Methodological aspects of measuring amino acid uptake in studies with porcine jejunal brush border membrane vesicles

With L-glutamine, as a representative amino acid this study was undertaken to examine the effects of substrate concentrations on initial and equilibrium amino acid uptake and intravesicular volume determined with porcine jejunal brush border membrane vesicles prepared by Mg2+-aggregation and differential centrifugation. Transport measurements (24 degrees C) were conducted by the rapid filtration manual procedure. Glutamine uptake was shown to occur into an osmotically-active space ranging between 1.09-1.58 microl/mg protein with little non-specific membrane binding. At different concentrations (in parentheses), the duration of initial glutamine uptake in both Na+ gradient and Na+-free conditions was 10 s (0.01 mM), 15 s (0.17 mM), and 20 s (1.9 and 9.4 mM), respectively. Substrate concentrations affected the duration of initial uptake, with lower substrate concentrations giving shorter duration for initial amino acid uptake. At different substrate concentrations (in parentheses), the time required to reach equilibrium glutamine uptake was 5 min (0.01 mM), 10 min (0.17 mM), and 60 min (1.9 and 9.4 mM), respectively. Thus, substrate concentrations also affected the time required to reach equilibrium uptake. The higher the substrate concentration, the longer the incubation time needed to reach equilibrium amino acid uptake. At the glutamine concentrations of 0.01, 0.17, 1.9, and 9.4 mM, the average intravesicular volume was estimated to be 1.58+/-0.21, 1.09+/-0.28, 1.24+/-0.18, and 1.36+/-0.21 microl/mg protein, respectively. Substrate concentrations had no effect (p>0.05) on the intravesicular volume of membrane vesicles. In conclusion, in the experiments on amino acid transport kinetics measured with the rapid filtration manual procedure, the incubation time used for measuring the initial uptake rate should be determined from the time course experiments conducted at the lowest substrate concentration used, whereas the intravesicular volume can be obtained from equilibrium uptake measured at any substrate concentrations.     

Regio-selective preparation of vinyl adenosine ester catalyzed by alkaline protease

The transesterification of divinyladipate with adenosine in DMF containing 20% (v/v) DMSO was catalyzed by Streptomyces sp. alkaline protease and esterification occurred exclusively at the 3-position of hydroxyl group of ribofuranose in adenosine to give 3-O-vinyladipoyl adenosine without other products.     

Pressure swing reactor for converting suspension of solid substrate p-hydroxyphenylhydantoin by immobilized d-hydantoinase

A reactor consisting of filter-separated two stirred compartments was developed to carry out the conversion of suspension of solid substrate d,l-p-hydroxyphenyl-hydantoin (pHPH) into d-n-carbamoyl-p-hydroxyphenylglycine (d-CpHPG) by immobilized d-hydantoinase (IDH). The immobilized enzyme and substrate suspensions were separated by a 60?μm filter to prevent the IDH from contamination with the insoluble impurities present in the substrate. The poor mass transfer between the two compartments limited the overall enzymatic reaction. It took 180?min for this reactor to accomplish the hydrolysis of 4% (w/v) pHPH. However, it took only 45?min for the reactor without using the filter to separate the two stirred compartments. The performance of the filter-separated reactor was significantly improved by applying pressure swing to the system. The pressure swing was generated by cyclically pressurizing the substrate compartment with nitrogen that caused the solution of the two compartments to flow back and forth through the filter. The reaction time for accomplishing the 4% (w/v) pHPH hydrolysis was reduced to 90?min when the pressure swing was applied with a frequency of 20?cycles/hr. The conversion of pHPH suspension of concentration as high as 15% (w/v) was easily accomplished in this pressure swing operated reactor. The used IDH of this reactor showed the same appearance as the fresh one. On the other hand, the used IDH in conventional stirred tank reactor was fouled with insoluble impurities present in the substrate.     

Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons

A video-rate (30 frames/s) scanning two-photon excitation microscope has been successfully tested. The microscope, based on a Nikon RCM 8000, incorporates a femtosecond pulsed laser with wavelength tunable from 690 to 1050 nm, prechirper optics for laser pulse-width compression, resonant galvanometer for video-rate point scanning, and a pair of nonconfocal detectors for fast emission ratioing. An increase in fluorescent emission of 1.75-fold is consistently obtained with the use of the prechirper optics. The nonconfocal detectors provide another 2.25-fold increase in detection efficiency. Ratio imaging and optical sectioning can therefore be performed more efficiently without confocal optics. Faster frame rates, at 60, 120, and 240 frames/s, can be achieved with proportionally reduced scan lines per frame. Useful two-photon images can be acquired at video rate with a laser power as low as 2.7 mW at specimen with the genetically modified green fluorescent proteins. Preliminary results obtained using this system confirm that the yellow "cameleons" exhibit similar optical properties as under one-photon excitation conditions. Dynamic two-photon images of cardiac myocytes and ratio images of yellow cameleon-2.1, -3.1, and -3.1nu are also presented.     

Using a galactose library for exploration of a novel hydrophobic pocket in the receptor binding site of the Escherichia coli heat-labile enterotoxin

The binding of the B subunits of Escherichia coli heat-labile enterotoxin (LT) to epithelial cells lining the intestines is a critical step for the toxin to invade the host. This mechanism suggests that molecules which possess high affinity to the receptor binding site of the toxin would be good leads for the development of therapeutics against LT. The natural receptor for LT is the complex ganglioside GM1, which has galactose as its terminal sugar. A chemical library targeting a novel hydrophobic pocket in the receptor binding site of LT was constructed based on galactose derivatives and screened for high affinity to the receptor binding site of LT. This screening identified compounds that have 2-3 orders of magnitude higher affinity toward the receptor binding site of LT than the parent compound, galactose. The present findings will pave the way for developing simple and easily synthesizable molecules, instead of complex oligosaccharides, as drugs and/or prophylactics against LT-caused disease.     

BALB/c mice produce blister-causing antibodies upon immunization with a recombinant human desmoglein 3

Pemphigus vulgaris (PV) is an Ab-mediated autoimmune blistering disease of mucotaneous surfaces. Over 95% of the patients with PV express DR4 or DRw6, and the disease is characterized by the presence of autoantibodies directed against desmoglein 3 (Dsg 3), a protein expressed on keratinocytes. An appropriate animal model is required to understand immunoregulation and to address the role of immunogenetic components in the production of pathogenic Abs that are characteristic of PV. Therefore, we turned to the development of a mouse model. Four strains of female mice (BALB/c, DBA/1, SJL/J, and HRS/J) were screened for their ability to produce pathogenic anti-Dsg 3 Abs. We demonstrated that only BALB/c mice immunized with a full-length Dsg 3 can produce pathogenic Abs capable of causing acantholysis of human foreskin in culture and blistering in neonatal mice. This observation suggested that either H-2d or the BALB background contains the immunogenetic makeup necessary for the production of pathogenic anti-Dsg 3 Abs. No correlation was noted between a given isotype and the pathogenic potential of autoantibodies from different strains of mice. Similarly, the pattern of reactivity of Abs with a panel of 46 synthetic peptides that span the entire Dsg 3 failed to reveal any association between binding specificity and the pathogenic potential, and suggested that pathogenic Abs might recognize conformational epitopes. Moreover, our studies showed that the epitopes recognized by pathogenic Abs are contained within the extracellular Dsg 3.     

Cloning, expression and characterization of a UDP-galactose 4-epimerase from Escherichia coli

The gene galE encoding UDP-galactose 4-epimerase was cloned into E. coli BL21(DE3) from the chromosomal DNA of E. coli strain K-12. High expression of the soluble recombinant epimerase was achieved in the cell lysate. In order to evaluate the use of this epimerase in enzymatic synthesis of important -Gal epitopes (oligosaccharides with a terminal Gal1,3Gal sequence), a new radioactivity assay (1,3-galactosyltransferase coupled assay) was established to characterize its activity in producing UDP-galactose from UDP-glucose. Approximately 2700 units (100 mg) enzyme with a specific activity of 27 U mg^–1 protein could be obtained from one liter of bacterial culture. The epimerase was active in a wide pH range with an optimum at pH 7.0. This expression system established a viable route to the enzymatic production of -Gal oligosaccharides to support xenotransplantation research.     

Role of Cysteine Residues in Human Plasma Phospholipid Transfer Protein

Phospholipid transfer protein (PLTP) belongs to a family of human plasma lipid transfer proteins that bind to small amphophilic molecules. PLTP contains cysteines at residues 5, 129, 168, and 318. Bactericidal/permeability-increasing protein, which is a member of the same gene family, contains an essential disulfide bond between Cys₁₃₅ and Cys₁₇₅; these residues, which correspond to Cys₁₂₉ and Cys₁₆₈ in PLTP, are conserved among all known members of the gene family. To identify the importance of these and the remaining cysteine residues to PLTP secretion and activity, each was replaced by a glycine by site-directed mutagenesis. The mutant as well as wild-type PLTP cDNAs were cloned into the mammalian expression vector pSV·SPORT1, and the PLTP cDNAs were transfected to COS-6 cells for expression. PLTP Cys₁₂₉ Gly and PLTP Cys₁₆₈ Gly were secretion incompetent. Neither PLTP mass nor activity was detectable in cell lysates and culture medium. Relative to wild-type PLTP, PLTP Cys₅ Gly and PLTP Cys₃₁₈ Gly exhibited similar specific activities but partially impaired PLTP synthesis and secretion. Intracellular PLTP appeared as two bands of 75 and 51 kDa corresponding to reported molecular masses for the glycosylated and nonglycosylated forms. The specific activities of PLTP Cys₅ Gly and PLTP Cys₃₁₈ Gly were similar in the cell lysates and medium, suggesting that glycosylation does not affect transfer activity.     

Antagonism of CD95 signaling blocks butyrate induction of apoptosis in young adult mouse colonic cells

There is greatinterest in utilizing butyrate as a chemopreventive agent for colontumorigenesis because of its ability to promote apoptosis in colontumor cell lines. Because CD95 (APO-1/Fas) transduces signals resultingin apoptosis, we tested the hypothesis that butyrate-dependentcolonocyte apoptosis is mediated by this death receptor. Butyrate (1 mM) exposure for 24 h upregulated expression of Fas andits ligand in young adult mouse colon (YAMC) cells. To delineate theproapoptotic effect of butyrate and to avoid the confounding effects ofdetachment from the extracellular matrix, adherent cell apoptosis wasmonitored as loss of plasma membrane asymmetry and dissipation ofmitochondrial membrane potential (_mt) by lasercytometry. Soluble Fas receptor protein (Fas:Fc chimera) and caspaseinhibitors (z-VAD-fmk and z-IETD-fmk) blocked butyrate induction ofapoptosis. Treatment with Fas agonistic antibody (clone Jo-2)significantly induced cell death, indicating that Fas in colonocytes isfunctional. In addition, butyrate promoted apoptosis by inducing lossof _mt and phospholipidasymmetry of the plasma membrane after 12 and 24 h of exposure,respectively, before cell detachment. Therefore, Fas receptor-dependentsignal transduction is involved in butyrate induction of apoptosis in colonocytes.