Parkinson's disease: activity of L-dopa decarboxylase in discrete brain regions.

The activity of L-dopa decarboxylase was greatly reduced in the striatum, less so in the hypothalamus, and unchanged in the cortex of brains of patients with Parkinson's disease. However, it appears that even in the striatum enough activity remained to allow for the formation of dopamine from L-dopa in patients treated with large doses of L-dopa.

Rat brain synaptic vesicles are devoid of Mg2+-ATPase activity and contain beta-amyloid precursor protein.

Rat brain synaptic vesicles (SVs) isolated by gel filtration on Sephacryl S-500 had little Mg2+(H+)-ATPase activity, though it was identified by Western blots with antibodies against the H+-ATPase A-subunit and other vesicle proteins. In contrast, tyrosine hydroxylase and dopa decarboxylase activities in the SVs were substantial, suggesting that the absence of Mg2+(H+)-ATPase activity was not due to inactivation during isolation but rather to the nature of the SVs. The vesicle component reactive to H+-ATPase antibody was also identified in the synaptosomal cytosol, so the antibody for the A-subunit seemed unnecessary to detect H+-ATPase. The SVs contained beta-amyloid precursor protein of approximately 100 kDa. Based on these observations, SVs without Mg2+(H+)-ATPase seemed to play a role(s) in the delivery of cytoplasmic and plasma membrane proteins to nerve terminals as well as in neurotransmission.

DNA methylation patterns of the calcitonin gene in human lung cancers and lymphomas.

Generalized hypomethylation of the genome and of specific genes has been described in human tumors. We now report that in human lung cancers, especially in the most aggressive form, small cell lung carcinoma, and in lymphomas, the 5'-region of the calcitonin (CT) gene exhibits methylation of increased numbers of CCGG sites in comparison with normal adult tissues. These unusual methylation patterns are found much less frequently in other tumor types examined. In the spectrum of the four major types of lung cancer (small cell, adeno-, squamous, and large cell carcinomas), the frequency of occurrence of hypermethylation in the 5'-region of the CT gene parallels that for presence of the neuroendocrine related biochemistry which characterizes small cell lung carcinoma. In medullary thyroid carcinoma, a tumor which expresses high levels of CT gene mRNA, the 5'-region of the CT gene is hypomethylated. Our findings provide a potential new molecular marker for two important human cancers (lung cancer and lymphomas) and suggest that there is a close relationship between abnormal CT gene methylation and developmental events for these tumors.

IA-1, a new marker for neuroendocrine differentiation in human lung cancer cell lines.

IA-1 is a recently isolated novel complementary DNA which encodes a protein of 510 amino acids that contains both a zinc finger DNA-binding domain and a putative prohormone domain. mRNA expression of IA-1 has been found thus far only in tumors of neuroendocrine origin. In this report we describe the expression of IA-1 mRNA in a panel of 64 human lung cancer cell lines. IA-1 mRNA was detected by Northern blot analysis in 97% (30 of 31) of small cell lung cancer cell lines. In contrast, IA-1 mRNA was detected in only 13% (4 of 30) of non-small cell lung cancer cell lines. Nine of the 30 (30%) expressed either chromogranin A mRNA or produced L-dopa decarboxylase. Four of these 9 (44%) had detectable levels of IA-1 mRNA. In most of the lung cancer cell lines examined, IA-1 showed high concordance with the other neuroendocrine markers, L-dopa decarboxylase, and chromogranin A. The one exception was a variant small cell lung cancer cell line which expressed low or nondetectable levels of L-dopa decarboxylase. IA-1 is a candidate marker of neuroendocrine differentiation of human lung tumors.

Establishment and identification of small cell lung cancer cell lines having classic and variant features.

Using a chemically defined medium containing hydrocortisone, insulin, transferrin, 17 beta-estradiol and selenium, with or without serum supplementation (2.5% v/v), continuous cell lines can be established from 72% of all fresh biopsy specimens of small cell lung cancer (SCLC) containing tumor cells. No differences were observed in the rate of establishing cell lines from newly diagnosed untreated patients, or from patients who have relapsed from prior therapy, or from a variety of different organ sites. Biochemical characterization of 50 SCLC cell lines for the expression of L-dopa decarboxylase; bombesin-like immunoreactivity; neuron-specific enolase, and the brain isozyme of creatine kinase, revealed that SCLC cell lines can be subdivided into two distinct classes: classic SCLC cell lines (35 lines), which express elevated levels of all four biomarkers; and variant SCLC cell lines (15 lines) which have undetectable levels of L-dopa-decarboxylase and bombesin-like immunoreactivity, but continue to express neuron-specific enolase and the brain isozyme of creatine kinase. The presence of the latter two markers distinguishes variant lines fron non-SCLC cell lines. In addition, four distinct classes were identified morphologically. The biomedical differences among established SCLC cell lines may account for the differences in response rates to cytotoxic therapy observed in newly diagnosed SCLC patients. A prospective study of biomarker characterization of SCLC tumors will determine if clinical differences exist between classic and variant SCLC tumors.

Characterization of variant subclasses of cell lines derived from small cell lung cancer having distinctive biochemical, morphological, and growth properties.

We have described the establishment and biochemical characterization of 50 small cell lung carcinoma (SCLC) cell lines. Further analysis of these data, combined with studies of morphology and growth characteristics, indicates that 35 (70%) of the lines retained typical morphology (SCLC, intermediate subtype), growth characteristics (growth as tightly packed floating cellular aggregates, long doubling times and low colony-forming efficiencies), and biochemical profile (presence of L-dopa decarboxylase, bombesin-like immunoreactivity, neuron-specific enolase, and high concentrations of brain isoenzyme of creatine kinase). They are referred to as classic SCLC lines. The remaining 15 (30%) lines had discordant expression of the biochemical markers; they retained high concentrations of brain isozyme of creatine kinase, but had significantly lower concentrations of neuron-specific enolase and lacked L-dopa decarboxylase and bombesin-like immunoreactivity. These cell lines are called variants. SCLC variant lines could further be divided into (a) biochemical variant lines having variant biochemical profile but retaining typical SCLC morphology and growth characteristics; and (b) morphological variant (SCLC-MV) lines having variant biochemical profile, altered morphology (features of large cell undifferentiated carcinoma) and altered growth characteristics (growth as loosely attached floating aggregates, relatively short doubling times and cloning efficiencies). Fifty-five clones derived from the three SCLC subclasses retained their parental phenotypes. In SCLC-MV lines there was a near constant relationship between variant morphology, altered growth characteristics and amplification of the c-myc oncogene; classic SCLC and biochemical variant SCLC lines were not amplified. Variant morphologies frequently are present in SCLC tumors at autopsy, and most SCLC-MV lines reflect changes that had occurred in the tumors from which they were derived. Because SCLC-MV tumors behave more virulently in the patient and are radioresistant in vitro, these findings are of considerable biological and clinical interest.

Characteristics of cell lines established from human colorectal carcinoma.

We have characterized 14 human colorectal carcinoma cell lines established from primary and metastatic sites by us during the years 1982 to 1985. Five lines were established in fully defined ACL-4 medium and 9 in serum supplemented R10 medium. However, after establishment, cultures could be grown interchangeably in either medium. The lines grew as floating cell aggregates in ACL-4 medium, while most demonstrated substrate adherence in R10 medium. The lines had relatively long doubling times and low cloning efficiencies. Twelve were tumorigenic in athymic nude mice when injected s.c., and two grew i.p. as well. Based on culture, xenograft, and ultrastructural morphologies, the 14 lines could be subtyped as follows: 4 were well differentiated; 5 were moderately differentiated; 4 were poorly differentiated; and 1 was a mucinous carcinoma. Membrane associated antigens characteristic for gastrointestinal cells (carcinoembryonic antigen, CA 19-9, and TAG-72 antigens) were expressed by 50-71% of the lines. Lines expressing carcinoembryonic antigen and CA 19-9 actively secreted these antigens into the supernatant fluids while TAG-72 antigen was not secreted. Surprisingly, 5 of 7 of the original tumor samples tested and 13 of 14 cultured lines expressed L-dopa decarboxylase activity, which is a characteristic enzyme marker of neuroendocrine cells and tumors. In addition, one poorly differentiated cell line contained dense core granules, characteristic of endocrine secretion. Preliminary cytogenetic analyses indicated that 9 of 11 lines examined contained double minute chromosomes. In addition, 3 of the 9 lines with double minutes also had homogeneously staining regions. These findings indicate a high incidence of amplification of one or more as yet unidentified genes.

A comparison of synaptophysin, chromogranin, and L-dopa decarboxylase as markers for neuroendocrine differentiation in lung cancer cell lines.

Synaptophysin is a Mr 38,000 integral membrane glycoprotein expressed by a variety of normal and neoplastic neuroendocrine cells. We studied synaptophysin as an immunocytochemical marker for neuroendocrine differentiation in lung cancer and compared it to the immunocytochemical expression of chromogranin A, a marker for dense core (endocrine) granules, and the biochemical activity of L-dopa decarboxylase (DDC), the key amine-handling enzyme. Of the 250 cell lines available to us, we selected examples representative of the following cell types: bronchial carcinoids (n = 4), small cell lung cancer (SCLC) (n = 7), extrapulmonary small cell carcinomas (n = 4), and non-small cell lung cancers (n = 18) whose neuroendocrine status had been previously determined on the basis of electron microscopy and DDC activity. We demonstrated (a) there was a higher incidence of synaptophysin than chromogranin A immunoreactivity in carcinoid (100 versus 75%), classic SCLC (70 versus 50%), and variant SCLC (57 versus 29%) cell lines; (b) 3 of the 4 (75%) extrapulmonary small cell lung cancer cell lines expressed synaptophysin and chromogranin A; (c) 5 of the 7 (71%) non-small cell lung cancer cell lines previously shown to express multiple neuroendocrine markers were positive for synaptophysin, chromogranin A, and DDC activity; (d) none of the other 11 non-small cell lung cancer cell lines expressed synaptophysin or chromogranin A; and (e) formalin fixation and paraffin embedding reduced synaptophysin immunoreactivity in 11 of 14 (79%) of the cell lines, as compared to freshly prepared specimens fixed in 95% ethanol. Western blot analysis using the synaptophysin antibody (SY38) demonstrated immunoreactive proteins ranging from Mr 43,000 to 45,000 in five representative cell lines. The concordance of expression of all three neuroendocrine markers was statistically significant when values for all cell lines were totalled. Synaptophysin was a more commonly expressed marker for variant SCLC cell lines, which rarely showed DDC activity. We conclude that synaptophysin may be a more sensitive and specific marker for neuroendocrine differentiation, when compared to chromogranin A and DDC in lung cancer cell lines which express only part of the neuroendocrine program.

Individualized chemotherapy for patients with non-small cell lung cancer determined by prospective identification of neuroendocrine markers and in vitro drug sensitivity testing.

We attempted to prospectively select individualized chemotherapy for 165 non-small cell lung cancer patients based on in vitro analysis of neuroendocrine (NE) markers and drug sensitivity testing (DST) using fresh tumor. The chemotherapy used for small cell lung cancer (SCLC) was selected when NE marker expression determined by L-dopa decarboxylase assay was documented. Selection of chemotherapy for other patients was guided by DST results using a modified dye exclusion assay when available; otherwise etoposide and cisplatin was administered. A total of 112 of 165 (68%) specimens were assayed for L-dopa decarboxylase and 36 patients (22%) had DST. In vitro data directed management for 27 of 96 (28%) patients given chemotherapy: 6 with NE markers were treated with the SCLC regimen; and 21 (58% of those with DST) received their DST-selected chemotherapy regimen. There were no significant differences in response rate among all 3 treatment arms (P = 0.076). However, response to chemotherapy for the patients treated prospectively with a SCLC regimen was 3 of 6 (50%), marginally better than patients given their DST-selected chemotherapy regimen (2 of 21; 9%; P = 0.056) or those treated with etoposide and cisplatin (10 of 69; 14%; P = 0.061). When patients whose NE markers were identified retrospectively are included, 4 of 9 (44%) responded to administered chemotherapy, compared to 7 of 55 (13%) with no NE markers present (P = 0.04). There were no differences in survival among the three treatment groups. Cisplatin and etoposide comprised the most active regimen in vitro for tumors from 16 of 36 (44%) patients, potentially limiting the benefit of DST since this is often the empiric therapy for non-SCLC. Furthermore, the correlation between in vitro and clinical response is nonsignificant for all drugs tested, highlighting the overall relative resistance of non-SCLC tumors to currently available chemotherapy.

[Radiological innovations in the screening and diagnosis of the inborn errors of metabolism]. / Nouveautés radiologiques dans le dépistage et le diagnostic des erreurs innées du métabolisme.

New metabolic diseases are regularly identified by a genetic or biochemical approach. Indeed, the metabolic diseases result from an enzymatic block with accumulation of a metabolite upstream to the block and deficit of a metabolite downstream. The characterization of these abnormal metabolites by MRI spectroscopy permitted to identify the deficient enzyme in two new groups of diseases, creatine deficiencies and polyol anomalies. Creatine deficiency is implicated in unspecific mental retardation. A low peak of creatine at MRI spectroscopy is evocating of creatine deficiency which is treatable by creatine administration. Deficiency of synthesis of polyols, metabolites on the pentose pathway, represent new described metabolic diseases with variable symptoms including a neurological distress, liver disease, splenomegaly, cutis laxa and renal insufficiency. The deficit of ribose-5-phosphate isomerase, one of the enzymes whose diagnosis is evoked in front of the accumulation of ribitol, arabitol and xylitol leads to a leucodystrophy in adults. This new deficit was highlighted by the identification of an abnormal peak in cerebral MRI-spectroscopy corresponding to the abnormal accumulation of polyols in brain. Congenital hyperinsulinism (HI) is characterized by profound hypoglycaemia related to inappropriate insulin secretion. Focal and diffuse forms of hyperinsulinism share a similar clinical presentation but their treatment is dramatically different. Until recently, preoperative differential diagnosis was based on pancreatic venous sampling, an invasive and technically demanding technique. Positron emission tomography (PET) after injection of [18F]Fluoro-L-DOPA has been evaluated for the preoperative differentiation between focal and diffuse HI, by imaging uptake of radiotracer and the conversion of [18F]Fluoro-L-DOPA into dopamine by DOPA decarboxylase. PET with [18F]Fluoro-L-DOPA has been validated as a reliable test to differentiate diffuse and focal HI and is now a major differential diagnosis tool in infantile hyperinsulinemic hypoglycaemia.

Difficulties in comparing catecholamine-related enzymes from the brains of schizophrenics and controls.

The catecholamine-forming and metabolizing enzyme tyrosine hydroxylase, dopa decarboxylase, dopamine-beta-hydroxylase, phenylethanolamine N-methyltransferase, and catecholamine-O-methyltransferase, as well as the endogenous inhibitor of dopamine-beta-hydroxylase were compared in the brains of schizophrenics and controls. While there were no statistically significant differences in the enzyme or inhibitor activity between groups, tbre was a decided trend toward a decreased enzyme activity in the brains of the schizophrenics. From another set of control brains it was found that changes in human enzyme activity following death are variable and may be dependent on how the brains were handled. Thus, it is unclear whether the apparent differences between schizophrenics and controls were present when they were alive or occurred after death.

High midbrain [18F]DOPA accumulation in children with attention deficit hyperactivity disorder.

OBJECTIVE: Attention deficit hyperactivity disorder (ADHD) is a highly prevalent childhood psychiatric disorder characterized by impaired attention, excessive motor activity, and impulsivity. Despite extensive investigation of the neuropathophysiology of ADHD by a wide array of methodologies, the neurobiochemical substrate of this disorder is still unknown. Converging evidence, however, suggests a primary role of the dopaminergic system. METHOD: This study examined the integrity of presynaptic dopaminergic function in children with ADHD through use of positron emission tomography and the tracer [18F]fluorodopa ([18F]DOPA). Accumulation of [18F]DOPA in synaptic terminals, a measure of dopa decarboxylase activity, was quantified in regions rich in dopaminergic innervation, including caudate nucleus, putamen, frontal cortex, and midbrain (i.e., substantia nigra and ventral tegmentum). RESULTS: Accumulation of [18F]DOPA in the right midbrain was higher by 48% in 10 children with ADHD than in 10 normal children. Despite its magnitude, this difference would not have reached statistical significance if corrected by the Bonferroni test for multiple comparisons. However, [18F]DOPA in the right midbrain was correlated with symptom severity. No other dopamine-rich regions significantly differed between groups. CONCLUSIONS: These findings are suggestive of dopaminergic dysfunction at the level of the dopaminergic nuclei in children with ADHD. Abnormality in dopa decarboxylase activity may be primary or secondary to deficits in other functional units of the dopamine pathway (e.g., receptor, uptake transporter, vesicular transporter, degradation enzymes). Efforts toward defining the origin of this abnormality should help delineate mechanisms of midbrain control of attention and motor behavior important for the understanding of the causes and treatment of ADHD.

Comparative anatomy of the histaminergic and other aminergic systems in zebrafish (Danio rerio).

The histaminergic system and its relationships to the other aminergic transmitter systems in the brain of the zebrafish were studied by using confocal microscopy and immunohistochemistry on brain whole-mounts and sections. All monoaminergic systems displayed extensive, widespread fiber systems that innervated all major brain areas, often in a complementary manner. The ventrocaudal hypothalamus contained all monoamine neurons except noradrenaline cells. Histamine (HA), tyrosine hydroxylase (TH), and serotonin (5-HT) -containing neurons were all found around the posterior recess (PR) of the caudal hypothalamus. TH- and 5-HT-containing neurons were found in the periventricular cell layer of PR, whereas the HA-containing neurons were in the surrounding cell layer as a distinct boundary. Histaminergic neurons, which send widespread ascending and descending fibers, were all confined to the ventrocaudal hypothalamus. Histaminergic neurons were medium in size (approximately 12 microm) with varicose ascending and descending ipsilateral and contralateral fiber projections. Histamine was stored in vesicles in two types of neurons and fibers. A close relationship between HA fibers and serotonergic raphe neurons and noradrenergic locus coeruleus neurons was evident. Putative synaptic contacts were occasionally detected between HA and TH or 5-HT neurons. These results indicate that reciprocal contacts between monoaminergic systems are abundant and complex. The results also provide evidence of homologies to mammalian systems and allow identification of several previously uncharacterized systems in zebrafish mutants.

Distribution of neurones containing DOPA decarboxylase and dopamine-beta-hydroxylase in some sympathetic ganglia of the dog: a quantitative study.

Using a technique by which binding sites for two antibodies can be visualized in single tissue sections, we have studied the distribution of neurones containing DOPA decarboxylase-like and dopamine beta-hydroxylase-like immunoreactivity in ganglia of dog sympathetic chain. Three types of neurones could be distinguished: those that contained both enzymes, and were presumably noradrenergic; those that contained neither enzyme, and were presumably not catecholaminergic; and a group that contained DOPA decarboxylase but lacked dopamine beta-hydroxylase. The numbers of cells of each type were counted in serially-sectioned ganglia from regions of the sympathetic chain thought to contain dopaminergic neurones (T12-L1 and L7-S2). The percentages of total cell numbers contributed by the DOPA decarboxylase-positive, dopamine beta-hydroxylase-negative cells in these regions were similar to the estimates of dopaminergic neurone numbers that can be made from previously obtained biochemical data. Our results are consistent with the presence of dopaminergic neurons in regions of the paravertebral chain supplying the kidney and the distal hindlimb.

Studies on dopamine-, tyrosine hydroxylase- and aromatic L-amino acid decarboxylase-containing cells in the rat diencephalon: comparison between formaldehyde-induced histofluorescence and immunofluorescence.

The morphology, number and distribution of catecholaminergic neurons, as visualized either with the aluminum-catalysed formaldehyde method for catecholamines or with the immunohistochemical method for the catecholamine-synthesizing enzymes tyrosine hydroxylase and aromatic L-amino acid decarboxylase, respectively, were analysed within the rat dorsal hypothalamus, ventral thalamus and adjoining regions (A11 and A13 cell groups). Both polyclonal rabbit and monoclonal mouse tyrosine hydroxylase antibodies were used in elution-restaining and double-staining experiments, respectively. Some of the animals also received spinal injections of the fluorescent tracer True Blue in order to retrogradely label cells projecting to the spinal cord. With respect to the number and distribution of catecholaminergic neurons in the A11 and medial A13 cell groups, including the spinal-projecting subpopulation, the results obtained with the two methods were very similar, indicating that within these regions of the CNS the two methods in principle visualize identical cell populations. However, the catecholaminergic cells were distinctly larger and their processes appeared more extensive with the immunohistochemical method. Animals processed for immunohistochemistry exhibited a lower total number of retrogradely labelled cells in the A11 area than those analysed with aldehyde-induced fluorescence despite the fact that both methods revealed similar numbers of retrogradely labelled tyrosine hydroxylase-positive and catecholamine-containing cells, respectively. The reason for these discrepancies, which are probably of methodological nature, are discussed. While this study shows that the results obtained with the two methods within the A11 and medial A13 cell group are very similar and thus strengthens the earlier proposed concept of the organization of the diencephalospinal dopaminergic system, it also documents that in intermingling and nearby CNS regions there are cell bodies which cannot be demonstrated with the aldehyde fluorescence method, but which still contain tyrosine hydroxylase and/or aromatic L-amino acid decarboxylase-like immunoreactivity. One explanation is low levels of enzyme and/or dopamine combined with a comparatively low sensitivity of the histochemical method. Thus, neurons containing both enzymes are probably dopaminergic, even if catecholamine fluorescence cannot be demonstrated. Neurons containing tyrosine hydroxylase, but lacking both aldehyde induced fluorescence and aromatic L-amino acid decarboxylase, may also still be dopaminergic.(ABSTRACT TRUNCATED AT 400 WORDS)

Markers and characteristics of human SCLC cell lines. Neuroendocrine markers, classical tumor markers, and chromosomal characteristics of permanent human small cell lung cancer cell lines.

Permanent human small cell lung cancer (SCLC) cell lines established in our laboratory were investigated for their expression of the enzymatic neuroendocrine markers L-DOPA decarboxylase (DDC), neuron-specific enolase (NSE), and creatine kinase (CK), including its BB isoenzyme (CK-BB), the classical tumor markers carcinoembryonic antigen (CEA), the alpha and beta subunits of human chorionic gonadotropin (alpha-HCG, beta-HCG), and alpha-fetoprotein (alpha-FP), and their chromosomal characteristics. DDC activities were detectable in 5/6 SCLC cell lines and absent in non-SCLC. NSE levels ranged from 160 to 1422 ng/mg soluble protein and were less than 290 ng/mg soluble protein in non-SCLC. Activities of CK and levels of CK-BB clearly distinguished SCLC from non-SCLC with CK activities greater than 1000 munits/mg soluble protein and CK-BB levels greater than 3000 ng/mg soluble protein in SCLC and less than 300 munits/mg soluble protein and less than 2000 ng/mg soluble protein in non-SCLC. CEA was detectable in 5/6 SCLC cell lines but absent in non-SCLC, and its level seemed to correlate with those of DDC, NSE, and CK. One cell line, SCLC-16H, lost some of its neuroendocrine properties and CEA after 1 year of in vitro cultivation. Generally, marker levels were low in fast growing cell lines and high in slow growing cell lines. HCG alpha and beta subunit and alpha-FP were not detectable in SCLC cell lines. All SCLC cell lines examined had near diploid DNA indices and modal chromosome numbers. Double minute chromosomes and homogeneously staining regions were found in 2/5 and 4/5 SCLC cell lines respectively. With respect to chromosomal aberrations, we found a deletion of the short arm of at least one chromosome 3 in all SCLC cell lines (5/5). These data show that SCLC expresses neuroendocrine markers and CEA; CK is the most sensitive marker, and DDC and CEA are the most specific markers for SCLC in vitro; individual marker levels correlate with each other and the in vitro malignancy of SCLC; and SCLC cell lines have relatively uniform chromosomal characteristics. Our results suggest that patients whose tumors have high levels of DDC, NSE, CK-BB, and CEA have a better prognosis than those with low marker levels. This hypothesis could be proved by comparing pairs of patients that are matched for all known prognostic parameters, in particular tumor spread, for their serum and tumor marker levels with respect to the patients' outcome and prognosis.

Molecular detection of dopamine decarboxylase expression by means of reverse transcriptase and polymerase chain reaction in bone marrow and peripheral blood: utility as a tumor marker for neuroblastoma.

A highly sensitive molecular method was used to evaluate the presence of dopamine decarboxylase (DDC) mRNA in the bone marrow and peripheral blood of patients with neuroblastoma (NB). DDC, like tyrosine hydroxylase (TH), is an enzyme involved in the catecholamine synthesis pathway and has recently been proposed as a specific marker of NB among pediatric malignancies. DDC transcript was detected in five of five NB cell lines, 10 of 10 NB primary tumors, 17 of 18 (94%) bone marrow samples, and 12 of 18 (66%) blood samples drawn at diagnosis in 18 patients affected by disseminated NB. In contrast, no PCR signal was found in 20 bone marrow samples obtained from patients with other malignancies or in eight of nine marrow and blood samples drawn from patients with localized NB (two stage 2 and seven stage 3). In addition, all marrow and blood samples obtained from NB patients at relapse revealed DDC mRNA. Furthermore, the percentage of DDC-positive samples was lower among the samples drawn from these patients during treatment. By comparison with conventional methods for disease evaluation, DDC transcript research can increase the sensitivity of NB cell detection in marrow and blood samples at diagnosis and during the treatment and follow-up of NB patients. These results suggest that finding DDC mRNA in NB patients could be a potential marker for minimal residual disease study.

Demonstration of histaminergic neurons in horizontal cells of guinea pig retina.

The existence of L-histidine decarboxylase (HDC, EC 4.1.1.22)-like immunoreactive (HDC-I) cells in guinea pig retina was demonstrated using antiserum raised against HDC purified from fetal rat liver. The anti-HDC antiserum partially cross-reacted guinea pig L-DOPA decarboxylase (DDC, EC 4.1.1.28), so the histaminergic neurons were carefully identified. Comparison of HDC-I and DDC-like immunoreactive (DDC-I) cell types in adjacent sections revealed that HDC-I structures were found in some horizontal cells and amacrine cells, and double-staining procedures with anti-HDC antiserum and monoclonal anti-DDC antibody showed that HDC-I horizontal cells had no DDC-I structures, but all the HDC-I amacrine cells had DDC-I structures. From the results, some horizontal cells (with HDC-like immunoreactivities but without DDC-like immunoreactivities) were concluded to be histaminergic.

Transmitter histochemistry of the rat olfactory bulb. I. Immunohistochemical localization of monoamine synthesizing enzymes. Support for intrabulbar, periglomerular dopamine neurons.

The rat olfactory bulb was studied at the light and electron microscopic level with the indirect immunofluorescence technique and the unlabelled antibody enzyme method (PAP-technique), respectively. Antibodies to all 4 enzymes in the catecholamine synthesis were used. In the principal bulb the first two enzymes, tyrosine hydroxylase (TH) and DOPA decarboxylase (DDC), but not dopamine-beta-hydroxylase (DBH), were present in a proportion of periglomerular cell bodies and dendrites indicating that these neurons synthesize dopamine (DA). This amine may therefore be released as a transmitter substance at some of the intraglomerular dendrodendritic synapses which periglomerular cells form with the mitral cells. There is evidence to suggest that some periglomerular cells use GABA as their transmitter. Thus, a morphologically and physiologically homogenous population of neurons can be subdivided on the basis of transmitter histochemical criteria. There was an impression of more DDC-positive than TH-positive fibers in the glomeruli. Such presumably DDC-positive, but TH-negative processes may represent 5-hydroxytryptamine (5-HT) nerve terminals. DBH-positive fibers were seen in the granular, external plexiform, and very rarely, in the glomerular layers, probably representing noradrenaline (NA) nerve terminals ascending from the lower brain stem. Weakly fluorescent DDC-positive fibers may represent nerve terminals of ascending 5-HT neurons. No phenylethanolamine-N-methyltransferase (PNMT)-positive neurons were observed.

Ontogeny of epinephrine metabolic pathways in the rat: role of glucocorticoids.

Recent studies suggest that the initial expression of adrenal phenylethanolamine N-methyltransferase (PNMT) and epinephrine (E) are dependent upon stimulation of adrenal glucocorticoid receptors. However, evidence suggests that the expression of heart and brain PNMT is independent of glucocorticoids. We measured PNMT activity and E levels in adrenal, heart and head over the latter half of gestation in rat fetuses treated chronically with glucocorticoids, and in normal controls. Chronic glucocorticoid treatment ending on embryonic day (e)12 did not affect heart, head or trunk PNMT activity or E levels. In contrast, chronic glucocorticoid exposure ending e19 or e20 resulted in marked increases in both PNMT and E in adrenal, heart and head tissues. The elevation of E in all three tissues was unaffected by maternal adrenalectomy, indicating enhanced fetal E synthesis. In the absence of exogenous glucocorticoid treatment heart PNMT activity peaked on e12, prior to the earliest reported appearance of glucocorticoid receptors. We conclude that expression of PNMT in all three tissues is glucocorticoid independent until the latter part of gestation when it is readily enhanced by glucocorticoids.