The arabidopsis thaliana myo-inositol 1-phosphate synthase (EC 5.5.1.4).
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Arabidopsis↗
Biomedical subjects
M D Johnson
Publications and source records attributed to M D Johnson.
Plasmid-directed assembly of the lipid-containing membrane of bacteriophage phi 6.
The nucleocapsid of bacteriophage phi 6 is enveloped within a lipid-containing membrane. The membrane is composed of proteins P3, P6, P9, P10, and P13 and phospholipids. The relationship between membrane protein P9 and morphogenetic protein P12 was studied in the absence of phage infection. cDNA copies of genes 9 and 12 were expressed on plasmids in Pseudomonas syringae pv. phaseolicola. Immunoblotting demonstrated the presence of protein P9 in strains carrying both gene 9 and gene 12 but not in strains with gene 9 alone. In the absence of P12, P9 was found to be unstable. Simultaneous synthesis of proteins P9 and P12 led to the formation of a low-density P9 particle having a buoyant density similar to that of precursor structures composed of phospholipid and proteins isolated from phi 6-infected cells. These results are consistent with results of previous genetic experiments suggesting that P9 and P12 are necessary and sufficient for the formation of the phi 6 envelope. Extensions of P9 at the C terminus do not impair particle formation; however, N-terminal extensions or C-terminal deletions that extend into the hydrophobic region of P9 do impair particle formation.
Amino Acid Sequence↗
Isolation and characterization of nonsense mutations in gene 10 of bacteriophage phi 6.
Nonsense mutants of bacteriophage phi 6 were isolated by a procedure that involved directed mutagenesis of a cDNA copy of genomic segment M, transcription of this segment, in vitro packaging into procapsids, and transfection of spheroplasts to form viable mutant phage. Recombinant phi 6 viruses that contained amber mutations in two open reading frames, ORF 10 and ORF D, of genomic segment M were isolated. We show that phi 6 protein P10 is the gene product of ORF 10. Further characterization of the phi 6 ORF 10(Am) mutant revealed that phi 6 membrane-associated protein P10 is not required to make enveloped phage particles in infected cells. Enveloped phage particles isolated from a phi 6 ORF 10(Am) infection contained extremely low levels of phi 6 membrane-associated proteins P6 and P3. The low abundance is due to the very low level of P6 synthesis in phi 6 ORF 10(Am)-infected cells. The results suggest that P10 might play a role in regulating the translation of gene 6. Protein P10 was found to be required for host lysis.
Bacteriophage phi 6↗
Maintenance of training effects on the Wisconsin Card Sorting Test by patients with schizophrenia or affective disorders.
The authors used the Wisconsin Card Sorting Test to study 50 hospitalized psychiatric patients: 28 with schizophrenia, 17 with affective disorders, and five with schizoaffective disorder. The schizophrenic patients performed significantly more poorly than the patients with affective disorders. Both groups of patients improved when given additional instructions. The schizophrenic patients maintained their improvement when retested approximately 6 weeks later. The results suggest that factors other than frontal cortex dysfunction are involved in schizophrenic patients' performance on the Wisconsin Card Sorting Test.
Adult↗
Cytokines in experimental otitis media with effusion.
Studies in the authors' laboratory have recently demonstrated the presence of potent inflammatory cytokines such as interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF alpha) in human middle ear effusions. The clinical significance of this finding has not been fully elucidated because of the limitations of human studies. We hypothesized that the chinchilla model of otitis media may be an appropriate system with which to study the role of cytokines in otitis media with effusion. To begin to investigate this possibility, 30 chinchillas underwent surgical blockage of the eustachian tube (ET) to promote effusion development. After 2 weeks, examination by otoscopy demonstrated 27 ears to have developed an effusion. Next, all middle ear clefts, in random manner, were either injected with heat-killed Streptococcus pneumoniae 1 x 10(6) in 0.1 mL normal saline, injected with 0.1 mL normal saline alone, or received no injection at all. Middle ear effusions were obtained and analyzed for IL-1 beta and TNF alpha by enzyme-linked immunosorbent assay (ELISA). This study demonstrated a significant correlation between IL-1 beta and the presence of an effusion (P < .001). Additionally, increased TNF alpha levels correlated with bacterial component presence (P < .001), i.e., mean TNF alpha level was 108, 10.8, and 0 pg/mL in bacteria, normal saline, and noninjected ears, respectively. These findings would suggest that cytokine expression may relate to specific pathological conditions and that the chinchilla model for otitis media with effusion (OME) could be used to further explore the role of cytokines in OME.
Animals↗
Murine model of otitis media with effusion: immunohistochemical demonstration of IL-1 alpha antigen expression.
Recent studies have suggested that cytokines likely play a central role in the formation and maintenance of otitis media with effusion (OME). Currently, there is no immunologically defined animal model for the study of cytokines as they contribute to the formation of OME. In the present study, a murine model of OME, using eustachian tube blockage via an external surgical approach, was developed. The murine model temporal bone histology appears to mimic the histology found in chronic otitis media with effusion in humans. Additionally, using this murine model, interleukin-1 alpha (IL-1 alpha) expression was detected in the middle ear using standard immunohistochemical techniques. IL-1 alpha seemed localized to the epithelial lining of the middle ear as well as 5% to 10% of inflammatory cells. This model should provide the necessary tool to further study the immunologic aspects of OME.
Animals↗
Courteous service: Its assessment and modification in a human service organization.
We evaluated strategies to increase behaviors associated with courteous provision of service by 3 staff members of a human service agency. Training included written instructions, practice, and performance feedback. A lottery procedure was introduced to maintain courteous service after training. The results of a multiple baseline design across the 3 participants showed marked increases in courteous behaviors following training. These effects were maintained at 3-, 5-, and 8-month follow-ups. Consumers' satisfaction with service also increased. These findings suggest that simple training and reinforcement procedures can enhance courtesy afforded those who receive service from public and nonprofit organizations.
Journal Article↗
Indolent granulomatous angiitis. Case report.
Granulomatous angiitis is a rare, treatable central nervous system vasculitis. Prompt diagnosis may be thwarted by protean presenting symptoms, an indolent clinical course, and atypical neurological findings. The authors describe a case of indolent granulomatous angiitis in which the patient presented with cerebellar signs and tissue changes suggestive of an atypical cerebellar infarction. After several years of remissions and relapses, repeat evaluation and biopsy disclosed granulomatous angiitis both in remote infarctions and in new cortical lesions. The clinical course and neuroradiological and pathological findings are compared with previous reports of fulminant and indolent granulomatous angiitis.
Biopsy↗
Disordered eating in active and athletic women.
In an attempt to improve athletic performance, some female athletes develop patterns of disordered eating. The spectrum of disordered eating ranges from mild to severe, with the severe form resulting in anorexia nervosa or bulimia nervosa. Disordered eating can result in decreased athletic performance, increased morbidity, and occasional mortality.
Adult↗
T cell development in mice that lack the zeta chain of the T cell antigen receptor complex.
The zeta subunit of the T cell antigen receptor complex is required for targeting nascent receptor complexes to the cell surface and for receptor-mediated signal transduction. To examine the significance of the zeta subunit in T cell development, mice deficient for zeta expression were generated by gene targeting. These zeta-/- mice had few CD4+CD8+ thymocytes, and the generation of CD4+ and CD8+ single positive T cells was impaired but not completely abrogated. Peripheral T cells were present but were unusual in that they expressed small amounts of CD5 and few T cell receptors. Thus, zeta chain expression influences thymocyte differentiation but is not absolutely required for the generation of single positive T cells.
Animals↗
Localization of NADPH diaphorase activity in monoaminergic neurons of the rat brain.
Nitric oxide has recently been implicated as a neurotransmitter, and may modulate synaptic transmission, cerebral blood flow, and neurotoxicity. NADPH diaphorase histochemistry has been shown to be a reliable marker for nitric oxide synthase, the enzyme that synthesizes nitric oxide, in the nervous system. Because monoaminergic neurons frequently contain co-transmitters, we examined whether these cells also exhibit NADPH diaphorase activity. Frozen sections from postnatal and adult rat brains were stained for NADPH diaphorase activity and either serotonin-like immunoreactivity or tyrosine hydroxylase-like immunoreactivity. Numerous neurons in the mesopontine serotoninergic cell groups (including the caudal linear, dorsal, median, supralemniscal, and pontine raphe nuclei) contained both serotonin-like immunoreactivity and NADPH diaphorase activity. Within the dorsal raphe nucleus, approximately 70% of the serotoninergic neurons in the medial subnuclei displayed NADPH diaphorase activity, while less than 10% of the serotoninergic neurons in the lateral subnuclei were doubly labeled. Retrograde labeling with fluorescent microspheres indicated that many raphe-cortical neurons contained NADPH diaphorase activity. No NADPH diaphorase activity was detected in serotoninergic neurons in the medullary nuclei (including the raphe magnus, raphe pallidum, and raphe obscurus). Only a small proportion of tyrosine hydroxylase-like immunoreactive neurons in the periaqueductal gray, rostral linear nucleus, and rostrodorsal ventral tegmental area contained NADPH diaphorase activity. Tyrosine hydroxylase-like immunoreactive neurons in the substantia nigra, locus coeruleus, hypothalamus, olfactory bulb, and dorsal raphe nucleus did not contain detectable NADPH diaphorase activity. The observation that many mesopontine (but not medullary) serotoninergic neurons contain NADPH diaphorase activity suggests that these neurons may release both serotonin and nitric oxide.
Animals↗
G proteins in cardiovascular function and dysfunction.
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Aging↗
Quantitative demonstration of spontaneous metastasis by MCF-7 human breast cancer cells cotransfected with fibroblast growth factor 4 and LacZ.
We recently established transfectants of MCF-7 human breast cancer cells with fibroblast growth factor 4 (fgf-4) that showed rapid growth and spontaneous metastasis in ovariectomized and tamoxifen-treated nude mice. To establish a spontaneous metastatic model of human breast cancer cells in nude mice with a sensitive marker for detection of micrometastasis, the transfection of fgf-4 was combined with transfection of the bacterial lacZ gene encoding beta-galactosidase. MKL-4 cells, a lacZ transfectant of an fgf-4-transfected cell line, showed the same level of fgf-4 expression as parental cells and expressed a high level of beta-galactosidase activity. When MKL-4 cells were injected s.c. into female nude mice, rapidly growing tumors developed. Whole organ staining for beta-galactosidase activity was able to detect even small numbers of metastatic tumor cells. Micrometastases in lymph nodes, lung, and brain were detected 3 weeks after the tumor cell injections, the first time point tested. Within 12 weeks, metastases were observed in lymph nodes, lung, brain, kidney, perirenal fatty tissues, liver, spleen, retroperitoneum, heart, and gallbladder. The frequency of metastasis and number of foci were correlated with the volume of the primary tumors. The distribution of metastatic sites was similar to that in breast cancer patients. MKL-4 cells may be a useful model for studying the malignant progression of hormone-dependent breast cancer, antimetastatic drugs, or early events in metastasis.
Animals↗
The role of cathepsin D in the invasiveness of human breast cancer cells.
The aspartyl protease cathepsin D has been shown to be a marker of poor prognosis when found at high levels in primary breast tumors. It has been suggested that this is because the production of cathepsin D increases the invasive potential of the tumor cells, thus increasing the probability of metastasis. We have therefore conducted experiments to determine if secreted cathepsin D makes a significant contribution to the invasive phenotype of breast cancer cells in the Boyden chamber assay of invasion, which measures the ability of a cell to invade through an artificial basement membrane. Cathepsin D secretion and Boyden chamber invasiveness were measured in nine clones of the breast cancer cell line MCF-7, and no correlation was found between cathepsin secretion and invasive behavior. Invasion assays were also conducted in the presence of the aspartyl protease inhibitor pepstatin A, and no inhibition of the invasive behavior of cells was seen. Since low-pH environments are required for both the activation of pro-cathepsin D and the activity of the mature enzyme, assays were also conducted in the presence of chloroquine to neutralize the pH in the acidic compartments of the cells. This treatment did not inhibit invasiveness. Cathepsin D secretion by the breast cancer cell lines MDA-MB-231, MDA-MB-435, MDA-MB-435s, MDA-MB-468, SK-Br-3, and MCF-7-ADRr was also measured. Again, there was no correlation with invasion. In fact, cathepsin D levels were inversely correlated with aggressive behavior in vivo and in vitro in previously reported studies. These data suggest that cathepsin D secretion by tumor cells is not an important determinant of the invasiveness of the tumor cells per se. These data also reinforce the view that the poor prognosis in clinical breast cancer linked to high tumor levels of cathepsin D is probably due to high levels of cathepsin D in the stromal components of the tumor such as infiltrating inflammatory cells.
Breast Neoplasms↗
Breast cancer: influence of endocrine hormones, growth factors and genetic alterations.
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Breast↗
The invasive and metastatic properties of hormone-independent but hormone-responsive variants of MCF-7 human breast cancer cells.
We have previously isolated a series of MCF-7 human breast cancer cell variants which no longer require estrogen-supplementation for tumor growth in nude mice (Clarke et al. Proc Natl Acad Sci USA 86: 3649-3653, 1989). We now report that these hormone-independent and hormone-responsive variants (MIII, MCF7/LCC1) can invade locally from solid mammary fat pad tumors, and produce primary extensions on the surface of intraperitoneal structures including liver, pancreas, and diaphragm. Both lymphatic and hematogenous dissemination are observed, resulting in the establishing of pulmonary, bone, and renal metastases. The pattern of metastasis by MIII and MCF7/LCC1 cells closely resembles that frequently observed in breast cancer patients, and provides the first evidence of metastasis from MCF-7 cells growing in vivo without supplementary estrogen. The interexperimental incidence of metastases, and the time from cell inoculation to the appearance of metastatic disease are variable. The increased metastatic potential is not associated with an increase in either the level of laminin attachment, laminin receptor mRNA expression, or secreted type IV collagenolytic activity. We also did not detect a significant decrease in the steady-state mRNA levels of the metastasis inhibitor nm23 gene. However, when growing without estrogen in vitro, MCF7/LCC1 cells produce elevated levels of the estrogen-inducible cathepsin D enzyme.
Animals↗
Detection of genes that are differentially expressed during mouse embryogenesis by genetic trapping strategies.
OBJECTIVE: Our objective was to identify novel genes that are expressed in temporally and spatially restricted patterns during mouse embryonic development and organogenesis. STUDY DESIGN: Two genetic trapping reporter constructs that lack transcriptional regulatory sequences were introduced independently into transcriptionally active gene loci by electroporation into mouse embryonic stem cells. Patterns of host gene-reporter construct expression were investigated in differentiated embryonic stem cells, embryoid bodies, and chimeric embryos at various stages of development. RESULTS: Three patterns of host gene-reporter construct expression were observed from the developmental analysis of four vector-integrated cell lines. Reporter expression patterns reflecting developmental regulation, constitutive activity, and developmental inactivation of the host genes were observed. CONCLUSIONS: Two vector-integrated gene loci from cell lines CCE-1C1 and D3-B44 have expression patterns not previously described by genetic trapping. Molecular characterization of these interrupted genes will shed light on their developmental function.
Animals↗
Transforming growth factor-beta in neural embryogenesis and neoplasia.
The transforming growth factor-beta (TGF-beta) family of polypeptides includes three structurally and functionally related mammalian isoforms that influence cell proliferation, differentiation, and extracellular matrix production. Recent identification of these isoforms in the embryonic murine central nervous system suggests that these factors may regulate proliferation and differentiation of meningeal and neuroepithelial cells during development. Predominant expression of TGF-beta 1 in the leptomeninges compared with the brain of the murine and human central nervous system implicates this isoform in regulation of that mesodermal tissue. Thus, defective TGF-beta regulation may contribute to neoplastic transformation. Failure to activate latent TGF-beta s may contribute to the loss of autocrine regulation seen in meningiomas. Expression of TGF-beta 2 and TGF-beta 3 primarily in embryonic murine radial glia and adult human astrocytes suggests other roles for these isoforms, including glioblast differentiation and guidance of neuroblast migration. Although inhibitory to "normal" astrocyte proliferation, TGF-beta s demonstrate autocrine growth stimulation in vitro among hyperdiploid malignant gliomas, medulloblastomas, primitive neuroectodermal tumors, and anaplastic ependymomas. Hence, synthesis and release of active TGF-beta s by malignant brain tumors may create aberrant stimulatory autocrine loops. The mechanism of TGF-beta-induced growth stimulation is poorly understood. Future studies will likely clarify and identify additional roles for the TGF-beta isoforms in neuro-embryogenesis and neoplasia.
Animals↗