Deep-Learning-Based Detection of Vertebral Fracture and Osteoporosis Using Lateral Spine X-Ray Radiography.

Osteoporosis and vertebral fractures (VFs) remain underdiagnosed. The addition of deep learning methods to lateral spine radiography (a simple, widely available, low-cost test) can potentially solve this problem. In this study, we develop deep learning scores to detect osteoporosis and VF based on lateral spine radiography and investigate whether their use can improve referral of high-risk individuals to bone-density testing. The derivation cohort consisted of patients aged 50 years or older who underwent lateral spine radiography in Severance Hospital, Korea, from January 2007 to December 2018, providing a total of 26,299 lateral spine plain X-rays for 9276 patients (VF prevalence, 18.6%; osteoporosis prevalence, 40.3%). Two individual deep convolutional neural network scores to detect prevalent VF (VERTE-X pVF score) and osteoporosis (VERTE-X osteo score) were tested on an internal test set (20% hold-out set) and external test set (another hospital cohort [Yongin], 395 patients). VERTE-X pVF, osteo scores, and clinical models to detect prevalent VF or osteoporosis were compared in terms of the areas under the receiver-operating-characteristics curves (AUROCs). Net reclassification improvement (NRI) was calculated when using deep-learning scores to supplement clinical indications for classification of high-risk individuals to dual-energy X-ray absorptiometry (DXA) testing. VERTE-X pVF and osteo scores outperformed clinical models in both the internal (AUROC: VF, 0.93 versus 0.78; osteoporosis, 0.85 versus 0.79) and external (VF, 0.92 versus 0.79; osteoporosis, 0.83 versus 0.65; p < 0.01 for all) test sets. VERTE-X pVF and osteo scores improved the reclassification of individuals with osteoporosis to the DXA testing group when applied together with the clinical indications for DXA testing in both the internal (NRI 0.10) and external (NRI 0.14, p < 0.001 for all) test sets. The proposed method could detect prevalent VFs and osteoporosis, and it improved referral of individuals at high risk of fracture to DXA testing more than clinical indications alone. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Inhibition of EGR-1-dependent MMP1 transcription by ethanol extract of Ageratum houstonianum in HaCaT keratinocytes.

Matrix metalloproteinase 1 (MMP-1) initiates the breakdown of matrix networks by cleaving fibrillar collagen during the pathophysiological progression of skin aging. Ageratum houstonianum ethanol extract (AHE) has been used as a traditional herbal medicine to treat external wounds and skin diseases. However, the mechanism of action underlying A. houstonianum-mediated modulation of skin aging has not been investigated. In this study, we evaluated the effect of AHE on MMP-1 expression in HaCaT keratinocytes. Gene expression was analyzed by Reverse transcription-PCR (RT-PCR), Quantitative real-time PCR (Q-PCR), gene promoter-reporter assay, and immunoblotting. We found that AHE abrogated TNFα-induced MMP1 expression at the transcriptional level via the suppression of ERK1/2 mitogen-activated protein kinase (MAPK)-mediated Early Growth Response 1 (EGR1) expression. We also demonstrated that ß-caryophyllene, a cannabinoid receptor 2 (CB2) agonist, is a functional component of the AHE that inhibits TNFα-induced EGR-1 and MMP1 expression. AHE exerts inhibitory activity on TNFα-induced MMP1 expression at the transcription level through EGR-1 downregulation in keratinocytes. ß-Caryophyllene is a bioactive ingredient of AHE that is responsible for the inhibition of TNFα-induced EGR1 expression. ß-Caryophyllene can be used as a potential agent to prevent inflammation-induced skin aging.

Chrysoeriol Prevents TNFα-Induced CYP19 Gene Expression via EGR-1 Downregulation in MCF7 Breast Cancer Cells.

Estrogen overproduction is closely associated with the development of estrogen receptor-positive breast cancer. Aromatase, encoded by the cytochrome P450 19 (CYP19) gene, regulates estrogen biosynthesis. This study aimed to identify active flavones that inhibit CYP19 expression and to explore the underlying mechanisms. CYP19 expression was evaluated using reverse transcription PCR, quantitative real-time PCR, and immunoblot analysis. The role of transcription factor early growth response gene 1 (EGR-1) in CYP19 expression was assessed using the short-hairpin RNA (shRNA)-mediated knockdown of EGR-1 expression in estrogen receptor-positive MCF-7 breast cancer cells. We screened 39 flavonoids containing 26 flavones and 13 flavanones using the EGR1 promoter reporter activity assay and observed that chrysoeriol exerted the highest inhibitory activity on tumor necrosis factor alpha (TNFα)-induced EGR-1 expression. We further characterized and demonstrated that chrysoeriol inhibits TNFα-induced CYP19 expression through inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated EGR-1 expression. Chrysoeriol may be beneficial as a dietary supplement for the prevention of estrogen receptor-positive breast cancer, or as a chemotherapeutic adjuvant in the treatment of this condition.

Effect of 6,7-dimethoxy-2,2-dimethyl-2H-chromene (agerarin) on the recovery of filaggrin expression through targeting of Janus kinases in the inflammatory skin.

Filaggrin (FLG) is a structural component of the stratum corneum that is essential for maintaining the barrier function of the skin and for the formation of natural moisturizing factors. 6,7-Dimethoxy-2,2-dimethyl-2H-chromene (Agerarin) is a bioactive compound derived from Ageratum houstonianum, a plant that is used as a traditional medicine to treat skin diseases. This study aimed to evaluate the effect of agerarin on skin inflammation in a dinitrochlorobenzene (DNCB)-induced atopic dermatitis mouse model. We found that the topical administration of agerarin ameliorates atopic dermatitis-like skin lesions. We also showed that agerarin restores the reduced filaggrin (FLG) expression in DNCB-applied skin sections. Moreover, agerarin decreased phosphorylation of JAK1 and JAK2 kinases to enhance FLG expression, which was reduced by TNFα+IFNγ and IL4+IL13 treatment, in HaCaT keratinocytes. These results demonstrate the feasibility of agerarin as a possible therapeutic against conditions of skin inflammation, such as atopic dermatitis, by improving the upregulation of FLG expression.

Inhibition of TNFα-induced interleukin-6 gene expression by barley (Hordeum vulgare) ethanol extract in BV-2 microglia.

BACKGROUND: Inflammation in the central nervous system is closely associated with pathological neurodegenerative diseases as well as psychiatric disorders. Prolonged activation of microglia can produce many inflammatory mediators, which may result in pathological neurotoxic side effects. Interleukin (IL)-6 serves as a hallmark of the injured brain. OBJECTIVE: Whole grains are known to contain many bioactive components. However, little information is available about anti-neuroinflammatory effects of grains in the CNS. This study aims to investigate the effect of Hordeum vulgare ethanol extract (HVE) on the suppression of IL-6 expression in BV2 microglia. METHODS: Inhibitory effects of HVE on IL-6 expression were analyzed by immunoblot anaysis, immunofluoresce microscopic analysis, reverse transcription-polymerase chain reaction, and luciferase promoter reporter assay. RESULTS: HVE inhibited TNFα-induced phosphorylation of IKKα/ß, IκB, and p65/RelA NF-κB. TNFα-induced IL-6 mRNA expression and promoter activity were reduced by HVE. Point mutation of NF-κB-binding site within the IL-6 gene promoter abolished TNFα-induced reporter activity, whereas exogenous expression of p65 NF-κB enhanced IL-6 promoter activity. CONCLUSION: NF-κB-binding site within the IL-6 promoter region is a HVE target element involved in the inhibition of TNFα-induced IL-6 gene transcription. HVE inhibits TNFα-induced IL-6 expression via suppression of NF-κB signaling in BV2 microglial cells.

Comparison of T2* mapping between regular echo time and ultrashort echo time with 3D cones at 3 tesla for knee meniscus.

The objectives of this study were to compare the ultrashort T2* relaxation time with the T2* relaxation time using the 3 dimensional (3D) cones sequence in 3 groups of patients with normal, degenerated, and torn knee menisci, and to demonstrate the additional effect of the ultrashort echo time (UTE) signal intensity.Following institutional review board approval, 42 knee magnetic resonance imaging (MRI) scans of 42 patients who presented with knee pain and underwent knee MRIs, with the 3D Cones of UTE sequence (minimum TEs: 32 µs) and a 3T MRI scanner (Discovery 750, GE Healthcare, Waukesha, WI), were analyzed. The enrolled patients were classified into 3 subgroups:normal meniscus on conventional MRI, with no positive meniscus-related physical examination in medical records;meniscal degeneration with signal changes on conventional MRI; andmeniscal tear.For the quantitative assessment, the mean values inside user-drawn regions of interest (ROIs) of the medial menisci were drawn on UTE T2* map and T2* map. For statistical analyses, 1-way analysis of variance (ANOVA) with post-hoc analysis using the Tukey HSD test was conducted to compare groups, and effect size was used to compare the discrimination power.The ultrashort T2* relaxation times were higher in patients with meniscal tear than in those with normal and degeneration groups (P <.05, respectively) whereas T2* relaxation times were not statistically significantly different. The ultrashort T2* relaxation times showed higher effect sizes than the T2* times between tear and normal/degeneration.The ultrashort T2* relaxation times showed better delineation of meniscal degeneration or tears than T2* relaxation times. The ultrashort T2* relaxation times could be more sensitive at differentiating between normal and pathologic meniscal conditions in patients.

Inhibitory Effect of Alisma canaliculatum Ethanolic Extract on NF-κB-Dependent CXCR3 and CXCL10 Expression in TNFα-Exposed MDA-MB-231 Breast Cancer Cells.

CXC motif chemokine ligand 10 (CXCL10) and its receptor CXC motif chemokine receptor 3 (CXCR3), play important roles in the motility of breast cancer cells. Alisma canaliculatum is a herb that has been used as a traditional medicine for thousands of years in Korea and China. Whether A. canaliculatum inhibits the motility of metastatic breast cancer cells is not clear yet. In this study, we show that A. canaliculatum ethanolic extract (ACE) prevented tumor necrosis factor-alpha (TNFα)-induced migration of MDA-MB-231 cells. ACE significantly attenuated TNFα-induced upregulation of CXCL10 and CXCR3 expression at the gene promoter level. Mechanistically, ACE inhibits TNFα-induced phosphorylation of inhibitor of κB (IκB) kinase (IKK), IκB and p65/RelA, leading to the suppression of nuclear translocation of p65/RelA nuclear factor kappa-B (NF-κB). Also, ACE inhibited NF-κB-dependent CXCR3 and CXCL10 promoter activities. These results suggest that ACE abrogates TNFα-induced migration of MDA-MB-231 breast cancer cells through down-regulation of IKK-NF-κB-dependent CXCR3 and CXCL10 expression. Our results suggest that ACE has potential as a herbal supplement for the inhibition of breast cancer metastasis.

Downregulation of α-Melanocyte-Stimulating Hormone-Induced Activation of the Pax3-MITF-Tyrosinase Axis by Sorghum Ethanolic Extract in B16F10 Melanoma Cells.

Ultraviolet irradiation-induced hyperpigmentation of the skin is associated with excessive melanin production in melanocytes. Tyrosinase (TYR) is a key enzyme catalyzing the rate-limiting step in melanogenesis. TYR expression is controlled by microphthalmia-associated transcription factor (MITF) expression. Sorghum is a cereal crop widely used in a variety of foods worldwide. Sorghum contains many bioactive compounds and is beneficial to human health. However, the effects of sorghum in anti-melanogenesis have not been well characterized. In this study, the biological activity of sorghum ethanolic extract (SEE) on α-melanocyte-stimulating hormone (α-MSH)-induced TYR expression was evaluated in B16F10 melanoma cells. SEE attenuated α-MSH-induced TYR gene promoter activity through the downregulation of the transcription factor MITF. We found that paired box gene 3 (Pax3) contributes to the maximal induction of MITF gene promoter activity. Further analysis demonstrated that SEE inhibited α-MSH-induced Pax3 expression. The collective results indicate that SEE attenuates α-MSH-induced TYR expression through the suppression of Pax3-mediated MITF gene promoter activity. Targeting the Pax3-MITF axis pathway could be considered a potential strategy to increase the efficacy of anti-melanogenesis.

Agerarin, identified from Ageratum houstonianum, stimulates circadian CLOCK-mediated aquaporin-3 gene expression in HaCaT keratinocytes.

The juice of Ageratum houstonianum is used in folk medicine as an external wound healing aid for skin injuries. However, the active component of A. houstonianum and its mode of action in skin wound healing has not been investigated. This study was conducted to investigate the effect of A. houstonianum ethanolnolic extract (AHE) on the expression of aquaporin-3 (AQP3), an integral membrane protein for water and glycerol transport in keratinocytes, and to identify the structure of the A. houstonianum bioactive compound. Here, we show that AHE increased AQP3 gene expression at the transcriptional level through the p38 MAPK pathway in HaCaT cells. Furthermore, AHE ameliorated suppression of AQP3 expression caused by ultraviolet B (UVB) irradiation. Agerarin (6,7-dimethoxy-2,2-dimethyl-2H-chromene) was identified as the bioactive compound responsible for the up-regulation of AQP3 expression by enhancing the expression of the transcription factor circadian locomotor output cycles kaput (CLOCK). In conclusion, agerarin is a bioactive compound in AHE responsible for CLOCK-mediated AQP3 expression in keratinocytes.

γ-Oryzanol-Rich Black Rice Bran Extract Enhances the Innate Immune Response.

The innate immune response is an important host primary defense system against pathogens. γ-Oryzanol is one of the nutritionally important phytoceutical components in rice bran oil. The goal of this study was to investigate the effect of γ-oryzanol-rich extract from black rice bran (γORE) on the activation of the innate immune system. In this study, we show that γORE increased the expression of CD14 and Toll-like receptor 4 and enhanced the phagocytic activity of RAW264.7 macrophages. Furthermore, γORE and its active ingredient γ-oryzanol promoted the secretion of innate cytokines, interleukin-8, and CCL2, which facilitate phagocytosis by RAW264.7 cells. These findings suggest that γ-oryzanol in the γORE enhances innate immune responses.

Euphorbia humifusa Willd exerts inhibition of breast cancer cell invasion and metastasis through inhibition of TNFα-induced MMP-9 expression.

BACKGROUND: Breast cancer is the most common type of malignancy in women worldwide. Euphorbia humifusa Willd (EuH) is a plant that is widely used as a traditional medicine. However, no systemic studies on the anti-cancer effects of EuH have been reported. The aim of this study is to evaluate the anti-metastatic effect of the EuH. METHODS: Ethyl acetate fraction was prepared from EuH methanol extracts (EA/EuH). Inhibitory effect of EA/EuH on cell migration was determined using an in vitro scratch-wound healing assay. The anti-invasive activity was determined by in vitro three-dimensional spheroid culture system and in vivo syngenic experimental lung metastasis experiment. Gene expression profiles were analyzed by using RT-PCR, real-time PCR, and luciferase reporter assay systems. RESULTS: Ethyl acetate fraction from the EuH extract (EA/EuH) inhibited the migration and invasive capabilities of highly metastatic MDA-MB-231 breast cancer cells and attenuated syngeneic lung metastasis of mouse 4 T1 breast cancer cells in vivo. Mechanistically, EA/EuH decreased tumor necrosis factor alpha (TNFα)-induced matrix metalloproteinase (MMP)-9 mRNA expression through the inhibition of NF-κB activity in MDA-MB-231 cells. CONCLUSION: EuH may be beneficial in the prevention of invasion and metastasis of early stage breast cancer and can be served as an anti-metastatic agent or adjuvant therapy against metastatic breast cancer.

Inhibition of PDGF-induced migration and TNF-α-induced ICAM-1 expression by maltotetraose from bamboo stem extract (BSE) in mouse vascular smooth muscle cells.

SCOPE: Expression of intercellular adhesion molecule-1 (ICAM-1) on vascular smooth muscle cells (VSMCs) plays an important role in the progression of atherosclerosis. We investigated the effects of bamboo stem extract (BSE) on motility and ICAM-1 expression by using mouse MOVAS-1 cells. Active constituents of BSE exhibiting an inhibitory activity on TNF-α-induced ICAM1 expression were identified using HPLC. METHODS AND RESULTS: The effects of BSE on platelet-derived growth factor (PDGF)-BB-induced migration, tumor necrosis factor alpha (TNF-α)-induced expression of ICAM-1, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation were investigated. BSE inhibited migration of MOVAS-1 cells and sprout formation by mouse aorta explants. Reverse transcription PCR analysis and promoter reporter assays revealed that BSE suppressed ICAM-1 expression by inhibiting NF-κB activity. In addition, BSE reduced adhesion between VSMCs and monocytes. Several oligosaccharides were identified in BSE. Among the oligosaccharides contained in BSE, maltotetraose and stachyose were potent inhibitors of TNF-α-induced ICAM-1 expression. We confirmed that maltotetraose reduced PDGF-induced sprout formation by mouse aorta explants and inhibited TNF-α-induced NF-κB activation and ICAM-1 expression in MOVAS-1 cells. CONCLUSION: The BSE constituent maltotetraose may be beneficial in the suppression of early atherosclerosis development and could be developed as a dietary supplement for cardiovascular health.

Structural properties of polyphenols causing cell cycle arrest at G1 phase in HCT116 human colorectal cancer cell lines.

Plant-derived polyphenols are being tested as chemopreventive agents; some polyphenols arrest the cell cycle at G1 phase, whereas others inhibit cell cycle proliferation at G2/M phase. Therefore, polyphenols have been proposed to inhibit cell cycle progression at different phases via distinct mechanisms. Indeed, our previous studies showed that small structural differences in polyphenols cause large differences in their biological activities; however, the details of the structural properties causing G1 cell cycle arrest remain unknown. In this study, we prepared 27 polyphenols, including eight different scaffolds, to gain insight into the structural conditions that arrest the cell cycle at G1 phase in a quantitative structure-activity relationship study. We used cell cycle profiles to determine the biophores responsible for G1 cell cycle arrest and believe that the biophores identified in this study will help design polyphenols that cause G1 cell cycle arrest.

EGCG inhibits recepteur d'origine nantais expression by suppressing Egr-1 in gastric cancer cells.

Abnormal accumulation and activation of the recepteur d'origine nantais (RON) has been implicated in epithelial tumor carcinogenesis. In the present study, we examined the effect of epigallocatechin-3-gallate (EGCG), the major green tea catechin, on the induction of RON and tumor growth in human gastric cancer. EGCG inhibited phorbol 12-myristate 13-acetate (PMA)-induced RON expression and reduced RON transcriptional activity. However, (-)-epigalloca-techin (EGC), (-)-epicatechin gallate (ECG) and (-)­epicatechin (EC) did not affect RON expression. Experiments with deleted and site-directed mutagenesis of the RON promoter indicated that Egr-1 binding sites in the RON promoter may be the EGCG­response element acting as a cis-element in gastric cancer cells. EGCG also inhibited PMA-induced Egr-1 expression and DNA binding in a dose-dependent manner. Furthermore, gastric cancer cells pretreated with PMA showed markedly enhanced invasiveness, which was partially abrogated by EGCG and siRNA-targeted RON and Egr-1. EGCG significantly reduced tumor growth in an in vivo tumor model, whereas RON expression was downregulated. These results suggest that EGCG may exert at least part of its anticancer effect by controlling RON expression through suppression of Egr-1 activation.

2'-Hydroxyflavanone induces apoptosis through Egr-1 involving expression of Bax, p21, and NAG-1 in colon cancer cells.

SCOPE: Natural flavanones exhibit cancer preventive and/or therapeutic effects. The objective of this study was to investigate the molecular mechanism underlying the action of the antitumor activity of hydroxyflavanone using the HCT116 colon cancer cell line. METHODS AND RESULTS: We investigated the effect of hydroxyflavanones on antitumor activity. We found that 2'-hydroxyflavanone (2'-HF) potently inhibited the clonogenicity of HCT116 cells. 2'-HF triggered apoptosis in both wild-type and p53-null HCT116 cells, as revealed by DNA fragmentation and caspase activation. 2'-HF upregulated nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1) expression through induction of Egr-1. Silencing of NAG-1 or Egr-1 using small interfering RNA (siRNA) could attenuate 2'-HF-induced apoptosis. Egr-1 also upregulated the proapoptotic gene Bax and the cell cycle inhibitor p21. CONCLUSION: Dietary 2'-HF may possess antitumor activity against human colon cancer.

Comparison of the content of bioactive substances and the inhibitory effects against rat plasma oxidation of conventional and organic hot peppers (Capsicum annuum L.).

The aim of this study was to evaluate the chemical compositions and antioxidative activities of hot pepper fruits cultivated with strict management by organic and conventional agricultural practices. The ascorbic acid content in the organically grown hot pepper (OGP) was significantly higher than that of conventionally grown hot pepper (CGP) in both green and red fruits. The content of other bioactive compounds such as flavonoids (apigenin, luteolin, quercetin) and total phenolics in OGP was typically higher than in CGP regardless of fruit color. In addition, the ABTS(+) radical-scavenging activity of OGP red fruits was significantly higher than that of CGP red fruits. Moreover, regardless of the color of the fruits, a higher antioxidative activity was observed in blood plasma from rats administered the OGP fruit extracts than in blood plasma from rats administered the CGP fruit extracts. It was hypothesized that the higher antioxidant activity of the OGP fruits may have resulted from the higher antioxidant content in the OGP fruits. These results suggest that consumption of pepper fruits may increase antioxidant activity in the blood, and OGP fruits may be more effective in increasing this antioxidant activity than CGP fruits.

Deoxypodophyllotoxin induces G2/M cell cycle arrest and apoptosis in HeLa cells.

The natural flavolignan deoxypodophyllotoxin (DPPT) inhibits tubulin polymerization and induces cell cycle arrest at G(2)/M, followed by apoptosis. However, the precise mechanism of DPPT action is currently unknown. Here, we investigated the mechanism by which DPPT treatment of HeLa cervical carcinoma cells induces cell cycle arrest and apoptosis. We show that DPPT treatment inhibits cell viability in a dose-dependent manner and that this reduction in cell viability results from cell cycle arrest at G(2)/M phase, accompanied by an increase in apoptotic cell death. The induction of apoptosis by DPPT was confirmed by visualization of morphologic changes and internucleosomal DNA fragmentation. In addition, DPPT causes p53 and Bax to accumulate, accompanied by activation of DNA damage-sensing kinases, including ataxia-telangiectasia mutated (ATM) kinase and Chk2. Furthermore, DPPT activates caspase-3 and -7, suggesting that caspase-mediated pathways are involved in DPPT-induced apoptosis. Levels of the tumor suppressor PTEN were up-regulated during DPPT treatment, coincident with Akt inhibition. Together, these data suggest that DPPT induces G(2)/M cell-cycle arrest followed by apoptosis through multiple cellular processes, involving the activation of ATM, upregulation of p53 and Bax, activation of caspase-3 and -7, and accumulation of PTEN resulting in the inhibition of the Akt pathway.

NF-kappaB Activation by compounds found in Platycodon grandiflorum extract.

Compounds extracted from Platycodon grandiflorum were evaluated for an activation effect on nuclear factor-kappa B (NF-kappaB). In its active state, NF-kappaB turns on the expression of genes related to cell proliferation or death. NF-kappaB activators promote growth of neuron cells and can be used to control neurodegenerative diseases. The biological activity of P. grandiflorum extracts toward NF-kappaB had not yet been studied. Although the biological activity of several compounds extracted from P. grandiflorum was evaluated, only three exhibited any significant activation effect on NF-kappaB.

Antitumor activity of deoxypodophyllotoxin isolated from Anthriscus sylvestris: Induction of G2/M cell cycle arrest and caspase-dependent apoptosis.

An active compound having antitumor activity was isolated from the root of Anthriscus sylvestris. Structural studies revealed that it was deoxypodophyllotoxin (DPPT), and its biological activity was evaluated in HeLa human cervix carcinoma cells. Flow cytometric analysis showed that DPPT arrests the cell cycle in the G2/M phase prior to apoptosis. The mechanisms of action of DPPT involve inhibition of tubulin polymerization, dysregulation of cyclin A and cyclin B1 expression, and activation of caspases-3 and -7.

Cloning of two cellulase genes from endophytic Paenibacillus polymyxa GS01 and comparison with cel 44C-man 26A.

Endophytic bacteria are acknowledged as a new source of genes, proteins and other biochemical compounds, which are often used in biochemical processes. In this study, Paenibacillus polymyxa GS01 was isolated from the interior of the roots of Korean cultivars of ginseng (Panax ginseng C. A. Meyer). Two cellulase genes, cel 5A and cel 5B, were cloned from GS01, and encode 334 aa and 573 aa proteins, respectively. Cel5A and Cel5B each contain a glycosyl hydrolase family 5 (GH5) catalytic domain. The molecular mass of Cel5A and Cel5B were estimated to be 33 kDa and 61 kDa, respectively, by CMC-SDS-PAGE. When purified from Escherichia coli Cel5A and Cel5B both displayed cellulase activity with pH optima of 7.0 and 6.0, respectively and shared a temperature optimum of 50 degrees C. Neither enzyme had detectable xylanase, lichenase, or mannase activity, in contrast to the multifunctional Cel44C-Man26A enzyme of P. polymyxa which displays cellulase, xylanase, lichenase and mannanase activities. However, Cel5A and Cel5B exhibited higher specific cellulase activity than Cel44C-Man26A (120% and 140%, respectively). Cel5A and Cel5B mutants with alanine substitutions at a conserved glutamic acid in the GH5 domain (Glu 179 of Cel5A and Glu184 of Cel5B) lacked cellulase activity, suggesting that this residue is important for GH5 domain function.