Crystal structure of tyrosine hydroxylase at 2.3 A and its implications for inherited neurodegenerative diseases

Tyrosine hydroxylase (TyrOH) catalyzes the conversion of tyrosine to L-DOPA, the rate-limiting step in the biosynthesis of the catecholamines dopamine, adrenaline, and noradrenaline. TyrOH is highly homologous in terms of both protein sequence and catalytic mechanism to phenylalanine hydroxylase (PheOH) and tryptophan hydroxylase (TrpOH). The crystal structure of the catalytic and tetramerization domains of TyrOH reveals a novel alpha-helical basket holding the catalytic iron and a 40 A long anti-parallel coiled coil which forms the core of the tetramer. The catalytic iron is located 10 A below the enzyme surface in a 17 A deep active site pocket and is coordinated by the conserved residues His 331, His 336 and Glu 376. The structure provides a rationale for the effect of point mutations in TyrOH that cause L-DOPA responsive parkinsonism and Segawa's syndrome. The location of 112 different point mutations in PheOH that lead to phenylketonuria (PKU) are predicted based on the TyrOH structure.     

99Tcm-MDP scintigraphy in high-voltage electrical burn patients

In high-voltage electrical burn injuries (> 1000 V), it is difficult to identify the site and extent of non-viable deep tissue damage for debridement to avoid further tissue injury from wound infection and the risk of sepsis. This prospective study was designed to evaluate the usefulness of 99Tcm-methylene di-phosphonate (99Tcm-MDP) scintigraphy in detecting the extent of tissue injury and determining the level of amputation required for electrical burn patients. Over a 5 year period, 33 high-voltage electrical burn patients were studied. Blood flow and blood pool studies revealed absent perfusion in 37 limbs, all of which eventually were amputated. In addition to a routine three-phase bone scan, images were obtained at 30-60 min (early images) to evaluate whether soft tissue injury could be detected better at that time. For comparison of the detection rate from the early images and bone (delayed) images, 164 corresponding spot views of both images were reviewed. Eighty-three and 125 tissue necrotic lesions were demonstrated by the early images and bone images respectively. All of the 83 lesions found by the early images were more clearly identified by the bone images. All but one of the 125 lesions underwent surgical debridement or amputation. We concluded that the blood flow and blood pool images correlated well with the level of amputation required. The site and extent of tissue necrotic lesions can be clearly identified on 99Tcm-MDP bone scans. Because the early images were less sensitive in detecting tissue necrosis, we suggest that early imaging is not necessary.     

The ability of the rat to metabolize myristoyl-methionine: an acylamino acid with potentially useful antibacterial properties

Two experiments with Sprague Dawley rats tested their ability to hydrolyse myristoyl-methionine (M-M) into myristic acid and L-methionine (M). In the first experiment, lasting for 3 days. male rats were orally administered [9,10-3H]myristoyl-L-[35S]methionine. The recovery of radioactivity was approximately 90% for both isotopes; 19% of the administered 3H was recovered in the urine and 16% in the faeces, while the recovered 35S activity was 13 and 12%, respectively. The balance of the radioactivity was found among the tissues, organs and blood. In the second experiment, male and female rats received soybean-based diets which were supplemented with either 0.305% M-M or 0.2% M (both diets contained equal amounts of M) for periods up to 4 weeks. The growth rate of the rats receiving the 0.305% M-M diets was slightly slower than that for the rats on the 0.2% M diet, but the difference was not statistically significant (P > 0.05). The M-M rats had a transitory decrease in feed consumption, suggesting that palatability may have contributed to the growth difference and that a somewhat greater amount of M-M was necessary for the rat to attain the same growth rate as that produced by 0.2% M. When the amount of dietary M-M was increased to 3.05% M-M, a greater reduction in feed consumption and body weight gain was observed. This latter diet was an initial attempt to study the potential toxicity of M-M. None of the haematological, clinical chemistry or organ weight data suggested that M-M was overtly toxic per se, but longer-term feeding studies are needed to evaluate the potential toxicity of M-M more fully.     

The gp200-MR6 molecule which is functionally associated with the IL-4 receptor modulates B cell phenotype and is a novel member of the human macrophage mannose receptor family

The human gp200-MR6 molecule has previously been shown to have either an antagonistic or agonistic effect on IL-4 function, demonstrated by inhibition of IL-4-induced proliferation of T cells or mimicking of IL-4-induced maturation of epithelium, respectively. We now show that gp200-MR6 ligation can also mimic IL-4 and have an anti-proliferative pro-maturational influence within the immune system, causing up-regulation of co-stimulatory molecules on B lymphocytes. Biochemical analysis and cDNA cloning reveal that gp200-MR6 belongs to the human macrophage mannose receptor family of multidomain molecules. It comprises 1722 amino acids in toto (mature protein, 1695 amino acids; signal sequence, 27 amino acids) organized into 12 external domains (an N-terminal cysteine-rich domain, a fibronectin type II domain and 10 C-type carbohydrate recognition domains), a transmembrane region and a small cytoplasmic C terminus (31 amino acids) containing a single tyrosine residue (Y1679), but no obvious kinase domain. Strong amino acid sequence identity (77%) suggests that gp200-MR6 is the human homologue of the murine DEC-205, indicating that this molecule has much wider functional activity than its classical endocytic role. We also show that the gp200-MR6 molecule is closely associated with tyrosine kinase activity; the link between gp200-MR6 and the IL-4 receptor may therefore be via intracellular signaling pathways, with multifunctionality residing in its extracellular multidomain structure.     

Postrepolarization refractoriness versus conduction slowing caused by class I antiarrhythmic drugs: antiarrhythmic and proarrhythmic effects

BACKGROUND: Conduction block may be both antiarrhythmic and proarrhythmic. Drug-induced postrepolarization refractoriness (PRR) may prevent premature excitation and tachyarrhythmia induction. The effects of propafenone and procainamide on these parameters, and their antiarrhythmic or proarrhythmic consequences, were investigated. METHODS AND RESULTS: In 11 isolated Langendorff-perfused rabbit hearts, monophasic action potentials (MAPs) were recorded simultaneously from six to seven different right and left ventricular sites, along with a volume-conducted ECG. All recordings were used to discern ventricular tachycardia (VT) or ventricular fibrillation (VF) induced by repetitive extrastimulation (S2-S5) or 10-second burst stimulation at 25 to 200 Hz at baseline and after addition of procainamide (20 micromol/L) or propafenone (1 micromol/L) to the perfusate. MAPs were analyzed for action potential duration at 90% repolarization (APD90), conduction times (CT) between the pacing site and the other MAPs, and PRR (effective refractory period-APD90=PRR) and related to the induction of VT or VF. During steady-state pacing, procainamide and propafenone prolonged APD90 by 12% and 14%, respectively. Procainamide slowed mean CT by 40% during S2-S5 pacing, whereas propafenone slowed mean CT by up to 400% (P<0.001 versus baseline and procainamide). Wavelength was not changed significantly by procainamide but was shortened fourfold by propafenone at S5. Both drugs produced PRR, which was associated with a 70% decrease in VF inducibility with procainamide and elimination of VF with propafenone. Despite this protection from VF, monomorphic VT was induced with propafenone in 57% of burst stimulations. CONCLUSIONS: Drug-induced PRR protects against VF induction. Propafenone promotes slow monomorphic VT, probably by use-dependent conduction slowing and wavelength shortening.     

Differential acetylcholine and GABA release from cultured chick retina cells

In the present work we investigated the mechanisms controlling the release of acetylcholine (ACh) and of gamma-aminobutyric acid (GABA) from cultures of amacrine-like neurons, containing a subpopulation of cells which are simultaneously GABAergic and cholinergic. We found that 81.2 +/- 2.8% of the cells present in the culture were stained immunocytochemically with an antibody against choline acetyltransferase, and 38.5 +/- 4.8% of the cells were stained with an antibody against GABA. Most of the cells containing GABA (87.0 +/- 2.9%) were cholinergic. The release of acetylcholine and GABA was mostly Ca2+-dependent, although a significant release of [3H]GABA occurred by reversal of its transporter. Potassium evoked the Ca2+-dependent release of [3H]GABA and [3H]acetylcholine, with EC50 of 31.0 +/- 1.0 mm and 21.6 +/- 1.1 mm, respectively. The Ca2+-dependent release of [3H]acetylcholine was significantly inhibited by 1 micrometer tetrodotoxin and by low (30 nm) omega-conotoxin GVIA (omega-CgTx GVIA) concentrations, or by high (300 nm) nitrendipine (Nit) concentrations. On the contrary, the release of [14C]GABA was reduced by 30 nm nitrendipine, or by 500 nm omega-CgTx GVIA, but not by this toxin at 30 nm. The release of either transmitters was unaffected by 200 nm omega-Agatoxin IVA (omega-Aga IVA), a toxin that blocks P/Q-type voltage-sensitive Ca2+ channels (VSCC). The results show that Ca2+-influx through omega-CgTx GVIA-sensitive N-type VSCC and through Nit-sensitive L-type VSCC induce the release of ACh and GABA. However, the significant differences observed regarding the Ca2+ channels involved in the release of each neurotransmitter suggest that in amacrine-like neurons containing simultaneously GABA and acetylcholine the two neurotransmitters may be released in distinct regions of the cells, endowed with different populations of VSCC.     

Mortality and platelet depletion occur independently of fibrinogen consumption in murine models of tumour necrosis factor-mediated systemic inflammatory responses

The link between left ventricular dysfunction and arrhythmogenesis is commonly known. However, so far, only the systolic left ventricular dysfunction has been evaluated. Because of the controversial results of those studies, we decided to assess if is there a link between late potentials (LP) and left ventricular diastolic dysfunction. Our material consisted of 56 patients: 11 women and 45 men, mean age was 61.12 +/- 10.07 years. Signal averaged ECG and ECHO were performed in each patient, 2-3 months after myocardial infarction. For high pass filter of 40 Hz, LP were defined as 2 or 3 abnormal SAECG variables (the averaged QRS > 114 ms, the low amplitude signal duration LAS > 38 ms and root mean square voltage of the terminal 40 ms RMS40 < 20 microV). During ECHO study, we assessed E and A waves E/A ratio, left ventricular end-diastolic volume (LVEDV), ejection fraction (EF), acceleration (AT) and deceleration times (DT). The patients were divided into 2 groups: group I-30 patients LP positive and group II-26 patients LP negative. There were no significant differences between the groups in terms of age, EF, and heart rate. We presented significant differences between group I and II in terms of E wave velocity (0.75 +/- 0.19 vs 0.64 +/- 0.19 p < 0.03) E/A ratio (2.13 +/- 1.56 vs 1.0 +/- 0.5 p < 0.05) respectively. We did not confirm significant differences as regards A wave velocity, AT, isovolumetric time (IVRT) and LVEDV between both tested groups. In group I we revealed a significant correlation between E wave (r = 0.45), E/A ratio (r = 0.62), AT (r = -0.42) E/A ratio (r = 0.56), DT (r = 0.55) and QRS, as well as DT and LPD (r = 0.40) and between IVRT and RMS40 (r = -0.43). The results of our study suggest that in patients after myocardial infarction: 1/incidence of LP depends on the degree of left ventricular filling pattern like in impaired relaxion, quite well correlated with filtered QRS time 3/in LP positive patients there was predominance of restrictive left ventricular filling pattern, quite well correlation with RMS40 amplitude.