Characterization of the flavoprotein moieties of NADPH-sulfite reductase from Salmonella typhimurium and Escherichia coli. Physicochemical and catalytic properties, amino acid sequence deduced from DNA sequence of cysJ, and comparison with NADPH-cytochrome P-450 reductase

NADPH-sulfite reductase flavoprotein (SiR-FP) was purified from a Salmonella typhimurium cysG strain that does not synthesize the hemoprotein component of the sulfite reductase holoenzyme. cysJ, which codes for SiR-FP, was cloned from S. typhimurium LT7 and Escherichia coli B, and both genes were sequenced. Physicochemical analyses and deduced amino acid sequences indicate that SiR-FP is an octamer of identical 66-kDa peptides and contains 4 FAD and 4 FMN per octamer. Potentiometric titrations of SiR holoenzyme, SiR-FP, and FMN-depleted SiR-FP yielded the following redox potentials for the prosthetic groups at pH 7.7: E'1 (FMNH./FMN) = -152 mV; E'2 (FMNH2/FMNH.) = -327 mV; E'3 (FADH./FAD) = -382 mV; E'4 (FADH2/FADH.) = -322 mV. Microcoulometric titration of SiR-FP at 25 degrees C yielded data which were in full agreement with these potentials. Spectroscopic and catalytic studies of native SiR-FP and of SiR-FP depleted of FMN support the following electron flow sequence: NADPH----FAD----FMN. FMN can then contribute electrons to the hemoprotein component of sulfite reductase, as well as to cytochrome c and various diaphorase acceptors. The FMN is postulated to cycle between the FMNH2 and FMNH. oxidation states during catalysis; in this sense SiR-FP shares a catalytic mechanism with NADPH-cytochrome P-450 oxidoreductase. SiR-FP domains involved in binding FMN, FAD, and NADPH are proposed from amino acid sequence homologies with Desulfovibrio vulgaris flavodoxin (Dubourdieu, M., and Fox, J.L. (1977) J. Biol. Chem. 252, 1453-1463) and spinach ferredoxin-NADP+ oxidoreductase (Karplus, P.A., Walsh, K.A., and Herriott, J. R. (1984) Biochemistry 23, 6576-6583). Comparison of the deduced amino acid sequences of SiR-FP and NADPH-cytochrome P-450 oxidoreductase (Porter, T. D., and Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U. S.A. 82, 973-977) also showed identities that suggest these two proteins are descended from a common precursor, which contained binding regions for both FMN and FAD.     

High level expression, purification, and characterization of the Kunitz-type protease inhibitor domain of protease nexin-2/amyloid beta-protein precursor.

The protease inhibitor, protease nexin-2 (PN-2), is the secreted isoform of the Alzheimer's amyloid beta-protein precursor (A beta PP) that contains the Kunitz-type protease inhibitor (KPI) domain. Here we describe the use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the KPI domain of PN-2/A beta PP. In addition to the 57 amino acid KPI domain, the expression product contained an additional four amino acid residues at its amino terminus that correspond to amino acids 285-288 of A beta PP (Ponte et al. 1988 Nature 311:525-527). This expression system generated yields of greater than 1.0 gram of KPI domain per liter of fermentation media. The secreted 61 amino acid product was purified to homogeneity and biochemically characterized. Amino acid analysis and sequencing of the entire expressed KPI domain verified its integrity. Similar to native PN-2/A beta PP, the purified KPI domain potently inhibited trypsin, chymotrypsin, and coagulation factor XIa. Although heparin augments the inhibition of factor XIa by native PN-2/A beta PP it had no effect on the inhibition of factor XIa by expressed KPI domain suggesting that heparin binds to regions on native PN-2/A beta PP outside of the protease inhibitory domain. This KPI domain expression product should be useful in studying the physiologic and pathophysiologic functions of PN-2/A beta PP.     

Bioreductive activation of mitomycin C by DT-diaphorase.

The role of DT-diaphorase (DTD, EC 1.6.99.2) in the bioreductive activation of mitomycin C was examined using purified rat hepatic DTD. The formation of adducts with reduced glutathione (GSH), binding of [3H]mitomycin C to DNA, and mitomycin C-induced DNA interstrand cross-linking were used as indicators of bioactivation. Mitomycin C was metabolized by DTD in a pH-dependent manner with increasing amounts of metabolism observed as the pH was decreased from 7.8 to 5.8. The major metabolite observed during DTD-mediated reduction of mitomycin C was 2,7-diaminomitosene. GSH adduct formation, binding of [3H]mitomycin C and mitomycin C-induced DNA interstrand cross-linking were observed during DTD-mediated metabolism. In agreement with the pH dependence of metabolism, increased bioactivation was observed at lower pH values. Temporal studies and experiments using authentic material showed that 2,7-diaminomitosene could be further metabolized by DTD resulting in the formation of mitosene adducts with GSH. DNA cross-linking during either chemical (sodium borohydride) or enzymatic (DTD) mediated reduction of mitomycin C could be observed at pH 7.4, but it increased as the pH was decreased to 5.8, showing the critical role of pH in the cross-linking process. These data provide unequivocal evidence that the obligate two-electron reductase DTD can bioactivate mitomycin C to reactive species which can form adducts with GSH and DNA and induce DNA cross-linking. The use of mitomycin C may be a viable approach to the therapy of tumors high in DTD activity, particularly when combined with strategies to lower tumor pH.     

Growth of Ectopic Dendrites on Cortical Pyramidal Neurons in Neuronal Storage Diseases Correlates with Abnormal Accumulation of GM2 Ganglioside

Abstract: Ganglioside analysis and quantitative Golgi studies of the cerebral cortex of cats with ganglioside and nonganglioside lysosomal storage diseases reveal a correlation between the amount of accumulated GM2 ganglioside and the extent of ectopic dendrite growth on cortical pyramidal neurons. This correlation was not observed with any of the other gangliosides assayed for, including GM1 ganglioside. These results suggest a specific role for GM2 ganglioside in the initiation of ectopic neurites on pyramidal cells in vivo and are consistent with the developing hypothesis that different gangliosides have specific roles in different cell types dependent upon the receptor or other effector molecules with which they may interact.     

Relationships among non-Acremonium sp. fungal endophytes in five grass species.

Many cool-season grasses (subfamily Pooideae) possess maternally transmitted fungal symbionts which cause no known pathology and often enhance the ecological fitness and biochemical capabilities of the grass hosts. The most commonly described endophytes are the Acremonium section Albo-lanosa spp. (Acremonium endophytes), which are conidial anamorphs (strictly asexual forms) of Epichloë typhina. Other endophytes which have been noted are a Gliocladium-like fungus in perennial ryegrass (Lolium perenne L.) and a Phialophora-like fungus in tall fescue (Festuca arundinacea Schreb.). Here, we report the identification of additional non-Acremonium sp. endophytes (herein designated p-endophytes) in three more grass species: Festuca gigantea, Festuca arizonica, and Festuca pratensis. In each grass species, the p-endophyte was cosymbiotic with an Acremonium endophyte. Serological analysis and sequence determinations of variable portions of their rRNA genes indicated that the two previously identified non-Acremonium endophytes are closely related to each other and to the newly identified p-endophytes. Therefore, the p-endophytes represent a second group of widely distributed grass symbionts.     

Agitation,aeration and perfusion modules for cell culture bioreactors

For an optimized bioreactor design which is adapted to the cultivation of sensitive animal cells different modular bioreactor components for gentle agitation, sufficient aeration and long-term perfusion were developed and investigated with respect to their suitability from laboratory to production scale. Aeration systems have been designed for both shear sensitive cells and cells which tolerate bubbles. The systems are based on either membranes for bubble-free aeration or stainless steel sparger systems. They were characterized by determination of their oxygen transfer capacity and optimized in cultivation processes of different cell lines under process conditions such as batch and perfusion mode.Different impellers for suspension cells and cells grown on carriers were investigated for their suitability to ensure homogeneous gentle mixing. A large pitch blade impeller as well as a novel 3-blade segment impeller are appropriate for homogeneous mixing at low shear rates. Especially with the 3-blade segment impeller fluid mechanical stress can be reduced at a given stirrer speed which is advantageous for the cultivation of cells attached to microcarriers or extremely shear sensitive suspension cells. However, our results indicate that shear sensitivity of animal cells has been generally overestimated.Continuous perfusion of both suspension cell cultures and cells cultivated on microcarriers could be successfully performed over extended periods of time using stainless steel spinfilters with appropriate pore sizes and systems based on microporous hydrophilic membranes. Spinfilters are suitable cell retention systems for technical scale bioreactors allowing continuous perfusion cultures of suspension cells (pore size 10 to 20 m) as well as anchorage dependent cells grown on microcarriers (pore size 75 m) over six weeks to 3 months.Applying the developed modules for agitation, aeration and perfusion process adapted bioreactor set-ups can be realized which ensure optimum growth and product formation conditions in order to maximize cell and product yields.     

Responsiveness of mesolimbic, mesocortical, septal and hippocampal cholecystokinin and substance P neuronal systems to stress, in the male rat

The effects of acute and subchronic stress upon discrete cholecystokinin (CCK) and Substance P (SP) neuronal systems have been studied. Adult male rats were exposed to foot-shock stress for periods of 2, 4, 10, 30 or 60 min, immediately following which they were decapitated; brains were rapidly removed and frozen, and subsequently microdissected and extracted. CCK and SP were determined by RIA. In the olfactory tubercule, stress had no effect upon CCK content, but induced a rapid depletion of SP. In the prefrontal cortex, increased CCK concentrations were found following 30 min of stress exposure. In the medial septum, foot-shock led to a rapid increase in CCK content, and to a similar but delayed change in SP levels. A rapid rise in CCK concentrations was also seen in the lateral septum, but no stress effect whatsoever upon SP occurred in this structure. In the dentate gyrus, CCK exhibited a biphasic responsiveness to stress, while SP levels were increased only at the later time intervals. These data demonstrate that discrete CCK and SP neuronal systems are responsive to stress, and thereby support a functional role for these peptides in the processing of neural and hormonal signals by the CNS.     

The diffusional and osmotic water permeability of the hindgut cuticle in larval Sarcophaga bullata

The diffusional water permeability (P_D) and the pressure filtration coefficient (L_P) of isolated larval hindgut cuticle of the fleshfly, Sarcophaga bullata, were measured using tracer techniques coupled with a simple mathematical model system based on equations of non-equilibrium thermodynamics. Data obtained from the model system were matched to experimental tracer data by means of a mathematical optimization scheme. The following parameter values were obtained: P_D for tritiated water = 1.02 × 10^?6 cm-sec^?1, and L_P = 9.18 × 10^?11 cm³-dyn^?1-sec^?1. These results are now being used to determine reflection coefficients (σ's) and solute mobilities (ω's) for the cuticle system in an attempt to gain an understanding of the mechanisms controlling solute and water movements across the hindgut wall.