Changes In Phosphatase Activity Research Articles

Phosphatase activity in rat adipocytes: effects of insulin and insulin resistance.

Insulin regulates the activity of both protein kinases and phosphatases. Little is known concerning the subcellular effects of insulin on phosphatase activity and how it is affected by insulin resistance. The purpose of this study was to determine insulin-stimulated subcellular changes in phosphatase activity and how they are affected by insulin resistance. We used an in vitro fatty acid (palmitate) induced insulin resistance model, differential centrifugation to fractionate rat adipocytes, and a malachite green phosphatase assay using peptide substrates to measure enzyme activity. Overall, insulin alone had no effect on adipocyte tyrosine phosphatase activity; however, subcellularly, insulin increased plasma membrane adipocyte tyrosine phosphatase activity 78 +/- 26% (n = 4, P < 0.007), and decreased high-density microsome adipocyte tyrosine phosphatase activity 42 +/- 13% (n = 4, P < 0.005). Although insulin resistance induced specific changes in basal tyrosine phosphatase activity, insulin-stimulated changes were not significantly altered by insulin resistance. Insulin-stimulated overall serine/threonine phosphatase activity by 16 +/- 5% (n = 4, P < 0.005), which was blocked in insulin resistance. Subcellularly, insulin increased plasma membrane and crude nuclear fraction serine/threonine phosphatase activities by 59 +/- 19% (n = 4, P < 0. 005) and 21 +/- 7% (n = 4, P < 0.007), respectively. This increase in plasma membrane fractions was inhibited 23 +/- 7% (n = 4, P < 0. 05) by palmitate. Furthermore, insulin increased cytosolic protein phosphatase-1 (PP-1) activity 160 +/- 50% (n = 3, P < 0.015), and palmitate did not significantly reduce this activity. However, palmitate did reduce insulin-treated low-density microsome protein phosphatase-1 activity by 28 +/- 6% (n = 3, P < 0.04). Insulin completely inhibited protein phosphatase-2A activity in the cytosol and increased crude nuclear fraction protein phosphatase-2A activity 70 +/- 29% (n = 3, P < 0.038). Thus, the major effects of insulin on phosphatase activity in adipocytes are to increase plasma membrane tyrosine and serine/threonine phosphatase, crude nuclear fraction protein phosphatase-2A, and cytosolic protein phosphatase-1 activities, while inhibiting cytosolic protein phosphatase-2A. Insulin resistance was characterized by reduced insulin-stimulated serine/threonine phosphatase activity in the plasma membrane and low-density microsomes. Specific changes in phosphatase activity may be related to the development of insulin resistance.

Treatment of mice with EGF and orthovanadate activates cytoplasmic and nuclear MAPK, p70S6k, and p90rsk in the liver

Background/Aims: Although signal transduction pathways activated by EGF have been extensively studied in cultured cells, few such studies have been done in whole animals. In this study, activation of hepatic kinases, phosphatases, and DNA-binding activity of AP-1 was examined after intraperitoneal injections of either EGF or sodium orthovanadate into mice.Methods: Cytoplasmic and nuclear proteins, extracted from isolated hepatocytes or whole liver tissue, were immunoprecipitated with either anti-ERK1/2, anti-70S6k, or anti-p90rsk antibodies and kinase activities were measured using specific substrates. Kinase protein levels was evaluated by Western blot analysis. AP-1 DNA binding activity was measured by electrophoretic mobility shift assay.Results: Systemic administration of EGF induced simultaneous increase in the activities of cytoplasmic and nuclear MAPK, p70S6k, and p90rsk. MAPK and p70S6k were more potently activated in the cytosol while p90rsk activation was more pronounced in the nucleus. Orthovanadate also activated these kinases but to a much lesser degree than EGF. In vitro phosphatase assays showed that neither EGF nor orthovanadate induced measurable changes in phosphatase activities. EGF, but not orthovanadate, activated nuclear AP-1 DNA-binding activity in intact liver, indicating that activation of MAPK, p70S6k, and p90rsk by orthovanadate is not sufficient to activate this transcription factor.Conclusion: These observations provide groundwork for future studies to examine the role of EGF-induced kinase cascades and transcription factors in liver regeneration and other growth factor-mediated hepatic processes.

Purification of porcine brain protein phosphatase 2A leucine carboxyl methyltransferase and cloning of the human homologue.

The carboxyl methyltransferase, which is claimed to exclusively methylate the carboxyl group of the C-terminal leucine residue of the catalytic subunit of protein phosphatase 2A (Leu(309)), was purified from porcine brain. On the basis of tryptic peptides, the cDNA encoding the human homologue was cloned. The cDNA of this gene encodes for a protein of 334 amino acids with a calculated M(r) of 38 305 and a predicted pI of 5.72. Database screening reveals the presence of this protein in diverse phyla. Sequence analysis shows that the novel methyltransferase is distinct from other known protein methyltransferases, sharing only sequence motifs supposedly involved in the binding of adenosylmethionine. The recombinant protein expressed in bacteria is soluble and the biophysical, catalytic, and immunological properties are indistinguishable from the native enzyme. The methylation of PP2A by the recombinant protein is restricted to Leu(309) of PP2A(C). No direct effects on phosphatase activity changes were observed upon methylation of the dimeric or trimeric forms of PP2A.

The role of the C-terminal region in phosphoglycerate mutase

Removal of the C-terminal seven residues from phosphoglycerate mutase from Saccharomyces cerevisiae by limited proteolysis is associated with loss of mutase activity, but no change in phosphatase activity. The presence of the cofactor 2, 3-bisphosphoglycerate, or of the cofactor and substrate 3-phosphoglycerate together, confers protection against proteolysis. The substrate alone offers no protection. Replacement of either or both of the two lysines at the C-terminus by glycines has only limited effects on the kinetic properties of phosphoglycerate mutase, indicating that these residues are unlikely to be involved in crucial electrostatic interactions with the substrate, intermediate or product in the reaction. However, the double-mutant form of the enzyme is more sensitive to proteolysis and is no longer protected against proteolysis by the presence of cofactor. The proteolysed wild-type and two of the mutated forms of the enzyme show a reduced response to 2-phosphoglycollate, which enhances the instability of the phospho form of the native enzyme. The phosphoglycerate mutase from Schizosaccharomyces pombe, which lacks the analogous C-terminal tail, has an inherently lower mutase activity and is also less responsive to stimulation by 2-phosphoglycollate. It is proposed that the C-terminal region of phosphoglycerate mutase helps to maintain the enzyme in its active phosphorylated form and assists in the retention of the bisphosphoglycerate intermediate at the active site. However, its role seems not to be to contribute directly to ligand binding, but rather to exert indirect effects on the transfer of the phospho group between substrate, enzyme, intermediate and product.

Increasing the Ratio of PP2A Core Enzyme to Holoenzyme Inhibits Tat-Stimulated HIV-1 Transcription and Virus Production

We demonstrated previously that PP2A exists in many cell types as two abundant forms: (1) holoenzyme composed of two regulatory subunits, A and B, and a catalytic subunit C; and (2) core enzyme consisting of the A and C subunits. These two forms have different substrate specificities. Since published data suggested that HIV-1 transcription may be regulated by a cellular protein phosphatase, it was of interest to determine whether changing the ratio between PP2A core and holoenzyme affects HIV-1 gene expression. This question was addressed by expression in COS cells of an N-terminal mutant of the A subunit, AΔ5, which binds the C but not the B subunit. This resulted in an increase in the amount of core enzyme and a decrease in the amount of holoenzyme concomitant with the expected change in phosphatase activity. Tat-stimulated transcription from the HIV-1 LTR was inhibited 5-fold by mutant AΔ5, whereas mRNA synthesis directed by the actin promoter was not affected. Furthermore, virus production in COS, HeLa, and Jurkat T cells was inhibited 45-, 5-, and 3-fold, respectively, by mutant AΔ5. These results demonstrate that the balance between PP2A holoenzyme and core enzyme is important for HIV-1 gene expression and virus production.

Cell density-dependent regulation of mdr-1 gene expression in murine colon cancer cells

We studied the regulation of mdr-1 and P-glycoprotein in sparse and confluent cultures of murine CT-26 colon carcinoma cells. The expression level of mdr-1 mRNA transcripts (analyzed by Northern blot and in situ hybridization) and P-glycoprotein (analyzed by flow cytometry) inversely correlated with cell density. The modulation of mdr gene expression in sparse and confluent cells was not related to cell division, nutrient depletion, inhibition of protein synthesis, gap junction status, extracellular ATP, or the presence of various extracellular matrixes, but may be related to cell-density and cell-contact mediated changes in phosphatase activity. The confluence-mediated downmodulation of mdr-1 increased the chemosensitivity of the cells to several anticancer drugs commonly associated with an in vitro MDR phenotype by increasing the intracellular accumulation of the drugs. These data may explain some of the discrepancies in results obtained when analyzing mdr gene expression in tumors growing in vivo or in vitro, and why mdi expression in tumors is localized to the periphery of the lesions.

Histochemical/histoenzymic studies in broiler chicks fed aflatoxin, ochratoxin and inoculated with inclusion body hepatitis virus singly and in concurrence.

The effect of feeding mycotoxins, i.e. aflatoxin B1 (1.25 ppm from 3 to 38 days of age) and ochratoxin A (0.5 ppm from 3 to 38 days of age) along with inclusion body hepatitis virus (IBHV) inoculation (at 10 days of age) singly and in combination was studied in broiler chicks. Birds in combined treatment groups, i.e. aflatoxin fed and virus inoculated and ochratoxin fed and virus inoculated, showed more changes in activities of phosphatases (AKPase, ATPase, G-6-Pase and ACPase) in liver and kidney tissues than their respective individual treatment groups with a few exceptions. Reduction in the activities of oxido-reductases in liver and kidney tissues were almost comparable in different treatment groups. The increase in muco-polysaccharides reaction was more marked in both the combined treatment groups than the single treatment groups. Intensity of lipid reaction was more in ochratoxin virus combination group than either alone.

Importance of tyrosine phosphatases in the effects of cell-cell contact and microenvironments on EGF-stimulated tyrosine phosphorylation.

We have compared the EGF responses of A431 cells when grown as monolayers at a variety of cell densities or as multicellular spheroids in order to investigate the effects of cell contact and 3-dimensional structure on signal transduction. Proliferation of the A431 squamous carcinoma cell line grown in our laboratory was unaffected by EGF when grown in monolayer culture. As 3-dimensional, multicellular spheroids, however, growth was stimulated by EGF. The maximum volume attainable in the presence of EGF was more than 30 times that in its absence. EGF-dependent tyrosine phosphorylation was compared under these conditions by immunohistochemistry and Western blotting. In initial experiments using published procedures, tyrosine phosphorylation was density-dependent in monolayers and undetectable in spheroids. However, the density-dependence was abolished by the addition of high concentrations of protein tyrosine phosphatase inhibitors (1 mM Zn++ and VO4(3)-). The density dependence of EGF-stimulated tyrosine phosphorylation in monolayers was, therefore, largely the result of changes in phosphatase activity rather than kinase. Using high concentrations of phosphatase inhibitors, phosphotyrosine was clearly visible by immunohistochemistry in the outermost cells of spheroids, but it was still not visible in the spheroid center. The lack of response within the spheroid was not related to the presence of EGF receptor nor diffusion of EGF. In companion experiments, we showed that staining for EGF receptor was present homogeneously throughout the spheroid and that EGF penetrated to its center under the conditions of the experiment. Thus, although an increase in tyrosine phosphatase activity was a major factor affecting tyrosine phosphorylation in the outer cells, other factors were important in the inner cells. We concluded that an increase of tyrosine phosphatase activity was the most important component of the adaptation of the EGF signal transduction system to high cell density in monolayer cultures. In spheroids, tyrosine phosphatases are also enhanced, but other factors, such as autocrine synthesis of TGF-alpha and possibly the cellular distribution of EGF receptors and cell shape, play a role.

Defective insulin response of phosphorylase phosphatase in insulin-resistant humans.

Insulin-stimulated glycogen synthase activity in human muscle is reduced in insulin-resistant subjects. Insulin regulation of human muscle glycogen synthase may require activation of a type-1 protein phosphatase (PP-1). We investigated the change of phosphorylase phosphatase and glycogen synthase activities in muscle biopsies obtained during a 2-h hyperinsulinemic euglycemic clamp in 12 insulin-sensitive (group S) and 8 insulin-resistant (group R) subjects. Fasting phosphorylase phosphatase activity was lower in group R than in group S, and did not increase significantly with insulin infusion in group R until 20 min. In group S, phosphorylase phosphatase was significantly stimulated by 10 min, remaining significantly higher than in group R at all time points. The insulin-mediated changes in phosphatase activities were not decreased by 3 nM okadaic acid but were completely inhibited by 1 microM okadaic acid, thereby verifying that insulin-stimulated phosphorylase phosphatase is accounted for by a PP-1. Subcellular fractionation demonstrated reduced fasting PP-1 activities in both the glycogen and cytosolic fractions of muscle obtained from subjects in group R compared to those in group S. These results suggest that insulin activation of PP-1 could contribute to the stimulation of glycogen synthase by this hormone in human muscle. Lower fasting PP-1 activity in cytosol and glycogen fractions plus lower insulin-stimulated PP-1 activity could explain, in part, reduced insulin-stimulated glycogen synthase in skeletal muscle of insulin-resistant subjects.

Gametophytic self-incompatibility inPapaver rhoeas L.

We have developed an in vitro system whereby we can reproduce the self-incompatibility (SI) reactions ofP. rhoeas in pollen grown in vitro, using stigmatic extracts. This has enabled us to investigate a number of aspects of SI, which would otherwise be difficult. On the stigma side of the reaction, the in vitro system has enabled us to characterize and partially purify the stigmatic S-component, following S-specific activity. It has also enabled us to establish that, in contrast to the S-linked glycoprotein ofNicotiana alata, no detectable ribonuclease activity correlates with the presence of the functional stigmatic S-gene product in this species. Turning to look at the pollen side, we have used the in vitro system to study the metabolic events occurring in the pollen ofP. rhoeas as a consequence of the SI reaction. We have determined that it requires both de novo glyco-sylation and RNA transcription for full inhibition of pollen-tube growth during the SI reaction. Transcription products of pollen SI response genes, which are produced specificially in an incompatible reaction, have been identified. These pollen response genes have been cloned and are currently being characterized. Since the extracellular pollen-stigma interaction results directly in gene transcription in the pollen, it seems likely that a signal transduction mechanism may be operating in the SI response. The in vitro system has allowed us to begin to investigate this possibility. We have detected rapid and transient phosphorylation of certain pollen proteins, together with changes in phosphatase activity during the SI reaction. These studies provide evidence for a role for signal transduction in the SI reaction. Thus, our in vitro system has enabled us to begin to examine, not only stigma and pollen components, but also the interaction between them in the SI reaction.

Biocompatibility of a prolonged-action antialcohol preparation

Acid and alkaline phosphatase activities in liver lysosomes and liver mitochondria, respectively, as well as in connective tissue capsule were studied for an antialcohol preparation (polyurethane carrier and disulfiram at 150, 300 and 625 mg/kg body weight) implanted subcutaneously. Increased acid phosphatase activity was observed in connective tissue capsule on days 14, 30 and 90 after implantation and in liver lysosome fraction on days 14 and 30 with preparation containing disulfiram at 300 and 625 mg/kg body weight. Alkaline phosphatase activity increased both in connective tissue capsule and in liver mitochondria up to day 30 only when sample with maximal disulfiram dose was implanted. Acid phosphatase activity is an adequate indicator for biocompatibility of prolonged-action medicine preparations. The data obtained show that the suggested medical form containing disulfiram 150 mg/kg body weight is biocompatible. Interaction between acid and alkaline phosphatase activity changes and biocompatibility and destruction of polymer implants is discussed.

The effects of metabolism on Na(+)‐K(+)‐Cl‐ co‐transport in ferret red cells.

1. The effects of altering metabolism on Na(+)-K(+)-Cl- co-transport were studied in ferret red cells. Na(+)-K(+)-Cl- co-transport was measured as the bumetanide-sensitive uptake of 86Rb. 2. Glucose, but not inosine or adenosine, sustained metabolism and maintained cell ATP content ([ATP]i) at the physiological level. [ATP]i could be reduced by prolonged incubation of cells in a substrate-free medium or more quickly by incubating cells with 2-deoxyglucose or with a mixture of iodoacetamide and glucose. 3. Na(+)-K(+)-Cl- co-transport activity was inhibited when [ATP]i was reduced to below 100 mumol (1 cell)-1 by starvation or by treatment with 2-deoxyglucose. However, a unique relationship between [ATP]i and activity could not be found. [ATP]i and the method and time course of ATP depletion all influenced activity. The inhibition of Na(+)-K(+)-Cl- co-transport, caused by reducing [ATP]i could be partially reversed by restoring [ATP]i to normal. 4. Increasing the concentration of intracellular ionized magnesium [( Mg2+]i) did not stimulate co-transport activity in ATP-depleted cells. This contrasts with the substantial stimulation seen in cells with normal [ATP]i. 5. Vanadate stimulated Na(+)-K(+)-Cl- co-transport activity in ATP-depleted cells but not in cells with normal [ATP]i. Fluoride did not affect activity at any [ATP]i. 6. The effects of some sulphydryl reagents on Na(+)-K(+)-Cl- co-transport were also examined. n-Ethylmaleimide (1 mM) inhibited Na(+)-K(+)-Cl- co-transport while it stimulated bumetanide-resistant potassium transport. Dithiothreitol (1 mM) did not affect activity. Iodoacetamide (6 mM) appeared to reduce the inhibition of cotransport activity seen at low [ATP]i but also greatly increased cell fragility. 7. The data suggest that activity of the Na(+)-K(+)-Cl- co-transport system is controlled by a cycle of phosphorylation and dephosphorylation with the phosphorylated form being active. Phosphorylation and transport appear to be almost maximal in ferret red cells with normal [ATP]i. Reduction of [ATP]i may allow changes in phosphatase activity to manifest as changes in transport rate. Differences in the balance between phosphorylation and dephosphorylation may explain tissue-dependent variations in the response of the system to various stimuli.

Phosphatase activity in relation to phytoplankton composition and pH in Swedish lakes

SUMMARY. 1. Potential phosphatase activity and phytoplankton from several lakes of different character were compared in order to evaluate the importance of lake water pH and phytoplankton composition for the activity and pH optimum of lake water phosphatases.2. In oligotrophic lakes, in which phytoplankton biomass was most often dominated by Ochromonadaceae spp., optimum phosphate activity was found at pH values &lt;6. In eutrophic lakes, where species of Cyanophyceae and Bacillariophyceae dominated the phytoplankton biomass, optimum phosphatase activity was found at pH 7.5 or 8.5.3. The pH optimum of phosphatase activity often differed from the corresponding lake water pH.4. Experimental variation in phosphorus availability resulted in predictable changes in phosphatase activity. However, specific phosphatase activity, calculated per biomass of phytoplankton, was dependent on plankton species composition.

Glucose-6-phosphate stimulation of human muscle glycogen synthase phosphatase

The influence of glucose-6-phosphate (glucose-6-P) on skeletal muscle glycogen synthase phosphatase was examined in normal glucose-tolerant Southwest American Indians. Phosphatase was stimulated with physiological concentrations of glucose-6-P and inhibited by ATP (5 mmol/L) and glycogen (0.1%). Phosphatase activity was measured before and after insulin infusion using the euglycemic clamp technique. Although glycogen synthase fractional activity increased in all subjects, this increase was not related to a change in phosphatase activity in the absence or presence of glucose-6-P. These results suggest that glycogen synthase phosphatase from human muscle can be regulated by physiological concentrations of glucose-6-P, ATP, and glycogen, and that insulin does not alter glucose-6-P stimulation of the enzyme in normal subjects.

Interference with soil phosphatase activity by maize herbicidal treatment and incorporation of maize residues

This study is concerned with the way alachlor, atrazine and metolachlor interfered with phosphatase activity in a clay loam soil unenriched and enriched with maize residues. Enrichment caused an increase in all phosphatase activities (acid and alkaline phosphomonoesterase, phosphodiesterase, phosphotriesterase) tested. Interference with phosphatase activity following herbicidal treatment was found in both unenriched and enriched soil samples. Statistically significant interference was dependent on soil enrichment, the type of herbicide and its rate of application and the time elapsed since the herbicidal treatment. The observed changes in phosphatase activities are attributed to herbicidal action on phosphatase-producing microorganisms. Among the herbicides tested, the acetanilide derivatives exerted a lesser inhibiting effect than atrazine. Nevertheless, all the altered phosphatase activities showed a tendency, more or less rapid, to reach the levels in the corresponding untreated soil samples.

Intestinal hydrolysis of pyridoxal 5′-phosphate in vitro and in vivo in the rat: effect of ethanol

The effect of ethanol on intestinal hydrolysis of pyridoxal 5′-phosphate (PLP), the first step in PLP absorption, was evaluated in the rat. Models included PLP decay during in vitro incubation and PLP luminal disappearance during in vivo perfusion of jejunal segments. Ethanol inhibited PLP in vitro decay (pH = 7.4 and 3.0) and in vivo disappearance (pH = 7.4) in a concentration-dependent manner (1-4% w/v). At pH = 7.4, 4% w/v ethanol inhibited in vitro PLP decay by 48% (p < 0.001) and in vivo PLP disappearance by 55% (p < 0.001). This inhibition was reproduced in vitro with other alcohols but not with osmotic controls, paralleled in vitro changes in phosphatase activity in the presence of ethanol, and was completely reversed in vivo after ethanol was removed from the perfusing solution. The inhibition of intestinal PLP hydrolysis may possibly be one of the mechanisms by which a high percentage of alcoholics become biochemically vitamin B6 deficient.

&lt;b&gt;IMMUNOCYTOCHEMICAL LOCALIZATION OF COLLAGENASE IN ISOLATED MOUSE BONE &lt;/b&gt;&lt;b&gt;CELLS &lt;/b&gt;

Immunoreactive collagenase was localized -in isolated mouse bone cells by indirect immunofluorescent and immunoperoxidase antibody techniques using goat antimouse bone collagenase antibody. Immunoreactive collagenase was localized primarily in cells with osteoblastic characteristics (osteoblast-like cells) based on biochemical parameters; i.e., cAMP production and alkaline and acid phosphatase activity changes in response to parathyroid hormone. There was no collagenase immunofluorescence in cell populations rich in osteoclastic cells. Both parathyroid hormone and heparin appeared to stimulate the synthesis and/or release of immunoreactive collagenase by isolated osteoblast-like cells. These findings were consistent with the results of our biochemical studies (subsequent paper) using the same isolated bone cell preparations. Collagenase immunofluorescence of isolated bone cells was observed initially as fine granular fluorescence packed at perinuclear regions. This fine granular fluorescence then appeared to diffuse through the cytoplasm and to be secreted extracellularly.

Long-term effects of crude oil on microbial processes in subarctic marine sediments: Studies on sediments amended with organic nutrients

Subarctic marine sediments amended with various organic compounds were exposed to fresh Cook Inlet crude oil at a concentration of 50 ppt for either 6 or 8 months. After the sediments were initially treated, they were returned to the approximate location where they were collected and left to be exposed to natural environmental conditions until they were retrieved for analysis. As a result of crude oil treatment, the activities of the enzymes that hydrolyse structural polysaccharides were reduced and the activities of the enzymes that hydrolyse storage polysaccharides were stimulated. In addition to these changes, we observed changes in phosphatase activity, nitrogen fixation rates, potential denitrification rates, methane concentrations, CO 2 production rates, and the glucose uptake and mineralization rates. The effect of the crude oil perturbation was different depending on the organic compound used in the amended sediments. Many of these differences could be explained by the effect of crude oil on the hydrolases which were responsible for degrading the compound in question. The results of this study suggest the effect of crude oil on microbial processes may be affected by the type of organic material present in the impacted marine sediment. This study also illustrates the value of measuring hydrolase activity in studies designed to determine the effects of this or any other pollutant on microbial processes in marine sediments.

Effect of cell density and senescence of WI-38 cells on cytochemically demonstrable phosphatases

Exponentially growing and stationary phase young and old cultures of the human cell line WI-38 were studied using cytochemistry at the ultrastructural level. 5′-Nucleotidase activity was present in the plasma membrane of all cells examined; in exponentially growing cultures the reaction was more intense in mitotic cells than in interphase cells. An increase in the amount of the reaction product was observed at confluency, especially in older cells. The reaction for Mg 2+-activated adenosine 5′-triphosphatase was absent or very weak in exponentially growing cells and increased at confluency, especially in older cells. Alkaline phosphatase was detectable only in the cell membranes and intercellular spaces of young cells at confluency. Acid phosphatase activity was increased in old cells, especially at confluency. In these old cells, positive reactions appeared in numerous small lysosomes, autophagic vacuoles and in some flattened sacs of the Golgi apparatus. The obtained results confirm and extend previous biochemical observations and indicate that changes in phosphatase activity are associated with proliferative activity and senescence of cells growing in vitro.

Histochemical and histopathological changes in skeletal muscle in late-onset hereditary distal myopathy (Welander)

Histochemical and histopathological staining methods were applied to muscle biopsy material from 13 patients with distal myopathy of late onset. Six cases showed slight to moderate histopathological changes and the normal distinction between Type I and Type II muscle fibres, based on their staining characteristics for myofibrillar ATPase, was well preserved. A selective Type I atrophy and an irregular distribution of oxidative enzyme and fat staining in Type I fibres were evident. In the other 7 cases. with moderate to advanced histopathological changes, there was a marked blurring of the normal difference observed in ATPase activity between Type I and Type II fibres. Thus, both types of fibre exhibited a high intensity of staining for myofibrillar ATPase at pH 9.4 without inhibition by acid preincubation (pH 4.3). These changes in phosphatase activity were found not only in atrophic fibres but also in normalsized fibres without other signs of degeneration. Nuclear proliferation in chains and “ring fibres” were found. The early histopathological and histochemical changes in distal myopathy are strikingly similar to those of myotonic dystrophy.