Impaired Th2 development and increased mortality during Schistosoma mansoni infection in the absence of CD40/CD154 interaction

The role of CD40/CD154 interaction during infection has primarily focused on pathogens that drive inflammatory Th1 responses. In this study, we show that CD40/CD154 interaction is a fundamental requirement for Th2 response development to the parasitic helminth Schistosoma mansoni. Compared with infected wild-type mice, greatly reduced levels of Th2-associated cytokines were measured both in vitro and in vivo, and no IgE or IgG1 was detected in infected CD154(-/-) mice. In the absence of an overt Th2 response, no exaggerated Th1 response was mounted by CD154(-/-) mice. Infected CD154(-/-) mice suffered severe morbidity and mortality, even though parasitemias in wild-type and CD154(-/-) mice did not differ significantly. These data indicate that CD40/CD154 interaction is required to allow development of a Th2-dominated immune response to S. mansoni and support the view that failure to develop such a response can have fatal consequences.     

Activation of cytomegalovirus in pig-to-primate organ xenotransplantation

Xenotransplantation of porcine organs carries the risk of reactivation of latent virus in donor and recipient tissues as well as transmission of viruses between species. We have investigated the activation of baboon cytomegalovirus (BCMV) and porcine CMV (PCMV) in a pig-to-primate model of xenotransplantation. Tissues originating from a series of six swine-to-baboon composite thymokidney xenotransplants were investigated. Four immunosuppressed baboons died (survival range, 7 to 27 days) with the graft in situ. Increases in BCMV DNA copy numbers occurred in three (75%) of these baboons and was thought to be responsible for pneumonitis and the death of one animal. In two baboons, disseminated intravascular coagulation was successfully treated by graftectomy and discontinuation of immunosuppression. PCMV was upregulated in five of six xenografts (83%). PCMV infection was associated with ureteric necrosis in one xenograft. Although significantly increased in native tissues, low levels of BCMV and PCMV were also detected in tissues other than that of the native viral host species. The cross-species presence of CMV did not appear to cause clinical or histological signs of invasive disease. Thus, viral infections with clinical disease were restricted to tissues of the native species of each virus. Intensive immune suppression currently required for xenotransplantation results in a significant risk of reactivation of latent infections by BCMV and PCMV. It is not yet known whether viral DNA detected across species lines represents cellular microchimerism, ongoing viral infection, or uptake of free virus. The observation of graft injury by PCMV demonstrates that CMV will be an important pathogen in immunosuppressed xenograft recipients. Strategies must be developed to exclude CMV from porcine organ donors.     

Insulin receptor-mediated organ overgrowth in <Emphasis Type="Italic">Drosophila</Emphasis> is not restricted by body size

Recent studies have demonstrated that insulin receptor (Inr) signalling plays an important role in determining organ size and maintaining proportionality in normal animals. However, it is unclear whether the activity of this pathway in a developing organ is invariably a dominant determinant of its mass or whether size can be restricted by other non-autonomous growth regulatory mechanisms if a tissue starts to outgrow the rest of the body. To test this in Drosophila, we induced excess Inr-dependent growth by removal of the Inr signalling antagonist, DPTEN, in the eyes of flies with dramatically different body sizes. Although our data suggest a very limited level of growth competition between organs in animals with giant eyes, there do not appear to be mechanisms by which neighbouring structures substantially inhibit eye overgrowth, even when the resulting organ is many times enlarged relative to the rest of the animal. Overall, our results support a simple model in which organs are normally maintained in proportion to each other, primarily because they all respond similarly to levels of insulin-like factors and other growth regulators. Given the evolutionarily conserved role of insulin receptor signalling in growth control, our data may also help to explain why this pathway is so frequently hyperactivated in mammalian tumours.     

Fgf3 and Fgf8 are required together for formation of the otic placode and vesicle

Fgf3 has long been implicated in otic placode induction and early development of the otocyst; however, the results of experiments in mouse and chick embryos to determine its function have proved to be conflicting. In this study, we determined fgf3 expression in relation to otic development in the zebrafish and used antisense morpholino oligonucleotides to inhibit Fgf3 translation. Successful knockdown of Fgf3 protein was demonstrated and this resulted in a reduction of otocyst size together with reduction in expression of early markers of the otic placode. fgf3 is co-expressed with fgf8 in the hindbrain prior to otic induction and, strikingly, when Fgf3 morpholinos were co-injected together with Fgf8 morpholinos, a significant number of embryos failed to form otocysts. These effects were made manifest at early stages of otic development by an absence of early placode markers (pax2.1 and dlx3) but were not accompanied by effects on cell division or death. The temporal requirement for Fgf signalling was established as being between 60% epiboly and tailbud stages using the Fgf receptor inhibitor SU5402. However, the earliest molecular event in induction of the otic territory, pax8 expression, did not require Fgf signalling, indicating an inductive event upstream of signalling by Fgf3 and Fgf8. We propose that Fgf3 and Fgf8 are required together for formation of the otic placode and act during the earliest stages of its induction.     

A role for nuclear localised proteasomes in mediating auxin action

A number of important cellular events in animals and yeast are regulated by protein degradation, and it is becoming apparent that such regulated proteolysis is involved in many facets of plant physiology and development. We have investigated the role of protein degradation by proteasomes in plants using NtPSA1, a tobacco gene that is predominantly expressed in young developing tobacco tissues and has extensive homology to yeast and human alpha-type proteasome subunit genes. The NtPSA1 cDNA was used to complement a lethal mutation of the yeast PRC1 alpha subunit gene indicating that NtPSA1 encodes a functional proteasome subunit, and transient expression of an NtPSA1::GUS protein fusion in onion cells confirmed that the nuclear localisation signal that is present in the NtPSA1 peptide sequence is active in plant cells. Plants transformed with an antisense NtPSA1 gene had reduced levels of NtPSA1 mRNA and exhibited reduced apical dominance. In addition, these low NtPSA1 plants displayed several morphological defects associated with auxin resistance such as reduced stamen length, and showed increased tolerance to high concentrations of auxin. These results support a role for nuclear localised proteasomes in floral development and auxin responses.     

Molecular characterization of a cystathionine beta-synthase gene,CBS1, in Magnaporthe grisea

CBS1 from Magnaporthe grisea is a structural and functional homolog of the cystathionine β-synthase (CBS) gene from Saccharomyces cerevisiae. Our studies indicated that M. grisea can utilize homocysteine and methionine through a CBS-independent pathway. The results also revealed responses of M. grisea to homocysteine that are reminiscent of human homocystinuria.     

Cathepsin B stability,but not activity,is affected in cysteine:cystine redox buffers

In order to test the hypothesis that the lysosomal cysteine protease cathepsin B may be redox regulated in vivo, cathepsin B activity and stability were measured in cysteine- and/or cystine-containing buffers. Cathepsin B activity in cysteine-containing buffers was similar at pH 6.0 and pH 7.0, over all thiol concentrations tested. In contrast, the stability of the enzyme was greater at pH 6.0 than at pH 7.0. This suggests that the enzyme's operational pH in vivo may be < pH 7.0. The activity of the enzyme was depressed in glutathione-containing buffers. When assessed in cysteine:cystine redox buffers (pH 6.0-7.0) cathepsin B was active over a broad redox potential range, suggesting that cathepsin B activity may not be redox regulated. However, at pH 7.0, the stability of cathepsin B decreased with increasing reduction potential and ambient cystine concentration. This suggests that the stability of the enzyme at neutral pH is dependent on redox potential, and on the presence of oxidising agents.     

Changes in gene expression in Arabidopsis shoots during phosphate starvation and the potential for developing smart plants

Our aim was to generate and prove the concept of "smart" plants to monitor plant phosphorus (P) status in Arabidopsis. Smart plants can be genetically engineered by transformation with a construct containing the promoter of a gene up-regulated specifically by P starvation in an accessible tissue upstream of a marker gene such as beta-glucuronidase (GUS). First, using microarrays, we identified genes whose expression changed more than 2.5-fold in shoots of plants growing hydroponically when P, but not N or K, was withheld from the nutrient solution. The transient changes in gene expression occurring immediately (4 h) after P withdrawal were highly variable, and many nonspecific, shock-induced genes were up-regulated during this period. However, two common putative cis-regulatory elements (a PHO-like element and a TATA box-like element) were present significantly more often in the promoters of genes whose expression increased 4 h after the withdrawal of P compared with their general occurrence in the promoters of all genes represented on the microarray. Surprisingly, the expression of only four genes differed between shoots of P-starved and -replete plants 28 h after P was withdrawn. This lull in differential gene expression preceded the differential expression of a new group of 61 genes 100 h after withdrawing P. A literature survey indicated that the expression of many of these "late" genes responded specifically to P starvation. Shoots had reduced P after 100 h, but growth was unaffected. The expression of SQD1, a gene involved in the synthesis of sulfolipids, responded specifically to P starvation and was increased 100 h after withdrawing P. Leaves of Arabidopsis bearing a SQD1::GUS construct showed increased GUS activity after P withdrawal, which was detectable before P starvation limited growth. Hence, smart plants can monitor plant P status. Transferring this technology to crops would allow precision management of P fertilization, thereby maintaining yields while reducing costs, conserving natural resources, and preventing pollution.     

Swelling-activated chloride channels in cardiac physiology and pathophysiology

Characteristics and functions of the cardiac swelling-activated Cl current (I_Cl,swell) are considered in physiologic and pathophysiologic settings. I_Cl,swell is broadly distributed throughout the heart and is stimulated not only by osmotic and hydrostatic increases in cell volume, but also by agents that alter membrane tension and direct mechanical stretch. The current is outwardly rectifying, reverses between the plateau and resting potentials (E_m), and is time-independent over the physiologic voltage range. Consequently, I_Cl,swell shortens action potential duration, depolarizes E_m, and acts to decrease cell volume. Because it is activated by stimuli that also activate cation stretch-activated channels, I_Cl,swell should be considered as a potential effector of mechanoelectrical feedback. I_Cl,swell is activated in ischemic and non-ischemic dilated cardiomyopathies and perhaps during ischemia and reperfusion. I_Cl,swell plays a role in arrhythmogenesis, myocardial injury, preconditioning, and apoptosis of myocytes. As a result, I_Cl,swell potentially is a novel therapeutic target.     

Ssk1p response regulator binding surface on histidine-containing phosphotransfer protein Ypd1p

Ypd1p, a histidine-containing phosphotransfer protein, plays an important role in a branched His-Asp phosphorelay signal transduction pathway that regulates cellular responses to hyperosmotic stress in Saccharomyces cerevisiae. Ypd1p is required for phosphoryl group transfer from the membrane-bound Sln1p sensor histidine kinase to two downstream response regulator proteins, Ssk1p and Skn7p. To investigate the molecular basis for interaction of Ypd1p with these response regulator domains, we used an approach that coupled alanine-scanning mutagenesis of surface-exposed residues in Ypd1p with a yeast two-hybrid interaction screen. Mutated residues that adversely affected the interaction of Ypd1p with the C-terminal response regulator domain of Ssk1p were identified and found to cluster on or near the αA helix in Ypd1p. Our results, supported by analysis of a modeled complex, identify a binding site on Ypd1p for response regulators that is composed of a cluster of conserved hydrophobic residues surrounded by less conserved polar residues. We propose that molecular interactions involving Ypd1p are mediated primarily through hydrophobic contacts, whereas binding specificity and strength of interaction may be influenced by select polar side chain interactions.