Analysis of Flow Phenomena in Gastric Contents Induced by Human Gastric Peristalsis Using CFD

This paper uses computational fluid dynamics to simulate and analyze intragastric fluid motions induced by human peristalsis. We created a two-dimensional computational domain of the distal stomach where peristalsis occurs. The motion of the gastric walls induced by an antral contraction wave (ACW) on the wall of the computational domain was well simulated using a function defined in this study. Retropulsive flow caused by ACW was observed near the occluded region, reaching its highest velocity of approximately 12 mm/s in the narrowest region. The viscosity of the model gastric contents applied in this study hardly affected the highest velocity, but greatly affected the velocity profile in the computational domain. The shear rate due to gastric fluid motion was calculated using the numerical output data. The shear rate reached relatively high values of approximately 20 s⁻¹ in the most occluded region. The shear rate profile was almost independent of the fluid viscosity. We also simulated mass transfer of a gastric digestive enzyme (pepsin) in model gastric content when peristalsis occurs on the gastric walls. The visualized simulation results suggest that gastric peristalsis is capable of efficiently mixing pepsin secreted from the gastric walls with an intragastric fluid.     

Free radical mechanisms in enzyme reactions

Free radicals are formed in prosthetic groups or amino acid residues of certain enzymes. These free radicals are closely related to the activation process in enzyme catalysis, but their formation does not always result in the formation of substrate free radicals as a product of the enzyme reactions. The role of free radicals in enzyme catalysis is discussed.     

Monoclonal antibodies against Pseudomonas aeruginosa elastase: a neutralizing antibody which recognizes a conformational epitope related to an active site of elastase.

We have established seven murine hybridoma cell lines which produce monoclonal antibodies (mAbs) against Pseudomonas aeruginosa elastase. The seven mAbs recognized at least six different epitopes on the elastase molecule. All mAbs inhibited both enzymatic activities of elastase and protease, in which elastin fluorescein and hide powder azure were used as substrates, respectively. One of them, mAb E-4D3, strongly neutralized enzymatic activities of peptidase in which furylacryloyl-glycyl-leucinamide was used as a substrate, as well as of elastase and protease. In contrast, the other six mAbs did not neutralize peptidase activity at all. The Ki value for furylacryloyl-glycl-leucinamide of E-4D3, as well as its Fab fragment, was comparable to those for metalloprotease inhibitors such as phosphoramidon and Zincov inhibitor. The binding of mAb E-4D3 was inhibited by phosphoramidon and Zincov inhibitor, but not by metal chelators such as EDTA and o-phenanthroline. A line of evidence suggests that mAb E-4D3 directly interacts with active site and highly neutralizes enzymatic activity of P. aeruginosa elastase. Data of Western blotting and ELISA suggest that mAb E-4D3 is likely to recognize an elastase molecule in a conformation-dependent manner as an epitope. In contrast, the neutralizing activity of the other mAbs against elastase and protease seems to be caused by a low accessibility of an enzyme to insoluble and high-molecular-mass substrates through the binding and steric hindrance of the mAbs to an enzyme.     

Hydrogen evolution by co-immobilized Chlorella vulgaris and Clostridium butyricum cells

Whole cells of Chlorella vulgaris and Clostridium butyricum were co-immobilized in 2% agar gel. NADP was suitable as an electron carrier. The rate of hydrogen evolution increased with increasing NADP concentration. The optimum conditions for hydrogen evolution were pH 7.0 and 37°C. The immobilized C. vulgaris-NADP-immobilized Cl. butyricum system continuously evolved hydrogen at a rate of 0.29–1.34 μmol/h per mg Chl for 6 days. On the other hand, the system without NADP evolved only a trace amount of hydrogen.     

Carbonic anhydrase inhibitors: Inhibition of the tumor-associated isozymes IX and XII with polyfluorinated aromatic/heterocyclic sulfonamides

The tumor-associated transmembrane carbonic anhydrase (CA, EC 4.2.1.1) isozymes IX (CA IX) and XII (CA XII) are involved in acidification of hypoxic tumors, a process correlated with poor prognosis and clinical outcome of patients harboring such tumors. This process may be reversed by inhibiting these enzymes with potent sulfonamide/sulfamate inhibitors. A series of such aromatic/heterocyclic sulfonamides incorporating 2,3,5,6-tetrafluorobenzoyl-, 2,3,5,6-tetrafluoro- phenylsulfonyl- and pentafluorophenylureido moieties has been investigated for its interaction with the catalytic domain of the human isozymes hCA IX and hCA XII. Some of these compounds showed excellent inhibitory properties against both isozymes IX and XII, with several subnanomolar inhibitors detected for the first time. These sulfonamides may constitute valuable candidates for the development of novel antitumor therapies based on the inhibition of such tumor-associated CA isozymes.     

Sequence-dependent hydrolysis of RNA using modified oligonucleotide splints and RNase H

To cleave RNA molecules using RNase H in a site-specific manner, a short deoxyoligonucleotide (3-5mer) joining with 2'-O-methyl oligonucleotide(s) was designed as a DNA splint to be used. Model experiments were carried out using ribooligonucleotide substrates (9 and 18 mer). It was found that the use of this type of splints (9 mer) causes a unique cleavage by RNase H. For example, when 3'm (GA)d(AGAA)m(GGU)5' was used as a hybridization strand, 32pUCUUUCUUCUUCCAGGAU was cleaved specifically between U11 and C12 to yield 32pUCUUUCUUCUU. This method will have a variety of applications for the study of RNA.     

Identification of I:A mismatch base-pairing structure in DNA

Deoxyoligonucleotides containing deoxyinosine residues at positions corresponding to ambiguous nucleotides derived from an amino acid sequence have been successfully used as hybridization probes. It is assumed that the hypoxanthine residue can make base pairs with multiple bases. In order to obtain direct evidence for I:A base-pairing, a self-complementary deoxyoligonucleotide, d(G-G-I-A-C-C), was synthesized and its properties were examined by NMR spectroscopy. Three hydrogen-bonded imino proton resonances are observed at low temperatures in H2O suggesting the formation of a self-duplex with complete base pairing. Nuclear Overhauser effect (NOE) experiments showed that a signal at 15.1 ppm originated from the imino proton (H1) of the dI residue (I3) which is hydrogen-bonded to the dA residue (A4). Both the I3 and A4 residues were assumed to have taken an anti glycosidic conformation since irradiating the H1 of I3 gave NOEs both to its own H2 and to that of A4, an NOE also being observed between the H2 protons of I3 and A4. Comparison of the 31P NMR spectra of d(G-G-I-A-C-C) and d(G-G-I-C-C-C) showed the backbone structure of d(G-G-I-A-C-C) to have been disturbed by the presence of purine:purine base pairs in the middle of the hexamer duplex.     

The in vivo stability, maturation and aminoacylation of anticodon-substituted Escherichia coli initiator methionine tRNAs

We have constructed eight anticodon-modified Escherichia coli initiator methionine (fMet) tRNAs by insertion of synthetic ribotrinucleotides between two fragments ('half molecules') derived from the initiator tRNA. The trinucleotides, namely CAU (the normal anticodon), CAA, CAC, CAG, GAA, GAC, GAG and GAU, were joined to the 5' and 3' tRNA fragments with T4 RNA ligase. The strategy of reconstruction permitted the insertion of radioactive 32P label between nucleotides 36 and 37. tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes, and the following properties were evaluated: the stability of these eubacterial tRNA variants in the eukaryotic oocytes; the enzymatic modification of the adenosine at position 37 (3' adjacent to the anticodon) and aminoacylation of the chimeric tRNAs by endogenous oocyte aminoacyl-tRNA synthetases. In contrast to other variants, the two RNAs having CAU and GAU anticodons were stable and underwent quantitative modification at A-37. These results show that the enzyme responsible for the modification of A-37 to N-[N-(9-beta-D-ribofuranosylpurine-6-yl)carbamoyl]threonine (t6A) is present in the cytoplasm of oocytes and is very sensitive to the anticodon environment of the tRNA. Also, these same GAU and CAU anticodon-containing tRNAs are fully aminoacylated with the heterologous oocyte aminoacyl-tRNA synthetases in vivo. During the course of this work we developed a generally applicable assay for the aminoacylation of femtomole amounts of labelled tRNAs.     

A new solid-phase synthesis of oligoribonucleotides by the phosphoro-p-anisidate method using tetrahydrofuranyl protection of 2''-hydroxyl groups.

Six nonaribonucleotides containing the 5'-splice site, one complementary nonamer and an octadecamer containing the 3'-splice site have been synthesized on a polymer support using the phosphoro-p-anisidate method. A 5'-linked 2'-O-tetrahydrofuranyl-N-protected nucleoside 3'-(o-chlorophenyl)phosphoro-p-anisidate was used as the starting nucleotide, and the chain elongated in the 3'-direction by removing the p-anisidate protecting group with isoamyl nitrite under neutral conditions. The octadecamer has been synthesized using dinucleotide blocks and a 3'-terminal trinucleotide.