Regulation of alternative splicing by SRrp86 and its interacting proteins

SRrp86 is a unique member of the SR protein superfamily containing one RNA recognition motif and two serine-arginine (SR)-rich domains separated by an unusual glutamic acid-lysine (EK)-rich region. Previously, we showed that SRrp86 could regulate alternative splicing by both positively and negatively modulating the activity of other SR proteins and that the unique EK domain could inhibit both constitutive and alternative splicing. These functions were most consistent with the model in which SRrp86 functions by interacting with and thereby modulating the activity of target proteins. To identify the specific proteins that interact with SRrp86, we used a yeast two-hybrid library screen and immunoprecipitation coupled to mass spectrometry. We show that SRrp86 interacts with all of the core SR proteins, as well as a subset of other splicing regulatory proteins, including SAF-B, hnRNP G, YB-1, and p72. In contrast to previous results that showed activation of SRp20 by SRrp86, we now show that SAF-B, hnRNP G, and 9G8 all antagonize the activity of SRrp86. Overall, we conclude that not only does SRrp86 regulate SR protein activity but that it is, in turn, regulated by other splicing factors to control alternative splice site selection.     

The expanding interleukin-1 family and its receptors: do alternative IL-1 receptor/signaling pathways exist in the brain?

Interleukin-1 (IL-1) has been implicated in neuroimmune responses and has pleiotropic actions in the brain. Compelling evidence has shown that IL-1 is a major mediator of inflammation and the progression of cell death in response to brain injury and cerebral ischemia. Its expression is strongly increased in these pathological conditions, and central administration of exogenous IL-1 significantly exacerbates ischemic brain damage. In contrast, inhibiting IL-1 actions (by intracerebroventricular [icv] injection of IL-1ra, neutralizing antibody to IL-1 or caspase-1 inhibitor) significantly reduces ischemic brain damage. IL-1 acts by binding to the IL-1 type-1 receptor (IL-1RI), which is to date, the only known functional receptor for IL-1. However, our recent investigations suggest that IL-1 can act independently of IL-1RI, raising the possibility that additional, as yet undiscovered, receptor(s) for IL-1 exist in the brain. The recent characterization of putative, new IL-1 ligands and new IL-1 receptor-related molecules leads to the hypothesis that there might be alternative IL-1 signaling pathway(s) in the central nervous system (CNS).     

A genomewide survey of developmentally relevant genes in Ciona intestinalis. X. Genes for cell junctions and extracellular matrix

Cell junctions and the extracellular matrix (ECM) are crucial components in intercellular communication. These systems are thought to have become highly diversified during the course of vertebrate evolution. In the present study, we have examined whether the ancestral chordate already had such vertebrate systems for intercellular communication, for which we have searched the genome of the ascidian Ciona intestinalis. From this molecular perspective, the Ciona genome contains genes that encode protein components of tight junctions, hemidesmosomes and connexin-based gap junctions, as well as of adherens junctions and focal adhesions, but it does not have those for desmosomes. The latter omission is curious, and the ascidian type-I cadherins may represent an ancestral form of the vertebrate type-I cadherins and desmosomal cadherins, while Ci-Plakin may represent an ancestral protein of the vertebrate desmoplakins and plectins. If this is the case, then ascidians may have retained ancestral desmosome-like structures, as suggested by previous electron-microscopic observations. In addition, ECM genes that have been regarded as vertebrate-specific were also found in the Ciona genome. These results suggest that the last common ancestor shared by ascidians and vertebrates, the ancestor of the entire chordate clade, had essentially the same systems of cell junctions as those in extant vertebrates. However, the number of such genes for each family in the Ciona genome is far smaller than that in vertebrate genomes. In vertebrates these ancestral cell junctions appear to have evolved into more diverse, and possibly more complex, forms, compared with those in their urochordate siblings.     

Characterization of the hipA7 allele of Escherichia coli and evidence that high persistence is governed by (p)ppGpp synthesis

The ability of a high frequency (10(-2)) of Escherichia coli to survive prolonged exposure to penicillin antibiotics, called high persistence, is associated with mutations in the hipA gene. The hip operon is located in the chromosomal terminus near dif and consists of two genes, hipA and hipB. The wild-type hipA gene encodes a toxin, whereas hipB encodes a DNA-binding protein that autoregulates expression of the hip operon and binds to HipA to nullify its toxic effects. We have characterized the hipA7 allele, which confers high persistence, and established that HipA7 is non-toxic, contains two mutations (G22S and D291A) and that both mutations are required for the full range of phenotypes associated with hip mutants. Furthermore, expression of hipA7 in the absence of hipB is sufficient to establish the high persistent phenotype, indicating that hipB is not required. There is a strong correlation between the frequency of persister cells generated by hipA7 strains and cell density, with hipA7 strains generating a 20-fold higher frequency of persisters as cultures approach stationary phase. It is also demonstrated that relA knock-outs diminish the high persistent phenotype in hipA7 mutants and that relA spoT knock-outs eliminate high persistence altogether, suggesting that hipA7 facilitates the establishment of the persister state by inducing (p)ppGpp synthesis. Consistent with this proposal, ectopic expression of relA' from a plasmid was shown to increase the number of persistent cells produced by hipA7 relA double mutants by 100-fold or more. A model is presented that postulates that hipA7 increases the basal level of (p)ppGpp synthesis, allowing a significantly greater percentage of cells in a population to assume a persistent, antibiotic-insensitive state by potentiating a rapid transition to a dormant state upon application of stress.     

Characterization of the rat alpha(1,3)galactosyltransferase: evidence for two independent genes encoding glycosyltransferases that synthesize Galalpha(1,3)Gal by two separate glycosylation pathways

The important xenoepitope Galalpha(1,3)Gal was thought to be exclusively synthesized by a single alpha(1,3)galactosyltransferase. However, the cloning of the distant family member rat iGb3 synthase, which is also capable of synthesizing Galalpha(1,3)Gal as the glycolipid structure iGb3, challenges the notion that alpha(1,3)galactosyltransferase is the sole Galalpha(1,3)Gal-synthesizing enzyme. We describe the cloning of the rat homolog of alpha(1,3)galactosyltransferase, showing that indeed the rat expresses two distinct alpha(1,3)galactosyltransferases, alpha(1,3)GT and iGb3 synthase. Rat alpha(1,3)galactosyltransferase shows a high amino acid sequence identity with the alpha(1,3)galactosyltransferase of mouse (90%), pig (76%), and ox (75%), in contrast to the low amino acid sequence identity (42%) with iGb3 synthase. The rat alpha(1,3)galactosyltransferase is expressed in heart, brain, spleen, kidney, and liver and has a similar intron/exon structure to the mouse alpha(1,3)galactosyltransferase. Transfection studies show that in contrast to the iGb3 synthase, rat alpha(1,3)galactosyltransferase can synthesize Galalpha(1,3)Gal on glycoproteins but cannot synthesize the glycolipid iGb3, defining two separate glycosylation pathways for the synthesis of Galalpha(1,3)Gal. Furthermore iGb3 synthase was found to be distinct from alpha(1,3)GT with its ability to synthesize poly-alpha-Gal glycolipid structures.     

Phenylbutyrates as potent,orally bioavailable vitronectin receptor (integrin alphavbeta3) antagonists

In our continuing efforts to identify small molecule vitronectin receptor antagonists, we have discovered a series of phenylbutyrate derivatives, exemplified by 16, which have good potency and excellent oral bioavailability (approximately 100% in rats). This new series is derived conceptually from opening of the seven-membered ring of SB-265123.     

Myocardial adrenergic and cholinergic receptor function in hypoxia: correlation with O(2) transport in exercise

The time course of changes in rat myocardial alpha(1)- and beta-adrenoceptors and of muscarinic cholinergic (M-Ach) receptor characteristics was studied parallel with the changes in exercise systemic O(2) transport during a 21-day period of hypoxia (barometric pressure 380 Torr) to assess the effects of receptor modification during acclimatization on maximal exercise capacity. Hypoxia resulted in polycythemia, pulmonary hypertension, right ventricular hypertrophy, and transient left ventricular weight loss. Maximal O(2) consumption at 30 min of hypoxia was reduced to 60% of the normoxic value and remained unchanged. This was partly due to a gradual decrease in maximal cardiac output and heart rate (HR(max)), which offset the increase in blood O(2) content. HR(max) correlated positively (r = 0.994) with beta-adrenoceptor density and negatively (r = -0.964) with M-Ach-receptor density, suggesting that HR(max) reduction results from intrinsic changes in myocardial receptor characteristics leading to reduced responses to adrenergic stimulation and elevated responses to cholinergic stimulation. alpha-Adrenoceptor density in both ventricles increased initially to eventually fall below normoxic values. The dissociation between the different patterns of right and left ventricular weight and the similar pattern of alpha-adrenoceptor change in both ventricles do not support a role for these receptors on right ventricular myocardial hypertrophy.     

Recent studies on the biological action of parathyroid hormone (PTH)-related peptide (PTHrP) and PTH/PTHrP receptor in cartilage and bone

Mice with a targeted deletion of parathyroid hormone (PTH)-related peptide (PTHrP) develop a form of dyschondroplasia resulting from diminished proliferation and premature maturation of chondrocytes. Abnormal, heterogeneous populations of chondrocytes at different stages of differentiation were seen in the hypertrophic zone of the mutant growth plate. Although the homozygous null animals die within several hours of birth, mice heterozygous for PTHrP gene deletion reach adulthood, at which time they show evidence of osteopenia. Therefore, PTHrP appears to modulate cell proliferation and differentiation in both the pre and post natal period. PTH/PTHrP receptor expression in the mouse is controlled by two promoters. We recently found that, while the downstream promoter controls PTH/PTHrP receptor gene expression in bone and cartilage, it is differentially regulated in the two tissues. 1alpha,25-dihydroxyvitamin D3 downregulated the activity of the downstream promoter in osteoblasts, but not in chondrocytes, both in vivo and in vitro. Most of the biological activity of PTHrP is thought to be mediated by binding of its amino terminus to the PTH/PTHrP receptor. However, recent evidence suggests that amino acids 87-107, outside of the amino terminal binding domain, act as a nucleolar targeting signal. Chondrocytic cell line, CFK2, transfected with wild-type PTHrP cDNA showed PTHrP in the nucleoli as well as in the secretory pathway. Therefore, PTHrP appears to act as a bifunctional modulator of both chondrocyte proliferation and differentiation, through signal transduction linked to the PTH/PTHrP receptor and by its direct action in the nucleolus.