Deletion analysis of the starch binding domain from Aspergillus glucoamylase

Glucoamylase (GA) from Aspergillus (EC 3.2.1.3) exists in two forms: GAI (amino acids 1-616) and GAII (amino acids 1-512). GAII is identical to GAI but lacks the C-terminal region (amino acids 513-616). While both forms of glucoamylase are able to digest soluble starch, only GAI has the ability to bind to and therefore hydrolyze native starch granules. To further examine the location of the starch binding domain at the C-terminus of GAI and to determine how many amino acid residues of GAI are required for starch binding, six [beta]-galactosidase fusion proteins were constructed. Affinity purified fusion proteins were tested for native starch binding. Fusion proteins containing 119 and 133 amino acids from the C-terminus of GAI, respectively, had much higher affinity to starch granules than [beta]-galactosidase and a size-control fusion protein. The results confirmed the hypothesis that the starch binding domain of Aspergillus GAI resides in the C-terminus of the enzyme and indicated that the C-terminal 119 amino acids of GAI is near the optimum functional size when fused to [beta]-galactosidase and expressed in E. coli. The binding specificity of the fusion proteins native starch granules was also tested. The results showed that the fusion proteins could specifically as well as strongly bind to native starch granules in the presence of crude soluble cell extracts and could be eluted from starch granules with similar purity to that achieved by affinity chromatography, suggesting the potential application of native starch as an adsorbent to facilitate the purification or immobilization of fusion proteins containing the starch binding region;In order to test the effect of deletions in the starch binding domain on glucoamylase itself, five deletion mutants of glucoamylase were constructed and expressed in Saccharomyces cerevisiae. Progressive loss of starch binding and starch hydrolytic activity was observed upon removal of 8 to 52 amino acid residues from the C-terminus of GAI. Removal of the C-terminal 103 amino acids resulted in nearly complete loss of starch binding and hydrolytic activity of GAI. These deletions in the starch binding domain did not affect enzyme activity on soluble starch or thermostability of the enzyme, confirming the independence of the catalytic domain from the starch binding domain. Study of the starch binding domain will facilitate understanding of the interaction between glucoamylase and starch and also the future utilization of the domain

Further validation of the Health Scale of Traditional Chinese Medicine (HSTCM)

<p>Abstract</p> <p>Background</p> <p>Few health measurement scales are based on Chinese medicine theory. The Health Scale of Traditional Chinese Medicine (HSTCM) was developed to fill this gap. The aim of this study is to validate the HSTCM.</p> <p>Methods</p> <p>A convenience sample of 630 participants was recruited in 11 settings. All participants were asked to complete the HSTCM and World Health Organization Quality of Life Measure-Abbreviated Version (WHOQOL-BREF).</p> <p>Results</p> <p>Properties of the HSTCM were tested. Intra-class correlation coefficient representing the inter-interviewer reliability was 0.99 (95%CI) for the overall instrument. Spearman-Brown correlation coefficient and Cronbach's coefficient alpha were 0.81 and 0.94 respectively, indicating satisfactory internal reliability and inter-interviewer reliability. Spearman's rho correlation coefficient between the HSTCM and WHOQOL-BREFF was -0.67. A receiver operating characteristic (ROC) curve analysis was performed to test the discriminate validation. Areas under the ROC curve analysis for the HSTCM and its domains ranged 0.71–0.87 and all the lower levels of 95%CI were greater than 0.50.</p> <p>Conclusion</p> <p>The HSTCM was validated as a generic health scale and may complement existing health measurement scales in Chinese medicine health care.</p

Inhibition of COP9-signalosome (CSN) deneddylating activity and tumor growth of diffuse large B-cell lymphomas by doxycycline

In searching for small-molecule compounds that inhibit proliferation and survival of diffuse large B-cell lymphoma (DLBCL) cells and may, therefore, be exploited as potential therapeutic agents for this disease, we identified the commonly used and well-tolerated antibiotic doxycycline as a strong candidate. Here, we demonstrate that doxycycline inhibits the growth of DLBCL cells both in vitro and in mouse xenograft models. In addition, we show that doxycycline accumulates in DLBCL cells to high concentrations and affects multiple signaling pathways that are crucial for lymphomagenesis. Our data reveal the deneddylating activity of COP-9 signalosome (CSN) as a novel target of doxycycline and suggest that doxycycline may exert its effects in DLBCL cells in part through a CSN5-HSP90 pathway. Consistently, knockdown of CSN5 exhibited similar effects as doxycycline treatment on DLBCL cell survival and HSP90 chaperone function. In addition to DLBCL cells, doxycycline inhibited growth of several other types of non-Hodgkin lymphoma cells in vitro. Together, our results suggest that doxycycline may represent a promising therapeutic agent for DLBCL and other non-Hodgkin lymphomas subtypes

Deletion analysis of the starch binding domain from Aspergillus glucoamylase

Glucoamylase (GA) from Aspergillus (EC 3.2.1.3) exists in two forms: GAI (amino acids 1-616) and GAII (amino acids 1-512). GAII is identical to GAI but lacks the C-terminal region (amino acids 513-616). While both forms of glucoamylase are able to digest soluble starch, only GAI has the ability to bind to and therefore hydrolyze native starch granules. To further examine the location of the starch binding domain at the C-terminus of GAI and to determine how many amino acid residues of GAI are required for starch binding, six [beta]-galactosidase fusion proteins were constructed. Affinity purified fusion proteins were tested for native starch binding. Fusion proteins containing 119 and 133 amino acids from the C-terminus of GAI, respectively, had much higher affinity to starch granules than [beta]-galactosidase and a size-control fusion protein. The results confirmed the hypothesis that the starch binding domain of Aspergillus GAI resides in the C-terminus of the enzyme and indicated that the C-terminal 119 amino acids of GAI is near the optimum functional size when fused to [beta]-galactosidase and expressed in E. coli. The binding specificity of the fusion proteins native starch granules was also tested. The results showed that the fusion proteins could specifically as well as strongly bind to native starch granules in the presence of crude soluble cell extracts and could be eluted from starch granules with similar purity to that achieved by affinity chromatography, suggesting the potential application of native starch as an adsorbent to facilitate the purification or immobilization of fusion proteins containing the starch binding region;In order to test the effect of deletions in the starch binding domain on glucoamylase itself, five deletion mutants of glucoamylase were constructed and expressed in Saccharomyces cerevisiae. Progressive loss of starch binding and starch hydrolytic activity was observed upon removal of 8 to 52 amino acid residues from the C-terminus of GAI. Removal of the C-terminal 103 amino acids resulted in nearly complete loss of starch binding and hydrolytic activity of GAI. These deletions in the starch binding domain did not affect enzyme activity on soluble starch or thermostability of the enzyme, confirming the independence of the catalytic domain from the starch binding domain. Study of the starch binding domain will facilitate understanding of the interaction between glucoamylase and starch and also the future utilization of the domain.</p

A role for NF-κB activity in skin hyperplasia and the development of keratoacanthomata in mice.

Previous studies have implicated NF-κB signaling in both cutaneous development and oncogenesis. However, these studies have been limited in part by the lethality that results from extreme over- or under-expression of NF-κB in available mouse models. Even cre-driven tissue specific expression of transgenes, or targeted deletion of NF-κB can cause cell death. Therefore, the present study was undertaken to evaluate a novel mouse model of enhanced NF-κB activity in the skin.A knock-in homologous recombination technique was utilized to develop a mouse model (referred to as PD mice) with increased NF-κB activity.The data show that increased NF-κB activity leads to hyperproliferation and dysplasia of the mouse epidermis. Chemical carcinogenesis in the context of enhanced NF-κB activity promotes the development of keratoacanthomata.Our findings support an important role for NF-κB in keratinocyte dysplasia. We have found that enhanced NF-κB activity renders keratinocytes susceptible to hyperproliferation and keratoacanthoma (KA) development but is not sufficient for transformation and SCC development. We therefore propose that NF-κB activation in the absence of additional oncogenic events can promote TNF-dependent, actinic keratosis-like dysplasia and TNF-independent, KAs upon chemical carcinogensis. These studies suggest that resolution of KA cannot occur when NF-κB activation is constitutively enforced

PKK deletion in basal keratinocytes promotes tumorigenesis after chemical carcinogenesis

Squamous cell carcinoma (SCC) of the skin is a keratinocyte malignancy characterized by tumors presenting on sun-exposed areas with surgery being the mainstay treatment. Despite advances in targeted therapy in other skin cancers, such as basal cell carcinoma and melanoma, there have been no such advances in the treatment of SCC. This is partly due to an incomplete knowledge of the pathogenesis of SCC. We have recently identified a protein kinase C-associated kinase (PKK) as a potential tumor suppressor in SCC. We now describe a novel conditional PKK knockout mouse model, which demonstrates that PKK deficiency promotes SCC formation during chemically induced tumorigenesis. Our results further support that PKK functions as a tumor suppressor in skin keratinocytes and is important in the pathogenesis of SCC of the skin. We further define the interactions of keratinocyte PKK with TP63 and NF-κB signaling, highlighting the importance of this protein as a tumor suppressor in SCC development

Proliferation and Dysplasia of PD mouse skin.

<p>A. Homozygous PD mouse skin at 10 days showing focus of epidermal dysplasia with aggregates of basaloid cells in the epidermis without penetration of the basement membrane. B. Examination of proliferation of epidermal keratinocytes in mouse skin with Ki-67. Nuclear Ki-67 is restricted to the basal keratinocytes in wild-type littermate controls (top) whereas the PD homozygous mouse has positive staining at all levels of the epidermis (bottom). Skin treated with a rat isotype control shows no non-specific staining in the epidermis (not shown).</p

Keratoacanthoma development in the PD mice.

<p>Chemical carcinogenesis study in heterozygous PD and wild-type littermates. A. Papilloma formation in a PD heterozygote during TPA treatment shows no difference between experimental and control groups of mice. B. Clinical appearance of a typical papilloma. C. PD heterozygous mouse that has developed a typical keratoacanthoma showing a keratinaceous core and well-defined tumor. D. Histologic examination of keratoacanthomata from PD heterozygous mice showing classic appearance of these tumors. E. Mutational analysis of PD mice for <i>Hras</i> codons 12,13,61 and <i>Tp53</i>.</p

Generation of PD mice.

<p>Targeted knock-in at <i>RelA</i> to generate a S276D amino acid transition from exon 7 in mice creating the PD targeting vector. A. Strategy for generating the targeted knock-in including the coding change at serine 276 and addition of an EagI restriction site to allow for genotyping. B. Genotyping of the PD mouse shows a 722 base pair (bp) cDNA in the wild-type mouse that can be cut in to two pieces (356 and 366 bp) with EagI in the presence of the PD allele. C. Homozygous PD mice are born in fewer numbers than expected by normal Mendelian ratios. C. Approximately 10 day old littermates from a male and female heterozygous PD breeding. Homozygous PD mice are runted, erythrodermic, alopecic, and scaly. At this point in development, wild-type littermates demonstrate normal hair growth and size. D. Immunohistochemistry staining of mouse skin showing few F4/80+ cells in wild-type tissue (left) compared to the large infiltrate in PD homozygous mouse skin (middle). Spleen shows typical F4/80+ staining as a control (right).</p