Continuous enzymatic production of peptide precursor in aqueous/organic biphasic medium

N-(benzyloxycarbonyl)-L-aspartic acid (Z-L-Asp) has generally been used as a carboxyl substrate for the enzymatic synthesis of a precursor of aspartame (synthetic sweetener); however, alternative inexpensive protection groups have been in demand for lowering the total cost of its industrial-scale production. A formyl group (F-) was found to be a more desirable protecting group for the N-terminus of amino acid derivatives due to its low cost of preparation, introduction, and removal. The yield of F-AspPheOMe (N-formyl-L-aspartyl-L-phe- nylalanine methylester), however, was found to be <10% in a conventional aqueous medium. We found that F-L-Asp and L-PheOMe were partitioned mainly to the aqueous phase in an aqueous/organic biphasic medium, whereas F-AspPheOMe partitioned to the organic phase, especially when some extracting agents were added. In this study, simultaneous operation of an enzymatic reaction and a product separation by liquid-liquid extraction was thus applied to the F-AspPheOMe synthesis. We succeeded in synthesizing F-AspPheOMe continuously in an aqueous/tributylphosphate (TBP) biphasic medium with >95% yield, which was about tenfold higher than that in an aqueous monophasic medium.     

Neutral taxoids from Taxus cuspidata as modulators of multidrug-resistant tumor cells

Two taxoids, taxinine NN-7 (1) and 3,11-cyclotaxinine NN-2 (2), were isolated from the neutral fraction of the EtOAc extract of a mixture of needles and young stems of Taxus cuspidata. The structures were determined by spectroscopic analysis. Both compounds showed some activity as modulators of multidrug-resistant tumor cells.     

A positive screening for drugs that specifically inhibit the Ca2+-signaling activity on the basis of the growth promoting effect on a yeast mutant with a peculiar phenotype

An inappropriate activation of a signaling pathway in yeast often has a deleterious physiological effect and causes various defects, including growth defects. In a certain genetic background (deltazds1) of Saccharomyces cerevisiae, the cell-cycle progression in G2 is specifically blocked in the medium with CaCl2 by the hyperactivation of the Ca2+-signaling pathways. Here, we developed a novel drug screening procedure designed to detect the active compounds that specifically attenuate the Ca2+-signaling activity on the basis of the ability to abrogate the growth defect of the cells suffering from the hyperactivated Ca2+ signal. Using known calcineurin inhibitors as model compounds, we have established the screening conditions for the drugs that suppress the Ca2+-induced growth inhibition. An indicator strain with an increased drug sensitivity was constructed with a syr1/erg3 null mutation.     

Control of bacteriophage T4 tail lysozyme activity during the infection process

Bacteriophage T4 has an efficient mechanism for injecting the host Escherichiacoli cell with genomic DNA. Its gene product 5 (gp5) has a needle-like structure attached to the end of a tube through which the DNA passes on its way out of the head and into the host. The gp5 needle punctures the outer cell membrane and then digests the peptidoglycan cell wall in the periplasmic space. gp5 is normally post-translationally cleaved between residues 351 and 352. The function of this process in controlling the lysozyme activity of gp5 has now been investigated. When gp5 is over-expressed in E.coli, two mutants (S351H and S351A) showed a reduction of cleavage products and five other mutants (S351L, S351K, S351Y, S351Q, and S351T) showed no cleavage. Furthermore, in a complementation assay at 20 degrees C, the mutants that had no cleavage of gp5 produced a reduced number of plaques compared to wild-type T4. The crystal structure of the non-cleavage phenotype mutant of gp5, S351L, complexed with gene product 27, showed that the 18 residues in the vicinity of the potential cleavage site (disordered in the wild-type structure) had visible electron density. The polypeptide around the potential cleavage site is exposed, thus allowing access for an E.coli protease. The lysozyme activity is inhibited in the wild-type structure by a loop from the adjacent gp5 monomer that binds into the substrate-binding site. The same inhibition is apparent in the mutant structure, showing that the lysozyme is inhibited before gp5 is cleaved and, presumably, the lysozyme is activated only after gp5 has penetrated the outer membrane.     

Detection of antibodies to Neospora caninum in cattle by enzyme-linked immunosorbent assay with truncated NcSRS2 expressed in Escherichia coli

The surface antigen 1-related sequence 2 of Neospora caninum (NcSRS2) is considered as an immunodominant antigen. In this study, the gene encoding truncated NcSRS2 (NcSRS2t) lacking an N-terminal signal peptide and C-terminal hydrophobic regions was expressed in Escherichia coli, and its diagnostic potential in an enzyme-linked immunosorbent assay (ELISA) was evaluated. ELISA could discriminate clearly between known N. caninum-positive and -negative sera from cattle. Field serum samples collected from cattle in Brazil were examined for the diagnosis of N. caninum infection using ELISA. Of the 197 samples analyzed, 64 (32.5%) samples were positive for antibodies to N. caninum. Of the 64 ELISA-positive samples, 58 (90.6%) were confirmed as positive by Western blot analysis with whole-parasite antigens. These results suggest that ELISA with recombinant NcSRS2t is an effective method for diagnosis of N. caninum infection in cattle.     

Endothelial dysfunction and hypercontractility of vascular myocytes are ameliorated by fluvastatin in obese Zucker rats

Static exercise causes activation of the sympathetic nervous system, which results in increased blood pressure (BP) and renal vascular resistance (RVR). The question arises as to whether renal vasoconstriction that occurs during static exercise is due to sympathetic activation and/or related to a pressure-dependent renal autoregulatory mechanism. To address this issue, we monitored renal blood flow velocity (RBV) responses to two different handgrip (HG) exercise paradigms in 7 kidney transplant recipients (RTX) and 11 age-matched healthy control subjects. Transplanted kidneys are functionally denervated. Beat-by-beat analyses of changes in RBV (observed via duplex ultrasound), BP, and heart rate were performed during HG exercise in all subjects. An index of RVR was calculated as BP/RBV. In protocol 1, fatiguing HG exercise (40% of maximum voluntary contraction) led to significant increases in RVR in both groups. However, at the end of exercise, RVR was more than fourfold higher in control subjects than in the RTX group (88 vs. 20% increase over baseline; interaction, P < 0.001). In protocol 2, short bouts of HG exercise (15 s) led to significant increases in RVR at higher workloads (50 and 70% of maximum voluntary contraction) in the control subjects (P < 0.001). RVR did not increase in the RTX group. In conclusion, we observed grossly attenuated renal vasoconstrictor responses to exercise in RTX subjects, in whom transplanted kidneys were considered functionally denervated. Our results suggest that renal vasoconstrictor responses to exercise in conscious humans are mainly dependent on activation of a neural mechanism.     

Rapid and quantitative detection of blood Serratia marcescens by a real-time PCR assay: its clinical application and evaluation in a mouse infection model

Large-scale nosocomial outbreaks of Serratia marcescens septicaemia in Japan have had a fatality rate of 20-60% within 48 h. As a countermeasure, a real-time PCR assay was constructed for the rapid diagnosis of S. marcescens septicaemia. This assay indeed detected S. marcescens in clinical blood specimens (at ca. 10(2)CFU ml(-1)), at a frequency of 0.5% in suspected cases of septicaemia. In mice, the assay provided estimates of blood S. marcescens levels at various infectious stages: namely, 10(7) to 10(8)CFU ml(-1) at a fatal stage (resulting in 100% death), 10(4)-10(5)CFU ml(-1) at a moderately fatal stage (resulting in 50% or more death), and <10(3)CFU ml(-1) at a mild stage (resulting in 100% survival), consistent with actual CFU measurements. Blood bacterial levels could be an important clinical marker that reflects the severity of septicaemia. The simultaneous detection of S. marcescens and the carbapenem resistance gene was also demonstrated.     

Gene expression, cellular localization, and enzymatic activity of diacylglycerol kinase isozymes in rat ovary and placenta

Female reproductive organs show remarkable cyclic changes in morphology and function in response to a combination of hormones. Evidence has accumulated suggesting that phosphoinositide turnover and the consequent diacylglycerol (DG) protein kinase C (PKC) pathway are intimately involved in these mechanisms. The present study has been performed to investigate the gene expression, cellular localization, and enzymatic activity of the DG kinase (DGK) isozymes that control the DG-PKC pathway. Gene expression for DGK, -, -, and - was detected in the ovary and placenta. Intense expression signals for DGK and - were observed in the theca cells and moderate signals in the interstitium and corpora lutea of the ovary. On the other hand, signals for DGK were seen more intensely in granulosa cells. In the placenta, signals for DGK and - were observed in the junctional zone, whereas those for DGK were detected in the labyrinthine zone. At higher magnification, the signals for DGK were mainly discerned in giant cytotrophoblasts, and those for DGK were found in small cytotrophoblasts of the junctional zone. DGK signals were observed in all cellular components of the labyrinthine zone, including mesenchyme, trabecular trophoblasts, and cytotrophoblasts. DGK signals were detected in the junctional zone on day 13 and 15 of pregnancy and were diffusely distributed both in the labyrinthine and junctional zones at later stages. The present study reveals distinct patterns of mRNA localization for DGK isozymes in the rat ovary and placenta, suggesting that each isozyme plays a unique role in distinct cell types in these organs.This work was supported by Grants-in-Aids from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan, the Uehara Memorial Foundation, the ONO Medical Research Foundation, the Ciba-Geigy Foundation (Japan) for the Promotion of Science, the Kato Memorial Bioscience Foundation, and the Yamagata Health Support Foundation (to K.G.).