UDP-4-Keto-6-Deoxyglucose, a Transient Antifungal Metabolite, Weakens the Fungal Cell Wall Partly by Inhibition of UDP-Galactopyranose Mutase

ABSTRACT Can accumulation of a normally transient metabolite affect fungal biology? UDP-4-keto-6-deoxyglucose (UDP-KDG) represents an intermediate stage in conversion of UDP-glucose to UDP-rhamnose. Normally, UDP-KDG is not detected in living cells, because it is quickly converted to UDP-rhamnose by the enzyme UDP-4-keto-6-deoxyglucose-3,5-epimerase/-4-reductase (ER). We previously found that deletion of the er gene in Botrytis cinerea resulted in accumulation of UDP-KDG to levels that were toxic to the fungus due to destabilization of the cell wall. Here we show that these negative effects are at least partly due to inhibition by UDP-KDG of the enzyme UDP-galactopyranose mutase (UGM), which reversibly converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). An enzymatic activity assay showed that UDP-KDG inhibits the B. cinerea UGM enzyme with a K i of 221.9 µM. Deletion of the ugm gene resulted in strains with weakened cell walls and phenotypes that were similar to those of the er deletion strain, which accumulates UDP-KDG. Galf residue levels were completely abolished in the Δugm strain and reduced in the Δer strain, while overexpression of the ugm gene in the background of a Δer strain restored Galf levels and alleviated the phenotypes. Collectively, our results show that the antifungal activity of UDP-KDG is due to inhibition of UGM and possibly other nucleotide sugar-modifying enzymes and that the rhamnose metabolic pathway serves as a shunt that prevents accumulation of UDP-KDG to toxic levels. These findings, together with the fact that there is no Galf in mammals, support the possibility of developing UDP-KDG or its derivatives as antifungal drugs. IMPORTANCE Nucleotide sugars are donors for the sugars in fungal wall polymers. We showed that production of the minor sugar rhamnose is used primarily to neutralize the toxic intermediate compound UDP-KDG. This surprising finding highlights a completely new role for minor sugars and other secondary metabolites with undetermined function. Furthermore, the toxic potential of predicted transition metabolites that never accumulate in cells under natural conditions are highlighted. We demonstrate that UDP-KDG inhibits the UDP-galactopyranose mutase enzyme, thereby affecting production of Galf, which is one of the components of cell wall glycans. Given the structural similarity, UDP-KDG likely inhibits additional nucleotide sugar-utilizing enzymes, a hypothesis that is also supported by our findings. Our results suggest that UDP-KDG could serve as a template to develop antifungal drugs. IMPORTANCE Nucleotide sugars are donors for the sugars in fungal wall polymers. We showed that production of the minor sugar rhamnose is used primarily to neutralize the toxic intermediate compound UDP-KDG. This surprising finding highlights a completely new role for minor sugars and other secondary metabolites with undetermined function. Furthermore, the toxic potential of predicted transition metabolites that never accumulate in cells under natural conditions are highlighted. We demonstrate that UDP-KDG inhibits the UDP-galactopyranose mutase enzyme, thereby affecting production of Galf, which is one of the components of cell wall glycans. Given the structural similarity, UDP-KDG likely inhibits additional nucleotide sugar-utilizing enzymes, a hypothesis that is also supported by our findings. Our results suggest that UDP-KDG could serve as a template to develop antifungal drugs.

Construction of Generalized Integral Formulas by Means of Laplace Transformations

We present a method for the construction of integral identities that contain an undetermined function. Except for mild restrictions, this function can be chosen arbitrarily. Our method is illustrated by several examples leading to new integral identities.

A Class of Exact Solution of (3+1)-Dimensional Generalized Shallow Water Equation System

Shallow water wave equation has increasing use in many applications for its success in eliminating spurious oscillation, and has been widely studied. In this paper, we investigate (3+1)-dimensional generalized shallow water equation system. Based on the $(G'/G)$-expansion method and the variable separation method, we choose $\xi (x,y,z,t) = f(y + cz) + ax + h(t)$ and suppose that ${a_i}(i = 1,2, \ldots,m)$ is an undetermined function about $x,y,z,t$ instead of a constant in eq. (3), which are different from those in previous literatures. With the aid of symbolic computation, we obtain a family of exact solutions of the (3+1)-dimensional generalized shallow water equation system in forms of the hyperbolic functions and the trigonometric functions. When the parameters take special values, in addition to traveling wave solutions, we also get the nontraveling wave solutions by using our method; these obtained solutions possess abundant structures. The figures corresponding to these solutions are illustrated to show the particular localized excitations and the interactions between two solitary waves. The $(G'/G)$-expansion method is a very general and powerful tool that will lead to further insights and improvements of the nonlinear models.

HAWKING RADIATION OF DIRAC PARTICLES FROM A GENERAL DYNAMICAL SPHERICALLY SYMMETRICAL BLACK HOLE USING A NEW TORTOISE COORDINATE TRANSFORMATION

By utilizing the improved Damour–Ruffini method with a new tortoise transformation, we study the Hawking radiation of Dirac particles from a general dynamical spherically symmetric black hole. In the improved Damour–Ruffini method, the position of the event horizon of the black hole is an undetermined function, and the temperature parameter κ is an undetermined constant. By requiring the Dirac equation to be the standard wave equation near the event horizon of the black hole, κ can be determined automatically. Therefore, the Hawking temperature can be obtained. The result is consistent with that of the Hawking radiation of scalar particles.

Mutational Analysis of nocK and nocL in the Nocardicin A Producer Nocardia uniformis

ABSTRACT The nocardicins are a family of monocyclic β-lactam antibiotics produced by the actinomycete Nocardia uniformis subsp. tsuyamanensis ATCC 21806. The most potent of this series is nocardicin A, containing a syn-configured oxime moiety, an uncommon feature in natural products. The nocardicin A biosynthetic gene cluster was recently identified and found to encode proteins in keeping with nocardicin A production, including the nocardicin N-oxygenase, NocL, in addition to genes of undetermined function, such as nocK, which bears similarities to a broad family of esterases. The latter was hypothesized to be involved in the formation of the critical β-lactam ring. While previously shown to effect oxidation of the 2′-amine of nocardicin C to provide nocardicin A, it was uncertain whether NocL was the only N-oxidizing enzyme required for nocardicin A biosynthesis. To further detail the role of NocL in nocardicin production in N. uniformis, and to examine the function of nocK, a method for the transformation of N. uniformis protoplasts to inactivate both nocK and nocL was developed and applied. A reliable protocol is reported to achieve both insertional disruption and in trans complementation in this strain. While the nocK mutant still produced nocardicin A at levels near that seen for wild-type N. uniformis, and therefore has no obvious role in nocardicin biosynthesis, the nocL disruptant failed to generate the oxime-containing metabolite. Nocardicin A production was restored in the nocL mutant upon in trans expression of the gene. Furthermore, the nocL mutant accumulated the biosynthetic intermediate nocardicin C, confirming its role as the sole oxime-forming enzyme required for production of nocardicin A.

Isolation and Characterization Of Rice R Genes: Evidence for Distinct Evolutionary Paths in Rice and Maize

R and B genes and their homologues encode basic helix-loop-helix (bHLH) transcriptional activators that regulate the anthocyanin biosynthetic pathway in flowering plants. In maize, R/B genes comprise a very small gene family whose organization reflects the unique evolutionary history and genome architecture of maize. To know whether the organization of the R gene family could provide information about the origins of the distantly related grass rice, we characterized members of the R gene family from rice Oryza sativa. Despite being a true diploid, O. sativa has at least two R genes. An active homologue (Ra) with extensive homology with other R genes is located at a position on chromosome 4 previously shown to be in synteny with regions of maize chromosomes 2 and 10 that contain the B and R loci, respectively. A second rice R gene (Rb) of undetermined function was identified on chromosome 1 and found to be present only in rice species with AA genomes. All non-AA species have but one R gene that is Ra-like. These data suggest that the common ancestor shared by maize and rice had a single R gene and that the small R gene families of grasses have arisen recently and independently.

Dihydrocytochalasin B. Biological effects and binding to 3T3 cells.

Dihydrocytochalasin B (H2CB) does not inhibit sugar uptake in BALB/c 3T3 cells. Excess H2CB does not affect inhibition of sugar uptake by cytochalasin B (CB), indicating that it does not compete with CB for binding to high-affinity sites. As in the case of CB, H2CB inhibits cytokinesis and changes the morphology of the cells. These results demonstrate that the effects of CB on sugar transport and on cell motility and morphology involve separate and independent sites. Comparison of the effects of H2CB, CB, and cytochalasin D (CD) indicates that treatment of cells with any one of the compounds results in the same series of morphological changes; the cells undergo zeiosis and elongation at 2-4 microM CB and become arborized and rounded up at 10-50 microM CB. H2CB is slightly less potent than CB, whereas CD is five to eight times more potent than CB in causing a given state of morphological change. These results indicate that the cytochalasin-induced changes in cell morphology are mediated by a specific site(s) which can distinguish the subtle differences in the structures of the three compounds. Competitive binding studies indicate that excess H2CB displaces essentially all of the high-affinity bound [3H]CB, but, at less than 5 x 10(-5) M H2CB is not so efficient as unlabeled CB in the displacement reaction. In contrast, excess CD displaces up to 40% of the bound [3H]CB. These results suggest that three different classes of high-affinity CB binding sites exist in 3T3 cells: sites related to sugar transport, sites related to cell motility and morphology, and sites with undetermined function.

An Iterative Technique for Minimising a Double Integral with Applications in Elasticity

The solution of a problem in plane elasticity can be associated with a minimum energy principle, involving the minimisation of a double integral subject to certain boundary conditions. In 1933, Kantorovich proposed a method which reduced this problem to that of finding the minimum of a single integral. He chose part of the solution a priori (in accordance with the character of the problem) which left only one undetermined function in one of the variables. This unknown function could then be found through the solution to an ordinary differential equation. He then constructed a second approximation by introducing two unknown functions which led to the solution of two simultaneous differential equations. Again the accuracy of the solution was dependent on the initial choice for part of the function; so also was the rate of convergence.

THE COLLISION MATRIX FOR THE COMPOUND NUCLEUS

The nuclear collision matrix is calculated under the restrictions of the compound nucleus hypothesis. The matrix so found contains only one undetermined function, τ(E), a phase shift caused by nuclear interaction. Cross sections are evaluated in terms of τ(E), a set of constants, and functions determined by the non-nuclear potentials.