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.     

Neuropathology of human Alzheimer disease after immunization with amyloid-beta peptide: a case report

Amyloid-beta peptide (Abeta) has a key role in the pathogenesis of Alzheimer disease (AD). Immunization with Abeta in a transgenic mouse model of AD reduces both age-related accumulation of Abeta in the brain and associated cognitive impairment. Here we present the first analysis of human neuropathology after immunization with Abeta (AN-1792). Comparison with unimmunized cases of AD (n = 7) revealed the following unusual features in the immunized case, despite diagnostic neuropathological features of AD: (i) there were extensive areas of neocortex with very few Abeta plaques; (ii) those areas of cortex that were devoid of Abeta plaques contained densities of tangles, neuropil threads and cerebral amyloid angiopathy (CAA) similar to unimmunized AD, but lacked plaque-associated dystrophic neurites and astrocyte clusters; (iii) in some regions devoid of plaques, Abeta-immunoreactivity was associated with microglia; (iv) T-lymphocyte meningoencephalitis was present; and (v) cerebral white matter showed infiltration by macrophages. Findings (i)-(iii) strongly resemble the changes seen after Abeta immunotherapy in mouse models of AD and suggest that the immune response generated against the peptide elicited clearance of Abeta plaques in this patient. The T-lymphocyte meningoencephalitis is likely to correspond to the side effect seen in some other patients who received AN-1792 (refs. 7-9).     

An association between the acute phase response and patterns of antigen induced T cell proliferation in juvenile idiopathic arthritis

The aim of this research was to determine whether all memory T cells have the same propensity to migrate to the joint in patients with juvenile idiopathic arthritis. Paired synovial fluid and peripheral blood mononuclear cell proliferative responses to a panel of antigens were measured and the results correlated with a detailed set of laboratory and clinical data from 39 patients with juvenile idiopathic arthritis. Two distinct patterns of proliferative response were found in the majority of patients: a diverse pattern, in which synovial fluid responses were greater than peripheral blood responses for all antigens tested; and a restricted pattern, in which peripheral blood responses to some antigens were more vigorous than those in the synovial fluid compartment. The diverse pattern was generally found in patients with a high acute phase response, whereas patients without elevated acute phase proteins were more likely to demonstrate a restricted pattern. We propose that an association between the synovial fluid T cell repertoire and the acute phase response suggests that proinflammatory cytokines may influence recruitment of memory T cells to an inflammatory site, independent of their antigen specificity. Additionally, increased responses to enteric bacteria and the presence of αEβ7 T cells in synovial fluid may reflect accumulation of gut associated T cells in the synovial compartment, even in the absence of an elevated acute phase response. This is the first report of an association between the acute phase response and the T cell population recruited to an inflammatory site.     

The IIIb isoform of fibroblast growth factor receptor 2 is required for proper growth and branching of pancreatic ductal epithelium but not for differentiation of exocrine or endocrine cells

Fibroblast growth factors (Fgfs) and their receptors have been implicated in embryonic pancreas development. Recently it was shown that Fgf10, a major ligand for the IIIb isoform of fibroblast growth factor receptor 2 (Fgfr2b), has an important regulatory role in early pancreas development. The aim of our study was to define the role of Fgfr2b in pancreas development by analyzing the phenotype of Fgfr2b (-/-) mice. Pancreases of Fgfr2b (-/-) embryos were noticeably smaller than the wild type littermates during embryogenesis, and pancreatic ductal branching as well as duct cell proliferation was significantly reduced. However, both exocrine and endocrine pancreatic differentiation occurred relatively normally. Exogenous addition of Fgfr2b ligands (Fgf7 and Fgf10) stimulated duct cell proliferation and inhibited endocrine cell differentiation in the ex vivo embryonic organ cultures of wild type pancreas. Our results thus suggest that Fgfr2b-mediated signaling plays a major role in pancreatic ductal proliferation and branching morphogenesis, but has little effect on endocrine and exocrine differentiation.