The alcohol deprivation effect in the alcohol-preferring P rat under free-drinking and operant access conditions

The alcohol-deprivation effect (ADE) was examined under 4-hr operant and 24-hr free-choice alcohol access in the alcohol-preferring (P) rat after deprivation intervals from 2 to 4 weeks. Results indicated that adult male P rats responding for 6 weeks on a concurrent FR-5/FR-1 schedule of reinforcement for alcohol and water, respectively, and then deprived of alcohol for 2 weeks, demonstrated a 40% increase in alcohol responding during the first 60 min of alcohol reinstatement. The alcohol deprivation effect was temporary, however, as responding did not differ from baseline levels on the second day of reinstatement. In a second experiment, weanling male and female P rats received 7 weeks of continuous access to alcohol, beginning at 21 days of age, and were then deprived of alcohol for 4 weeks. On the first day of alcohol reinstatement, P rats exhibited a 40% to 45% increase from baseline alcohol drinking levels, with alcohol intake returning to baseline levels by the 3rd day of reinstatement. Although alcohol intake was higher in females than in males when adjustment was made for body weight, there were no gender differences in the magnitude of the alcohol deprivation effect. Taken together, these results indicate that the ADE is a long-lasting phenomenon that occurs under both operant and continuous access conditions in the P rat, and thus these rats may be useful models for the study of factors involved in relapse of alcohol drinking.     

Pathogenesis of an infectious mononucleosis-like disease induced by a murine gamma-herpesvirus: role for a viral superantigen?

The murine gamma-herpesvirus 68 has many similarities to EBV, and induces a syndrome comparable to infectious mononucleosis (IM). The frequency of activated CD8+ T cells (CD62L(lo)) in the peripheral blood increased greater than fourfold by 21 d after infection of C57BL/6J (H-2(b)) mice, and remained high for at least a further month. The spectrum of T cell receptor usage was greatly skewed, with as many as 75% of the CD8+ T cells in the blood expressing a Vbeta4+ phenotype. Interestingly, the Vbeta4 dominance was also seen, to varying extents, in H-2(k), H-2(d), H-2(u), and H-2(q) strains of mice. In addition, although CD4 depletion from day 11 had no effect on the Vbeta4 bias of the T cells, the Vbeta4+CD8+ expansion was absent in H-2IA(b)-deficient congenic mice. However, the numbers of cycling cells in the CD4 antibody-depleted mice and mice that are CD4 deficient as a consequence of the deletion of MHC class II, were generally lower. The findings suggest that the IM-like disease is driven both by cytokines provided by CD4+ T cells and by a viral superantigen presented by MHC class II glycoproteins to Vbeta4+CD8+ T cells.     

Integrated MRI assessment of regional function and perfusion in canine myocardial infarction

A single integrated examination using regional measurements of perfusion from contrast-enhanced MRI and three-dimensional (3D) strain from tissue-tagged MRI was developed to differentiate infarcted myocardium from adjacent tissue with functional abnormalities. Ten dogs were studied at baseline and 10 days after a 2-hour occlusion of the left anterior descending coronary artery (LAD). Strain was determined using a 3D finite element model. Two-dimensional measurements of hypoenhancing regions were highly correlated with myocardial viability (r = 0.96). Signal intensity versus time curves obtained from contrast-enhanced MRI were used for quantitative perfusion analysis. The remote and adjacent noninfarcted tissue of the dogs with LAD occlusion, as well as the infarcted tissue, exhibited abnormal deformation patterns as compared to normal dogs (positive predictive value (PPV) of strain determination of infarction = 66%). Integration of contrast-enhanced MRI results with 3D strain analysis enabled the delineation of the myocardial infarction (PPV = 100%) from functionally compromised myocardium. This integrated cardiac examination shows promise for noninvasive serial assessment of potentially jeopardized noninfarcted myocardium to study the process of infarct remodeling and expansion.     

Evidence for a Turner syndrome locus or loci at Xp11.2-p22.1

Turner syndrome is the complex human phenotype associated with complete or partial monosomy X. Principle features of Turner syndrome include short stature, ovarian failure, and a variety of other anatomic and physiological abnormalities, such as webbed neck, lymphedema, cardiovascular and renal anomalies, hypertension, and autoimmune thyroid disease. We studied 28 apparently nonmosaic subjects with partial deletions of Xp, in order to map loci responsible for various components of the Turner syndrome phenotype. Subjects were carefully evaluated for the presence or absence of Turner syndrome features, and their deletions were mapped by FISH with a panel of Xp markers. Using a statistical method to examine genotype/phenotype correlations, we mapped one or more Turner syndrome traits to a critical region in Xp11.2-p22.1. These traits included short stature, ovarian failure, high-arched palate, and autoimmune thyroid disease. The results are useful for genetic counseling of individuals with partial monosomy X. Study of additional subjects should refine the localization of Turner syndrome loci and provide a rational basis for exploration of candidate genes.     

Protein thermostability above 100 degreesC: a key role for ionic interactions

The discovery of hyperthermophilic microorganisms and the analysis of hyperthermostable enzymes has established the fact that multisubunit enzymes can survive for prolonged periods at temperatures above 100 degreesC. We have carried out homology-based modeling and direct structure comparison on the hexameric glutamate dehydrogenases from the hyperthermophiles Pyrococcus furiosus and Thermococcus litoralis whose optimal growth temperatures are 100 degreesC and 88 degreesC, respectively, to determine key stabilizing features. These enzymes, which are 87% homologous, differ 16-fold in thermal stability at 104 degreesC. We observed that an intersubunit ion-pair network was substantially reduced in the less stable enzyme from T. litoralis, and two residues were then altered to restore these interactions. The single mutations both had adverse effects on the thermostability of the protein. However, with both mutations in place, we observed a fourfold improvement of stability at 104 degreesC over the wild-type enzyme. The catalytic properties of the enzymes were unaffected by the mutations. These results suggest that extensive ion-pair networks may provide a general strategy for manipulating enzyme thermostability of multisubunit enzymes. However, this study emphasizes the importance of the exact local environment of a residue in determining its effects on stability.     

Familial Mediterranean fever at the millennium. Clinical spectrum, ancient mutations, and a survey of 100 American referrals to the National Institutes of Health

Regarded as the most common and best understood of the hereditary periodic fever syndromes, familial Mediterranean fever (FMF) is a recessively inherited disease of episodic fever with some combination of severe abdominal pain, pleurisy, arthritis, and a characteristic ankle rash. The flares typically last for up to 3 days at a time, and most patients are completely asymptomatic between attacks; if untreated with prophylactic colchicine, some patients later develop amyloidosis and renal failure. The recent cloning of the FMF gene on the short arm of chromosome 16p, and the subsequent finding that its tissue expression is limited to granulocytes, has helped to explain the dramatic accumulation of neutrophils at the symptomatic serosal sites; the wild-type gene likely acts as an upregulator of an anti-inflammatory molecule or as a downregulator of a pro-inflammatory molecule. For nearly half a century, FMF was thought to cluster primarily in non-Ashkenazi Jews, Arabs, Armenians, and Turks, although the screening of the 8 known mutations in an American cohort has identified substantial numbers of people from the Ashkenazi Jewish and Italian populations in the United States who also have this disease. Nevertheless, the symptoms often go unrecognized and patients remain undiagnosed for years, not receiving the highly efficacious colchicine therapy; their histories often include multiple laparotomies, laparoscopies, and psychiatric evaluations. The combinations of clinical manifestations among FMF patients are quite heterogeneous, but our American cohort did not establish any connections between individual mutations and specific clinical pictures--as is seen in other diseases like cystic fibrosis, in which distinct genotypes target certain organ systems. Specifically, the data from our American series are insufficient to evaluate the hypothesis that the M694V/M694V genotype confers a more severe phenotype, or increases the risk of amyloidosis; but both our data and the recent literature (160) indicate that amyloidosis can occur in FMF patients with only 1 copy, or no copies, of the M694V mutation. It appears that specific MEFV mutations are probably not the sole determinants of phenotype, and that unknown environmental factors or modifying genes act as accomplices in this disease. Although we hope the discovery of the FMF gene will allow the diagnosis of FMF to become genetically accurate, the reality is that both clinical and genetic tools must still be used together unless mutations are identified on both of a patient's chromosomes. Physicians should be careful not to rule out the diagnosis in patients of high-risk ethnic backgrounds just because of atypical clinical features, as our data indicate that MEFV mutations are sometimes demonstrable in such patients. At the same time, physicians cannot yet rely solely on a genetic diagnosis because we have not yet identified a sufficient spectrum of mutations, and it is not currently feasible to examine every patient's full DNA sequence for the entire gene; screening an ethnically consistent and clinically positive patient for the 8 known mutations frequently identifies a mutation on only 1 chromosome, and genetic analysis of other classic cases will often reveal none of the 8 mutations. Still, our data suggest that ethnic background is an important predictor of finding 1 of the presently known mutations, and the knowledge of ancestries atypical for FMF can suggest the diagnosis of other hereditary periodic fever syndromes. As the list of FMF-associated MEFV mutations is expanded, and/or new sequencing technologies permit more rapid screening, the value and interpretation of genetic testing for FMF will become more straightforward. Moreover, as the pathophysiology of this disorder becomes less of a hypothesis and more of an understood entity, it is likely that treatment options will broaden beyond the use of daily prophylactic colchicine. (ABSTRACT TRUNCATED)     

Molecular mapping with functional antibodies localizes critical sites on the human IL receptor common gamma (gamma c) chain

The IL receptor common gamma (gamma c) chain is required for the formation of high affinity cytokine receptor complexes for IL-2, IL-4, IL-7, IL-9, and IL-15, and for signals regulating cell survival, growth, and differentiation. Our current understanding of how gamma c chain associates with multiple ligands and receptor subunits is drawn largely from its structural homology to the human growth hormone (hGH) receptor and known structure of the hGH/hGH receptor complex. These receptors share distinct features in their extracellular portions and are believed to function by a mechanism of ligand-induced association of receptor subunits. Here, we report the first directed mutational analysis of the human gamma c chain by alanine scanning conducted across seven regions likely to contain residues required for intermolecular contact. Functionally distinct, neutralizing anti-gamma c mAbs were employed to define critical residues. One particular mAb, CP.B8, unique in its ability to inhibit IL-2-, IL-4-, IL-7-, and IL-15-induced proliferation and high affinity cytokine binding of normal T cells as an intact mAb and as a Fab fragment, localized critical residues to four noncontinuous stretches, namely residues in loops AB and EF of domain 1, in the interdomain segment, and in loop FG of domain 2. Notably, these residues form a contiguous patch on the gamma c chain surface in a three-dimensional structural model. These results provide functional evidence for the location of contact points on gamma c chain required for its association with multiple ligands.