Recent Research Progress in the Applied Microbiology Laboratory : Enzymes, Toxins and Cell-Envelope Structure of Environmental and Pathogenic Bacteria(Recent Topics of the Agricultunal Biological Science in Tohoku University)

Here we describe the current status of our studies of xylanases of Paenibacillus sp. W-61, poly (D-lactic acid) hydrolases of Amycolatopsis sp. K104-1 and Brevibacterium sp. 93, pectin lyase of plant pathogenic Pectobacterium carotovora and bi-component toxins of Staphylococcus aureus. Paenibacillus sp. W-61 utilizes xylan as a carbon source via three enzymes, of which Xyn5 plays a crucial role both in xylan utilization and in the synthesis of the other two xylanases. Understanding the roles of each xylanase in xylan degradation will help to develop an enzymatic preparation of xylose from xylan. The poly (D-lactic acid) [PLA] hydrolases of Amycolatopsis sp. K104-1 and Brevibacterium sp. 93 appear to be novel enzymes with potential for PLA recycling. We recently purified and characterized the PLA hydrolases produced by these bacteria. The plant-soft rotting bacterium P. carotovora produces pectin lyase, a potential pathogenic factor, and a bacteriocin when exposed to DNA-damaging agents. We recently determined the cis-acting regulatory sequences responsible for the DNA-damage-inducible synthesis of the enzyme and bacteriocin. Leukocidin and γ-hemolysin are bi-component toxins that lyse leukocytes and erythrocytes respectively. We describe the molecular structure and pore-forming mechanism of the toxin components encoded by the chromosome and by a phage genome. Finally, we briefly describe the cell-envelope structure of the rumen bacterium Selenomonas ruminantium and its outer membrane protein Mep45 that possibly bridges the outer membrane and the peptidoglycan. The objects of our studies include environmental bacteria (xylanolytic Paenibacillus sp. W-61 and poly (D-lactic acid)-hydrolyzing bacteria Amycolatopsis sp. K104-1 and Brevibacterium sp. 93), as well as the plant and animal pathogens, Pectobacterium carotovora and Staphylococcus aureus, respectively. Here we briefly describe the current status of our investigations into these bacteria, their extracellular enzymes (or toxins), and an outer membrane protein in the rumen bacterium Selenomonas ruminantium subsp. lactilytica. Our research also includes polyglutamate biosynthesis and the degradation enzymes of a Bacillus subtilis Natto starter, acetan (a polysaccharide produced by Acetobacter and Gluconobacter)-degrading enzymes of Paenibacillus sp. and catabolic enzymes of amino acid and polyamine in Pseudomonas aeruginosa PAOl. The topics of these projects are available on our Web site

Comparison between Cases of Total Hip Arthroplasty Followed by Colonna Capsular Arthroplasty and Lorenz Cast Reduction in Patients with Developmental Dysplasia of the Hip

Most patients with developmental dysplasia of the hip (DDH) now receive closed-reduction treatment within 6 months after birth. The long-term outcomes of patients with late-detection DDH have remained unclear. We reviewed the clinical records of 18 patients who underwent Colonna capsular arthroplasty (n=8) or closed reduction (n=10) for developmental dysplasia of the hip as infants or young children and underwent total hip arthroplasty approximately in midlife. Both the Colonna capsular arthroplasty and closed reduction groups achieved good clinical results after total hip arthroplasty. However, the operating time was longer and the improvements of hip range of motion and clinical score were significantly worse in the Colonna capsular arthroplasty group than in the closed reduction group

Initiation of skin basement membrane formation at the epidermo-dermal interface involves assembly of laminins through binding to cell membrane receptors

To study the mechanism of basement membrane formation, we determined by immunochemistry temporal and spatial expression of laminin-5 (Ln-5), laminin-1 (Ln-1) and their integrin receptors during early skin morphogenesis. A 3-dimensional skin culture was used that allows the study of the sequential molecular events of basement membrane formation at the epidermodermal interface. During early anchorage of keratinocytes to the extracellular matrix there is expression of Ln-5, BP-230 antigen and &#945;3, &#946;1 integrin subunits. During epidermal stratification and prior to the formation of the lamina densa there is assembly of Ln-5, Ln-1, collagen IV and nidogen accompanied by keratinocyte basal clustering of &#945;2, &#945;3, &#945;6, &#946;1, and &#946;4 integrin subunits. The assembly pattern of Ln-1 and Ln-5 can be disturbed with functional antibodies against the &#946;1 (AIIB2) and &#945;6 (GoH3) integrin subunits. Ln-1 assembly can also be disturbed with antibodies against its E8 domain and by competitive inhibition with a synthetic peptide (AG-73) derived from its G-4 domain. Quantitative RT-PCR showed that the dermis contributes about 80% of the laminin &#947;1 chain mRNA while 20% is produced by the epidermis which emphasizes its dual tissue origin and the major contribution of the mesenchyma in laminin production. The laminin &#947;2 chain mRNA, present in Ln-5, was mostly of epidermal origin. This study presents evidence that during the initiation of basement membrane formation, laminins bind to keratinocyte plasma membrane receptors and thus may serve as nucleation sites for further polymerization of these compounds by a self-assembly process.</p

Difference between carbohydrate antigen 19-9 and fluorine-18 fluorodeoxyglucose positron emission tomography in evaluating the treatment efficacy of neoadjuvant treatment in patients with resectable and borderline resectable pancreatic ductal adenocarcinoma: Results of a dual-center study

kita, H, Takahashi, H, Eguchi, H, et al. Difference between carbohydrate antigen 19‐9 and fluorine‐18 fluorodeoxyglucose positron emission tomography in evaluating the treatment efficacy of neoadjuvant treatment in patients with resectable and borderline resectable pancreatic ductal adenocarcinoma: Results of a dual‐center study. Ann Gastroenterol Surg. 2020; 00: 1– 9. https://doi.org/10.1002/ags3.12418

The application of methylation specific electrophoresis (MSE) to DNA methylation analysis of the 5' CpG island of mucin in cancer cells

<p>Abstract</p> <p>Background</p> <p>Methylation of CpG sites in genomic DNA plays an important role in gene regulation and especially in gene silencing. We have reported mechanisms of epigenetic regulation for expression of mucins, which are markers of malignancy potential and early detection of human neoplasms. Epigenetic changes in promoter regions appear to be the first step in expression of mucins. Thus, detection of promoter methylation status is important for early diagnosis of cancer, monitoring of tumor behavior, and evaluating the response of tumors to targeted therapy. However, conventional analytical methods for DNA methylation require a large amount of DNA and have low sensitivity.</p> <p>Methods</p> <p>Here, we report a modified version of the bisulfite-DGGE (denaturing gradient gel electrophoresis) using a nested PCR approach. We designated this method as methylation specific electrophoresis (MSE). The MSE method is comprised of the following steps: (a) bisulfite treatment of genomic DNA, (b) amplification of the target DNA by a nested PCR approach and (c) applying to DGGE. To examine whether the MSE method is able to analyze DNA methylation of mucin genes in various samples, we apply it to DNA obtained from state cell lines, ethanol-fixed colonic crypts and human pancreatic juices.</p> <p>Result</p> <p>The MSE method greatly decreases the amount of input DNA. The lower detection limit for distinguishing different methylation status is < 0.1% and the detectable minimum amount of DNA is 20 pg, which can be obtained from only a few cells. We also show that MSE can be used for analysis of challenging samples such as human isolated colonic crypts or human pancreatic juices, from which only a small amount of DNA can be extracted.</p> <p>Conclusions</p> <p>The MSE method can provide a qualitative information of methylated sequence profile. The MSE method allows sensitive and specific analysis of the DNA methylation pattern of almost any block of multiple CpG sites. The MSE method can be applied to analysis of DNA methylation status in many different clinical samples, and this may facilitate identification of new risk markers.</p

Effects of high magnetic field on Euglena gracilis

The effects of high magnetic field on Euglena gracilis Z were examined. When a horizontal magnetic field gradient (ca. 400 T2m-1) was applied, living E. gracilis moved to a higher field (positive magnetotaxis), whereas dead one gathered in a lower field. Magnetotaxis was not observed in a uniform magnetic field of 8 T. E. gracilis was oriented almost perpendicularly to the magnetic field regardless of life and death. Magnetotaxis of E. gracilis would be explained by taking into account inhomogeneous magnetic forces on and magnetic orientation of E. gracilis

Spatiotemporal characteristics of hemodynamic changes in the human lateral prefrontal cortex during working memory tasks

Abstract The prefrontal cortex (PFC) is widely believed to subserve mental manipulation and monitoring processes ascribed to the central executive (CE) of working memory (WM). We attempted to examine and localize the CE by functional imaging of the frontal cortex during tasks designed to require the CE. Using near-infrared spectroscopy, we studied the spatiotemporal dynamics of oxygenated hemoglobin (oxy-Hb), an indicator of changes in regional cerebral blood flow, in both sides of lateral PFC during WM intensive tasks. In most participants, increases in oxy-Hb were localized within one subdivison during performance of the n-back task, whereas oxy-Hb increased more diffusely during the random number generation (RNG) task. Activation of the ventrolateral PFC (VLPFC) was prominent in the n-back task; both sustained and transient dynamics were observed. Transient dynamics means that oxy-Hb first increases but then decreases to less than 50% of the peak value or below the baseline level before the end of the task. For the RNG task sustained activity was also observed in the dorsolateral PFC (DLPFC), especially in the right hemisphere. However, details of patterns of activation varied across participants: subdivisions commonly activated during performance of the two tasks were the bilateral VLPFCs, either side of the VLPFC, and either side of the DLPFC in 4, 2, and 4 of the 12 participants, respectively. The remaining 2 of the 12 participants had no regions commonly activated by these tasks. These results suggest that although the PFC is implicated in the CE, there is no stereotyped anatomical PFC substrate for the CE

Androgen replacement therapy for cancer-related symptoms in male advanced cancer patients : study protocol for a randomised prospective trial (ARTFORM study)

Recent studies reveal that hypogonadism with low serum androgen levels is associated with advanced cancer and induction of most cancer-related symptoms.We designed an ARTFORM study to evaluate the efficacy of androgen replacement therapy in male advanced cancer patients. The ARTFORM study is an investigatorinitiated, randomised controlled trial comparing intramuscle injection of testosterone enanthate with non-administration in male advanced cancer patients with non-curative locally advanced or metastatic lesions. Serum total and free testosterone levels are measured and patients with low testosterone level are randomised. The primary endpoint is the difference in validated health-related quality of life questionnaires at week 12. Trial registration of the ARTFORM study is assigned to University hospital Medical Information Network, Center identifier UMIN 000010939

Hepatopulmonary syndrome-discussion of cardiopulmonary parameters

We report a 70-year-old man with hepatopulmonary syndrome (HPS) in C liver cirrhosis. Hypoxemia worsened markedly, especially on exertion, while the hepatic function was clinically stable. Contrast echocardiography, 99mTc macroaggregated albumin (99mTcMAA) lung scan, and pulmonary angiography were performed. The findings suggested the presence of both intrapulmonary vascular dilatation and substantial right-to-left shunt. The contribution of intrapulmonary vascular abnormalities in patients with severe liver cirrhosis without abnormal chest radiography and spirometry tests when marked hypoxemia is present should be investigated