Comparative study of gene expression of cholinergic system-related molecules in the human spinal cord and term placenta.

By reverse transcription-polymerase chain reaction, Southern blot analysis, direct sequencing, and immunohistochemistry, we studied the expression of cholinergic neuronal markers (choline acetyltransferase [ChAT], vesicular acetylcholine transporter [VAChT], and a high-affinity choline transporter [CHT1]), and gene regulatory molecules (repressor element-1 silencing transcription factor/neuron-restrictive silencer factor [REST/NRSF] and CoREST) in the human spinal cord and term placenta, both of which are well known to contain cells synthesizing acetylcholine. H-type, M-type, N2-type, and R-type ChAT mRNAs, VAChT mRNA, and CHT1 mRNA were detected in the spinal cord, but only H-type, M-type, and N2-type ChAT mRNAs, in the term placenta. REST/NRSF and CoREST were detected in the spinal cord and the placenta, but the amounts of both mRNAs were greater in the placenta than in the spinal cord. Further microdissection analyses revealed that the placental trophoblastic cells contained more REST/NRSF and CoREST transcripts than the spinal large motor neurons. Large motor neurons in the anterior horn of the spinal cord were immunohistochemically stained for ChAT and VAChT. In the placenta, stromal fibroblasts, endothelial cells, and trophoblastic cells of the chorionic villi were positively stained with anti-ChAT antibody but not with anti-VAChT antibody. These findings suggest that transcriptions of the R-type ChAT and VAChT mRNAs are coordinately suppressed in the human term placenta, which might be regulated in part by a REST/NRSF complex that binds to a consensus sequence of repressor element 1/neuron-restrictive silencer element (RE1/NRSE) in the 5' region upstream from exon R, whereas transcriptions of the H-type, M-type, and N2-type ChAT mRNAs might be independent of control by RE1/NRSE. It is possible that at least two separate regulatory mechanisms of gene expression are present for the human cholinergic gene locus, which might be selected by different combinations of DNA motifs and binding proteins to function in neuronal and non-neuronal cells.

Clinicopathological study of involvement of granulocyte colony stimulating factor and granulocyte-macrophage colony stimulating factor in non-lymphohematopoietic malignant tumors accompanied by leukocytosis.

Involvement of granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) in non-lymphohematopoietic malignant tumors accompanied by leukocytosis was clinicopathologically investigated. Among 1,778 autopsy cases in the last 20 years, 485 lesions of 439 cases with non-lymphohematopoietic malignant tumors accompanied by leukocytosis with a white blood cell count of 10,000/mm3 or greater during the course were immunohistologically examined for G-CSF and GM-CSF. Three (0.7%) and two cases (0.5%) were G-CSF- and GM-CSF-positive, respectively. GM-CSF mRNA was confirmed by using non-fixed cryopreserved tumor tissues in one case positive for GM-CSF. G-CSF-positive cases were large cell carcinoma of the lung, adenocarcinoma of the colon, and adenocarcinoma of the stomach, and GM-CSF-positive cases were spindle cell carcinoma of the lung and malignant thymoma. In the case with stomach carcinoma, the primary lesion showing moderately differentiated adenocarcinoma was negative, but the lung metastatic lesion showing less differentiated adenocarcinoma was G-CSF-positive. The survival period was six months or less in four out of five positive cases. The highest white blood cell count in five CSF-positive cases was markedly elevated: 29,400-103,500/mm3 (mean: 59,700/mm3). In four cases, excluding one case which may have been markedly affected by chemotherapy, the bone marrow showed hyperplasia, and the number of the granulocyte series cells significantly increased. There were three cases (0.7%) negative for both G-CSF and GM-CSF, although they showed marked leukocytosis (60,000/mm3 or higher) which were higher than the mean count of CSF-positive cases and was not observed in autopsy cases with non-tumorous diseases. Other stimulating factors may be involved in the development of leukocytosis in such cases.

Immunohistochemical and in situ hybridization studies of choline acetyltransferase in large motor neurons of the human spinal cord.

The localization of choline acetyltransferase (ChAT) protein and mRNA was investigated in large motor neurons of the lumbar spinal cord of 10 autopsied individuals without neurological diseases, by immunohistochemistry and in situ hybridization. In the immunohistochemistry using 20 serial tissue sections with a total thickness of 80 microm, about approximately 58-85% (average 67%) of the large motor neurons (30 microm and more in somal minimal diameter) in the ventral horn were stained with the anti-human ChAT antibody. In the positive neurons, most immunoreactive products were observed focally in the perikarya. Occasionally, the perikarya of some neurons were stained diffusely. In situ hybridization with a single 4 microm-thick tissue section showed that almost all large motor neurons had positive signals (approximately 93-100%, average 98%), which were distributed diffusely in the perikarya. The positivity rate in the in situ hybridization was higher than that in the immunohistochemistry for all 10 cases. These results indicate that ChAT mRNA is transcribed in almost all large motor neurons in the ventral horn of the human spinal cord, but ChAT protein cannot always be detected in the cytoplasm by immunohistochemistry.

Neurons with choline acetyltransferase immunoreactivity and mRNA are present in the human cerebral cortex.

We examined the cerebral cortex of five autopsied individuals without neurological and psychiatric diseases by immunohistochemistry using an anti-human recombinant choline acetyltransferase (ChAT) polyclonal antibody and in situ hybridization with 35S-labeled human ChAT riboprobes. The immunohistochemistry detected positive neurons which were medium-sized or large pyramidal neurons located predominantly in layers III and V. The density of such neurons was higher in the motor and secondary sensory areas than in other cortical areas; the immunoreactive neurons in layer V were more densely distributed in the motor area and those in layer III were distributed in the secondary sensory areas. Positively stained, non-pyramidal neurons were observed in the superficial layer of the cingulate gyrus and parahippocampus. No immunoreactive neurons were found in the primary sensory areas. The in situ hybridization detected some neurons with signals for ChAT mRNA in the cerebral cortex, most of which were distributed in layer V of the motor area and in layer III of the secondary visual area. These results indicate that the human cerebral cortex contains cholinergic neurons and displays regional and laminal variations in their distribution.

In situ mRNA hybridization study of the distribution of choline acetyltransferase in the human brain.

We examined the distribution of choline acetyltransferase (ChAT) mRNA in the brain of six autopsied individuals by in situ hybridization with 35S-labeled human ChAT riboprobes. Neurons containing hybridization signal for ChAT mRNA were observed in the nucleus of the diagonal band of Broca, the basal nucleus of Meynert, the caudate nucleus, the putamen, the pedunculopontine tegmental nucleus, the laterodorsal tegmental nucleus, the parabigeminal nucleus, the oculomotor nucleus and the trochlear nucleus. These findings were in good agreement with previous ChAT-immunohistochemical data. In contrast, labeled neurons were not observed in the medial septal and medial habenular nuclei, in which previously ChAT-immunoreactive neurons have been identified in many mammalian species, including the human. An unexpected result of the present study was the demonstration of neurons with ChAT mRNA signal in restricted areas of the human cerebral cortex.

Translation initiation sites and relative activity of large and small forms of human choline acetyltransferase.

We have previously shown that translation of human choline acetyltransferase (ChAT) mRNA starts at least at two sites and produces two enzyme proteins with different molecular weights. In this study, translation initiation sites and relative activity of large and small forms of ChAT were determined by site-directed mutagenesis, followed by expression and immunoblotting analyses. The large and small forms were translated at the first and second ATG codons of ChAT cDNA, respectively, and the specific activity was almost the same between the two forms of the enzyme.

Localization of choline acetyltransferase and acetylcholine in the chorion of early human pregnancy.

This report concerns the activity of choline acetyltransferase (ChAT), and the localization of ChAT and acetylcholine (ACh) in the human chorion during the 5th to 10th weeks of gestation. Radio-enzyme, immunohistochemical, and in situ hybridization assays were used. We found that ChAT activity increases as a function of gestational age between the 5th and the 9th weeks of pregnancy. At 5 weeks' gestation, ChAT was detected by immunohistochemical means mainly in cytotrophoblast and villous stromal cells, particularly in the cytotrophoblastic shell and cell columns. With the development of blood vessels at the 6th week, their endothelial cells also expressed ChAT. The amount of ChAT immunoreaction product deposits decreased in the villous cytotrophoblast by the 9th and 10th weeks when the layer of the cell became flat. By comparison, ChAT protein was detected only rarely in the syncytiotrophoblast layer during the gestation period studied. The ChAT gene transcript was demonstrated in most of the constitutive cells of the chorion. However, in contrast to the results of the immunohistochemical assays, positive hybridization signals for ChAT mRNA were evenly distributed over both cytotrophoblast and syncytiotrophoblast. On the other hand, the distribution of ACh immunoreaction products was almost coincident with that of ChAT.