Hibernation confers resistance to intestinal ischemia-reperfusion injury

The damaging effects of intestinal ischemia-reperfusion (I/R) on the gut and remote organs can be attenuated by subjecting the intestine to a prior, less severe I/R insult, a process known as preconditioning. Because intestines of hibernating ground squirrels experience repeated cycles of hypoperfusion and reperfusion, we examined whether hibernation serves as a model for natural preconditioning against I/R-induced injury. We induced intestinal I/R in either the entire gut or in isolated intestinal loops using rats, summer ground squirrels, and hibernating squirrels during natural interbout arousals (IBA; body temperature 37-39 degrees C). In both models, I/R induced less mucosal damage in IBA squirrels than in summer squirrels or rats. Superior mesenteric artery I/R increased MPO activity in the gut mucosa and lung of rats and summer squirrels and the liver of rats but had no effect in IBA squirrels. I/R in isolated loops increased luminal albumin levels, suggesting increased gut permeability in rats and summer squirrels but not IBA squirrels. The results suggest that the hibernation phenotype is associated with natural protection against intestinal I/R injury.     

Experimental Exposures of Boreal Toads (Bufo boreas) to a Pathogenic Chytrid Fungus (Batrachochytrium dendrobatidis)

One of the major causes of worldwide amphibian declines is a skin infection caused by a pathogenic chytrid fungus (Batrachochytrium dendrobatidis). This study documents the interactions between this pathogen and a susceptible amphibian host, the boreal toad (Bufo boreas). The amount of time following exposure until death is influenced by the dosage of infectious zoospores, duration of exposure, and body size of the toad. The significant relation between dosage and the number of days survived (dose-response curve) supports the hypothesis that the degree of infection must reach a particular threshold of about 10⁷–10⁸ zoosporangia before death results. Variation in air temperature between 12°C and 23°C had no significant effect on survival time. The infection can be transmitted from infected to healthy animals by contact with water containing zoospores; no physical contact between animals is required. These results are correlated with observations on the population biology of boreal toads in which mortalities associated with B. dendrobatidis have been identified.     

Systematics and Evolution of the Dasyurid Marsupial Genus Sminthopsis: II. The Murina Species Group

Genetic variation within the Murina species group, which includes S. murina, S. gilberti, S. leucopus, S. dolichura and S. archeri, was examined through analyses of complete 12S rRNA, partial control region mitochondrial DNA sequences and partial omega-globin nuclear DNA sequences. Sminthopsis butleri was found to be an additional member of the Murina group, and appears to be most closely related to S. leucopus rather than the morphologically similar S. archeri. This latter species appears to be the most divergent member of the group, and there is a possible sister relationship between S. murina and S. gilberti, as suggested by previous allozyme evidence. It appears that the systematic affinities of the taxonomically problematic northeastern Queensland populations of both S. murina and S. leucopus and a disjunct population of S. gilberti (from the Western Australia/South Australia border) are indeed with those respective species; although each appears to belong to a distinct morphological and genetic lineage. A specimen of S. leucopus from Queensland was found to be as divergent from each of the southeastern Australian S. leucopus subspecies as they are from each other, suggesting that this northern population of S. leucopus may also warrant recognition as a distinct taxon. Specimens of S. murina murina were found to be genetically divergent from each other, and this subspecies appears to be paraphyletic, as suggested by previous morphological evidence. ^* This paper is the second part of a series dealing with the systematics and evolution of the dasyurid marsupial genus Sminthopsis. Part one covered the Macroura species group and was published in 2001 in the Journal of Mammalian Evolution 8, 149–170.     

A high-throughput <Emphasis Type="Italic">de novo</Emphasis> sequencing approach for shotgun proteomics using high-resolution tandem mass spectrometry

Background  

High-resolution tandem mass spectra can now be readily acquired with hybrid instruments, such as LTQ-Orbitrap and LTQ-FT, in high-throughput shotgun proteomics workflows. The improved spectral quality enables more accurate de novo sequencing for identification of post-translational modifications and amino acid polymorphisms.     

Identification of qRT-PCR reference genes for analysis of opioid gene expression in a hibernator

Previous work has suggested that central and peripheral opioid signaling are involved in regulating torpor behavior and tissue protection associated with the hibernation phenotype. We used quantitative real-time PCR (qRT-PCR) to measure mRNA levels of opioid peptide precursors and receptors in the brain and heart of summer ground squirrels (Ictidomys tridecemlineatus) and winter hibernating squirrels in the torpid or interbout arousal states. The use of appropriate reference genes for normalization of qRT-PCR gene expression data can have profound effects on the analysis and interpretation of results. This may be particularly important when experimental subjects, such as hibernating animals, undergo significant morphological and/or functional changes during the study. Therefore, an additional goal of this study was to identify stable reference genes for use in qRT-PCR studies of the 13-lined ground squirrel. Expression levels of 10 potential reference genes were measured in the small intestine, liver, brain, and heart, and the optimal combinations of the most stable reference genes were identified by the GeNorm Excel applet. Based on this analysis, we provide recommendations for reference genes to use in each tissue that would be suitable for comparative studies among different activity states. When appropriate normalization of mRNA levels was used, there were no changes in opioid-related genes in heart among the three activity states; in brain, DOR expression was highest during torpor, lowest in interbout arousal and intermediate in summer. The results support the idea that changes in DOR expression may regulate the level of neuronal activity in brain during the annual hibernation cycle and may contribute to hibernation-associated tissue protection.     

Manipulating Each MreB of Bdellovibrio bacteriovorus Gives Diverse Morphological and Predatory Phenotypes

We studied the two mreB genes, encoding actinlike cytoskeletal elements, in the predatory bacterium Bdellovibrio bacteriovorus. This bacterium enters and replicates within other Gram-negative bacteria by attack-phase Bdellovibrio squeezing through prey outer membrane, residing and growing filamentously in the prey periplasm forming an infective “bdelloplast,” and septating after 4 h, once the prey contents are consumed. This lifestyle brings challenges to the Bdellovibrio cytoskeleton. Both mreB genes were essential for viable predatory growth, but C-terminal green fluorescent protein tagging each separately with monomeric teal-fluorescent protein (mTFP) gave two strains with phenotypic changes at different stages in predatory growth and development. MreB1-mTFP cells arrested growth early in bdelloplast formation, despite successful degradation of prey nucleoid. A large population of stalled bdelloplasts formed in predatory cultures and predation proceeded very slowly. A small proportion of bdelloplasts lysed after several days, liberating MreB1-mTFP attack-phase cells of wild-type morphology; this process was aided by subinhibitory concentrations of an MreB-specific inhibitor, A22. MreB2-mTFP, in contrast, was predatory at an almost wild-type rate but yielded attack-phase cells with diverse morphologies, including spherical, elongated, and branched, the first time such phenotypes have been described. Wild-type predatory rates were seen for all but spherical morphotypes, and septation of elongated morphotypes was achieved by the addition of A22.The predatory bacterium Bdellovibrio bacteriovorus shows novel filamentous growth within the periplasm of the Gram-negative prey bacterium on which it feeds. This study focuses on the cytoskeletal protein MreB and the role that two homologues of it play in B. bacteriovorus predatory or host-dependent (HD) growth. The HD B. bacteriovorus life cycle can be split into two phases: an attack phase and a growth phase (Fig. ​(Fig.1).1). The attack-phase B. bacteriovorus is a small free-swimming, highly motile cell within which replication has been arrested and which does not take up organic nutrients from the environment or grow extensively (24, 26). Once an attack-phase cell has collided with a suitable prey bacterium, B. bacteriovorus opens and squeezes through a small hole, formed in the outer membrane, using type IV pili to pull itself inside (7, 10). The B. bacteriovorus reseals the hole upon entering the periplasm. Once inside, the prey is killed rapidly within 15 min, and the prey cell wall is partially digested (35), forming a rounded structure called the bdelloplast (see Fig. ​Fig.11 and 4Ac and d). The HD B. bacteriovorus cell then enters the second, growth phase, part of the life cycle, (Fig. ​(Fig.1),1), whereby it grows filamentously while simultaneously coordinating the digestion and transportation of monomers from the prey cytoplasm. The mature growth-phase cell is multiploid and elongates typically 3 to 10 times the length of an attack-phase cell, its length being a reflection of the nutritional resources available in the prey (20). Once resources within the bdelloplast are depleted, the mature filament septates sequentially from one pole to form multiple progeny. These lyse the exhausted bdelloplast, mature into attack-phase cells, and repress growth once again (Fig. ​(Fig.1).1). B. bacteriovorus can be cultured slowly, without prey, as host-independent (HI) cells growing upon peptone-rich medium (30). In these conditions they grow pleiomorphically as mainly long filamentous or serpentine cells, from which some small attack-phase cells septate (30).Open in a separate windowFIG. 1.Schematic host-dependent (HD) predatory cycle for B. bacteriovorus on E. coli prey, showing the different phases of growth and inferred demands on the B. bacteriovorus cell cytoskeleton. References where the roles of the cytoskeleton in cell development have been proven for other bacteria are provided in parentheses. The status of the prey genome is both drawn from an earlier study (26) and confirmed by our work in the present study (see Fig. ​Fig.44).The predatory lifestyle of B. bacteriovorus presents a number of novel developmental challenges to the B. bacteriovorus cell and its cytoskeleton. It is not known how the attack-phase cells deform, allowing the B. bacteriovorus to squeeze through a pore it makes in the prey outer membrane that is narrower than the width of an attack-phase cell, as was imaged by Burnham et al. in the 1960s and more recently by Evans et al. (7, 10). It is also not known how the growth-phase filamentous cell within the bdelloplast is generated and remains resistant to division until terminal sequential septation begins, despite having multiple potential sites for septation along its length while elongating.The processes of cell elongation in rod-shaped bacteria are coordinated by an internal MreB cell cytoskeleton (9). MreB is a eukaryotic actin homologue and has been well studied in Escherichia coli, Bacillus subtilis, and Caulobacter crescentus (38). MreB monomers polymerize on ATP binding, forming helical structures in vivo that appear to associate with the cytoplasmic side of the bacterial cytoplasmic membrane (11, 18, 31). Bacterial two-hybrid experiments in Escherichia coli suggest that MreB forms a transmembrane complex with the two proteins MreC and MreD, each of which have been shown to form helical structures in vivo (21). A complex of MreBCD, together with the RodA protein, influences the shape of the peptidoglycan cell wall and thus the shape of the cell by positioning the peptidoglycan biosynthetic machinery so that its action is directionally specific (9, 19, 37). The MreB filament has also been shown to have roles in chromosome segregation, septation, and cell polarity (13, 14, 22, 37).Depletion of the MreB protein levels in E. coli and B. subtilis led to cells taking on a spherical morphology and eventual loss of viability since new peptidoglycan is not synthesized evenly along the cell wall (9, 36). Uneven incorporation of new peptidoglycan is potentially driven by the tubulin homologue FtsZ (4, 36). A similar phenotype is achieved by addition of the MreB inhibitor A22 that causes the reversible loss of MreB filament localization in vivo (14, 17). A22 was discovered in a chemical library being screened for the ability to generate anucleate minicells from E. coli (16, 17). It has been used extensively by others to examine MreB function in bacteria of many different genera. The addition of A22 to E. coli cells at 3.13 μg/ml leads to the breakdown of MreB filaments, spheroplasting and the generation of minicells (16). In B. subtilis, A22 at concentrations in excess of 100 μg/ml is required to generate spheroplasts; in C. crescentus 6-h incubations of A22 at 10 μg/ml are needed before any change to the cell shape can be observed—in this case cells take on a characteristic “lemon shape” (14, 16). Isolation and sequencing of A22-resistant mutants of C. crescentus, as well as biochemical evidence from purified MreB from Thermotoga maritima, revealed that A22 binds in the nucleotide binding pocket of MreB (3, 14). In vitro light scattering assays of MreB filamentation showed that A22 acted as a competitive inhibitor of ATP binding and was able in inhibit the formation of MreB filaments, presumably by sequestering and inactivating MreB monomers preventing their recycling (3). This study also demonstrated that in vitro A22 can have a role in stabilizing ADP-bound MreB (3).In the present study we investigated the functions of the two MreB homologues found in B. bacteriovorus, testing the role each has in predation and cell morphology by a combination of genetic approaches and A22 treatment. A reduction in function in both MreB proteins, achieved by C-terminal teal fluorescent protein (TFP) tagging, suggests that the MreB1 (Bd0211) protein was required in the growth-phase cell in bdelloplasts, whereas the MreB2 (Bd1737) protein is required later in the predatory process, for maintaining and allowing successful resolution of the growth-phase filament into short attack-phase vibroid cells.     

The First Bite— Profiling the Predatosome in the Bacterial Pathogen Bdellovibrio

Bdellovibrio bacteriovorus is a Gram-negative bacterium that is a pathogen of other Gram-negative bacteria, including many bacteria which are pathogens of humans, animals and plants. As such Bdellovibrio has potential as a biocontrol agent, or living antibiotic. B. bacteriovorus HD100 has a large genome and it is not yet known which of it encodes the molecular machinery and genetic control of predatory processes. We have tried to fill this knowledge-gap using mixtures of predator and prey mRNAs to monitor changes in Bdellovibrio gene expression at a timepoint of early-stage prey infection and prey killing in comparison to control cultures of predator and prey alone and also in comparison to Bdellovibrio growing axenically (in a prey-or host independent “HI” manner) on artificial media containing peptone and tryptone. From this we have highlighted genes of the early predatosome with predicted roles in prey killing and digestion and have gained insights into possible regulatory mechanisms as Bdellovibrio enter and establish within the prey bdelloplast. Approximately seven percent of all Bdellovibrio genes were significantly up-regulated at 30 minutes of infection- but not in HI growth- implicating the role of these genes in prey digestion. Five percent were down-regulated significantly, implicating their role in free-swimming, attack-phase physiology. This study gives the first post- genomic insight into the predatory process and reveals some of the important genes that Bdellovibrio expresses inside the prey bacterium during the initial attack.