Regulation of tyrosine hydroxylase and aromatic L-amino acid decarboxylase by dopaminergic drugs

We provide evidence that dopamine receptors differentially modulate tyrosine hydroxylase and aromatic L-amino acid decarboxylase in the mouse striatum. The dopamine D1 receptor family (D1-like) antagonist, R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1 H-3-benazepine (SCH 23390), elevated aromatic L-amino acid decarboxylase activity and protein content in striatum, as well as the mRNA for the enzyme in midbrain. The dopamine D1-like receptor agonist, (+/-)-1-phenyl-2,3,4,5-tetrahydro-(1 H)-3-benzazepine-7,8-diol (SKF 38393), had no effect on aromatic L-amino acid decarboxylase. The dopamine D1-like drugs had no effect on tyrosine hydroxylase. In contrast, the dopamine D2 receptor family (D2-like) antagonists haloperidol and spiperone elevated both tyrosine hydroxylase and aromatic L-amino acid decarboxylase activities. The increase in aromatic L-amino acid decarboxylase activity was accompanied by elevated enzyme protein content but not mRNA. The dopamine D2-like receptor agonists, bromocriptine, quinpirole and (+/-)-7-hydroxydipropylaminotetralin (7-OH-DPAT), all decreased striatal tyrosine hydroxylase. Under the conditions used, bromocriptine and 7-OH-DPAT, but not quinpirole, decreased aromatic L-amino acid decarboxylase activity of striatum. Both the dopamine D1- and D2-like receptor antagonists enhanced the turnover of striatal dopamine to differing degrees, as judged by the ratio of acid metabolites of dopamine to dopamine. Taken together our results indicate that aromatic L-amino acid decarboxylase can be modulated independently of tyrosine hydroxylase.     

Role of aromatic L-amino acid decarboxylase for dopamine replacement by genetically modified fibroblasts in a rat model of Parkinson's disease

Investigations of gene therapy for Parkinson's disease have focused primarily on strategies that replace tyrosine hydroxylase. In the present study, the role of aromatic L-amino acid decarboxylase in gene therapy with tyrosine hydroxylase was examined by adding the gene for aromatic L-amino acid decarboxylase to our paradigm using primary fibroblasts transduced with both tyrosine hydroxylase and GTP cyclohydrolase I. We compared catecholamine synthesis in vitro in cultures of cells with tyrosine hydroxylase and aromatic L-amino acid decarboxylase together versus cocultures of cells containing these enzymes separately. L-DOPA and dopamine levels were higher in the cocultures that separated the enzymes. To determine the role of aromatic L-amino acid decarboxylase in vivo, cells containing tyrosine hydroxylase and GTP cyclohydrolase I were grafted alone or in combination with cells containing aromatic L-amino acid decarboxylase into the 6-hydroxydopamine-denervated rat striatum. Grafts containing aromatic L-amino acid decarboxylase produced less L-DOPA and dopamine as monitored by microdialysis. These findings indicate that not only is there sufficient aromatic L-amino acid decarboxylase near striatal grafts producing L-DOPA, but also the close proximity of the enzyme to tyrosine hydroxylase is detrimental for optimal dopamine production. This is most likely due to feedback inhibition of tyrosine hydroxylase by dopamine.     

D2 dopamine receptor antisense increases the activity and mRNA of tyrosine hydroxylase and aromatic L-amino acid decarboxylase in mouse brain

OBJECTIVE: Cytolytic activity of TNF was analysed at L929 and K562 tumor cell lines. METHODS: TNF-mediated cytotoxicity was studied within 10(-6)-10(-17) M concentration range after 18 h of incubation with target cells. RESULTS: TNF caused reliable cytotoxicity values in both cell lines, while L929 cells were more sensitive to cytolytic action of the protein than K562 cells. Three cytotoxicity maxima were detected at each cell line: at concentrations of 10(-6) M, 10(-17) M and 10(-15) M in K562 cells and at concentrations of 10(-7) M, 10(-11) M, 10(-14), 10(-16) M in L929 cells. CONCLUSIONS: DNA fragmentation analysis demonstrated that all cytolytic processes induced by TNF in L929 cells are associated with apoptotic mechanism of cell death, while cytolytic process induced in K562 cells differed in DNA fragmentation patterns: cytolytic processes induced by 10(-6) M of TNF was of apoptotic type, while the other processes were not associated with internucleosomal DNA cleavage.     

A dopamine-synthesizing cell group demonstrated in the human basal forebrain by dual labeling immunohistochemical technique of tyrosine hydroxylase and aromatic L-amino acid decarboxylase

The human basal forebrain has been known to contain many neurons immunoreactive (ir) to tyrosine hydroxylase (TH; the first dopamine-synthesizing enzyme). We examined whether these neurons might contain aromatic L-amino acid decarboxylase (AADC; the second step dopamine-synthesizing enzyme) by dual labeling immunohistochemistry and confocal laser-scanning microscopy. Neurons dually-labeled for TH and AADC were found in the anterior olfactory nucleus, olfactory tubercle and the ventral margin of the rostral nucleus accumbens. The examination in the basal forebrain of the macaque monkey also gave substantially the same results. These neurons appear to constitute an independent dopaminergic cell group in the primate basal forebrain.     

Differential effects of NMDA and non-NMDA antagonists on the activity of aromatic L-amino acid decarboxylase activity in the nigrostriatal dopamine pathway of the rat

Exhaled nitric oxide (NO) is elevated in asthmatics, and varies with disease severity. We postulated that a respiratory virus infection increases exhaled NO levels in asthma, and examined the relationship between the virus-induced changes in exhaled NO and in airway hyperresponsiveness to histamine. In a parallel study, seven patients underwent experimental rhinovirus 16 (RV16) inoculation at days 0 and 1, whilst seven patients received placebo. Exhaled NO was measured at baseline (day 0) and at days 1, 2 and 3 after inoculation. Histamine challenges were performed prior to (day -7) and after inoculation (day 3), and were expressed as provocative concentration causing a 20% fall in forced expiratory volume in one second (FEV1) (PC20). Following RV16 infection there was a significant increase in NO at days 2 and 3 as compared to baseline (median change (range): 4.2 (7.5) parts per billion (ppb), p=0.03, and 3.0 (10.1) ppb, p=0.02, respectively). Furthermore, PC20 decreased significantly following RV16 infection (mean+/-SD change in doubling dose: -0.65+/-0.54, p=0.02), whereas PC20 did not change in the placebo group (p=0.1). There was a significant correlation between the RV16-induced changes in exhaled NO levels at day 2 and the accompanying changes in PC20 at day 3 (rank correlation coefficient (rs): 0.86, p=0.01). Hence, the greater the increase in exhaled NO, the smaller the decrease in PC20. We conclude that rhinovirus infection increases exhaled nitric oxide levels in asthmatics, and that this increase is inversely associated with worsening of airway hyperresponsiveness to histamine. These results suggest that viral induction of nitric oxide synthase within the airways may play a protective role in exacerbations of asthma.     

Behavioral recovery in 6-hydroxydopamine-lesioned rats by cotransduction of striatum with tyrosine hydroxylase and aromatic L-amino acid decarboxylase genes using two separate adeno-associated virus vectors

Parkinson's disease (PD) is characterized by the progressive loss of the dopaminergic neurons in the substantia nigra and a severe decrease in dopamine in the striatum. A promising approach to the gene therapy of PD is intrastriatal expression of enzymes in the biosynthetic pathway for dopamine. Tyrosine hydroxylase (TH) catalyzes the synthesis of L-dopa, which must be converted to dopamine by aromatic L-amino acid decarboxylase (AADC). Since the endogenous AADC activity in the striatum is considered to be low, coexpression of both TH and AADC in the same striatal cells would increase the dopamine production and thereby augment the therapeutic effects. In the present study, the TH gene and also the AADC gene were simultaneously transduced into rat striatal cells, using two separate adeno-associated virus (AAV) vectors, AAV-TH and AAV-AADC. Immunostaining showed that TH and AADC were coexpressed efficiently in the same striatal cells in vitro and in vivo. Moreover, cotransduction with these two AAV vectors resulted in more effective dopamine production and more remarkable behavioral recovery in 6-hydroxydopamine (6-OHDA)-lesioned rats, compared with rats receiving AAV-TH alone (p < 0.01). These findings suggest an alternative strategy for gene therapy of PD and indicate that the simultaneous transduction with two AAV vectors can extend their utility for potential gene therapy applications.     

Tyrosine hydroxylase- and/or aromatic L-amino acid decarboxylase-containing cells in the suprachiasmatic nucleus of the Syrian hamster (Mesocricetus auratus)

We assessed forearm vascular and blood pressure responses to dynamic leg exercise in patients 7 and 28 days postmyocardial infarction. To determine a possible association between abnormal exercise vascular responses and baroreflex dysfunction, integrated and carotid baroreflex sensitivity and forearm vascular responses (during application of subhypotensive lower body negative pressure) were assessed. On day 7, 42 patients were compared with 21 age- and sex-matched controls. All subjects were assessed for (1) forearm vascular resistance during semierect exercise, (2) blood pressure measurements during erect treadmill exercise, and (3) integrated, cardiopulmonary, and carotid baroreceptor sensitivity. These studies were repeated in 13 patients on day 28. Forearm vascular resistance increased during exercise by 36% +/- 63% in patients versus 121% +/- 105% in controls (P = 0.0001), and fell in 15 patients, a response seen in none of the controls. Exercise hypotension was demonstrated in 5 patients, all of whom had abnormal vasodilator vascular responses. Those patients with vasodilator responses had a lower left ventricular ejection fraction (52% +/- 12% vs 62% +/- 9%; P = 0.007), and lower cardiopulmonary mechanoreceptor sensitivity (-6.6 +/- 3.9 units vs +6.4 +/- 10.4 units, P = 0.02) than those with constrictor responses, respectively. In the 13 patients studied on day 28, the change in forearm vascular resistance was similar to that observed on day 7 (36% +/- 63% vs 46% +/- 73%). Paradoxical vasodilation of forearm vessels during leg exercise is common in patients studied 7 and 28 days postmyocardial infarction, and is associated with lower left ventricular ejection fraction and abnormal vascular responses during subhypotensive lower body negative pressure.     

Aromatic L-amino acid decarboxylase is present in serotonergic fibers of the striatum of the rat. A double-labeling immunofluorescence study

The aim of the present study is to examine whether serotonergic fibers of the striatum of the rat contain aromatic L-amino acid decarboxylase (AADC). By use of a double-labeling immunofluorescence method, we showed that AADC was localized in serotonergic fibers of the striatum and cerebral cortex as well as in serotonergic cell bodies of the midbrain raphe nuclei. We previously demonstrated that serotonergic fibers of the rat striatum contained dopamine after intraperitoneal injection of L-dopa. These findings suggest that dopamine is produced from the injected L-dopa in serotonergic fibers of the rat striatum.     

The oxidative deamination of L-aminoethylcysteine sulfoxide and sulfone by snake venom L-amino acid oxidase

The oxidation of L-aminoethylcysteine (AEC) by L-amino acid oxidase has been compared with that of the respective sulfoxide (AEC-SO) and sulfone (AEC-SO2). Spectral and HPLC analyses of the incubates reveal the formation of the respective cyclic ketimines. While the ketimine coming from AEC is subjected to autooxidation yielding the sulfoxide and other products, the ketimines produced from AEC-SO and AEC-SO2 are more stable and account for almost the total conversion of the substrate in the product. Spectrophotometric and HPLC properties of the ketimine produced from AEC-SO are identical to those reported earlier for the main product of the autooxidation of AEC ketimine, thus confirming its identification. These results could explain the presence of chondrine in biological materials as a product of reduction of AEC-SO ketimine.     

The instability of polyhydroxylated aromatic protein tyrosine kinase inhibitors in the presence of manganese

The aim of the trial was to determine the diagnostic accuracy of scintimmammography with technetium-99m methoxyisobutylisonitrile (99mTc-MIBI) in the detection of primary breast cancer and to verify its clinical usefulness. A total of 246 patients with a suspicious breast mass or positive mammogram were included in this prospective European multicentre trial. At 5 min and 60 min (optional) p.i. two lateral prone images were acquired for 10 min each; 30 min p.i. one anterior image was acquired for 10 min. There were 253 lesions (195 palpable and 58 non-palpable), in respect of which histology revealed 165 cancers and 88 benign lesions. Institutional and blinded read results were correlated to core laboratory histopathology results obtained during excisional biopsy. Diagnostic accuracy for the detection of breast cancer was calculated per lesion. The overall sensitivity and specificity of blinded read scintimammography were 71% and 69%, respectively. For palpable lesions, the sensitivity of blinded read and institutional read scintimammography was 83% and 91%, respectively. Sensitivity was not dependent on the density of the breast tissue. Invasive ductal and invasive lobular cancers showed similar sensitivity. The sensitivity and specificity of mammography were 91% and 42%, respectively, and did not depend on the tumour size. In 60% of false-negative mammograms, 99mTc-MIBI was able to diagnose malignancy (true-positive). High-quality imaging with 99mTc-MIBI has a high diagnostic accuracy for the detection of primary breast cancer. Used as a complementary method, scintimammography with 99mTc-MIBI can help to diagnose breast cancer at an earlier stage in patients with dense breasts.     

Glutamic acid decarboxylase and other autoantigens in IDDM

Most family policy implicitly or explicitly focuses on families with young children, but the revolution in longevity suggests the value of a life course focus, aimed at promoting the effectiveness of families and individuals at all ages and stages. Gerontologists can make a contribution by documenting and describing the gaps between needs and resources of families at all life stages, developing family indicators of social change, and sensitizing both decision makers and the public to the unintended consequences of existing or proposed policies.     

The regulation of calcium homeostasis in nerve cells

We have reviewed the major cellular elements related to the release and buffering of calcium in neurons. Voltage-operated, chemical-operated calcium channels and mechanisms of stability of intercellular calcium homeostasis (mitochondria, endoplasmic reticulum, calcium binding proteins, calcium exchange and calcium pump) are demonstrated in normal and pathological condition (105 ref.).     

H(+)-coupled alpha-methylaminoisobutyric acid transport in human intestinal Caco-2 cells

Numerous theoretical as well as pharmacological arguments lead to the assumption that anxiety and memory are two closely linked concepts. Nevertheless, the study of this relationship is full of complexities because neither memory nor anxiety are unitary phenomena. Indeed, the term memory covers a large number of concepts, and anxiety has been divided in two main classes, "state" and "trait" anxiety. Recently the neophobic responses exhibited by Balb/c mice confronted to the free exploratory paradigm have been proposed as a "trait anxiety" model while response exhibited in the light/dark choice procedure as a "state anxiety" one. The aim of this study was to further clarify the link between these two anxiety types and memory of emotional events assessed in the passive avoidance test. The relationship between the variables measured in these three tests were assessed by a principal component analysis that confirmed that the behavior recorded in the two anxiety tests does not reflect the same psychological state, and showed that emotional memory is linked to "state" but not "trait" anxiety.     

The presence of protein-bound gamma-carboxyglutamic acid in calcium-containing renal calculi

The amino acid gamma-carboxyglutamic acid (Gla) is found in four blood-clotting proteins, in a bone protein, in kidney protein, and in the protein present in various ectopic calcifications. This paper reports the presence of Gla in the EDTA-soluble, nondialyzable proteins of calcium-containing renal calculi including calcium oxalate, hydroxyapatite, and mixed stores of apatite and struvite (MgNH4PO4). Calculi composed of pure struvite and those composed of only uric acid or cystine do not contain Gla. From calcium oxalate and hydroxyapatite stontes, a protein of about 17,000 daltons was obtained which contained about 40 residues of Gla per 1,000 amino acids. The amino acid composition of this protein had no apparent relationship to the Gla-containing bone protein or to the similarly-sized F1 fragment of prothrombin which contains about 64 residues of Gla per 1,000 amino acid residues. The Gla-rich protein in calcium-containing renal stones thus may be a different Gla-containing protein. These data as well as other studies demonstrating the presence of Gla in pathologically calcified tissues not normally containing Gla suggest that the Gla-containing proteins may be of considerable pathophysiological significance.     

Modification of fluorescent properties of norfloxacin in the presence of certain antacids

Norfloxacin (NFLX), a broad spectrum antibacterial quinolone, is a very thermostable but photosensitive drug, especially in solution leading to the formation of an ethylenediamine degradate. The modification of the fluorescent properties of NFLX in acid solution after exposure to fluorescent light and the degradation mechanisms were studied. Two analytical methods were previously developed and validated for NFLX, ultraviolet spectrophotometry (UV) and spectrofluorimetry (FL). Data obtained using both methods in the analysis of remaining NFLX in terms of percent recoveries revealed that there was no statistically significant modifications of the UV signal and of the recoveries obtained by the method. However, an important increase of the fluorescent signal after light exposure of NFLX solutions appeared, which led to an increase of the average recovery up to 270% over 15 months. Using a previously validated HPLC method for the photostability studies of NFLX, a loss of 5% with respect to the initial drug amount was observed. The study of UV and fluorescence spectra evidenced the formation of the degradation product, which induced significant modification of the fluorescent properties of NFLX samples. These results clearly indicated that FL analysis definitively is the method of choice and can be used to study the photodegradation of NFLX.     

Diagnostic usefulness of glutamic acid decarboxylase antibodies in stiff-man syndrome

Stiff-man syndrome (SMS) is a rare neurological disorder characterized by progressive rigidity of the axial musculature with superimposed spasms. Frequently, SMS remains undiagnosed for prolonged periods or the patients are diagnosed of a primary psychiatric disorder. 60% of the SMS patients harbor GAD-autoantibodies (GAD-Ab). We have analyzed the diagnostic value of GAD-Ab in a syndrome whose clinical expression is not well known, but its diagnosis is performed by clinical criteria. Five patients were studied following the established clinical criteria for diagnosis of SMS. GAD-Ab were analyzed by radioimmunoassay (RIA) and immunohistochemistry, and confirmed by immunoblot. The GAD-Ab titers were compared with those of 49 patients with insulin-dependent diabetes mellitus (IDDM), 322 with other neurological disorders, 14 non-IDDM first-degree relatives of IDDM patients with antibodies anti-islet cells and 91 normal subjects. Three patients fulfilled all clinical criteria (typical SMS). Unilateral limb symptoms alone, and acute onset with rapid progression involving the distal limb muscles constituted the atypical features of SMS in the remaining 2 patients. The 5 patients presented several serum organ-specific autoantibodies. All but one also presented autoimmune diseases. By RIA, GAD-Ab titers from all patients were elevated (mean: 24,532 +/- 26,892 U/ml) and significantly higher than the titers of IDDM patients without neurological disorders (mean: 48 +/- 112 U/ml) (p < 0.0001). GAD-Ab were absent in the non-SMS patients and in normal subjects. These findings suggest that clinical expression of SMS is more extensive than that recognized by the established criteria. GAD-Ab are helpful to define the clinical spectrum of SMS.