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Biomedical subjects

M J Spencer

Publications and source records attributed to M J Spencer.

At least 19 recordsLinked to original sources
PubMed1996

Calcium influx into human neuroblastoma cells induces ALZ-50 immunoreactivity: involvement of calpain-mediated hydrolysis of protein kinase C.

Calcium influx into SH-SY5Y human neuroblastoma cells after ionophore treatment or transient permeabilization in calcium-containing medium increased ALZ-50 immunoreactivity markedly. This increase was prevented by inhibitors active against calpain or against protein kinase C (PKC), suggesting that both of these enzymes were required to mediate the effect of calcium influx on ALZ-50 immunoreactivity. Treatment with PKC activator TPA increased ALZ-50 immunoreactivity in the absence of calcium influx or after intracellular delivery of the specific calpain inhibitor calpastatin, indicating that the influence of PKC was downstream from that of calpain. Calcium influx also resulted in mu-calpain autolysis (one index of calpain activation) and the transient appearance of PKM (i.e., free PKC catalytic subunits, generated by calpain-mediated cleavage of the regulatory and catalytic PKC domains). Inhibition of calpain within intact cells resulted in a dramatic increase in steady-state levels of total tau (migrating at 46-52 kDa) but resulted in a relatively minor increase in 68-kDa ALZ-50-immunoreactive tau isoforms. Although calcium influx into intact cells resulted in accumulation of ALZ-50 immunoreactivity, total tau levels were, by contrast, rapidly depleted. Incubation of isolated fractions with calpain in the presence of calcium indicated that ALZ-50-immunoreactive tau isoforms were more resistant to calpain-mediated proteolysis than were non-ALZ-50 reactive tau isoforms. These data therefore indicate that calpain may regulate tau levels directly via proteolysis and indirectly through PKC activation. A consequence of the latter action is altered tau phosphorylation, perhaps involving one or more kinase cascades, and the preferential accumulation of ALZ-50-immunoreactive tau isoforms due to their relative resistance to degradation. These findings provide a basis for the possibility that disregulation of calcium homeostasis may contribute to the pathological levels of conversion of tau to A68 by hyperactivation of the calpain/PKC system.

Antigens↗
PubMed1996

Re-epithelialization of normal human excisional wounds is associated with a switch from alpha v beta 5 to alpha v beta 6 integrins.

During cutaneous wound repair, keratinocytes move laterally across the wound surface. For this lateral movement epidermal cells must disassemble their tenacious connections to the basement membrane and their neighbouring cells, and express surface receptors that permit translocation over the wound surface extracellular matrix. If the basement membrane is disrupted, the epidermis migrates over a provisional matrix that contains fibrinogen, fibronectin, vitronectin and tenascin. Although alpha 5 beta 1 integrin, a fibronectin receptor, is expressed by human epidermis during re-epithelialization of excisional and incisional wounds, the spatial and temporal patterns of vitronectin, tenascin, and other fibronectin receptors are less clear. Other potential receptors include alpha v beta 5 for vitronectin and alpha v beta 6 for fibronectin and tenascin. To study provisional matrix integrin expression during human wound healing, full-thickness 4-mm punch biopsies were performed on the inner surface of the upper arm in adult volunteers. At 3, 7 and 14 days after injury wound sites were excised, bisected, quick frozen in liquid nitrogen, and examined for the expression of alpha 5, beta 1, alpha v, beta 5 and beta 6. At 3 days, alpha 5 beta 1 and alpha v beta 5, but not alpha v beta 6, appeared around the basal and suprabasalar cells of the migrating epidermis. At 7 days, alpha 5 beta 1, alpha v beta 5, and alpha v beta 6 appeared around the perimeter of the basal cells of the migrating epidermis. By 14 days, when re-epithelialization was complete, all basal and suprabasalar cells overlying the wound expressed alpha 5 beta 1 and alpha v beta 6, but not alpha v beta 5. Thus, alpha v appeared to switch its heterodimeric association from beta 5 to beta 6 subunit during re-epithelialization.

Adult↗
PubMed1995

Calpains are activated in necrotic fibers from mdx dystrophic mice.

Death of dystrophin-deficient muscle purportedly results from increases in [Ca]in that cause the activation of calpains. We have tested whether calpains play a role in this process by assaying for changes in calpain concentration and activation in peak necrotic mdx mice (4 weeks of age) and in completely regenerated mdx mice (14 weeks of age). Biochemical fractionation and immunoblotting with epitope-specific antisera allowed measurement of the concentrations of m- and mu-calpains and the extent of autoproteolytic modification. Our findings show that total calpain concentration is elevated in both 4-week and 14-week mdx mice. This increase in concentration was shown to result primarily from a significant increase in m-calpain concentration at 4 weeks. Northern analysis demonstrated that neither m- nor mu-calpain mRNA concentrations differed between mdx and controls suggesting that the increased calpain concentration results from post-translational regulation. Immunoblotting with antibodies directed against amino-terminal peptides revealed an increase in autoproteolysis of mu-calpain, indicative of increased activation. The extent of autoproteolysis of mu-calpain returns to control levels during regeneration. This is not a consequence of increased calpastatin mRNA or protein. The findings reported here support a role for calpains in both the degenerative and regenerative aspects of mdx dystrophy.

Age Factors↗
PubMed1995

Enhancement of neurite outgrowth following calpain inhibition is mediated by protein kinase C.

We examined the interdependence of calpain and protein kinase C (PKC) activities on neurite outgrowth in SH-SY-5Y human neuroblastoma cells. SH-SY-5Y cells elaborated neurites when deprived of serum or after a specific thrombin inhibitor, hirudin, was added to serum-containing medium. The extent of neurite outgrowth under these conditions was enhanced by treatment of cells with the cell-permeant cysteine protease inhibitors N-acetyl-leucyl-leucyl-norleucinal ("C1") and calpeptin or by the phospholipid-mediated intracellular delivery of either a recombinant peptide corresponding to a conserved inhibitory sequence of human calpastatin or a neutralizing anti-calpain antisera. Calpain inhibition in intact cells was confirmed by immunoblot analysis showing inhibition of calpain autolysis and reduced proteolysis of the known calpain substrates fodrin and microtubule-associated protein 1. The above inhibitory peptides and antiserum did not induce neurites in medium containing serum but lacking hirudin, suggesting that increased surface protein adhesiveness is a prerequisite for enhancement of neurite outgrowth by calpain inhibition. Treatment of cells with the PKC inhibitor H7, staurosporine, or sphingosine induced neurite outgrowth independently of serum concentration. Because calpain is thought to regulate PKC activity, we examined this potential interrelationship during neurite outgrowth. Simultaneous treatment with calpain and PKC inhibitors did not produce additive or synergistic effects on neurite outgrowth. PKC activation by 2-O-tetradecanoylphorbol 13-acetate (TPA) prevented and reversed both neurite initiation by serum deprivation and its enhancement by calpain inhibitors. Treatment of cells with the calpain inhibitor C1 retarded PKC down-regulation following TPA treatment. Cell-free analyses demonstrated the relative specificity of various protease and kinase inhibitors for calpain and PKC and confirmed the ability of millimolar calcium-requiring calpain to cleave the SH-SY-5Y PKC regulatory subunit from the catalytic subunit, yielding a free catalytic subunit (protein kinase M). These findings suggest that the influence of PKC on neurite outgrowth is downstream from that of surface adhesiveness and calpain activity.

Blood Physiological Phenomena↗
PubMed1995

Diet-induced obesity in mice can be treated without energy restriction using exercise and/or a low fat diet.

Diet-induced obesity was treated with a high carbohydrate, low fat diet and/or increased voluntary exercise in mice. All mice had free access to food and water during the two stage experiment. In Stage 1, 20 female mice were fed a high carbohydrate diet and 50 were made obese by consumption of a diet providing 40.8% of energy from fat. At the end of Stage 1, obese mice had significantly greater body fat stores (22.9 +/- 0.9 g/100 g body wt) than mice fed the high carbohydrate diet (12.9 +/- 1.2) (P < 0.001), yet there was no significant difference in lean body mass. In Stage 2, half of the mice were given activity wheels to increase their voluntary activity and half of the obese mice were switched to a high carbohydrate diet resulting in six groups with treatment designations of obese or lean; exercise or nonexercise, and carbohydrate or fat diets. Body fat was significantly reduced by consumption of the high carbohydrate diet (P < 0.005) and by exercise (P < 0.001), but neither treatment affected lean body mass. Exercising mice consumed significantly more energy than nonexercising mice, yet experienced a decrease in body fat and energy stores.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Apoptosis precedes necrosis of dystrophin-deficient muscle.

The current view that death of dystrophin-deficient muscle fibers is a necrotic process relies primarily upon the histological appearance of the tissue after the degenerative process is well advanced. Here, we tested this view by examining the possibility that apoptosis is a component of dystrophin-deficient muscle cell death. Three assays for apoptosis were employed in analyzing prenecrotic, peak necrotic and regenerated hindlimb muscle of mdx mice: (1) terminal deoxynucleotidyl transferase (TdT) mediated end-labeling of DNA in nuclei in tissue sections; (2) assays for DNA ladders; and (3) electron microscopic assays for the presence of organelles undergoing structural changes characteristic of apoptosis. At all ages sampled, mdx muscle contained apoptotic nuclei, according to TdT-mediated dUTP labeling of tissue sections. Nuclei in regenerated mdx muscle fibers did not display apoptosis. dUTP-labeled nuclei in control C57 muscles were rare or absent at all ages sampled. DNA from 4-week-old mdx mice was found to be cleaved into fragments indicative of preferential cleavage at internucleosomal sites. Electron microscopic analysis showed that organelle structural changes indicating apoptosis appear before pathological changes diagnostic of necrosis. For example, condensed mitochondria, fragmented sarcoplasmic reticulum and nuclei with chromatin condensations resembling apoptosis appear in fibers that otherwise possess normal morphology. Together, the findings show that apoptosis precedes any detectable necrotic change in mdx muscle, and that apoptotic events continue into the stage of dystrophic pathology that is currently viewed as necrosis. Thus, apoptosis characterizes the onset of pathology in dystrophin-deficient muscle which is followed secondarily by necrotic processes.

Animals↗
PubMed1995

PTH-mediated osteoblast retraction: possible participation of the calpain pathway.

Parathyroid hormone (PTH)-induced osteoblast retraction may play a pivotal role a role in bone resorption by providing osteoclasts direct access to mineralized bone surface. We have been working on the hypothesis that the calpains participate in this retractile response through a calcium-dependent process. We have first demonstrated the presence of calpain activities in MC3T3-E1 osteoblastic cells and that these activities can be stimulated by PTH. Second, we have demonstrated that the PTH-induced osteoblast retraction is dramatically attenuated by two different cysteine protease inhibitors. Finally, initial immunofluorescent cytochemical studies suggest that this PTH-induced osteoblastic retraction is mediated through a calpain-dependent, proteolytic modification of the cytoskeletal organization.

Amino Acid Sequence↗
PubMed1993

PDGF stimulation induces phosphorylation of talin and cytoskeletal reorganization in skeletal muscle.

Modifications in the interactions of the muscle cytoskeleton with the cell membrane occur during cell growth and adaptation, although the mechanisms regulating these interactions are unknown. We have observed that myotendinous junctions (MTJs), which are the primary sites of turnover of the thin filament-membrane associations in skeletal muscle, are greatly enriched in receptors for PDGF. The high concentration of PDGF receptors at MTJs suggested to us that receptor binding may initiate cytoskeletal remodeling in skeletal muscle. We tested this possibility by examining the organization and phosphorylation of cytoskeletal components of L6 myocytes after PDGF stimulation. We have found that 10 min after PDGF stimulation, L6 myoblasts exhibit no stress fibers discernible by phalloidin binding, and that vinculin relocates from focal contacts into a diffuse cytoplasmic distribution. After 60 min of incubation, these changes are largely reversed. Indirect immunofluorescence shows that at 10-min PDGF stimulation, there are no changes in the distribution of talin, the beta 1 subunit of integrin, pp125FAK or desmin. Phosphotyrosine distribution changes upon stimulation from focal contacts to being located both in focal contacts and granules concentrated in perinuclear regions. These granules also immunolabel with anti-PDGF receptor Immunoprecipitations with anti-phosphotyrosine show that polypeptides at 180 and 230 kD show the greatest increase in tyrosine phosphorylation after PDGF stimulation. Immunoblots of anti-phosphotyrosine precipitates show that these polypeptides are the PDGF receptor and talin. We also examined the possibility that the cytoskeletal reorganization observed may result from calpain activation caused by elevated intracellular calcium induced by PDGF stimulation. However, immunoblots of control and stimulated cells show no decrease in the inactive calpain proenzyme or increase in the proteolytic, autolyzed forms of calpain pursuant to stimulation. Furthermore, stimulation produces no increase in the proportion of the 190-kD talin fragment characteristic of calpain-mediated cleavage. The retention of talin and integrin at focal contacts after talin phosphorylation, while vinculin is redistributed, indicate that phosphorylation of talin in PDGF-stimulated cells leads to separation of talin-vinculin associations but not talin-integrin associations. We propose that PDGF binding to PDGF receptors at MTJs may provide one means of regulating myofibril associations with the muscle cell membrane.

Animals↗
PubMed1993

Major histocompatibility class II antigen expression on the surface of epidermal cells from normal and ultraviolet B irradiated subjects.

The influence of ultraviolet B irradiation in therapeutic doses on MHC II-positive epidermal cell numbers and their surface MHC II antigen expression was studied quantitatively using light microscopic immunoperoxidase and immunogold electron microscopic techniques. In multiple ultrathin sections through many MHC II-positive epidermal cells from five healthy subjects, prior to ultraviolet exposure, Langerhans cells and indeterminate cells were found to express similar densities of surface MHC II antigens, which were uniformly distributed over the cell surface. The variation in surface MHC II antigen expression on 97 dendritic epidermal cells from one subject was normally distributed. Following a 6-week course of ultraviolet B irradiation, in the same doses as those normally used for the treatment of psoriasis, MHC II-positive epidermal cell numbers were significantly reduced (mean decrease to 51% of the pre-UVB sample; p < 0.001 analysis of variance), but their surface MHC class II antigen density was significantly increased (p < 0.05 analysis of variance). No MHC II-negative Langerhans cells were detected in either the pre- or post-UVB samples.

Adult↗
PubMed1993

Fluoxetine hydrochloride (Prozac) toxicity in a neonate.

A case of fluoxetine toxicity in a newborn of 38 weeks' gestation has been presented. The total drug concentration in cord blood was 80 ng/mL. The fluoxetine level, 26 ng/mL, is below the adult therapeutic level; the norfluoxetine cord blood level, 54 ng/mL, is at the adult therapeutic level. At 96 hours the fluoxetine level was not measurable and the norfluoxetine level was 55 ng/mL. The parent compound is fluoxetine, which is metabolized in the liver to norfluoxetine. The half-life of fluoxetine is 2 to 3 days, and that of norfluoxetine is 7 to 9 days. Interestingly, in our patient the fluoxetine was absent in the blood at 4 days, but norfluoxetine was present. The most common side effects of Prozac in adult patients involve primarily the central nervous system and include nervousness, tremor, jitteriness, and occasionally seizures. Central nervous system symptoms were most prominent in this newborn. He also had an increased heart rate. Cardiovascular side effects are less prominent in adults who are taking Prozac. The neonate in this case was asymptomatic at 96 hours of age, indicating that the parent compound, fluoxetine, may be the active part of the drug and side effects may be caused by the parent compound.

Female↗
PubMed1993

Identification of calcium-activated neutral protease activity and regulation by parathyroid hormone in mouse osteoblastic cells.

Calcium-activated neutral protease activity was detected in mouse MC3T3-E1 cell extracts. Inclusion of the cysteine protease inhibitor, E64c, reduced the activity, while pretreatment of intact cells with 10 nM parathyroid hormone for 90 minutes increased it. The presence of calpains in solubilized cells was confirmed by Western blotting using an antibody specific for the 80 K catalytic subunit. These results, combined with the observation that preincubation with a membrane-permeable cysteine protease inhibitor ablates 50% of the PTH-induced osteoblastic retraction, suggest that calpain-catalyzed hydrolysis of regulatory enzymes or structural proteins plays a role in mediating its short-term effects in bone.

Animals↗
PubMed1992

Calpain concentration is elevated although net calcium-dependent proteolysis is suppressed in dystrophin-deficient muscle.

The concentration, activity, and distribution of calcium-dependent proteases (calpains) are compared in dystrophin-deficient (mdx) and control mouse muscle. Calpains have been implicated previously as the protease responsible for the observed necrosis in dystrophin-deficient human muscle. Although these mouse and human muscular dystrophies have been attributed to similar genetic defects, the mouse dystrophy shows a brief necrotic episode while the human deficiency results in progressive, lethal muscle necrosis. Findings of the present study show that control mouse muscle contains more calcium-dependent proteolytic activity than dystrophin-deficient muscle. Paradoxically, adult, dystrophin-deficient mouse muscle contains higher concentrations of calpain than found in controls. Furthermore, indirect immunofluorescence using antisera produced against an oligopeptide found in the proteolytic domain of calpain shows that calpain distribution in dystrophin-deficient muscle is dispersed throughout the cytoplasm while immunolabeling of control muscle shows calpain concentrated at Z-discs. This redistribution is consistent with calpain activation in dystrophic muscle. These findings indicate that mdx mice possess the capability of suppressing calpain-mediated proteolysis. We speculate that this suppression may enable dystrophin-deficient mouse muscle to arrest necrosis and regenerate successfully.

Aging↗
PubMed1992

PDGF-receptor concentration is elevated in regenerative muscle fibers in dystrophin-deficient muscle.

Dystrophin-deficient muscle undergoes sudden, postnatal onset of muscle necrosis that is either progressive, as in Duchenne muscular dystrophy, or successfully arrested and followed by regeneration, as in most muscles of mdx mice. The mechanisms regulating regeneration in mdx muscle are unknown, although the possibility that there is renewed expression of genes regulating embryonic muscle cell proliferation and differentiation may provide testable hypotheses. Here, we examine the possibility that necrotic and regenerating mdx muscles exhibit renewed or increased expression of PDGF-receptors. PDGF-binding to receptors on muscle has been shown previously to be associated with myogenic cell proliferation and delay of muscle differentiation. We find that PDGF-receptors are present in 4-week-old mdx mice in muscles that undergo brief, reversible necrosis (hindlimb muscles) or progressive necrosis (diaphragm), as well as in 4-week-old control mouse muscles. Immunoblots indicate that the concentrations of PDGF-receptors in 4-week-old dystrophic (necrotic) and control muscles are similar. Prenecrotic, dystrophic fibers and control fibers possess some cell surface labeling of fibers treated with anti-PDGF-receptor and viewed by indirect immunofluorescence. Necrotic fibers in dystrophic muscle show cytoplasmic labeling for PDGF-receptors and labeling of perinuclear regions at the muscle cell surface. Adult dystrophic muscle displays higher concentrations of PDGF-receptor in both regenerated muscle (hindlimb) and progressively necrotic muscle (diaphragm) than found in controls. Anti-PDGF-receptor labeling of regenerated, dystrophic muscle is observed primarily in granules surrounding central nuclei or surrounding nuclei located at the surface of regenerated fibers. No labeling of perinuclear regions of control muscle or prenecrotic fibers was observed. Myonuclei fractionated from adult mdx hindlimb muscles contained no PDGF-receptor, indicating that PDGF-receptor-positive structures are not tightly associated with nuclei or within nuclei. L6 myoblasts show PDGF-receptor distributed diffusely on the cell surface. Stimulation of L6 myoblasts with 10 ng/ml of PDGF-BB causes receptor internalization and concentration in granules at perinuclear regions. Thus, PDGF stimulation of myoblasts causes a redistribution of PDGF-receptors to resemble receptor localization observed during muscle regeneration. These findings implicate PDGF-mediated mechanisms in regeneration of dystrophic muscle.

Amino Acid Sequence↗
PubMed1990

Dermatitis due to intravesical mitomycin C: a delayed-type hypersensitivity reaction?

Mitomycin C is an alkylating agent, used by intravesical instillation to treat carcinoma of the bladder. Repeated instillations can induce cystitis and an eczematous eruption affecting the palms, soles and face. If these effects are due to delayed hypersensitivity with sensitization to mitomycin C occurring in the bladder wall, it should be possible to demonstrate antigen-presenting cells in the bladder wall and positive patch tests to the drug. Using an immuno-alkaline phosphatase method we have identified CDI+ cells in bladder epithelium and submucosa and have demonstrated Birbeck granules in a few cells. In further support of our hypothesis it was also possible to demonstrate delayed type hypersensitivity in 13 out of 26 patients who had received mitomycin instillations by applying the allergen as a patch test. These results indicate that the eczematous eruption in this group of patients is most likely a hypersensitivity reaction and that it may be mediated transvesically.

Administration, Intravesical↗
PubMed1990

Papuloerythroderma--clinical and ultrastructural features.

Papuloerythroderma is a newly described entity. The clinical features of a case and the therapeutic response to systemic corticosteroids are described, together with the immunohistochemical and ultrastructural characteristics of the associated inflammatory infiltrate.

Aged↗
PubMed1990

The Laugier-Hunziker syndrome--a clinical review of six cases.

The Laugier-Hunziker syndrome is an acquired, benign, macular hyperpigmentation of the lips and buccal mucosa. The nails are often involved with the development of melanonychia. Twenty-two previous cases have been recorded in the literature. We present details of six Caucasian patients with the Laugier-Hunziker syndrome who are the first recorded from Britain. They all had acquired, macular hyperpigmentation of the lips and buccal mucosa. In five of these patients longitudinal pigmented bands were found on the nails. None had other family members affected. Although this is the first report of British patients with this syndrome, we believe that the condition is probably more common than is generally recognized.

Adult↗
PubMed1990

The UCLA-University of Utah epidemiologic survey of autism: prenatal, perinatal, and postnatal factors.

In a recent epidemiologic survey conducted in Utah, 241 autistic patients (DSM-III criteria) were found. Medical records of 233 autistics were surveyed for the presence of 36 potentially pathologic prenatal, perinatal, and postnatal factors. These results were compared with those of an identical survey of 62 of their nonautistic siblings, with the results of four previously published surveys, and with normative data. No potentially pathologic factor or group of factors occurred significantly more frequently among the autistic patients. Also, previous observations of significant differences in the occurrence of certain factors in the histories single vs multiple siblings with autism were not confirmed, with the exception of increased viral-type illness during gestation in single-incidence cases. Thus, the etiology of the brain pathology that characteristically disrupts normal development and produces the syndrome of autism remains obscure. Other data from the epidemiologic survey, however, suggest that the role of genetic factors needs to be explored further.

Apgar Score↗