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Hereditary persistence of fetal hemoglobin is an uncommon, benign disorder in which the expression of gamma-globin genes persists into adult life. Several point mutations have been associated with the increased gamma-globin gene promoter activity. We evaluated the -195 (C->G) mutation by a functional in vitro assay based on the luciferase reporter gene system. The results indicated that the increased promoter activity observed in vivo could not be reproduced in vitro under the conditions employed, suggesting that other factors may be involved in the overexpression of the gamma-globin gene containing the -195 (C->G) mutation. Furthermore, this is the first time that the -195 (C->G) mutation of the Agamma-globin gene has been evaluated by in vitro gene expression.

Restriction fragment length polymorphism (RFLP) was used to examine the extent of mtDNA polymorphism among six strains of rats (Rattus norvegicus) - Wistar, Wistar Munich, Brown Norway, Wistar Kyoto, SHR and SHR-SP. A survey of 26 restriction enzymes has revealed a low level of genetic divergence among strains. The sites of cleavage by EcoRI, NcoI and XmnI were shown to be polymorphic. The use of these three enzymes allows the 6 strains to be classified into 4 haplotypes and identifies specific markers for each one. The percentage of sequence divergence among all pairs of haplotypes ranged from 0.035 to 0.33%, which is the result of a severe population constriction undergone by the strains. These haplotypes are easily demonstrable and therefore RFLP analysis can be employed for genetic monitoring of rats within animal facilities or among different laboratories.

Sympathetic hyperactivity (SH) and renin angiotensin system (RAS) activation are commonly associated with heart failure (HF), even though the relative contribution of these factors to the cardiac derangement is less understood. The role of SH on RAS components and its consequences for the HF were investigated in mice lacking U2A and OL2c adrenoceptor knockout ([[alpha].sub.2A]/[[alpha].sub.2C]ARKO) that present SH with evidence of HF by 7 mo of age. Cardiac and systemic RAS components and plasma norepinephrine (PN) levels were evaluated in male adult mice at 3 and 7 mo of age. In addition, cardiac morphometric analysis, collagen content, exercise tolerance, and hemodynamic assessments were made. At 3 mo, [[alpha].sub.2A]/[[alpha].sub.2C]ARKO mice showed no signs of HF, while displaying elevated PN, activation of local and systemic RAS components, and increased cardiomyocyte width (16%) compared with wild-type mice (WT). In contrast, at 7 too, [[alpha].sub.2A]/[[alpha].sub.2C] ARKO mice presented clear signs of HF accompanied only by cardiac activation of angiotensinogen and ANG II levels and increased collagen content (twofold). Consistent with this local activation of RAS, 8 wk of ANG II [AT.sub.1] receptor blocker treatment restored cardiac structure and function comparable to the WT. Collectively, these data provide direct evidence that cardiac RAS activation plays a major role underlying the structural and functional abnormalities associated with a genetic SH-induced HF in mice. sympathetic nervous system; AT1 receptor blocker; [[alpha].sub.2a]/[[alpha].sub.2c] adrenergic knockout mice

We evaluated the effects of swimming and anabolic steroids (AS) on ventricular function, collagen synthesis, and the local renin-angiotensin system in rats. Male Wistar rats were randomized into control (C), steroid (S; nandrolone decanoate; 5 mg/kg sc, 2x/wk), steroid + losartan (SL; 20 mg x [kg.sup.-1] x [day.sup.-1]), trained (T), trained + steroid (T+S), and trained + steroid + losartan (T+SL; n = 14/group) groups. Swimming was performed 5 times/wk for 10 wk. Serum testosterone increased in S and T+S. Resting heart rate was lower in T and T+S. Percent change in left ventricular (LV) weight-to-body weight ratio increased in S, T, and T+S. LV systolic pressure declined in S and T+S. LV contractility increased in T (P < 0.05). LV relaxation increased in T (P < 0.05). It was significantly lower in T+S compared with C. Collagen volumetric fraction (CVF) and hydroxyproline were higher in S and T+S than in C and T (P < 0.05), and the CVF and LV hypertrophy were prevented by losartan treatment. LV-ANG I-converting enzyme activity increased (28%) in the S group (33%), and type III collagen synthesis increased (56%) in T+S but not in T group. A positive correlation existed between LV-ANG I-converting enzyme activity and collagen type III expression ([r.sup.2] = 0.88; P < 0.05, for all groups). The ANG II and angiotensin type 1a receptor expression increased in the S and T+S groups but not in T group. Supraphysiological doses of AS exacerbated the cardiac hypertrophy in exercise-trained rats. Exercise training associated with AS induces maladaptive remodeling and further deterioration in cardiac performance. Exercise training associated with AS causes loss of the beneficial effects in LV function induced by exercising. These results suggest that aerobic exercise plus AS increases cardiac collagen content associated with activation of the local renin-angiotensin system. cardiac hypertrophy; collagen; renin-angiotensin system; ventricular function

A study was conducted to characterize a sensitive adenosine 3',5'-cyclic monophosphate (cAMP) responsive endothelial cell line. The cell lien was determined through a stable transfection of a luciferase enhancer trap into the aortic endothelial cells of rabbits. Results indicated that a cAMP-responsive endothelial cell line supporting a luciferase reporter gene could be utilized to analyze patterns of gene activation by cAMP and by Ca2+ after complex stimuli.

Tubular fluids collected from rat nephron and urine were analyzed to determine the activity of the angiotensin-converting enzyme (ACE) or kinninase II on kidney function. Analysis of rat renal tubular fluid based on micropuncture techniques indicated the presence of ACE activity in the initial and terminal parts of the nephron. However, ACE activity decreased in the early proximal tubule and late distal tubule. Furthermore, ACE activity was modified along the distal nephron of rat kidneys.

Takahashi T, Schreiber R, Krieger JE, Saad STO, Costa FF. Analysis of the mechanism of action of the Brazilian type (Aγ−195 C → G) of hereditary persistence of fetal hemoglobin. Eur J Haematol 2003: 71: 418–424. © Blackwell Munksgaard 2003. We report an in vitro expression study of the A γ-globin gene promoter containing the A γ−195 C → G mutation that causes the Brazilian type of hereditary persistence of fetal hemoglobin (HPFH). To demonstrate that this mutation results in increased promoter strength, we evaluated the mutant promoter linked to the hypersensitive site-2 of the locus control region with the luciferase reporter gene system and examined protein interactions by eletrophoretic mobility shift assay. The transient expression was studied in three cell lines: K562, HEL and 293, and indicated increased promoter activity of the promoter containing the Brazilian mutation in all cell lines. The protein–DNA interaction showed that, in contrast to the A γ−198 T → C mutation which has increased affinity for the Sp1 protein and creates a motif that behaves like a novel CACCC box in the γ promoter, the Brazilian HPFH mutation decreases the affinity at the Sp1 protein and does not act as a CACCC motif. These results suggest that this mutation may act to increase the A γ-globin chain production. In addition, the mechanism by which this increased production occurs is different to that of the −198 mutation. Other proteins may be involved in the overexpression of the γ-globin chain and/or may be dependent upon the DNA structure. [ABSTRACT FROM AUTHOR]

Studies the association and linkage between bipolar affective disorder (BPAD) and the polymorphic CAG/CTG repeat loci. Implication of the presence of anticipation in bipolar affective disorder; Association of BPAD and longer CAG/CTG repeats; Result of the extended transmission disequilibrium test performed in the controls.