A point mutation in the IL-12R beta 2 gene underlies the IL-12 unresponsiveness of Lps-defective C57BL/10ScCr mice

Lps-defective C57BL/10ScCr (Cr) mice are homozygous for a deletion encompassing Toll-like receptor 4 that makes them refractory to the biological activity of LPS. In addition, these mice exhibit an inherited IL-12 unresponsiveness resulting in impaired IFN-gamma responses to different microorganisms. By positional cloning methods, we show here that this second defect of Cr mice is due to a mutation in a single gene located on mouse chromosome 6, in close proximity to the Igkappa locus. The gene is IL-12Rbeta2. Cr mice carry a point mutation creating a stop codon that is predicted to cause premature termination of the translated IL-12Rbeta2 after a lysine residue at position 777. The truncated beta2 chain can still form a heterodimeric IL-12R that allows phosphorylation of Janus kinase 2, but, unlike the wild-type IL-12R, can no longer mediate phosphorylation of STAT4. Because the phosphorylation of STAT4 is a prerequisite for the IL-12-mediated induction of IFN-gamma, its absence in Cr mice is responsible for their defective IFN-gamma response to microorganisms.     

Effect of postal prompts to patients and general practitioners on the quality of primary care after a coronary event (POST): randomised controlled trial

ObjectivesTo determine whether postal prompts to patients who have survived an acute coronary event and to their general practitioners improve secondary prevention of coronary heart disease.DesignRandomised controlled trial.Setting52 general practices in east London, 44 of which had received facilitation of local guidelines for coronary heart disease.Participants328 patients admitted to hospital for myocardial infarction or unstable angina.InterventionsPostal prompts sent 2 weeks and 3 months after discharge from hospital. The prompts contained recommendations for lowering the risk of another coronary event, including changes to lifestyle, drug treatment, and making an appointment to discuss these issues with the general practitioner or practice nurse.ResultsPrescribing of β bockers (odds ratio 1.7, 95% confidence interval 0.8 to 3.0, P>0.05) and cholesterol lowering drugs (1.7, 0.8 to 3.4, P>0.05) did not differ between intervention and control groups. A higher proportion of patients in the intervention group (64%) than in the control group (38%) had their serum cholesterol concentrations measured (2.9, 1.5 to 5.5, P<0.001). Secondary outcomes were significantly improved for consultations for coronary heart disease, the recording of risk factors, and advice given. There were no significant differences in patients’ self reported changes to lifestyle or to the belief that it is possible to modify the risk of another coronary event.ConclusionsPostal prompts to patients who had had acute coronary events and to their general practitioners in a locality where guidelines for coronary heart disease had been disseminated did not improve prescribing of effective drugs for secondary prevention or self reported changes to lifestyle. The prompts did increase consultation rates related to coronary heart disease and the recording of risk factors in the practices. Effective secondary prevention of coronary heart disease requires more than postal prompts and the dissemination of guidelines.

Key messages

• Postal prompts to patients and their general practitioners about effective secondary prevention after a myocardial infarction did not improve the prescribing of cholesterol lowering drugs and β blockers
• The prompts did improve general practice recording of cardiovascular risk factors and lifestyle advice given to patients, but they made no difference to patients’ reports of changes to lifestyle
• Other methods are needed to improve the quality of secondary prevention of coronary heart disease in general practice

     

Three-dimensional structure of an unusual Kunitz (STI) type trypsin inhibitor from Copaifera langsdorffii

The crystallographic structure of a novel trypsin inhibitor (CTI) from Copaifera langsdorffii is reported. The structure was solved by MIRAS procedure and refined to a crystallographic residual of 17.3% (R(free) = 20.3%) at 1.8 A resolution. Two isomorphous derivatives were obtained by quick cryo-soaking approach. CTI is the first structure of a member of Kunitz (STI) family formed by two noncovalently bound polypeptide chains and only one disulfide bridge. A standard Kunitz-type inhibitor has a single polypeptide chain and two disulfide bridges. Structural features granting CTI high inhibitory activity are discussed.     

Rapid evolution of RNA editing sites in a small non-essential plastid gene

Chloroplast RNA editing proceeds by C-to-U transitions at highly specific sites. Here, we provide a phylogenetic analysis of RNA editing in a small plastid gene, petL, encoding subunit VI of the cytochrome b₆f complex. Analyzing representatives from most major groups of seed plants, we find an unexpectedly high frequency and dynamics of RNA editing. High-frequency editing has previously been observed in plastid ndh genes, which are remarkable in that their mutational inactivation does not produce an obvious mutant phenotype. In order to test the idea that reduced functional constraints allow for more flexible evolution of RNA editing sites, we have created petL knockout plants by tobacco chloroplast transformation. We find that, in the higher plant tobacco, targeted inactivation of petL does not impair plant growth under a variety of conditions markedly contrasting the important role of petL in photosynthesis in the green alga Chlamydomonas reinhardtii. Together with a low number of editing sites in plastid genes that are essential to gene expression and photosynthetic activity, these data suggest that RNA editing sites may evolve more readily in those genes whose transitory loss of function can be tolerated. Accumulated evidence for this ‘relative neutrality hypothesis for the evolution of plastid editing sites’ is discussed.     

Polyamine derivatives as selective RNaseA mimics

Site-selective scission of ribonucleic acids (RNAs) has attracted considerable interest, since RNA is an intermediate in gene expression and the genetic material of many pathogenic viruses. Polyamine-imidazole conjugates for site-selective RNA scission, without free imidazole, were synthesized and tested on yeast phenylalanine transfer RNA. These molecules catalyze RNA hydrolysis non-randomly. Within the polyamine chain, the location of the imidazole residue, the numbers of nitrogen atoms and their relative distances have notable influence on cleavage selectivity. A norspermine derivative reduces the cleavage sites to a unique location, in the anticodon loop of the tRNA, in the absence of complementary sequence. Experimental results are consistent with a cooperative participation of an ammonium group of the polyamine moiety, in addition to it’s binding to the negatively charged ribose-phosphate backbone, as proton source, and the imidazole moiety as a base. There is correlation between the location of the magnesium binding sites and the RNA cleavage sites, suggesting that the protonated nitrogens of the polycationic chain compete with some of the magnesium ions for RNA binding. Therefore, the cleavage pattern is specific of the RNA structure. These compounds cleave at physiological pH, representing novel reactive groups for antisense oligonucleotide derivatives or to enhance ribozyme activity.     

Evaluation of procedures for amplification of small-size samples for hybridization on microarrays

Various approaches have been developed for the preparation of samples for gene expression monitoring. For Affymetrix chips, a standard protocol is widely used; however, this is inefficient for small samples such as laser capture microdissections. Several amplification procedures for such samples already exist, and our goal was to test two of them: the first is based on random PCR amplification, and the second, linear amplification, involves performing the standard protocol twice. We analyzed a dilution of a commercially available mouse brain total RNA preparation and microdissections from mouse hippocampus and striatum. We evaluated the quality of microarray data by analyzing several chip parameters and performing multiple comparisons. At the biological level, brain microdissections prepared with either method gave similar expression results. At the technical level, analysis of the commercial sample showed that random PCR amplification is more reproducible, requires smaller RNA input, and generates cRNA of higher quality than linear amplification.     

Cardioprotection by chronic estrogen or superoxide dismutase mimetic treatment in the aged female rat

Aging and estrogen deficiency increase the risk for developing cardiovascular disease (CVD). Oxidative stress has also been implicated in the pathophysiology of CVD and in ischemia-reperfusion (I/R) injury. We tested the hypothesis that chronic in vivo estrogen treatment or superoxide inhibition with the SOD mimetic EUK-8 improves cardiac functional recovery after I/R in the aged female rat. Sprague-Dawley rats (12-14 mo) were used as follows: intact (n = 6), ovariectomized + placebo (OVX, n = 6), OVX + EUK-8 (EUK-8, 3 mg/kg, n = 6), and OVX + estrogen (1.5 mg/pellet, 60 days release, n = 6). Perfused isolated hearts were subjected to global ischemia (25 min) followed by reperfusion (40 min). Functional recovery after I/R and myocardial protein expression of NADPH oxidase (p22, p67, and gp91(phox)), inducible nitric oxide synthase (NOS), endothelial NOS, and SOD1, as well as nitrotyrosine levels (as a marker for peroxynitrite), were assessed. Compared with OVX, EUK-8 and estrogen markedly improved functional recovery after I/R, which was associated with a decrease in NADPH oxidase expression and nitrotyrosine staining. However, estrogen increased inducible NOS expression, whereas EUK-8 had little effect. There were no significant changes in endothelial NOS and SOD1 expression among the groups. These results indicate that EUK-8 and estrogen improved cardiac recovery after I/R. Given the controversy surrounding hormone replacement therapy, EUK-8 may be an alternative to estrogen in protecting those at risk for myocardial ischemia in the aging population.     

Alpha2-adrenergic regulation of NO production alters postoperative intestinal smooth muscle dysfunction in rodents

Alpha2-adrenergic receptor activation plays an important role in the development of postoperative ileus. Alpha2-adrenergic receptors also regulate nitric oxide (NO) production by the mononuclear phagocyte system. We have previously shown that intestinal manipulation leads to a significant increase in NO production by infiltrating monocytes within the intestinal muscularis. The purpose of this study was to investigate whether alpha2-adrenergic blockade with yohimbine would alter postsurgical intestinal smooth muscle dysfunction and NO production by infiltrating monocytes and macrophages within the intestinal muscularis. Rats underwent small bowel intestinal manipulation with or without yohimbine. In vivo gastrointestinal transit and in vitro jejunal circular muscle contractility was measured 24 h postoperatively. RT-PCR was used to detect inducible NO synthase (iNOS) expression. NO levels in tissue culture supernatants were measured. Immunohistochemistry was used to localize alpha2-adrenergic receptor expression in the intestinal muscularis. Yohimbine significantly decreased manipulation-induced delay in gastrointestinal transit and reversed the postoperative decrease in intestinal muscle contractility. Intestinal manipulation resulted in significant iNOS mRNA induction in the intestinal muscularis, which was markedly attenuated after yohimbine treatment. Yohimbine also significantly decreased the postoperative increase in NO released into intestinal muscularis tissue culture supernatant. Immunohistochemistry identified alpha2-adrenergic receptors on monocytes recruited postoperatively into the intestinal muscularis. This study demonstrates that alpha2-adrenergic receptor stimulation of the inflamed postoperative intestinal muscularis plays a significant role in aggravating postoperative ileus through an enhanced induction of iNOS mRNA and increased release of NO from manipulated intestinal muscularis.     

Imidazo[1,2-b]pyridazines: a potent and selective class of cyclin-dependent kinase inhibitors

Modification of imidazo[1,2-a]pyridine CDK inhibitors lead to identification of less lipophilic imidazo[1,2-b]pyridazine series of CDK inhibitors. Although several equivalent compounds from these two series have similar structure and show similar CDK activity, the SAR of the two series differs significantly. Protein inhibitor structure determination has confirmed differences in binding mode and given some understanding of these differences in SAR. Potent and selective imidazo[1,2-b]pyridazine inhibitors of CDK2 have been identified, which show >1 microM plasma levels following a 2mg/kg oral dose to mice.