Cyclopalladated Compound 7a Induces Apoptosis- and Autophagy-Like Mechanisms in Paracoccidioides and Is a Candidate for Paracoccidioidomycosis Treatment.

Paracoccidioidomycosis (PCM), caused by Paracoccidioides species, is the main cause of death due to systemic mycoses in Brazil and other Latin American countries. Therapeutic options for PCM and other systemic mycoses are limited and time-consuming, and there are high rates of noncompliance, relapses, toxic side effects, and sequelae. Previous work has shown that the cyclopalladated 7a compound is effective in treating several kinds of cancer and parasitic Chagas disease without significant toxicity in animals. Here we show that cyclopalladated 7a inhibited the in vitro growth of Paracoccidioides lutzii Pb01 and P. brasiliensis isolates Pb18 (highly virulent), Pb2, Pb3, and Pb4 (less virulent) in a dose-response manner. Pb18 was the most resistant. Opportunistic Candida albicans and Cryptococcus neoformans were also sensitive. BALB/c mice showed significantly lighter lung fungal burdens when treated twice a day for 20 days with a low cyclopalladated 7a dose of 30 µg/ml/day for 30 days after intratracheal infection with Pb18. Electron microscopy images suggested that apoptosis- and autophagy-like mechanisms are involved in the fungal killing mechanism of cyclopalladated 7a. Pb18 yeast cells incubated with the 7a compound showed remarkable chromatin condensation, DNA degradation, superoxide anion production, and increased metacaspase activity suggestive of apoptosis. Autophagy-related killing mechanisms were suggested by increased autophagic vacuole numbers and acidification, as indicated by an increase in LysoTracker and monodansylcadaverine (MDC) staining in cyclopalladated 7a-treated Pb18 yeast cells. Considering that cyclopalladated 7a is highly tolerated in vivo and affects yeast fungal growth through general apoptosis- and autophagy-like mechanisms, it is a novel promising drug for the treatment of PCM and other mycoses.

Effects of immunization with natural and recombinant lysine decarboxylase on canine gingivitis development.

Periodontal disease, gingival inflammation (gingivitis) and periodontal attachment loss (periodontitis), causes tooth loss and susceptibility to chronic inflammation. Professionally scaling and cleaning the teeth regularly controls the disease, but is expensive in companion animals. Eikenella corrodens is common in canine oral cavities where it is a source of lysine decarboxylase (LDC). In human dental biofilms (plaques), LDC converts lysine to cadaverine and impairs the gingival epithelial barrier to bacteria. LDC vaccination may therefore retard gingivitis development. Year-old beagle dogs provided blood samples, and had weight and clinical measurements (biofilm and gingivitis) recorded. After scaling and cleaning, two dogs were immunized subcutaneously with 0.2mg native LDC from E. corrodens and 2 sets of four dogs with 0.2mg recombinant LDC purified from Escherichia coli. A third set of 4 dogs was immunized intranasally. Rehydragel(®), Emulsigen(®), Polygen™ or Carbigen™ were used as adjuvant. Four additional pairs of dogs were sham-immunized with each adjuvant alone (controls). Immunizations were repeated twice, 3 weeks apart, and clinical measurements were obtained after another 2 weeks, when the teeth were scaled and cleaned again. Tooth brushing was then stopped and the diet was changed from hard to soft chow. Clinical measurements were repeated after 1, 2, 3, 4, 6 and 8 weeks. Compared with sham-immunized dogs, gingivitis was reduced over all 8 weeks of soft diet after subcutaneous immunization with native LDC, or after intranasal immunization with recombinant LDC in Carbigen™, but for only 6 of the 8 weeks after subcutaneous immunization with recombinant LDC in Emulsigen(®) (repeated measures ANOVA). Subcutaneous vaccination induced a strong serum IgG antibody response that decreased during the soft diet period, whereas intranasal immunization induced a weak serum IgA antibody response that did not decrease. Immunization with recombinant LDC may provide protection from gingivitis if procedures are optimized.

Systems-wide metabolic pathway engineering in Corynebacterium glutamicum for bio-based production of diaminopentane.

In the present work the Gram-positive bacterium Corynebacterium glutamicum was engineered into an efficient, tailor-made production strain for diaminopentane (cadaverine), a highly attractive building block for bio-based polyamides. The engineering comprised expression of lysine decarboxylase (ldcC) from Escherichia coli, catalyzing the conversion of lysine into diaminopentane, and systems-wide metabolic engineering of central supporting pathways. Substantially re-designing the metabolism yielded superior strains with desirable properties such as (i) the release from unwanted feedback regulation at the level of aspartokinase and pyruvate carboxylase by introducing the point mutations lysC311 and pycA458, (ii) an optimized supply of the key precursor oxaloacetate by amplifying the anaplerotic enzyme, pyruvate carboxylase, and deleting phosphoenolpyruvate carboxykinase which otherwise removes oxaloacetate, (iii) enhanced biosynthetic flux via combined amplification of aspartokinase, dihydrodipicolinate reductase, diaminopimelate dehydrogenase and diaminopimelate decarboxylase, and (iv) attenuated flux into the threonine pathway competing with production by the leaky mutation hom59 in the homoserine dehydrogenase gene. Lysine decarboxylase proved to be a bottleneck for efficient production, since its in vitro activity and in vivo flux were closely correlated. To achieve an optimal strain having only stable genomic modifications, the combination of the strong constitutive C. glutamicum tuf promoter and optimized codon usage allowed efficient genome-based ldcC expression and resulted in a high diaminopentane yield of 200 mmol mol(-1). By supplementing the medium with 1 mgL(-1) pyridoxal, the cofactor of lysine decarboxylase, the yield was increased to 300 mmol mol(-1). In the production strain obtained, lysine secretion was almost completely abolished. Metabolic analysis, however, revealed substantial formation of an as yet unknown by-product. It was identified as an acetylated variant, N-acetyl-diaminopentane, which reached levels of more than 25% of that of the desired product.

Biogenic amines in restructured beef steaks as affected by added walnuts and cold storage.

This article evaluates changes in biogenic amines and how these relate to microbiological growth in chilled, fresh restructured beef steaks containing transglutaminase as a cold binding agent and different amounts of walnut. Added walnut and chilling favored higher total and lactic acid bacteria counts during storage, whereas Enterobacteriaceae were not affected. The highest initial biogenic amine concentrations were identified as spermidine, spermine, and tyramine. Both added walnut and cold storage generally favored the formation of amines (tyramine, histamine, putrescine, and cadaverine), which was more obviously apparent by the end of the storage period. Agmatine, on the other hand, was not generally affected by the walnut.

Dietary guar gum and pectin stimulate intestinal microbial polyamine synthesis in rats.

The effects of two highly fermentable dietary fibers (guar gum and pectin) on the type and concentrations of cecal polyamines as affected by the intestinal microflora were studied in groups of germ-free (n = 10/group) and conventional rats (n = 6/group). Both germ-free and conventional rats were randomly assigned to one of three treatments as follows: 1) fiber-free control diet, 2) control diet + 10% guar gum and 3) control diet + 10% pectin. In germ-free rats, guar gum and pectin had no effect on cecal polyamine concentrations. Putrescine was confirmed to be the major endogenous polyamine within the gut lumen. In cecal contents of conventional rats, both guar gum and pectin led to the appearance of cadaverine and to elevated putrescine concentrations in comparison with the fiber-free control diet (1.35 +/- 0.15 and 2.27 +/- 0.32, respectively, vs. 0.20 +/- 0.03 micromol/g dry weight, P < 0.05). The cecal cadaverine concentration was higher in pectin- than in guar-fed rats (8.20 +/- 0.89 vs. 1.92 +/- 0.27 micromol/g dry weight, P < 0.05). Counts of total bacteria, bacteroides, fusobacteria and enterobacteria were higher (P < 0.05) in rats fed guar gum and pectin. Bifidobacteria were found exclusively in guar-fed rats. In vitro studies on selected species representing the numerically dominant population groups of the human gut flora (bacteroides, fusobacteria, anaerobic cocci and bifidobacteria) were examined for their ability to synthesize intracellular polyamines. These experiments demonstrated the ability of bacteroides, fusobacteria and anaerobic cocci to synthesize high amounts of putrescine and spermidine. Calculations based on these results suggest that the intestinal microflora are a major source of polyamines in the contents of the large intestine.

A new polyamine 4-aminobutylcadaverine. Occurrence and its biosynthesis in root nodules of adzuki bean plant Vigna angularis.

Root nodules of adzuki bean plant (Vigna angularis) contained a novel polyamine. The chemical structure of the new polyamine was determined to be NH2(CH2)5-NH(CH2)4NH2 (4-aminobutylcadaverine) based on gas chromatography-mass spectrometry. The occurrence of 4-aminobutylcadaverine was specific to the root nodules, since the unusual triamine was not detected in other organs of the adzuki bean plant. Bacteroids, isolated from root nodules, contained both sym-homospermidine and 4-aminobutylcadaverine, whereas the plant cytosol fraction contained large quantities of putrescine and cadaverine. A cell-free extract of bacteroids showed the ability to form this triamine from putrescine and cadaverine under the presence of NAD+ and K+. 1,3-Diaminopropane and NADH were inhibitory for the synthesis of both sym-homospermidine and 4-aminobutylcadaverine. [1,4-15N]Putrescine was incorporated not only into sym-homospermidine but also into 4-aminobutylcadaverine by the cell-free extract of bacteroids when incubated with excess cadaverine. Analysis of the fragment ion peaks in the 15N-enriched 4-aminobutylcadaverine indicated the transfer of a aminobutyl moiety to the amino terminus of cadaverine. These results suggest that, in adzuki bean, 4-aminobutylcadaverine is formed through the action of homospermidine synthase in nodule bacteroids under a cadaverine-rich environment.

Catabolism of arginine by the mixed bacteria in human salivary sediment under conditions of low and high glucose concentration.

The catabolism of glucose by the oral mixed bacteria results in a lowering of the pH whereas arginine degradation favours a rise. In the mouth, low and high levels of glucose cause different plaque pH conditions which, in turn, might affect the rate and mode of degradation of arginine. This possibility was examined in the suspended salivary-sediment system where these pH conditions can be simulated. With the pH, the metabolic parameters examined were arginine utilization, ammonia, carbon dioxide and putrescine formation, utilization of glucose and changes in levels of L(+)- and D(-)-lactic acid. At the lower glucose concentration, the pH rapidly fell and then slowly rose whereas, with the higher glucose level, the pH showed a greater fall and no subsequent rise. The more acidic pH conditions favoured by the higher glucose level inhibited arginine degradation and the appearance of its various end-products and intermediates. Arginine degradation with arginine-[U-14C] and paper chromatography-autoradiography showed successive appearance of citrulline, ornithine and putrescine and, depending upon the pH, some succinate. When the pH was held constant at several different values, arginine degradation was optimal when the pH was near neutrality. In supplementary experiments, arginine had little effect on the ability of the oral mixed bacteria to utilize glucose and produce and utilize lactic acid, whereas the arginine peptide, arginylisoleucine and saliva supernatant stimulated these processes. Thus glycolysis enhancement and a more rapid clearance of fermentable carbohydrate by the oral bacteria would accompany pH-rise activity with arginine peptide and saliva but would not accompany pH-rise activity with arginine.