Repeated batch and continuous syntheses of N-(benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester with immobilized thermolysin

Summary N-(Benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester was synthesized from N-(benzyloxycarbonyl)-l-phenylalanine and l-phenylalanine methyl ester in an aqueous solution (aqueous phasic reaction), in an aqueous/organic biphasic system (biphasic reaction), and in an organic solvent (organic phasic reaction) with immobilized thermolysin. In the aqueous phasic reaction with thermolysin immobilized on Amberlite XAD-7, the whole product was trapped inside the support; extraction with ethyl acetate was needed to recover the product, and the equilibrium yield was low (about 65%). With the biphasic and organic phasic reactions with ethyl acetate as an organic solvent, the yield was around 95%. Because of the high yield and feasibility of operation, repeated batch and continuous reactions were done in the biphasic and organic phasic systems, respectively. The half-lives of the activity for the immobilized enzyme used in the biphasic system at 40°C by repeated batch operation and in a plug flow reactor fed with substrate dissolved in ethyl acetate at 40°C and 30°C were estimated to be about 200 h (67 batches), 420 h, and 1100 h, respectively.     

Early detection of gastric cancer after Helicobacter pylori eradication due to endoscopic surveillance

Background

Helicobacter pylori eradication therapy is commonly performed to reduce the incidence of gastric cancer. However, gastric cancer is occasionally discovered even after successful eradication therapy. Therefore, we examined the prognosis of gastric cancer patients, diagnosed after successful H. pylori eradication therapy.

Materials and Methods

All‐cause death rates and gastric cancer‐specific death rates in gastric cancer patients who received successful H. pylori eradication treatment was tracked and compared to rates in patients who did not receive successful eradication therapy.

Results

In total, 160 gastric cancer patients were followed‐up for up to 11.7 years (mean 3.5 years). Among them, 53 gastric cancer patients received successful H. pylori eradication therapy prior to gastric cancer diagnosis. During the follow‐up period, 11 all‐cause deaths occurred. In the successful eradication group, the proportion of patients with cancer stage I was higher. The proportions of patients who received curative endoscopic therapy and endoscopic examination in the 2 years prior to gastric cancer diagnosis were also higher in the successful eradication group. Kaplan–Meier analysis of all‐cause death and gastric cancer‐specific death revealed a lower death rate in patients in the successful eradication group (P = .0139, and P = .0396, respectively, log‐rank test). The multivariate analysis showed that endoscopy within 2 years before cancer diagnosis is associated with stage I cancer.

Conclusions

Possible early discovery of gastric cancer after H. pylori eradication due to regular endoscopic surveillance may contribute to better prognosis of patients with gastric cancer.     

Tumor‐suppressive roles of ΔNp63β‐miR‐205 axis in epithelial–mesenchymal transition of oral squamous cell carcinoma via targeting ZEB1 and ZEB2

We previously revealed that epithelial‐to‐mesenchymal transition (EMT) was mediated by ΔNp63β, a splicing variant of ΔNp63, in oral squamous cell carcinoma (OSCC). Recent studies have highlighted the involvement of microRNA (miRNA) in EMT of cancer cells, though the mechanism remains unclear. To identify miRNAs responsible for ΔNp63β‐mediated EMT, miRNA microarray analyses were performed by ΔNp63β‐overexpression in OSCC cells; SQUU‐B, which lacks ΔNp63 expression and displays EMT phenotypes. miRNAs microarray analyses revealed miR‐205 was the most up‐regulated following ΔNp63β‐overexpression. In OSCC cells, miR‐205 expression was positively associated with ΔNp63 and negatively with zinc‐finger E‐box binding homeobox (ZEB) 1 and ZEB2, potential targets of miR‐205. miR‐205 overexpression by miR‐205 mimic transfection into SQUU‐B cells led to decreasing ZEB1, ZEB2, and mesenchymal markers, increasing epithelial markers, and reducing cell motilities, suggesting inhibition of EMT phenotype. Interestingly, the results opposite to this phenomenon were obtained by transfection of miR‐205 inhibitor into OSCC cells, which express ΔNp63 and miR‐205. Furthermore, target protector analyses revealed direct regulation by miR‐205 of ZEB1 and ZEB2 expression. These results showed tumor‐suppressive roles of ΔNp63β and miR‐205 by inhibiting EMT thorough modulating ZEB1 and ZEB2 expression in OSCC.     

Novel physical chemistry approaches in biophysical researches with advanced application of lasers: Detection and manipulation

Novel methodologies utilizing pulsed or intense CW irradiation obtained from lasers have a major impact on biological sciences. In this article, recent development in biophysical researches fully utilizing the laser irradiation is described for three topics, time-resolved fluorescence spectroscopy, time-resolved thermodynamics, and manipulation of the biological assemblies by intense laser irradiation. First, experimental techniques for time-resolved fluorescence spectroscopy are concisely explained in Section 2. As an example of the recent application of time-resolved fluorescence spectroscopy to biological systems, evaluation of the viscosity of lipid bilayer membranes is described. The results of the spectroscopic experiments strongly suggest the presence of heterogeneous membrane structure with two different viscosity values in liposomes formed by a single phospholipid. Section 3 covers the time-resolved thermodynamics. Thermodynamical properties are important to characterize biomolecules. However, measurement of these quantities for short-lived intermediate species has been impossible by traditional thermodynamical techniques. Recently, development of a spectroscopic method based on the transient grating method enables us to measure these quantities and also to elucidate reaction kinetics which cannot be detected by other spectroscopic methods. The principle of the measurements and applications to some protein reactions are reviewed. Manipulation and fabrication of supramolecues, amino acids, proteins, and living cells by intense laser irradiation are described in Section 4. Unconventional assembly, crystallization and growth, amyloid fibril formation, and living cell manipulation are achieved by CW laser trapping and femtosecond laser-induced cavitation bubbling. Their spatio-temporal controllability is opening a new avenue in the relevant molecular and bioscience research fields. This article is part of a Special Issue entitled “Biophysical Exploration of Dynamical Ordering of Biomolecular Systems” edited by Dr. Koichi Kato.     

Use of a surface plasmon resonance biosensor for the determination of binding constants of transiently expressed recombinant antibody to its antigen

Summary By using a commercially available surface plasmon resonance (SPR) biosensor, the values of the association rate constant (kass), dissociation rate constant (kdiss), and association constant (KA = kass / kdiss) for binding to the antigens were determined. They were almost the same for the recombinant antibody expressed in COS cells, CHO cells, and mouse hybridoma cells. The system of transient expression of the recombinant antibody (Ab) in COS cells and SPR analysis of the supernatant should be useful for rapid expression and evaluation of the binding ability of large numbers of engineered Abs.     

A novel immunohistochemical technique for the detection of human oral carcinomas transplanted in nude mice using mouse monoclonal antibodies

 We have developed a new immunostaining technique specifically for the detection of human tumors transplanted into nude mice using mouse monoclonal antibodies (MoAbs) produced in our laboratory. The formation of a molecular complex consisting of three components (mouse MoAb or hybridoma supernatant, biotin-labeled anti-mouse immunoglobulins, and normal nude mouse serum) markedly reduced background staining and enhanced specific reaction with the transplanted tumors in nude mice. When MoAb production by electrofusion was screened with this new method, the incidence of hybridoma supernatant reactive with sections of transplanted tumor was 2.3 per 100 wells immunostained. These results suggest that production of MoAbs using transplanted tumors is immunohistochemically possible and that this method may provide a new means for developing useful tumor markers. Accepted: 30 September 1996     

Fluorescence energy transfer in tryptophanase

Excitation of apotryptophanase from Escherichia coli$\text{B}\text{lt7-A}$ at 290 nm yielded a fluorescence emission centered at 340 nm. Binding of pyridoxal phosphate to apoenzyme induced quenching of protein fluorescence concomitant with an appearance of another peak at 510 nm by way of energy transfer from tryptophan. Based on the results, an approximate distance between the coenzyme and tryptophan was estimated to be 18–24 Å according to the Förster's theory. The ozone-inactivated enzyme yielded only the 340 nm-peak upon excitation at 290 nm following reconstitution with the coenzyme. The fluorescence decay time of the tryptophyl residue was somewhat increased by ozone-inactivation. These results suggest that the tryptophyl residue essential for the activity is involved in a direct interaction with the coenzyme.     

F Pilus as f+ Antigen

Specific aggregate formation of F pili was observed, by electron microscopy, in a mixture of male Escherichia coli (or of isolated F pili) and anti-f(+) serum. Cellular appendages other than F pili never showed such aggregation when mixed with anti-f(+) serum. The f(+) agglutinability of male cells, as well as F piliation, was sensitive to mechanical agitation. The f(+) agglutination was inhibited when appropriate numbers of phage M12, capable of attaching to F pili, were mixed with the male culture before the addition of anti-f(+) serum. Correlation between f(+) agglutinability and the extent of F piliation was observed. It was concluded that the F pilus is the structure of the f(+) antigen and is responsible for f(+) agglutination.     

New orally bioavailable 2-aminobenzamide-type histone deacetylase inhibitor possessing a (2-hydroxyethyl)(4-(thiophen-2-yl)benzyl)amino group

New 2-aminobenzamide-type histone deacetylase (HDAC) inhibitors were synthesized. They feature a sulfur-containing bicyclic arylmethyl moiety—a surface recognition domain introduced to increase in cellular uptake—and a substituted tert-amino group which affects physicochemical properties such as aqueous solubility. Compound 22 with a (2-hydroxyethyl)(4-(thiophen-2-yl)benzyl)amino group reduced the volume of human colon cancer HCT116 xenografts in nude mice to T/C 67% by oral administration at 45 mg/kg, which was comparable to the rate (T/C 62%) for a positive control, MS-275. Western blot analyses as well as cell cycle and TUNEL assays by flow cytometry suggested that the two compounds inhibited the growth of cancer cells via similar mechanisms.