard one-trait-locus, one-marker-locus linkage analysis with and without allowance for heterogeneity. We also compare the utility of the two-trait-locus, two-marker-locus analysis to two-trait-locus, one-marker-locus linkage analysis. For common diseases, pedigrees are often bilineal, with disease genes entering via two or more unrelated pedigree members. Since such pedigrees often are avoided in linkage studies, we also investigate the relative information content of unilineal and bilineal pedigrees. For the dominant-or-recessive and threshold models that we consider, we find that two-trait-locus, two-marker-locus linkage analysis can provide substantially more linkage information, as measured by expected maximum lod score, than standard one-trait-locus, one-marker-locus methods, even allowing for heterogeneity, while, for a dominant-or-dominant generating model, one-locus models that allow for heterogeneity extract essentially as much information as the two-trait-locus methods. For these three models, we also find that bilineal pedigrees provide sufficient linkage information to warrant their inclusion in such studies. We also discuss strategies for assessing the significance of the two linkages assumed in two-trait-locus, two-marker-locus models.     

Molecular and statistical approaches to the detection and correction of errors in genotype databases.

Errors in genotyping data have been shown to have a significant effect on the estimation of recombination fractions in high-resolution genetic maps. Previous estimates of errors in existing databases have been limited to the analysis of relatively few markers and have suggested rates in the range 0.5%-1.5%. The present study capitalizes on the fact that within the Centre d'Etude du Polymorphisme Humain (CEPH) collection of reference families, 21 individuals are members of more than one family, with separate DNA samples provided by CEPH for each appearance of these individuals. By comparing the genotypes of these individuals in each of the families in which they occur, an estimated error rate of 1.4% was calculated for all loci in the version 4.0 CEPH database. Removing those individuals who were clearly identified by CEPH as appearing in more than one family resulted in a 3.0% error rate for the remaining samples, suggesting that some error checking of the identified repeated individuals may occur prior to data submission. An error rate of 3.0% for version 4.0 data was also obtained for four chromosome 5 markers that were retyped through the entire CEPH collection. The effects of these errors on a multipoint map were significant, with a total sex-averaged length of 36.09 cM with the errors, and 19.47 cM with the errors corrected. Several statistical approaches to detect and allow for errors during linkage analysis are presented. One method, which identified families containing possible errors on the basis of the impact on the maximum lod score, showed particular promise, especially when combined with the limited retyping of the identified families. The impact of the demonstrated error rate in an established genotype database on high-resolution mapping is significant, raising the question of the overall value of incorporating such existing data into new genetic maps.     

A strategy for the characterization of minute chromosome rearrangements using multiple color fluorescence in situ hybridization with chromosome-specific DNA libraries and YAC clones

The identification of marker chromosomes in clinical and tumor cytogenetics by chromosome banding analysis can create problems. In this study, we present a strategy to define minute chromosomal rearrangements by multicolor fluorescence in situ hybridization (FISH) with whole chromosome painting probes derived from chromosome-specific DNA libraries and Alu-polymerase chain reaction (PCR) products of various region-specific yeast artificial chromosome (YAC) clones. To demonstrate the usefulness of this strategy for the characterization of chromosome rearrangements unidentifiable by banding techniques, an 8p+ marker chromosome with two extra bands present in the karyotype of a child with multiple anomalies, malformations, and severe mental retardation was investigated. A series of seven-color FISH experiments with sets of fluorochrome-labeled DNA library probes from flow-sorted chromosomes demonstrated that the additional segment on 8p+ was derived from chromosome 6. For a more detailed characterization of the marker chromosome, three-color FISH experiments with library probes specific to chromosomes 6 and 8 were performed in combination with newly established telomeric and subtelomeric YAC clones from 6q25, 6p23, and 8p23. These experiments demonstrated a trisomy 6pter6p22 and a monosomy 8pter8p23 in the patient. The present limitations for a broad application of this strategy and its possible improvements are discussed.Dedicated to Professor Dr. U. Wolf on the occasion of his 60th birthday     

Faster induction of photosynthesis increases biomass and grain yield in glasshouse-grown transgenic Sorghum bicolor overexpressing Rieske FeS

Sorghum is one of the most important crops providing food and feed in many of the world's harsher environments. Sorghum utilizes the C₄ pathway of photosynthesis in which a biochemical carbon-concentrating mechanism results in high CO₂ assimilation rates. Overexpressing the Rieske FeS subunit of the Cytochrome b₆f complex was previously shown to increase the rate of photosynthetic electron transport and stimulate CO₂ assimilation in the model C₄ plant Setaria viridis. To test whether productivity of C₄ crops could be improved by Rieske overexpression, we created transgenic Sorghum bicolor Tx430 plants with increased Rieske content. The transgenic plants showed no marked changes in abundances of other photosynthetic proteins or chlorophyll content. The steady-state rates of electron transport and CO₂ assimilation did not differ between the plants with increased Rieske abundance and control plants, suggesting that Cytochrome b₆f is not the only factor limiting electron transport in sorghum at high light and high CO₂. However, faster responses of non-photochemical quenching as well as an elevated quantum yield of Photosystem II and an increased CO₂ assimilation rate were observed from the plants overexpressing Rieske during the photosynthetic induction, a process of activation of photosynthesis upon the dark–light transition. As a consequence, sorghum with increased Rieske content produced more biomass and grain when grown in glasshouse conditions. Our results indicate that increasing Rieske content has potential to boost productivity of sorghum and other C₄ crops by improving the efficiency of light utilization and conversion to biomass through the faster induction of photosynthesis.     

Long-distance migration: evolution and determinants

Long‐distance migration has evolved in many organisms moving through different media and using various modes of locomotion and transport. Migration continues to evolve or become suppressed as shown by ongoing dynamic and rapid changes of migration patterns. This great evolutionary flexibility may seem surprising for such a complex attribute as migration. Even if migration in most cases has evolved basically as a strategy to maximise fitness in a seasonal environment, its occurrence and extent depend on a multitude of factors. We give a brief overview of different factors (e.g. physical, geographical, historical, ecological) likely to facilitate and/or constrain the evolution of long‐distance migration and discuss how they are likely to affect migration. The basic driving forces for migration are ecological and biogeographic factors like seasonality, spatiotemporal distributions of resources, habitats, predation and competition. The benefit of increased resource availability will be balanced by costs associated with the migratory process in terms of time (incl. losses of prior occupancy advantages), energy and mortality (incl. increased exposure to parasites). Furthermore, migration requires genetic instructions (allowing substantial room for learning in some of the traits) about timing, duration and distance of migration as well as about behavioural and physiological adaptations (fuelling, organ flexibility, locomotion, use of environmental transport etc) and control of orientation and navigation. To what degree these costs and requirements put constraints on migration often depends on body size according to different scaling relationships. From this exposé it is clear that research on migration warrants a multitude of techniques and approaches for a complete as possible understanding of a very complex evolutionary syndrome. In addition, we also present examples of migratory distances in a variety of taxons. In recent years new techniques, especially satellite radio telemetry, provide new information of unprecedented accuracy about journeys of individual animals, allowing re‐evaluation of migration, locomotion and navigation theories.     

Effects of UV-B radiation and nitrogen starvation on enzyme activities in isolated plasma membranes of Euglena gracilis

Circadian rhythms are characteristic of many physiological and biochemical processes in the freshwater flagellate Euglena gracilis. Earlier, we found that the rhythms of photosynthesis, phototaxis and cell shape followed the same pattern in control organisms, but were differently affected by stress such as UV-B irradiation and nitrogen deficiency. Here we extend our studies to use isolated plasma membranes to characterize the rhythms of some plasma membrane-bound enzymes. Also, we wanted to see whether stress-induced changes of these rhythms could be detected at the subcellular level and possibly be coupled to the changes seen in photosynthesis, phototaxis and cell shape. The isolation of plasma membranes using aqueous polymer two-phase partitioning was successful, as judged by the large enrichment of the plasma membrane-marker 5′-nucleotidase, and the difference in the polypeptide pattern compared with the microsomal fraction from which it was prepared. Two other enzymes were analyzed, K⁺, Mg²⁺-ATPase, and adenylyl cyclase. The specific activities of all three enzymes were decreased by UV-B radiation by ca 30–50%, compared with the control cultures. On the other hand, nitrogen deficiency not only reduced the activity of the K⁺.Mg²⁺-ATPase but also increased the activities of the 5′-nucleotidase and adenylyl cyclase. The different treatments also resulted in differences in polypeptide pattern, e.g., a polypeptide around 30 kDa seemed to be specific to plasma membranes of nitrogen-deficient cultures and one at 39 kDa for the UV-B radiated ones. All three enzymes showed diurnal rhythms that were affected by UV-B radiation. The peak in the rhythm of the ATPase was shifted by UV-B radiation, the rhythm of the 5′-nucleotidase nearly eliminated. The first peak of adenylyl cyclase activity was delayed, so that it looked more like a broad peak between 2 and 11 h after the onset of light. The rhythm of ATPase activity could be correlated with that of photosynthesis in both control and UV-B irradiated cultures. Also, the rhythms of adenylyl cyclase activity and cell shape changes showed some similarities.     

Liming induced stimulation of the amino acid metabolism in mycorrhizal roots of Norway spruce (Picea abies [L.] Karst.)

Localization and activity of three enzymes involved in the amino acid metabolism of ectomycorrhizas were investigated within an interdisciplinary experiment performed in a mature Norway spruce stand in Southern Germany (Höglwald). The enzymes NAD-glutamate dehydrogenase and aspartate aminotransferase were present in root cells, whereas aminopeptidase was found in mycorrhizas of Norway spruce such as Piceirhiza nigra and those with the fungi Cenococcum geophilum, Elaphomyces sp., Russula ochroleuca and Tylospora sp. Mycorrhizas growing in the humus layer contained about double the amount of protein found in those taken from the upper mineral soil (0–5 cm).Acid irrigation of the soil had no effect on the activity of any of the investigated enzymes, soluble protein or total N-contents irrespective of whether roots were taken from the organic layer or from the upper mineral soil. Liming, however, stimulated the activity of the three enzymes in mycorrhizas of the organic layer (O_f+O_h) whereas it had no effect on the activity of the investigated enzymes of mycorrhizas in the upper mineral soil. This effect is attributed to increased contents of soluble organic nitrogen compounds in the soil of the limed plots as compared to the unlimed plots.