Biomedical subjects
Publications and source records attributed to S Chen.
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Aromatase, a cytochrome P450, catalyzes three consecutive hydroxylation reactions converting C19 androgens to aromatic C18 estrogenic steroids. On the basis of a recent computer modeling of the active site of aromatase, a hydrophobic pocket, thought to be important for the binding of some aromatase inhibitors, was predicted to extend roughly in the plane of the steroid substrate, from the position that would be occupied by its C4 and C7 atoms. Four mutants, G121A, I125N, F235N, and I474F, were generated to test this model. Although the mutagenesis results have shown that the current model for the active site of aromatase almost certainly contains a number of errors, the results are in general very satisfactory in that they suggest how the model should be altered by local realignments of the aromatase sequence with that of cytochrome P450cam. Among the mutants, I474F is the most interesting one. Its Km value for androstenedione was found to be lower than the wild type enzyme, and the kinetic analysis exhibited a substrate inhibition-like kinetic profile through an "in-cell" assay. In addition, this mutation reduces the binding affinity of an aromatase inhibitor, 4-hydroxyandrostenedione, and increases the binding affinity of two aromatase inhibitors, aminoglutethimide and CGS 16949. This study demonstrates a useful approach, by a combination of computer modeling, site-directed mutagenesis, and inhibitor binding studies, to examine the structure of the active site of aromatase and the binding nature of various aromatase inhibitors.
In cerebella of neonatal rats, immunoreactivity of protein kinase C delta (PKC delta) was moderate to strong in radial glia, Bergmann fibers and astrocytes but was absent in neurons. Beginning on the 7th day, a few small clusters of Purkinje cells expressed weak PKC delta reactions in caudal lobules of the vermis. From 10 to 20 days, clusters of labeled Purkinje cells increased in number, size and intensity while astrocytes and palisades of aligned Bergmann glia gradually diminished in intensity. At 21 days, glial cells had weak reactions while Purkinje and stellate-basket cells had intensity and distribution patterns found in adults. A transient occurrence of PKC delta in glia and later appearance in selective groups of neurons strongly support a significant role for this enzyme in signal transduction.
Experimentation on human stem cells is hampered by the relative paucity of this population and by the lack of assays identifying multilineage differentiation, particularly along the lymphoid lineages. In our current study, phenotypic analysis of low-density fetal bone marrow cells showed two distinct populations of CD34+ cells: those expressing a high density of CD34 antigen on their surface (CD34hi) and those expressing an intermediate level of CD34 antigen (CD34lo). Multiple tissues were used to characterize the in vitro and in vivo potential of these subsets and showed that only CD34hi cells support long-term B lymphopoiesis and myelopoiesis in vitro and mediate T, B, and myeloid repopulation of human tissues implanted into SCID mice. CD34lo cells repeatedly failed to provide long-term hematopoietic activity in vivo or in vitro. These results indicate that a simple fractionation based on well-defined CD34 antigen levels can be used to reproducibly isolate cells highly enriched for in vivo long-term repopulating activity and for multipotent progenitors, including T- and B-cell precursors. Additionally, given the limited variability in the results and the high correlation between in vitro and in vivo hematopoietic potential, we propose that the CD34hi population contains virtually all of the stem cell activity in fetal bone marrow and therefore is the population of choice for future studies in hematopoietic stem cell development and gene therapy.
The effect of hydrostatic pressure of up to 15 bars on the aggregability of rat and human red blood cells (RBC), i.e., their capability to form aggregates, was studied using computerized image analysis. The aggregate size distribution was determined under ambient pressure, following application of hydrostatic pressure for various durations up to 2 h. It was found that RBC aggregability markedly increases, up to three-fold, as the pressure which had been applied was increased. Accordingly, higher shear stress is required for dispersing the aggregates of pressure-treated RBC than those of untreated cells. The median size of human RBC aggregates was about three times higher than that of rat RBC, and this ratio was maintained following pressure treatment. RBC aggregability is a major determinant in blood flow, especially in the microcirculation. Pressure at the levels used in this study occurs in physiological states such as hyperbaric treatment or diving. The enhanced aggregability induced by application of such pressure implies that blood flow in microvessels might be altered under conditions associated with elevated hydrostatic pressure.
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The expression of aromatase in JAR cells, human placental choriocarcinoma cells, was found to be induced by the treatment of phorbol 12,13-diacetate (PDA), phorbol 12,13-didecanoate (PDD), or phorbol 12-myristate 13-acetate (TPA), but not 4 alpha-phorbol, 12,13-didecanoate (4 alpha PDD). At 1 microM or higher concentrations, these phorbol esters increased the level of aromatase mRNA and aromatase activity in a dose- and time-dependent fashion. Since the rates of the decrease of aromatase mRNA in phorbol ester treated and untreated cells were not significantly different in the presence of actinomycin D, the induction was not due to an increase in the stability of aromatase mRNA, but rather due to an increase in the synthesis of aromatase mRNA. The stimulation was not inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). It is thought that the induction either follows a protein kinase C-independent manner or results from a down-regulation of protein kinase C pathway. Studies from several laboratories have revealed that the regulation of the expression of aromatase in estrogen-producing cells involves very complex processes. The apparent induction of aromatase expression in JAR cells by phorbol esters represent a mechanism modulating estrogen production in human placental choriocarcinoma cells, that may or may not be utilized in other estrogen-producing cells.
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The relationship of neurons that express nitric oxide synthase type I (NOS-I), the synthetic enzyme of the free radical nitric oxide (NO), with cells that contain the low affinity p75 nerve growth factor receptor (NGFr) was examined in the basal forebrain region of the rat using double immunohistofluorescence. NOS and NGFr were found to be co-localized in a neuronal population that displayed a selective distribution. Double immunopositive neurons were evident in the medial septum and in rostral levels of the diagonal band nuclei where the vast majority (more than 80%) of NOS-immunoreactive cells were also NGFr-positive; the two cell populations were separate at more caudal levels of the basal forebrain. These findings support the chemical heterogeneity of septal and basal forebrain neurons which contain NGFr and indicate that in the rat a subset of neurons of the septum-diagonal band complex may release NO.
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The recipient vessels are of prime importance in free flap transfers to the lower limbs. To determine the incidence and pattern of vascular trauma, a study was carried out in 126 patients who had Gustillo Type III open fractures of the distal legs and feet that required free flaps for wound coverage. In comparison with the posterior tibial artery, the anterior tibial artery has a much higher incidence of injury with more extensive damage. It can be injured at a more proximal level than estimated from gross inspection during surgery. This should be borne in mind when the anterior tibial artery is selected as the recipient artery in order to prevent reexploration and failure of the flaps. However, the posterior tibial artery is much less vulnerable to damage in most injuries and is more reliable as the recipient artery. In this series there was no problem in finding proper recipient veins in the legs. A general principle is proposed for selecting recipient vessels in crushed legs, with a warning against the pitfalls that have been encountered.
NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2) (DT-diaphorase) is a FAD-containing reductase that catalyzes a unique 2-electron reduction of quinones. It consists of 2 identical subunits. In this study, it was found that the carboxyl-terminal portion of the 2 subunits can be cleaved by various proteases, whereas the amino-terminal portion cannot. It was also found that proteolytic digestion of the enzyme can be blocked by the prosthetic group FAD, substrates NAD(P)H and menadione, and inhibitors dicoumarol and phenindione. Interestingly, chrysin and Cibacron blue, 2 additional inhibitors, cannot protect the enzyme from proteolytic digestion. The results obtained from this study indicate that the subunit of the quinone reductase has a 2-domain structure, i.e., an amino-terminal compact domain and a carboxyl-terminal flexible domain. A structural model of the quinone reductase is generated based on results obtained from amino-terminal and carboxyl-terminal protein sequence analyses and electrospray mass spectral analyses of hydrolytic products of the enzyme generated by trypsin, chymotrypsin, and Staphylococcus aureus protease. Furthermore, based on the data, it is suggested that the binding of substrates involves an interaction between 2 structural domains.
The amino acid sequence of mouse liver NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2) has been determined by tandem mass spectrometry and deduced from the nucleotide sequence of the cDNA encoding for the enzyme. The electrospray mass spectral analyses revealed, as previously reported (Prochaska HJ, Talalay P, 1986, J Biol Chem 261:1372-1378), that the 2 forms--the hydrophilic and hydrophobic forms--of the mouse liver quinone reductase have the same molecular weight. No amino acid sequence differences were found by tandem mass spectral analyses of tryptic peptides of the 2 forms. Moreover, the amino-termini of the mouse enzymes are acetylated as determined by tandem mass spectrometry. Further, only 1 cDNA species encoding for the quinone reductase was found. These results suggest that the 2 forms of the mouse quinone reductase have the same primary sequences, and that any difference between the 2 forms may be attributed to a labile posttranslational modification. Analysis of the mouse quinone reductase cDNA revealed that the enzyme is 273 amino acids long and has a sequence homologous to those of rat and human quinone reductases. In this study, the mouse quinone reductase cDNA was also ligated into a prokaryotic expression plasmid pKK233.2, and the constructed plasmid was used to transform Escherichia coli strain JM109. The E. coli-expressed mouse quinone reductase was purified and characterized. Although mouse quinone reductase has an amino acid sequence similar to those of the rat and human enzymes, the mouse enzyme has a higher NAD(P)H-menadione reductase activity and is less sensitive to flavones and dicoumarol, 2 known inhibitors of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
A wide range of glycoalkaloids from potato shoots and tomatoes, including trisaccharide-containing glycoalkaloids (alpha-chaconine, alpha-solanine, and alpha-solasonine), tetrasaccharide-containing glycoalkaloids (alpha-tomatine and demissine), and disaccharide-containing glycoalkaloids (beta 1-chaconine, beta 2-chaconine, and beta-solamargine), have been studied by both positive and negative ion liquid secondary ion and four-sector tandem mass spectrometry with scanning-array detection. In positive ion mode, collisionally induced dissociation tandem mass spectra of the [M + H]+ ions induce three major fragmentation processes, Z cleavage, Y cleavage, and 1,5X cleavage, which are structurally informative. Signals resulting from Z0, Y0, and 1,5X0 cleavages provide information on the nature of various aglycone moieties in all glycoalkaloids. Linkages and positions of the sugars in trisaccharide- and tetrasaccharide-containing glycoalkaloids are indicated by the presence or absence of the ions corresponding to Z alpha/beta and Y alpha/beta cleavages and intensity differences of the peaks due to 1,5X alpha and 1,5X beta cleavages, respectively. In negative ion mode, collisionally induced dissociation tandem mass spectra of the [M - H]- ions induce Y cleavage as the major fragmentation process. The location of the terminal sugars in branched trisaccharide and tetrasaccharide glycoalkaloids is indicated by the difference in intensity of the ions due to Y alpha cleavage and Y beta cleavage. Isomeric structures cannot, however, be differentiated unambiguously; complete structural assignment is only possible by NMR of purified components. Both positive and negative ion tandem mass spectrometry are considered to be suitable for the characterisation of glycoalkaloids in mixtures. The positive ion method has the advantage of (i) a lower detection limit than in conventional mass spectrometry; (ii) numerous and intense fragment ions which are structurally informative; and (iii) the capability of analyzing minor components in crude extracts. Comparable analysis by other analytical means would not have provided the amount of structural information on the components in the glycoalkaloid mixtures.
Intracerebroventricular (icv) injection of propidium iodide (PI) in the rat results in a transient movement disorder characterized by nystagmus, ataxia, and shaking. In the present study we used c-Fos as a marker for neuronal activation to investigate the neural substrate underlying this movement disorder. PI was injected into the lateral cerebral ventricle of freely moving rats through a previously implanted cannula. Animals were perfused 3 h after the injection and the brains were processed for c-Fos immunocytochemistry. Paired control animals were injected with saline. After PI injection, a significant Fos expression was seen in the cerebral cortex, thalamic midline nuclei, thalamic intralaminar nuclei, hypothalamus, central gray, pontine nuclei, locus coeruleus, vestibular complex, inferior olive, ventrolateral medulla, nucleus of solitary tract, and deep cerebellar nuclei. Few or no Fos immunoreactive cells were seen in the above structures of the control animals. The present study indicates that a large number of neurons located in many different neural structures are activated following icv injection of PI. Second, consistent with the cerebellar feature of the movement disorder, a major Fos expression was found in the cerebellar circuitry (deep cerebellar nuclei, pontine nuclei, vestibular complex, and inferior olive). It reinforces further the assumption that the movement disorder is due to cerebellar dysfunction caused by PI.
NADPH diaphorase (NADPH-D) histochemistry was examined following cerebellar lesions induced by stab or knife cuts. A population of Purkinje cells adjacent to the injury became NADPH-D positive after either type of lesion. These cells appeared by 3 days postlesion and were still seen after 6 weeks. Somata of reactive cells shrank gradually and were reduced about 38% in size by 6 weeks after lesion. In the inferior olive of animals subjected to cerebellar knife cut, NADPH-D-positive cells were found in the beta nucleus, dorsal cap, and subnucleus C. These cells became NADPH-D positive 5 days postlesion, were shrunken at 4 weeks postlesion, and were reduced about 26% in somata size by 6 weeks after lesion. Other cerebellar afferents did not exhibit obvious NADPH-D induction following these lesions. In unlesioned animals, no Purkinje cells or inferior olivary neurons were NADPH-D positive. The present study demonstrates that cerebellar damage induces NADPH-D, a marker for nitric oxide (NO) synthase, in Purkinje cells and inferior olivary neurons. In addition, these reactive neurons exhibit long-term degenerative changes. Taken together with previous studies, these findings indicate that NO, produced by the lesion-induced nitric oxide synthase (NOS), may contribute to the degeneration of brain neurons after injury. The lack of NADPH-D-positive neurons in either Purkinje or the inferior olivary neurons in nonlesioned animals indicates that this may provide a useful model system to study injury-induced NOS in brain.
This study examined the expression of the sensory neuropeptides, calcitonin gene-related peptide (CGRP) and substance P (SP), in the lumbar 4 and 5 dorsal root ganglion (DRG) and spinal cord of spontaneously diabetic BB rats and non-diabetic controls using quantitative immunohistochemical analysis. In both animal groups immunoreactivities for CGRP and SP were widely distributed within the neurons of DRG and in nerve fibres of the dorsal spinal cord. Image analysis of each neuropeptide subpopulation in the DRG showed that in diabetic rats the cell diameter of immunostained CGRP neurons was significantly decreased compared with controls, while no difference could be found for SP-immunoreactive (IR) neurons. The decrease in the CGRP-IR cell diameter appeared to occur mainly in medium to large neurons (30-50 microns diameter; 2.2% controls, < 1% diabetes), this change being parallel to an increased frequency of small-size neurons (< 20 microns diameter) in diabetic rats (62% controls, 69% diabetes; P < 0.05). However, there was no statistical difference in the total number of cells immunostained for either CGRP or SP between control and diabetic rats. The ratio of CGRP or SP neurons compared to total cells in the ganglion was similar in control and diabetic groups. No difference could be observed for peptide immunoreactivity in the dorsal and ventral horns of either control or diabetic animals. The observed changes of perikaryal size in diabetic rats might relate to the reduced axonal calibre and conduction velocity observed in these animals, and indicate that subpopulations of sensory neurons are affected differently by diabetes.
Cells of healthy maize plants as well as those infected by maize dwarf mosaic potyvirus were examined by electron microscopy for the location of ATPase activity. In healthy and virus infected plants, ATPase activity was found in plasma membranes, chloroplast thylakoid membranes, nuclear membranes and in mitochondria. In virus-infected cells, ATPase activity was also observed in cytoplasmic vesicles which were found in close proximity to the virus-specific cytoplasmic inclusion bodies (CI), at the ends of the arms of the CI and in plasmodesmata.