Cyclic AMP in ruminal and other anaerobic bacteria

Abstract An examination of cAMP levels in predominant species of ruminal bacteria and other anaerobic bacteria was conducted. Cellular cAMP concentrations of glucose-grown cultures of Butyrivibrio fibrisolvens 49, Prevotella ruminicola D31d, Selenomonas ruminantium HD4 and D, Megasphaera elsdenii B159, Sreptococcus bovis JB1, Bacteroides thetaiotaomicron 5482, and Clostridium acetobutylicum ATCC 824 were determined at various times during growth by a competitive binding radioimmunoassay procedure. The results were compared to those for Escherichia coli NRRL B3704. The levels of cAMP ranged from undetectable for B. thetaiotaomicron to approximately 15 pmol/mg cell protein for P. ruminicola D31d. Varying the growth substrate in a manner previously shown to elicit regulatory response did not alter the level of cAMP in these organisms. In general, cAMP concentrations present in these organisms were much lower than the 6–25 pmol/mg cell protein observed for E. coli . The levels of cAMP in P. ruminicola were consistently higher than levels in other anaerobes, particularly during the early exponential and stationary phases of growth. Based on these data it seems unlikely that cAMP is involved in regulation of substrate catabolism in the anaerobic bacteria examined except in P. ruminicola where it may have an unknown regulatory function.     

Distribution of xylanase genes and enzymes among strains of Prevotella (Bacteroides) ruminicola from the rumen

The distribution of two xylanase genes was examined by Southern hybridization among 26 strains of the rumen anaerobic bacterium Prevotella (Bacteroides) ruminicola. Hybridization with a xylanase/endoglucanase gene from the type strain 23 was found in six strains while hybridization with a xylanase gene from strain D31d was found in 14 strains. Sequences related to both genes were present, on different restriction fragments, in six strains, whereas no hybridization to either gene was detected in five other strains capable of hydrolysing xylan, or in seven strains that showed little or no xylanase activity. Zymogram analyses of seven xylanolytic strains of P. ruminicola demonstrated interstrain variation in the apparent molecular masses of the major xylanases and carboxymethylcellulases that could be renatured following SDS polyacrylamide gel electrophoresis.     

Cloning, sequencing and complementation analysis of the recA gene from Prevotella ruminicola

Abstract Degenerate PCR primers based on conserved RecA protein regions were used to amplify a portion of recE from Prevotella ruminicola strain 23, which was used as a probe to isolate the full-length recA gene from the P. ruminicola genomic library. The P. ruminicola recA gene encoded a protein of 340 amino acids with a molecular mass of 36.81 kDa. P. ruminicola RecA was highly similar to other RecA proteins and most closely resembled that of Bacteroides fragilis (75% identity). It alleviated the methyl methanesulfonate and mitomycin C sensitivities of Escherichia coli recA mutants, but did not restore the resistance to UV-light irradiation. Mitomycin C treatment of otherwise isogenic E. coli strains showed a higher level of prophage induction in a recA harboring lysogen.     

Determination of adenylate cyclase activity in a variety of organisms: Evidence against the occurrence of the enzyme in higher plants

Adenylate cyclase activity was examined in a variety of organisms using a highly sensitive assay. Activity was found in a blue-green alga, four green algae, two cellular slime molds, a fungus and moss protonemata. Fern prothalli and fronds gave variable results. No activity was detected in any of the higher plant tissues tested. The results throw further doubt on the existence of adenosine 3:5-cyclic monophosphate in higher plants.Abbreviation cAMP adenosine 3:5-cyclic monophosphate     

A pepD-like peptidase from the ruminal bacterium, Prevotella albensis

Peptidases of Prevotella spp. play an important role in the breakdown of protein to ammonia in the rumen. This study describes a peptidase cloned from Prevotella albensis M384. DNA from P. albensis was used to complement a peptidase-deficient strain of Escherichia coli, CM107. A cloned fragment, Pep581, which enabled growth of E. coli CM107, contained an ORF of 1452 bp, encoding a 484 amino acid residue protein with a calculated molecular weight of 53.2 kDa and a theoretical pI of 4.90. Pep581 shared similar sequence identity of 47% with PepD from E. coli, and it was also a metallo-aminopeptidase. A putative catalytic metal binding region was identified in Pep581, similar to that found in the related PepT (a tripeptidase) and PepA (an oligopeptidase). Gel filtration indicated Pep581 was a dimer in its native state, similar to PepD of E. coli. PepD is a broad specificity dipeptidase that has been found in several prokaryotes. The enzyme expressed from Pep581 differed from PepD enzymes previously characterised in that it hydrolysed tri- and oligopeptides in addition to dipeptides, cleaving single amino acids from the N terminus.     

Immunohistochemical localization of cyclic AMP and ultrastructural demonstration of adenylate cyclase activity in the testis of Esox lucius at time of spermiation

Summary In the testis of Esox lucius at the time of spermiation, activity of cyclic adenosine 3,5-monophosphate (cAMP) was immunocytochemically localized at the level of the Sertoli cells. In these cells adenylate cyclase activity was also ultracytochemically demonstrated by using adenylyl imidodiphosphate as a substrate. Reaction products of adenylate cyclase were primarily detectable on the basal and adluminal plasma membranes and on the surface of protrusions of the cell body into the lumen.     

Isolation of genes encoding β-D-xylanase, β-D-xylosidase and α-L-arabinofuranosidase activities from the rumen bacterium Prevotella ruminicola B14

Abstract Prevotella ruminicola B₁4 is a strictly anaerobic, Gram-negative, polysaccharide-degrading rumen bacterium. Xylanase activity in this strain was found to be inducible, the specific activity of cells grown on xylan being increased at least 20-fold by comparison with cells grown on glucose. Ten bacteriophage clones expressing xylanase activity were isolated from a A EMBL3 genomic DNA library of P. ruminicola B₁4. These clones were shown to represent four distinct chromosomal regions, based on restriction enzyme analysis and DNA hybridisation. Three groups of clones encoded activity against oat spelt xylan but not carboxymethylcellulose (CMC). In one of these groups, represented by clone 5, activities against pNP-arabinofuranoside and pNP-xyloside were found to be encoded separately from endoxylanase activity. The fourth region encoded activity against CM cellulose and lichenan, in addition to xylan, and contains an endoglucanase/xylanase gene isolated previously.     

Susceptibility of primary human airway epithelial cells to Bordetella pertussis adenylate cyclase toxin in two- and three-dimensional culture conditions

The human pathogen Bordetella pertussis targets the respiratory epithelium and causes whooping cough. Its virulence factor adenylate cyclase toxin (CyaA) plays an important role in the course of infection. Previous studies on the impact of CyaA on human epithelial cells have been carried out using cell lines derived from the airways or the intestinal tract. Here, we investigated the interaction of CyaA and its enzymatically inactive but fully pore-forming toxoid CyaA-AC^– with primary human airway epithelial cells (hAEC) derived from different anatomical sites (nose and tracheo-bronchial region) in two-dimensional culture conditions. To assess possible differences between the response of primary hAEC and respiratory cell lines directly, we included HBEC3-KT in our studies. In comparative analyses, we studied the impact of both the toxin and the toxoid on cell viability, intracellular cAMP concentration and IL-6 secretion. We found that the selected hAEC, which lack CD11b, were differentially susceptible to both CyaA and CyaA-AC^–. HBEC3-KT appeared not to be suitable for subsequent analyses. Since the nasal epithelium first gets in contact with airborne pathogens, we further studied the effect of CyaA and its toxoid on the innate immunity of three-dimensional tissue models of the human nasal mucosa. The present study reveals first insights in toxin–cell interaction using primary hAEC.     

Protective role of adenylate cyclase in the context of a live pertussis vaccine candidate

Despite high vaccination coverage, pertussis remains an important respiratory infectious disease and the least-controlled vaccine-preventable infectious disease in children. Natural infection with Bordetella pertussis is known to induce strong and long-lasting immunity that wanes later than vaccine-mediated immunity. Therefore, a live attenuated B. pertussis vaccine, named BPZE1, has been developed and has recently completed a phase I clinical trial in adult human volunteers. In this study, we investigated the contribution of adenylate cyclase (CyaA) in BPZE1-mediated protection against pertussis. A CyaA-deficient BPZE1 mutant was thus constructed. Absence of CyaA did not compromise the adherence properties of the bacteria onto mammalian cells. However, the CyaA-deficient mutant displayed a slight impairment in the ability to survive within macrophages compared to the parental BPZE1 strain. In vivo, whereas the protective efficacy of the CyaA-deficient mutant was comparable to the parental strain at a vaccine dose of 5 × 10⁵ colony forming units (CFU), it was significantly impaired at a vaccine dose of 5 × 10³ CFU. This impairment correlated with impaired lung colonization ability, and impaired IFN-γ production in the animal immunized with the CyaA-deficient BPZE1 mutant while the pertussis-specific antibody profile and Th17 response were comparable to those observed in BPZE1-immunized mice. Our findings thus support a role of CyaA in BPZE1-mediated protection through induction of cellular mediated immunity.     

Dissociation by cooling of hormone and cholera toxin activation of adenylate cyclase in intact cells

Cholera toxin, through adenylate cyclase activation reproduced cyclic AMP-mediated effects of thyroid-stimulating hormone (TSH) in dog thyroid slices, i.e protein iodination, [1-¹⁴C]glucose-oxidation and hormone secretion. Iodide and carbamylcholine decreased the cyclic AMP accumulation induced by cholera toxin as well as by TSH, which supports the hypothesis of an action of these agents beyond the steps of hormone-receptor and receptor-adenylate cyclase interaction. Cooling to 20°C did not impair the TSH induced cyclic AMP accumulation in thyroid slices, but completely suppressed the cholera toxin effect.This observation has been extended to other hormones and target tissues, such as the parathyroid hormone (PTH) (kidney cortex), adrenocorticotropic hormone (ACTH) (adrenal cortex)_and luteinizing hormone (LH) (ovary systems). As in thyroid, cooling dissociated the cholera toxin and hormonal effects on cyclic AMP accumulation. In homogenate, cooling decreased cyclic AMP generation in the presence of cholera toxin but at 20°C and 16°C a cholera toxin stimulation was still observed. These results bear strongly against the hypothesis that the glycoprotein hormones TSH and LH activate adenylate cyclase by a mechanism identical to cholera toxin.     

Forskolin stimulates adenylate cyclase and iodine metabolism in thyroid

Forskolin is a potent activator of the cyclic AMP-generating system in many tissues. In dog thyroid slices, the enhancement of cyclic AMP level was rapid, sustained in the presence of forskolin, but easily reversible after its withdrawal. Contrary to TSH, forskolin induced little apparent desensitization. Forskolin potentiated the effects of TSH, PGE1 and cholera toxin. However, the forskolin-induced cyclic AMP accumulation was still sensitive to inhibitors of dog thyroid adenylate cyclase such as iodide, norepinephrine and adenosine. As fluoride, but contrary to TSH and PGE1, forskolin stimulated adenylate cyclase in a medium where Mg2+ was replaced by Mn2+. This suggests that in thyroid, as in other tissues, forskolin acts beyond the receptor level but, as it potentiates hormone action and does not impair modulation by inhibitors, it may interact with the nucleotide-binding regulatory proteins. Forskolin mimicked the effect of TSH on iodide organification and secretion.     

Supra-molecular organization of adenylate cyclase in a neuroblastoma × glioma hybrid

Studies on the thermal inactivation of adenylate cyclase from neuroblastoma x glioma hybrid cells have been carried out. Inactivation curves show marked deviation from first-order kinetics, and as a first approximation can be adequately described as a sum of two negative exponentials. Half-lives of the rapidly decaying component have been estimated to be 5, 3.4,1.2 and 0.5 min at 37, 40, 44 and 48°C, respectively. The corresponding values for the slow-decaying component are found to be 90, 30, 11 and 5 min. Plausible inactivation pathways responsible for multi-exponential decay curves are discussed. Kinetic curves describing fractional loss of stimulatory response of adenylate cyclase to prostaglandin E₁ are shifted downwards with reference to basal activity. In contrast, an upward shift is observed for the inhibitory response of the enzyme to etorphine. A quantitative analysis of the inactivation curves for prostaglandin and etorphine-responsiveness has led to definitive predictions regarding the heat-sensitivity of the ‘hypothetical’ temperature-labile component responsible for the observed shifts.     

Decreased adenylate cyclase responsiveness of transformed cells correlates with the presence of a viral transforming protein

The adenylate cyclase responsiveness of transformed fibroblastic and epithelial cell lines to forskolin, fluoride, guanine nucleotides and cholera toxin was reduced compared to their parental counterparts. This phenomenon was observed in lines transformed by either RNA or DNA tumor viruses, and in the case of polyoma virus, coincided with the expression of middle T antigen. The data suggest that decreased responsiveness of adenylate cyclase to non-hormone activators is a general consequence of viral transformation and may be related to viral regulation of protein kinase activity.     

Adenylyl cyclase isoforms in rat testis and spermatozoa from the cauda epididymidis

Expression of adenylyl cyclase genes in rat testis and spermatozoa from the cauda epididymidis was investigated using RT-PCR analysis. Genes encoding the transmembrane adenylyl cyclases (tmAC) II, III, IV, V, VI, VII, and VIII were expressed in the testis, whereas only the gene for tmAC III was expressed in caudal spermatozoa. Immunocytochemistry was used to investigate which tmAC were translated into putative, functional proteins in spermatozoa. Indirect immunofluorescence localized the tmAC II enzyme to a region on the head occupied by the acrosome. The tmAC III enzyme was localized to the posterior margin of the head and to the flagellum, whereas tmAC V and/or VI was localized to the region where the ventral surface of the acrosomal equatorial segment is located. The tmAC VII and VIII enzymes were localized to the convex margin of the head, covering the dorsal region of the acrosomal crescent. To our knowledge, this is the first demonstration that five apparently different tmAC enzymes are localized to discrete subcellular regions of mammalian spermatozoa. These findings provide a fundamental basis for future studies, to determine the physiological roles of tmAC in testis and mature spermatozoa.This work was supported by a grant from the Australian Research Council/Department of Education, Training and Youth Affairs (ARC A40001141)     

Ultrastructural cytochemical localization of adenylate cyclase in the early chick embryo

Summary Adenylate cyclase activity was localized in various tissues of the early chick embryo using an ultrastructural histochemical technique. Reaction product was deposited on the lateral plasma membrane of all cells, but with a preferential localization at the apical terminal complex in the epiblast. There was no activity associated with the free surfaces of these or other cells in the embryo. Intracellular deposits were found in all cells associated with the endoplasmic reticulum, nuclear envelope and Golgi bodies. In the last organelle, the deposit was sometimes observed to be distributed through the stack in a non-uniform way, with the heaviest deposits occurring at the forming face. No clear difference could be detected between the cytochemical activity associated with cells in various regions of the embryo, or with embryos at different stages of early development.