Substance P but not vasoactive intestinal peptide modulates immunoglobulin secretion in murine schistosomiasis.

Neuropeptides including SP and VIP modulate Ig secretion by in vitro stimulated lymphocyte cultures. It is not known whether these neuropeptides effect the B cell directly, or if they significantly alter humoral immune responses to pathogens. We have previously shown that granulomas derived from schistosome-infected mice contain immunoglobulin secreting B cells (ISC) as well as eosinophils that secrete substance P (SP) and vasoactive intestinal peptide (VIP). It therefore seemed plausible that B cells derived from infected animals might respond to these neuropeptides, and that such responses might effect immunoregulatory signals. In this study, we addressed these issues in the murine Schistosoma mansoni model, at the level of immunoglobulin secretion in single B cells. Spontaneous ISC were observed in both splenic and granuloma cell preparations. The addition of SP resulted in a dose-dependent reduction in the number and size of plaques (a 50% reduction was observed at 10(-9) M). This effect was blocked with SP antagonists. Similar results were observed in T cell-depleted cell cultures. VIP had no effect on ISC number or plaque size. We conclude that SP, but not VIP, decreases spontaneous ISC number and Ig secretion in short-term cultures of spleen and granuloma cells. SP appears to exert its effects at the level of single B cells through a receptor-mediated mechanism and may thus play an immunoregulatory role in schistosomiasis.     

Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases.

We describe the development of an expression-secretion system in Bacillus subtilis to improve the quality and quantity of the secreted foreign proteins. This system consists of a strain (WB600) deficient in six extracellular proteases and a set of sacB-based expression vectors. With the inactivation of all six chromosomal genes encoding neutral protease A, subtilisin, extracellular protease, metalloprotease, bacillopeptidase F, and neutral protease B, WB600 showed only 0.32% of the wild-type extracellular protease activity. No residual protease activity could be detected when WB600 was cultured in the presence of 2 mM phenylmethylsulfonyl fluoride. By using TEM beta-lactamase as a model, we showed that WB600 can significantly improve the stability of the secreted enzyme. To further increase the production level we constructed an expression cassette carrying sacY, a sacB-specific regulatory gene. This gene was placed under the control of a strong, constitutively expressed promoter, P43. With this cassette in the expression vector, an 18-fold enhancement in beta-lactamase production was observed. An artificial operon, P43-sacY-degQ, was also constructed. However, only a partial additive enhancement effect (24-fold enhancement) was observed. Although degQ can stimulate the production of beta-lactamase in the system, its ability to increase the residual extracellular protease activity from WB600 limits its application. The use of the P43-sacY cassette and WB600 would be a better combination for producing intact foreign proteins in high yield.     

Selective Impairment of Respiration in Mitochondria Isolated from Brain Subregions Following Transient Forebrain Ischemia in the Rat

Using Percoll density gradient centrifugation, free (nonsynaptosomal) mitochondria were isolated from the dorsal-lateral striatum and paramedian neocortex of rats during complete forebrain ischemia and reperfusion. Mitochondria prepared from either region after 30 min of ischemia showed decreased state 3 (ADP and substrate present) and uncoupled respiration rates (19-45% reductions) with pyruvate plus malate as substrates, whereas state 4 respiration (no ADP present) was preserved. At 6 h of recirculation, state 3 and uncoupled respiration rates for mitochondria from the paramedian neocortex (a region resistant to ischemic damage) were similar to or even increased compared with control values. By contrast, in mitochondria from the dorsal-lateral striatum (a region containing neurons susceptible to global ischemia), decreases in state 3 and uncoupled respiration rates (25 and 30% less than control values) were again observed after 6 h of recirculation. With succinate as respiratory substrate, however, no significant differences from control values were found in either region at this time point. By 24 h of recirculation, respiratory activity with either pyruvate plus malate or succinate was greatly reduced in samples from the dorsal-lateral striatum, probably reflecting complete loss of function in some organelles. In contrast with these marked changes in free mitochondria, the respiratory properties of synaptosomal mitochondria, assessed from measurements in unfractionated homogenates, were unchanged from controls in the dorsal-lateral striatum at each of the time points studied, but showed reductions (19-22%) during ischemia and after 24 h of recirculation in the paramedian neocortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pathogenesis of feline leukemia virus T17: contrasting fates of helper, v-myc, and v-tcr proviruses in secondary tumors.

A naturally occurring feline thymic lymphosarcoma (T17) provided the unique observation of a T-cell antigen receptor beta-chain gene (v-tcr) transduced by a retrovirus. The primary tumor contained three classes of feline leukemia virus (FeLV) provirus, which have now been characterized in more detail as (i) v-tcr-containing recombinant proviruses, (ii) v-myc-containing recombinant proviruses, and (iii) apparently full-length helper FeLV proviruses. The two transductions appear to have been independent events, with distinct recombinational junctions and no sequence overlap in the host-derived inserts. The T17 tumor cell line releases large numbers of FeLV particles of low infectivity; all three genomes are encapsidated, but passage of FeLV-T17 on feline fibroblast and lymphoma cells led to selective loss of the recombinant viruses. The oncogenic potential of the T17 virus complex was, therefore, tested by infection of neonatal cats with virus harvested directly from the primary T17 tumor cell line. A single inoculation of FeLV-T17 caused persistent low-grade infection culminating in thymic lymphosarcoma and acute thymic atrophy, which was accelerated by coinfection with the weakly pathogenic FeLV subgroup A (FeLV-A)/Glasgow-1 helper. Molecularly cloned FeLV-tcr virus (T-31) rescued for replication by a weakly pathogenic FeLV-A/Glasgow-1 helper virus was similarly tested in vivo and induced thymic atrophy and thymic lymphosarcomas. Most FeLV-T17-induced tumors manifested either v-myc or an activated c-myc allele and had undergone rearrangement of endogenous T-cell antigen receptor beta-chain genes, supporting the proposition that the oncogenic effects of c-myc linked to the FeLV long terminal repeat are targeted to a specific window in T-cell differentiation. However, neither the FeLV-T17-induced tumors nor the T-31 + FeLV-A-induced tumors contained clonally represented v-tcr sequences. Only one of the FeLV-T17-induced tumors contained detectable v-tcr proviruses, at a low copy number. While v-tcr does not have a readily transmissible oncogenic function, a more restricted role is not excluded, perhaps involving antigenic peptide-major histocompatibility complex recognition by the T-cell receptor complex. Such a function could be obscured by the genetic diversity of the outbred domestic cat host.     

Mapping of the calpain proteolysis products of the junctional foot protein of the skeletal muscle triad junction

Summary The Ca²⁺ activated neutral protease calpain II in a concentration-dependent manner sequentially degrades the Junctional foot protein (JFP) of rabbit skeletal muscle triad junctions in either the triad membrane or as the pure protein. This progression is inhibited by calmodulin. Calpain initially cleaves the 565 kDa JFP monomer into peptides of 160 and 410 kDa, which is subsequently cleaved to 70 and 340 kDa. The 340 kDa peptide is finally cleaved to 140 and 200 kDa or its further products. When the JFP was labeled in the triad membrane with the hydrophobic probe 3-(trifuoromethyl) 3-(m) [¹²⁵I]iodophenyl diazirine and then isolated and proteolysed with calpain II, the [¹²⁵I] was traced from the 565 kDa parent to M _r, 410 kDa and then to 340 kDa, implying that these large fragments contain the majority of the transmembrane segments. A 70-kDa frament was also labeled with the hydrophobic probe, although weakly suggesting an additional transmembrane segment in the middle of the molecule. These transmembrane segments have been predicted to be in the C-terminal region of the JFP. Using an ALOM program, we also predict that transmembrane segments may exist in the 70 kDa fragment. The JFP has eight PEDST sequences; this finding together with the calmodulin inhibition of calpain imply that the JFP is a PEDST-type calpain substrate. Calpain usually cleaves such substrates at or near calmodulin binding sites. Assuming such sites for proteolysis, we propose that the fragments of the JFP correspond to the monomer sequence in the following order from the N-terminus: 160, 70, 140 and 200 kDa. For this model, new calmodulin sequences are predicted to exist near 160 and 225 kDa from the N-terminus. When the intact JFP was labeled with azidoATP, label appeared in the 160 and 140 kDa fragments, which according to the above model contain the GXGXXG sequences postulated as ATP binding sites. This transmembrane segment was predicted by the ALOM program. In addition, calpain and calpastatin activities remained associated with triad component organelles throughout their isolation. These findings and the existence of PEDST sequences suggest that the JFP is normally degraded by calpain in vivo and that degradation is regulated by calpastatin and calmodulin     

Dark inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase in legumes: A biosystematic study

2-carboxyarabinitol 1-phosphate (CA 1-P) is a naturally occurring inhibitor of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Members of the Fabaceae exhibit a particularly wide range in the extent of CA 1-P accumulation during darkness and include Phaseolus vulgaris, whose dark/light regulation of Rubisco activity is principally achieved by synthesis/degradation of CA 1-P. An extensive survey of the degree of dark inhibition of Rubisco was undertaken for the subfamily Papilionoideae to elucidate evolutionary patterns in the occurrence of this regulatory mechanism. Seventy-five species from 21 tribes were examined. Dark inhibition of Rubisco was found in ancestral tribes such as the Sophoreae, but was substantially reduced or absent in representative species of three more recently evolved tribes, Cicereae, Hedysareae and Vicieae. We conclude that regulation of Rubisco by CA 1-P is neither of recent origin nor of restricted distribution among the Papilionoideae. On the contrary, it becomes lost or less pronounced only in a minority of the more evolutionarily advanced species in this important subfamily.     

DNA binding and dimerization determinants for thyroid hormone receptor alpha and its interaction with a nuclear protein.

The gel retardation assay was used to analyze the role of the thyroid hormone receptor alpha (TR alpha) ligand-binding domain (LBD) in controlling receptor interaction with a thyroid hormone responsive element (TRE). While wild type receptor TR alpha binds to the TRE mainly as monomer, deletion of 85 amino acids from its C-terminus results in a mutant receptor with enhanced DNA binding that forms several slow mobility complexes as revealed by gel retardation assay. Receptor deletion mutants that lack most of the LBD show significantly elevated DNA binding and are still able to bind to DNA as two complexes. Thus, the C-terminal end of TR alpha appears to interfere with the dimerization/oligomerization function and DNA binding of TR alpha. All C-terminal deletion mutants have lost their T3-responsive activator function, but some show constitutive activity. Nuclear factor from several cell lines, including CV-1, F9, and GC cells, interacts with TR alpha receptor to form a larger molecular weight complex as determined by gel retardation assay. This factor could not be detected in HeLatk- cells, where TR alpha does not activate a TRE-containing reporter gene. The nuclear factor is heat sensitive and does not bind to TRE itself but can interact with TR alpha in the absence of DNA. Deletion analysis demonstrates that the leucine zipper-like sequence located in the LBD of TR alpha is involved in this interaction. Together, our data suggest that TR alpha contains a dimerization function outside the LBD which is inhibited by the carboxy-terminal region, while the leucine zipper-like sequence in the LBD is required for interaction with a nuclear factor.     

Comparison of amide proton exchange in reduced and oxidizedRhodobacter capsulatus cytochrome c2: A1H−15N NMR study

Summary The hydrogen-deuterium exchange rates of the reduced and oxidized forms ofRhodobacter' capsulatus cytochrome c₂ were studied by¹H–¹⁵N homonuclear multiple quantum correlation spectroscopy. Minimal differences were observed for the N- and C-terminal helices on changing redox state suggesting that although these helices are structurally important they do not affect the relative stability of the two redox states and hence may not be important in determining the redox potential differences observed amongst the class I c-type cytochromes. However, significant differences were observed for other regions of the protein. For example, all slow exchanging protons of the helix spanning Phe⁸² to Asp⁸⁷ are similarly affected on reduction indicating that the unfolding equilibrium of this helix is altered between the two redox states. Other regions are not as simple to interpret; however, the difference in NH exchange rates between the redox states for a number of residues including His¹⁷, Leu³⁷, Arg⁴³, Ala⁴⁵, Gly⁴⁶, Ile⁵⁷, Val⁵⁸, Leu⁶⁰, Gly⁶¹ and Leu¹⁰⁰ suggest that interactions affecting the causes of these differences may be important factors in determining redox potential.Abbreviations NMR nuclear magnetic resonance - HMQC homonuclear multiple quantum correlation - NOESY nuclear Overhauser effect spectroscopy     

Impact of mild experimental acidification on short term invertebrate drift in a sensitive British Columbia stream

We report daytime drift behavior of lotic macroinvertebrates following short term (12 h) additions of HCl or HCl plus AlCl₃ to a circumneutral softwater (alkalinity ca. 100 µeq 1^-1) mountain stream in British Columbia, Canada. Addition of HCl (pH reduced from 7.0 to 5.9) resulted in an overall tripling of invertebrate drift density with rapid (< 1 h) increases in chironomid Diptera and Trichoptera. Small Ephemeroptera also entered the drift at high densities, but were delayed about 6 h. Addition of AlCl₃ (0.71 to 0.95 mg 1^-1 total Al³⁺) in HCl (stream pH reduced to 5.9) resulted in an overall 6-fold increase in invertebrate drift, with rapid increases by Ephemeroptera and delayed responses by chironomids and Trichoptera. These results suggest that the behavior of several macroinvertebrates from low alkalinity, unacidified streams can be altered by simulations of short-term, mild acidic deposition events. Further, the magnitude and timing of entry into the drift varies among taxonomic groups with the presence or absence of low concentrations of aluminum ions.     

Determination of growth and coupled nitrification/denitrification by immobilized Thiosphaera pantotropha using measurement and modeling of oxygen profiles

An oxygen microsensor in combination with mathematical modeling was used to determine the behavior of immobilized Thiosphaera pantotropha. This organism can convert ammonia completely to nitrogen gas under aerobic conditions (coupled nitrification/denitrification) and denitrifies nitrate at highest rates under anaerobic conditions. Immobilization of T. pantotropha can result in aerobic and anaerobic zones inside the biocatalyst particle which will be advantageous for the conversion of ammonia and nitrate from wastewater. However, information of the effects of immobilization on the physiology of T. pantotropha is necessary for the development of such a system. This article gives the extension of a model developed to describe the behavior of chemostat cultures of T. pantotropha so that it can be used for immobilized cells. The original model was based on metabolic reaction equations. Kinetic and diffusion equations have now been added. Experimental verification was carried out using a stirred tank reactor and a Kluyver flask. After immobilization in agarose, the cells were grown in the particles under continuous culture conditions for 3 days. After 24 h the oxygen penetration depth showed a constant value of 100 mu, indicating that a steady state was reached. Scanning electron micrographs showed that large colonies of cells were present in this 100-mum aerobic layer.From the dynamics of the start-up phase, several parameters were determined from measurements of the oxygen concentration profiles made every few hours. The profiles simulated by the model were fitted to the measured data. The average value for the maximum specific growth rate was 0.52 h(-1), and the maximum oxygen conversion rate was 1.0 mol Cmol(-1) h(-1). The maximum specific acetate uptake rate was 2.0 mol Cmol(-1) h(-1), and the Monod constant for acetate was 2.9 x 10(-2) mol m(-3). The maximum specific nitrification rate was 0.58 x 10(-1) mol Cmol(-1) h(-1), and the amount of oxygen necessary for nitrification was 11% of the total oxygen uptake rate. Most of the kinetic parameters determined for the immobilized cells were in good agreement with those for the suspended cells. Only the maximum specific growth rate was significantly higher, and the maximum specific nitrification rate was some what lower than for suspended cells. The experimental results clearly show that an oxygen microsensor, in combination with mathematical modeling, can successfully be used to elucidate the kinetic behavior of immobilized, oxygen-consuming, cells.