Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications

Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of individuals representing 54 species of frogs, two species of salamanders, a caecilian, and a lungfish. Eight of these sites were present in all species examined, and two were found in all but one species. Alignment of these conserved restriction sites revealed, among anuran 28S rRNA genes, five regions of major length variation that correspond to four of 12 previously identified divergent domains of this gene. One of the divergent domains (DD8) consists of two regions of length variation separated by a short segment that is conserved at least throughout tetrapods. Most of the insertions, deletions, and restriction-site variations identified in the 28S gene will require sequence-level analysis for a detailed reconstruction of their history. However, an insertion in DD9 that is coextensive with frogs in the suborder Neobatrachia, a BstEII site that is limited to representatives of two leptodactylid subfamilies, and a deletion in DD10 that is found only in three ranoid genera are probably synapomorphies.      

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Evidence for "twisted plane" undulations in golden hamster sperm tails

Motile spermatozoa from the golden hamster have been arrested by rapid freezing and then fixed with glutaraldehyde at low temperature after substitution with ethylene glycol. As far as can be judged, the flagellar waveforms thus stabilized are similar to those seen in living sperm; in contrast, fixation in glutaraldehyde, without prior freezing, induces agonal changes in flagellar conformation. The characteristics waveform after freeze substitution contains three bends. Approx. half of these flagella are entirely planar. The rest are three dimensional, with the third bend displaced in a regular way from the plane containing the proximal two bends. From the geometry of these flagella, it is concluded that the plane of action of a given bending cycle undergoes a clockwise twist (from a forward viewpoint) as the cycle is succeeded by new bending cycles. This "twisted plane" undulation is quite different from helical movement. The twisting seems to occur abruptly, between cycles, as if each bending cycle has a preferred plane of action. The mechanism underlying the twisting is uncertain. However, on the basis of the angular displacements between the preferred planes, and the findings from electron microscopy, the following idea is presented as a working hypothesis: that, if the most proximal plane of bending is topographically determined by peripheral doublet 1, then successive distal planes of action are influenced predominantly by doublets 2, 3, etc., in clockwise sequence. The merits and weaknesses of this hypothesis are discussed.     

Impact of human activities on the reproduction of Hooded Vultures Necrosyrtes monachus in Burkina Faso

During the last decades, the critically endangered Hooded Vulture Necrosyrtes monachus has strongly declined across its African range. Although direct persecution has been suggested as a major cause of this decline, little is known about the impact of humans on reproductive output in West Africa. We studied the impact of human activities on the reproductive output of Hooded Vultures in the Garango area of Burkina Faso. Twenty and 56 nesting attempts were monitored, respectively, during the breeding season in 2013/14 and 2014/15, to determine reproductive success and identify causes of nest failure. Annual breeding success varied between 0.68 and 0.71 chicks fledged per breeding pair per year and productivity was assessed at 0.57 chicks fledged per territorial pair in 2014/15. The main threats imposed by humans were poaching of eggs, chicks and collection of nest materials, leading to 20% (13 out of 64 breeding attempts) of nest failures over the two years. An additional important reason for nest failure was the pruning and (partial) cutting of nest trees. Despite this high level of human interference, we found that Hooded Vulture nest success increased with proximity to human settlements, probably because breeding vultures benefit from protection by people against persecution and disturbance.     

Production, recovery and immunogenicity of the protective antigen from a recombinant strain of Bacillus anthracis

The protective antigen (PA) is one of the three components of the anthrax toxin. It is a secreted nontoxic protein with a molecular weight of 83 kDa and is the major component of the currently licensed human vaccine for anthrax. Due to limitations found in the existing vaccine formulation, it has been proposed that genetically modified PA may be more effective as a vaccine. The expression and the stability of two recombinant PA (rPA) variants, PA-SNKE-ΔFF-E308D and PA-N657A, were studied. These proteins were expressed in the nonsporogenic avirulent strain BH445. Initial results indicated that PA-SNKE-ΔFF-E308D, which lacks two proteolysis-sensitive sites, is more stable than PA-N657A. Process development was conducted to establish an efficient production and purification process for PA-SNKE-ΔFF-E308D. pH, media composition, growth strategy and protease inhibitors composition were analyzed. The production process chosen was based on batch growth of B. anthracis using tryptone and yeast extract as the only source of carbon, pH control at 7.5, and antifoam 289. Optimal harvest time was 14–18 h after inoculation, and EDTA (5 mM) was added upon harvest for proteolysis control. Recovery of the rPA was performed by expanded-bed adsorption (EBA) on a hydrophobic interaction chromatography (HIC) resin, eliminating the need for centrifugation, microfiltration and diafiltration. The EBA step was followed by ion exchange and gel filtration. rPA yields before and after purification were 130 and 90 mg/l, respectively. The purified rPA, without further treatment, treated with small amounts of formalin or adsorbed on alum, induced, high levels of IgG anti-PA with neutralization activities. Journal of Industrial Microbiology & Biotechnology (2002) 28, 232–238 DOI: 10.1038/sj/jim/7000239 Received 28 August 2001/ Accepted in revised form 20 December 2001     

Effects of Alzheimer's amyloid-beta and tau protein on mitochondrial function -- role of glucose metabolism and insulin signalling

Alzheimer's disease (AD) is the most frequent form of dementia among the elderly and is characterized by neuropathological hallmarks of extracellular amyloid-beta (Abeta) plaques and intracellular neurofibrillary tangles composed of abnormally hyperphosphorylated microtubular protein tau in the brains of AD patients. Of note, current data illustrate a complex interplay between the amyloid and tau pathology during the course of the disease. We hypothesize a direct impact of abnormally phosphorylated tau and Abeta on proteins/enzymes involved in metabolism, respiratory chain function and cellular detoxification. Probably at the level of mitochondria, both Alzheimer proteins exhibit synergistic effects finally leading to/accelerating neurodegenerative mechanisms. Moreover, accumulating evidence that mitochondria failure, reduced glucose utilization and deficient energy metabolism occur already very early in the course of the disease suggests a role of impaired insulin signalling in the pathogenesis of AD. Thus, this review addresses also the question if mitochondrial dysfunction may represent a link between diabetes and AD.     

Uber die Ernährung der Arbeitermade vonApis Mellifica L., insbesondere in der Altersperiode

Zusammenfassung der Ergebnisse Verf. sucht in dieser Arbeit experimentell zu klären, wie die Bienen das Mischfutter zusammensetzen, mit welchem sie die Arbeitermaden in der Altersperiode füttern.Nachdem es sich in früheren Versuchen ergeben hatte, daß bei einer Fütterung lediglich mit Pollen+Honig+Aqua dest. keine Verpuppung zustande kommt, ein Futtersaftzusatz demnach unerläßlich ist, verwendete Verf. bei seinen Brutschrankversuchen die drei von ihm, nachgewiesenen Futtersaftarten: 1.) den Arbeiterfuttersaft, 2.) den Königinjungmaden-Futtersaft, 3.) den Königinaltmaden-Futtersaft. Der Erwartung entsprechend, wurde die Metamorphose durch 3.) am stärksten gefördert, dagegen durch 1.) verhindert. Die Verpuppung einiger mit 2.) gefütterten Maden war ein unerwartetes Ergebnis.Verf. weist nach, daß die biologische Bedeutung des Futterwechsels des Überganges zur Mischfutterernährung, vor allem in der Änderung der Futtersaftqualität liegt, weil diese erst die Metamorphose ermöglicht, ferner, daß die Mitverwendung von Pollen und Honig keine determinative Bedeutung hat, vielmehr dem ebenfalls wichtigen Zwecke dient, unter Einsparung von Futtersaft alle Maden auf ein Gewicht zu bringen, bei welchem sie verwandlungsfähig sind.

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Summary The bee-larvae, in their early state, are fed, with pure jelly till they weigh approx. 35 mg. This is, as to its quality, not identical with the jelly for the queen-larvae. Yet with the jelly for queen-young-larvae, which serves to predetermine the female larva for a queen (v.Rhein, 1951), it has in common, that it is only good for the larva in its early state. By this characteristic it differs from the jelly for the queen-old-larvae, by which the predetermined larvae are determined queens; by means of this latter very young worker-larvae can be bred in thermostates. The results are, however, since those are not predetermined, either normal workers or medium forms with more or less enlarged ovaries and spermatic vessels (v.Rhein, 1933). This observation proves, that, for determining the larva for a worker, no pure pollen is necessary.This pollen is found, as it is well known, aside honey, in the mixed food, which is received by the bee-larvae from their hatching-nurses in their later state. That this food also contains jelly was already supposed byRösch (1925). I was able to proof this thesis by way of experiment. I began to feed larvae after their early stage with a mixture of pollen, honey, aqua dest. in relation 32065. Although the larvae grew normally and even reached a weight of 214 mg (norm:approx. 158 mg), they all died before excrementing (v.Rhein, 1933). During the years 1953 and 1954 I was able to solve this problem by adding to the just mentioned mixture in different test-groups 100 and 200 mg jelly for worker-young-larvae, jelly for queen-young-larvae and jelly for queen-old-larvae; the addition of water was decreased, corresponding to the used quantity of jelly, to 670 respectively to 570 mg. The result was as follows:Table (see German text).Thus we can conclude, that the jelly is the most suitable as additional food, for queen-old-larvae while the jelly is not suitable at all for worker-young-larvae.The chrysalis, arised from this experiment, were, judged from the morphologie and anatomic point of view, normal chrysalis of workers, except one chrysalis originated from a particular heavy larva (238 mg), which showed enlarged ovaries (74 ovaries in the average).

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Résumé Les larves des Abeilles reçoivent une alimentation particulière jusqu'à ce qu'elles atteignent un poids de 35 mg environ. La gelée qui sert de base à cette alimentation n'est pas qualitativement identique à la gelée royale; le seul caractère commun à ces deux substances est de ne convenir qu'aux très jeunes larves. Ce caractère les sépare de la gelée servie aux vieilles larves de reines grâce à laquelle les larves prédéterminées achèvent leur détermination: cette gelée en effet, peut permettre d'élever complètement de jeunes larves d'ouvrières dans un thermostat. Comme ces larves n'ont pas été prédéterminées, on obtient soit des ouvrières normales, soit des formes intermédiaires, avec ovaires et vésicules séminales plus ou moins hypertrophiés (von Rhein, 1933). Cette observation prouve que le pollen à l'état brut n'est pas indispensable à la détermination d'une larve ouvrière; pourtant on en trouve dans le miel et dans la nourriture mixte servie aux larves d'Abeilles âgées par les nourrices. Depuis longtemps, on a émis l'hypothèse (Resch, 1925) que cette nourriture mixte renferme aussi de la gelée royale. J'ai réussi à en fournir la preuve expérimentale. A la fin de la première phase de l'existence des larves (phase de jeunesse), j'ai commencé à les alimenter avec du pollen, du miel et de l'eau distillée mélangés dans les proportions suivantes: 3/20/65. Les larves se développèrent normalement et atteignirent un poids de 214 mg (le poids normal est d'environ 158 mg), mais elles mouraient toutes avant la défécation (von Rhein, 1933). En 1953 et 1954, je trouvais la solution du problème en ajoutant dans plusieurs groupes expérimentés, au mélange sus-mentionné, 100 ou 200 mg de gelée de jeunes larves d'ouvrières, de gelée de jeunes larves de reines, de gelée de vieilles larves de reines; en même temps, l'apport d'eau correspondant à la quantité de gelée utilisée était réduit respectivement à 670 et 570 mg. Le résultat était le suivant:Tableau (texte allemand).On peut conclure de ce tableau que la gelèe des vieilles larves de reines est la plus propre à servir de nourriture complémentaire; la gelée des jeunes larves d'ouvrières y est totalement impropre. Les nymphes obtenues à la suite de l'expérience étaient, du point de vue morphologique et anatomique, des nymphes normales d'ouvrières, excepté une particulièrement lourde (238 mg), qui présentait des ovaires hypertrophiés (74 avarioles en moyenne).

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Durchgeführt mit Unterstützung der Deutschen Forschungsgemeinschaft.