Reflection on the proposed changes to dose quantities-an industrial perspective.

In 2021, the ICRP initiated the revision of the general recommendations of the system of radiation protection, and part of it will focus on dose quantities. The recently published ICRP Publication 147 and ICRU Report 95 have described the extent of the proposed modifications and paved the way for the strategy to be adopted. These revisions would seek to simplify, improve the accuracy and extend the field of use of dose quantities. While the Radiological Protection Working Group of the World Nuclear Association recognises the notable improvement in the estimation of the protection quantities and the usefulness of such changes for the medical and research sector, the benefits of the proposed new system seem very limited for the nuclear industry and industries involving naturally occurring radioactive materials. The complexity associated with changing a long-standing and robust system and the risk incurred by the human factor seem unjustified, bearing in mind the likely cost.

Pharmacological inhibition of mTORC1 but not mTORC2 protects against human disc cellular apoptosis, senescence, and extracellular matrix catabolism through Akt and autophagy induction.

OBJECTIVE: The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that integrates nutrients to execute cell growth. We hypothesized that mTOR is influential in the intervertebral disc-largest avascular, low-nutrient organ. Our objective was to identify the optimal mTOR inhibitor for treating human degenerative disc disease. DESIGN: mTOR complex 1 (mTORC1) regulates p70/ribosomal S6 kinase (p70/S6K), negatively regulates autophagy, and is controlled by Akt. Akt is controlled by phosphatidylinositol 3-kinase (PI3K) and mTOR complex 2 (mTORC2). mTORC1 inhibitors-rapamycin, temsirolimus, everolimus, and curcumin, mTORC1&mTORC2 inhibitor-INK-128, PI3K&mTOR inhibitor-NVP-BEZ235, and Akt inhibitor-MK-2206-were applied to human disc nucleus pulposus (NP) cells. mTOR signaling, autophagy, apoptosis, senescence, and matrix metabolism were evaluated. RESULTS: mTORC1 inhibitors decreased p70/S6K but increased Akt phosphorylation, promoted autophagy with light chain 3 (LC3)-II increases and p62/sequestosome 1 (p62/SQSTM1) decreases, and suppressed pro-inflammatory interleukin-1 beta (IL-1ß)-induced apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity (versus rapamycin, 95% confidence interval (CI) -0.431 to -0.194; temsirolimus, 95% CI -0.529 to -0.292; everolimus, 95% CI -0.477 to -0.241; curcumin, 95% CI -0.248 to -0.011) and poly (ADP-ribose) polymerase (PARP) and caspase-9 cleavage, senescent senescence-associated beta-galactosidase (SA-ß-gal) positivity (versus rapamycin, 95% CI -0.437 to -0.230; temsirolimus, 95% CI -0.534 to -0.327; everolimus, 95% CI -0.485 to -0.278; curcumin, 95% CI -0.210 to -0.003) and p16/INK4A expression, and catabolic matrix metalloproteinase (MMP) release and activation. Meanwhile, dual mTOR inhibitors decreased p70/S6K and Akt phosphorylation without enhanced autophagy and suppressed apoptosis, senescence, and matrix catabolism. MK-2206 counteracted protective effects of temsirolimus. Additional disc-tissue analysis found relevance of mTOR signaling to degeneration grades. CONCLUSION: mTORC1 inhibitors-notably temsirolimus with an improved water solubility-but not dual mTOR inhibitors protect against inflammation-induced apoptosis, senescence, and matrix catabolism in human disc cells, which depends on Akt and autophagy induction.

Shochu slop is an excellent medium for Escherichia coli K-12.

We found that shochu slop, the residue generated during the production of distilled shochu liquor, which must be treated as industrial waste, can be used as an excellent medium for Escherichia coli culture. LB medium is generally used in laboratories for culturing E. coli. However, it is not the optimal medium for E. coli culture because the bacterial cells cannot grow to very high densities in LB medium. On the other hand, E. coli can grow to higher densities in Terrific broth and this medium is used when researchers want to grow E. coli to high density or to obtain a protein with high yield. In this study, we removed solid matter from shochu slop, adjusted the pH of the mixture to 7 and subsequently used the slop for E. coli culture. The ability of shochu slop to support E. coli growth was compared with those of LB Miller medium and Terrific broth. The results indicate that sweet potato shochu slop as culture medium for E. coli is comparable to Terrific broth and much better than LB Miller medium in terms of supporting cell proliferation, and plasmid and enzyme production. SIGNIFICANCE AND IMPACT OF THE STUDY: Shochu manufacturers incur a cost to dispose shochu slop, which is recognized as food manufactural residues. Escherichia coli has been used in laboratories and in industry. However, culture media used in the laboratories are expensive and those used in industry are expensive because of their large scale. We found that sweet potato shochu slop is an excellent culture medium for E. coli. This finding is not only useful for laboratories and industry, but also beneficial to the effective utilization of this renewable resource to create a sustainable society.

Selective interference of mTORC1/RAPTOR protects against human disc cellular apoptosis, senescence, and extracellular matrix catabolism with Akt and autophagy induction.

OBJECTIVE: The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that integrates nutrients to execute cell growth and protein synthesis. We hypothesized that mTOR is essential for the intervertebral disc, the largest avascular, low-nutrient organ. Our objective was to elucidate roles of mTOR signaling in human disc cells. DESIGN: The mTOR exists in two complexes: mTORC1 containing the regulatory-associated protein of mTOR (RAPTOR) and mTORC2 containing the rapamycin-insensitive companion of mTOR (RICTOR). To analyze their functions in human disc nucleus pulposus cells, RNA interference (RNAi) of mTOR targeting mTORC1 and mTORC2, RAPTOR targeting mTORC1, or RICTOR targeting mTORC2 or rapamycin, a pharmacological mTORC1 inhibitor, was applied. First, mTOR signaling including Akt, p70/ribosomal S6 kinase (p70/S6K), and autophagy were assessed. Then, apoptosis, senescence, and matrix metabolism were evaluated under pro-inflammatory interleukin-1 beta (IL-1ß) stimulation. RESULTS: Western blotting showed significant decreases in specific proteins by each RNAi (all P < 0.0001). In mTOR signaling, RNAi of mTOR and RICTOR decreased p70/S6K and Akt phosphorylation, whereas RAPTOR RNAi decreased p70/S6K but increased Akt phosphorylation. All RNAi treatments increased light chain 3 (LC3)-II and decreased p62/sequestosome 1 (p62/SQSTM1), indicating enhanced autophagy. In apoptosis, IL-1ß-induced terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells and poly (ADP-ribose) polymerase (PARP) and caspase-9 cleavage decreased by RAPTOR RNAi. In senescence, IL-1ß-induced senescence-associated beta-galactosidase (SA-ß-gal)-positive cells and p16/INK4A expression also decreased by RAPTOR RNAi. In matrix metabolism, RAPTOR RNAi reduced IL-1ß-induced catabolic matrix metalloproteinase (MMP) release and activation and up-regulated anabolic gene expression. These findings were all consistent with rapamycin administration. Additional disc-tissue analysis detected expression and phosphorylation of mTOR-signaling molecules in varying ages. CONCLUSION: Selective interference of mTORC1/RAPTOR protects against inflammation-induced apoptosis, senescence, and matrix catabolism possibly through Akt and autophagy induction in human disc cells.

Para-psychobiotic Lactobacillus gasseri CP2305 ameliorates stress-related symptoms and sleep quality.

AIMS: To confirm the stress-relieving effects of heat-inactivated, enteric-colonizing Lactobacillus gasseri CP2305 (paraprobiotic CP2305) in medical students taking a cadaver dissection course. METHODS AND RESULTS: Healthy students (21 males and 11 females) took paraprobiotic CP2305 daily for 5 weeks during a cadaver dissection course. The General Health Questionnaire and the Pittsburgh Sleep Quality Index were employed to assess stress-related somatic symptoms and sleep quality respectively. The aggravation of stress-associated somatic symptoms was observed in female students (P = 0·029). Sleep quality was improved in the paraprobiotic CP2305 group (P = 0·038), particularly in men (P = 0·004). Among men, paraprobiotic CP2305 shortened sleep latency (P = 0·035) and increased sleep duration (P = 0·048). Diarrhoea-like symptoms were also effectively controlled with CP2305 (P = 0·005) in men. Thus, we observed sex-related differences in the effects of paraprobiotic CP2305. In addition, CP2305 affected the growth of faecal Bacteroides vulgatus and Dorea longicatena, which are involved in intestinal inflammation. CONCLUSIONS: CP2305 is a potential paraprobiotic that regulates stress responses, and its beneficial effects may depend on specific cell component(s). SIGNIFICANCE AND IMPACT OF THE STUDY: This study characterizes the effects of a stress-relieving para-psychobiotic in humans.

The effects of administration of the Lactobacillus gasseri strain CP2305 on quality of life, clinical symptoms and changes in gene expression in patients with irritable bowel syndrome.

AIMS: To clarify the effects of Lactobacillus gasseri CP2305 (CP2305) on quality of life and clinical symptoms and its functional mechanisms in patients with irritable bowel syndrome (IBS). METHODS AND RESULTS: After the patients were administered CP2305 daily for 4 weeks, the IBS-severity index score was significantly improved compared with that of the placebo group, and this improvement was accompanied by a reduction in health-related worry and changes in intestinal microbiota. The gene expression profiling of the peripheral blood leucocytes showed that CP2305 treatment significantly up-regulated genes related to eukaryotic initiation factor 2 (EIF2) signalling. Eighty-two genes were down-regulated in IBS patients compared with healthy controls. The expression of 23 of these genes exhibited a CP2305-dependent increase associated with an improvement in IBS severity. The majority of the restored genes were related to EIF2 signalling. CONCLUSIONS: CP2305 administration is a potential candidate therapeutic option for patients with IBS. SIGNIFICANCE AND IMPACT OF THE STUDY: Although probiotics have been proposed to benefit IBS patients, objective clinical evidence and elucidation of the functional mechanism remain insufficient. Our study demonstrated that CP2305 administration beneficially influences IBS patients in both subjective and objective evaluations, and gene expression profiling provided insights into the functional mechanism.

Expression of hOvol2 in the XY body of human spermatocytes.

Male infertility is common at infertile clinics, and 10-20% of infertile males are azoospermic. Non-obstructive azoospermia is a complex multifactorial disease, and the process of spermatogenesis remains largely unknown. Ovol1 and Ovol2, a family of zinc finger transcription factors, are expressed in spermatocytes at the pachytene stage and are suggested to be critical regulators of pachytene progression in male germ cells. In this study, we examined the expression of human Ovol2 (hOvol2) in the seminiferous tubes of patients subjected to testicular sperm extraction. We first cloned hOvol1 and hOvol2 from the testis of one of the patients and found no alteration in these nucleotide sequences of this patient. While hOvol1 and hOvol2 were detected by RT-PCR in the testis of patients capable of spermatogenesis, they were not detected in those with Sertoli cell-only syndrome. We recently succeeded in preparing anti-Ovol2 antibody by immunising rats with recombinant mouse Ovol2 (mOvol2) and confirmed the specificity and cross-reactivity of this antibody with hOvol2 in cells transfected with hOvol1 or hOvol2 cDNA. hOvol2 expression was restricted to the XY body of spermatocytes at the pachytene stage. This study demonstrates that hOvol2 is expressed in germ cells and may be involved in spermatogenesis.

Identification of a novel carotenoid, 2'-isopentenylsaproxanthin, by Jejuia pallidilutea strain 11shimoA1 and its increased production under alkaline condition.

Carotenoids are a class of naturally occurring pigment, carrying out important biological functions in photosynthesis and involved in environmental responses including nutrition in organisms. Saproxanthin and myxol, which have monocyclic carotenoids with a γ-carotene skeleton, have been reported to show a stronger antioxidant activity than those with ß-carotene and zeaxanthin. In this research, a yellow-orange bacterium of strain 11shimoA1 (JCM19538) was isolated from a seaweed collected at Nabeta Bay (Shizuoka, Japan). The 16S rRNA gene sequence of strain 11shimoA1 revealed more than 99.99 % similarity with those of Jejuia pallidilutea strains in the family Flavobacteriaceae. Strain 11shimoA1 synthesized two types of carotenoids. One of them was (3R, 3'R)-zeaxanthin with dicyclic structure and another was identified as (3R, 2'S)-2'-isopentenylsaproxanthin, a novel monocyclic carotenoid with pentenyl residue at C-2' position of saproxanthin, using FAB-MS, (1)H NMR, and CD analyses. Culturing strain 11shimoA1 in an alkaline medium at pH 9.2 resulted in a markedly increased in production of 2'-isopentenylsaproxanthin per dry cell weight, but a decreased in zeaxanthin production as compared to their respective production levels in medium with pH 7.0. These carotenoids are likely to play some roles in the adaptation of the bacterium to the environmental conditions.

Histone deacetylase inhibitors suppress mechanical stress-induced expression of RUNX-2 and ADAMTS-5 through the inhibition of the MAPK signaling pathway in cultured human chondrocytes.

OBJECTIVE: To investigate the inhibitory effects and the regulatory mechanisms of histone deacetylase (HDAC) inhibitors on mechanical stress-induced gene expression of runt-related transcription factor (RUNX)-2 and adisintegrin and metalloproteinase with thrombospondin motif (ADAMTS)-5 in human chondrocytes. METHODS: Human chondrocytes were seeded in stretch chambers at a concentration of 5 × 10(4)cells/chamber. Cells were pre-incubated with or without HDAC inhibitors (MS-275 or trichostatin A; TSA) for 12h, followed by uniaxial cyclic tensile strain (CTS) (0.5Hz, 10% elongation), which was applied for 30 min using the ST-140-10 system (STREX, Osaka, Japan). Total RNA was extracted and the expression of RUNX-2, ADAMTS-5, matrix metalloproteinase (MMP)-3, and MMP-13 at the mRNA and protein levels were examined by real-time polymerase chain reaction (PCR) and immunocytochemistry, respectively. The activation of diverse mitogen-activated protein kinase (MAPK) pathways with or without HDAC inhibitors during CTS was examined by western blotting. RESULTS: HDAC inhibitors (TSA: 10 nM, MS-275: 100 nM) suppressed CTS-induced expression of RUNX-2, ADAMTS-5, and MMP-3 at both the mRNA and protein levels within 1h. CTS-induced activation of p38 MAPK (p38), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) MAPKs was downregulated by both HDAC inhibitors. CONCLUSION: The CTS-induced expression of RUNX-2 and ADAMTS-5 was suppressed by HDAC inhibitors via the inhibition of the MAPK pathway activation in human chondrocytes. The results of the current study suggested a novel therapeutic role for HDAC inhibitors against degenerative joint disease such as osteoarthritis.

Identification of the gene responsible for gelatinous drop-like corneal dystrophy.

Gelatinous drop-like corneal dystrophy (GDLD; OMIM 204870) is an autosomal recessive disorder characterized by severe corneal amyloidosis leading to blindness, with an incidence of 1 in 300,000 in Japan. Our previous genetic linkage study localized the gene responsible to a 2.6-cM interval on chromosome 1p. Clinical manifestations, which appear in the first decade of life, include blurred vision, photophobia and foreign-body sensation. By the third decade, raised, yellowish-grey, gelatinous masses severely impair visual acuity, and lamellar keratoplasty is required for most patients. Here we report DNA sequencing, cDNA cloning and mutational analyses of four deleterious mutations (Q118X, 632delA, Q207X and S170X) in M1S1 (formerly TROP2 and GA733-1), encoding a gastrointestinal tumour-associated antigen. The Q118X mutation was the most common alteration in the GDLD patients examined, accounting for 33 of 40 (82.5%) disease alleles in our panel of families. Protein expression analysis revealed aggregation of the mutated, truncated protein in the perinuclear region, whereas the normal protein was distributed diffusely in the cytoplasm with a homogenous or fine granular pattern. Our successful identification of the gene that is defective in GDLD should facilitate genetic diagnosis and potentially treatment of the disease, and enhance general understanding of the mechanisms of amyloidosis.

Macular corneal dystrophy type I and type II are caused by distinct mutations in a new sulphotransferase gene.

Macular corneal dystrophy (MCD; MIM 217800) is an autosomal recessive hereditary disease in which progressive punctate opacities in the cornea result in bilateral loss of vision, eventually necessitating corneal transplantation. MCD is classified into two subtypes, type I and type II, defined by the respective absence and presence of sulphated keratan sulphate in the patient serum, although both types have clinically indistinguishable phenotypes. The gene responsible for MCD type I has been mapped to chromosome 16q22, and that responsible for MCD type II may involve the same locus. Here we identify a new carbohydrate sulphotransferase gene (CHST6), encoding an enzyme designated corneal N-acetylglucosamine-6-sulphotransferase (C-GlcNAc6ST), within the critical region of MCD type I. In MCD type I, we identified several mutations that may lead to inactivation of C-GlcNAc6ST within the coding region of CHST6. In MCD type II, we found large deletions and/or replacements caused by homologous recombination in the upstream region of CHST6. In situ hybridization analysis did not detect CHST6 transcripts in corneal epithelium in an MCD type II patient, suggesting that the mutations found in type II lead to loss of cornea-specific expression of CHST6.

Akt phosphorylation in human chondrocytes is regulated by p53R2 in response to mechanical stress.

OBJECTIVE: The p53 tumor-suppressor protein p53R2 is activated in response to various stressors that act on cell signaling. When DNA is damaged, phosphorylation of p53 at its Ser 15 residue induces p53R2 production. The role of p53R2 in chondrocytes remains poorly understood. In this study, we evaluated in chondrocytes, p53R2 expression and its regulation in response to mechanical stress. Furthermore, we investigated the function of p53R2 in relation to mechanotransduction. METHODS: Osteoarthritis (OA) cartilage obtained from total knee replacements and normal cartilage obtained from femoral neck fractures was used to measure p53R2 expression by using immunohistochemistry, western blotting, and real-time polymerase chain reaction (PCR). The OA chondrocytes were subjected to a high magnitude of cyclical tensile strain by using an FX-2000 Flexercell system. Next, sulfated glycosaminoglycan (sGAG) production was quantified in these cells. Protein expression of p53R2, and phosphorylation of Akt, p38MAPK, ERK1/2, and JNK was also detected using western blotting. Moreover, Akt phosphorylation was detected after transfecting the cells with p53R2-specific small interfering RNA (siRNA). RESULTS: Expression of p53R2 was significantly increased in OA chondrocytes and in chondrocytes after applying 5% tensile strain to the cells. However, Akt phosphorylation was down-regulated in OA chondrocytes after the strain, and was up-regulated after transfection of p53R2. sGAG protein as well as collagen type II and aggrecan mRNA was increased following transfection of p53R2-specific siRNA after 5% tensile strain. CONCLUSIONS: p53R2 could regulate matrix synthesis via Akt phosphorylation during chondrocyte mechanotransduction. Down-regulation of p53R2 may be a new therapeutic approach in OA therapy.

Salvage lymphadenectomy for cervical lymph node recurrence after esophagectomy for squamous cell carcinoma of the thoracic esophagus.

Prognosis of patients with recurrent esophageal cancer is usually unsatisfactory. We have successfully treated five patients with cervical node recurrence after esophagectomy with multimodal treatment including salvage lymphadenectomy. In order to clarify the efficacy of salvage surgery for cervical node recurrence, we have reviewed the clinical course and prognosis of these patients. From August 2004 to December 2007, 30 patients with 33 recurrent sites were treated in the Department of Surgery, Iizuka Hospital. Among these patients, there were five patients with recurrence limited within the cervical nodes. Salvage cervical lymphadenectomy was performed for all five patients. Curative resection was achieved in four patients and reduction surgery followed by planned chemoradiotherapy was performed in another patient. All stations including the suspicious node were dissected and a partial sternotomy was added for one patient whose recurrent tumor was located in the right recurrent nerve node. There was no mortality and one minor complication (subcutaneous hemorrhage) was observed. Median duration of hospital stay was 7 days. Adjuvant chemotherapy was performed for all patients. Median follow-up period was 54 months and all patients are alive without relapse of the disease. Salvage cervical lymphadenectomy is a safe and effective treatment for patients with cervical node recurrence after esophagectomy.

Regulation of mechanical stress-induced MMP-13 and ADAMTS-5 expression by RUNX-2 transcriptional factor in SW1353 chondrocyte-like cells.

OBJECTIVE: To investigate the mechanism of mechanical stress-induced expression and regulation of aggrecanases and examine the role of runt-related transcription factor 2 (RUNX-2) in chondrocyte-like cells. METHODS: SW1353 cells were seeded onto stretch chambers at a concentration of 5×104 cells/chamber, and a uni-axial cyclic tensile strain (CTS) (0.5 Hz, 10% stretch) was applied for 30 min. Total RNA was extracted, reverse transcribed, and analyzed by polymerase chain reaction (PCR) and real-time PCR. RUNX-2 overexpression and small interfering RNA (siRNA) targeting RUNX-2 were used to investigate the role of RUNX-2 in CTS-induced gene expression. The involvement of diverse mitogen-activated protein kinase (MAPK) pathways in the activation of RUNX-2, MMP-13 and ADAMTS-5 during CTS was examined by Western blotting. RESULTS: CTS induced expression of RUNX-2, MMP-13, ADAMTS-4, -5, and -9. Overexpression of RUNX-2 up-regulated expression of MMP-13 and ADAMTS-5, whereas RUNX-2 siRNA resulted in significant down-regulation of mechanically-induced MMP-13 and ADAMTS-5 expression. CTS induced activation of p38 MAPK, and CTS induction of RUNX-2, MMP-13 and ADAMTS-5 mRNA was down-regulated by the selective p38 MAPK inhibitor SB203580 but not by the p44/42 MAPK inhibitor U0126, or the JNK MAPK inhibitor JNK inhibitor II. CONCLUSIONS: RUNX-2 might have a role as a key downstream mediator of p38's ability to regulate mechanical stress-induced MMP-13 and ADAMTS-5 expression.

Optical imaging of mouse articular cartilage using the glycosaminoglycans binding property of fluorescent-labeled octaarginine.

OBJECTIVE: The aim of the current study was to examine the cartilage-specific binding property of polyarginine peptides (R4, 8, 12, and 16) and specifically to test octaarginine peptides for the optical imaging of articular cartilage in experimentally induced arthritis in mice. METHODS: Four rhodamine-labeled polyarginine peptides each with a different-length arginine chain (R4, 8, 12, or 16) were injected into the knee joints of C57BL/6J mice (n=20). The joints were excised 1h later and the fluorescent signal intensity in cartilage cryosections was compared for the four peptides. To examine the substrate of R8 in cartilage, femoral condyles obtained from another set of mice were treated with chondroitinase ABC (Ch'ase ABC), keratanase or heparitinase then immersed in R8-rhodamine. Fluorescent signals were examined by fluorescent microscopy. Next, R8-rhodamine was injected into the right knee joints of three control and three collagen antibody-induced arthritis (CAIA) mice, and fluorescent intensity in normal and degenerative cartilage was semi-quantitatively analysed on the histological sections using image software. Finally, femoral condyles from normal mice (n=2) and CAIA mice (n=2) were immersed in R8-rhodamine and calcein, then imaged using optical projection tomography (OPT). RESULTS: Fluorescent signals were specifically detected in the cartilage pericellular matrix from the surface to the tide mark but were completely absent in the calcified layer or bone marrow. The number of arginine residues significantly influenced peptide accumulation in articular cartilage, with R8 accumulating the most. The fluorescent signal in the femoral condylar cartilage diminished when it was treated with Ch'ase ABC. R8 accumulation was significantly decreased in the degenerative cartilage of CAIA mice, and this was demonstrated both histologically and in three-dimensional (3D)-reconstruction image by OPT. CONCLUSION: R8 may be a useful new experimental probe for optical imaging of normal and arthritic articular cartilage.

Integrin-regulated secretion of interleukin 4: A novel pathway of mechanotransduction in human articular chondrocytes.

Chondrocyte function is regulated partly by mechanical stimulation. Optimal mechanical stimulation maintains articular cartilage integrity, whereas abnormal mechanical stimulation results in development and progression of osteoarthritis (OA). The responses of signal transduction pathways in human articular chondrocytes (HAC) to mechanical stimuli remain unclear. Previous work has shown the involvement of integrins and integrin-associated signaling pathways in activation of plasma membrane apamin-sensitive Ca2+-activated K+ channels that results in membrane hyperpolarization of HAC after 0. 33 Hz cyclical mechanical stimulation. To further investigate mechanotransduction pathways in HAC and show that the hyperpolarization response to mechanical stimulation is a result of an integrin-dependent release of a transferable secreted factor, we used this response. Neutralizing antibodies to interleukin 4 (IL-4) and IL-4 receptor alpha inhibit mechanically induced membrane hyperpolarization and anti-IL-4 antibodies neutralize the hyperpolarizing activity of medium from mechanically stimulated cells. Antibodies to interleukin 1beta (IL-1beta) and cytokine receptors, interleukin 1 receptor type I and the common gamma chain/CD132 (gamma) have no effect on me- chanically induced membrane hyperpolarization. Chondrocytes from IL-4 knockout mice fail to show a membrane hyperpolarization response to cyclical mechanical stimulation. Mechanically induced release of the chondroprotective cytokine IL-4 from HAC with subsequent autocrine/paracrine activity is likely to be an important regulatory pathway in the maintenance of articular cartilage structure and function. Finally, dysfunction of this pathway may be implicated in OA.

Akt activation induces epidermal hyperplasia and proliferation of epidermal progenitors.

Various common signaling pathways maintain tissue stem cells, including Notch and Wnt/beta-catenin signals. Phosphoinositide-3 kinase (PI3K)/Akt signaling regulates the 'stemness' of several stem cells in culture, specifically in maintaining embryonic stem and neural stem cells, and in deriving embryonic germ cells from primordial germ cells. We examined the effect of Akt signaling in epidermal cells in transgenic mice expressing an Akt-Mer fusion protein whose kinase activity was conditionally activated by treatment with 4-hydroxytamoxifen (4OHT). The topical application of 4OHT to adult skin of the transgenic mice induced new hair growth in resting phase follicles. In addition, the mice showed hyperplasia in interfollicular epidermis (IFE) and hair follicles, which was presumably caused by the extensive proliferation of keratinocytes in basal layer of IFE and outer root sheath of hair follicles, respectively. The progenitor cell population increased consistently in 4OHT-treated transgenic mice. Our results show that PI3K/Akt signaling induces epidermal hyperplasia and proliferation of epidermal progenitors.

Differential expression of MUC16 in human oral mucosal epithelium and cultivated epithelial sheets.

Cultivated oral mucosal epithelial sheet transplantation is a new surgical strategy to treat severe ocular surface disorders such as chemical burns, ocular cicatricial pemphigoid, and Stevens-Johnson syndrome. MUC16 is thought to be the most important membrane-associated mucin on the ocular surface because it forms a protective barrier on the epithelial cell surface. In this study, we studied MUC16 expression in mRNA and protein levels and compared the expression patterns between cultivated oral mucosal epithelial cell sheet and oral mucosal tissue. Specimens (5x5 mm) of oral mucosal tissue harvested from healthy volunteers were used. The oral mucosal epithelial cells were cultured on temperature-responsive culture dishes to generate stratified cell sheets. Cultivated oral mucosal epithelial cells formed three- to five-cell thick stratified sheets for 2 weeks. Scanning electron micrographs revealed that the apical surfaces of the oral mucosal tissue and the oral mucosal sheets were covered with dense microvilli/microplicae. Real-time PCR showed significantly more MUC16 transcripts in the cultivated oral mucosal sheets and corneal epithelial sheets than in the oral mucosal tissue (P=0.023 and 0.008, respectively, Mann-Whitney rank sum test). These findings were confirmed by immunohistochemical examination using an MUC16 antibody to the protein. MUC16 protein was localized to the apical cells of the oral mucosal sheets, but the human oral mucosal tissue did not express MUC16 protein in any cell layers. In this study, interestingly, the expression of membrane-associated mucin MUC16 differs between human oral mucosal epithelia and cultivated epithelial sheets. MUC16 expressed in the oral mucosal sheets may contribute to ocular surface reconstruction after oral mucosal sheet transplantation.