Major linear antibody epitopes and capsid proteins differentially induce protective immunity against Theiler's virus-induced demyelinating disease.

Theiler's murine encephalomyelitis virus-induced immunologically mediated demyelinating disease (TMEV-IDD) in susceptible mice provides a relevant infectious model for multiple sclerosis. Previously, we have identified six major linear antibody epitopes on the viral capsid proteins. In this study, we utilized fusion proteins containing individual capsid proteins and synthetic peptides containing the linear antibody epitopes to determine the potential role of antibody response in the course of virus-induced demyelination. Preimmunization of susceptible mice with VPI and VP2 fusion proteins, but not VP3, resulted in the protection from subsequent development of TMEV-IDD. Mice free of clinical symptoms following preimmunizations with fusion proteins displayed high levels of antibodies to the capsid proteins corresponding to the immunogens. In contrast, the level of antibodies to a particular linear epitope, A1C (VP1(262-276)), capable of efficiently neutralizing virus in vitro increased with the progression of disease. Further immunization with synthetic peptides containing individual antibody epitopes indicated that antibodies to the epitopes are differentially effective in protecting from virus-induced demyelination. Taken together, these results suggest that antibodies to only certain linear epitopes are protective and such protection may be restricted during the early stages of viral infection.     

Combination of the tod and the tol pathways in redesigning a metabolic route of Pseudomonas putida for the mineralization of a benzene, toluene, and p-xylene mixture.

Construction of a hybrid strain which is capable of mineralizing components of a benzene, toluene, and p-xylene mixture simultaneously was attempted by redesigning the metabolic pathway of Pseudomonas putida. Genetic and biochemical analyses of the tod and the tol pathways revealed that dihydrodiols formed from benzene, toluene, and p-xylene by toluene dioxygenase in the tod pathway could be channeled into the tol pathway by the action of cis-p-toluate-dihydrodiol dehydrogenase, leading to complete mineralization of a benzene, toluene, and p-xylene mixture. Consequently, a hybrid strain was constructed by cloning todC1C2BA genes encoding toluene dioxygenase on RSF1010 and introducing the resulting plasmid into P. putida mt-2. The hybrid strain of P. putida TB105 was found to mineralize a benzene, toluene, and p-xylene mixture without accumulation of any metabolic intermediate.     

Ginkgolide B production in cultured cells derived from Ginkgo biloba L. leaves

Callus cultures and cell suspension cultures derived from Ginkgo biloba L. leaves produced ginkgolidc B. In cell suspension cultures, the production reached a maximum by the 13th day of subculture and followed by a sharp decrease. The medium of Murashige and Skoog induced the highest ginkgolide B content in cultures while the medium of Schenk and Hildebrandt promoted cell growth. For the maximal production of ginkgolide B, cells were cultured in Murashige and Skoog medium modified to contain 1.0 mg/l of -naphthaleneacetic acid, 0.1 mg/1 of kinetin, 30 g/1 sucrose and 1.25 mM potassium phosphate with a molar ratio of ammonium to nitrate ions of 1 3.Abbreviations B5 Gamborg et al (1968) medium - GKB Ginkgolide B - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic aicd - SH Schenk and Hildebrandt (1972) medium     

Structure-function analysis of the DNA binding domain of Saccharomyces cerevisiae ABF1.

To localize the DNA binding domain of the Saccharomyces cerevisiae Ars binding factor 1 (ABF1), a multifunctional DNA binding protein, plasmid constructs carrying point mutations and internal deletions in the ABF1 gene were generated and expressed in Escherichia coli. Normal and mutant ABF1 proteins were purified by affinity chromatography and their DNA binding activities were analyzed. The substitution of His61, Cys66 and His67 respectively, located in the zinc finger motif in the N-terminal region (amino acids 40-91), eliminated the DNA binding activity of ABF1 protein. Point mutations in the middle region of ABF1, specifically at Leu353, Leu399, Tyr403, Gly404, Phe410 and Lys434, also eliminated or reduced DNA binding activity. However, the DNA binding activity of point mutants of Ser307, Ser496 and Glu649 was the same as that of wild-type ABF1 protein and deletion mutants of amino acids 200-265, between the zinc finger region and the middle region (residues 323-496) retained DNA binding activity. As a result, we confirmed that the DNA binding domain of ABF1 appears to be bipartite and another DNA binding motif, other than the zinc finger motif, is situated between amino acid residues 323 and 496.     

New taxol assay method using tubulin-assembly stimulation

Summary A novel taxol determination method which involves the tubulin-assembly stimulation is described. The tubulin-assembly was monitored by turbidity change at 350nm. In a limited range of taxol concentration (0 to 24 M), taxol stimulated tubulin-assembly linearly. And this linear relation was observed from 20min to 30min after the reaction started. Bioactive derivatives of taxol, such as cephalomanin and 7-epi-10-deacetyltaxol also stimulated the tubulin-assembly. However, baccatin III, which was known as less active taxol derivative did not stimulate tubulin assembly. This result showed that the stimulation of tubulin assembly has a relationship with the antimiotic activity. This assay method have several advantages. 1) Time required for the measurement is relatively short. 2) Multiple samples can be measured simultaneously. 3) It can remove interference of less active taxane compounds more selectively than immuno-assay. Consequently, this method can be used to determine taxol concentration in biological samples. Especially, this method can be used for large scale selection of cell line and primary screening of new antimiotic compounds.     

Cold-induced hemolysis in a hypertonic milieu

Summary Suspension of human erythrocytes at 37° C in an environment made hypertonic by increasing concentrations of sodium chloride and sucrose was followed by hemolysis when the temperature was lowered to 0° C. Two distinct stages were involved in this hemolytic phenomenon, the first being incubation with hypertonic solute at some temperature above 20° C with an increasing effect up to 45° C, and the second stage consisting of lowering the temperature below 15° C with increasing hemolysis down to 0° C. The rate of cooling was not an important factor, but the presence of ions reduced the extent of cold-induced hemolysis in hypertonic sucrose. No significant release of membrane phospholipid and cholesterol accompanied this hemolysis. The solubilization of membrane protein components was investigated, with some differences appearing on sodium dodecyl sulfate polyacrylamide gel electrophoresis between hypertonic and isotonic supernatants. Spectrin could not be identified in solubilized form. Correlation of the temperatures of note in these studies with results from the literature on other biological effects of temperature-induced phase transitions in membrane lipids strongly points to the conclusion that such transitions are involved in the mechanism of cold-induced hypertonic hemolysis. It is postulated that the hypertonic milieu has resulted in membrane-protein alteration damage which prevents normal adaption to the new physical state of the membrane lipids during cooling.     

gamma-Vinyl GABA (4-amino-hex-5-enoic acid), a new selective irreversible inhibitor of GABA-T: effects on brain GABA metabolism in mice

Abstract— γ-Vinyl GABA (4-amino-hex-5-enoic acid, RMI 71754) is a catalytic inhibitor of GABA-T in vitro. When given by a peripheral route to mice, it crosses the blood-brain barrier and induces a long-lasting, dose-dependent, irreversible inhibition of brain GABA transaminase (GABA-T). Glutamate decarboxylase (GAD) is only slightly affected even at the highest doses used. γ -Vinyl GABA has little or no effect on brain succinate semialdehyde dehydrogenase, aspartate transaminase and alanine transaminase activities. GABA-T inhibition is accompanied by a sustained dose-dependent increase of brain GABA concentration. From the rate of accumulation of GABA it was estimated that GABA turnover in brain was at least 6.5 μmol/g/h. Based on recovery of enzyme activity the half-life of GABA-T was found to be 3.4 days, that of GAD was estimated to be about 2.4 days. γ -Vinyl GABA should be valuable for manipulations of brain GABA metabolism.     

The use of percoll gradients, elutriator rotor elution, and mithramycin staining for the isolation and identification of intraerythrocytic stages of plasmodium berghei

Intraerythrocytic parasites of Plasmodium vinckei and Plasmodium berghei were separated according to their developmental stages using discontinuous Percoll gradients. Contaminating nucleated blood cells such as leukocytes were removed by elutriation centrifugation. The stages were unequivocally identified in smears using a newly developed DNA-specific staining procedure with mithramycin and fluorescence microscopy. This stain can also be used to detect parasites in human blood of very low parasitemias. The combination of methods described has many possible applications in immunologic and biochemical parasite research.     

Uptake of neutral and acidic amino acids by Lemna gibba correlated with the H+-electrochemical gradient at the plasmalemma

Uptake rates of L-alanine, L-serine and L-aspartate and trans-membrane electrical potentials (Δψ) were determined for a pH range in the external medium between 3.5 and 9.0. The proton electrochemical gradients (     ) were calculated from Δψ, pH of the medium, and an assumed cytoplasmic pH of 7.5. At external amino-acid concentrations of 0.1 mol m⁻³, where carrier-mediated uptake dominates total uptake, a linear correlation between uptake rates and     is obtained, which extrapolates to zero uptake at zero     . This corroborates the contention that neutral and acidic amino acids are taken up by Lemna gibba L. by H⁺-cotransport.