Oxidative Stress Inhibits Endotoxin Tolerance and May Affect Periodontitis.

Periodontal disease is caused by dysbiosis of the dental biofilm and the host inflammatory response. Various pathogenic factors, such as proteases and lipopolysaccharides (LPSs) produced by bacteria, are involved in disease progression. Endotoxin tolerance is a function of myeloid cells, which sustain inflammation and promote tissue regeneration upon prolonged stimulation by endotoxins such as LPS. The role of endotoxin tolerance is gaining attention in various chronic inflammatory diseases, but its role in periodontal disease remains elusive. Oxidative stress, one of the major risk factors for periodontal disease, promotes disease progression through various mechanisms, of which only some are known. The effect of oxidative stress on endotoxin tolerance has not yet been studied, and we postulated that endotoxin tolerance regulation may be an additional mechanism through which oxidative stress influences periodontal disease. This study aimed to reveal the effect of oxidative stress on endotoxin tolerance and that of endotoxin tolerance on periodontitis progression. The effect of oxidative stress on endotoxin tolerance was analyzed in vitro using peritoneal macrophages of mice and hydrogen peroxide (H2O2). The results showed that oxidative stress inhibits endotoxin tolerance induced by Porphyromonas gingivalis LPS in macrophages, at least partially, by downregulating LPS-elicited negative regulators of Toll-like receptor (TLR) signaling. A novel oxidative stress mouse model was established using SMP30KO mice incapable of ascorbate biosynthesis. Using this model, we revealed that oxidative stress impairs endotoxin tolerance potential in macrophages in vivo. Furthermore, gingival expression of endotoxin tolerance-related genes and TLR signaling negative regulators was decreased, and symptoms of ligature-induced periodontitis were aggravated in the oxidative stress mouse model. Our findings suggest that oxidative stress may contribute to periodontitis progression through endotoxin tolerance inhibition.

Utility of the Berlin Initiative Study-1 equation for the prediction of serum vancomycin concentration in elderly patients aged 75 years and older.

Accurate assessment of renal function is essential for determining serum vancomycin (VCM) concentration. Creatinine clearance (Ccr)-calculated using the Cockcroft and Gault (CG) equation-can be used to evaluate renal function for determining VCM dosage. However, Ccr-based evaluation may not be an accurate representation of the renal function in the elderly. Herein, we examine the effectiveness of estimated glomerular filtration rate (eGFR) calculated using the Berlin Initiative Study-1 (BIS1) equation, for predicting the serum VCM concentration. Herein, we retrospectively analyzed patients (aged ≥ 75 years) who had received VCM. Serum VCM concentration was predicted based on Ccr and eGFR. eGFR was calculated using the Japanese equation for eGFR (eGFRJAP), Modification of Diet in Renal Disease (MDRD) equation (eGFRMDRD), chronic kidney disease epidemiology collaboration (CKD-EPI) equation (eGFRCKD-EPI), and BIS1 equation (eGFRBIS1). The predicted serum VCM concentration was compared with the measured values. Prediction bias, accuracy, and precision were evaluated by calculating the mean prediction error (ME), mean absolute prediction error (MAE), and root mean squared prediction error (RMSE). Our results showed that the ME between the measured and the predicted values calculated using Ccr and each eGFR was the largest and smallest when calculated based on Ccr and eGFRMDRD, respectively. MAE and RMSE were the largest and smallest when calculated based on Ccr and eGFRBIS1, respectively. A significant difference was observed in the MAE associated with eGFRJAP, eGFRMDRD, and eGFRCKD-EPI compared to that associated with eGFRBIS1. In conclusion, our results suggest that the BIS1 equation might be useful for determining the VCM dosage in the elderly.

Citrullinated inter-alpha-trypsin inhibitor heavy chain 4 in arthritic joints and its potential effect in the neutrophil migration.

The citrullinated inter-alpha-trypsin inhibitor heavy chain 4 (cit-ITIH4) was identified as its blood level was associated with the arthritis score in peptide glucose-6-phosphate-isomerase-induced arthritis (pGIA) mice and the disease activity in patients with rheumatoid arthritis (RA). This study aimed to clarify its citrullination pathway and function as related to neutrophils. In pGIA-afflicted joints, ITIH4 and cit-ITIH4 levels were examined by immunohistochemistry (IHC), immunoprecipitation (IP) and Western blotting (WB), while peptidylarginine deiminase (PAD) expression was measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), IHC and immunofluorescent methods. The pGIA mice received anti-lymphocyte antigen 6 complex locus G6D (Ly6G) antibodies to deplete neutrophils and the expression of cit-ITIH4 was investigated by WB. The amounts of ITIH4 and cit-ITIH4 in synovial fluid (SF) from RA and osteoarthritis (OA) patients were examined by I.P. and W.B. Recombinant ITIH4 and cit-ITIH4 were incubated with sera from healthy volunteers before its chemotactic ability and C5a level were evaluated using Boyden's chamber assay and enzyme-linked immunosorbent assay (ELISA). During peak arthritic phase, ITIH4 and cit-ITIH4 were increased in joints while PAD4 was over-expressed, especially in the infiltrating neutrophils of pGIA mice. Levels of cit-ITIH4 in plasma and joints significantly decreased upon neutrophil depletion. ITIH4 was specifically citrullinated in SF from RA patients compared with OA patients. Native ITIH4 inhibited neutrophilic migration and decreased C5a levels, while cit-ITIH4 increased its migration and C5a levels significantly. Cit-ITIH4 is generated mainly in inflamed joints by neutrophils via PAD4. Citrullination of ITIH4 may change its function to up-regulate neutrophilic migration by activating the complement cascade, exacerbating arthritis.

Sesame lignans suppress age-related disorders of the kidney in mice.

OBJECTIVE: Sesamin is a functional ingredient in sesame (Sesamum indicum) seeds and has many physiological effects. This study investigated whether sesame lignans, sesamin and episesamin (1:1), can suppress age-related disorders of the kidney. MATERIALS AND METHODS: Twenty-month-old mice were divided into three groups, and each group received a regular diet (O-C), diet containing sesame lignans (O-SE), and diet containing sesame lignans and α-tocopherol (VE; O-SE+VE), respectively, for 5 months. Six-month-old young mice (Y-C) were compared to the older mice. RESULTS: Renal lipofuscin deposition was increased in the O-C group compared to that in the Y-C group and its deposition with aging was significantly decreased in both O-SE and O-SE+VE groups. Plasma blood urea nitrogen levels in the O-C group increased compared to those in the Y-C group; however, those in both O-SE and O-SE+VE groups did not differ from those in the Y-C group. The number of podocytes in the O-C group decreased compared to that in the Y-C group and this effect was attenuated in the O-SE and O-SE+VE groups. The effect was strongest in the O-SE+VE group. Histological examinations showed that glomerular hypertrophy accompanied by mesangial hyperplasia and renal tubular degeneration was less severe in the O-SE and O-SE+VE groups than in the O-C group. Moreover, age-related increases in the mRNA expression of NADPH oxidase- and inflammation-related genes, including p67phox, p40phox, TNFα, and IL-6, in the kidney were suppressed in the O-SE and O-SE+VE groups. CONCLUSIONS: Sesame lignans might be useful to suppress age-related kidney disorders, and these effects could be enhanced with VE.

Vimentin citrullination probed by a novel monoclonal antibody serves as a specific indicator for reactive astrocytes in neurodegeneration.

AIMS: Vimentin citrullination, the calcium (Ca2+ )-dependent peptidylarginine deiminase (PAD)-mediated conversion of an arginine residue of vimentin to a citrulline residue, has emerged as a pathophysiological outcome of autoimmune diseases and neurodegeneration. However, the roles, functions, and expression of citrullinated vimentin have not yet been elucidated because available antibodies are limited. METHODS: We developed mouse monoclonal IgG1 and IgM specific for vimentin citrullinated at position R440 or R450 and applied Western blotting, immunohistochemistry, and immunofluorescent staining to investigate the pathogenesis of prion diseases in animal models, in patients with prion diseases, and in vitro. RESULTS: Vimentin was found to be highly citrullinated at R440 and R450, and these citrullinated forms were mainly expressed in reactive astrocytes in the brain tissues of scrapie-infected mice. Full-length and cleaved forms of citrullinated vimentin were found in the cerebral cortices of sporadic Creutzfeldt-Jakob disease (sCJD) patients. The distribution of citrullinated vimentin was mainly confirmed in vimentin-, GFAP-, and YKL-40-positive reactive astrocytes. Biochemically, citrullination promoted resistance to the caspase-3- and caspase-9-mediated fragmentation of vimentin. Additionally, citrullination led to increased cytoplasmic and integral membrane/organelle vimentin enrichment, which indicated changes in the intrinsic solubility and distribution of vimentin. CONCLUSIONS: Our observations suggest that citrullinated vimentin acts as a specific indicator for the reactive state of astrocytes under abnormal neurological conditions. In addition, these novel antibodies will be helpful for studying the role of citrullinated vimentin in the pathogenesis of human disorders.

Bone Degeneration and Its Recovery in SMP30/GNL-Knockout Mice.

Senescence marker protein-30 (SMP30) decreases androgen-independently with aging and is a lactone-hydrolyzing enzyme gluconolactonase (GNL) that is involved in vitamin C biosynthesis. In the present study, bone properties of SMP30/GNL knockout (KO) mice with deficiency in vitamin C synthesis were investigated to reveal the effects of SMP30/GNL and exogenous vitamin C supplementation on bone formation. Mineral content (BMC) and mineral density (BMD) of the mandible and femur of SMP30/GNL KO and wild-type mice at 2 and 3 months of age with or without vitamin C supplementation were measured by dual-energy X-ray absorptiometry. Body and bone weight of both age groups decreased and became significantly lower than those of wild-type mice. The bones of SMP30/GNL KO mice were rough and porous, with BMC and BMD significantly below wild-type. Oral supplementation with vitamin C eliminated differences in body weight, bone weight, BMC, and BMD between SMP30/GNL KO and wild-type mice at each age. These results indicate that bone degeneration in SMP30/GNL KO mice was caused by lack of vitamin C, and that this mouse strain is an appropriate model for bone metabolism in humans, which have no ability to synthesize vitamin C.

Susceptibility quantitative trait loci for pathogenic leucocytosis in SCG/Kj mice, a spontaneously occurring crescentic glomerulonephritis and vasculitis model.

The spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj) mouse, a model of human crescentic glomerulonephritis (CrGN) and systemic vasculitis, is characterized by the production of myeloperoxidase-specific anti-neutrophil cytoplasmic autoantibody (MPO-ANCA) and marked leucocytosis. This study was performed to identify the specific populations of leucocytes associated with CrGN and susceptibility loci for pathogenic leucocytosis. Four hundred and twenty female (C57BL/6 × SCG/Kj) F2 intercross mice were subjected to serial flow cytometry examination of the peripheral blood (PB). Kidney granulocytes and monocytes were examined histopathologically. Linkage analyses were performed with 109 polymorphic microsatellite markers. Correlation studies revealed that increase of the granulocytes, F4/80(+) cells, CD3(+) CD4(-) CD8(-) T cells and dendritic cells (DCs) in peripheral blood (PB) were associated significantly with glomerulonephritis, crescent formation and vasculitis. In kidney sections, F4/80(low) cells were observed in crescent, while F4/80(high) cells were around the Bowman's capsules and in the interstitium. Numbers of F4/80(+) cells in crescents correlated significantly with F4/80(+) cell numbers in PB, but not with numbers of F4/80(+) cells in the interstitium. Genome-wide quantitative trait locus (QTL) mapping revealed three SCG/Kj-derived non-Fas QTLs for leucocytosis, two on chromosome 1 and one on chromosome 17. QTLs on chromosome 1 affected DCs, granulocytes and F4/80(+) cells, but QTL on chromosome 17 affected DCs and granulocytes. We found CrGN-associated leucocytes and susceptibility QTLs with their positional candidate genes. F4/80(+) cells in crescents are considered as recruited inflammatory macrophages. The results provide information for leucocytes to be targeted and genetic elements in CrGN and vasculitis.

Volcanic sulfur dioxide and acute respiratory symptoms on Miyakejima island.

OBJECTIVES: Following a volcanic eruption in 2000, high concentrations of ambient sulfur dioxide (SO2) are still observed on Miyakejima, Japan despite the reversal 2 years ago of the ban on residents living on the island. This study examines the association between current levels of volcanic SO2 and the incidence of acute subjective symptoms in volunteers on Miyakejima. METHODS: The authors conducted a follow-up study on 611 healthy volunteers, on a person-hour basis (28 413 person-hours), who visited the island to provide support to residents from February to July 2005. Adverse health symptoms were measured by self-administered diary and exposure was approximated using monitoring data across 14 sites. Associations between incidence rates and increasing SO2 levels (reference (the lowest), very low, low, middle and high) were examined using Poisson regression. RESULTS: Hourly incidence of cough, scratchy throat, sore throat and breathlessness showed clear exposure-response relationships with SO2 concentrations. There were statistically significant risks of those symptoms at relatively low SO2 levels. Thus, rate ratios in the 0.6-2.0 ppm exposure band (vs <0.01 ppm) were: for cough, 3.4 (95% CI 1.8 to 6.6) in men and 9.8 (3.9 to 24.9) in women; for sore throat, 3.2 (1.7 to 6.2) in men and 5.8 (2.0 to 16.5) in women; and for breathlessness, 10.5 (4.2 to 26.6) in men and 18.5 (4.6 to 74.3) in women. Little evidence of SO2 effects on sputum and nasal discharge/congestion was observed in this study. Eye and skin irritations showed inconsistent results between hourly maximal and hourly mean SO2 concentrations. CONCLUSION: The authors observed strong evidence of an exposure-response relationship between volcanic SO2 and subjective acute respiratory symptoms among a healthy population on Miyakejima. The results are consistent with reports that females and non-smokers are more sensitive to irritant gas than males and smokers, respectively.

Accelerated tubular cell senescence in SMP30 knockout mice.

An experimental model with accelerated but not drastic renal senescence seemed useful to recognize the mechanisms of how kidney function deteriorates with age. Senescence marker protein-30 (SMP30), whose expression decreased with age and was sex-independent, is mainly expressed in hepatocytes and proximal tubular cells. Therefore, we established a SMP30 deficient strain of mice with a C57BL/6 background by gene targeting to investigate whether this molecule is involved in renal tubular cell senescence. Male SMP30 knockout (SMP30Y/-) mice and male wild-type (SMPY/+) mice (n=5) aged 12 months were examined histologically. Their tubular epithelia showed the deposition of lipofuscin and the presence of senescence-associated beta-galactosidase (SA-beta-GAL). However, no tubular cells were atrophic. In electron microscopy, SMP30-KO mice showed markedly enlarged lysosomes containing an electron dense substance. These are convincing hallmarks of senescence. We recognized the early manifestation of senescence hallmarks in SMP30-KO mice at 12 months old. Thus, this model represents the first report of a mouse strain that manifests accelerated ordinal senescence in a kidney after gene manipulation.

Immunohistochemical localization of senescence marker protein-30 (SMP30) in the submandibular gland and ultrastructural changes of the granular duct cells in SMP30 knockout mice.

Senescence Marker Protein-30 (SMP30) is a calcium-regulating protein that decreases in an androgen-independent manner as aging occurs. An enzyme-labeled antibody technique has demonstrated that SMP30 localized to the ducts (granular, intercalated, and striated ducts) of mouse submandibular glands. Immunoelectronmicroscopy demonstrated that the granular duct cells were strongly positive for SMP30, but that pillar cells in the granular duct were negative for the protein. In SMP30-knockout (KO) mice, the granular ducts were smaller in diameter. Swelling of mitochondria in the granular duct cells was observed; however, this phenomenon was not observed in the pillar cells. After administration of alpha-isoproterenol, a beta-adrenergic stimulant, a large numbers of small secretory granules were present in the granular duct cells and an expansion of the rough endoplasmic reticulum in SMP30-wild type (WT) mice; in contrast, little change was observed in SMP30-KO mice. These results suggest that SMP30 may be closely related to a signal transduction pathway in the granular duct cells of submandibular glands.

Suppressing effects of bisphenol A on the secretory function of ovine anterior pituitary cells.

We investigated the action of bisphenol A (BPA) on cellular GH release and content, cell number, GHmRNA expression, and concentrations of cellular cyclic AMP ([cAMP]c) and calcium ion ([Ca2+]c) in primary cultured ovine anterior pituitary cells. The following results were found: (1) BPA as well as nonylphenol (NP) at 10(-6) to 10(-3) M significantly and concentration-dependently suppressed basal and GHRH-stimulated GH release, and the cellular GH content, (2) BPA suppressed the cell number in a time- and concentration-dependent manner, (3) 10(-4)M BPA suppressed GHmRNA expression to 68% of control (BPA-free), and abolished GHRH (10(-8) M)-induced increases in [cAMP]c and [Ca2+]c. From these findings we conclude that BPA possesses a suppressing action on GH synthesis and release, and this suppressing action is probably related to impairment of cellular signal transduction systems in ovine anterior pituitary cells.

Regulatory effects of senescence marker protein 30 on the proliferation of hepatocytes.

Senescence marker protein 30 (SMP 30) is preferentially expressed in the liver. One of its remarkable functions is the protection of cells against various injuries by enhancement of membrane calcium-pump activity. We analyzed the role of SMP 30 in hepatocyte proliferation. SMP 30 expression was decreased initially, then increased along with hepatic regeneration, after carbon tetrachloride (CCl4) administration. SMP 30 expression was decreased in the necrotic phase and then gradually increased. Its increase was slightly delayed just after the mitotic phase. These results lead us to speculate that mitoses of hepatic cells induce enhanced SMP 30 expression. In contrast, administration of lead nitrate (LN) as a hepatic mitogen induced a more stable increase of SMP 30 expression. To estimate the effect of SMP 30 on cell proliferation, we evaluated hepatic mitosis in wild-type and SMP 30-deficient knockout (KO) mice after CCl4 administration. We found an increase in mitotic numbers in hepatocytes of KO mice. This result suggests that SMP 30 has a suppressive effect on cell proliferation. Suppressive activity of SMP 30 cDNA was shown in cultured hepatoblastic cells. Our results suggest that SMP 30 performs a regulatory function in liver regeneration.

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.

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.

Neuronal changes in the arcuate and hypoglossal nuclei of brain stem induced by head injury.

In head injury, assessing the damage not only to the cerebrum and the cerebellum but also to the brain stem is very important. In this paper, we report neuronal changes of the arcuate nucleus (ARC) and the hypoglossal nucleus (HN) in the brain stem. We investigated these changes immunohistochemically with antibodies against microtubule-associated protein 2 (MAP2), muscarinic acetylcholine receptor (mAChR), c-fos gene product (c-Fos), and the 72 kD heat-shock protein (HSP70). We measured the percentage of immunopositive neurons among the total neurons of the ARC and the HN. The investigation of neuronal changes in relation to the type of head injury showed different results. In cases of tonsillar herniation, immunoreactivity to MAP2 and mAChR in the ARC was significantly lower than in the HN (p < 0.01). Moreover, MAP2, HSP70 and c-Fos reactivities in the ARC were significantly lower than in other types of head injuries (p < 0.01). In the HN, diffuse axonal injury produced slightly higher immunoreactivity to mAChR and c-Fos (p < 0.1). Our observations indicate that immunohistochemical examination of brain stem nuclei can provide useful information for estimating damage to the brain stem.

Studies on specificity of peptidylarginine deiminase reactions using an immunochemical probe that recognizes an enzymatically deiminated partial sequence of mouse keratin K1.

Citrulline residues are detected in keratins and filaggrin in the cornified layers of mammalian epidermis. Such citrulline residues are formed by the enzymatic deimination of arginine residues by peptidylarginine deiminases (EC 3.5.3.15). Major deiminated keratins are derived from keratin K1. Two arginine residues identified as preferred deimination sites in mouse K1 are located in its V subdomains. To develop an immunochemical probe which recognizes the deiminated peptide sequence specifically, we enzymatically deiminated an undecapeptide corresponding to the deiminated peptide sequence identified in the V2 subdomain for immunizing rabbits. An IgG fraction obtained from the antiserum was affinity-purified using an immobilized peptide column. The affinity-purified IgG showed high specificity towards partially degraded keratin K1 obtained from the cornified layer of 3-day-old mouse epidermis. It also yielded intense signals of unidentified minor components localized in the cornified layers of late embryonic and early postnatal mouse epidermis. Comparative studies using different types of the enzymes suggested that peptidylarginine deiminase type I acted on the arginine residue in the V2 subdomain of keratin K1 more readily than peptidylarginine deiminase type II. The data are discussed in conjunction with possible factors influencing the specificity of the enzyme reaction.

Molecular characterization of peptidylarginine deiminase in HL-60 cells induced by retinoic acid and 1alpha,25-dihydroxyvitamin D(3).

Three types of peptidylarginine deiminase (PAD), which converts a protein arginine residue to a citrulline residue, are widely distributed in animal tissues. Little is known about PAD of hemopoietic cells. We found that PAD activity in human myeloid leukemia HL-60 cells was induced with the granulocyte-inducing agents retinoic acid and dimethyl sulfoxide and with the monocyte-inducing agent 1alpha,25-dihydroxyvitamin D(3). We cloned and characterized a PAD cDNA from retinoic acid-induced cells. The cDNA was 2,238 base pairs long and encoded a 663-amino acid polypeptide. The HL-60 PAD had 50-55% amino acid sequence identities with the three known enzymes and 73% identity with the recently cloned keratinocyte PAD. The recombinant enzyme differs in kinetic properties from the known enzymes. Immunoblotting and Northern blotting with an antiserum against the enzyme and the cDNA, respectively, showed that a protein of approximately 67 kDa increased concomitantly with increase of mRNA of approximately 2.6 kilobases during granulocyte differentiation. During monocyte differentiation the same mRNA and protein increased as in granulocyte differentiation. Neither the enzyme activity nor the protein was found in macrophage-induced cells. These results suggested that expression of the PAD gene is tightly linked to myeloid differentiation.

Molecular cloning of two novel types of peptidylarginine deiminase cDNAs from retinoic acid-treated culture of a newborn rat keratinocyte cell line.

Peptidylarginine deiminases (PADs) are a group of enzymes which convert protein arginine residues to citrulline residues. Using rat muscle PAD cDNA as a probe, we obtained two novel cDNAs, PAD-R11 and PAD-R4, from immortalized rat keratinocytes treated with all-trans retinoic acid. Comparison of the deduced amino acid sequences with those of muscle and hair follicle enzymes showed high conservation in the C-terminal region. Recombinant proteins encoded by both PAD-R11 and PAD-R4 showed the enzyme activities. That of PAD-R11 showed a characteristic feature of the enzyme found in the epidermis.

Aging reduces the numbers of hepatocytes synthesizing DNA in response to EGF and epinephrine.

Primary cultures of hepatocytes were prepared from young (6 month) and old (24 month) Wistar rats and exposed to epinephrine or epidermal growth factor. Incorporation of [3H]thymidine into DNA was determined both radiochemically and autoradiographically. The numbers of responding cells and degree of response per cell were determined and the results confirmed by FACScan analysis. Such analyses clearly demonstrate a reduced number of hepatocytes capable of responding to the above stimuli in cultures obtained from old rats. Thus, changes in numbers of responding cells may be an important mechanism involved in reduced responsiveness of the aged liver to agents which stimulate DNA synthesis and cell division.

All-trans retinoic acid increases peptidylarginine deiminases in a newborn rat keratinocyte cell line.

We studied the expression of peptidylarginine deiminases (EC 3.5.3.15) in an immortalized newborn rat keratinocyte cell line. No measurable enzyme activities were noted in either growing or confluent cultures. The enzyme activity was increased by all-trans retinoic acid in dose- and time-dependent manners. The enzyme activity was resolved into two peaks by anion exchange chromatography. The minor peak resembled enzyme preparations obtained from the epidermis in earlier studies. The major peak was indistinguishable from rat muscle peptidylarginine deiminase in the chromatographic and Western blotting profiles. Northern blot hybridization showed a major band in retinoic acid-treated cells migrating slightly behind muscle peptidylarginine deiminase mRNA.