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A 65-year-old woman presented with right elbow pain after a fall. Imaging showed an avulsion fracture of the olecranon. The patient subsequently underwent surgery using the suture bridge technique with anchors. However, loosening was observed intraoperatively. Therefore, a McLaughlin cerclage with an artificial ligament was added, resulting in a rigid fixation. Bone union was achieved at 4 months postoperatively. At 18 months postoperatively, no limitation was observed in the range of motion of the elbow joint; the disabilities of the arm, shoulder and hand score was 0. McLaughlin cerclage with an artificial ligament provided additional fixation, demonstrating greater strength compared with suture anchors and minimizing the risk of cut-through in the osteoporotic bone. This approach offers a promising alternative for such cases by combining firm fixation with a reduced risk of complications, particularly in older patients with osteoporosis., Competing Interests: None declared., (Published by Oxford University Press and JSCR Publishing Ltd. © The Author(s) 2024.)

High-speed atomic force microscopy (HS-AFM) is a unique approach that allows direct real-time visualization of biological macromolecules in action under near-physiological conditions, without any chemical labeling. Typically, the temporal resolution is sub-100 ms, and the spatial resolution is 2-3 nm in the lateral direction and ∼0.1 nm in the vertical direction. A wide range of biomolecular systems and their dynamic processes have been studied by HS-AFM, providing deep mechanistic insights into how biomolecules function. However, the level of mechanistic detail gleaned from an HS-AFM experiment critically depends on the spatiotemporal resolution of the system. In this review article, we explain the principle of HS-AFM and describe how the resolution is determined. We also discuss recent attempts to improve the resolution of HS-AFM to further extend the observable range of biological phenomena., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Japanese Society of Microscopy.)

A case of infant botulism of unknown origin, not involved in honey consumption, occurred in Osaka, Japan in 2020. A Clostridium botulinum type B strain named Osaka2020 was isolated from a stool sample of the patient. To clarify the epidemiology of the case, we performed whole-genome sequencing (WGS) of the isolate and compared it with strains from other sources. WGS analysis revealed that isolate Osaka2020 was classified into ST133 of a new sequence type, B5 subtype, and its toxin gene was encoded in a ∼274 kb plasmid. This plasmid was closely related to the pCLJ plasmid from strain 657Ba in the USA, reported to be conjugatively transferable to other strains. Moreover, isolate Osaka2020 also possesses another smaller plasmid that was common with some type A(B) infant botulism isolates in Japan. The phylogenetic tree from whole-genome SNP analysis showed that isolate Osaka2020 was the most closely related to a type B infant botulism isolate that occurred in Japan 10 years ago. Although no epidemiological connection among the two cases was confirmed, there is possibility that the cases are attributed to common causes such as some environmental substance., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Background: Under non-antimicrobial selective pressure, antimicrobial-resistant bacteria do not easily become dominant in the microbiota. Furthermore, their low levels prevent detection by isolation, resulting in an underestimation of the prevalence of antimicrobial-resistant bacteria., Objectives: We evaluated the infiltration of antimicrobial-resistant bacteria and their related β-lactamase genes among healthy people in non-clinical settings., Methods: Cephalosporin- and fluoroquinolone-resistant Escherichia coli and bla genes were quantified in 217 faecal samples from healthy people in non-clinical settings in Japan. E. coli colonies grown on deoxycholate hydrogen sulphide-lactose (DHL) agar, with and without antimicrobials (cefotaxime and ciprofloxacin), were quantified, and E. coli isolates were analysed for their susceptibility to antimicrobials and the presence of bla genes. DNA extracted from faecal samples was used to quantify bla genes using quantitative PCR (qPCR)., Results: The isolation rates of cefotaxime- and ciprofloxacin-resistant E. coli were 6.9% and 12.4%, respectively, using agars without antimicrobials, and 12.0% and 24.4%, respectively, using agars with antimicrobials. For samples from which cefotaxime- and ciprofloxacin-resistant E. coli were isolated only using agars with antimicrobials, the ratios of cfu on DHL agars with and without antimicrobials were below -2 log. E. coli harbouring bla genes were isolated from 35.0% of the faecal samples using agars, and bla genes were detected in 65.0% of faecal DNA samples using qPCR., Conclusions: Among people carrying cefotaxime- and ciprofloxacin-resistant E. coli in non-clinical settings, cefotaxime- and ciprofloxacin-resistant E. coli were not dominant in half of the subjects. These individuals may play a role as reservoirs of antimicrobial-resistant bacteria., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

The prognosis of patients with relapsed osteosarcoma is extremely poor and the optimal treatment remains to be identified. Here, we retrospectively analysed the clinical outcomes of nine patients with relapsed osteosarcoma treated with temozolomide/etoposide. Of the two patients who received temozolomide/etoposide as palliative therapy for unresectable tumours, one remained alive with stable disease for >4 years. The remaining seven patients received temozolomide/etoposide as adjuvant therapy following resection of relapsed metastatic disease; of these, one was free from disease for 41 months. Potentially beneficial effects were observed in two of three O6-methylguanine-DNA methyltransferase protein-negative patients, whereas all five O6-methylguanine-DNA methyltransferase-positive patients experienced subsequent relapse. None of the patients experienced severe adverse effects requiring hospitalization. Temozolomide/etoposide is a feasible candidate as salvage therapy for relapsed osteosarcoma. Further studies are needed to verify the utility of O6-methylguanine-DNA methyltransferase protein expression as a biomarker for predicting the response to this treatment., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Background: Toxoplasmosis, a parasitic disease caused by Toxoplasma gondii, is an important cause of miscarriage or adverse fetal effects, including neurological and ocular manifestations in humans. Current anti-Toxoplasma drugs have limited efficacy against toxoplasmosis and also have severe side effects. Therefore, novel efficacious drugs are urgently needed. Here, we identified metacytofilin (MCF) from a fungal Metarhizium species as a potential anti-Toxoplasma compound., Methods: Anti-Toxoplasma activities of MCF and its derivatives were evaluated in vitro and in vivo using nonpregnant and pregnant mice. To understand the mode of action of MCF, the RNA expression of host and parasite genes was investigated by RNAseq., Results: In vitro, MCF inhibited the viability of intracellular and extracellular T. gondii. Administering MCF intraperitoneally or orally to mice after infection with T. gondii tachyzoites increased mouse survival compared with the untreated animals. Remarkably, oral administration of MCF to pregnant mice prevented vertical transmission of the parasite. Interestingly, RNA sequencing of T. gondii-infected cells treated with MCF showed that MCF inhibited DNA replication and enhanced RNA degradation in the parasites., Conclusions: With its potent anti-T. gondii activity, MCF is a strong candidate for future drug development against toxoplasmosis., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The vitamin D receptor (VDR) is a nuclear receptor for the active form of vitamin D, 1α,25-dihydroxyvitamin D 3 , and regulates various physiologic processes, such as bone and calcium metabolism, cellular proliferation and differentiation, and immunity. VDR is highly expressed in the intestine, kidney, bone, and macrophages, but is expressed at a low level in the liver. The liver is a major metabolic organ and also acts as an immune gateway for dietary nutrients and xenobiotics. In this study, we investigated the function of VDR in hepatic immune cells, such as Kupffer cells/macrophages, utilizing VDR knockout (KO) mice. We showed that VDR is functionally expressed in hepatic mononuclear cells, specifically resident Kupffer cells. We examined the role of VDR in acute hepatitis induced by concanavalin A (Con-A) and found that Con-A-induced hepatitis is attenuated in VDR-KO mice compared to wild-type (WT) mice. Con-A-induced hepatitis is known to be mediated by NKT cell activation, cytokine production, and reactive oxygen species (ROS) production in Kupffer cells/macrophages. However, the proportions of Kupffer cells/macrophages and the NKT cell activation were similar in the liver of WT and VDR-KO mice and inflammatory cytokine gene expression was increased in VDR-KO mice. On the other hand, plasma and hepatic ROS levels were decreased in the liver of VDR-KO mice compared to WT mice. The phagocytic activity of resident Kupffer cells and hepatic neutrophils were also decreased in VDR-KO mice. Therefore, VDR is necessary for Con-A-induced acute hepatitis and plays an important role in hepatic immune cell functions., (©2019 Society for Leukocyte Biology.)

Liver X receptor (LXR) α expression is mainly localized to metabolic tissues, such as the liver, whereas LXRβ is ubiquitously expressed. LXRα is activated by oxysterols and plays an important role in the regulation of lipid metabolism in metabolic tissues. In macrophages, LXRs stimulate reverse cholesterol transport and regulate immune responses. Although a high-cholesterol diet induces severe steatohepatitis in LXRα-knockout (KO) mice, the underlying mechanisms linking lipid metabolism and immune responses remain largely unknown. In this study, we investigated the role of LXRα in the pathogenesis of steatohepatitis by assessing the effects of a high-fat and high-cholesterol diet (HFCD) on hepatic immune cell proportion and function as well as lipid metabolism in wild-type (WT) and LXRα-KO mice. HFCD feeding induced severe steatohepatitis in LXRα-KO mice compared with WT mice. These mice had higher cholesterol levels in the plasma and the liver and dysregulated expression of LXR target and proinflammatory genes in both whole liver samples and isolated hepatic mononuclear cells. Flow cytometry showed an increase in CD68+CD11b+ Kupffer cells/macrophages and a decrease in invariant natural killer T cells in the liver of HFCD-fed LXRα-KO mice. These mice were more susceptible to lipopolysaccharide-induced liver injury and resistant to inflammatory responses against α-galactosylceramide or concanavalin-A treatment. The findings provide evidence for activation of bone marrow-derived Kupffer cells/macrophages and dysfunction of invariant natural killer T cells in LXRα-KO mouse liver. These findings indicate that LXRα regulates hepatic immune function along with lipid metabolism and protects against the pathogenesis of nonalcoholic steatohepatitis.

Roxadustat (FG-4592, Rox) is a stabilizer for hypoxia-inducible transcription factors (HIFs), which induce production of the erythroid growth factor erythropoietin, and has been listed by the World Anti-Doping Agency as a prohibited substance for athletes since 2011. Although the detection technologies for Rox and its glucuronide-conjugated metabolite (Rox-Gluc) have been developed exploiting triple quadrupole mass spectrometry (MS/MS), the production of metabolites from Rox in the human body remains to be clarified. Here, we established a protocol for the detection of unknown metabolites in plasma and urine samples from Rox-doping mice by global metabolomics using an ultra high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS). We identified methylated Rox (Rox-Methyl), a novel metabolite, and Rox-Gluc in mouse urine by principal component analysis and orthogonal partial least squares discriminant analysis based on detected features by UHPLC-QTOF/MS analysis. The estimated pharmacokinetic parameters of Rox-Methyl and Rox-Gluc in mouse plasma showed similar profiles to that of Rox and both compounds showed similar biological activities. Of note, Rox-Methyl showed shorter half-life than Rox-Gluc in vivo, implying the easy escape from anti-doping screen. These results demonstrate that the global metabolomics method introduced in this study will contribute to the identification and detection of HIF-analog doping., (© The Author(s) 2018. Published by Oxford University Press on behalf of The Journal of Biochemistry.)

Immobilization and sedimentation of liposomes (lipid vesicles) are used in liposome-protein binding assays, facilitated by avidin/streptavidin/NeutrAvidin and biotinylated phospholipid-containing liposomes. Here, we examined the effects of three spacers [six-carbon (X), polyethylene glycol (PEG) 180 (molecular weight 180) and PEG2000 (molecular weight 2,000)] between biotin and the phospholipid headgroup on the immobilization and sedimentation of small unilamellar liposomes/vesicles (SUVs). PEG180 and PEG2000 showed more efficient immobilization of biotinylated SUVs on NeutrAvidin-coated plates than X, but X and PEG180 showed more efficient sedimentation of biotinylated SUVs upon NeutrAvidin addition than PEG2000. Thus, the most appropriate spacers differed between immobilization and sedimentation. A spacer for biotinylated SUVs must be selected according to the particular liposome-protein binding assays examined., (© The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Serum is an important component of cell culture media. The present study demonstrates contamination of intracellular protein extract by bovine serum albumin from the culture media and illustrates how this contamination can cause the misinterpretation of western blot results. Preliminary experiments can prevent the misinterpretation of some experimental results, and optimization of the washing process may enable specific protein detection.

Conditions that influence the selective development or recruitment of connective tissue-type and mucosal-type mast cells (MCs) are not well understood. Here, we report that cynomolgus monkey embryonic stem (ES) cells cocultured with the murine aorta-gonad-mesonephros-derived stromal cell line AGM-S1 differentiated into cobblestone (CS)-like cells by day 10-15. When replated onto fresh AGM-S1 with the addition of stem cell factor, interleukin-6, and Flt3 ligand, these CS-like cells displayed robust growth and generated almost 100% tryptase/chymase double-positive MCs within 3 weeks. At all time points, the percentage of tryptase-positive cells did not exceed that of chymase-positive cells. These ES-derived MCs were CD45+/Kit+/CD31+/CD203c+/HLA-DR- and coexpressed a high-affinity IgE receptor on their surface, which was upregulated after IgE exposure. Electron microscopy showed that they contained many electron dense granules. Moreover, ES-derived MCs responded to stimulation by via IgE and substance P by releasing histamine. These results indicate that ES-derived MCs have the phenotype of functionally mature connective tissue-type MCs. The rapid maturation of ES-derived MCs suggests a unique embryonic pathway in primates for early development of connective tissue-type MCs, which may be independent from the developmental pathway of mucosal-type MCs.

Multipotent germline stem (mGS) cells have been established from neonatal mouse testes. Here, we compared mGS, embryonic stem (ES), and embryonic germ (EG) cells with regard to their ability to differentiate into mesodermal cells, namely, cardiomyocytes and endothelial cells. The in situ morphological appearances of undifferentiated mGS, ES, and EG cells were similar, and 4 days after being induced to differentiate, approximately 30%-40% of each cell type differentiated into Flk1(+) cells. The sorted Flk1(+) cells differentiated efficiently into cardiomyocytes and endothelial cells. By day 10 after differentiation induction, the three cell types generated equal number of endothelial colonies. However, by day 13 after differentiation induction, the Flk1(+) mGS cells generated more contractile colonies than did the Flk1(+) ES cells, whereas the Flk1(+) EG cells generated equivalent numbers as the Flk1(+) mGS cells. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of differentiation markers such as Rex1, FGF-5, GATA-4, Brachyury, and Flk1 revealed that mGS cells expressed these markers more slowly during days 0-4 after differentiation induction than did ES cells, but that this mGS cell pattern was similar to that of the EG cells. RT-PCR analysis also revealed that the three differentiation cell types expressed various cardiac markers. Moreover, immunohistochemical analysis revealed that the contractile colonies derived from Flk1(+) mGS cells express mature cardiac cell-specific markers. In conclusion, mGS cells are phenotypically similar to ES and EG cells and have a similar potential to differentiate into cardiomyocytes and endothelial cells. Disclosure of potential conflicts of interest is found at the end of this article.

Few studies on the long-term culture of postnatal mouse hepatic stem/progenitor cells have been reported. We successfully adapted a serum-free culture system that we employed previously to expand fetal mouse hepatic stem/progenitor cells and maintained them in culture over long periods. The expanded postnatal cells contained immature alpha-fetoprotein-positive cells along with hepatocytic and cholangiocytic lineage-committed cells. These cells expressed CD49f but not CD45, CD34, Thy-1, c-kit, CD31, or flk-1, and oncostatin M induced their differentiation. This heterogeneous population contained side population (SP) cells, which express the ATP-binding cassette transporter ABCG2, and sca-1+ cells. As mice aged, the frequency of SP and sca-1+ cells decreased along with the ability of cultured cells to expand. Approximately 20%-40% of the SP cells expressed sca-1, but only a few sca-1+ cells were also SP cells. Analysis of colonies derived from single SP or sca-1+ cells revealed that, although both cells had dual differentiation potential and self-renewal ability, SP cells formed colonies more efficiently and gave rise to SP and sca-1+ cells, whereas sca-1+ cells generated only sca-1+ progeny. Thus, SP cells are more characteristic of stem cells than are sca-1+ cells. In regenerating livers, ABCG2+ cells and sca-1+ cells were detected around or in the portal area (the putative hepatic stem cell niche). The expanded cells share many features of fetal hepatic stem/progenitor cells or oval cells and may be useful in determining the mechanisms whereby hepatic stem cells self-renew and differentiate.

The temporal pattern of embryonic, fetal, and adult globin expression in the alpha (zeta --> alpha) and beta (epsilon --> gamma and gamma --> beta) clusters were quantitatively analyzed at the transcriptional and translational levels in erythrocytes induced from primate embryonic stem cells in vitro. When vascular endothelial growth factor receptor-2(high) CD34(+) cells were harvested and reseeded onto OP9 stromal cells, two-wave erythropoiesis occurred sequentially. Immunostaining and real-time reverse transcription-polymerase chain reaction analyses of floating mature erythrocytes revealed that globin switches occurred in parallel with the erythropoietic transition. Colony-forming assays showed replacement of primitive clonogenic progenitor cells with definitive cells during culturing. A decline in embryonic zeta- and epsilon-globin expression at the translational level occurred in individual definitive erythroid progenitors. Expression of beta-globin in individual definitive erythroid progenitors was upregulated in the presence of OP9 stromal cells. Thus, this system reproduces early hematopoietic development in vitro and can serve as a model for analyzing the mechanisms of the globin switch in humans.

We identified intermediate-stage progenitor cells that have the potential to differentiate into hematopoietic and endothelial lineages from nonhuman primate embryonic stem (ES) cells. Sequential fluorescence-activated cell sorting and immunostaining analyses showed that when ES cells were cultured in an OP9 coculture system, both lineages developed after the emergence of two hemoangiogenic progenitor-bearing cell fractions, namely, vascular endothelial growth factor receptor (VEGFR)-2(high) CD34(-) and VEGFR-2(high) CD34(+) cells. Exogenous vascular endothelial growth factor increased the proportion of VEGFR-2(high) cells, particularly that of VEGFR-2(high) CD34(+) cells, in a dose-dependent manner. Although either population of VEGFR-2(high) cells could differentiate into primitive and definitive hematopoietic cells (HCs), as well as endothelial cells (ECs), the VEGFR-2(high) CD34(+) cells had greater hemoangiogenic potential. Both lineages developed from VEGFR-2(high) CD34(-)or VEGFR-2(high) CD34(+) precursor at the single-cell level, which strongly supports the existence of hemangioblasts in these cell fractions. Thus, this culture system allows differentiation into the HC and EC lineages to be defined by surface markers. These observations should facilitate further studies both on early developmental processes and on regeneration therapies in human.

Because T7 RNA polymerase has a strong preference for particular sequences to initiate transcription, some RNAs having pyrimidine-rich sequences at their 5'-end (yeast tRNA(Tyr), for example) are hardly transcribed by this enzyme. To circumvent this inconvenience, we have developed an efficient method for in vitro preparation of such tRNAs. The RNA of interest is first transcribed as a precursor form that has purine-rich extra sequences at its 5'-end, then processed with RNase P to generate the objective tRNAs. By using this protocol, we were able to prepare easily and efficiently yeast tRNA(Tyr) transcript and its mutants harboring base substitutions within the anticodon loop and/or acceptor stem regions. Aminoacylation analyses of these tRNA transcripts with yeast tyrosyl-tRNA synthetase revealed that the replacement of G34 by C34 (mutation to amber suppressor) severely impaired the aminoacylation, whereas the replacement of the U4:G69 wobble base-pair in the acceptor stem region by C4:G69 normal Watson-Crick type base-pair improved it.

Recent studies have indicated that bone marrow cells can regenerate damaged muscles and that they can adopt phenotypes of other cells by cell fusion. Our direct visualization system gave evidence of massive muscle regeneration by green fluorescent protein (GFP)-labeled CD45+c-Kit+Sca-1+Lin- cells (KSL cells), and we investigated the role of KSL cells in muscle regeneration after transplantation with or without lethal irradiation. In the early phase, GFP signals were clearly observed in all the muscles of only irradiated mice. Transverse cryostat sections showed GFP+myosin+ muscle fibers, along with numerous GFP+ hematopoietic cells in damaged muscle. These phenomena were temporary, and GFP signals had dramatically reduced 30 days after transplantation. After 6 months, GFP+ fibers could hardly be detected, but GFP+c-Met+ mononuclear cells were located beneath the basal lamina where satellite cells usually exist in both conditioned mice. Immunostaining of isolated single fibers revealed GFP+PAX7+, GFP+MyoD+, and GFP+Myf5+ satellite-like cells on the fibers. Single-fiber cultures from these mice showed proliferation of GFP+ fibers. These results indicate two different roles of KSL cells: one leading to regeneration of damaged muscles in the early phase and the other to conversion into satellite cells in the late phase.

During 1996, 1997, and 1999, studies were conducted in cotton, sugar beets, alfalfa, yardlong bean, and peanut fields to compare insect catches in CC traps equipped with different trap base colors. The studies were conducted in southwestern United States, China, and India. The nine colors, white, rum, red, yellow, lime green, spring green, woodland green (dark green), true blue, and black, varied in spectral reflectance in the visible (400-700 nm) and near-infrared (700-1050 nm) portions of spectrum. Lime green, yellow, and spring green were the three most attractive trap base colors for silverleaf whitefly, Bemisia argentifolii Bellows & Perring, and leafhopper, Empoasca spp. adults. The three trap base colors were moderately high in the green, yellow, and orange spectral regions (490-600 nm), resembling the spectral reflectance curve of the abaxial (underleaf) surfaces of green cotton leaves. True blue and white were the most attractive trap base colors for western flower thrips, Frankliniella occidentalis (Pergande), adults. The true blue and white trap bases were moderately high in the blue spectral region (400-480 nm).

Fifty-eight patients (mean age 18.4 years) who had received splint therapy for internal derangement of the temporomandibular joint (TMJ) were examined retrospectively to investigate the efficacy of occlusal reconstruction by orthodontic treatment. The subjects were divided into three groups: 18 patients (mean age 18.6 years) who underwent orthodontic treatment combined with the use of splints (ST group); 27 patients (mean age 18.2 years) who underwent orthodontic treatment without the use of splints (NST group); and 13 patients (mean age 17.9 years) who received only splint therapy for temporomandibular joint disorders (TMD; control group). TMJ sound, pain on movement and restriction of mandibular movement were examined at the initial examination (T1), at the end of the splint therapy for TMD or beginning of orthodontic treatment (T2), at the end of orthodontic treatment (T3), and at recall or 1 year after orthodontic treatment (T4). The following results were found. (1) The percentage of patients with no joint sound at T2 was 20-30 per cent. The percentage of such patients in both the ST and NST groups increased to over 50 per cent at T3, but slightly decreased to 39-50 per cent at T4. There were no significant inter-group differences at any time point. (2) The number of patients who had no pain on movement at T2 was 60-80 per cent. The percentage of such patients in both the ST and NST groups increased to over 90 per cent at T3, but then slightly decreased to 80 per cent at T4. There were no significant inter-group differences at any time point. (3) None of the patients showed restriction of movement of the TMJ at T2 or T4. One patient in the ST group was found to have restriction at T3. There were no significant inter-group differences at any time point. (4) The most frequent type of malocclusion in both ST and NST groups was anterior open bite. These results suggest that TMD symptoms that have been eliminated by splint therapy are not likely to recur due to subsequent orthodontic treatment, but it cannot be concluded that orthodontic treatment itself had a positive effect on TMD symptoms. The results also indicate that there is a relationship between anterior open bite and TMD.

Four acidic amino acid residues, Asp97, Asp101, Glu118, and Glu202, were located in the cleft from the X-ray crystallographic analysis of FI-CMCase, endo-1,4-beta-glucanase (EC: 3.2.1.4) of Aspergillus aculeatus No. F-50. To identify the catalytic residues of the FI-CMCase, these residues were mutated to Glu or Ser from Asp97 and Asp101, and to Asp or Ser from Glu118 and Glu202 by site-directed mutagenesis, and totally 8 single mutant enzymes expressed in Escherichia coli were prepared: D97E, D97S, D101E, D101S, E118D, E118S, E202D, and E202S. Mutant enzymes E118S and E202S were not shown to have any detectable activity. Kinetic parameters of other mutant enzymes were measured after purification. The Km of mutant enzymes were not much different from that of wild type FI-CMCase, while the Vmax of mutant enzymes D97E, D97S, D101E, D101S, E118D, and D202E were much decreased to 1/50, 1/20, 1/4000, 1/2000, 1/800, and 1/1600 of the wild type FI-CMCase, respectively. From these results we concluded that Glu118 and Glu202 were most probable candidates for a catalytic pair of acidic amino acids in FI-CMCase.

We have previously reported the effect of hydrocortisone (HC) on the adenylate cyclase system of pig epidermis. HC had no effect on the basal level of cyclic AMP but there was an increased adrenaline-induced cyclic AMP accumulation when epidermal slices were incubated for more than 6 h with HC. This incubation did not alter the responsiveness to histamine. We are interested in the effects on epidermal cells of HC, which may act through the adenylate cyclase system. It is well documented that adrenaline, histamine and adenosine stimulate adenylate cyclase and cause an accumulation of cyclic AMP in pig epidermis. An increased cyclic AMP results in the inhibition of mitosis and epidermal outgrowth. Employing the rate of epidermal outgrowth and mitotic index as indicators of cyclic AMP effect, we studied the effect of HC on the adenylate cyclase system. The pretreatment with HC (100 microM) magnified the inhibitory effect of adrenaline on epidermal outgrowth and mitosis, whereas the inhibitory effect of histamine or adenosine was not affected by HC. It is suggested that HC induced an increased responsiveness of adenylate cyclase to adrenaline, resulting in a magnified inhibitory effect of adrenaline on epidermal outgrowth and mitosis.