Biotechnology, physiology and genetics of the yeast Pichia anomala

The ascomycetous yeast Pichia anomala is frequently associated with food and feed products, either as a production organism or as a spoilage yeast. It belongs to the nonSaccharomyces wine yeasts and contributes to the wine aroma by the production of volatile compounds. The ability to grow in preserved food and feed environments is due to its capacity to grow under low pH, high osmotic pressure and low oxygen tension. A new application of P. anomala is its use as a biocontrol agent, which is based on the potential to inhibit a variety of moulds in different environments. Although classified as a biosafety class-1 organism, cases of P. anomala infections have been reported in immunocompromised patients. On the other hand, P. anomala killer toxins have a potential as antimicrobial agents. The yeast can use a broad range of nitrogen and phosphor sources, which makes it a potential agent to decrease environmental pollution by organic residues from agriculture. However, present knowledge of the physiological basis of its performance is limited. Recently, the first studies have been published dealing with the global regulation of the metabolism of P. anomala under different conditions of oxygenation.     

Novel bis(1-acyl-2-pyrazolines) of potential anti-inflammatory and molluscicidal properties

A variety of bis[3-aryl-4,5-dihydro-1H-pyrazol-1-carboxaldehydes] 4a-h were obtained via reaction of bis[1-aryl-2-propen-1-ones] 3a-h with hydrazine hydrate in refluxing formic acid. In addition, the corresponding bis[1-acetyl-3-aryl-4,5-dihydro-1H-pyrazoles] 4i-m were formed through conducting the reaction of 3 with hydrazine hydrate in refluxing acetic acid. The starting bis(2-propen-1-ones) 3a-h were prepared stereoselectively as E,E'-geometric isomer via condensation of bisbenzaldehydes 1a,b with (un)substituted acetophenones 2 in ethanolic KOH solution. Anti-inflammatory as well as ulcerogenic activities of the prepared pyrazolines were evaluated in vivo and compared with that of a standard drug (indomethacin). Many of the tested compounds show remarkable anti-inflammatory properties with an ulcerogenic liability (especially 4f, g, j, and k) lower than that of the standard used drug. Compound 4f was established to be the best effectively prepared anti-inflammatory active pyrazoline derivative and safer than indomethacin with respect to its ulcerogenic liability. Molluscicidal activity of the prepared compounds against Biomphalaria alexandrina snails (the intermediate host of Schistosoma mansoni) was screened. Where, some of the prepared compounds show considerable activities.     

Novel synthesis of nicotinamide derivatives of cytotoxic properties

A variety of 2-substituted-4,6-diaryl-3-pyridinecarboxamides 5 were synthesized through aromatic nucleophilic substitution reaction of secondary amines with 2-bromo analogues 4. The latter were obtained via bromination of 2-cyano-3,5-diaryl-5-oxo-N-substituted pentamides 3 in glacial acetic acid. Moreover, pentamide derivatives 3 were prepared through base-catalyzed Michael addition of cyanacetanilides 2 with 1,3-diaryl-2-propen-1-ones 1. Otherwise, reaction of 2-bromo-3-pyridinecarboxamides 4 with primary aromatic amines in refluxing pyridine afforded the corresponding 2-(arylamino)-3-pyridinecarboxamides 6 besides the unexpected 2-unsubstituted amino analogues 7. Antitumor properties of the synthesized pyridinecarboxamides utilizing 59 different human tumor cell lines, representing leukemia, melanoma, and cancers of the lung, colon, brain, ovary, breast, prostate as well as kidney, were screened. Many of the tested compounds show considerable in vitro antitumor properties especially 5c and 7a, which reveal moderate activities against most of the used human tumor cell lines. It has also been achieved that, all the tested nicotinamide derivatives reveal promising antitumor properties against MDA-MB-231/ATCC (breast cancer).     

Synthesis of novel vasodilatory active nicotinate esters with amino acid function

A variety of N-[(ethyl-4,6-diaryl-3-pyridinecarboxylate)-2-yl]amino acid esters 6a–h were synthesized through the reaction of 2-bromonicotinates 4 with a number of primary amino acid ester hydrochlorides 5 in refluxing tetrahydrofuran in the presence of triethylamine as dehydrohalogenating agent. Similarly, reaction of 4 with N-glycylglycine ethyl ester hydrochloride 7 ‘as a representative example of dipeptide derivative’ afforded smoothly the corresponding N-[(ethyl-4,6-diaryl-3-pyridinecarboxylate)-2-yl]-N′-glycylglycine ethyl ester analogues 8. However, reaction of 4 with 5 in refluxing pyridine yielded the unexpected 2-aminonicotinate esters 9. Vasodilation activity screening for the synthesized nicotinate esters was investigated in vitro on the contractile response of vascular thoracic aorta smooth muscle from Wistar rats, where all the tested compounds exhibit considerable vasodilatory properties. In addition, few prepared compounds especially, 6b, 6h and 9b reveal remarkable vasodilation potency (IC₅₀).     

Role of glutamine 148 of human 15-hydroxyprostaglandin dehydrogenase in catalytic oxidation of prostaglandin E2

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short-chain dehydrogenase/reductase (SDR) family, catalyzes the first step in the catabolic pathways of prostaglandins and lipoxins. This enzyme oxidizes the C-15 hydroxyl group of prostaglandins and lipoxins to produce 15-keto metabolites which exhibit greatly reduced biological activities. A three-dimensional (3D) structure of 15-PGDH based on the crystal structures of the levodione reductase and tropinone reductase-II was generated and used for docking study with NAD+ coenzyme and PGE2 substrate. Three well-conserved residues among SDR family which correspond to Ser-138, Tyr-151, and Lys-155 of 15-PGDH have been shown to participate in the catalytic reaction. Based on the molecular interactions observed from 3D structure of 15-PGDH, we further propose that Gln-148 in 15-PGDH is important in properly positioning the 15-hydroxyl group of PGE2 by hydrogen bonding with the side-chain oxygen atom of Gln-148. This residue is found to be less conserved and replaceable by glutamyl, histidinyl, and asparaginyl residues in SDR family. Accordingly, site-directed mutagenesis of Gln-148 of 15-PGDH to alanine, glutamic acid, histidine, and asparagine (Q148A, Q148E, Q148H, and Q148N) was carried out. The activity of mutant Q148A was not detectable, whereas those of mutants Q148E, Q148H, and Q148N were comparable to or higher than the wild type. This indicates that the side-chain oxygen or nitrogen atom at position 148 of 15-PGDH plays an important role in anchoring C-15 hydroxyl group of PGE2 through hydrogen bonding for catalytic reaction.     

Conserved and non-conserved loci of the glucagon gene in old world ruminating ungulates

The homology and diversification of genomic sequence encoding glucagon gene among native Egyptian buffalos, camel and sheep were tested using cattle as model. Oligodeoxynucleotide primers designed from the available GenBank data were used for PCR probing of the glucagon gene encoding sequence at different loci. The DNA oligomer probes were constructed to flank either the whole gene encoding sequence or different intra-gene encoding sequences. The PCR products were visualized using agarose gel electrophoresis. All species showed a same size band of prepro-glucagon when PCR was used to amplify the whole gene encoding sequence. In contrary, amplifications of different intra-gene loci failed to give the same results. The results indicated variable degrees of diversity among old world ruminating ungulates in the glucagon gene encoding sequence. Compared with other ruminants, the variation appears predominantly in camel. Surprisingly, the similarity in size between both amplification products of whole gene encoding sequence and the proposed size of glucagon cDNA definitely excludes the possibility of large intervening introns spanning the genomic sequence of the glucagon gene in these species. This indicates that, in contrast to other tested mammals, the glucagon gene includes an essentially full-length copy of glucagon mRNA. The study revealed a possible new aspect of glucagon gene evolution in order to correlate its corresponding protein function among different ruminant species.     

In vitro organogenesis and alkaloid accumulation in Datura innoxia

The kinetics of tropane alkaloids accumulation in different organs such as roots, leaves, stems, flowers and seeds of Datura innoxia was investigated by GC-MS. Twenty-six tropane alkaloids were detected. The ester derivatives of tropine (3alpha-tigloyloxytropine and 3-tigloyloxy-6-hydroxytropine) are the major compounds. Undifferentiated callus were established from the stem explants of Datura innoxia using Murashige and Skoog (MS) medium supplied with 6-benzylaminopurine (BA, 1 mg l(-10) and indole-3-acetic acid (IAA, 0.5 mg l(-1)) in combination for 6 weeks. Callus differentiation was initiated by subculture onto solid MS medium, free from hormones, for more than 10 months. Initially, shoots were formed after four weeks from subculture. Further subculturing in basal MS medium without growth regulators initiated the rooting of a shooty callus after 6 weeks. Investigation of the alkaloid content of the unorganized and organized callus revealed that callus (either green or brown) yielded only trace amounts of alkaloids. On the other hand, re-differentiated shoots contained mainly scopolamine while re-differentiated roots biosynthesized hyoscyamine as the main alkaloid.     

Pyrrolidine bis-cyclic guanidines with antimicrobial activity against drug-resistant Gram-positive pathogens identified from a mixture-based combinatorial library

The rapid rise in antibiotic-resistant Gram-positive bacterial infections prompted us to explore the development of novel strategies for synthesis of large chemical libraries amenable to high-throughput screening for antimicrobial activities. Here we report the solid-phase synthesis of a 738,192 member pyrrolidine bis-cyclic guanidine chemical library with 26 different amino acids at three positions of diversity and 42 carboxylic acids at the fourth position. This synthetic combinatorial library was developed for positional scanning and screened for bacteriostatic and bactericidal activities against the important human pathogen methicillin-resistant Staphylococcus aureus (MRSA). The eight compound mixtures exhibiting bactericidal activity (10 microg/mL) against MRSA were used to direct the synthesis of 36 individual compounds that were then screened for activity against MRSA, vancomycin-resistant Enterococcus faecalis (VRE), and two Gram-negative bacterial species. At least 20 individual compounds were bactericidal for MRSA at 2.5 microg/mL, with a subset of these compounds showing bactericidal activities (10 microg/mL) against the other species tested. This approach demonstrates the capability to synthesize and screen a complex library to yield promising antimicrobials that address a critical need for novel infectious disease therapeutics.     

Inhibitory effect of flavonoids on mutant H-Rasp protein

Mutant form of H-Ras (Harvey-Ras) proteins are found in almost 10%-25% of human tumours. Mutational activation transforms it into an oncogenic form, which results in the loss of intrinsic GTPase function and therefore the protein is constitutively in the active, GTP-bound state and is continuously sending signals for cell growth and proliferation. In the present insilico study, the inhibitory effect of different flavonoid compounds on mutant H Ras protein p21 has been assessed. In addition, inhibitory effect of flavonoids is compared with 3 known anticancer drugs. Upon docking, it was found that flavonoids such as Naringenin, Daidzein, and Hesperetin showed highest affinity (most negative ΔG), while Rutin showed no affinity towards mutant H Ras. The 3 clinical anticancer agents (Erlotinib, Letrozole and Exemestane) showed binding energies in the range of -1.11 to -5.51 kcal/mol which is comparatively lower than the flavonoids indicating efficacy of flavonoids in the treatment of cancer with little or no cytotoxicity. Our study demonstrates that flavonoids (particularly Naringenin, Daidzein, and Hesperetin) are the effective drugs to inhibit function of mutant H-Ras P21 protein, which in turn arrests the process of cell growth and proliferation of the cancer cell.     

Rapid Hybridization Probe Assay and PCR for Detection of Chlamydia trachomatis in Urinary Tract Infections: A Prospective Study

Chlamydia trachomatis is a widespread bacterium that causes trachoma and genital tract infections in humans. The fact that the growth of this pathogen does not normally occur outside living cells poses a challenge in its diagnosis. The present study aimed to compare the efficacies of different molecular and cultural methods in the detection of C. trachomatis in urine samples collected from patients with urinary tract infections. Examined detection methods involved the Gen-Probe C. trachomatis (GP-CT) assay, direct antigen detection by enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) method. The efficacies of these methods were compared to that of the cell culture technique depending on sensitivity, specificity, and accuracy. C. trachomatis was detected in 25 out of 50 (50%) of examined urine samples using the cell culture method. Compared with this standard technique, the GP-CT assay was the most sensitive procedure, being able to detect the pathogen in all positive samples, followed by PCR and ELISA, which showed 60% and 40% sensitivities, respectively. PCR and ELISA displayed the highest level of specificity (100%) compared to the cell culture method with the GP-CT assay showing 40% specificity. The rate of accuracy was comparable between the GP-CT, PCR, and ELISA methods ranging from 70–80% of the accuracy of the cell culture method. The above results suggest that C. trachomatis is a frequent pathogen associated with upper and lower urinary tract infections. Both the GP-CT assay and PCR method can be recommended as reliable detection methods for C. trachomatis, and the GP-CT can be used as a screening tool.