Primary cultures of rat pancreatic acinar cells in serum-free medium

Summary Rat pancreatic acinar cells were isolated and cultured in Ham's F12 medium with 15% bovine calf serum. Caerulein, insulin, somatostatin, and dexamethasone (DEX) had no effect on intracellular or secreted amylase in these cultured cells. A serum-free medium, using Waymouth's MB 752/1 supplemented with albumin, epidermal growth factor (EGF), DEX, and HEPES, was then developed to avoid serum factors that might mask hormonal effects. In this SF medium, pancreatic acinar, cells maintained the morphological and ultrastructural characteristics of freshly isolated cells and secreted amylase in response to the secretagogue, carbamyl choline. Insulin, at a concentration of 1 μg/ml, significantly increased intracellular and secreted amylase activity after 3 d. This model cell system can be used to study the regulation of the synthesis of amylase and other pancreatic enzymes in vitro.     

Direct microscopic studies of clamp connection formation in growing hyphae of Schizophyllum commune

Summary Quantitative measurements of apical growth, nuclear movements and pseudo-clamp connection formation were compared with photomicroscopic details of cytoplasmic events in live A-mutant hyphae of Schizophyllum commune. Nuclear behavior was described during pseudo-clamp connection formation in uninucleate, binucleate and trinucleate hyphal apices and intercalation was shown in sub-terminal regions of these hyphae. Conventional (i.e. rearward)rd) pseudo-clamp connection formation was contrasted with forward pseudo-clamp initiation. Primary branches were shown to be initiated from uninucleate, septate pseudo-clamps. The ultrastructural aspects of A-mutant septa were delineated.     

REMARKABLE CONSERVATION OF INTERNALLY TRANSCRIBED SPACER SEQUENCES OF ARTHROSPIRA (“SPIRULINA” ) (CYANOPHYCEAE,CYANOBACTERIA) STRAINS FROM FOUR CONTINENTS AND OF RECENT AND 30‐YEAR‐OLD DRIED SAMPLES FROM AFRICA1

The internally transcribed spacer (ITS) sequences of 21 Arthrospira clonal strains from four continents and assigned to four different species (A. platensis, A. maxima, A. fusiformis, A. indica) in the culture collections were determined. Two main clusters, I and II, were differentiated by 49 positions out of 475 nt or 477 nt, respectively. Each cluster was further subdivided into two subclusters. Subclusters I.A and I.B were separated by two substitutions, whereas subclusters II.A and II.B were distinguished by four substitutions. After direct sequencing of the PCR products, three dried samples from Chad aged between 3 months and 35 years yielded a sequence belonging to subcluster I.A, as did a recent commercial product. The strains grown in production plants belonged to the same (sub)clusters as strains from culture collections, mainly I.A and II. PCR primers specific for each cluster and subcluster were designed and tested with crude cell lysates of Arthrospira strains. One dried sample (“dihé” 1) and a herbarium sample from Lake Sonachi (Kenya) only contained I.A sequences, whereas the commercial product was a mixture of the four genotypes and the other two dried samples contained minor polymorphisms characteristic of different clusters. Five clonal Arthrospira strains, thought to be duplicates, showed the simultaneous presence of the two forms of the four diagnostic positions that distinguish subclusters genotype II.A and genotype II.B. This is likely to be caused by multiple copies of the rDNA operon, in a intermediate stage of homogenization between subcluster II.A and subcluster II.B. The high conservation of ITS sequences is in contrast with the assignment to four different species, the great morphological variability of the strains, and their wide geographic distribution.     

Genetic heterogeneity in Bacillus sporothermodurans as demonstrated by ribotyping and repetitive extragenic palindromic-PCR fingerprinting

Thirty-eight strains of Bacillus sporothermodurans isolated from ultra-high-temperature (UHT)-treated milk or sterilized milk (UHT isolates) and from animal feed or raw milk (farm isolates) were characterized by automated ribotyping and by repetitive extragenic palindromic (REP)-PCR fingerprinting. By investigating the genetic relationships among isolates from these various sources, the relative importance of different contamination sources could be evaluated. The results of the separate clustering analyses of the PvuII and EcoRI ribopatterns and the REP-PCR patterns were largely consistent with each other and revealed the existence of two main clusters; there was one homogeneous group containing all (REP-PCR) or most (ribotyping) of the UHT isolates, and there was a second more diverse group comprising the farm isolates. A combined three-dimensional analysis of all data showed that three German UHT isolates did not belong to the compact group containing the majority of the UHT isolates. These results demonstrate that B. sporothermodurans is more heterogeneous than previously assumed and that most of the UHT isolates form a genetically distinct subgroup and are capable of producing highly heat-resistant spores. The close genetic relationship of these UHT isolates suggests a clonal origin of a few predominant strains of B. sporothermodurans that can be found in UHT-treated or sterilized milk products.     

The effect of autocorrelation in environmental variability on the persistence of populations: an experimental test

Despite its significance regarding the conservation and management of biological resources, the body of theory predicting that the correlation between successive environmental states can profoundly influence extinction has not been empirically validated. Identical clonal populations from a model experimental system based on the collembolan Folsomia candida were used in the present study to investigate the effect of environmental autocorrelation on time to extinction. Environmental variation was imposed by variable implementation (present/absent) of a culling procedure according to treatments that represented six patterns of environmental autocorrelation. The average number of culling events was held constant across treatments but, as environmental autocorrelation increased, longer runs of both favourable and unfavourable culling tended to occur. While no difference was found among the survival functions for the various treatments, the time taken for 50% of the component populations to become extinct decreased significantly with increasing environmental autocorrelation. Similarly, analysis of all extinct populations demonstrated that time to extinction was shortened as environmental autocorrelation increased. However, this acceleration of extinction can be fully offset if sequential introduction is used in place of simultaneous introduction when founding the populations.     

Establishment success and extinction risk in autocorrelated environments

We consider establishment success (and extinction risk of small populations) in fluctuating environments, by means of an inhomogeneous branching process model. In this model it is assumed that individuals reproduce asexually during discrete reproduction periods. Within each period individuals reproduce independently and have random numbers of offspring. Expected numbers of offspring vary over reproduction periods due to random environmental changes. Previous simulation results indicated that there is a positive autocorrelation between the establishment probabilities of invaders in successive reproduction periods when environmental states are independently distributed. This result was never formally proved. In this paper we prove that this is indeed true, regardless of the form of the distribution of environmental states or the offspring distribution (under a monotonicity condition, which holds for biologically realistic models). Furthermore, we prove that it is also true for positively autocorrelated environmental states. We show by a counterexample that in environments with a strong negative autocorrelation establishment probabilities can be negatively autocorrelated. This was further examined through simulations. Our results imply that in independent, positively autocorrelated and weakly negatively autocorrelated environments the probability of success of invasion in different independently varying sites is the highest, followed by sequential invasion. For environments with a strong negative autocorrelation, sequential invasion has the highest probability of success. Effects of autocorrelation were further examined with simulations. From the results it appears that the expected length of 'runs of bad luck' is the most crucial factor for establishment success.     

Modes of reproduction and the accumulation of deleterious mutations with multiplicative fitness effects

Mutational load depends not only on the number and nature of mutations but also on the reproductive mode. Traditionally, only a few specific reproductive modes are considered in the search of explanations for the maintenance of sex. There are, however, many alternatives. Including these may give radically different conclusions. The theory on deterministic deleterious mutations states that in large populations segregation and recombination may lead to a lower load of deleterious mutations, provided that there are synergistic interactions. Empirical research suggests that effects of deleterious mutations are often multiplicative. Such situations have largely been ignored in the literature, since recombination and segregation have no effect on mutation load in the absence of epistasis. However, this is true only when clonal reproduction and sexual reproduction with equal male and female ploidy are considered. We consider several alternative reproductive modes that are all known to occur in insects: arrhenotoky, paternal genome elimination, apomictic thelytoky, and automictic thelytoky with different cytological mechanisms to restore diploidy. We give a method that is based on probability-generating functions, which provides analytical and numerical results on the distributions of deleterious mutations. Using this, we show that segregation and recombination do make a difference. Furthermore, we prove that a modified form of Haldane's principle holds more generally for thelytokous reproduction. We discuss the implications of our results for evolutionary transitions between different reproductive modes in insects. Since the strength of Muller's ratchet is reduced considerably for several forms of automictic thelytoky, many of our results are expected to be also valid for initially small populations.     

Identification and cardiotropic actions of brain/gut-derived tachykinin-related peptides (TRPs) from the American lobster Homarus americanus

Two tachykinin-related peptides (TRPs) are known in decapods, APSGFLGMRamide and TPSGFLGMRamide. The former peptide appears to be ubiquitously conserved in members of this taxon, while the latter has been suggested to be a genus (Cancer)- or infraorder (Brachyura)-specific isoform. Here, we characterized a cDNA from the American lobster Homarus americanus (infraorder Astacidea) that encodes both TRPs: six copies of APSGFLGMRamide and one of TPSGFLGMRamide. Mass spectral analyses of the H. americanus supraoesophageal ganglion (brain) and commissural ganglia confirmed the presence of both peptides in these neural tissues; both isoforms were also detected in the midgut. Physiological experiments showed that both APSGFLGMRamide and TPSGFLGMRamide are cardioactive in H. americanus, eliciting identical increases in both heart contraction frequency and amplitude. Collectively, our data represent the first genetic confirmation of TRPs in H. americanus and of TPSGFLGMRamide in any species, demonstrate that TPSGFLGMRamide is not restricted to brachyurans, and show that both this peptide and APSGFLGMRamide are brain-gut isoforms, the first peptides thus far confirmed to possess this dual tissue distribution in H. americanus. Our data also suggest a possible role for TRPs in modulating the output of the lobster heart.     

Establishment Probability in Fluctuating Environments: A Branching Process Model

We study the establishment probability of invaders in stochastically fluctuating environments and the related issue of extinction probability of small populations in such environments, by means of an inhomogeneous branching process model. In the model it is assumed that individuals reproduce asexually during discrete reproduction periods. Within each period, individuals have (independent) Poisson distributed numbers of offspring. The expected numbers of offspring per individual are independently identically distributed over the periods. It is shown that the establishment probability of an invader varies over the reproduction periods according to a stable distribution. We give a method for simulating the establishment probabilities and approximations for the expected establishment probability. Furthermore, we show that, due to the stochasticity of the establishment success over different periods, the expected success of sequential invasions is larger then that of simultaneous invasions and we study the effects of environmental fluctuations on the extinction probability of small populations and metapopulations. The results can easily be generalized to other offspring distributions than the Poisson.