Use of a codon alteration strategy in a novel approach to cloning the Mycobacterium tuberculosis diaminopimelic acid epimerase.

Previous attempts to express the diaminopimelate epimerase gene dapF of Mycobacterium tuberculosis in Escherichia coli resulted in undetectable enzyme yields. We used silent mutation of the first 10 codons of the recombinant ORF in an attempt to reduce the formation of secondary structures that might occur near the 5' end of the mRNA and inhibit translation. This significantly increased the yield of the enzyme, which was purified and characterized biochemically. This strategy could be generally applied to other mycobacterial genes that are difficult to express hetero-specifically and here provided pure M. tuberculosis DapF, a good foundation for future research in antimycobacterial agents.

Parasite polyclonal activators: new targets for vaccination approaches?

Taking into consideration that the immune response following infection promotes the expansion of lymphocyte clones that are essentially non-specific, ensuring both parasite evasion and persistence inside the host, what would be the major consequences of this polyclonal response to the development of immunopathology? We favor the hypothesis that the polyclonal B cell responses triggered by the infection is responsible of the host susceptibility and is a major contributor to the maintenance of a progressive disease. In particular, the activation of B cells by parasite mitogens would contribute to the class determination of T cell responses and to the inhibition of macrophages - target cells for parasite multiplication and also responsible for parasite clearance. We also envisage that the activation of T cells by parasite 'superantigens', and the ensuing energy and deletion of these cells, processes that are frequently observed, would contribute for the immunosuppression as well as to parasite escape and persistence in the host. We had concentrated our efforts on the study of the non-specific aspects of the immune response following Trypanosoma cruzi infection. We aimed at finding new strategies to modulate and control the mechanisms leading to both the immunosuppression and the development of chronic auto-immunity leading to rational vaccine approaches against parasite infection and immunopathology.

Isolation of the murI gene from Brevibacterium lactofermentum ATCC 13869 encoding D-glutamate racemase.

The murI gene encoding D-glutamate racemase plays an important role in the biosynthesis of D-glutamic acid, an essential component of cell wall peptidoglycan of almost all eubacteria. A DNA fragment that could rescue the auxotrophy of D-glutamic acid in the Escherichia coli murI mutant strain WM335 was isolated from Brevibacterium lactofermentum ATCC 13869 belonging to the coryneform bacteria. DNA sequencing reveals that it encodes a protein of 284 amino acid residues, which shows a high level of homology with D-glutamate racemases from several other bacteria.

Enhancing the effect of secreted cyclophilin B on immunosuppressive activity of cyclosporine.

BACKGROUND: Cyclophilin B (CyPB) is a cyclosporine (CsA)-binding protein, located within intracellular vesicles and secreted in biological fluids. In previous works, we reported that CyPB specifically interacts with the T-cell membrane and potentiates the ability of CsA to inhibit CD3-induced proliferation of T lymphocytes. METHODS: CyPB levels were measured in plasma from healthy donors and transplant patients. The role of extracellular CyPB on the distribution and activity of CsA was investigated first by studies on the uptake of free and CyPB-complexed drug by blood cells, and second by studies on the inhibitory effects of these two compounds on the CD3-induced proliferation of peripheral blood mononuclear cells. RESULTS: A significant increase in plasma CyPB level was observed for CsA-treated patients (13+/-6.4 nM, n=42) in comparison with untreated donors (4.3+/-2.1 nM, n=34). In vitro, extracellular CyPB dose dependently modified CsA distribution between plasma, erythrocyte, and lymphocyte contents, by both retaining the complexed drug extracellularly and promoting its specific accumulation within peripheral blood mononuclear cells. Moreover, the enhanced ability of CyPB-complexed CsA to suppress CD3-induced T-cell proliferation was preserved in the presence of other blood cells, implying specific targeting of the drug to sensitive cells. Furthermore, although a large interindividual variability of sensitivity to the drug was confirmed for 18 individuals, we found that CyPB potentiated the activity of CsA in restoring a high sensitivity to the immunosuppressant. CONCLUSION: These results suggest that plasma CyPB may contribute to the acceptance and the good maintenance of organ transplantation by enhancing the immunosuppressive activity of CsA through a receptor-mediated incorporation of CyPB-complexed CsA within peripheral blood lymphocytes.

Compensation for D-glutamate auxotrophy of Escherichia coli WM335 by D-amino acid aminotransferase gene and regulation of murI expression.

D-glutamate, an indispensable component of peptidoglycans of bacteria, is provided by glutamate racemase in E. coli cells. Compensation for D-glutamate auxotrophy of E. coli WM335 cells lacking the glutamate racemase gene, murI, with the D-amino acid aminotransferase gene suggests that presence of a threshold concentration for the D-glutamate required by E. coli cells, as well as a regulation system for murI expression.

Stage-specific expression of the immunophilin FKBP59 messenger ribonucleic acid and protein during differentiation of male germ cells in rabbits and rats.

FKBP59 is an immunophilin that binds the immunosuppressant drugs FK506 and rapamycin. It is a 90-kDa heat shock protein (hsp90)-binding protein that was originally discovered as a member of steroid receptor complexes. FKBP59 is ubiquitous and well conserved, and it appears to be a multifunctional protein. It has peptidylprolyl cis-trans-isomerase activity and therefore may be involved in protein folding as a molecular chaperon. FKBP59 also includes in its structure a tetratricopeptide repeat (TPR) motif and could have a function in the cell division process. In situ hybridization experiments revealed an overexpression of FKBP59 mRNA in rabbit and rat testes in comparison with other organs. This high level of expression was restricted to germ cells of the seminiferous epithelium. Increasing levels of FKBP59 mRNA became obvious from the midpachytene stage, and the strongest signal was observed in the late pachytene, diplotene, and diakinesis primary spermatocytes. The expression then declined progressively in postmeiotic early spermatids. High expression of FKBP59 mRNA did not occur in earlier and later germ cell stages. During prepubertal development, Northern blot and in situ hybridization of rat testes examined at various postnatal ages revealed that FKBP59 mRNA was not expressed at over a basal level until the pachytene stage. High expression of the FKBP59 protein was demonstrated in the rabbit testis by Western blot and was localized by immunohistochemistry from late pachytene spermatocytes to round spermatids. The cell type-specific and developmental stage-specific expression of FKBP59 at a restricted period of male germ cell differentiation suggests that FKBP59 is involved in a specific function during the cell division process.

Crystal structure of cytoplasmic Escherichia coli peptidyl-prolyl isomerase: evidence for decreased mobility of loops upon complexation.

The structure of the unliganded form of the Escherichia coli cytoplasmic peptidyl-prolyl isomerase (ppiB gene product) in a new crystal form was determined by the molecular replacement method and refined to an R-factor of 16.1% at 2.1 A resolution. The enzyme crystallized in the orthorhombic C2221 space group with unit cell dimensions of a=44.7 A, b=68.2 A and c=102.0 A. Comparison with the reported structure of the enzyme complexed with the tripeptide substrate succinyl-Ala-Pro-Ala-p-nitroanilide revealed subtle changes that occur upon complex formation. There is evidence to suggest that two surface loops have significantly reduced mobility in the complexed structure.

Cyclophilins are induced by hypoxia and heat stress in myogenic cells.

This is a novel study demonstrating that cyclophilins are heat and stress inducible proteins in eukaryotic myogenic cells. We investigated the expression of cyclophilins in embryonal rat heart derived H9c2 myocytes following heat stress and chronic hypoxia. We report here that cyclophilins, the proteins capable of catalysing the interconversion of cis and trans isomers (PPIses) in proteins and peptides, are heat and stress inducible, and are involved in the complex stress response, as their level is significantly elevated after heat stress and hypoxia. A time course analysis showed the gradual increase in expressed levels of cyclophilin after heat stress of cells, with maximal expression as measured by Western blot at 48 hours after the actual treatment. Rat myogenic cells exposed to chronic hypoxia followed by 5 hours reoxygenation resulted in approximately threefold expression of PPI-ases. The results showing that cyclophilins are heat and stress inducible suggest a multiple role for cyclophilins in ischemia: a potential functional association with the different heat shock proteins, with the established protective role in ischaemic injury, as well as the possible involvement of cyclophilins in the protein folding in cooperation with molecular chaperones.

Basal and stimulated C-fos mRNA expression in the rat brain: effect of chronic dietary lithium.

The mechanisms underlying the therapeutic efficacy of lithium in affective disorders are poorly understood; however, previous studies have established an influence of lithium on receptor-coupled and postreceptor signal transduction mechanisms, including the transcription factor c-fos. We investigated the effect of chronic lithium on basal, stress-, muscarinic-, and haloperidol-induced c-fos mRNA expression in various rat brain regions. Chronic lithium produced significant reductions in basal c-fos expression in the frontal cortex and hippocampus, confirming our previous report. Stress-induced c-fos was significantly attenuated in the frontal cortex, hippocampus, and pituitary, was increased in the occipital cortex, and unchanged in the hypothalamus by chronic lithium. Pilocarpine-induced c-fos was significantly reduced in the frontal cortex and hippocampus by chronic lithium, but was enhanced in the occipital cortex and hypothalamus. Haloperidol-induced c-fos was augmented in the striatum and pituitary, but reduced in the frontal cortex by chronic lithium treatment. In regions in which haloperidol did not induce fos expression in control animals, fos levels after haloperidol were reduced after chronic lithium. One week after discontinuation of the lithium treatment, basal c-fos levels remained significantly lower in the frontal cortex and hippocampus, whereas the effects of stress, pilocarpine, or haloperidol on fos were normalized in most regions, except in the hippocampus, where the attenuated fos response to injection stress persisted. We suggest that repression of basal fos expression and inhibition and activation of inducible fos may be factors to be considered in the longer-term effects of lithium, leading to changes in expression of genes that regulate fos and are regulated by Fos, and ultimately to alterations in the functional activity of neural systems involved in the pathophysiology of affective disorder.

Estrogen inhibits hypothalamic pro-opiomelanocortin gene expression in hypothalamic neuronal cultures.

Many in vitro studies show estrogen regulation of the hypothalamic pro-opiomelanocortin (POMC) system, including a decrease in hypothalamic POMC mRNA after estradiol treatment. Because such in vivo experiments do not allow one to determine whether peripheral, interacting systems or extra-hypothalamic brain regions are involved in this regulation, we sought to establish whether estrogen acts directly in hypothalamus to decrease POMC mRNA. Using an in vitro approach, we studied effects of estradiol (E2) on POMC/cyclophilin mRNA concentrations (RNAse protection assays) in neuronal cultures derived from day 17 fetal rat hypothalamus. Chemically defined medium was deprived of progesterone for 2 days prior to E2 treatment and for the duration of the study. E2 (10(-13)-10(-9) M) dose-dependently decreased POMC mRNA concentrations during a 2-day treatment. Whereas the lowest dose (10(-13) M) of E2 resulted in a statistically significant 44% decrease in POMC mRNA concentrations relative to control cultures, this inhibitory effect was lost because higher doses (10(-11) and 10(-9) M) did not produce statistically significant decrements (22 and 16%, respectively) in POMC mRNA concentrations. Additional time course studies revealed that this decrease in POMC mRNA can be seen as early as 4 h after E2 (10(-13) M) treatment. We conclude that E2 inhibition of POMC mRNA concentrations in hypothalamic neuronal cultures indicates that this inhibition can occur directly in hypothalamus.

Trigger factor is induced upon cold shock and enhances viability of Escherichia coli at low temperatures.

Trigger factor (TF) in Escherichia coli is a molecular chaperone with remarkable properties: it has prolyl-isomerase activity, associates with nascent polypeptides on ribosomes, binds to GroEL, enhances GroEL's affinity for unfolded proteins, and promotes degradation of certain polypeptides. Because the latter effects appeared larger at 20 degrees C, we studied the influence of temperature on TF expression. Unlike most chaperones (e.g., GroEL), which are heat-shock proteins (hsps), TF levels increased progressively as growth temperature decreased from 42 degrees C to 16 degrees C and even rose in cells stored at 4 degrees C. Upon temperature downshift from 37 degrees C to 10 degrees C or exposure to chloramphenicol, TF synthesis was induced, like that of many cold-shock proteins. We therefore tested if TF expression might be important for viability at low temperatures. When stored at 4 degrees C, E. coli lose viability at exponential rates. Cells with reduced TF content die faster, while cells overexpressing TF showed greater viability. Although TF overproduction protected against cold, it reduced viability at 50 degrees C, while TF deficiency enhanced viability at this temperature. By contrast, overproduction of GroEL/ES, or hsps generally, while protective against high temperatures, reduced viability at 4 degrees C, which may explain why expression of hsps is suppressed in the cold. Thus, TF represents an example of an E. coli protein which protects cells against low temperatures. Moreover, the differential induction of TF at low temperatures and hsps at high temperatures appears to provide selective protection against these opposite thermal extremes.

Peptidyl prolyl cis-trans isomerase activity of cyclophilin A in functional homo-oligomeric receptor expression.

The functional expression of homo-oligomeric alpha7 neuronal nicotinic and type 3 serotonin receptors is dependent on the activity of a cyclophilin. In this paper we demonstrate that the mechanism of cyclophilin action during functional homo-oligomeric receptor expression in Xenopus oocytes is distinct from the calcineurin-dependent immunosuppressive mechanism by showing that a nonimmunosuppressive analog of cyclosporin A (CsA), SDZ 211-811, reduces functional receptor expression to the same extent as CsA. The cytoplasmic subtype of cyclophilin, cyclophilin A (CyPA), appears to be required for functional receptor expression. This is because overexpression of CyPA and a CyPA mutant that is deficient in CsA binding activity reverses CsA-induced reduction in functional receptor expression. The mechanism of action of CyPA is likely to involve its prolyl isomerase activity because a mutant CyPA with a single amino acid substitution (arginine 55 to alanine) that is predicted to produce a 1000-fold attenuation in isomerase activity fails to reverse the cyclosporin A effect. Our data also suggest that CyPA does not form a stable complex with receptor subunits.

X-ray-mediated reduction in basic fibroblast growth factor expression in primary rat astrocyte cultures.

Injury of the normal central nervous system is a major concern in the radiotherapy of brain tumors, but the pathogenesis of injury remains poorly understood. Modulation of the production of growth factors is associated with ischemia and traumatic injury in the central nervous system. Ionizing radiation has been shown to induce basic fibroblast growth factor in endothelial cells and in cells of a human breast carcinoma cell line. The inducibility of basic fibroblast growth factor after irradiation and its potential role in the recovery response of the central nervous system led us to investigate the effects of radiation on the expression of this growth factor in primary cultures of normal rat type 1 astrocytes. Astrocyte monolayers were exposed to ionizing radiation (1 to 10 Gy). Northern blot analysis revealed that doses of 2 to 10 Gy markedly reduced the expression of basic fibroblast growth factor as early as 1 h after irradiation, and that it remained below levels in unirradiated cells for at least 24 h. The effect was not associated with astrocyte cytotoxicity, and it appears to have some specificity for basic fibroblast growth factor since the levels of mRNA coding for ciliary neurotrophic factor and glial fibrillary acidic protein were not affected.

Expression of a functional non-ribosomal peptide synthetase module in Escherichia coli by coexpression with a phosphopantetheinyl transferase.

BACKGROUND: Non-ribosomal peptide synthetases (NRPSs) found in bacteria and fungi are multifunctional enzymes that catalyze the synthesis of a variety of biologically important peptides. These enzymes are composed of modular units, each responsible for the activation of an amino acid to an aminoacyl adenylate and for the subsequent formation of an aminoacyl thioester with the sulfhydryl group of a 4'-phosphopantetheine moiety. Attempts to express these modules in Escherichia coli have resulted in recombinant proteins deficient in 4'-phosphopantetheine. The recent identification of a family of phosphopantetheinyl transferases (P-pant transferases) associated with NRPS have led us to investigate whether coexpression of NRPS modules with P-pant transferases in E. coli would lead to the incorporation of 4'-phosphopantetheine. RESULTS: A truncated module of gramicidin S synthetase, PheAT(His6), was expressed as a His6 fusion protein in E. coli with and without Gsp, the P-pant transferase associated with gramicidin S synthetase. Although PheAT(His6) expressed alone in E. coli catalyzed Phe-AMP formation from Phe and ATP, <1% was converted to the Phe thioester. In contrast, >80% of the PheAT(His6) that was coexpressed with Gsp could form the Phe thioester in the presence of Phe and ATP. CONCLUSIONS: Our finding indicates the presence of an almost equimolar amount of 4'-phosphopantetheine covalently bound to the NRPS module PheAT(His6), and that the functional expression of NRPS modules in E. coli is possible, provided that they are coexpressed with an appropriate P-pant transferase.

An internal FK506-binding domain is the catalytic core of the prolyl isomerase activity associated with the Bacillus subtilis trigger factor.

Two major families of peptidylprolyl cis-trans-isomerases, the cyclophilins and the structurally unrelated FK506-binding proteins (FKBPs), have been identified as cellular factors involved in protein folding in vitro. Here we report on the biochemical characterization of a second prolyl isomerase of Bacillus subtilis that was purified from a cyclophilin-negative (ppiB null) mutant and was shown to be the trigger factor (TigBS). N-terminal sequencing of 27 amino acid residues of the purified protein revealed 100% identity to the deduced sequence encoded by the tig gene, sequenced as a part of the B. subtilis genome project. The tigBS gene, located at 246 degrees on the genetic map upstream of the clpX and lonA,B genes, encodes an acidic protein (pI 4.3) of 47.5 kDa. Purified and recombinant TigBS-His proteins share the same substrate specificity and catalytic activity (Kcat/K(m) of 1.5 microM-1 s-1); both are inhibited by the macrolide FK506 with IC50 the range of 500 nM. We also demonstrate that the prolyl isomerase activity of TigBS is mediated by an internal domain of about 13 kDa (homologous to FKPB12) that represents the catalytic core of the trigger factor.

Trigger factor associates with GroEL in vivo and promotes its binding to certain polypeptides.

Trigger factor (TF) is a putative molecular chaperone recently found to function together with GroEL in the degradation of the fusion protein, CRAG. TF overproduction enhanced the ability of GroEL to form complexes with CRAG, as well as fetuin or histone. To define further this effect on GroEL binding, affinity columns containing a variety of denatured proteins were used. When cell extracts were applied onto a fetuin column, both TF and GroEL bound but not GroES. Upon ATP addition, TF and GroEL were eluted together and remained tightly associated (even in presence of GroES) in complexes containing one TF per GroEL 14-mer. Overproduction of TF enhanced the capacity of GroEL to bind to many denatured proteins. Moreover, GroEL-TF complexes isolated from such cells showed much greater binding capacity than GroEL from TF-deficient cells. Furthermore, the addition of pure TF to pure GroEL also enhanced markedly its binding capacity. The affinity of GroEL for CRAG also rises during heat shock due to GroEL phosphorylation. TF expression, however, did not promote GroEL phosphorylation. Moreover, heat shock and TF overproduction affected GroEL binding to other denatured polypeptides in distinct ways; only TF promoted binding to certain polypeptides, whereas only phosphorylation increased binding to others. Thus, association with TF and phosphorylation are independent regulators of GroEL function. This enhanced affinity of TF-GroEL complexes for unfolded proteins may also be important in protein folding, because TF has prolyl isomerase activity and associates with nascent polypeptides.

Neurotrophin receptor and tyrosine hydroxylase gene expression in aged sympathetic neurons.

Ribonuclease protection measurements revealed decreases of 26% in p75 neurotrophin receptor mRNA and 30% in trkA mRNA in superior cervical ganglia (SCG) of aged Long-Evans rats. These declines were not related to the presence of a spatial memory impairment, whose presence is known to strongly predict increased hypothalamic-pituitary-adrenal axis activity in these aged animals. A similar decrease with age was observed in p75, but not cyclophilin mRNA levels in SCG from F-344 inbred rats. In situ hybridization with paired sections from mature and aged F-344 rats revealed a 25% decline in the mean neuronal labeling index (LI) for p75 mRNA. In other paired sections, mean trkA LI decreased 16%, tyrosine hydroxylase (TH) LI increased 74% and cyclophilin LI did not change. Neuronal hypertrophy, p75 decreases and TH increases all occurred to a greatest extent in intermediate-sized neurons, resembling those innervating the pineal and cerebral vessels. In contrast to other SCG targets, this innervation is known to decline nearly 50% with aging. Retrograde tracer/in situ hybridization studies will be required to establish whether decreased p75 represents a marker for selective axonal regression and also to determine the significance of increased TH and neuronal hypertrophy.

The effect of GABA stimulation on GABAA receptor subunit protein and mRNA expression in rat cultured cerebellar granule cells.

1. After 8 days in vitro, rat cerebellar granule cells were exposed to 1 mM gamma-aminobutyric acid (GABA) for periods of 1, 2, 4, 6, 8 and 10 days. The effect of the GABA exposure on GABAA receptor alpha 1, alpha 6 and beta 2,3 subunit protein expression and alpha 1 and alpha 6 subunit steady-state mRNA levels, was examined using Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. 2. GABA exposure for 2 days decreased alpha 1 (35 +/- 10%, mean +/- s.e.mean), beta 2,3 (21 +/- 9%) and alpha 6 (28 +/- 10%) subunit protein expression compared to control levels. The GABA-mediated reduction in alpha 1 subunit expression after 2 days treatment was abolished in the presence of the GABAA receptor antagonist, Ru 5135 (10 microM). 3. GABA exposure for 8 days increased alpha 1 (26 +/- 10%, mean +/- s.e.mean) and beta 2,3 (56 +/- 23%) subunit protein expression over control levels, whereas alpha 6 subunit protein expression remained below control levels (by 38 +/- 10%). However, after 10 days GABA exposure, alpha 6 subunit protein expression was also increased over control levels by 65 +/- 29% (mean +/- s.e.mean). 4. GABA exposure did not change the alpha 1 or alpha 6 subunit steady-state mRNA levels over and 8 day period, nor did it alter the expression of cyclophilin mRNA over 1-8 days. 5. These results suggest that chronic GABA exposure of rat cerebellar granule cells has a bi-phasic effect on GABAA receptor subunit expression that is independent of changes to mRNA levels. Therefore, the regulation of the GABAA receptor expression by chronic agonist treatment appears to involve post-transcriptional and/or post-translational processes.

Down-regulation of the GABA receptor subunits mRNA levels in mammalian cultured cortical neurons following chronic neurosteroid treatment.

We have recently shown that chronic neurosteroid, 5 alpha 3 alpha, treatment produced down-regulation of the GABA receptor binding and function, and heterologous uncoupling on the GABAA receptor complex in cultured mammalian cortical neurons. In order to explore the underlying mechanism of these observed down-regulation and heterologous uncoupling phenomenon, we investigated the effect of chronic 5 alpha 3 alpha (1 microM; 5 days) treatment on the GABAA receptor subunits mRNA levels, using RNase protection assay. We found that chronic neurosteroid, 5 alpha 3 alpha, treatment decreased the beta- and alpha-subunits mRNA levels while not altering the gamma 2S-subunit mRNA levels in the cortical neurons. The decrease in the beta-subunits mRNA levels suggests a decrease in the presence of the beta-subunits in the composition of GABAA receptors. This phenomenon may explain the down-regulation of the GABAA receptor binding and function. A decrease in the alpha 3-subunit mRNA level suggests a corresponding decrease in the alpha 3-subunit in the composition of GABAA receptor isoforms, relative to other isoforms. This observation may be responsible for the chronic neurosteroid-induced uncoupling and decreased efficacy. In summary, chronic 5 alpha 3 alpha treatment produced down-regulation of the GABAA receptor beta- and alpha-subunit mRNA levels, and these changes may be associated with the down-regulation, heterologous uncoupling, and decreased efficacy of GABAA receptor complex in the cultured mammalian cortical neurons.