Electron-microscopic immunocytochemistry of neuropeptide Y immunoreactive innervation of vasopressin neurons in the paraventricular nucleus of the rat hypothalamus

The neuropeptide Y (NPY) immunoreactive synaptic input to neurons containing neurophysin II (NP II), the carrier protein of vasopressin (VP), was observed in the paraventricular nucleus (PVN) of the rat hypothalamus by double-labeling immunocytochemistry combining the preembedding peroxidase-antiperoxidase (PAP) method with the postembedding immunogold staining method at the electron-microscopic level. NPY-like immunoreactivities were detected by the PAP method in the dense granular vesicles (70-100 nm in diameter) in the immunoreactive presynaptic axon terminals. NP II-like immunoreactive large neurosecretory granules labeled with gold particles were found in the neurons receiving synaptic input of the NPY-like immunoreactive terminals. This suggests that NPY may be a neurotransmitter or neuromodulator and that NPY neurons may, through synaptic contacts, regulate the secretion of VP neurons.     

DNA cleavage by hydroxyl radicals generated in a vanadyl ion-hydrogen peroxide system.

Vanadyl ion (+4 oxidation state) has been shown to be an effective agent for chemoprotection of cancers in animals. For understanding the mechanism, distribution of vanadium was studied. More vanadium was found to accumulate in the nuclei of the liver of rats when it was given as vanadyl sulfate than when it was given as sodium vanadate (+5 oxidation state). The reactivity of vanadyl ion with DNA was investigated by the DNA cleavage technique and the reaction mechanism by ESR spectroscopy. Incubation of double-strand DNA with vanadyl ion and hydrogen peroxide resulted in marked concentration- and pH-dependent DNA cleavage. Studies by the ESR spin-trap method demonstrated that hydroxyl radicals are generated during the reactions of vanadyl ion with hydrogen peroxide. Thus the antineoplastic action of vanadyl ion is proposed to be due to DNA cleavage by hydroxyl radicals generated in the cells.     

Errata

The online version of the original article can be found at     

Tissue Distribution and Immunocytochemical Localization of Neurotrophin-3 in the Brain and Peripheral Tissues of Rats

Abstract: The tissue distribution of neurotrophin-3 (NT-3) was investigated in rats at 1 month of age using a newly established, sensitive two-site enzyme immunoassay system for NT-3, as well as the immunocytochemical localization of this protein. The immunoassay for NT-3 enabled us to quantify NT-3 at levels > 3 pg per assay. In the rat brain, NT-3 was detectable only in the olfactory bulb (0.54 ng/g wet weight), cerebellum (0.71 ng/g), septum (0.91 ng/g), and hippocampus (6.3 ng/g). By contrast, NT-3 was widely distributed in peripheral tissues. Appreciable levels of NT-3 were also found in the thymus (31 ng/g), heart (38 ng/g), diaphragm (21 ng/g), liver (45 ng/g), pancreas (892 ng/g), spleen (133 ng/g), kidney (40 ng/g), and adrenal gland (46 ng/g). An antibody specific for NT-3 bound to pyramidal cells in the CA2-CA4 regions of the hippocampus, to A cells in the islets of Langerhans in the pancreas, to unidentified cells in the red pulp of the spleen, to liver cells, and to muscle fibers in the diaphragm from rats at 1 month of age. Molecular masses of NT-3-immunoreactive proteins in the hippocampus and pancreas were 14 and 12 kDa, respectively. Thus, in rats, NT-3 was detected in restricted regions of the brain and in the visceral targets of the nodose ganglia at high concentrations. Our present results suggest that NT-3 not only functions as a classical target-derived neurotrophic factor but also can play other roles.     

ClpX protein of Escherichia coli activates bacteriophage Mu transposase in the strand transfer complex for initiation of Mu DNA synthesis.

During transposition bacteriophage Mu transposase (MuA) catalyzes the transfer of a DNA strand at each Mu end to target DNA and then remains tightly bound to the Mu ends. Initiation of Mu DNA replication on the resulting strand transfer complex (STC1) requires specific host replication proteins and host factors from two partially purified enzyme fractions designated Mu replication factors alpha and beta (MRFalpha and beta). Escherichia coli ClpX protein, a molecular chaperone, is a component required for MRFalpha activity, which removes MuA from DNA for the establishment of a Mu replication fork. ClpX protein alters the conformation of DNA-bound MuA and converts STC1 to a less stable form (STC2). One or more additional components of MRFalpha (MRFalpha2) displace MuA from STC2 to form a nucleoprotein complex (STC3), that requires the specific replication proteins and MRFbeta for Mu DNA synthesis. MuA present in STC2 is essential for its conversion to STC3. If MuA is removed from STC2, Mu DNA synthesis no longer requires MRFalpha2, MRFbeta and the specific replication proteins. These results indicate that ClpX protein activates MuA in STC1 so that it can recruit crucial host factors needed to initiate Mu DNA synthesis by specific replication enzymes.     

Purification and Characterization of Membrane Protein (90 kDa) from Mycoplasma salivarium,Which Binds Immunoglobulin (Ig) G Fc Fragment

A 90 kDa protein of Mycoplasma salivarium was released from cell membranes of the organism with Triton X-100 and purified by ion-exchange chromatography and chromatofocusing. The protein was eluted at pH 5.5 by chromatofocusing. The protein was shown to react with the Fc fragments of IgG from human and nine different animal species and did not distinguish between IgG from different species, while protein A, tested for comparative purposes, displayed a strong specificity for human and swine IgG. Furthermore, the protein reacted with antigen specific goat IgG (specific for gamma chains of human IgG), sheep red blood cells (SRBC) sensitized with rabbit antiserum to SRBC, that is, the Fc part of rabbit IgG, and concanavalin A as well. These findings may suggest that the protein is a lectin which binds the carbohydrate moiety of the Fc part of IgG.     

Laminin A, B1, and B2 Chain Gene Expression in Transected and Regenerating Nerves: Regulation by Axonal Signals

Abstract: Laminin A, B1, and B2 chain mRNA levels in degenerating and regenerating mouse sciatic nerves were examined using northern blot analysis. In normal intact nerves, B1 and B2 mRNA steady-state levels were high, but when the nerves were crushed, the steady-state levels of B1 and B2 mRNA per milligram wet tissue weight of the distal segments of the nerves increased five- to eightfold over that of control levels as the total RNA and β-actin mRNA levels increased, suggesting that these increases were the consequence of Schwann cell proliferation after axotomy. When the steady-state levels of B1 and B2 mRNA were normalized as the ratio to total RNA or β-actin mRNA levels, however, they drastically decreased to about 20% of the normal nerve levels in the nerve segments distal to both the crush and transaction sites 1 day after injury. In the crushed nerves, B1 and B2 mRNA levels gradually increased as the regenerating nerves arrived at the distal segments and reestablished normal axon–Schwann cell contact, and then returned to normal levels on the 21 st day. In the transected nerves, where Schwann cells continued to be disconnected from axons, both B1 and B2 mRNA levels remained low. Cultured Schwann cells expressed detectable levels of B1 and B2 chain mRNA which significantly increased when the cells were cocultured with sensory neurons. However, mRNA for A chain was not detectable in the normal, axotomized nerves or in cultured Schwann cells. These data indicate that Schwann cells express laminin B1 and B2 chain mRNA that are up-regulated by axonal or neuronal contact, but they do not express A chain mRNA.     

Embryonic Development and Postnatal Changes in Free d-Aspartate and d-Serine in the Human Prefrontal Cortex

Abstract: We have analyzed free chiral amino acids (aspartate and serine) in the human frontal cortex at different ontogenic stages (from 14 weeks of gestation to 101 years of age) by HPLC with fluorometric detection after derivatization with N-tert -butyl-oxycarbonyl- l -cysteine and o -phthaldialdehyde. Exceptionally high levels of free d -aspartate and d -serine were demonstrated in the fetal cortex at gestational week 14. The ratios of d -aspartate and of d -serine to the total corresponding amino acids were also high, at 0.63 and 0.27, respectively. The concentration of d -aspartate dramatically decreased to a trace level by gestational week 41 and then remained very low during all postnatal stages. In contrast, the frontal tip contained persistently high levels of d -serine throughout embryonic and postnatal life, whereas the d -amino acid content in adolescents and aged individuals was about half of that in the fetuses. Because d -aspartate and d -serine are known to have selective actions at the NMDA-type excitatory amino acid receptor, the present data suggest that these d -amino acids might play a pivotal role in cerebral development and functions that are related to the NMDA receptor.     

Ontogeny of pituitary cell-types and the hypothalamo-hypophysial relationship during early development of chum salmon,Oncorhynchus keta

Tissue non-specific alkaline phosphatase is a membrane-bound glycoprotein enzyme which is characterized by its phosphohydrolytic, protein phosphatase, and phosphotransferase activities. This enzyme is distributed virtually in all mammalian tissues, particularly during embryonic development. Its expression is stagespecific and can be demonstrated in the developing embryo as early as the 2-cell stage. It has been suggested that tissue non-specific alkaline phosphatase might play a role in tissue formation. In the study reported here, a genetransfer approach was employed to investigate possible roles for this enzyme by inserting the cDNA for rat tissue non-specific alkaline phosphatase into CHO and LLC-PK1 cells. Permanently transfected cell-lines expressing varying levels of alkaline phosphatase were estblished. The data showed that functional enzyme was expressed in the transfected cells. Cell spreading and attachment were enhanced in transfected CHO cells expressing high levels of tissue non-specific alkaline phosphatase but not in the LLC-PK1 cells. Further, in CHO cells, proliferation was shown to be inversely proportional to the level of the tissue non-specific alkaline phosphatase expression. Homotypic cell association was demonstrated in both alkaline phosphatase-positive and alkaline phosphatase-negative cells in both CHO and LLC-PK1 celllines. Taken together, these findings suggest that in addition to a role in mineralization of bone, tissue nonspecific alkaline phosphatase might also play a role in other cell activities, including those related to differentiation, such as cell-cell or cell-substrate interaction and proliferation.