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Biomedical subjects

T Saitoh

Publications and source records attributed to T Saitoh.

At least 469 records · Page 26Linked to original sources

Comparative studies of RNA polymerase subunits from various bacteria.

The molecular structure of RNA polymerases from Escherichia coli, Salmonella typhimurium, Salmonella anatum,serratia marcescens, Aerobacter aerogens, Proteus mirabilis and Bacillus subtilis were compared based on:i) inhibition of the enzyme activity by treatment with antibodies against E. coli RNA polymerase subunits;ii) analysis of antibody precipitates by sodium ododecyl sulfatepolyacrylamide gel electrophoresis; and iii) analysis of antibody precipitates by urea-isoelectrofocusing followed by sodium dodecyl sulfate-slab gel electrophoresis in the second dimension. All the bacterial RNA polymerases examined cross-react equally with anti-E. COLI HOLOPOLYMERASE BUT EXHIbit different extents of cross-reaction with antibodies against individual subunits. Except for B. subtilis RNA polymerase, the molecular weight and isoelectric point of the enzyme subunits are close to those of E. coli polymerase. However, minor difference were found at least within the resolution of the techniques employed:S. anatum polymerase has sigma subunit larger than E. coli sigma subunit; P. mirabilis enzyme has sigma subunit larger in size and more acidic in charge, and alpha subunit smaller and more basic than corresponding E. coli subunits. The electrophoretic map of B. subtilis enzyme subunits is completely different from that of E. coli enzyme.

Antibodies, Bacterial↗

F deoxyribonucleic acid transferred to recipient cells in the presence of rifampin.

When Escherichia coli F+ cells resistant to rifampin and streptomycin are mated with F- cells sensitive to the antibiotics, a large fraction of F factor deoxyribonucleic acid (DNA), which is transferred to the recipient cells in the presence of either of the antibiotics, can be recovered as covalently closed, circular double-stranded DNA, as in the control mating in the absence of antibiotic. Similar results were obtained in the presence of chloramphenicol or chloramphenicol plus rifampin. It is suggested that the transferred single-stranded F DNA can be converted to the covalently closed, circular double-stranded form in the absence of protein synthesis, and that rifampin-sensitive transcription is not required for the conversion.

Centrifugation, Density Gradient↗

F deoxyribonucleic acid superinfected into phenocopies of donor strains.

When F+ donor cells of Escherichia coli are conjugated with F-, F+, or Hfr recipients under the conditions of phenocopy mating, the male recipients are found capable of accepting the F episome as effectively as the F- recipients. The F deoxyribonucleic acid (DNA) superinfected into the male recipients is converted to the covalently closed, circular duplex form, as in the F- recipients. It is also found that the synthesis of the strand complementary to the transferred single strand and its subsequent conversion to the covalently closed, circular duplex occur effectively in male recipients as well as in female recipients. Under these mating conditions, F-ilv+ episome superinfected to F+ and Hfr cells is diluted out during growth, whereas F-ilv+ transferred into F-cells is replicated and established in almost all progeny cells. These results suggest that the incompatibility of the F episome is not due to the reduction in the rate of the conversion of transferred single-straned F DNA to covalently closed, circular duplex, but, rather, to an inhibition of further replication of the covalently closed, circular F DNA.

Centrifugation, Density Gradient↗

Rejection of renal allografts: specific immunologic suppression.

Kidneys were transplanted across a major genetic barrier (Ag-B locus), from LewisxBN F(1) hybrid rats into bilaterally nephrectomized Lewis rats. Survival of grafts is prolonged (indefinite?) in rats treated with a combination of (i) intravenous injection of donor spleen cells 1 day before the graft, and (ii) passive immunization with antiserum prepared in rats of the recipient strain against donor spleen and lymph-node cells. The recipient's immune response to other antigens is not impaired.

Animals↗

Glycogen synthase kinase 3 alteration in Alzheimer disease is related to neurofibrillary tangle formation.

Highly phosphorylated tau protein is the main component of paired helical filaments (PHF), which comprise the neurofibrillary tangles (NFT) in some neurons of patients with Alzheimer disease (AD). Glycogen synthase kinase 3 (GSK3) phosphorylates tau in vitro at several sites also found to be phosphorylated in PHF-tau; tau is phosphorylated at these sites in both AD and normal control (NC) brains, although the extent of phosphorylation is far greater in tau from AD. If GSK3 levels are increased in AD, then tau phosphorylation and perhaps PHF formation may occur. To quantify GSK3, blots of AD and NC brain supernatant and particulate fractions were probed with antibodies to GSK3. In particulate fractions of AD compared to NC, GSK3 alpha immunoreactivity did not increase, but in fact, decreased 40%, and GSK3 beta immunoreactivity decreased 30%. GSK3 alpha and GSK3 beta levels correlated well with each other. GSK3 levels correlated negatively with numbers of NFT.

Aged↗

Accumulation of amyloid precursor fragment in Alzheimer plaques.

Regenerative and degenerative neurites are components of classical senile plaques found in brain tissue of patients with Alzheimer's disease (AD). Amyloid beta/A4-protein derived from its precursor, amyloid beta/A4-protein precursor (APP/ABPP), constitutes the major portion of the amyloid core of senile plaques. A large N-terminal portion of APP (approximately Mr 100,000) is released from cells, leaving a minor C-terminal portion (approximately Mr 15,000) behind. A series of antisera against various sequences of APP were prepared and used to study the localization of each sequence in brain tissue. Plaque neurites stained as intensely as neuronal cell bodies with three antisera against the N-terminal portion of APP (N-terminal to a.a. 225), whereas five other antisera directed against the other C-terminal portions of APP (a.a. 284 to C-terminal) and antisera against the Kunitz-type protease inhibitor portion of APP stained plaque neurites less intensely than neuronal cell bodies in the hippocampus. These results suggest that a major part of the APP present in the neuritic component of senile plaques is a fragment representing the N-terminal one-third of the molecule.

Aged↗

PDGF is associated with neuronal and glial alterations of Alzheimer's disease.

In the present study we observed that while platelet-derived growth factor (PDGF)-BB is exclusively expressed by neurons in the human brain, PDGF-AA is expressed in neurons and blood vessels. In Alzheimer's disease (AD), antibodies against PDGF-BB (but not PDGF-AA) recognized the neurofibrillary alterations of this disease. The levels of PDGF-BB correlated with the patterns of synaptic loss and sprouting while PDGF-AA immunostaining of the vessels was correlated with glial proliferation. Immunostaining was completely abolished when the antibodies were preincubated with their respective purified recombinant PDGF. Western blot analysis showed that antibodies against PDGF recognized a 31 kDa protein that was mildly increased in AD. These data suggest that PDGF, as well as other neurotrophic factors, play an important role in the mechanisms of neurofibrillary pathology in AD.

Aged↗

SCG10, a neuron-specific growth-associated protein in Alzheimer's disease.

Neuronal growth-associated proteins (nGAPs) are markers of neuronal process outgrowth and are associated with both degenerative and sprouting responses in Alzheimer's disease (AD) brain. To study possible involvement of SCG10, an nGAP, in AD, we cloned human SCG10 cDNA and analyzed SCG-10 at mRNA and protein levels in control and AD brains. The deduced amino acid sequence of human SCG10 was 69% identical to stathmin, another nGAP. By in situ hybridization, both SCG10 and stathmin mRNAs were detected in selected neuronal populations in aged human brains. Quantitative analysis by RNase protection revealed that levels of neither SCG10 nor stathmin mRNAs were significantly altered in AD. Using an SCG10-specific antibody, Western blot analysis did not reveal any quantitative changes of SCG10 in AD. However, when the concentration of SCG10 protein was plotted against the number of tangles, a positive correlation was found. SCG10 levels did not correlate with plaque numbers. Furthermore, immunohistochemical study revealed that neuronal SCG10 protein accumulated in the cell bodies in AD-affected regions. Thus, SCG10 compartmentalization and metabolism may be altered in AD possibly due to mechanisms related to tangle formation in this disease.

Alzheimer Disease↗

Neurofibrillary tangle-associated alteration of stathmin in Alzheimer's disease.

Stathmin (p19), a 19-kDa cytosolic phosphorotein, plays a key role in converting extracellular signals into intracellular biochemical changes. Antibodies and cDNA specific for stathmin were used to study its levels and localization in normal and Alzheimer's disease (AD) brain tissue. The stathmin protein concentration was reduced in AD neocortex as assessed by Western blotting, whereas the concentration of its mRNA detected by both in situ hybridization and slot blot were increased in AD. The alteration of the stathmin protein concentration was negatively correlated with neurofibrillary tangle numbers but not with plaque numbers. Immunoreactivity was evenly localized to the cytoplasm of neurons in control cortical sections, whereas in AD it was preferentially localized to some of the neurofibrillary tangle-bearing neurons. Numbers of stathmin-positive neurons were inversely correlated with tangle numbers but not with plaque numbers in the frontal cortex of AD patients.

Alzheimer Disease↗

Detection of VP-16-treated HL-60 cell apoptosis by TUNEL electron microscopy.

Apoptosis is essential to many physiological processes, including maturation of cells and the immune system. Deficient regulation of apoptosis may play an important role in many pathological conditions, such as autoimmunity, AIDS, and myelodysplastic syndrome. Several methods have been described to identify apoptotic cells. DNA strand breaks can be identified by labeling free 3'-OH termini with modified nucleotides by enzymatic reaction (TUNEL method). In this study, apoptosis was introduced in cultured HL-60 cells treated with VP-16, and the TUNEL method was adapted for electromicroscopy, called the EM TUNEL method. The results of the EM TUNEL method were compared with those of light microscopy and flow cytometry. Apoptotic cells following VP-16 (10 micrograms/mL) treatment were detected after 3 h by all methods (light microscopy, Erythrocin B, electron microscopy, flow cytometry, TUNEL, and EM TUNEL). EM TUNEL was the most sensitive method of detection, with a detection rate of 32%. Furthermore, EM TUNEL was more suitable for distinguishing cell lineage than light microscopy TUNEL. The results indicate that EM TUNEL can be used to detect apoptosis in bone marrow species in vivo.

Apoptosis↗

Enhancement of lipid phagocytosis by cloned cells derived from rat malignant fibrous histiocytoma under hyperlipemic conditions.

The histogenesis of malignant fibrous histiocytoma (MFH) was studied by observing the influence of hyperlipemic (HL) conditions on the behavior of cloned MT-8 cells with an undifferentiated mesenchymal character and MT-9 cells with histiocytic and fibroblastic features, both derived from a spontaneous rat MFH. The cells were grown in medium containing 10% normal rat serum (CSM) or 10% HL rat serum (HSM) for 1, 3, 8 and 24 h. The cells grown in HSM showed a time-dependent, significant increase in numbers of cytoplasmic lipid droplets per cell and cells positive for acid phosphatase (ACP) and non-specific esterase (NSE) compared with cells grown in CSM. The number of cells positive for two different anti-rat monocyte/macrophage monoclonal antibodies (ED-1 and ED-2) also increased significantly in HSM. This histiocytic nature was confirmed by electron microscopy. Tumors were induced by inoculating MT-8 and MT-9 cells into rats on a standard diet and rats on a HL diet. MT-8 and MT-9 tumor cells in HL rats phagocytized lipid droplets and showed stronger positive reactions for ACP and NSE compared with tumors in control rats. These results imply that xanthomatous cells in MFH may be a phenotype of MFH cells expressing an enhanced histiocytic nature. MFH cells seem to alter their behavior depending on cultural or microenvironmental conditions.

Animals↗