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Biomedical subjects

P Davies

Publications and source records attributed to P Davies.

At least 19 recordsLinked to original sources
PubMed1992

Inhibition of human leukocyte elastase. 4. Selection of a substituted cephalosporin (L-658,758) as a topical aerosol.

Human leukocyte elastase (HLE) is a serine protease which has been implicated as a causative agent in several pulmonary diseases. The continued modification of our previously reported cephalosporin-based HLE inhibitors has led to the identification of a series of C-2 amides with potent, topical activity in an in vivo hamster lung hemorrhage model. While the most potent in vitro HLE inhibition had previously been obtained with lipophilic ester derivatives, it was found that the less active, but more polar and stable, amide derivatives were much more effective in vivo. The development of the structure--activity relations for optimization of these activities is discussed. These results led to the selection of 3-(acetoxymethyl)-2-[(2(S)-carboxypyrrolidino)carbonyl]-7 alpha-methoxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene, 5,5-dioxide (3, L-658,758) as a selective, potent, time-dependent HLE inhibitor suitable for formulation as a topical aerosol drug for possible clinical use.

Administration, Topical↗
PubMed1992

In vivo circumvention of human colon carcinoma resistance to bleomycin.

Metabolic inactivation of bleomycin (BLM) by cysteine proteinase-like enzymes is thought to be a major mechanism of BLM tumor resistance. We now report that the human colon carcinoma COLO-205 is highly resistant to BLM and that E-64, a cysteine proteinase inhibitor, sensitizes COLO-205 to BLM. Treatment of COLO-205-bearing nude mice with either E-64 (40 mg/kg) or BLM (10 mg/kg) alone did not inhibit COLO-205 growth. However, pretreatment with E-64 prior to BLM prevented these xenografts from growing. Analysis by high performance liquid chromatography of in vivo BLM metabolism following [3H]BLM A2 treatment of COLO-205-bearing nude mice showed a different metabolic profile among the various organs and the tumor. Whereas [3H]BLM A2 was the only major radioactive peak detected in sera and tumors, several metabolites, including deamido-BLM A2, were found in kidney, liver, and lung as early as 15 min. Pretreatment of mice with E-64 inhibited tumor, kidney, and lung BLM A2 metabolism. Furthermore, pretreatment with E-64 increased BLM A2 accumulation in tumors (6.1-fold), kidney (4.0-fold), lung (2.8-fold), liver (1.8-fold), and serum (1.7-fold). E-64 pretreatment did not enhance the major toxicity of BLM, pulmonary fibrosis, as determined by both lung hydroxyproline levels and histopathology. Thus, the cysteine proteinase inhibitor E-64 affects the metabolic fate and the levels of accumulation of BLM in vivo. These results demonstrate that resistance of human COLO-205 tumors to BLM can be circumvented by E-64 without enhancement of the major side effect of BLM, suggesting a possible clinical use of this combination therapy.

Animals↗
PubMed1992

Regional localization of the regulatory subunit (RII beta) of the type II cAMP-dependent protein kinase in human brain.

The distribution of the regulatory (RII beta) subunits of type II cAMP-dependent protein kinase in cortical and subcortical areas was examined in human control and Alzheimer's disease (AD) brains. Four monoclonal antibodies generated against bovine brain RII, which cross-reacted with human brain RII beta, detected RII-immunoreactivity in pyramidal neurons of the hippocampus and frontal, occipital, parietal and superior temporal cortices and in non-pyramidal neurons of the amygdala and putamen. RII beta immunoreactivity was localized to neuronal perikarya, proximal dendrites and cell processes. With the exception of rare processes in the ventroposterior lateral nucleus, RII-immunoreactivity was not seen in the thalamus. Other areas lacking RII-immunoreactivity included the midbrain, caudate nucleus and globus pallidus. RII-immunoreactivity was not detected in endothelia or glia. Except for the neocortex, the distribution of RII beta immunoreactivity was the same in AD and non-demented control brains; however, cell bodies and their processes stained more intensely and uniformly in the neocortical regions of non-demented controls compared to AD. In the neocortex of AD, RII beta immunoreactivity was substantially decreased in the superior temporal and occipital cortices, but not in the frontal cortex. Our data suggest that RII subunits are regionally distributed in the human brain. RII-immunoreactivity was decreased in some regions of neocortex in AD, but it did not preferentially colocalize with neurofibrillary tangles (NFT), senile plaques, or neuropil threads.

Alzheimer Disease↗
PubMed1992

A protein kinase associated with paired helical filaments in Alzheimer disease.

We have identified a protein kinase in immunoaffinity-purified preparations of paired helical filaments from brain tissue of individuals with Alzheimer disease. The kinase phosphorylates the filament proteins in vitro in a manner independent of second messenger regulation or of modulation by heparin and polyamines. Physiological concentrations of hemin, an oxidized heme porphyrin, inhibit the kinase and abolish Alz-50 immunoreactivity of the proteins. Since paired helical filaments are composed of hyperphosphorylated proteins, association of a protein kinase with the filaments provides a mechanism for abnormal processing of the proteins in disease.

Adenosine Triphosphate↗
PubMed1992

Characterization of a synaptic antigen of interest in neuropsychiatric illness.

Monoclonal antibodies exhibiting relative differences in binding to brain homogenates from diseased versus control brains may be useful probes into the molecular pathology of neuropsychiatric illness. To be of value, the antibodies must be useful in characterization of the antigens putatively involved in the illness. An antibody called EP10 showed some differences in binding to homogenates from a small sample of schizophrenia brains compared with controls. In the present study, the antigen for this antibody was characterized in control brains using quantitative, immunocytochemical, and biochemical techniques. The antigen is a 38,000 dalton synaptic vesicle protein, which is identical to synaptophysin by immunological criteria. However, the EP10 epitope is of interest, as this binding site does not appear to be present in rat or bovine synaptophysin. Brain regional studies using EP10 indicate that the antigen may be present in only a subset of synaptic terminals. Further studies are required to fully characterize the epitope, and to determine the significance of the earlier findings related to schizophrenia.

Animals↗
PubMed1992

Purification and characterization by fast-atom-bombardment mass spectrometry of the polymorphonuclear-leucocyte-elastase-generated A alpha (1-21) fragment of fibrinogen from human blood after incubation with calcium ionophore A23187.

The stimulation of human blood with a Ca2+ ionophore, A23187, leads to activation of polymorphonuclear leucocytes (PMN) with release of small amounts of catalyticaly active elastase, as demonstrated by the formation of a characteristic product, the N-terminal A alpha (1-21) peptide of the Aa subunit of fibrinogen. The identity of the peptide was initially established by radioimmunoassay (r.i.a.) with an antibody raised to A alpha (1-21). We now provide independent confirmation of the formation of A alpha (1-21) by fast-atom-bombardment-m.s. analysis of the fractions separated chromatographically after spiking of plasma samples with peptide labelled with [2H8]Phe at position 8. Identity of the peptides was established on the basis of their chromatographic retention time and by the distinct peaks in the mass spectra of these fractions. The relative intensities of the molecular ions of natural and labelled peptides were measured. On the basis of a comparison of the peaks of similar intensities, the concentration of the natural peptide at the time of spiking was close (79%) to the amount obtained by r.i.a. An additional peptide, des-alanyl-A alpha (2-21), was also seen. The total amount of material measured by r.i.a. could be accounted for by the sum of these two provides. The addition of label and assay by m.s. has provided an independent physical-chemical method for identifying A alpha (1-21) as a characteristic product of PMN elastase release in whole blood, but which is absent in freshly drawn blood.

Amino Acid Sequence↗
PubMed1992

Hydrofluoric acid-treated tau PHF proteins display the same biochemical properties as normal tau.

Tau (tau) is a major constituent of paired helical filaments (PHF) found in Alzheimer's disease. The current study examines the possibility that the distinct properties of PHF-associated tau proteins (tau PHF) result from post-translational modifications of normal soluble tau (tau s). Following hydrofluoric acid (HF) treatment, tau PHF proteins are heat- and acid-stable, soluble in 2-(N-morpholino)ethanesulfonic acid buffers and display the same molecular weight, pI, and immunochemical properties as normal tau s. Alkaline phosphatase treatment of dissociated PHF results in similar, although less extensive, electrophoretic changes and a reduction in PHF-1 immunoreactivity. Therefore, phosphorylation of normal tau s appears to be responsible for the distinct properties of tau PHF. Although our results suggest that all of the normal tau isoforms are in PHF, the relative abundance of individual tau species differs in HF-treated PHF and tau s samples. Moreover, the loss of PHF following HF treatment suggests that post-translational modifications contribute to the structural stability of PHF.

Adult↗
PubMed1992

Induction of emphysema and bronchial mucus cell hyperplasia by intratracheal instillation of lipopolysaccharide in the hamster.

The aim of this study was to determine whether lipopolysaccharide-induced elastase release from recruited neutrophils in the hamster lung would induce emphysema, measured by mean linear intercept (Lm) and bronchial mucus cell hyperplasia (BMCH), scored in tissue sections stained with periodic acid-Schiff. Lipopolysaccharide (LPS) was instilled transorally twice a week for up to 5 weeks in hamsters. At 4 weeks after seven LPS instillations, Lm amounted to 87.6 +/- 1.2 microns, while it was 68.3 +/- 1.5 microns after seven saline instillations (P less than 0.01). At 6 months after the sixth LPS instillation, the Lm of these lungs was 83.3 +/- 1.6 microns, indicating irreversible tissue destruction. LPS-treated hamsters showed marked to severe BMCH, which was most evident in large intrapulmonary airways. Instillations of highly selective inhibitor of hamster PMN elastase resulted in 50 per cent inhibition of LPS-induced emphysema. The development of BMCH was inhibited by approximately 35 per cent by this agent. To study the response in time of cellular infiltration after a single LPS instillation, the lungs of groups of four hamsters were lavaged at different time points. PMN recruitment showed peak values at 4 and 48 h after LPS instillation and returned to baseline values at 96 h. Simultaneous intratracheal instillation of LPS and anti-TNF alpha antiserum resulted in a considerable reduction of neutrophil influx into bronchoalveolar spaces in the first 6 h after instillation.

Animals↗
PubMed1992

Immunocytochemistry of neurofibrillary tangles with antibodies to subregions of tau protein: identification of hidden and cleaved tau epitopes and a new phosphorylation site.

Antibodies to multiple epitopes spanning the length of the tau molecule were used to study Alzheimer neurofibrillary tangles (NFT) using immunocytochemical methods and several different methods of fixation and tissue processing, including staining of vibratome sections, hydrated autoclaving of paraffin sections and immunofluorescence of NFT isolated from fresh brain tissue. Smears and sections were pretreated with trypsin and/or phosphatase to further characterize antibody binding. In tissue fixed briefly in periodate-lysine-paraformaldehyde, tau immunoreactivity was detected in astrocytes, but only a few tau epitopes were detected in NFT with this fixation method. In contrast, all tau epitopes were detected in NFT in tissue fixed in formaldehyde for prolonged periods of time. In the hippocampus, the number of NFT detected in the dentate fascia was in proportion to the duration of dementia, as we previously noted. Dentate fascia NFT were intracellular (i-NFT) and were reactive with antibodies recognizing epitopes in both the carboxy- and amino-terminal regions of tau, but not the microtubule-binding domain of tau, suggesting that microtubule-binding domain epitopes are hidden in i-NFT. In contrast, NFT in the subiculum and layer II of the parahippocampal cortex were mostly extracellular (e-NFT), especially in severe cases of long duration. e-NFT were immunoreactive with antibodies to the microtubule-binding domain, but only weakly reactive with antibodies to carboxy- or amino-terminal epitopes, suggesting that e-NFT may contain fragments of tau. In both isolated NFT and NFT in sections, amino-terminal epitopes, including the Alz-50 epitope, were sensitive to trypsin proteolysis, which suggests that the lack of staining of e-NFT by antibodies to the amino-terminal regions of tau is due to proteolysis. Antibodies reactive with amino-terminal epitopes also stained fewer NFT following hydrated autoclaving, while those reacting with the carboxy half of tau stained more NFT after hydrated autoclaving. Thus, although carboxy-terminal regions are not detected in e-NFT, they are probably masked, rather than proteolytically cleaved, since they can be revealed by hydrated autoclaving. Finally, phosphatase treatment of isolated NFT revealed enhanced immunostaining not only with Tau-1, as in previous studies demonstrating abnormal phosphorylation of tau proteins in NFT, but also with an antibody to exon 2, which reveals yet another phosphorylation site in tau of NFT.

Aged↗
PubMed1992

Persistent innervation of the rat neocortex by basal forebrain cholinergic neurons despite the massive reduction of cortical target neurons. I. Morphometric analysis.

In Alzheimer's disease, a characteristic neurochemical abnormality is the loss of cholinergic enzymes in the neocortex, reflecting the degeneration of basal forebrain neurons responsible for cholinergic innervation of the neocortex. It is hypothesized that basal forebrain neuronal degeneration results from a reduction in the level of trophic factors synthesized by neurons in the target regions of cholinergic projections. Data from a large number of animal lesioning studies tend to support this theory; however, most of these lesions also induce widespread, nonspecific injury responses in the CNS. To directly test the dependence of basal forebrain cholinergic neurons on target-derived neurotrophic support, pregnant Sprague-Dawley rats were injected with moderate doses of methylazoxymethanol acetate (MAM) on Gestational Days 14 and 15. Extensive morphometric analysis of offspring reveals that the prenatal administration of MAM during this period of neurogenesis results in the ablation of 40-70% of cortical neurons, without significant effects on the hippocampus or the genesis of basal forebrain cholinergic neurons. Examination of MAM-treated animals at several ages reveals no significant differences in neuronal density of cholinergic neurons as compared to controls. Extensive analysis of animal brains at several time points has failed to reveal any evidence of classical injury responses which might be responsible for preservation of basal forebrain neurons. These results contradict the theory that mature basal forebrain cholinergic neurons are critically dependent on the availability of target-derived neurotrophic factors and are therefore unlikely to be the major etiological factor in basal forebrain neuronal degeneration characteristic of Alzheimer's disease.

Acetylcholine↗
PubMed1992

Persistent innervation of the rat neocortex by basal forebrain cholinergic neurons despite the massive reduction of cortical target neurons. II. Neurochemical analysis.

A significant reduction in the activity of cholinergic enzymes in the neocortex is one of the characteristic neurochemical abnormalities in Alzheimer's disease. The withdrawal of cholinergic innervation is thought to be due to atrophy and degeneration of the cholinergic neurons of the basal forebrain, postulated to occur as a consequence of the loss of postsynaptic target neurons and trophic factors synthesized by these target cells. To directly test the dependence of basal forebrain cholinergic innervation on the presence of target neurons, pregnant rats were administered moderate doses of methylazoxymethanol acetate on Gestational Days 14 and 15. This results in a 40-70% reduction of cortical neurons in offspring of treated dams, but has no significant effect on the genesis of basal forebrain cholinergic neurons. In this paper, neurochemical analysis of cholinergic enzymes (ChAT and AchE) in 2-month-old offspring reveals that the basal forebrain innervates the hypocellular cortex in apparently normal fashion, despite the loss of target neurons. The cholinergic innervation of basal forebrain target regions was examined at various times through 20 months of age and was found to retain the initial high levels of enzymatic activity, without any evidence of loss of cholinergic innervation. The preservation of cholinergic innervation over 20 months despite the massive loss of target neurons contradicts the theory of dependence of basal forebrain cholinergic neurons on target-derived trophic support. These results suggest that the degeneration of basal forebrain cholinergic neurons in Alzheimer's disease is due to phenomena more complex than a reduction in the availability of trophic factors.

Acetylcholinesterase↗
PubMed1992

Metabolic half-life of somatostatin and peptidase activities are altered in Alzheimer's disease.

Several reports have described decreased immunoreactive somatostatin levels in specific regions of post-mortem brain tissue from patients diagnosed with senile dementia of the Alzheimer type (SDAT). In an attempt to determine if the metabolism of somatostatin is also altered as a result of SDAT, we examined the regional metabolic half-life of somatostatin-28 (SS-28) and somatostatin-14 (SS-14). The activity of the following peptidases was also determined: neutral endopeptidase E.C. 3.4.24.11; metalloendopeptidase E.C. 3.4.24.15; carboxypeptidase E (E.C. 3.4.17.10); and trypsin-like serine protease. The metabolic half-life of SS-28 was significantly reduced in post-mortem Brodmann Area 22 of SDAT tissue. This decrease in SS-28 metabolic half-life was correlated with a significant increase in trypsin-like serine protease activity in the same SDAT brain region. The formation rate of SS-14 from SS-28 incubated with Brodmann Area 22 homogenates was also increased in SDAT tissues as compared to controls. A regional variation in neutral endopeptidase E.C. 3.4.24.11 was also noted in both controls and SDAT samples. Although postmortem intervals of samples varied significantly, no effect was seen on any biochemical parameter measured. Results from this study provide evidence that a correlation can be made between changes in metabolic half-life somatostatin and alterations in neuropeptidase activities due to SDAT. As these data show alterations in both proteolytic metabolism and peptidase activities, many other biologically active peptide substrates could also be affected in SDAT.

Aged↗
PubMed1992

Potency of antileukoprotease and alpha 1-antitrypsin to inhibit degradation of fibrinogen by adherent polymorphonuclear leukocytes from normal subjects and patients with chronic granulomatous disease.

We have studied the relative efficacy of antileukoprotease (ALP) and alpha 1-antitrypsin (alpha 1AT) to inhibit the degradation of substrate by polymorphonuclear leukocytes (PMN) attached onto a fibrinogen matrix. PMN elastase activity was assayed by radioimmunoassay of a specific 21-residue cleavage product from the amino terminus of the A alpha chain, A alpha (1-21), of fibrinogen. The adherence of PMN (1.0 x 10(6)) to a fibrinogen matrix was facilitated by incubation with recombinant tumor necrosis factor-alpha (1 nM). Subsequently, the cells were exposed to inhibitors before stimulation with cytochalasin B and formylmethionyl-leucylphenylalanine. Under these conditions, ALP inhibited A alpha (1-21) formation with an IC50 of 85 +/- 30 nM and alpha 1AT gave an IC50 of 220 +/- 98 nM (mean +/- SD). The effect of oxidant production on A alpha (1-21) formation was evaluated by comparing the effect of PMN from normal subjects with PMN from subjects with X-linked NADPH oxidase deficiency. Stimulation of PMN from the latter subjects in a similar fashion as described above resulted in the formation of 40 +/- 4 pmol/ml A alpha (1-21), or approximately twice the amount seen with cells from normal subjects. Preincubation with ALP or alpha 1AT in a concentration range between 10 to 900 nM resulted in an IC50 of 50 +/- 13 nM for ALP compared with 150 +/- 21 nM for alpha 1AT. Both inhibitors are more effective to prevent fibrinogen degradation caused by chronic granulomatous disease (CGD) PMN than by normal PMN despite the fact that CGD PMN generated more A alpha (1-21) than did normal PMN.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochalasin B↗
PubMed1992

Cytokines and lipopolysaccharide induce nitric oxide synthase in cultured rat pulmonary artery smooth muscle.

In the current study, we describe cytokine and Escherichia coli lipopolysaccharide (LPS) induction of nitric oxide (NO) synthase mRNA levels in cultured smooth muscle from rat pulmonary artery (RPASM). Exposure of RPASM to interleukin-1 beta, interferon-gamma, or LPS alone did not significantly affect NO synthesis, as determined by nitrite concentrations in media. Exposure to tumor necrosis factor-alpha caused a modest (2x) increase in nitrite production. In contrast, exposure to a combination of the above three cytokines and LPS caused a large increase in NO synthesis. Exposure of RPASM to this combination caused an increase in mRNA levels of NO synthase (as described by Northern blot analysis with 32P-cDNA probe to an inducible form of NO synthase present in murine macrophages) that was apparent as early as 4 h. Expression of the induced gene product after exposure to the cytokine and LPS mixture was evident by significant increases in nitrite production at 12 h. Production of nitrite was completely abolished in the presence of NG-monomethyl-L-arginine (NMA), and this inhibition was reversible by the addition of excess L-arginine. NO synthase mRNA levels were not affected by NMA. The nitrite production induced by the combination of cytokines and LPS was abolished by pretreating cells with cycloheximide. These data indicate that a combination of cytokines and LPS affect expression of the gene for the inducible form of NO synthase in cultured RPASM.

Amino Acid Oxidoreductases↗
PubMed1992

Transforming growth factor-alpha gene expression in late-gestation fetal rat lung.

Transforming growth factor-alpha (TGF-alpha) is a structural homologue of epidermal growth factor and competes for binding on a common transmembrane protein receptor/kinase. Although TGF-alpha appears to be more important than epidermal growth factor in embryogenesis and mammalian organogenesis, there is little information regarding its expression in developing lung. Accordingly, we measured levels of immunoreactive TGF-alpha and its gene expression in late-term fetal rat lung during the transition from the canalicular (19-20 d) to the saccular (21 d) stage. We report that at 19 d gestation intrapulmonary levels of TGF-alpha were 1.4 +/- 0.3 pmol/mg protein as determined by RIA, but had decreased by 50% at 21 d. To determine if the TGF-alpha gene is expressed in lung, RNA isolated from fetal rat lung was reverse transcribed, and a 302-bp fragment corresponding to a portion of the TGF-alpha gene was amplified by polymerase chain reaction. Southern blot hybridization with a 32P-labeled 2.3-kb EcoRI fragment of rat TGF-alpha cDNA clone showed a pattern of declining expression during late gestation. Therefore, fetal rat lung expresses TGF-alpha, as is evidenced by the synthesis of both the message and the protein. Because levels of protein were highest in the period of canalicular lung development when the respiratory acinus is formed and vascularized, a potential role for this intrapulmonary growth factor in pulmonary remodeling is suggested.

Animals↗
PubMed1992

Expression of pulmonary metallothionein genes in late gestational lambs.

Metallothioneins (MT) are low molecular weight proteins that are important in providing protection against heavy metals such as cadmium. Other precise physiologic roles for this family of proteins are less clear, but fetal hepatic cell proliferation and differentiation may be regulated through changes in MT levels and attendant MT-mediated regulation of zinc levels. The role of MT in other developing tissue, most notably lung, is far less clear. Although expression of MT has been reported to be extremely low in early postnatal and mature lung, we hypothesized that MT has a more ubiquitous role in organ development and that pulmonary MT levels may be elevated during periods of rapid lung growth. Thus, we studied expression of MT in late-gestation fetal lambs. Sheep are particularly useful because alveolarization of lung parenchyma occurs before birth (by d 120 of a normal 147-d gestation). Immunoreactive MT was localized to bronchial epithelium of fetal, newborn, and mature sheep. The intensity of staining was greatest in the 130-d gestational age (saccular) lung, where positive reaction product was noted in the cytoplasm and nucleus of alveolar epithelial and interstitial cells. We next evaluated MT expression in developing lung tissue using Northern blot analysis and 32P-cDNA probes against the 3'-untranslated regions of mRNA encoding each of four known functional sheep MT (sMT) isoforms. Expression of sMT-II, sMT-Ia, and sMT-Ib was restricted to the saccular stage (120-132 d gestational age), and sMT-Ic mRNA was not detected in pulmonary samples from any stage of development.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Complete androgen insensitivity due to deletion of exon C of the androgen receptor gene highlights the functional importance of the second zinc finger of the androgen receptor in vivo.

Androgen-dependent gene transcription is mediated by the androgen receptor (AR) through interaction of its central zinc finger region with specific DNA sequences on target genes. Failure of this receptor-mediated gene transcription results in end organ resistance to androgens-the androgen insensitivity syndromes. In a pair of siblings with complete androgen insensitivity who had supranormal levels of androgen binding in genital skin fibroblasts, polymerase chain reaction and Southern blot analysis of the androgen receptor gene confirmed by polymerase chain reaction and sequence analysis of AR cDNA, revealed an in-frame deletion of exon C encoding the second zinc finger of the receptor. The mutant receptor in cultured genital skin fibroblasts had normal androgen binding affinity and was localized in the nucleus but had markedly reduced DNA-binding affinity. When recreated in vitro and tested in a cotransfection assay system the mutant receptor failed to activate transcription of an androgen-responsive reporter gene. This naturally occurring mutation highlights the functional dependence of the AR upon its second zinc finger in vivo and explains the complete insensitivity to androgen manifest by the affected individuals despite increased androgen binding. The elevated AR levels in the subjects' genital skin fibroblasts further suggests a possible role for the second zinc finger in autoregulation of receptor levels in vivo.

Androgen-Insensitivity Syndrome↗