Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow.

Bone marrow stem cells give rise to a variety of hematopoietic lineages and repopulate the blood throughout adult life. We show that, in a strain of mice incapable of developing cells of the myeloid and lymphoid lineages, transplanted adult bone marrow cells migrated into the brain and differentiated into cells that expressed neuron-specific antigens. These findings raise the possibility that bone marrow-derived cells may provide an alternative source of neurons in patients with neurodegenerative diseases or central nervous system injury.

Non-neuronal dopamine in the gastrointestinal system.

1. Dopamine (DA) is a protective agent in the gastrointestinal (GI) tract in both rats and humans. Therefore, we have studied the site of DA production in rat and human GI tract using a variety of techniques, including immunocytochemistry (ICC), in situ hybridization histochemistry, reverse transcription-polymerase chain reaction, HPLC, western blotting and immunoelectron microscopy. 2. We found very high concentrations of DA that persisted after chemical sympathectomy (CS) in the gastric juice, the stomach mucosa and in the pancreas. Both the stomach mucosa and the pancreas also had tyrosine hydroxylase (TH) activity, most of which remained after CS. Double-labelling ICC showed that acid-producing parietal cells and the exocrine pancreas must also be capable of producing DA. 3. We isolated rat stomach parietal cells by cell fractionation and found that both DA and TH activity are present in isolated (denervated) parietal cells. These cells also have other features of aminergic cells: they are immuno- (and mRNA) positive for the DA plasma membrane transporter and vesicular monoamine transporter(s). In both gastric and duodenal mucosa, we demonstrated the presence of significant amounts of the D5 receptor that could serve as a target for locally produced DA. 4. Because DA, its biosynthetic enzymes and its transporters are also found in parietal cells in the human stomach, a mucosal protective system involving DA could be important clinically.

Distribution of the parathyroid hormone 2 receptor in rat: immunolocalization reveals expression by several endocrine cells.

The PTH2 receptor is a G protein-coupled receptor selectively activated by PTH. We are studying the receptors distribution to guide the investigation of its physiological function. We have now generated an antibody from a C-terminal peptide sequence of the PTH2 receptor and used this to study its cellular distribution. Labeling with the antibody identified a number of endocrine cells expressing the PTH2 receptor, including thyroid parafollicular cells, pancreatic islet D cells, and some gastrointestinal peptide synthesizing cells. There was complete overlap of PTH2 receptor labeling with somatostatin in pancreatic islets, and partial overlap with somatostatin in thyroid parafollicular cells and in the gastrointestinal tract. Furthermore, observations made previously by in situ hybridization histochemistry, including expression throughout the cardiovascular system, as well as by discrete populations of cells within the gastrointestinal tract and reproductive system were confirmed. These data suggest a broad role for the PTH2 receptor, especially within the endocrine system, and provide a basis for experimental exploration of its physiology.

Alpha synuclein is present in Lewy bodies in sporadic Parkinson's disease.

A missense mutation in the human alpha synuclein gene was recently identified in some cases of familial Parkinson's disease (FPD). We have developed an antibody that recognizes the C-terminal 12 amino acids of the human alpha synuclein protein and have demonstrated that alpha synuclein is an abundant component of the Lewy bodies found within the degenerating neurons of patients with Parkinson's disease (PD). The presence of alpha synuclein in Lewy bodies of sporadic PD patients suggests a central role for alpha synuclein in the pathogenesis of PD.

Expression of the CB1 and CB2 receptor messenger RNAs during embryonic development in the rat.

We mapped the distribution of CB1 and CB2 receptor messenger RNAs in the developing rat to gain insight into how cannabinoids may affect embryogenesis. In situ hybridization histochemistry studies were done using riboprobes specific for CB1 or CB2 receptor messenger RNAs. We found that CB1 and CB2 receptor messenger RNAs are expressed in the placental cone and in the smooth muscle of the maternal uterus at the earliest gestational periods studied [from eight days of gestation (E8) through E12]. In the embryo, as early as E11, CB1 receptor messenger RNA is expressed in some cells of the neural tube and, at later embryological stages (from E15 to E21), in several distinct structures within the central nervous system. In addition, high levels of CB1 receptor messenger RNA were also found in areas of the peripheral nervous system such as the sympathetic and parasympathetic ganglia, in the retina and in the enteric ganglia of the gastrointestinal tract. In addition to neural structures, high levels of the CB1 receptor messenger RNA were also present in two endocrine organs, the thyroid gland and the adrenal gland. On the other hand, CB2 receptor messenger RNA is expressed exclusively in the liver of the embryo as early as E13. The region-specific expression of CB1 and CB2 receptor messenger RNAs suggests that these receptors have a functional role during embryogenesis.

Immunohistochemical signal amplification by catalyzed reporter deposition and its application in double immunostaining.

The biotinyl-tyramide substrate of the horseradish peroxidase enzyme has been recently introduced to amplify immunohistochemical signals. We applied either fluorochromeor biotin-conjugated tyramine to improve the detection of different antigens in sections of rat stomach, pancreas, and hypothalamus. A ten- to 100-fold increase in staining efficiency was achieved, depending on the antibody, with either fluorescent or peroxidase detection systems. The amplification method was particularly useful for increasing a weak signal of conventional immunostaining caused by suboptimal tissue fixation. At a very low concentration of the primary antibody, the antigen can no longer be detected by a conventional fluorescent secondary antibody but is still detectable after amplification. When an antibody is used at this very low concentration and is detected by a fluorescent amplification method, another primary antibody, raised in the same host species, can be used and demonstrated with a different fluorochrome in subsequent conventional immunostaining of the same section. In this way it becomes possible to immunostain the same section with two different primary antibodies raised in the same host species. Samples for such double immunostaining are demonstrated here using pairs of monoclonal antibodies (to tyrosine hydroxylase and oxytocin) in the hypothalamus and polyclonal antibodies (to glucagon and neurofilament M) in sections of rat pancreas. Because in many cases the availability of antibodies is limited, the amplification method can be a quick and efficient tool for double immunostaining with antibodies from the same host species.

Distribution of parathyroid hormone-2 receptor messenger ribonucleic acid in rat.

The PTH2 receptor is a recently identified G protein-coupled receptor activated by PTH. Its amino acid sequence is most similar to the PTH/PTHrP receptor, but unlike the PTH/PTHrP receptor, it is activated by PTH and not by PTH-related peptide. We previously demonstrated using Northern blots that expression of PTH2 receptor messenger RNA was greatest within the brain and occurred at lower levels in pancreas, testis, and placenta. We have now obtained a complementary DNA encoding the rat PTH2 receptor and used it to study the distribution of the PTH2 receptor using in situ hybridization histochemistry. PTH2 receptor messenger RNA is abundantly expressed in arterial and cardiac endothelium and at lower levels in vascular smooth muscle. It is also abundant in the lung, both within bronchi and in the parenchyma, and is present within the exocrine pancreas. It is expressed by sperm in the head of the epididymis. A small number of cells associated with the vascular pole of renal glomeruli express the receptor. These data suggest that the PTH2 receptor may be responsible for PTH effects in a number of physiological systems.

A novel nonneuronal catecholaminergic system: exocrine pancreas synthesizes and releases dopamine.

Cells of the exocrine pancreas produce digestive enzymes potentially harmful to the intestinal mucosa. Dopamine has been reported to protect against mucosal injury. In looking for the source of dopamine in the small intestine, we found that the duodenal juice contains high levels of dopamine and that the pancreas itself has a high dopamine [and dihydroxyphenylalanine (dopa)] content that does not change significantly after chemical sympathectomy. Furthermore, we were able to demonstrate tyrosine hydroxylase (TH) activity in control pancreas as well as in pancreas from rats after chemical sympathectomy. Immunostaining and in situ hybridization histochemistry confirmed both the presence of TH, dopamine, and the dopamine transporter, and the mRNAs encoding TH and dopamine transporter, and the presence of both types of vesicular monoamine transporters in the exocrine cells of the pancreas. Since there are no catecholaminergic enteric ganglia in the pancreas, the above results indicate that pancreatic cells have all the characteristics of dopamine-producing cells. We suggest that the pancreas is an important source of nonneuronal dopamine in the body, and that this dopamine has a role in protecting the intestinal mucosa and suggests that dopamine D1b receptor agonists might be used to help mucosal healing in the gastrointestinal tract.

PACAP acts through VIP type 2 receptors in the rat testis.

Pituitary adenylate cyclase-activating peptide (PACAP) is present and synthesized in the testis in large amounts. Messenger RNA encoding the peptide is expressed in a stage specific manner in the developing germ cells. PACAP regulates a variety of physiological actions, among them, paracrine modulation of spermatogenesis. The PACAP peptides are potential ligands of at least three receptor types, the type I PACAP receptor, VIP1 and VIP2 receptors. Although PACAP27 binding sites have found in the testis, the receptor at which it acts has not been identified. We used in situ hybridization with riboprobes to identify the PACAP binding receptor present in the testis. Neither type I PACAP receptor, nor VIP1 receptor mRNA was present within the germ cells. Using the VIP2 receptor probe there was strong labelling within some cross sections of the seminiferous tubuli, while others were not labelled. The in situ results were also confirmed using reverse-transcription PCR (RT-PCR). Our data suggest that PACAP mediates its possible paracrine effect in the testis through the VIP2 receptor.

Protein kinase C epsilon subcellular localization domains and proteolytic degradation sites. A model for protein kinase C conformational changes.

Protein kinase C (PCK) epsilon has been found to have unique properties among the PCK isozymes in terms of its membrane association, oncogenic potential, and substrate specificity. Recently we have demonstrated that PKC epsilon localizes to the Golgi network via its zinc finger domain and that both the holoenzyme and its zinc finger region modulate Golgi function. To further characterize the relationship between the domain organization and the subcellular localization of PKC epsilon, a series of NIH 3T3 cell lines were created, each overexpressing a different truncated version of PKC epsilon. The overexpressed proteins each were designed to contain an epsilon-epitope tag peptide at the COOH terminus to allow ready detection with an antibody specific for the tag. The subcellular localization of the recombinant proteins was analyzed by in vivo phorbol ester binding, immunocytochemistry, and cell fractionation followed by immunoblotting. Results revealed several regions of PKC epsilon that contain putative subcellular localization signals. The presence either of the hinge region or of a 33-amino-acid region including the pseudosubstrate sequence in the recombinant proteins resulted in association with the plasma membrane and cytoskeletal components. The catalytic domain was found predominantly in the cytosolic fraction. The accessibility and thus the dominance of these localization signals is likely to be affected by the overall conformation of the recombinant proteins. Regions with putative proteolytic degradation sites also were identified. The susceptibility of the overexpressed proteins to proteolytic degradation was dependent on the protein conformation. Based on these observations, a model depicting the interaction and hierarchy of the suspected localization signals and proteolytic degradation sites is presented.

Analysis of aldehyde oxidase and xanthine dehydrogenase/oxidase as possible candidate genes for autosomal recessive familial amyotrophic lateral sclerosis.

Recently, point mutations in superoxide dismutase 1 (SOD1) have been shown to lead to a subset of autosomal dominantly inherited familial amyotrophic lateral sclerosis (ALS). These findings have led to the hypothesis that defects in oxygen radical metabolism may be involved in the pathogenesis of ALS. Therefore, we decided to analyze other enzymes involved in oxygen radical metabolism for possible involvement in other forms of ALS. We report here analysis of two genes encoding the molybdenum hydroxylases aldehyde oxidase (AO) and xanthine dehydrogenase/oxidase (XDH) for involvement in ALS. Of particular interest, one gene identified as encoding aldehyde oxidase is shown to map to 2q33, a region recently shown to contain a gene responsible for a familial form of ALS with autosomal recessive inheritance (FALS-AR). The AO gene appears to be located within 280,000 bp of simple sequence repeat marker D2S116, which shows no recombination with the FALS-AR locus. The AO gene is highly expressed in glial cells of human spinal cord. In addition, we mapped a gene for XDH to 2p22, a region previously shown to contain a highly homologous but different form of XDH. Neither of these XDH genes appears to be highly expressed in human spinal cord. This evidence suggests that AO may be a candidate gene for FALS-AR.

Potential problems in using [35S]-dATP-tailed oligonucleotides for detecting mRNAs in certain cells of the immune system.

In this study we examined the cause of unusually intense signals obtained in immune cells by in situ hybridization histochemistry using 35S-labeled oligonucleotides. We verified that the phenomenon is an amplification of a specific signal due to a series of chemical interactions after the probe binds to a specific mRNA in the tissue. The presence of oxidative enzymes in the tissue seems to be necessary for this reaction to occur. Therefore, most cells of the immune system (e.g., macrophages, neutrophil and eosinophil leukocytes), being rich in oxidative enzymes, will show some signal amplification. The intensification of the signal can be avoided if MgCl2 is substituted for CoCl2 in the synthesis of [35S]-thiophosphate-labeled probes, if 2,3-dimercaptopropanol [British anti-Lewisite (BAL)] is added to the hybridization buffer, or if [33P]-phosphate is used instead of [35S]-thiophosphate in the labeling of the probes.