Ingestion of gelatin has differential effect on bone mineral density and body weight in protein undernutrition.

Malnutrition, particularly protein undernutrition, contributes to the occurrence of osteoporotic fracture by lowering bone mass. In this study, the effects of dietary protein on bone mineral density and body weight in protein undernutrition were compared between gelatin and milk casein. When mice were fed for 10 wk with a low protein diet containing 10(%) casein or 6% casein +4% gelatin, there was no significant difference in the final body weight between the 6% casein+4% gelatin group and the 10% casein group. In contrast, bone mineral content and bone mineral density of the femur were significantly higher in the 6% casein+4% gelatin group than in the 10% casein group. Bone mineral content and bone mineral density did not differ significantly in 14% protein groups between 14% casein and 6% casein +80% gelatin. These results suggest that gelatin has differential effects on bone mineral density and body weight in protein undernutrition.

Spatiotemporal changes of fibronectin, tenascin-C, fibulin-1, and fibulin-2 in the skin during the development of chronic contact dermatitis.

In order to elucidate how chronic inflammation affects the organization of the extracellular matrix in the skin, a prolonged allergic contact dermatitis was induced in a mouse by repeated application to the ear of 2,4-dinitrofluorobenzene every 3 d for 66 d. Subsequently, the spatiotemporal changes of fibronectin, tenascin-C, fibulin-1, and fibulin-2 in the skin were examined. In the acute phase of inflammation (day 3-day 12), the amount of fibronectin and tenascin-C increased markedly and were degraded, whereas the amount of fibulin-2 changed slightly. Abundant deposition of tenascin-C was observed in the connective tissue. Fibulin-1 and fibulin-2 distributed as fine fibrils. In contrast, the amounts of fibronectin and tenascin-C decreased and their degradation was suppressed in the chronic phase (day 15-day 66), but the amount of fibulin-2 increased. Tenascin-C was observed mainly at and underneath the epidermal basement membrane. In the subepidermal region, many fibulin-2-positive microfibrils were distributed. The amount and distribution of fibulin-1 did not change markedly in either phase. MMP-like enzymes of 62 kDa, probably activated MMP-2, were upregulated in the chronic phase, whereas components of 92, 85, or 67 kDa were highly induced in the acute phase. These results suggest that chronic inflammation in allergic contact dermatitis is associated with temporal changes in the expression, deposition, and degradation of inducible extracellular matrix components.

Effect of tenascin-C deficiency on chemically induced dermatitis in the mouse.

Tenascin-C is a large extracellular matrix glycoprotein characterized by its spatiotemporal expression during embryogenesis, carcinogenesis, and wound healing. Many in vitro studies on tenascin-C have revealed its multifunctional properties; however, disruption of the tenascin-C gene did not reveal any obvious abnormalities during development, and its function in vivo remains unclear. Here, we investigated whether tenascin-C is involved in inflammatory dermatitis in adults by studying chemically induced dermatitis in tenascin-C knockout mice. An epicutaneous application of a hapten, 2,4-dinitrofluorobenzene, to the ear skin of BALB/CA mice resulted in inflammation and induced the expression of tenascin-C. In congenic tenascin-C knockout mice, the dermatitis occurred more severely than in wild-type mice; infiltration of polymorphonuclear cells in knockout mice persisted longer than in wild-type mice, and the elastosis-like disorganized extracellular matrix was also seen in the ear. These results suggest that tenascin-C plays a role in vivo in inflammatory responses in the skin, and that the genetic background has profound effects on the function of tenascin-C in mouse dermatitis.

Differential expression of tenascin in the skin during hapten-induced dermatitis.

Tenascin is a large extracellular matrix glycoprotein which is found in limited regions of normal adult tissues including the skin. We investigated the induction of tenascin expression in mouse skin during hapten-induced dermatitis. In the dorsal skin, hapten application first induced a transient expression of tenascin in deeper regions of the skin. Its distribution then spread over the whole dermis corresponding to the infiltration of Mac-2-positive macrophages. In the ear, tenascin was consistently found in the subcutaneous tissue on the inner side, but very little was seen on the outer side. Tenascin did appear transiently, however, on both sides under hapten treatment. In the early phase of allergic contact dermatitis, no apparent induction of tenascin expression was observed in the swollen ear. However, there was an abundant tenascin expression on both sides during healing. Tenascin expressed under normal conditions was mostly the 180-kDa isoform, while the 230-kDa isoform was markedly induced during healing of the dermatitis. These results suggest that tenascin, particularly the larger 230-kDa isoform, may play important roles in the pathogenesis and healing of hapten-induced dermatitis.

Site-specific phosphorylation of synapsin I by mitogen-activated protein kinase and Cdk5 and its effects on physiological functions.

Posttranslational modifications of synapsin I, a major phosphoprotein in synaptic terminals, were studied by mass spectrometry. In addition to a well known phosphorylation site by calmodulin-dependent protein kinase II (CaM kinase II), a hitherto unrecognized site (Ser553) was found phosphorylated in vivo. The phosphorylation site is immediately followed by a proline, suggesting that the protein is an in vivo substrate of so-called proline-directed protein kinase(s). To identify the kinase involved, three proline-directed protein kinases expressed highly in the brain, i.e. mitogen-activated protein (MAP) kinase, Cdk5-p23, and glycogen synthase kinase 3beta, were tested for the in vitro phosphorylation of synapsin I. Only MAP kinase and Cdk5-p23 phosphorylated synapsin I stoichiometrically. The phosphorylation sites were determined to be Ser551 and Ser553 with Cdk5-p23, and Ser62, Ser67, and Ser551 with MAP kinase. Upon phosphorylation with MAP kinase, synapsin I showed reduced F-actin bundling activity, while no significant effect on the interaction was observed with the protein phosphorylated with Cdk5-p23. These results raise the possibility that the so-called proline-directed protein kinases together with CaM kinase II and cAMP-dependent protein kinase play an important role in the regulation of synapsin I function.

Mouse uterus peptidylarginine deiminase is expressed in decidual cells during pregnancy.

Peptidylarginine deiminase is localized in the cytosol of the luminal and glandular epithelia of the nonpregnant murine uterus and its expression is regulated by sex hormones [Takahara et al., [1989]: J Biol Chem 264, 13361-13368; Takahara et al. [1992]: J Biol Chem 267,520-525]. Here, we demonstrate that changes occur in the enzyme level in the mouse uterus during pregnancy and parturition. After a rapid decrease in enzymatic activity from day 1 to day 5 of pregnancy, the activity sharply increased during the middle stage of pregnancy (day 8 to day 10) and then gradually decreased during late pregnancy. Expression of the enzyme occurred only in the decidual cells that had differentiated from endometrial stroma cells surrounding the implantation site. The immunochemical properties of the enzyme expressed in the decidualized cells was indistinguishable from those in the uterine epithelia. These results suggest that peptidylarginine deiminase has important roles in decidual cells and not just in the epithelia of the nonpregnant uterus. Moreover, the level of enzyme activity increased slightly just before parturition (day 17), and then decreased during the 12 h period after parturition. The tissue localization of the enzyme expressed around the time of parturition changed from decidua to the luminal and glandular epithelia. Semiquantitative analyses of the enzyme mRNA content in the pregnant uteri showed a remarkable increase from day 7 leading to the onset of the enzyme synthesis in the decidual cells. After reaching the maximal level at day 12, small peaks in the mRNA level were observed at two times during late pregnancy. Since these serial changes in the mRNA level did not correlate with changes in sex hormones, the expression of decidual peptidylarginine deiminase seemed to be controlled by factors other than sex hormones.

Identification of phosphorylation sites on glial fibrillary acidic protein for cdc2 kinase and Ca(2+)-calmodulin-dependent protein kinase II.

We identified the phosphorylation sites of glial fibrillary acidic protein (GFAP) for cdc2 kinase and Ca(2+)-calmodulin (CaM)-dependent protein kinase II. GFAP was phosphorylated to approximately 0.2 mol of phosphate/mol of GFAP by cdc2 kinase, and this phosphorylation did not induce disassembly of the filament structure. On the other hand, GFAP was phosphorylated to approximately 1.9 mol of phosphate/mol of GFAP by Ca(2+)-CaM-dependent protein kinase II, and this phosphorylation did induce disassembly of the filament. Sequential analysis of the purified phosphopeptides revealed that only Ser8 on GFAP was phosphorylated by cdc2 kinase, whereas Ser13, Ser17, Ser34, and Ser389 on GFAP were phosphorylated by Ca(2+)-CaM-dependent protein kinase II.

Evidence for direct binding of intracellularly distributed ganglioside GM2 to isolated vimentin intermediate filaments in normal and Tay-Sachs disease human fibroblasts.

Although some intracellularly distributed glycosphingolipids are reported to be associated with vimentin intermediate filaments or colchicine sensitive cytoskeleton, no direct evidence for such an association has yet been shown. In this report we demonstrated that the intracellularly distributed ganglioside GM2 directly binds to isolated vimentin intermediate filaments in normal and Tay-Sachs disease human fibroblasts. Indirect immunofluorescence microscopy using a GM2-specific monoclonal antibody demonstrated filamentously distributed GM2 in the cytoplasm. A double staining of Tay-Sachs fibroblasts with anti-GM2 and anti-vimentin monoclonal antibodies strongly suggested that the GM2 positive filaments are vimentin intermediate filaments. We then isolated vimentin, in the presence of a detergent and urea, from the normal human skin fibroblasts and murine mastocytoma cells. In a solid phase enzyme-linked immunosorbent assay, the isolated vimentin dose-dependently reacted with both anti-vimentin and anti-GM2 monoclonal antibodies but not with anti-GM3 or anti-GM1 monoclonal antibody. The molar ratio of GM2 to vimentin was approximately 20:1. The lipid fraction extracted from the purified vimentin preparation was immunostained with anti-GM2 on a thin-layer chromatography plate. Furthermore, only one band was detected at the molecular weight of 57 kDa, after electroblotting and simultaneous immunostaining with anti-GM2 and anti-vimentin monoclonal antibodies. These results clearly indicated that ganglioside GM2 directly binds to vimentin.

cdc2 kinase phosphorylation of desmin at three serine/threonine residues in the amino-terminal head domain.

Phosphorylation of desmin filament by cdc2 kinase induced a transition toward the depolymerized state of the filament. Sequence analysis of purified phosphopeptides derived from cdc2 kinase-phosphorylated desmin revealed that Ser-6, Ser-22 and Thr-64 in the N-terminal head domain were the sites phosphorylated.

p13suc1 suppresses the catalytic function of p34cdc2 kinase for intermediate filament proteins, in vitro.

The regulation of p34cdc2 kinase activity controls the entry into and exit from mitosis. Although genetic and biochemical evidence suggested close interactions between cyclins, p13suc1 and p34cdc2 kinase, the roles of p13suc1 on p34cdc2 kinase functions remain unclear. To examine the effects of p13suc1 on p34cdc2 kinase function we developed a simple purification procedure for p34cdc2 kinase, unassociated with p13suc1. The key to the purification procedures we used was buffer containing 0.5 M NaCl and 50% ethylene glycol, as a specific elutant of p34cdc2 kinase from p13suc1-Sepharose. This purified p34cdc2 kinase stoichiometrically phosphorylated vimentin and desmin. Exogenous p13suc1 suppressed the phosphorylation of these filament proteins by the kinase and prevented disassembly, although histone H1 phosphorylation was not affected. Peptide mapping analysis showed a similar extent of inhibition by p13suc1 for all five phosphorylation sites by p34cdc2 kinase of vimentin and desmin, hence these p13suc1-induced inhibitions are probably not site-specific. It thus appears that p13suc1 has a selective effect on the catalytic activity of p34cdc2 kinase for these filament proteins.

Expression of peptidylarginine deiminase in the uterine epithelial cells of mouse is dependent on estrogen.

The effects of the steroid hormones estrogen and progesterone on peptidylarginine deiminase protein-L-arginine iminohydrolase, EC 3.5.3.15) levels in adult ovariectomized mouse uterus were studied. The amount of the enzyme in the uterus was considerably diminished by ovariectomy. When the mice were injected with a variety of estrogenic compounds, 17 beta-estradiol-3-benzoate, which was the most potent stimulator of uterine cell proliferation among the estrogens tested, dramatically elevated the enzyme formation of the uterus in a dose- and time-dependent fashion. Results of immunohistochemistry with the antiserum against mouse peptidylarginine deiminase demonstrated that the induction of the enzyme by the estradiol exclusively occurred at the luminal and glandular epithelia, corresponding with the previous findings in the normal estrous cycle. Furthermore, administration of the estradiol significantly increased the content of mRNA coding for peptidylarginine deiminase in uterus, indicating the evidence of regulation in pretranslation. On the other hand, progesterone alone did not restore the enzyme level of the ovariectomized mouse, but moderated the action of estrogen when given in concert with estrogen. Thus, the expression of peptidylarginine deiminase in luminal and glandular epithelia of mouse uterus is controlled by the amount of the steroid hormones estrogen and progesterone.

Phosphorylation of neurofilament H subunit at the tail domain by CDC2 kinase dissociates the association to microtubules.

We sought the mammalian neurofilament tail domain-specific kinase. Several well known kinases including cAMP-dependent protein kinase, protein kinase C, Ca(2+)-calmodulin-dependent protein kinase II, casein kinase I, and casein kinase II phosphorylated the high (NF-H) and middle molecular mass subunit (NF-M) of bovine neurofilaments, but they did not reduced the electrophoretic mobility of the dephosphorylated form of NF-M and NF-H by phosphorylation nor was the amount of phosphorylation increased by dephosphorylation of NF proteins, indicating that the phosphorylation sites by these kinases are not major in vivo phosphorylation sites at the tail domain. In contrast, cdc2 kinase phosphorylated specifically the dephosphorylated form of NF-H. 4 mol of phosphates were incorporated per mol of NF-H and this phosphorylation returned the electrophoretic mobility of the dephosphorylated form of NF-H to the position of the isolated, fully phosphorylated form of NF-H. Furthermore, the phosphorylation by cdc2 kinase dissociated the binding of dephosphorylated NF-H to microtubules. Phosphorylation sites were located at the carboxyl-terminal tail domain. The KSPXK motif, but not KSPXX, in the repetitive sequence was suggested to be the phosphorylation site by using synthetic peptides.

A monoclonal antibody to the phosphorylated form of glial fibrillary acidic protein: application to a non-radioactive method for measuring protein kinase activities.

Monoclonal antibody YC10 showed specificity for the phosphorylated form of human, bovine and porcine glial fibrillary acidic proteins (GFAPs) and negligible reactivity towards the dephosphorylated form of the GFAPs. Analysis of species specificity and of the epitope, determined using synthetic phosphopeptides, indicated that this antibody recognized the local phosphorylation-site sequence Thr-phosphoSer-Ala-Ala-Arg-Arg (residues 7-12 of GFAP). Making use of this antibody we developed a non-radioactive method to measure protein kinase activities. After incubation of a protein kinase with non-radioactive ATP in ninety-six wells coated with the synthetic peptide Arg-Arg-Arg-Val-Thr-Ser-Ala-Ala-Arg-Arg-Ser-Cys (residues 3-13 of GFAP), the phosphorylated product was detected by using this mouse antibody and peroxidase-labeled goat anti-mouse IgG. This method proved to be equally as sensitive as the radioactive method for the measurement of protein kinase activities and was less affected by concentrations of ATP present in the reaction mixture.

Peptidylarginine deiminase of the mouse. Distribution, properties, and immunocytochemical localization.

Peptidylarginine deiminase (protein-L-arginine iminohydrolase, EC 3.5.3.15) is widely distributed in various organs of the mouse. Activity in salivary glands, pancreas, and uterus is higher than that in the other organs. In submandibular gland and uterus, sex- and estrous cycle-related differences were observed, respectively. The activity in the submandibular gland from females was approximately four times higher than that in the male. In the uterus, the activity increased in proportion to the hyperplasia of the tissues. Peptidylarginine deiminase from the murine skeletal muscle resembles the enzyme obtained from other animal species with respect to enzymatic and chemical properties. Double immunodiffusion tests and immunoblotting analyses showed that the enzymes present in each murine tissue have the same molecular weight (81,000) and are immunologically indistinguishable. Immunohistochemical analyses of salivary glands and pancreas revealed an intense staining only of the exocrine cells. In uterus, the staining was restricted to the luminal and glandular epithelia of endometrium; the intensity of the staining changed during the course of the estrous cycle. Furthermore, immunoelectron microscopy showed that the enzyme is distributed diffusely in the cytoplasm of the exocrine cell. These observations indicate the general importance of peptidylarginine deiminase, presumably in a cytoplasmic secretory process of the exocrine cells.