Immunocytochemical localization of peptidylarginine deiminase in human eosinophils and neutrophils.

Peptidylarginine deiminase, registered as PAD V in the DDBJ/GenBank/EMBL data banks, is expressed in HL-60 cells differentiated into granulocytes or monocytes. We analyzed PAD activities in density-fractionated human peripheral blood cell fractions. PAD activity with similar substrate specificity to that of PAD V was found in the eosinophil and neutrophil fractions, which showed single bands comigrating with authentic PAD V on immunoblotting with an anti-PAD V antibody. Both the biochemical and immunoblotting analyses showed marked enrichment of PAD V in the eosinophil fraction. Its immunoreactivity appeared to localize in eosinophilic granules at high density and in myeloperoxidase-negative cytoplasmic granules of neutrophils at low density, as determined by confocal laser-scanning microscopy. Possible roles of PAD V in myeloid differentiation and granulocyte function are discussed. In addition, we present evidence for the presence of PAD(s) that are antigenically different from PAD V in monocytes and lymphocytes.

Detection of deiminated proteins in rat skin: probing with a monospecific antibody after modification of citrulline residues.

We performed a systematic study on deiminated proteins present in rat epidermis. Proteins extracted from various epidermal samples were resolved by either one- or two-dimensional gel electrophoresis and Western blotted to nitrocellulose membranes. Deiminated proteins were detected by modification of citrulline residues followed by probing with an anti-modified citrulline monospecific antibody. The cornified layer of adult plantar skin gave multiple series of isoelectric variants, most of which were found to be differentially deiminated type II keratins (60 kDa, and 67 kDa or above). The whole epidermis of 5-day-old rat back skin showed isoelectric variants of 60-kDa keratin as major deiminated components, and deiminated 55-kDa keratin and deiminated filaggrin as minor spots. In addition, we found highly deiminated proteins (200-220 kDa) thought to be derived from trichohyalin. The immunoreactivity of deiminated proteins was mainly localized in the granular and cornified layers of epidermis. Co-localization of deiminated filaggrin and keratins in the granular layer suggests the possible role of protein deimination during the terminal stage of epidermal differentiation.

Deimination of 70-kD nuclear protein during epidermal apoptotic events in vitro.

Peptidylarginine deiminase (PAD) is the enzyme responsible for converting protein-bound arginine residues to citrulline. It has recently been shown that a number of epidermal proteins, including filaggrin, trichohyalin, and keratins, are deiminated by the action of PAD, suggesting a possible role for protein deimination during the final stages of epidermal differentiation. We report here a novel PAD substrate found during the course of identifying deiminated proteins in cultured rat epidermal keratinocytes. We found that a 70-kD protein localized to the periphery of the nucleus was preferentially deiminated after ionomycin treatment in the presence of 2 mM calcium and was associated with apoptotic events in these cells. Furthermore, we discovered that the deimination of nuclear protein could be induced by transfection of a PAD cDNA into rat epidermal keratinocytes. These data suggest that PAD may act on the 70-kD nuclear protein to induce disassembly of the nuclear lamina and promote apoptosis during terminal epidermal differentiation.

Comparison between muscle and liver enolases and their behavior during differentiation and growth.

1. Rabbit liver enolase (EC 4.2.1.11) was purified about 200-fold and the enzyme was distinguished from crystalline muscle enolase by column isoelectrofocusing. It was found that the pI of muscle enolase was at about pH 8.8 and the pI of liver enolase was at about pH 6.7. Liver enolase was more liable to heat than muscle enolase. Anti-muscle enolase antibody did not react with liver enolase in double diffusion and immunoprecipitation tests. No substantial difference seemed to exist between muscle and liver enolases in pH optima, kinetic constants, and gel filtration. 2. It was observed by electrofocusing that the pI of rat muscle enolase was pH 7.2 to 7.9 and that of liver enolase was about pH 5.9. The main component of muscle enolase was designated as type A enolase, and liver enolase as type B enolase. Type A enolase was present in skeletal muscle and heart muscle. Type B enolase was widely distributed and present in liver, kidney, spleen, brain, lung, small intestine, and heart muscle. More acidic isozyme than type B enolase coexisted in the brain, and more basic isozyme than type A enolase, coexisted in the small intestine. A prototype of enolase in the early stage of differentiation was found to be type B enolase and, as differentiation progressed, type B decreased in muscle, while type A increased. On the other hand, liver enolase was retained as type B during differentiation. The enolase in regenerating liver was the same as in normal liver.

Nucleoside triphosphate and cation requirement for dopamine uptake by plain synaptic vesicles isolated from rat cerebrums.

Plain synaptic vesicles were partially purified from rat cerebrums and their [3H]dopamine uptake was investigated. The addition of MgCl2 plus ATP and of CaCl2 plus ATP increased the uptake 16 times and 5.5 times, respectively, higher than the control level, whereas the separate addition of these agents augmented the level at most 2.5 times. GTP stimulated the uptake as well as ATP, whereas UTP was one-third as effective and CTP, ADP, and AMP were all ineffective. Adenylyl imidodiphosphate was not only ineffective, but strongly reduced the ATP-dependent uptake. The half-maximal level of the ATP-dependent uptake was reached within 1.5 min after the start of incubation at 25 degrees C. The incorporation peaked at 5-10 min, then gradually declined to half-maximum at 45 min. This decline was prevented by the further addition of small amounts of CaCl2. There was no uptake at 0 degrees C, and the incorporation rate was at least 2 times faster at 37 degrees C than it was at 25 degrees C. The apparent Michaelis constant for [3H] dopamine was 1.6 muM. The half maximal inhibition of uptake was obtained at 0.1 muM reserpine; neither colchicine nor ouabain showed significant inhibition. The ATP-dependent uptake was not affected by K+, Na+, and Cl-, but was drastically decreased by isotonic phosphate buffer.

Protein deimination in the rat brain after kainate administration: citrulline-containing proteins as a novel marker of neurodegeneration.

Peptidylarginine deiminases (PADs) are a group of enzymes that convert protein arginine residues to citrulline residues in a Ca2+-dependent manner. In the central nervous system, PAD type II localizes in glial cells, but its biological role is little understood. We examined the timing and region dependence of protein deimination in the rat cerebrum after a systemic injection of kainic acid (KA). Citrulline-containing proteins were consistently found in neurodegenerating regions. Western blot analyses showed deimination of numerous proteins in a broad-molecular-weight range. By immunocytochemical scrutiny, deiminated protein-positive astrocytes were found at 2 h after KA administration, and they increased in number until the 6 h. Furthermore, shrunken neurons became deiminated protein-positive at 12-24 h. These data suggest that PAD type II becomes activated in regions undergoing neurodegeneration and functions to deiminate various proteins. Therefore, citrulline-containing proteins seem to be a useful marker of acute neurodegeneration.

Collaborative work to determine the optimal administration period and parameters to detect drug effects on male rat fertility--study on estradiol benzoate effects.

In order to examine the optimal administration period and parameters for male fertility assessment, male rats were subcutaneously administered 0.2, 2 or 20 micrograms/kg of estradiol benzoate (E2B), a known testicular toxicant, for 4 weeks or 9 weeks before mating. After 4 weeks administration, suppression of body weight gain and food consumption, decreases in prostate and seminal vesicle weights, atrophy of Leydig cells, and mature spermatid retention at stages IX, X and XI were observed in the 2 and 20 micrograms/kg groups. In the 20, micrograms/kg group, decreases in epididymides weight and copulation index were also found but the number of sperm and sperm motility were not affected. In the 0.2 micrograms/kg group, no changes were noted in any parameters. After 9 weeks administration, decreases in testis weight and the number and motility of sperm were observed in the 20, micrograms/kg group, in addition to the changes found after 4 weeks administration. These results suggest that detailed histopathological evaluation and determination of accessory sex organ weights are sensitive for evaluating the effects of E2B on male fertility. Results with the 4-weeks treatment were comparable to those with the 9-weeks treatment in terms of these parameters.

Combined biochemical and immunocytochemical analyses of postmortem protein deimination in the rat spinal cord.

Immunocytochemical staining of citrulline-containing proteins, i.e. products of endogenous peptidylarginine deiminase (EC 3.5.3.15) reaction, has been performed by chemical modification of citrulline residues in situ, followed by probing with IgG specific to the modified residues. Rat spinal cords that had been preincubated in vitro to accelerate the enzyme reaction were used as samples. Not only astrocytes but also the cytoplasm of some large neurons were stained. Usefulness of the method for studying nervous tissue damage was suggested.

Recognition of collagen by fibroblasts through cell surface glycoproteins reactive with Phaseolus vulgaris agglutinin.

The role of glycochains of cell surface glycoproteins in the cell to collagen interaction was examined by studying the effect of lectins on the fibroblast-mediated collagen gel contraction. Lectins of Phaseolus vulgaris agglutinin (PHA), concanavalin A (ConA), lentil seed agglutinin (LCA), pea agglutinin (PSA), Ricinus communis agglutinin-60 (RCA), and wheat germ agglutinin (WGA) dose-dependently inhibited gel contraction, while lectins of mushroom agglutinin (ABA), peanut agglutinin (PNA), pokeweed mitogen (PWM), and soybean agglutinin (SBA) did not. Of these lectins, PHA seemed to be worthy of further analysis, because PHA, but not other lectins, inhibited spreading of fibroblasts on collagen fibrils but not on plastic or gelatin, suggesting that cell-surface glycoproteins responsive to the lectin are involved in the specific binding of fibroblasts to native collagen fibrils. The inhibitory effect of PHA-E4, an isolectin of PHA, was more intense than that of PHA-L4, another isolectin of PHA. The collagen gel contraction was also inhibited by tunicamycin and monensin in a concentration-dependent and reversible manner. These results strongly suggest that PHA-E4-reactive glycoproteins of the fibroblast surface play an important role in cell to collagen binding during the gel contraction. Five membrane proteins including beta 1 subunits of the integrin family were obtained by affinity chromatography with PHA-E4.

Collagen gel contraction by fibroblasts requires cellular fibronectin but not plasma fibronectin.

Fibroblasts embedded in three-dimensional lattices of collagen fibrils have been known to require serum constituents to induce a cell-mediated contraction of collagen gels. The gel contraction was studied with human skin fibroblasts cultured in the presence of fetal bovine serum (FBS). Removal of bovine serum fibronectin (sFN) from FBS did not affect the extent of gel contraction. Gel contraction occurred in serum-free defined media. Therefore, it is concluded that sFN is not required for gel contraction. That cellular FN (cFN) synthesized and secreted by fibroblasts plays a crucial role in gel contraction was suggested by the following experiments: (1) We obtained monoclonal antibodies (mAb A3A5) against fibroblast surface antigens, which suppressed the fibroblast-mediated gel contraction. Immunoblot analyses showed that mAb A3A5 recognizes cFN secreted by human fibroblasts and human plasma FN (pFN), but not bovine sFN in FBS used for culture. (2) Addition of rabbit antisera, which recognize human cFN, to a serum-free gel culture inhibited contraction. Uninvolvement of human pFN in gel contraction was further confirmed by the fact that neither pretreatment of fibroblasts with excess amounts of human pFN nor the presence of excess amounts of human pFN in gels affected the extent of gel contraction. This study seems to be the first demonstration of functional difference between cFN and pFN (or sFN) and proposes a novel mode of binding of fibroblasts with collagen fibrils via cFN during cell-mediated collagen morphogenesis.

Selective deimination of vimentin in calcium ionophore-induced apoptosis of mouse peritoneal macrophages.

We found citrulline-containing proteins in mouse peritoneal macrophages undergoing calcium ionophore-induced apoptosis. Such proteins were products of deimination of arginine residues catalyzed by endogenous peptidylarginine deiminase (EC 3.5.3.15) activated by calcium influx. Western blotting analyses of the extract from macrophages incubated with 1 microM ionomycin showed selective deimination of vimentin without detectable degradation. Double immunofluorescence staining of deiminated proteins and vimentin suggested localization of deiminated vimentin around the periphery of round-shaped nucleus, which was thought to be an early morphological sign of apoptosis. The biological implication of vimentin deimination in macrophage apoptosis is discussed.

A case of skin metastasis from follicular thyroid carcinoma.

We present a case of skin metastasis from follicular thyroid carcinoma which developed on the scalp of a 72-year-old man. The lesion was noticed 1 month after a surgical excision of the primary thyroid carcinoma and gradually enlarged during the past 11 months. A biopsy from the nodule showed mostly well-differentiated thyroid follicular structures with colloid material. Tumor cells showed mild variation of nuclear size and shape in almost all areas. We performed immunohistochemistry using antithyroglobulin antibody, which established the diagnosis of a metastatic lesion from thyroid follicular carcinoma. Total thyroidectomy and 131I radiotherapy were performed. No further metastasis has been discovered during the last 18 months.

Casein kinase in cytosol of rat and mouse mammary epithelial cells isolated from histone kinase by MgCl2 treatment and influence of pregnancy and lactation on the enzyme activity.

Rat mammary glands contain cyclic AMP-independent casein kinase and cyclic AMP-dependent histone kinase. The former was easily isolated from cyclic AMP-dependent histone kinase by MgCl2 treatment. Mammary casein kinase was not activated by cyclic nucleotides, and Mg++ and ATP were required for activation. The specific activity of casein kinase in cytosol of rat mammary epithelial cells increased 2 to 3-fold during pregnancy and lactation. Cytosol of mouse mammary epithelial cells also contained cyclic AMP-independent casein kinase, and the activity of this enzyme was about three times that of the Golgi fraction.

Detection of citrulline residues in deiminated proteins on polyvinylidene difluoride membrane.

We have developed a new detection method of deiminated proteins on polyvinylidene difluoride membranes. Citrulline residues in enzymatically deiminated histones were modified by incubating with diacetyl monoxime and antipyrine in a strong acid mixture. The products were injected to rabbits, and the antibodies obtained were affinity-purified using a modified citrulline column. Sample proteins blotted to the membrane were modified in a similar manner and incubated successively with the purified antibody and an alkaline phosphatase-conjugated second antibody. Detection was performed using a chemiluminescent substrate. The method enabled detection of 3-10 fmol of citrulline residues dot blotted as deiminated model proteins. It visualized numerous rat pituitary soluble proteins that had been enzymatically deiminated and Western blotted to the membrane. The data suggest usefulness of the method for detecting deiminated proteins regardless of the backbone protein molecules. Search for deiminated proteins on the Western blots of various rat tissue homogenates detected a single band on that of spinal cord, another band on that of uterus, and multiple bands on those of skin and hair root. The bands in the former two tissue homogenates comigrated with glial fibrillary acidic protein and vimentin, respectively.