Overexpression of NRPS4 leads to increased surface hydrophobicity in fusarium graminearum

The plant pathogen Fusarium graminearum is the infamous cause of Fusarium head blight worldwide resulting in significant losses of yield and reduced grain feed quality. It also has the potential to produce a range of small bioactive peptides produced by the non ribosomal peptide synthetases (NRPSs). Most of these are unknown as F. graminearum contains 19 NRPS encoding genes, but only three have been assigned products. For the first time, we use deletion and overexpression mutants to investigate the functions and product of NRPS4 in F. graminearum. Deletion of NRPS4 homologues in Alternaria brassicicola and Cochloibolus heterostrophus has been shown to result in mutants unable to repel water. In a time study of surface hydrophobicity we observed that water droplets could penetrate 7 d old colonies of the NRPS4 deletion mutants. Loss in ability to repel water was first observed on 13 d old cultures of the wild type strain, whereas the overexpression strain remained water repellant throughout the 38 d time study. The conidia of both mutants were examined and those of the overexpression mutant showed distinct morphological differences in form of collapsed cells. These observations might suggest that the peptide product of NRPS4 could be an architectural factor in the cell walls of Fusarium or an indirect regulator of hydrophobicity.     

Ionophores and cytochalasins modulate branching in Achlya bisexualis

Hyphae of Achlya bisexualis growing on a medium deficient in amino acids elongated but produced relatively few branches. Branching was enhanced by three classes of compound: cytochalasins A and E, the calcium ionophores A23187 and ionomycin and proton ionophores such as tetrachlorosalicylanilide (TCS), carbonylcyanide m-chlorophenylhydrazone (CCCP), and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP). We suggest that the effects of cytochalasins reflect the disruption of a microfilament-based system for vesicle transport. Enhancement of branching by ionophores implicates cytoplasmic ions in the control of branch initiation. There may be links between these phenomena and the earlier discovery that a new point of proton entry precedes the emergence of a branch and predicts its locus.     

Allosteric ribozymes sensitive to the second messengers cAMP and cGMP.

We have engineered allosteric ribozymes by combining modular rational design with combinatorial strategies. This new procedure was used to create allosteric ribozymes that are activated by specific nucleoside 3',5'-cyclic monophosphates (cNMPs). A random-sequence domain was attached to stem II of hammerhead ribozymes via a communication module that serves as an interface between ribozyme and the effector binding site. Subjecting this initial random pool to in vitro selection methods produced populations that respond, or cleave, only in the presence of specific effector molecules. From generation 18, 20 and 23, cGMP, cCMP and cAMP-specific responsive ribozymes, respectively, were isolated and characterized. These methods show great promise for engineering allosteric ribozymes and for creating new ligand-specific aptamers.     

The increase in cAMP and cGMP levels in the oestrogenized rat hypophysis.

Male and female rats were given oestradiol benzoate (1 mg s.c. twice a week for 3 weeks) and/or sodium nitroprusside (SN), a donor of nitric oxide (NO), which was administered in their food in amounts of 0.2 or 0.6 mg/rat/day. Neither oestradiol-induced hypertrophy of the hypophysis, nor the serum prolactin (PRL) level, was affected by the simultaneous administration of SN. The PRL content of the hypophysis rose after oestradiol in the males, but the increase was again uninfluenced by the simultaneous administration of SN and the cAMP content of the hypophysis--raised after oestradiol--was likewise unaffected. The amount of cGMP in the hypophysis after oestradiol rose only in males. Both the serum and the hypophyseal prolactin level were found to be correlated to the cAMP and the cGMP content of the hypophysis. It was found that the simultaneous administration of SN together with oestradiol slightly reduced the increase in the cGMP content of the hypophysis elicited with oestradiol treatment only.     

Differential effects of cAMP and cGMP on in vitro epidermal cell growth.

Primary keratinocyte cultures free of dermal fibroblasts were used to investigate the effect of varying cyclic AMP (cAMP) concentrations on epidermal cell function. Addition of 10^?3, 10^?4 or 10^?5 M dibutyryl cAMP to plated cells (day 1) results by day 5 in a dose dependent increase of [³H]TdR incorporation into DNA as determined by increases in both the labeling index and incorporation of ³H label into an isolated DNA fraction. 8-Bromo cAMP, another cAMP analogue, likewise induced keratinocyte proliferation. The proliferative response was dose and time dependent, and 5- to 6-fold increases in ³H label incorporated into DNA were seen at day 6, 8 and up until day 15 of culture. Moreover, elevation of cellular cAMP by addition of cholera toxin, an irreversible stimulator of adenylate cyclase, also demonstrated a time dependent stimulation of [³H]TdR uptake into DNA and increased the labeling index. Specific histochemical staining for keratinaceous protein (Kreyberg technique) demonstrated that elevated cAMP levels also enhance the production of specialized (differentiated) epidermal cells. Determination of the level of cAMP and cyclic GMP (cGMP) by RIA of partially purified fractions of the cultures revealed that addition of 8-bromo cAMP or cholera toxin to the cultures increased the levels of cAMP but not of cGMP. Addition of 8-bromo cGMP to the keratinocytes on day 1 at concentrations of 10^?6, 10^?7 or 10^?8 M had no effect on culture proliferation on days 4, 6 and 8, although qualitative changes in the electron microscopic pattern of the culture stratification and specialization were observed. The results indicate (1) both large and moderate increases in cellular cAMP levels induce keratinocyte culture proliferation and specialization in the absence of fibroblasts or dermal influences, (2) the quantitative enhancement of keratinocyte growth and specialization occurs without apparent participation of cGMP, (3) cGMP may be a qualitative effector of epidermal cell differentiation.     

Diverse directional changes of cGMP relative to cAMP in E. coli.

The relationships of the changes of cAMP and cGMP concentrations in $\text{E. coli}$ varied as a function of experimental conditions. (1) Cells starved for carbon source for a short time period had high cAMP and low cGMP concentrations. Addition of carbon source (succinate, glucose or α-methyl glucoside) led to a decrease in cAMP and an increase in cGMP (bi-directional change). (2) Washed cells starved for glucose for long time periods had low cAMP levels which did not change on glucose addition. Addition of succinate or glucose to such cells led to a transient increase in cGMP levels (uncoupled change). The cGMP concentration peaked at 15 minutes or 1 hour after glucose or succinate addition, respectively. (3) Sham shift-up experiments (addition of α-methyl glucoside to cultures growing in succinate) in $\text{E. coli}$ 1100 and CA 8000 showed decreases in cGMP levels in both strains; however, cAMP levels increased in the former (bi-directional change) and decreased in the latter (unidirectional change).     

IL-4 induces cAMP and cGMP in human monocytic cells

Human monocytes, preincubated with IFN-gamma respond to IL-4 by a cGMP increase through activation of an inducible NO synthase. Here, IL-4 was found to induce an accumulation of cGMP (1 - 3 min) and cAMP (20 - 25 min) in unstimulated monocytes. This was impaired with NOS inhibitors, but also with EGTA and calcium/calmodulin inhibitors. These results suggest that: (1) IL-4 may stimulate different NOS isoforms in resting and IFN-gamma activated monocytes, and (2) cAMP accumulation may be partially dependent on the NO pathway. By RT-PCR, a type III constitutive NOS mRNA was detected in U937 monocytic cells. IL-4 also increased the [Ca(2+)](i) in these cells. Different NOS may thus be expressed in monocytic cells depending on their differentiation and the signals they receive.     

CSCDB: the cAMP and cGMP signaling components database

Adenylate cyclases, guanylate cyclases, cyclic nucleotide phosphodiesterases, and cyclic nucleotide-binding proteins constitute the core of cAMP and cGMP signaling components. Using a combination of BLAST and profile search methods, we found that cyclic nucleotide-binding proteins exhibited diverse domain architectures. In addition to the domain architectures involved in the characterized functional groups, a cyclic nucleotide-binding domain was also fused to various domains involved in pyridine nucleotide-disulfide oxidoreductase, acetyltransferase, thioredoxin reductase, glutaminase, rhodanese, ferredoxin, and diguanylate cyclase, implying the versatile functions of cyclic nucleotide-binding proteins. We constructed the CSCDB database to accumulate the components of cAMP and cGMP signaling pathways in the complete genomes. User-friendly interfaces were created for easier browsing, searching, and downloading the data. Besides harboring the sequence itself, each entry provided detailed annotation information, such as sequence features, chromosomal localization, functional domains, transmembrane region, and sequence similarity against several major databases. Currently, CSCDB contains 4234 entries covering 466 organisms, including 35 eukaryotes, 382 bacteria, and 29 archaea. CSCDB can be freely accessible on the web at http://cscdb.com.cn.     

Photoreceptor channel activation: interaction between cAMP and cGMP

cAMP activates a current in excised patches from rod outer segments. The current at saturating concentrations of cAMP is approximately 25% of the current activated with 200 microM cGMP, the terminal cytoplasmic messenger in phototransduction. The K0.5 for cAMP is greater than 1.5 mM, and the index of cooperativity is approximately 1.4. cAMP activates the same population of channels as activated by cGMP since currents in the presence of both nucleotides are less than the sum of the individual responses. When increasing concentrations of cAMP, less than its K0.5, are added to a fixed, subsaturating concentration of cGMP, cAMP significantly enhances the total current compared with the current produced by cGMP alone. These results are predicted by a three-site, linear, sequential binding scheme where either cAMP or cGMP may bind to the same site on the channel. At approximately 5 microM cGMP, which is estimated to be the steady-state dark level in vertebrate photoreceptors, cAMP between 1 and 100 microM produces a large increase in the photoreceptor current. A possible physiological role for cAMP-cGMP interaction in phototransduction is discussed.     

Effect of leukoagglutinating phytohemagglutinin on cAMP and cGMP levels in lymphocytes

In concentrations which do not affect the cellular concentrations of the cyclic nucleotides cAMP and cGMP, phytohemagglutinin (PHA) (crystalline, kidney-bean leukoagglutinin) can activate adenoidal lymphocytes. High concentrations of leukoagglutinin caused an increase in cAMP without stimulating mitosis in lymphocytes. Our results do not support the concept that cyclic nucleotides act as mediators for lymphocyte activation by lectins. Previous contradictory reports on the role of cyclic nucleotides may have been due to the use of impure bean extracts and lymphocytes.     

Differential regulation of Paramecium ciliary motility by cAMP and cGMP

cAMP and cGMP had distinct effects on the regulation of ciliary motility in Paramecium. Using detergent-permeabilized cells reactivated to swim with MgATP, we observed effects of cyclic nucleotides and interactions with Ca2+ on the swimming speed and direction of reactivated cells. Both cAMP and cGMP increased forward swimming speed two- to threefold with similar half-maximal concentrations near 0.5 microM. The two cyclic nucleotides, however, had different effects in antagonism with the Ca2+ response of backward swimming and on the handedness of the helical swimming paths of reactivated cells. These results suggest that cAMP and cGMP differentially regulate the direction of the ciliary power stroke.     

cGMP signals mainly through cAMP kinase in permeabilized murine aorta

GMP affects vascular tone by multiple mechanisms, including inhibition of the Rho/Rho kinase-mediated Ca(2+) sensitization, a process identified as Ca(2+) desensitization. Ca(2+) desensitization is mediated probably by both cGMP- and cAMP-dependent protein kinases (cGKI and PKA). We investigate to which extent Ca(2+) desensitization is initiated by cGKI and PKA. cGMP/cAMP-induced relaxation was studied at constant [Ca(2+)] in permeabilized aortas from wild-type and cGKI-deficient mice. [Ca(2+)] increased aortic tone in the absence and presence of 50 microM GTPgammaS with EC(50) values of 160 and 30 nM, respectively. In the absence of GTPgammaS, the EC(50) for [Ca(2+)] was shifted rightward from 0.16 microM to 0.43 and 0.82 microM by 1 and 300 microM 8-bromo-cGMP (8-Br-cGMP), and to 8 microM by 10 microM Y-27632. Contractions induced by 300 nM [Ca(2+)] were relaxed by 8-Br-cGMP with an EC(50) of 2.6 microM. Surprisingly, [Ca(2+)]-induced contractions were also relaxed by 8-Br-cGMP in aortas from cGKI(-/-) mice (EC(50) of 19 microM). Western blot analysis of the vasodilator-stimulated phosphoprotein indicated "cross"-activation of PKA by 1 mM 8-Br-cGMP in aortic smooth muscle cells from cGKI(-/-) mice. Indeed, the PKA inhibitor peptide (PKI 5-24) completely abolished the relaxant effect of 8-Br-cGMP in muscles from cGKI(-/-) mice and to 65% in wild-type aortas. The thromboxane analogue U-46619 induced contraction at constant [Ca(2+)], which was only partially relaxed by 8-Br-cGMP but completely relaxed by Y-27632. The effect of 8-Br-cGMP on U-46619-induced contraction was attenuated by PKI 5-24. These results show that cGKI has only a small inhibitory effect on Ca(2+) sensitization in murine aortas.     

cAMP, cGMP and their visualization in living cells using fluorescent microscopy

cAMP and cGMP are ubiquitous second messengers regulating a myriad of intracellular functions. Standard biochemical techniques to measure their levels in cells and tissues lack high temporal and any spatial resolution. To enable real-time monitoring of cAMP and cGMP in living cells and physiological systems, we and others have developed several biosensors based on fluorescence resonance energy transfer. This review will describe such novel techniques and discuss their application for various biological questions.     

cAMP and cGMP changes associated with the differentiation of cultured chick embryo muscle cells.

A thin-slab, SDS polyacrylamide gel electrophoresis system is described in which actin within whole cell homogenates can be quantitated within a wide range of protein values (0.05–1.4 μg/band). After demonstrating the absence of appreciable contaminants in the actin band, and the lack of appreciable reincorporation of label during pulse-chase experiments, the turnover of actin was examined in pre-labeled cells during normal log growth and during induced encystation in Acanthamoeba. During log growth, no actin degradation was detected. However, as the cells approached the end of log phase growth and entered stationary phase, a dramatic increase in the amount of actin/cell and the percentage of total protein represented by actin was recorded. The encystation process per se was accompanied by a rapid reduction in these values to preencystment levels.