Biomedical subjects

J F Morris

Publications and source records attributed to J F Morris.

At least 73 records · Page 4Linked to original sources

PubMed1996

Constitutive nitric oxide synthase in hypothalami of normal and hereditary diabetes insipidus rats and mice: role of nitric oxide in osmotic regulation and its mechanism.

Constitutive nitric oxide synthase (cNOS) was immunolocalized to study its role in osmotic regulation. Immunoreactivity was observed in all major hypothalamic osmoregulatory structures, the organum vasculosum laminae terminalis, subfornical organ, median preoptic nucleus, and supraoptic and paraventricular nuclei. These nuclei were compared in normal Long-Evans rats and homozygous Brattleboro rats with hereditary hypothalamic diabetes insipidus and in normal mice and mice with hereditary nephrogenic diabetes insipidus. About 50% of supraoptic neurons in Long-Evans rats and 90% in Brattleboro rats were cNOS immunopositive; a qualitatively similar difference occurred in the paraventricular nucleus. Mice with hereditary nephrogenic diabetes insipidus also showed a greater proportion of cNOS-positive supraoptic neurons (50%) than normal mice (20%). However, the number of cNOS-positive cells in the organum vasculosum laminae terminalis, subfornical organ, and median preoptic nucleus dis not differ significantly between diabetic and normal animals. The similar changes in cNOS in two mutant strains in which the only common feature is chronic osmotic stimulation shows that differences in vasopressin and oxytocin are not involved in the regulation of cNOS. The results suggest strongly that cNOS is involved in long term modulation of the hypothalamo-neurohypophysial system and, hence, body water and electrolyte homeostasis, and that cNOS is itself regulated by body osmotic status.

Animals↗

PubMed1996

Marinoic acid, a novel bufadienolide-related substance in the skin of the giant toad, Bufo marinus.

We found a novel substance, 3 beta-hydroxy-11,12-seco-5 beta, 14 beta-bufa-20,22-dienolide-11,14-olide-12-oic acid (1), which we called marinoic acid, in the skin of the toad, Bufo marinus. The structure was established from NMR and MS data. Like bufadienolides, marinoic acid contained an A/B ring structure in the cis configuration and a D/alpha-pyrone ring structure, but the structure of the C ring differed considerably from that of bufadienolides. Marinoic acid exhibited biological activity, as demonstrated by inhibition of Na+, K(+)-ATPase enzymatic activity, and by inhibition of [3H]ouabain binding to the digitalis receptor site on Na+, K(+)-ATPase, although marinoic acid was a less effective inhibitor than typical bufadienolides. Although marinoic acid cannot be classified as a bufadienolide, its chemical structure and its Na+, K(+)-ATPase inhibitory activity suggest that it is bufadienolide-related.

Animals↗

PubMed1995

The myeloid zinc finger gene, MZF-1, regulates the CD34 promoter in vitro.

MZF-1 is a C2H2 zinc finger gene encoding a putative transcriptional regulator of myeloid differentiation. The MZF-1 protein contains 13 C2H2 zinc fingers arranged in bipartite DNA binding domains containing zinc fingers through 4 and, in the carboxy-terminus, 5 through 13. We previously identified the DNA consensus binding site recognized by the two DNA binding domains. To assess the transcription regulatory function of MZF-1, the full-length MZF-1 coding region was fused to the DNA binding domain of the yeast transactivator GAL4. The expression vector was cotransfected with the chloramphenicol acetyl transferase (CAT) reporter gene regulated by the thymidine kinase promoter containing GAL4 DNA binding sites into NIH 3T3, 293, K562, and Jurkat cell lines. MZF-1 represses CAT reporter gene expression via GAL4 binding sites in the nonhematopoietic cell lines NIH 3T3 and 293. In contrast, MZF-1 activates CAT reporter gene expression in the hematopoietic cell lines K562 and Jurkat. The MZF-1 binding sites are present in the promoters of several genes expressed during myeloid differentiation, including the CD34 promoter. MZF-1 transcriptional regulation of this physiologically relevant promoter was assessed in both hematopoietic and nonhematopoietic cell lines. Recombinant MZF-1 protein specifically binds to the consensus binding sites in the CD34 promoter in mobility shift assays. MZF-1 expression vectors were cotransfected with the luciferase reporter plasmids regulated by the CD34 promoter into both nonhematopoietic and hematopoietic cell lines. As with the heterologous DNA binding domain, MZF-1 represses reporter gene expression in nonhematopoietic cell lines and activates expression in hematopoietic cell lines. Activation of CD34 expression in hematopoietic cell lines is dependent on the presence of intact MZF-1 binding sites. The cell type-specific regulation of the CD34 promoter by MZF-1 suggests the presence of tissue-specific regulators/adapters or differential MZF-1 modifications that determine MZF-1 transcriptional regulatory function.

3T3 Cells↗

PubMed1995

Differential subcellular mRNA targeting: deletion of a single nucleotide prevents the transport to axons but not to dendrites of rat hypothalamic magnocellular neurons.

It has previously been shown that mRNA encoding the arginine vasopressin (AVP) precursor is targeted to axons of rat magnocellular neurons of the hypothalamo-neurohypophyseal tract. In the homozygous Brattle-boro rat, which has a G nucleotide deletion in the coding region of the AVP gene, no such targeting is observed although the gene is transcribed. RNase protection and heteroduplex analyses demonstrate that, in heterozygous animals, which express both alleles of the AVP gene, the wild-type but not the mutant transcript is subject to axonal compartmentation. In contrast, wild-type and mutant AVP mRNAs are present in dendrites. These data suggest the existence of different mechanisms for mRNA targeting to the two subcellular compartments. Axonal mRNA localization appears to take place after protein synthesis; the mutant transcript is not available for axonal targeting because it lacks a stop codon preventing its release from ribosomes. Dendritic compartmentation, on the other hand, is likely to precede translation and, thus, would be unable to discriminate between the two mRNAs.

Animals↗

PubMed1995

Immunophenotypic evidence for distinct populations of microglia in the rat hypothalamo-neurohypophysial system.

The morphology, distribution and immunophenotype of microglia throughout the adult rat hypothalamo-neurohypophysial system was examined. Four macrophage-associated antibodies (OX-42, F4/80, ED1 and ED2) were used; the expression of major histocompatibility complex antigens was investigated by use of antibodies against OX-6, OX-17 (MHC class II) and OX-18 (MHC class I). Three distinct types of microglia were identified. The first was located in the magnocellular nuclei; these 'radially branched' ('ramified') microglia had round cell bodies and long branched processes, and were strongly immunoreactive only for OX-42. The second was located outside the blood-brain barrier in the median eminence, pituitary stalk and neurohypophysis often close to blood vessels; these 'compact' microglia had irregular cell bodies and shorter processes, and were strongly labelled by OX-42 and F4/80, weakly labelled by OX-18, and generally unlabelled by ED1, ED2, OX-6 and OX-17. The third type was found in small numbers throughout the system at the surface of the nervous tissue or around blood vessels; these 'perivascular' microglia were elongated cells with no branching processes, and were strongly labelled by ED1, ED2, OX-18, OX-6, OX-17 and F4/80 antibodies but showed variable OX-42 immunoreactivity. Cells in a perivascular location were heterogeneous with respect to their immunophenotype. The presence in the normal adult rat hypothalamo-neurohypophysial system of MHC class-II molecules (OX-6 and OX-17) on a sub-set of perivascular microglia suggests that these cells are capable of presenting antigen to T lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Oestradiol acutely stimulates exocytosis of oxytocin and vasopressin from dendrites and somata of hypothalamic magnocellular neurons.

Oestrogen has many direct or indirect actions upon the magnocellular system of the hypothalamus. We have examined the possibility of acute actions of oestrogen upon the magnocellular system by stimulating slices of supraoptic nucleus in vitro with various concentrations of oestrogen, for varying lengths of time, and assessing the intrahypothalamic release of oxytocin and vasopressin under these conditions. Slices were stimulated in the presence of tannic acid, which precipitates extracellular protein and thereby stabilizes exocytosed neurosecretory granule cores. Stimulation for 5 or 20 min of slices of hypothalamus containing the supraoptic nuclei with 2.66 nM-26.6 microM 17 beta-oestradiol benzoate caused the exocytosis of granules from both dendrites and cell bodies of the magnocellular neurons; exocytosis from the dendrites predominated. Granules of both oxytocin- and vasopressin-producing cells were exocytosed to a similar extent. The incidence of exocytosis of both hormones after stimulation by oestrogen was significantly higher than after exposure either to physiological saline or to 17 alpha-oestradiol, but significantly lower than after stimulation by 56 mM potassium. The various doses of steroid and durations of stimulation all resulted in similar amounts of captured exocytosis. Furthermore, the oestradiol-induced release was not inhibited by removal of extracellular calcium, whereas the potassium-stimulated release was abolished. Exposure for 20 min to either testosterone or progesterone did not induce intranuclear release of significant numbers of neurosecretory granules from the magnocellular neurons. In contrast to its effect on the hypothalamus, 26.6 microM oestradiol for either 20 min or 5 min did not induce exocytosis of neurosecretory granules from the posterior pituitary. We conclude that oestrogen can exert acute non-genomic actions on the magnocellular neurons to promote intrahypothalamic release of oxytocin and vasopressin. This effect is probably direct on the magnocellular neurons as it is not dependent on external calcium. Such actions may be important in the development of the functional and morphological plasticity of the magnocellular system that occurs in parturition and lactation.

Animals↗

PubMed1995

Inhibition of cellular proliferation by the Wilms' tumor suppressor WT1 is associated with suppression of insulin-like growth factor I receptor gene expression.

We have investigated the regulation of the insulin-like growth factor I receptor (IGF-I-R) gene promoter by the Wilms' tumor suppressor WT1 in intact cells. The levels of endogenous IGF-I-R mRNA and the activity of IGF-I-R gene promoter fragments in luciferase reporter constructs were found to be significantly higher in G401 cells (a Wilms' tumor-derived cell line lacking detectable WT1 mRNA) than in 293 cells (a human embryonic kidney cell line which expresses significant levels of WT1 mRNA). To study whether WT1 could suppress the expression of the endogenous IGF-I-R gene, WT1-negative G401 cells were stably transfected with a WT1 expression vector. Expression of WT1 mRNA in G401 cells resulted in a significant decrease in the rate of cellular proliferation, which was associated with a reduction in the levels of IGF-I-R mRNA, promoter activity, and ligand binding and with a reduction in IGF-I-stimulated cellular proliferation, thymidine incorporation, and anchorage-independent growth. These data suggest that a major aspect of the action of the WT1 tumor suppressor is the repression of IGF-I-R gene expression.

Cell Division↗

PubMed1995

Repression of Pax-2 by WT1 during normal kidney development.

The developmental, regulatory gene Pax-2 is activated during early kidney morphogenesis and repressed in mature renal epithelium. Persistent Pax-2 expression is also observed in a variety of kidney tumors. Yet, little is known about the signals regulating this transient expression pattern in the developing kidney. We have examined the spatial and temporal expression patterns of Pax-2 and the Wilm's tumor suppresser protein WT1 with specific antibodies in developing mouse kidneys. A marked increase in WT1 protein levels coincided precisely with down-regulation of the Pax-2 gene in the individual precursor cells of the visceral glomerular epithelium, suggesting a direct effect of the WT1 repressor protein on Pax-2 regulatory elements. To examine whether WT1 could directly repress Pax-2 transcription, binding of WT1 to three high affinity sites in the 5' untranslated Pax-2 leader sequence was demonstrated by DNAseI footprinting analysis. Furthermore, co-transfection assays using CAT reporter constructs under the control of Pax-2 regulatory sequences demonstrated WT1-dependent transcriptional repression. These three WT1 binding sites were also able to repress transcription, in a WT1-dependent manner, when inserted between a heterologous promoter and the reporter gene. The data indicate that Pax-2 is a likely target gene for WT1 and suggest a direct link, at the level of transcriptional regulation, between a developmental control gene, active in undifferentiated and proliferating cells, and a known tumor suppressor gene.

Animals↗

PubMed1995

Production of hybrid oxytocin/vasopressin precursors and accumulation of oxytocin precursors in the rough endoplasmic reticulum of rat magnocellular neurons.

Most magnocellular hypothalamic neurons synthesize the precursor for either vasopressin (AVP) or oxytocin (OT). The AVP precursor is cleaved to give AVP, AVP-associated neurophysin (AVP-NP) and a glycopeptide (GP), whereas the OT precursor gives OT and OT-NP. In Brattleboro rats a frame-shift mutation in the AVP-NP-encoding region of the gene prevents the secretion of AVP by the cells and, in most AVP neurons, AVP itself is virtually undetectable. A small number of magnocellular neurons in homozygous Brattleboro rats contain very large accumulations of peptide in distended saccules of rough endoplasmic reticulum (RER), and this peptide is immunoreactive for AVP and C-terminal OT-NP, but not for OT, AVP-NP or GP (Pow et al., 1992). We have now shown that this results from somatic non-homologous crossing over of the AVP and OT genes, resulting in the production of hybrid mRNA molecules with the 5'end of the AVP sequence and the 3' end of the OT sequence (AVP/OT transcripts). In most cases, the crossing over occurs within the highly homologous B exons (Mohr et al., 1994). In addition to the production of AVP/OT hybrid transcripts, polymerase chain reaction (PCR) amplification of mRNA from the hypothalami of homozygous rats also reveals OT/AVP hybrid transcripts, with 5' OT sequences and 3' AVP sequences. Furthermore, both types of hybrid transcript are not restricted to homozygous Brattleboro rats but can also be found in normal Long Evans animals. To date, we have not been able to locate cells in which the OT/AVP hybrids are produced; all the magnocellular neurons with hybrid peptide accumulations in the RER so far studied have been shown by immunocytochemistry to be of the AVP/OT type. In both normal and homozygous Brattleboro rats large accumulations of peptide do occur in the RER of OT-producing neurons but the peptide is immunoreactive for OT and OT-NP but not for AVP, AVP-NP or GP. Such cells increase in number 10-fold after injection of 20 micrograms estradiol daily for 7 days (Pow et al., 1991). Why this apparently normal gene product accumulates within the RER remains to be determined.

Aging↗

PubMed1994

Somatic nonhomologous crossing-over between neuropeptide genes in rat hypothalamic neurons.

Molecular biological and immunocytochemical data demonstrate nonhomologous crossing-over between the closely linked vasopressin (VP) and oxytocin (OT) genes in rat hypothalamic neuroendocrine neurons. Reverse transcription of hypothalamic total RNA from wild-type or homozygous Brattleboro aged rats combined with polymerase chain reaction (PCR) amplifications in the presence of appropriate 5' forward and 3' reverse primers deduced from the VP and OT cDNA sequences yielded PCR products that, upon cloning and sequencing, revealed several hybrid transcripts. They encode the N-terminal part of the VP precursor fused to the C-terminal part of the OT precursor (VP/OT transcripts) and vice versa (OT/VP transcripts). VP/OT hybrid precursor proteins have been identified immunocytochemically in enlarged cisternae of the rough endoplasmic reticulum, yet there is no evidence that the products can be secreted from affected cells. Recombination appears to be a rather frequent genetic event affecting about 0.06-0.1% of the rat vasopressinergic magnocellular neurons in aged rats.

Amino Acid Sequence↗

PubMed1994

Perivascular microglia in the rat neural lobe engulf magnocellular secretory terminals during osmotic stimulation.

The response of microglia in the rat neural lobe to osmotic stimulation has been studied. Microglia were identified by immunoreactivity for the macrophage markers OX-42 and F4/80. The numerical density of microglia did not change significantly with osmotic stimulation but microglia in the perivascular space partially or completely enclosed significantly greater numbers of neurosecretory terminals in osmotically stimulated animals.

Animals↗

PubMed1994

Intra- and extracellular amyloid fibrils are formed in cultured pancreatic islets of transgenic mice expressing human islet amyloid polypeptide.

Islet amyloid polypeptide (IAPP) is the constituent peptide of amyloid deposits found in the islets of non-insulin-dependent diabetic patients. Formation of islet amyloid is associated with a progressive destruction of insulin-producing beta cells. Factors responsible for the conversion of IAPP into insoluble amyloid fibrils are unknown. Both the amino acid sequence of human IAPP (hIAPP) and hypersecretion of hIAPP have been implicated as factors for amyloid fibril formation in man. We have generated transgenic mice using rat insulin promoter-hIAPP or rat IAPP (rIAPP) gene constructs. No fibrillar islet amyloid was detectable in vivo in these normoglycemic mice, although small amorphous perivascular accumulations of IAPP were observed in hIAPP mice only. To determine the effects of glucose on IAPP secretion and fibrillogenesis, pancreatic islets from transgenic and control mice were examined in vitro. Islet IAPP secretion and content were increased in transgenic islets compared with control islets. IAPP-immunoreactive fibrils were formed at both intra- and extracellular sites in isolated hIAPP islets cultured with glucose at 11.1 and 28 mM for only 7 days. At 28 mM glucose, fibrils were present in deep invaginations of beta cells as observed in non-insulin-dependent diabetic patients. No fibrils were present at low glucose concentrations in hIAPP islets or at any glucose concentration in rIAPP or control islets. Thus, glucose-induced expression and secretion of hIAPP in transgenic mouse islets can lead to formation of amyloid fibrils similar to that found in non-insulin-dependent diabetes mellitus.

Amyloid↗

PubMed1994

Necrotizing pneumonia caused by mixed infection with Actinobacillus actinomycetemcomitans and Actinomyces israelii: case report and review.

Actinobacillus actinomycetemcomitans is an important cause of human pulmonary infections, either alone or with Actinomyces species. It may be critical to isolate Actinobacillus in patients with pulmonary infection for selection of an effective antimicrobial regimen. Clindamycin has superseded penicillin as the sole antimicrobial drug for anaerobic bacterial necrotizing pneumonia and abscess. In the case presented herein, therapy with clindamycin failed to halt worsening necrotizing pneumonia or to prevent hematogenous dissemination. After clindamycin-resistant A. actinomycetemcomitans in addition to Actinomyces israelii were isolated, the patient was treated with penicillin, ciprofloxacin, and cefazolin and was ultimately cured.

Actinobacillus Infections↗

PubMed1994

Novel oncogenic mutations in the WT1 Wilms' tumor suppressor gene: a t(11;22) fuses the Ewing's sarcoma gene, EWS1, to WT1 in desmoplastic small round cell tumor.

These studies suggest that the WT1 tumor suppressor gene, originally identified as a recessive oncogene in Wilms' tumors, is capable of sustaining a gain-of-function mutation which results in its contribution to a completely different disease entity: desmoplastic small round cell tumor. Two independent biochemical functions of WT1, DNA-binding activity and mode of transcriptional regulation, are altered as a consequence of the chromosomal translocation and fusion with EWS. The fusion of EWS and WT1 genes in DSRCT thus provides a unique paradigm for a means by which different alterations of transcription factor function can lead to diverse oncogenic processes.

Amino Acid Sequence↗

PubMed1994

Effect of a single amino acid substitution in the V3 domain of the human immunodeficiency virus type 1: generation of revertant viruses to overcome defects in infectivity in specific cell types.

Proviral clones of human immunodeficiency virus type 1 which contained single amino acid changes in the envelope V3 region were constructed. PCR amplification of Sup-T1 T cells transfected with one such mutant, G312T, revealed low levels of virus that resulted in the generation of a revertant virus, in which an alanine replaced the threonine residue at amino acid 312. The revertant virus (rA312) was fully infectious in Sup-T1 cells but lacked the ability to infect AA5 cells. The presence of a second mutation in a subsequent revertant virus (rR306), in which arginine was substituted for serine at amino acid 306 within the V3 loop, restored the ability of the mutated virus to infect AA5 cells. Our data highlight the importance of the V3 loop in defining virus tropism for specific cell types in culture and further suggest that a degree of interplay exists among V3 loop residues that helps maintain or control its biological function of the virus.

Animals↗

PubMed1994

Characterization of the DNA-binding properties of the myeloid zinc finger protein MZF1: two independent DNA-binding domains recognize two DNA consensus sequences with a common G-rich core.

The myeloid zinc finger gene 1, MZF1, encodes a transcription factor which is expressed in hematopoietic progenitor cells that are committed to myeloid lineage differentiation. MZF1 contains 13 C2H2 zinc fingers arranged in two domains which are separated by a short glycine- and proline-rich sequence. The first domain consists of zinc fingers 1 to 4, and the second domain is formed by zinc fingers 5 to 13. We have determined that both sets of zinc finger domains bind DNA. Purified, recombinant MZF1 proteins containing either the first set of zinc fingers or the second set were prepared and used to affinity select DNA sequences from a library of degenerate oligonucleotides by using successive rounds of gel shift followed by PCR amplification. Surprisingly, both DNA-binding domains of MZF1 selected similar DNA-binding consensus sequences containing a core of four or five guanine residues, reminiscent of an NF-kappa B half-site: 1-4, 5'-AGTGGGGA-3'; 5-13, 5'-CGGGnGAGGGGGAA-3'. The full-length MZF1 protein containing both sets of zinc finger DNA-binding domains recognizes synthetic oligonucleotides containing either the 1-4 or 5-13 consensus binding sites in gel shift assays. Thus, we have identified the core DNA consensus binding sites for each of the two DNA-binding domains of a myeloid-specific zinc finger transcription factor. Identification of these DNA-binding sites will allow us to identify target genes regulated by MZF1 and to assess the role of MZF1 as a transcriptional regulator of hematopoiesis.

Amino Acid Sequence↗

PubMed1994

Structures of novel bufadienolides in the eggs of a toad, Bufo marinus.

In this paper, we report chemical structures of five compounds including four novel polyhydroxylated cardiac steroids in the eggs of a toad, Bufo marinus. These cardiac steroids were purified by high-performance liquid chromatography, and their structures were determined to be 11 alpha,19-dihydroxy-telocinobufagin (I), 11 alpha-hydroxytelocinobufagin (II), 11 alpha,19-dihydroxymarinobufagin (III), 11 alpha-hydroxymarinobufagin (IV) and 19-hydroxytelocinobufagin (V) on the basis of spectral data of nuclear magnetic resonance and mass spectroscopy. All the five compounds showed biological activity, as tested by inhibition of Na+,K(+)-ATPase activity and of [3H]ouabain binding to the receptor on Na+,K(+)-ATPase. This is the first finding of bufadienolides as cardiac steroids in animal eggs.

Animals↗

PubMed1994

Physiological changes due to age. Implications for respiratory drug therapy.

The pulmonary system is modified in various ways over time and it is particularly vulnerable to environmental insults. Of particular interest are the implications of aging for therapy of respiratory illnesses. The changes in pulmonary structure and function due simply to aging, and changes due to diseases, should be distinguished from each other. The great reserve function of the lung permits reasonable physical capacity in healthy individuals despite aging changes. In principle, loss of function equivalent to more than one lung is necessary to impair aerobic capacity at any age. Elderly people are subject to the same respiratory diseases as younger adults but may manifest them differently. They may present in atypical ways such as in bacterial pneumonia, tuberculosis, and asthma, all modified by anatomical alterations or deterioration of immunological defence mechanisms. Accumulation of toxic substances over time such as cigarette smoke or environmental pollutants may give rise to chronic bronchitis, emphysema, bronchogenic carcinoma and interstitial lung disease. Changes in the number or function of airway receptors modulate responses to bronchodilator drugs. Chronic inflammation of the bronchial wall has blurred the distinction between traditional asthma and chronic bronchitis and emphysema, and similar drug therapy can be useful for all. Adverse reactions to respiratory drugs such as theophylline, oral corticosteroids, and isoniazid increase with age. As more data accumulate, drug therapy of respiratory diseases in older patients will become more effective and safer.

Aged↗