Protein synthesis and axonal transport have been studied in regenerating peripheral nerves. Sciatic nerves of bullfrogs were unilaterally crushed or cut. The animals were killed 1, 2, or 4 weeks later, and 8th and 9th dorsal root ganglia removed together with sciatic nerves and dorsal roots. The ganglia were selectively labeled in vitro with [35S]-methionine. Labeled proteins, in dorsal root ganglia and rapidly transported to ligatures placed on the sciatic nerves and dorsal roots, were analyzed by two-dimensional polyacrylamide gel electrophoresis. Qualitative analysis of protein patterns revealed no totally new proteins synthesized or rapidly transported in regenerating nerves. However, quantitative comparison of regenerating and contralateral control nerves revealed significant differences in abundance for some of the proteins synthesized in dorsal root ganglia, and for a few of the rapidly transported proteins. Quantitative analysis of rapidly transported proteins in both the peripheral processes (spinal nerves) and central processes (dorsal roots) revealed similar changes despite the fact that the roots were undamaged. The overall lack of drastic changes seen in protein synthesis and transport suggests that the neuron in its program of normal maintenance synthesizes and supplies most of the materials required for axon regrowth.
After the goldfish optic nerve was crushed, the total amount of protein in the nerve decreased by about 45% within 1 week as the axons degenerated, began to recover between 2 and 5 weeks as axonal regeneration occurred, and had returned to nearly normal by 12 weeks. Corresponding changes in the relative amounts of some individual proteins were investigated by separating the proteins by two-dimensional gel electrophoresis and performing a quantitative analysis of the Coomassie Brilliant Blue staining patterns of the gels. In addition, labelling patterns showing incorporation of [3H]proline into individual proteins were examined to differentiate between locally synthesized proteins (presumably produced mainly by the glial cells) and axonal proteins carried by fast or slow axonal transport. Some prominent nerve proteins, ON1 and ON2 (50-55 kD, pI approximately 6), decreased to almost undetectable levels and then reappeared with a time course corresponding to the changes in total protein content of the nerve. Similar changes were seen in a protein we have designated NF (approximately 130 kD, pI approximately 5.2). These three proteins, which were labelled in association with slow axonal transport, may be neurofilament constituents. Large decreases following optic nerve crush were also seen in the relative amounts of alpha- and beta-tubulin, which suggests that they are localized mainly in the optic axons rather than the glial cells. Another group of proteins, W2, W3, and W4 (35-45 kD, pI 6.5-7.0), which showed a somewhat slower time course of disappearance and were intensely labelled in the local synthesis pattern, may be associated with myelin. A small number of proteins increased in relative amount following nerve crush. These included some, P1 and P2 (35-40 kD, pIs 6.1-6.2) and NT (approximately 50 kD, pI approximately 5.5), that appeared to be synthesized by the glial cells. Increases were also seen in one axonal protein, B (approximately 45 kD, pI approximately 4.5), that is carried by fast axonal transport, as well as in two axonal proteins, HA1 and HA2 (approximately 60 and 65 kD respectively, pIs 4.5-5.0), that are carried mainly by slow axonal transport. Other proteins, including actin, that showed no net changes in relative amount (but presumably changed in absolute amount in direct proportion to the changes in total protein content of the nerve), are apparently distributed in both the neuronal and nonneuronal compartments of the nerve.
at the Down's syndrome critical region, which in triplication is associated with diverse phenotypic characteristics of Down's syndrome (1). Patients with Down's syndrome show various neurological symptoms and a high incidence of leukemia (1, 2). Members of the SIM family include SIM1 and SIM2, which map to 6q16.3-q21 and 21q22.2, respectively (3), and belong to a family of transcription factors containing a basic helix-loop-helix motif, two period homologue (PER)͞ARNT͞SIM (ARNT, aryl hydrocarbon receptor nuclear translocator) domains, and the HIF1␣͞SIM͞TRH domains (4-6). In Drosophila, SIM is a master regulator of fruit-fly neurogenesis, regulating midline gene expression (6, 7). The SIM2 gene exists in two distinct forms, the long and short forms (SIM2-l and SIM2-s), due to alternative splicing (3). A putative cancer-related role of the SIM family of genes is their ability to transcriptionally regulate key metabolic enzymes to inactivate carcinogens (8). Binding of carcinogens to the aryl hydrocarbon receptor (AhR), which is kept sequestered in the cytoplasm by heat-shock protein (HSP) 90 (9), dissociates HSP 90. The ligand-bound AhR is then translocated into the nucleus, where it can dimerize with ARNT (10). This complex binds to the xenobiotic response element, present in the promoters of key oxidative enzymes, and activates gene transcription (8, 11), thus causing inactivation of the carcinogen. The SIM proteins can inhibit the dimerization of the ligandbound AhR͞ARNT complex (12) and hence prevent carcinogen metabolism, leading to cumulative DNA damage and cancer.The growth arrest and DNA damage (GADD) family of genes was originally isolated from UV radiation-treated cells and subsequently grouped according to their coordinate regulation by growth arrest and DNA damage (13). The GADD family members include GADD34, -45␣, -45, -45␥, and -153 (14, 15). These are stressresponse genes induced by both genotoxic and nongenotoxic stresses (16)(17)(18). GADD45␣ is the most extensively studied member of the family and is regulated in both a p53-dependent and -independent manner (13,19). GADD45␣-mediated apoptosis may involve activation of JNK and͞or p38 mitogen-activated protein kinase (MAPK) signaling pathways (14,20).We have recently demonstrated that the SIM2 short-form (SIM2-s) gene is specifically expressed in distinct solid tumors, including colon, pancreas, and prostate, but not in the corresponding normal tissues (21-23). Antisense inhibition of SIM2-s expression caused apoptosis in colon and pancreatic cancer-derived cells. In an effort to understand the molecular mechanism underlying the role of SIM2-s in tumor cell growth, we have embarked on mapping the key pathways linked to SIM2-s. We show that apoptosis is tumor cell-selective and requires GADD45␣ function. Further, we demonstrate that key pathways, including caspase, p38 MAPK activities, and WT p53 status, are critical to apoptosis. Our results link apoptosis to induction of tumor cell-selective differentiation. MethodsCell Culture. Isogenic colon ca...
Astrocytes can proliferate as a result of trauma to the brain, such as occurs in a variety of diseases. Understanding the normal distribution of astrocytes is necessary before the extent of astrogliosis can be clearly determined. However, little is known about the normal distribution of GFAP+ astrocytes especially during development. This study examined distribution of GFAP+ astrocytes in regions of the cortex, cerebellum, and brainstem of adult and rat pup brains by immunocytochemistry using antibodies against GFAP. The findings showed a differential distribution of GFAP+ astrocytes in the rat brain. A paucity of GFAP expression was found in most regions of the normal adult rat brainstem, whereas GFAP+ astrocytes were abundantly distributed in all areas of the cortex and cerebellum. A similar regional heterogeneity in the distribution of GFAP+ astrocytes was seen in the neonatal rat brain. These findings suggest that the development of the differential pattern of GFAP+ astrocytes seen in the rat brain does not occur postnatally, but instead is present at birth and appears to be determined during fetal development.
Damage to the sciatic nerve produces significant changes in the relative synthesis rates of some proteins in dorsal root ganglia and in the amounts of some fast axonally transported proteins in both the sciatic nerve and dorsal roots. We have now analyzed protein synthesis and axonal transport after cutting the other branch of dorsal root ganglia neurons, the dorsal roots. Two to three weeks after cutting the dorsal roots, [35S]methionine was used to label proteins in the dorsal root ganglia in vitro. Proteins synthesized in the dorsal root ganglia and transported along the sciatic nerve were analyzed on two-dimensional gels. All of the proteins previously observed to change after sciatic nerve damage were included in this study. No significant changes in proteins synthesized in dorsal root ganglia or rapidly transported along the sciatic nerve were detected. Axon regrowth from cut dorsal roots was observed by light and electron microscopy. Either the response to dorsal root damage is too small to be detected by our methods or changes in protein synthesis and fast axonal transport are not necessary for axon regrowth. When such changes do occur they may still aid in regrowth or be necessary for later stages in regeneration.
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