DNA microarray analysis of differential gene expression of 6-year-old rat neural striatal progenitor cells during early differentiation.

EGF-responsive striatal progenitor cells from rat brain have been maintained in culture in the form of neurospheres for six years without exhausting their renewal capacity. The events surrounding differentiation of stem cells in the brain after a long progenitorship remain a mystery. Using DNA microarray analysis we investigated differential gene expression, comparing progenitor cells in their neurosphere state with the cells 24 hours after induction of differentiation. Eighty-one genes showed increased expression in the differentiated condition. Genes associated with cellular growth, neurite outgrowth, and synaptogenesis were activated, including both anti-apoptotic and pro-apoptotic genes. Two transmitter- related genes, acetylcholine receptor-beta and glutamate receptor-beta-unit were also elevated-, these genes not only fit the profile of early neural development, but also reflect the characteristics of striatal neurons. In addition, there are approximately 30 expressed sequence tags (ES7) increased during neural differentiation. Forty-seven genes showed decreased expression; half of them are known genes related to the cell cycle, cell adhesion, transcription, and signaling. Tbe signaling and cell cycle genes may be responsible for the life-long self-renewal. These data demonstrate for the first time that life-long quiescent stem cells retain the potential to become activated and develop into specific types of brain cells. The six-year long-term neural stem cells are an excellent model for studying developmental neurobiological processes and aging.

Differential cellular and subcellular expression of the human multifunctional apurinic/apyrimidinic endonuclease (APE/ref-1) DNA repair enzyme.

The multifunctional mammalian apurinic/apyrimidinic endonuclease (APE) is responsible for the repair of apurinic/apyrimidinic sites in DNA. In addition, this enzyme has been shown to function as a redox factor facilitating the DNA-binding capability of JUN and FOS, as well as numerous other transcription factors through the alteration of the transcription factor redox state. Biochemical studies of organ homogenates have shown that APE is present in the different tissues studied at similar concentrations. The present study examines the immunohistochemical distribution of APE in several organs and demonstrates new and unexpected patterns of cellular and subcellular localization of this enzyme. In the hippocampus, the APE protein was highly expressed in neurons of the dentate gyrus and regions CA3 and CA4, and unexpectedly, the staining was primarily cytoplasmic. AP endonuclease immunoreactivity in the cerebellum was found in the granule and Purkinje cells, both cytoplasmic and nuclear. APE staining of the hypoglossal nucleus of the brainstem, where motor neurons that control tongue movement reside, showed reactivity in the cytoplasmic Nissl substance. Skin, liver, and duodenum demonstrated nuclear staining; however, in the duodenum, only the enterocyte nuclei of the proximal villus and the crypts of Lieberkuhn were stained, with no staining of the distal villus. These results suggest that APE has different regulatory and functional roles in different cells and organs of the body. This study shows the importance of correlating in vitro findings in tissue culture cells with the organism as a whole. The cytoplasmic staining seen in parts of the brain and in liver suggests that there may be additional functions for the APE yet to be described.

Differential cellular expression of the human MSH2 repair enzyme in small and large intestine.

The human MSH2 (hMSH2) protein is responsible for the initial recognition of mismatched nucleotides during the postreplication mismatch repair process. Loss of hMSH2 function has been demonstrated to lead to the accumulation of replication errors, resulting in a mutator phenotype, which may be responsible for the multiple mutations required for multi-stage carcinogenesis. Alterations of the hMSH2 gene has been linked to approximately 60% of hereditary nonpolyposis colon cancer cases. Colon tumors in hereditary nonpolyposis colon cancer patients originate within benign preneoplastic adenomas and display replication errors in the form of microsatellite instability. The aim of this study was to investigate the cellular expression of the hMSH2 protein in cells of the large and small intestines. Using antibody specific for hMSH2, we have determined that this protein is highly expressed in cells of the crypts of Lieberkühn that are undergoing rapid renewal in both the ileum and colon. Proliferative perifibroblasts in the colon also showed significant presence of the hMSH2 protein. These results confirm the hypothesis that hMSH2 is expressed in highly proliferative cells of the gut, and mutations in this gene could, therefore, be expected to expedite the progression of adenoma to carcinoma in this tissue.

Heterogeneity of brain gene expression in Alzheimer's disease.

We have examined the expression of several genes whose transcripts have increased levels in Alzheimer's disease and have found heterogeneity in these levels in different patients with this condition. The level of expression of these genes was compared to different clinical and pathological aspects of the disease. A case with markedly elevated alpha 1-antichymotrypsin mRNA levels demonstrated prominent neuronal accumulation of this protein. Many of the neurons which demonstrated alpha 1-antichymotrypsin staining did not have neurofibrillary tangles, and vice versa. This suggests that alpha 1-antichymotrypsin staining might identify a different facet of the pathology of Alzheimer's disease than does neurofibrillary tangle staining and may provide new information in the study of this condition.

Library subtraction of in vitro cDNA libraries to identify differentially expressed genes in scrapie infection.

We have developed a system where double-stranded cDNA can be amplified using a synthetic oligonucleotide primer and the polymerase chain reaction, generating cDNA libraries in vitro. Using a library subtraction strategy (1), scrapie and control brain in vitro cDNA libraries were used to identify sequences whose expression is modulated in scrapie infection. One of these sequences represents beta-2 microglobulin, while the other two have not been previously described. The use of in vitro libraries offers increased speed and efficiency of construction, and their subtraction is more efficient and powerful, compared with the previous system (1).

Changes in brain gene expression shared by scrapie and Alzheimer disease.

We have isolated two recombinant cDNAs whose corresponding RNAs have an increased abundance in scrapie-infected hamster brain. DNA sequence analysis has shown that these two recombinants represent the genes for sulfated glycoprotein 2 and transferrin. The abundance of sulfated glycoprotein 2 RNA is increased in hippocampus from patients with Alzheimer disease and Pick disease, whereas transferrin RNA is not strongly modulated in these conditions. Expression of two previously identified scrapie-modulated genes, encoding glial fibrillary acidic protein and metallothionein, is also increased in both of these neurodegenerative diseases.

Isolation of cDNAs of scrapie-modulated RNAs by subtractive hybridization of a cDNA library.

We have developed a subtractive cloning procedure based on the hybridization of single-stranded cDNA libraries constructed in pi H3M, a vector containing the phage M13 origin of replication. We have used this strategy to isolate three transcripts whose abundance is increased in scrapie-infected brain. DNA sequence analysis showed that they represent glial fibrillary acidic protein, metallothionein II, and the B chain of alpha-crystallin; the latter two may represent a response to stress.

Magnetic resonance imaging of the midbrain in Parkinson's disease.

We have analyzed magnetic resonance images of the midbrain in patients with Parkinson's disease, and have found that there is a narrowing of the signal from the pars compacta of the substantia nigra relative to controls. The nature of the histological changes that may be responsible for this effect is discussed. Magnetic resonance imaging has the potential of becoming a useful diagnostic tool in the management of parkinsonism.

Identification of the major polyadenylylated transcription products and the genes active in their synthesis in a rat insulinoma.

Three RNA fractions with molecular weights of 200,000, 280,000, and 360,000, have been identified as the major polyadenylylated transcription products in a transplantable rat pancreatic islet cell tumor that synthesizes insulin. These three RNAs share sequence homology as demonstrated by comparisons of both partial and complete RNase T1 digestion products. The 200,000 and 280,000 molecular weight species hybridize primarily with three EcoRI restriction fragments of the rat genome having molecular weights of 1.4 X 10(6), 1.6 X 10(6), and 6.0 X 10(6). The 360,000 molecular weight species hybridizes preferentially with the 6.0 X 10(6) molecular weight DNA fragment.

Detection and kinetic behavior of preproinsulin in pancreatic islets.

Newly synthesized rat islet proteins have been analyzed by polyacrylamide slab gel electrophoresis and fluorography. A minor component having an apparent molecular weight of 11,100 was identified as preproinsulin by the sensitivity of its synthesis to glucose, the pattern of NH2-terminal leucine residues, and the rapidity of its appearance and disappearance during incubation of islets or islet cell tumors. A small amount of labeled peptide material which may represent the excised NH2-terminal extension of preproinsulin or its fragment was also detected. The kinetics of formation and processing of the preproinsulin fraction were complex, consisting of a rapidly turning over component having a half-life of about 1 min and a slower minor fraction that may have bypassed the normal cleavage process. The electrophoretic resolution of the preproinsulin and proinsulin fractions into two bands each is consistent with the presence of two closely related gene products in rat islets rather than intermediate stages in the processing of these peptides.

Partial purification and characterization of the mRNA for rat preproinsulin.

Electrophoretically homogeneous messenger RNA for rat preproinsulin has been prepared from an islet cell tumor by the use of oligo(dT(-cellulose chromatography and sucrose density gradient centrifugation. The molecular weight of the mRNA is about 210,000, as determined by polyacrylamide gel electrophoresis in formamide, and its sedimentation coefficient is 9.3 S in sucrose gradients containing 0.2 M NaCl. These results indicate that the synthesis of preproinsulin is directed by a monocistronic mRNA 600 nucleotides in length and requires approximately 55% of the molecule's coding capacity.

Reconstitution of a purified acetylcholine receptor.

Dialysis of the purified acetylcholine receptor from Torpedo californica electroplax with lipids from the same organ results in a vesicular membrane system in which the receptor is embedded in the bilayer and oriented so that most of the neurotoxin-binding sites appear to be on the outer surface. The reconstituted vesicles are chemically excitable by acetylcholine and carbamylcholine, as measured by 22Na+ efflux. The excitability is specifically blocked by the antagonist alpha-bungarotoxin. These results demonstrate that the purified reconstituted receptor system not only can specifically bind neurotransmitter but can also trigger ion translocation. It therefore has the properties necessary to effect postsynaptic depolarization in vivo.