Effects of an expanded cardiac rehabilitation programme in patients treated for an acute myocardial infarction or a coronary artery by-pass graft operation.

OBJECTIVE: To investigate the effects of expanded cardiac rehabilitation with multifactorial interventions on metabolic and inflammatory markers, exercise performance and on established cardiovascular risk factors. DESIGN: Single-centre prospective randomized controlled trial. SETTING: A university hospital. SUBJECTS: Two hundred and twenty-four patients with an acute myocardial infarction or patients undergoing coronary artery by-pass grafting. INTERVENTION: Patients were randomized to expanded cardiac rehabilitation including stress management, increased physical training, staying at a 'patient hotel' and cooking sessions, or to usual cardiac rehabilitation. MAIN MEASURES: Biochemical risk markers and exercise performance; follow-up was one year. RESULTS: There were no significant differences between the two treatment groups in the changes of biochemical risk markers or in exercise performance. Thus, low-density lipoprotein (LDL)-cholesterol levels decreased from 3.00 (0.97) to 2.54 (0.66) mmol/L in the intervention group and from 3.20 (0.85) to 2.54 (0.63) mmol/L in the control group, fibrinogen levels decreased from 5.30 (2.00) to 4.25 (1.01) g/L in the intervention group and from 5.29 (1.89) to 4.33 (0.83) g/L in the control group and C-reactive protein (CRP) levels decreased from 3.04 (2.79) to 2.09 (2.13) mg/L in the intervention group and from 4.01 (3.49) to 2.39 (2.49) mg/L in the control group. Total workload (W) improved from 118 (35) to 136 (34) in the intervention group and from 117 (36) to 133 (39) in the control group. CONCLUSION: There was no further significant benefit in biochemical risk markers or in exercise performance among patients undergoing the expanded rehabilitation as compared to the control group which received usual cardiac rehabilitation.

Protein kinase C phosphorylates and activates GTP cyclohydrolase I in rat renal mesangial cells.

GTP cyclohydrolase I is the rate-limiting enzyme in the de novo synthesis pathway of tetrahydrobiopterin which is an essential cofactor for all NO synthase isoforms. The expression of GTP cyclohydrolase I is regulated on a transcriptional level by a variety of cytokines like interleukin 1beta or tumor necrosis factor alpha. The present paper reports that stimulation of protein kinase C by angiotensin II, platelet-derived growth factor BB or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate triggers the phosphorylation and activation of GTP cyclohydrolase I. These data establish that in addition to transcriptional regulation, there is a prominent post-transcriptional modulation of enzyme activity.

Rat GTP cyclohydrolase I is a homodecameric protein complex containing high-affinity calcium-binding sites.

Recombinant rat liver GTP cyclohydrolase I has been prepared by heterologous gene expression in Escherichia coli and characterized by biochemical and biophysical methods. Correlation averaged electron micrograph images of preferentially oriented enzyme particles revealed a fivefold rotational symmetry of the doughnut-shaped views with an average particle diameter of 10 nm. Analytical ultracentrifugation and quantitative scanning transmission electron microscopy yielded average molecular masses of 270 kDa and 275 kDa, respectively. Like the Escherichia coli homolog, these findings suggest that the active enzyme forms a homodecameric protein complex consisting of two fivefold symmetric pentameric rings associated face-to-face. Examination of the amino acid sequence combined with calcium-binding experiments and mutational analysis revealed a high-affinity, EF-hand-like calcium-binding loop motif in eukaryotic enzyme species, which is absent in bacteria. Intrinsic fluorescence measurements yielded an approximate dissociation constant of 10 nM for calcium and no significant binding of magnesium. Interestingly, a loss of calcium-binding capacity observed for two rationally designed mutations within the presumed calcium-binding loop of the rat GTP cyclohydrolase I yielded a 45% decrease in enzyme activity. This finding suggests that failure of calcium binding may be the consequence of a mutation recently identified in the causative GTP cyclohydrolase I gene of patients suffering from dopa responsive dystonia.

Molecular mechanisms of inducible nitric oxide synthase gene expression by IL-1beta and cAMP in rat mesangial cells.

Expression of the inducible nitric oxide synthase (iNOS) gene in rat mesangial cells is differentially triggered by IL-1beta and cAMP predominantly at the transcriptional level. The 5'-flanking region of the rat iNOS gene contains several binding sites for transcription factors potentially involved in cytokine and cAMP signaling such as nuclear factor-kappaB/Rel, CCAAT/enhancer-binding protein (C/EBP), and cyclic AMP-responsive element-binding protein/ATF. We tested promoter activities of serial and site-directed deletion mutants of iNOS-chloramphenicol acetyltransferase reporter genes after transient transfection and stimulation of mesangial cells. A region between bp -277 and -111 bearing a CCAAT/enhancer-binding protein-response element was found to be critical for cAMP-mediated gene induction but dispensable for IL-1beta inducibility. Moreover, a minimal promoter ranging from the transcriptional start site up to -111 containing a kappaB site is sufficient to confer IL-1beta-mediated iNOS promoter activation. Consistent with these findings, an electrophoretic mobility shift assay shows the appearance of an IL-1beta-inducible nuclear factor-kappaB p50/p65 heterodimeric complex. Using probes containing C/EBP-binding sites from the iNOS gene revealed further binding of different complexes, all of which were strongly inducible by cAMP and to a lower extent also by IL-1beta. Abs against cyclic AMP-responsive element-binding protein, C/EBPbeta, and C/EBPdelta were able to partially supershift single complexes, suggesting the participation of these transcription factors in the regulation of iNOS gene expression by cAMP and IL-1beta. Finally, we show that both cAMP and IL-1beta strongly induce steady-state levels of C/EBPbeta and C/EBPdelta mRNA levels. These data demonstrate that IL-1beta and cAMP use distinct as well as partially overlapping sets of transcriptional activators to modulate iNOS gene expression in rat mesangial cells.

Differential effect of dexamethasone on interleukin 1beta- and cyclic AMP-triggered expression of GTP cyclohydrolase I in rat renal mesangial cells.

1. Endogenous synthesis of tetrahydrobiopterin (BH4) is an essential requirement for cytokine-stimulated nitric oxide (NO) synthesis in rat mesangial cells. GTP cyclohydrolase I, the rate-limiting enzyme in BH4 synthesis, is expressed in renal mesangial cells in response to two principal classes of activating signals. These two groups of activators comprise inflammatory cytokines such as interleukin (IL)-1beta and agents that elevate cellular levels of cyclic AMP. 2. We examined the action of the potent anti-inflammatory drug dexamethasone on GTP cyclohydrolase I induction in response to IL-1beta and a membrane-permeable cyclic AMP analogue, N6, O-2'-dibutyryladenosine 3'-5'-phosphate (Bt2cyclic AMP). 3. Nanomolar concentrations of dexamethasone markedly attenuated IL-1beta-induced GTP cyclohydrolase I mRNA steady state level as well as IL-1beta-induced GTP cyclohydrolase I protein expression and enzyme activity. In contrast, dexamethasone did not inhibit Bt2cyclic AMP-triggered increase in GTP cyclohydrolase I mRNA level and protein expression, and low (1 nM) or high (1 and 10 microM) doses of dexamethasone consistently increased Bt2cyclic AMP-induced GTP cyclohydrolase activity. 4. In summary, these results suggest that glucocorticoids act at several levels, critically dependent on the stimulus used, to control GTP cyclohydrolase I expression.

Interleukin 1 beta and cAMP trigger the expression of GTP cyclohydrolase I in rat renal mesangial cells.

Endogenous synthesis of tetrahydrobiopterin (BH4) is an important requirement for cytokine-stimulated nitric oxide (NO) production in mesangial cells. We have shown that inducible NO synthase is expressed in mesangial cells in response to two principal classes of activating signals, inflammatory cytokines such as interleukin 1 beta (IL-1 beta) and agents that elevate cellular levels of cAMP [Kunz, Mühl, Walker and Pfeilschifter (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 5387-5391]. In the present paper we demonstrate that IL-1 beta and cAMP similarly increase the steady-state mRNA levels of GTP cyclohydrolase I (EC 3,5,4,16), the rate-limiting enzyme in BH4 biosynthesis, as measured by a sensitive and quantitative nuclease protection assay. Stimulation of cells with a combination of IL-1 beta plus cAMP revealed an additive induction profile of GTP cyclohydrolase I mRNA. Message stability studies established that GTP cyclohydrolase I mRNA induced by cAMP has a longer half-life than the IL-1 beta-induced message. Moreover, cAMP exposure markedly prolonged the half-life of GTP cyclohydrolase I mRNA, from 1.5 to 3.4 h. In a next step we generated a rabbit polyclonal antibody against rat GTP cyclohydrolase I expressed in Escherichia coli and demonstrated that IL-1 beta and cAMP elevated GTP cyclohydrolase I protein levels in mesangial cells. Furthermore, IL-1 beta and cAMP led to a marked increase in GTP cyclohydrolase I activity and to increased accumulation of biopterin in mesangial cells. Combinations of IL-1 beta and cAMP resulted in a synergistic stimulation of GTP cyclohydrolase I activity. This may suggest that, in addition to transcriptional and post-transcriptional regulation, there is a prominent post-translational modulation of enzyme activity.

Therapeutic strategies for the inhibition of inducible nitric oxide synthase--potential for a novel class of anti-inflammatory agents.

In recent years, NO, a gas previously considered a potentially toxic chemical, has become established as a diffusible universal messenger mediating cell-cell communication throughout the body. In mammals, NO is a recognized mediator of blood vessel relaxation that helps to maintain blood pressure. In the central nervous system NO acts as a non-conventional neurotransmitter and participates in the establishment of long-term plasticity required for memory formation. In addition, NO is responsible for some parts of the host response to sepsis and inflammation and contributes to certain disease states. A number of strategies have emerged with regard to a pharmacological control of pathological NO overproductions. This review will discuss these novel therapeutic approaches that may provide new means for clinical medicine.

[Disease course in 20 patients with an early diagnosis of phenylketonuria and hyperphenylalaninemia]. / Verlauf bei 20 Patienten mit frühentdeckter Phenylketonurie und Hyperphenylalaninämie.

Twenty patients with PKU or hyperphenylalaninemia at ages 0.1 to 15.6 years (median age 6.2 years) were studied prospectively. In all children the condition had been diagnosed when they were neonates on the basis of an abnormal Guthrie test. To maintain plasma phenylalanine levels between 0.2-0.5 mM, dietary restriction of phenylalanine to 20-80 mg/kg daily (median 40 mg/kg) was necessary in 14 children. In children above 8 years, however, these plasma levels were frequently exceeded. In 6 children plasma phenylalanine levels were higher than normal diet. Height, weight and head circumference were within normal range in all patients at all ages. Determinations of DQ/IQ were done at 2, 4, 6 and 8 years of age and revealed values between 90-120 with a median of 102 in the 14 patients who were tested. Only 1 patient had IQ levels between 75-85 and attended special school. Nine other patients were in grade school performing averagely or above. This study confirms that early treatment and long-term follow-up of patients with PKU yield good results. Unsolved problems include duration of dietary treatment and the management of pregnancy in women with PKU.