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Candida cells can form biofilms that frequently are sources of infections and are less susceptible to antifungal drugs. Some authors have reported that Candida orthopsilosis and Candida metapsilosis isolates are not able to produce biofilms in vitro and there are no studies available on biofilm susceptibility for these species to antifungals. The aims of this study were to (i) quantify Candida spp. biofilms in vitro, and (ii) test the in vitro susceptibilities of Candida spp. biofilms to fluconazole (FLC) and amphotericin B (AMB). Isolates studied included four Candida albicans, six C. tropicalis, seven C. parapsilosis, eight C. orthopsilosis, and five C. metapsilosis. We compared two different methods to evaluate biofilm production, i.e., crystal violet (CV) staining and XTT-reduction assays (XTT). Scanning electron microscopy (SEM) was used to observe high, medium and low biofilm producing isolates screened by these two methods. To determine the minimum biofilm eradication concentration (MBEC) for FLC and AMB, XTT-reduction assay was used to measure cell metabolic activity. Biofilm quantification by CV and XTT showed that C. tropicalis isolates were the highest biofilm producer, followed by C. albicans, C. parapsilosis, C. orthopsilosis and C. metapsilosis. Examination of SEM images revealed that the extent of biofilms formed by high, medium, and low producers was highly correlated to the results generated by CV assay. Biofilm of all the isolates evaluated were resistant to FLC (MBEC(80) ≥ 256 ug/ml) but, in general, susceptible to AMB, except for six C. parapsilosis strains (MBEC(80) ≥ 8 ug/ml).

Pure cultures of a trypanosomatid isolated from tomato fruits infected laboratory‐raised tomatoes and nymphs of the hemipieran coreid Phthia picta. The flagellate could be transmitted back and forth from tomatoes to insects. Light and electron microscopy studies were done on culture, tomato and insect forms. Examination of enzymes of the ornithine‐arginine metabolism revealed absence of arginase and presence of arginine deiminase and citrulline hydrolase. Monoclonal antibodies specific for Phytomonas spp. reacted positively with tomato and insect forms. Endonuclease digestion of the k‐DNA of various Phytomonas spp. revealed a unique, distinctive pattern for the tomato flagellate. This flagellate thus seems to constitute a separate species of Phytomonas which we now call Phytomonas serpens (Gibbs).

Paradoxical growth (PG) has been described for echinocandins and is characterized by cell growth at drug concentrations above the MIC. In this study, two isolates each of Candida albicans, C. tropicalis, C. orthopsilosis, and C. parapsilosis, all of which displaying PG in response to caspofungin, were subjected to MIC, minimal fungicidal concentration (MFC), and time-kill curve assays to evaluate the levels of PG. Cell wall components and ultrastructural modifications of the PG cells were also investigated. The results showed that when cell growth and survival were evaluated by MFC or time-kill curve assays, high concentrations of caspofungin did not show fungicidal activity against PG cells. Furthermore, for C. parapsilosis and C. orthopsilosis, time-kill curves were more discriminatory than MFCs in detecting the PG effect. The four different Candida species studied demonstrated similar alterations in cell wall components and ultrastructure associated with PG. In PG cells, ␤-1,3-glucan content decreased from 2.7-to 7.8-fold, whereas chitin content increased from 4.0-to 6.6-fold. An electron microscopy study of the PG cells revealed morphological alterations, clumping of cells, enlarged cells, the absence of filamentation, abnormal septa, and accumulation of chitin in the cell wall. Also, PG cells basically exhibited a single dark high-density layer in the cell wall, indicating the loss of the ␤-1,3-glucan layer. Our results present novel details about the ultrastructural alterations that occur in C. albicans, C. parapsilosis, C. orthopsilosis, and C. tropicalis during PG and show that chitin is the major component of the cell walls of PG cells. Stimulation of chitin synthesis may represent a rescue mechanism against caspofungin activity.

Etoposide is a podophyllotoxin semiderivative that is used in a variety of chemotherapy treatments, including therapy for children tumors. This drug promotes the formation of a ternary DNA-topoisomerase II-etoposide complex that triggers apoptosis. The purpose of this work was to analyze the occurrence of apoptosis in the seminiferous epithelium of prepubertal, pubertal, and adult rats treated with 10, 20, and 40 mg/Kg of etoposide during the prepubertal phase, as well as the role of apoptosis in etoposide-induced testicular damage. The rat testes were fixed in Bouin's liquid, and the apoptotic cells were quantified by means of the hematoxylin and eosin (H&E) technique (all groups) and the terminal dUTP nick end labeling (TUNEL) method (prepubertal groups only). The results obtained from both the H&E and TUNEL methods showed an increased frequency of apoptosis in the seminiferous epithelium of treated animals, except for the subgroup that received the 10-mg/Kg dose and was sacrificed 12 hr after the treatment and for the etoposide-treated pubertal group, that did not show cells suggesting apoptosis during H&E analysis. The labeled cells were mainly primary spermatocytes and differentiated spermatogonia. The prepubertal rats showed an etoposide-dose-dependent diminution of differentiated spermatogonia. Etoposide treatment during the prepubertal phase increases the frequency of apoptosis in the seminiferous epithelium, and causes serious harm to male fertility. © 2004 Wiley-Liss, Inc. Key words: testis; etoposide; apoptosis; rat; spermatogenesisEtoposide is a podophyllotoxin semiderivative agent that has been used in a variety of chemotherapeutic treatments, including therapy for pediatric tumors. Previous reports have proposed that etoposide antimitotic activity is mediated by its interaction with topoisomerase II, an ATP-dependent nuclear enzyme that regulates DNA topology by transiently breaking and uniting doublestranded DNA (Arnold, 1979;Hande, 1998). Topoisomerase II is essential for DNA replication, transcription, recombination, and chromosomal segregation. Etoposide promotes the formation of a ternary DNA-topoisomeraseetoposide complex; thus, topoisomerase II is not allowed to rejoin the DNA fragment that is produced during the cell cycle (Creaven and Allen, 1975). This action converts topoisomerase II into physiological toxins that induce irreversible damage to DNA by generating insertions, deletions, chromosomal aberrations, and translocations. When a sufficient concentration of permanent DNA breaks is present in the cells, a series of events occurs that culminates in apoptosis (Creaven and Allen, 1975;Russell et al., 1998).Apoptosis is a common physiological phenomenon that occurs in proliferating and constantly renewing tissues, including the seminiferous epithelium (Russell et al., 1987;Kerr, 1992;Brinkworth et al., 1995; Blanco-Rodrí-guez and Martínez-Garcia, 1996). Spontaneous cell death during spermatogenesis is an essential means of maintaining homeostasis in the testis, and is responsible...

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