A New Model of Vaginal Infection by <Emphasis Type=Italic>Candida albicans</Emphasis> in Rats

Vulvovaginal candidiasis (VVC) is regarded as an important public health issue, and several aspects of its pathogenesis are not yet sufficiently clear. Experimental in vivo models of vaginal infection with Candida albicans have been extremely useful in the identification of factors concerning hormonal influences on the infection, the virulence of the yeasts, the susceptibility, and the treatment of the infection. The development of easily manageable, reproducible, and economically viable animal models of VVC is highly important. We describe a simple experimental model of VVC in rats, using a pharmaceutical brand of estradiol hexa-hydrobenzoate for human treatment. All the steps of this model were standardized; and after the experiments, the rats were euthanized for further examination of their tissues by scanning and transmission electron microscopy. Standardized features included the use of non-ovariectomized rats, sterile distilled water as the hormone vehicle, estradiol hexa-hydrobenzoate administered at 0.20 mg/week/rat fractionated three times/week, and a yeast suspension of 5 × 10⁸ yeasts/ml in a single vaginal administration 1 week after hormone induction. In this way, 100% of the rats were in pseudo-estrus and developed and maintained the infection until the third week of the experiment. Electron microscopy observation of the vagina of the rats confirmed the presence of both pseudo-estrus and vaginal infection. The standardized experimental model proved inexpensive, reproducible, and easily feasible for the induction of vaginal infection with C. albicans and may help to clarify important aspects of the pathogenesis and treatment of VVC.     

Differential Resistance to Oxidants and Production of Hydrolytic Enzymes in <Emphasis Type="Italic">Candida albicans</Emphasis>

Resistance to the toxic effects of reactive oxygen species produced by phagocytes and production of hydrolytic enzymes are important aspects of Candida albicans virulence. In this report, we compared twelve C. albicans isolates for their in vitro capacity to resist oxidants—hydrogen peroxide, menadione and paraquat; and to produce hydrolytic enzymes—phospholipase and protease. Different C. albicans isolates showed different degrees of resistance to oxidants as well as differences in production of hydrolytic enzymes. Resistance to oxidative stress did not correlate with production of hydrolytic enzymes. This reinforces the view that C. albicans differentially regulates the expression of virulence factors in response to local environmental conditions.     

Proteomic analysis of hyphae-specific proteins that are expressed differentially in <Emphasis Type=Italic>cakem1/cakem1</Emphasis> mutant strains of <Emphasis Type=Italic>Candida albicans</Emphasis>

The yeast-to-hyphal transition is a major virulence factor in the fungal pathogen Candida albicans. Mutations in the CaKEM1 gene, which encodes a 5′-3′ exoribonuclease responsible for mRNA degradation, show a defect in hyphal growth. We applied two-dimensional gel electrophoresis to identify hyphae-specific proteins that have altered expressions in the presence of the cakem1 mutation. Eight proteins, Eno1, Eps1, Fba1, Imh3, Lpd1, Met6, Pdc11, and Tsa1 were upregulated during hyphal transition in wild-type but not in cakem1/cakem1 mutant cells. A second group of proteins, Idh1, Idh2, and Ssb1, showed increased levels of expression in cakem1/cakem1 mutant cells when compared to wild-type cells. Overexpression of Lpd1, a component of the pyruvate dehydrogenase complex, caused slight hyperfilamentation in a wild-type strain and suppressed the filamentation defect of the cakem1 mutation. The Ssb1 protein, which is a potential heat shock protein, and the Imh3 protein, which is a putative enzyme in GMP biosynthesis also showed the filamentation-associated phenotypes.     

Attenuation of fibrosis <Emphasis Type=Italic>in vitro</Emphasis> and <Emphasis Type=Italic>in vivo</Emphasis> with <Emphasis Type=Italic>SPARC</Emphasis> siRNA

Introduction  

SPARC is a matricellular protein, which, along with other extracellular matrix components including collagens, is commonly over-expressed in fibrotic diseases. The purpose of this study was to examine whether inhibition of SPARC can regulate collagen expression in vitro and in vivo, and subsequently attenuate fibrotic stimulation by bleomycin in mouse skin and lungs.     

Efficient separation and purification of allophycocyanin from <Emphasis Type=Italic>Spirulina</Emphasis> (<Emphasis Type=Italic>Arthrospira</Emphasis>) <Emphasis Type=Italic>platensis</Emphasis>

Allophycocyanin (APC) is a minor component of phycobiliproteins in cyanobacteria and red algae. This paper describes a simple and inexpensive extracting method for isolating APC from Spirulina (Arthrospira) platensis with high efficiency. The crude phycobiliprotein extract was pretreated by ammonium sulfate fractionation. Then, by adding hydroxylapatite into crude phycobiliprotein extract dissolved in 20 mM phosphate buffer (pH 7.0), APC was selectively adsorbed by hydroxylapatite but C-phycocyanin (C-PC) was not. The hydroxylapatite was collected and APC was extracted from the crude phycobiliprotein extract. Then, the enriched APC was washed off from the hydroxylapatite using 100 mM phosphate buffer (pH 7.0). In this simple extracting method it was easy to remove C-PC and isolate APC in large amounts. The absorbance ratio A ₆₅₀/A ₂₈₀ of extracted APC reached 2.0. The recovery yield was 70%, representing 4.61 mg · g⁻¹ wet weight. The extracted APC could be further purified by a simple anion-exchange chromatography with a pH gradient from 5.6 to 4.0. The absorbance ratio A ₆₅₀/A ₂₈₀ of the purified APC reached 5.0, and the overall recovery yield was 43%, representing 2.83 mg · g⁻¹ wet weight. Its purity was confirmed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE.     

Interaction of <Emphasis Type=Italic>Bdellovibrio bacteriovorus</Emphasis> with bacteria <Emphasis Type=Italic>Campylobacter jejuni</Emphasis> and <Emphasis Type=Italic>Helicobacter pylori</Emphasis>

Interaction of Bdellovibrio bacteriovorus 100NCJB with bacteria Campylobacter jejuni (strains 1, 2, 3, 4, and 5) and Helicobacter pylori, strain TX30a, was confirmed. The results indicate that lytic activity of bdellovibrios both in liquid media and cells attached to a surface was observed. The potential use of the antimicrobial activity of predatory bacteria for environmental bioprotection and public health is discussed.     

Probiotic <Emphasis Type=Italic>Lactobacillus</Emphasis> strains: <Emphasis Type=Italic>in vitro</Emphasis> and <Emphasis Type=Italic>in vivo</Emphasis> studies

Three Lactobacillus strains (LOCK 0900, LOCK 0908, LOCK 0919) out of twenty-four isolates were selected according to their antagonistic activity against pathogenic bacteria, resistance to low pH and milieu of bile salts. Intragastric administration of a mixture of these strains to Balb/c mice affected cytokine T_H1-T_H2 balance toward nonallergic T_H1 response. Spleen cells, isolated from lactobacilli-treated mice and re-stimulated in vitro with the mixture of heat-inactivated tested strains, produced significantly higher amounts of anti-allergic tumor necrosis factor- and interferon-γ than control animals whereas the level of pro-allergic interleukin-5 was significantly lower. Lactobacillus cells did not translocate through the intestinal barrier into blood, liver and spleen; a few Lactobacillus cells found in mesenteric lymph nodes could create antigenic reservoir activating the immune system. The mixture of Lactobacillus LOCK 0900, LOCK 0908 and LOCK 0919 strains represents a probiotic bacterial preparation with possible use in prophylaxis and/or therapy of allergic diseases.     

Disruption of Homocitrate Synthase Genes in <Emphasis Type=Italic>Candida albicans</Emphasis> Affects Growth But Not Virulence

Two genes, LYS21 and LYS22, encoding isoforms of homocitrate synthase, an enzyme catalysing the first committed step in the lysine biosynthetic pathway, were disrupted in Candida albicans using the SAT1 flipper strategy. The double null lys21Δ/lys22Δ mutant lacked homocitrate synthase activity and exhibited lysine auxotrophy in minimal media that could be fully rescued by the addition of 0.5–0.6 mM l-lysine. On the other hand, its virulence in vivo in the model of disseminated murine candidiasis appeared identical to that of the mother, wild-type strain. These findings strongly question a possibility of exploitation of homocitrate synthase and possibly also other enzymes of the lysine biosynthetic pathway as targets in chemotherapy of disseminated fungal infections.     

<Emphasis Type=Italic>In vitro</Emphasis> organogenesis of <Emphasis Type=Italic>Passiflora alata</Emphasis>

Passiflora alata in vitro organogenesis was studied based on explant type, culture medium composition, and incubation conditions. The results indicated that the morphogenic process occurred more efficiently when hypocotyl segment-derived explants were cultured in media supplemented with cytokinin and AgNO₃ incubated under a 16-h photoperiod. The shoot bud elongation and plant development were obtained by transferring the material to MSM culture medium supplemented with GA₃ and incubated in flasks with vented lids. Histological analyses of the process revealed that the difficulties in obtaining plants could be related to the development of protuberances and leaf primordia structures, which did not contain shoot apical meristem. Roots developed easily by transferring elongated shoots to 1/2 MSM culture medium. Plant acclimatization occurred successfully, and somaclonal variation was not visually detected. The efficiency of this organogenesis protocol will be evaluated for genetic transformation of this species to obtain transgenic plants expressing genes that can influence the resistance to Cowpea aphid borne mosaic virus.     

Mutants of <Emphasis Type=Italic>Candida albicans</Emphasis> hypersensitive to calcofluor white display susceptibility to antifungal drugs

An increased infection incidence of Candida albicans (most common human fungal pathogen) contributes to the need of further functional genetic studies and development of new antifungal drugs. We developed a method to create mutants of C. albicans using an antisense cDNA library to interfere with gene expression, followed by screening for hypersensitivity to Calcofluor White (CFW) and the antifungal drugs caspofungin and itraconazole. Mutants with these properties have with a high probability defects in cell-wall integrity. Fifty out of 200 transformant colonies analyzed (25 %) showed hypersensitivity to CFW compared with the parental strain C. albicans CAI-4. Most of those CFW-hypersensitive mutants further displayed the susceptibility to antifungal drugs itraconazole and caspofungin using microbroth dilution method M27-A and an agar-diffusion test. The mutants obtained through this procedure could provide a potential model for screening antifungal pro-drugs which show weak action when standard C. albicans strain is used and may also aid in further identifying genes involved in cell integrity. In addition, we describe the effect of varying several parameters in electroporation transformation, including treatment with lithium acetate, upon the efficiency of transformation in C. albicans.     

Regulation of the <Emphasis Type=Italic>ALBINO3</Emphasis>-mediated transition to flowering in <Emphasis Type=Italic>Arabidopsis</Emphasis> depends on the expression of <Emphasis Type=Italic>CO</Emphasis> and <Emphasis Type=Italic>GA1</Emphasis>

ALBINO3, a homologue of PPF1 in Arabidopsis, encodes a chloroplast protein, and is essential for chloroplast differentiation. In the present study, ALBINO3(−) transgenic plants exhibited a significant decrease in both the number of rosette leaves at bolting and the days before bolting, suggesting the important roles of ALBINO3 in regulating flowering during non-inductive short-day photoperiods. ALBINO3 mRNA was apparently accumulated in shoot apical meristem and floral meristems around the shoot apical meristem in wild-type plants. ALBINO3 might be predominantly involved in inducing the floral repression pathway by activating the expression of TFL1, and by suppressing the expression of LFY, respectively, in the shoot apical meristem. Moreover, the function of ALBINO3 in regulating flowering transition depended on the expression of CO and GA1, because ALBINO3 might function in the downstream integration of the photoperiod-dependent and the photoperiod-independent pathways. These results suggest that ALBINO3 may have an important integrative function in the flowering process in Arabidopsis.     

Evaluation of the V404I and V509M amino acid substitutions of <Emphasis Type=Italic>ERG11</Emphasis> gene in <Emphasis Type=Italic>Candida albicans</Emphasis> isolates by pyrosequencing

The molecular mechanisms underlying fluconazole resistance in C. albicans involve mutations and the overexpression of the ERG11 gene and membrane transport proteins. We examined the relationship between the reduced fluconazole susceptibility of C. albicans and mutations of V404I and V509M in the ERG11 gene in 182 C. albicans clinical isolates using the Pyrosequencing™ method. DNAs from these clinical isolates with different levels of in-vitro fluconazole susceptibility — one resistant, five susceptible dose-dependent (SDD), four trailer, and 172 susceptible — were analyzed. None of the fluconazole-susceptible, SDD, trailer or resistant isolates had mutations of V404I or V509M. Our results showed that no correlation can be found between the V404I or V509M mutation and fluconazole susceptibility in C. albicans.     

Transgenic Rice Plants Expressing a Modified <Emphasis Type=Italic>cry1Ca1</Emphasis> Gene are Resistant to <Emphasis Type=Italic>Spodoptera litura</Emphasis> and <Emphasis Type=Italic>Chilo</Emphasis><Emphasis Type=Italic>suppressalis</Emphasis>

Nucleotide sequence encoding the truncated insecticidal Cry1Ca1 protein from Bacillus thuringiensis was extensively modified based on the codon usage of rice genes. The overall G + C contents of the synthetic cry1Ca1 coding sequence were raised to 65% with an additional bias of enriching for G and C ending codons as preferred by monocots. The synthetic gene was introduced into the Chinese japonica variety, Xiushui 11, by Agrobacterium-mediated transformation. Transgenic rice plants harboring this gene were highly resistant to Chilo suppressalis and Spodoptera litura larvae as revealed by insect bioassays. High levels of Cry1Ca1 protein were obtained in the leaves of transgenic rice, which were effective in achieving 100% mortality of S. litura and C. suppressalis larvae. The levels of Cry1Ca1 expression in the leaves of these transgenic plants were up to 0.34% of the total soluble proteins. The larvae of C. suppressalis and S. litura could consume a maximum of 1.89  and 4.89 mm² of transgenic leaf area whereas the consumption of non-transgenic leaves by these larvae was significantly higher; 58.33 and 61.22 mm², respectively. Analysis of R₁ transgenic plants indicated that the cry1Ca1 was inherited by the progeny plants and provided complete protection against C. suppressalis and S. litura larvae.     

Presence of <Emphasis Type=Italic>C. albidus</Emphasis>, <Emphasis Type=Italic>C. laurentii</Emphasis> and <Emphasis Type=Italic>C. uniguttulatus</Emphasis> in Crop and Droppings of Pigeon Lofts (<Emphasis Type=Italic>Columba livia</Emphasis>)

Columba livia is an important reservoir and carrier of Cryptococcus neoformans, Cryptococcus uniguttulatus, Cryptococcus laurentii and Cryptococcus albidus. Upper digestive tract of this species is also known as a habitat for Cryptococcus neoformans. Given the increasing clinical interest of this microorganism, 331 swabs from crop and 174 dropping samples from pigeon lofts in Grand Canary Island have been studied. The obtained results show an extensive presence samples 81 positive (24.47%) of Cryptococcus spp. in analysed crops: 32 (9.66%) for C. neoformans, 24 (7.2%) for C. uniguttulatus, 23 (6.9%) for C. albidus and 2 (0.6%) for C. laurentii. In the same way, Cryptococcus spp was also isolated in 82 (47.13%), dropping samples: C. neoformans in 59 (33.9%), C. uniguttulatus, in 9 (5.17%), C. laurentii in 8 (4.59%) and C. albidus in 6 (3.44%) of the investigated samples, respectively. The cryptococcosis produced by species of cryptococci other than C. neoformans has become more important during the last decade, supporting the study on the role of pigeon in the epidemiology of this disease.