Tolerance of a Phage Element by Streptococcus pneumoniae Leads to a Fitness Defect during Colonization

The pathogenesis of the disease caused by Streptococcus pneumoniae begins with colonization of the upper respiratory tract. Temperate phages have been identified in the genomes of up to 70% of clinical isolates. How these phages affect the bacterial host during colonization is unknown. Here, we examined a clinical isolate that carries a novel prophage element, designated Spn1, which was detected in both integrated and episomal forms. Surprisingly, both lytic and lysogenic Spn1 genes were expressed under routine growth conditions. Using a mouse model of asymptomatic colonization, we demonstrate that the Spn1⁻ strain outcompeted the Spn1⁺ strain >70-fold. To determine if Spn1 causes a fitness defect through a trans-acting factor, we constructed an Spn1⁺ mutant that does not become an episome or express phage genes. This mutant competed equally with the Spn1⁻ strain, indicating that expression of phage genes or phage lytic activity is required to confer this fitness defect. In vitro, we demonstrate that the presence of Spn1 correlated with a defect in LytA-mediated autolysis. Furthermore, the Spn1⁺ strain displayed increased chain length and resistance to lysis by penicillin compared to the Spn⁻ strain, indicating that Spn1 alters the cell wall physiology of its host strain. We posit that these changes in cell wall physiology allow for tolerance of phage gene products and are responsible for the relative defect of the Spn1⁺ strain during colonization. This study provides new insight into how bacteria and prophages interact and affect bacterial fitness in vivo.     

Regulation of human endometrial function: mechanisms relevant to uterine bleeding

This review focuses on the complex events that occur in the endometrium after progesterone is withdrawn (or blocked) and menstrual bleeding ensues. A detailed understanding of these local mechanisms will enhance our knowledge of disturbed endometrial/uterine function--including problems with excessively heavy menstrual bleeding, endometriosis and breakthrough bleeding with progestin only contraception. The development of novel strategies to manage these clinically significant problems depends on such new understanding as does the development of new contraceptives which avoid the endometrial side effect of breakthrough bleeding.     

Immune stimulation and malaria infection impose reproductive costs in Anopheles gambiae via follicular apoptosis

The employment of defense mechanisms is recognized as a costly life-history trait. In the malaria vector Anopheles gambiae, reproductive costs have been associated with both humoral and cellular innate immune responses and also with malaria infection. The resorption of developing oocytes associated with malaria infection is preceded by the programmed cell death, or apoptosis, of follicular cells. Here we demonstrate that apoptosis in ovarian follicular epithelial cells also occurs when mosquitoes are subjected to artificial immune-elicitors that induce a melanization response or humoral antimicrobial activity. Caspases are key cysteine proteases involved in apoptosis. Caspase-like activity was detected in epithelial cells in approximately 4.0% of the developing ovarian follicles of untreated, blood-fed, mosquitoes. Lipopolysaccharide injection resulted in a significant increase in anti-Micrococcus luteus humoral activity and a significant increase of 257.7% of follicles exhibiting apoptosis compared to results after saline injections. Melanization also triggered follicular apoptosis, which increased by 106.25% or 134.37% in Sephadex C-25 or G-25 bead-inoculated mosquitoes, respectively, compared to that in sham-injected ones. Ovaries from Plasmodium yoelii nigeriensis-infected mosquitoes exhibited a significant increase in follicular apoptosis of 440.9% compared to non-infected ones. Thus, at the time point investigated, infection had a much greater effect than artificial immune-elicitors. Death of follicular epithelial cells has been shown to lead to follicle resorption and hence a decrease in egg production. We propose the trade-off between reproductive fitness and immune defense in A. gambiae operates via the induction of apoptosis in ovarian follicles and that different immune responses impose costs via the same pathway.     

Understanding mammalian genetic systems: the challenge of phenotyping in the mouse

Understanding mammalian genetic systems is predicated on the determination of the relationship between genetic variation and phenotype. Several international programmes are under way to deliver mutations in every gene in the mouse genome. The challenge for mouse geneticists is to develop approaches that will provide comprehensive phenotype datasets for these mouse mutant libraries. Several factors are critical to success in this endeavour. It will be important to catalogue assay and environment and where possible to adopt standardised procedures for phenotyping tests along with common environmental conditions to ensure comparable datasets of phenotypes. Moreover, the scale of the task underlines the need to invest in technological development improving both the speed and cost of phenotyping platforms. In addition, it will be necessary to develop new informatics standards that capture the phenotype assay as well as other factors, genetic and environmental, that impinge upon phenotype outcome.     

Cloacal and buccal swabs are a reliable source of DNA for microsatellite genotyping of reptiles

In this study, a minimally invasive method for DNA sampling of reptiles and amphibians using cloacal and buccal swabs is described. High molecular weight DNA was isolated from the swabs, which were collected from tuatara (Sphenodon punctatus), and stored in 70% ethanol at room temperature for approximately 1 week. Amplification of mitochondrial and microsatellite DNA loci was successful from both cloacal and buccal swabs, and in all cases the genotypes matched those obtained from blood samples. These results show that cloacal and/or buccal swabbing is a useful alternative to blood sampling and toe clipping for genetic studies on reptiles. This method is rapid, inexpensive and easy to implement in field situations.     

The interpretation of morphogen gradients

Morphogens act as graded positional cues that control cell fate specification in many developing tissues. This concept, in which a signalling gradient regulates differential gene expression in a concentration-dependent manner, provides a basis for understanding many patterning processes. It also raises several mechanistic issues, such as how responding cells perceive and interpret the concentration-dependent information provided by a morphogen to generate precise patterns of gene expression and cell differentiation in developing tissues. Here, we review recent work on the molecular features of morphogen signalling that facilitate the interpretation of graded signals and attempt to identify some emerging common principles.     

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. MHC Class I genes in the Tuatara (Sphenodon spp.): evolution of the MHC in an ancient reptilian order

The major histocompatibility complex (MHC) is an extremely dynamic region of the genome, characterized by high polymorphism and frequent gene duplications and rearrangements. This has resulted in considerable differences in MHC organization and evolution among vertebrate lineages, particularly between birds and mammals. As nonavian reptiles are ancestral to both mammals and birds, they occupy an important phylogenetic position for understanding these differences. However, little is known about reptile MHC genes. To address this, we have characterized MHC class I sequences from the tuatara (Sphenodon spp.), the last survivor of an ancient order of reptiles, Sphenodontia. We isolated two different class I cDNA sequences, which share 93% sequence similarity with each other but are highly divergent from other vertebrate MHC genes. Southern blotting and polymerase chain reaction amplification of class I sequences from seven adult tuatara plus a family group indicate that these sequences represent at least two to three loci. Preliminary analysis of variation among individuals from an island population of tuatara indicates that these loci are highly polymorphic. Maximum likelihood analysis of reptile MHC class I sequences indicates that gene duplication has occurred within reptilian orders. However, the evolutionary relationships among sequences from different reptilian orders cannot be resolved, reflecting the antiquity of the major reptile lineages.     

Assay of protein kinases using radiolabeled ATP: a protocol

Protein kinase activity results in the incorporation of radiolabeled phosphate from [gamma-32P]ATP into a peptide or protein substrate. The measurement of the amount of radioactivity incorporated into a substrate as a function of time and enzyme concentration allows enzyme activity to be quantified. The activity is expressed as a 'unit', where 1 unit corresponds to the amount of protein kinase that catalyzes the incorporation of 1 nanomole of phosphate into the standard substrate in 1 minute. Specific activity is defined as units of activity per milligram protein. The assay format described here is quick, simple, inexpensive, sensitive and accurate, provides a direct measurement of activity and remains the 'gold standard' for the quantification of protein kinase activity. Up to 40 samples can be assayed manually at one time, and the assay takes one person less than 1 hour to complete.     

A novel leptin signalling pathway via PTEN inhibition in hypothalamic cell lines and pancreatic beta-cells

In obesity and diabetes, the ability of hypothalamic neurons to sense and transduce changes in leptin and insulin levels is compromised. The effects of both hormones require intracellular signalling via the PI3-kinase pathway, which is inhibited by the phosphatase PTEN. We show that leptin-stimulated F-actin depolymerization in mouse hypothalamic cells is inhibited by PTEN, a process involving independent effects of both its lipid and protein phosphatase activities. Potentially mediating this F-actin depolymerization, leptin, but not insulin, stimulated the phosphorylation of PTEN in a CK2 dependent manner, and inhibited its phosphatase activity. Similarly, hyperpolarization of mouse pancreatic beta-cells by leptin also requires coincident PtdIns(3,4,5)P3 generation and actin depolymerization, and could be inhibited by mechanisms requiring both the lipid and protein phosphatase activities of PTEN. These results demonstrate a critical role for PTEN in leptin signalling and indicate a mechanism by which leptin and insulin can produce PI3K dependent differential cellular outputs.