Development of the Overlapping Oligonucleotide Database and its application to signal sequence search of the human genome

We have developed ODS (Overlapping Oligonucleotide Databasefor Signal Sequence Search)—the first relational databasethat integrates information on biological features into thesearch for signal sequences. In existing biological sequencedatabases, even relational ones, retrieving nucleotide sequencesbased on their biological features involves much labour andtime or even the development of a new program. GenBank sequencedata, including FEATURES records, are organized into three relationaltables in ODS. Nucleotide sequences are transformed into overlappingoligonucleotides in order to facilitate the signal sequencesearch rapidly without the need for specific alignment programs.This transformation leads to a one-to-one correspondence betweenthe nucleotide sequence and its biological feature. The signalsequence search by ODS is done in SQL queries and ODS obviatesthe need for molecular biologists to write computer programs.The application of ODS to searches of promoter regions revealedputative cis-acting elements and basic statistical analysesof occurrences of oligonucleotides showed interesting findingsconcerning the ‘cg’ dinucleotide.     

Increased production of alpha-amylase by Bacillus amyloliquefaciens in the presence of glycine

The production of alpha-amylase by Bacillus amyloliquefaciens increased by a factor of 300 when glycine was added to a chemically defined simple medium at the early-logarithmic phase of growth. Glycine was not metabolized to a significant extent under the conditions used, but it considerably prevented the lowering of the pH of the culture.     

Effects of Phosphate in Medium on Protein Secretion in a Protein-Producing Bacterium, Bacillus brevis 47

Bacillus brevis 47 secreted up to 1 mg of protein per ml in a chemically defined medium, depending on phosphate concentration. The composition of exoproteins was altered quantitatively by the concentration of external phosphate. Morphologically, B. brevis 47 showed a distinct three-layered cell wall structure and shed the outer two layers during growth.     

Morphological alterations of cell wall concomitant with protein release in a protein-producing bacterium, Bacillus brevis 47.

Bacillus brevis 47 secreted vast amounts of protein into the medium and had a characteristic three-layered cell wall. The three layers are designated, from the outermost to the innermost layer, as the outer wall (4.2 nm), the middle wall(8.5 nm), and the inner wall (2.1-3.7 nm). The inner wall might be a peptidoglycan layer. The fine cell wall structure was morphologically altered to various extents, depending on the growth period. At the early stationary phase of growth, cells began to shed the outer two layers of a limited area of the surface. This shedding was complete after further cell growth. The morphological alterations in the cell wall occurred concomitantly with a prominent increase in protein excretion. When protein secretion was severely inhibited by growing cells with Mg2+, morphological alterations in the cell wall were not observed, even at the late stationary phase of growth. This was also the case with a nonprotein-producing mutant, strain 47-5-25. When cells were incubated in buffers, the outer two layers of the cell wall were specifically removed, leaving cells surrounded only by the inner wall layer. The layers removed by incubation were recovered by high-speed centrifugation. This fraction consisted of two layers resembling the outer and middle wall layers. Protein secreted by B. brevis 47-5 consisted mainly of two proteins with approximate molecular weights of 150,000 and 130,000. Proteins released by incubating cells in buffers and proteins in the outer- and middle-wall-enriched fraction were also composed mainly of two proteins with the same molecular weights as those secreted into the medium. Therefore, we conclude that B. brevis 47 secretes proteins derived from the outer two layers of cell wall and these components are synthesized even after the shedding of the outer two layers.     

Toward more comprehensive environmental impact assessments: interlinked global models of LCIA and IAM applicable to this century

The International Journal of Life Cycle Assessment - Despite the long-standing demand for research on dynamic lifecycle assessment (LCA) for policymaking, only a few studies have addressed this...     

Fluorescent-Based Methods for Gene Knockdown and Functional Cardiac Imaging in Zebrafish

A notable advantage of zebrafish as a model organism is the ease of gene knockdown using morpholino antisense oligonucleotide (MO). However, zebrafish morphants injected with MO for a target protein often show heterogeneous phenotypes, despite controlling the injection volume of the MO solution in all embryos. We developed a method for estimating the quantity of MO injected into each living morphant, based on the co-injection of a control MO labeled with the fluorophore lissamine. By applying this method for knockdown of cardiac troponin T (tnnt2a) in zebrafish, we could efficiently select the partial tnnt2a-depleted zebrafish with a decreased heart rate and impairment of cardiac contraction. To investigate cardiac impairment of the tnnt2a morphant, we performed fluorescent cardiac imaging using Bodipy-ceramide. Cardiac image analysis showed moderate reduction of tnnt2a impaired diastolic distensibility and decreased contraction and relaxation velocities. To the best of our knowledge, this is the first report to analyze the role of tnnt2a in cardiac function in tnnt2a-depleted living animals. Our combinatorial approach can be applied for analyzing the molecular function of any protein associated with human cardiac diseases.     

Prognostic Impact of Hyaluronan and Its Regulators in Pancreatic Ductal Adenocarcinoma

Background

Although pancreatic ductal adenocarcinoma is characterized by an abundant stroma enriched with hyaluronan (HA), the prognostic impact of HA and its regulators remains unknown.

Methods

Using immunohistochemistry, expression patterns of HA and its regulators, including a synthesizing enzyme (HAS2), and a degrading enzyme (HYAL1) were investigated in patients who received surgical resection. The prognostic significance of these markers and other clinicopathological variables was determined using univariate and multivariate analyses. The HA levels were determined quantitatively by enzyme-linked immunosorbent assay (ELISA).

Results

We found that strong expressions of HA (P=0.008) and HAS2 (P=0.022) were significantly associated with shorter survival time after surgery. By contrast, weak expression of HYAL1 was significantly associated with poor survival (P=0.001). In multivariate analysis, tumor stage (hazard ratio (HR)=2.76, 95% confidence interval (CI): 1.14-6.66 P=0.024), strong HA expression (HR=6.04, 95%CI: 1.42-25.69 P=0.015), and weak HYAL1 expression (HR=3.16, 95%CI: 1.19-8.40 P=0.021) were independent factors predicting poor survival. ELISA revealed higher concentration of HA in pancreatic cancer tissues than in normal pancreatic tissues (P=0.001).

Conclusion

These findings suggest, for the first time, that HA and its regulators may have prognostic impact in patients with pancreatic cancer.