The GH26 β-mannanase RsMan26H from a symbiotic protist of the termite Reticulitermes speratus is an endo-processive mannobiohydrolase: Heterologous expression and characterization

Symbiotic protists in the gut of termites are prominent natural resources for enzymes involved in lignocellulose degradation. Here we report expression, purification, and biochemical characterization of a glycoside hydrolase family 26 mannanase RsMan26H from the symbiotic protist of the lower termite, Reticulitermes speratus. Biochemical analysis of RsMan26H demonstrates that this enzyme is an endo-processive mannobiohydrolase producing mannobiose from oligo- and polysaccharides, followed by a minor accumulation of oligosaccharides larger than mannobiose. To our knowledge, this is the first report describing the unique mannobiohydrolase enzyme from the eukaryotic origin.     

Deafness induced by Connexin 26 (GJB2) deficiency is not determined by endocochlear potential (EP) reduction but is associated with cochlear developmental disorders

Connexin 26 (Cx26, GJB2) mutations are the major cause of hereditary deafness and are responsible for >50% of nonsyndromic hearing loss. Mouse models show that Cx26 deficiency can cause congenital deafness with cochlear developmental disorders, hair cell degeneration, and the reduction of endocochlear potential (EP) and active cochlear amplification. However, the underlying deafness mechanism still remains undetermined. Our previous studies revealed that hair cell degeneration is not a primary cause of hearing loss. In this study we investigated the role of EP reduction in Cx26 deficiency-induced deafness. We found that the EP reduction is not associated with congenital deafness in Cx26 knockout (KO) mice. The threshold of auditory brainstem response (ABR) in Cx26 KO mice was even greater than 110 dB SPL, demonstrating complete hearing loss. However, the EP in Cx26 KO mice varied and not completely abolished. In some cases, the EP could still remain at higher levels (>70 mV). We further found that the deafness in Cx26 KO mice is associated with cochlear developmental disorders. Deletion of Cx26 in the cochlea before postnatal day 5 (P5) could cause congenital deafness. The cochlea had developmental disorders and the cochlear tunnel was not open. However, no congenital deafness was found when Cx26 was deleted after P5. The cochlea also displayed normal development and the cochlear tunnel was open normally. These data suggest that congenital deafness induced by Cx26 deficiency is not determined by EP reduction and may result from cochlear developmental disorders.     

Uric acid inhibition of dipeptidyl peptidase IV in vitro is dependent on the intracellular formation of triuret

Uric acid affects endothelial and adipose cell function and has been linked to diseases such as hypertension, metabolic syndrome, and cardiovascular disease. Interestingly uric acid has been shown to increase endothelial progenitor cell (EPC) mobilization, a potential mechanism to repair endothelial injury. Since EPC mobilization is dependent on activity of the enzyme CD26/dipeptidyl peptidase (DPP)IV, we examined the effect uric acid will have on CD26/DPPIV activity. Uric acid inhibited the CD26/DPPIV associated with human umbilical vein endothelial cells but not human recombinant (hr) CD26/DPPIV. However, triuret, a product of uric acid and peroxynitrite, could inhibit cell associated and hrCD26/DPPIV. Increasing or decreasing intracellular peroxynitrite levels enhanced or decreased the ability of uric acid to inhibit cell associated CD26/DPPIV, respectively. Finally, protein modeling demonstrates how triuret can act as a small molecule inhibitor of CD26/DPPIV activity. This is the first time that uric acid or a uric acid reaction product has been shown to affect enzymatic activity and suggests a novel avenue of research in the role of uric acid in the development of clinically important diseases.     

Investigating anticancer properties of the sesquiterpene ferutinin on colon carcinoma cells,in vitro and in vivo

Aims

In this research, ferutinin was evaluated for its possible cytotoxic and apoptotic inducing effects in vitro and in vivo.

Main methods

To determine IC₅₀ values of ferutinin, CT26, HT29 and NIH/3T3 cells were treated with different concentrations of ferutinin. In addition to morphological changes in cells, the DNA damage was studied using DAPI staining, comet assay and PI staining. Ferutinin was also tested for its in vivo activity.

Key findings

Analyses of cell survival by MTT assay showed that the IC₅₀ values of ferutinin on CT26 and HT29 cells were 26 and 29 μg/ml, respectively, while after treating nontumoural mouse cells even with 50 μg/ml ferutinin, 70% of cells was still surviving. The results of DAPI staining and comet assay revealed that ferutinin significantly induced DNA damage in treated cells. Induction of sub-G₁ peak after PI staining was also indicative of apoptotic effects of ferutinin in cancerous cells. In vivo studies showed a significant regression in tumour size in mice treated with ferutinin as compared to control groups. Its antitumour effects were very similar to the cisplatin treated group. Histological studies demonstrated that apoptosis rate in tumour cells was increased in comparison to tumour cells in control mice without ferutinin treatment. Interestingly, haematoxylin and eosin staining showed no damage in the spleen and liver of ferutinin treated mice.

Significance

As ferutinin showed less toxic effects in nontumoural cells, and induced its effects via apoptosis induction, it could be considered as an effective anticancer agent for future preclinical experiments.     

高压氧联合化疗药物对结肠腺癌细胞 T 26 小C鼠移植瘤生长的影响

目的：探讨高压氧联合化疗药物对结肠腺癌细胞CT26小鼠移植瘤生长的影响。方法：建立小鼠结肠腺癌移植瘤模型,观察0．2MpaHBO及联合化疗药物5．FU或紫杉醇对荷瘤小鼠肿瘤生长的影响,计算肿瘤抑制率;用流式细胞仪观察0．2MpaHBO对CT26细胞周期的影响。结果：动物实验结果显示：HBO组小鼠肿瘤体积抑制率为：22．39％,肿瘤质量抑制率为：25．77％;5-Fu组分别为：42.38％,43．61％;5-FU＋HBO组分别为：72．10％,71．47％;紫杉醇组分别为：26．31％,23．04％;紫杉醇＋HBO分别为：33．24％。30．96％,5-FU＋HBO组与5-FU组及HB0组相比较有显著性差异（p〈0．01）;紫杉醇＋HB0组与紫杉醇组及HBO组相比差异不明显。流式细胞仪检测结果显示：0,2MpaHBO诱导CT26细胞在S期积蓄（G0／G1期（55．89％）、S期（40．79％）、G2／M期（3-32％））。结论：0．2MpaHBO可抑制了CT26结肠腺癌小鼠移植瘤的生长。并能增强细胞周期特异性化疗药物的敏感性。     

Comparison of a monoclonal antibody-based capture/enrichment sandwich enzyme linked immunosorbent assay with immunomagnetic bead separation for the detection of attachment effacement Escherichia coli O26 strains from cattle faeces

AIMS: A monoclonal antibody (Mab 2F3)-based sandwich enzyme linked immunosorbent assay (sELISA) format for the detection of Escherichia coli O26 that improves the sensitivity of the assay by combining enrichment with the capture stage has been developed. Culture of the enriched contents of wells before completion of the sELISA was compared with immunomagnetic bead separation (IMS) as a means of specific isolation of the target organism. METHODS AND RESULTS: Bovine faecal samples, c. 10% in buffered peptone water (BPW), were pre-enriched for 6 h before testing by capture/enrichment sELISA and by IMS. The sELISA consisted of a 1-2 h capture stage followed by addition of BPW to the wells and an overnight enrichment stage before completion of the assay. The capture/enrichment stage of the assay was repeated a second time on the enriched contents removed from the wells before completion of the first sELISA. From 204 cattle faeces samples, 30x O26 strains [20x attachment effacement Escherichia coli (AEEC) and 10x non-AEEC] were isolated from the enriched wells of the sELISA, in comparison with 11 (9x AEEC and 2x non-AEEC) that were isolated by IMS. Examination of the use of enterohaemolysin activity and rhamnose utilization on 1% rhamnose McConkey's (RMAC) agar with or without cefixime and potassium tellurite demonstrated that the selection based on enterohaemolysin production and growth on RMAC with cefixime and potassium tellurite would largely differentiate the AEEC strains from the non-AEEC strains. CONCLUSIONS: The capture/enrichment sELISA protocol used compared favourably with the IMS for the isolation of E. coli O26 from faeces samples. The ELISA optical density readings obtained in the procedure were used as a screening indicator for selection of samples for further culture examination, and the selective culture methods examined to assist strain isolation did have potential. SIGNIFICANCE AND IMPACT OF THE STUDY: The capture/enrichment format of an Mab-based sELISA protocol has the potential to provide a suitable screening assay for the specific detection of pathogenic strains from mixed culture samples like faeces.