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The incidence of fungal infections is increasing in different countries. The current available therapy of these infections does not satisfy all requirements in terms of specificity and therapeutic index, a fact that has stimulated the scientific community to identify fungal virulence determinants. Several pathogenic fungi are opportunistic and, therefore, identification of virulence genes is difficult, given their close relationship with host cells. In recent years, the development of genetic tools in several pathogenic fungi has enabled the development of genetic strategies for their identification. These include several strategies based on the phenotypic analysis of strains or environmental conditions in which the expression of the putative gene(s) is either altered or deleted; and this is accomplished through the development of in vitro or in vivo systems. In the near future, this research will produce a better picture of fungal pathogenesis and therefore define novel promising targets in antifungal therapy.

The capability of biopartitioning Micellar Chromatography, BMC, to describe and estimate pharmacokinetic and pharmacodynamic parameters of central nervous system drugs is reviewed in this article. BMC is a mode of micellar liquid chromatography, MLC, that uses micellar mobile phases of Brij35 (polyoxyethilene(23) lauryl ether) prepared in physiological conditions (pH, ionic strength). The retention of a drug in this system depends on its hydrophobic, electronic and steric properties, which also determine its biological activity. The results of BMC studies suggest that this in vitro approach is an attractive useful tool to be implemented into the lead optimization step of drug development scheme.

In recent years, the incidence of fungal infections has been rising all over the world. Although the amount of research in the field of pathogenic fungi has also increased, there is still a need for the identification of reliable determinants of virulence. In this review, we focus on identified Candida albicans genes whose deletant strains have been tested in experimental virulence assays. We discuss the putative relationship of these genes to virulence and also outline the use of new different systems to examine the precise effect in virulence of different genes.

Yeast cells are surrounded by the cell wall, a rigid but dynamic structure that is essential for their viability. The complexity and functionality of this structure suggest that a high number of proteins must be involved in the biogenesis of the cell wall architecture and, as a consequence, in the maintenance of cell integrity. Among them, a high percentage is assumed to be located at the cell surface, mostly as structural or enzymatic components of the cell wall. Therefore, the presence of a protein in the cell wall is suggestive of its cell wall-related function. Different techniques can be used to specifically detect the cell wall localisation of a given protein or to identify cell wall proteins in large-scale analyses. These include the detection of proteins in whole cells or specific cell wall fractions by immunological, biochemical, microscopic, or genetic approaches, as well as the emerging proteomic technology. The advantages, limitations, and usefulness of these techniques are discussed and illustrated with some examples., (Copyright 2000 Wiley-Liss, Inc.)

An analysis of 25 microsatellite loci in 210 animals has been used to define the genetic structure of the Iberian pig, traditionally classified into several varieties. In addition, a sample of 20 Duroc pigs was used as an outgroup for topology trees. Inter-variety genetic variation was estimated by unbiased average heterozygosity and the number of alleles observed. Significant deviations from the Hardy-Weinberg equilibrium (HWE) were shown for 19 loci across the whole population. By contrast, equilibrium deviation within varieties was much lower. Genetic variation measures, genetic distance values and a neighbour-joining tree were used to estimate subdivision. In addition, an individual tree was constructed to contrast the assignation of animals into varieties. Despite the low bootstrap values obtained in the varieties neighbour-joining tree, the degree of genetic variation found was sufficient to support the division of the Iberian pig into varieties, although in some cases the traditional classification cannot be accepted. These results have shown the value of this marker panel in the study of intra-breed genetic structures.

The frequency of opportunistic infections caused by the fungus Candida albicans is very high and is expected to continue to increase as the number of immunocompromised patients rises. Research initiatives to study the biology of this organism and elucidate its pathogenic determinants have therefore expanded significantly during the last 5-10 years. The past few years have also brought continuous improvement in the techniques to study gene function by gene inactivation and by regulated gene expression and to study gene expression and protein localization by using gene reporter systems. As steadily more genomic sequence information from this human fungal pathogen becomes available, we are entering a new era in antimicrobial research. However, many of the currently available molecular genetics tools are poorly adapted to a genome-wide functional analysis in C. albicans, and further development of these tools is hampered by the asexual and diploid nature of this organism. This review outlines recent advances in the development of molecular tools for functional analysis in C. albicans and summarizes current knowledge about the genomic and genetic variability of this important human fungal pathogen.

Protoplasts of Saccharomyces cerevisiae incubated in regenerating conditions secrete cell wall components in order to allow the biosynthesis of this structure. During the first hours of incubation, many of these are not retained in the forming cell wall but remain in the medium. We have developed a method for collecting the secreted proteins and have analysed these by two-dimensional electrophoresis to obtain a reference map of putative cell wall proteins. Several proteins were identified by microsequencing or immunoblotting; namely, cell wall hydrolytic enzymes, heat shock proteins, glycolytic enzymes and others. Some beta-1,3- and beta-1, 6-glucosylation was detected in the proteins secreted by regenerating protoplasts.

This paper reports the usefulness of two-dimensional gel electrophoresis followed by Western blotting with sera from patients with systemic candidiasis in the identification of the major Candida albicans antigens. In order to have different patterns of protein expression and subcellular localization, three types of protein preparations were obtained: cytoplasmic extracts, protoplast lysates and proteins secreted by protoplasts regenerating their cell wall. These proteins were separated by high-resolution two-dimensional electrophoresis using an immobilized pH gradient. Western blotting with sera from patients with systemic candidiasis allowed the detection of more than 18 immunoreactive proteins. Some of these proteins had different isoforms. All sera reacted with at least three C. albicans proteins and the most reactive serum detected up to eleven proteins. Some of these antigens, e.g., enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), have been identified on the 2-D map. The most reactive proteins were enolase and a 34 kDa protein in the acidic part of the gel (pI 4-4.4) that was only detected in regenerating protoplast-secreted proteins. The identification of all these antigens would be useful for the development of diagnostic strategies.

The JEM-1 gene, recently identified in acute promyelocytic leukemia (APL) cells, codes for a novel nuclear factor (Duprez et al Oncogene 1997; 14: 1563-1570). JEM-1 is kept silent in the APL cell line NB4, but up-regulated (3 kb transcript) during cell maturation. Here, we show that retinoic acid (RA)-induced JEM-1 expression is biphasic (peaks at 6 h and 48 h) and associated with the later stages of maturation. Retinoids, which cooperates with cAMP to induce maturation, also cooperates with cAMP to up-regulate JEM-1, either in maturation-responsive NB4 cells or in NB4-R1 resistant subclones. APL patients showed a low, yet variable, level of JEM-1 mRNA in bone marrow. RA treatment induced an increase in the level of JEM-1 mRNA, as detected by a semi-quantitative PCR. This increase can result from both gene up-regulation or replacement of leukemia cells by differentiated ones. Analysis of JEM-1 expression patterns in normal and tumor cells revealed that JEM-1 expression was ubiquitous. Cell lines derived from monocytic and erythroid leukemias, expressed low and high amounts of JEM-1 mRNA, respectively. Using a JEM cDNA probe, distinct profiles of expression and different transcript sizes (4 kb, 3 kb and 2 kb) were also identified in tumour and normal non-hematopoietic tissues, while interestingly only the 3kb transcript was up-regulated in NB4 cells. This work identifies JEM-1 as a novel ubiquitous gene whose expression is low in APL cells, but can be restored by RA treatment, concomitant with cell maturation.

Candida albicans is a dimorphic fungus that causes severe opportunistic infections in humans. Recent advances in molecular biology techniques applied to this organism (transformation systems, gene disruption strategies, new reporter systems, regulatable promoters) allow a better knowledge of both the molecular basis of dimorphism and the role of specific genes in Candida morphogenesis. These same molecular approaches together with the development of appropriate experimental animal models to analyze the virulence of particular mutants, may help to understand the molecular basis of Candida virulence.

The Candida albicans CDR1 gene encodes a member of the ABC-type family of multidrug transporters which has been shown to be involved in azole resistance. Using an in-frame gene fusion between the CDR1 open reading frame and the green fluorescent protein allele yEGFP3, an optimized derivative for its use in C. albicans, we show here how the CDR1-yEGFP3 gene expression is induced in response to azoles as well as to other structurally unrelated drugs like cycloheximide. Moderate increases were observed for calcofluor, canavanine, 5'-fluorcytosine, cilofungin and caffeine, while no induction was found for the antifungals benomyl and amphotericin B or hydrogen peroxide at subinhibitory concentrations. The use of confocal microscopy enabled us to localize the Cdr1p fusion protein at the cell periphery, thus suggesting a cytoplasmic membrane localization. These results suggest deregulation of CDR1 gene as a putative mechanism for the generation of azole resistance in this clinically important pathogenic fungus.

The Candida albicans MKC1 gene encodes a mitogen-activated protein (MAP) kinase, which has been cloned by complementation of the lytic phenotype associated with Saccharomyces cerevisiae slt2 (mpk1) mutants. In this work, the physiological role of this MAP kinase in the pathogenic fungus C. albicans was characterized and a role for MKC1 in the biogenesis of the cell wall suggested based on the following criteria. First, C. albicans mkc1 delta/mkc1 delta strains displayed alterations in their cell surfaces under specific conditions as evidenced by scanning electron microscopy. Second, an increase in specific cell wall epitopes (O-glycosylated mannoprotein) was shown by confocal microscopy in mkc1 delta/mkc1 delta mutants. Third, the sensitivity to antifungals which inhibit (1,3)-beta-glucan and chitin synthesis was increased in these mutants. In addition, evidence for a role for the MKC1 gene in morphological transitions in C. albicans is presented based on the impairment of pseudohyphal formation of mkc1 delta/mkc1 delta strains on Spider medium and on the effect of its overexpression on Sacch. cerevisiae colony morphology on SLADH medium. Using the two-hybrid system, it was also demonstrated that MKC1 is able to interact specifically with Sacch. cerevisiae Mkk1p and Mkk2p, the MAP-kinase kinases of the PKC1-mediated route of Sacch. cerevisiae, and to activate transcription in Sacch. cerevisiae when bound to a DNA-binding element. These results suggest a role for this MAP kinase in the construction of the cell wall of C. albicans and indicate its potential relevance for the development of novel antifungals.

Fluconazole is one of the most useful drugs in the treatment of fungal systemic infections which frequently affect non immunocompetent individuals. However, the emergence of resistant strains in recent years may severely limit its usefulness in future. Although there are several described mechanisms involved in resistance to azoles, recent genetic studies demonstrate the role of specific genes in clinical resistance. Currently, the best characterized are the MDR1 and CDR1 genes, which code members of the MFS or ABC family of drug transporters, respectively. These proteins respond to the membrane potential (MFS) or hydrolyse ATP (ABC) thus promoting drug efflux and therefore reducing its intracellular accumulation. It has been shown that the mRNA from these genes is frequently increased in some Candida albicans resistant strains from patients receiving long term azole treatment. The development of molecular genetic tools in C. albicans is allowing characterization of their role in this and other important processes in the fungal cell.

Both alleles of the XOG1 gene of Candida albicans, which encodes a protein with exoglucanase activity, were sequentially disrupted. Enzymic analysis of either cell extracts or culture supernatants of disrupted strains revealed that this gene is responsible for the major exoglucanase activity in C. albicans, although residual exoglucanase activity could still be detected. xog1 null mutants showed similar growth rates in both rich and minimal liquid medium as compared to the wild-type strain, indicating that the enzyme is not essential for C. albicans growth. In addition, no differences were observed between wild-type and xog1 null mutants with respect to their ability to undergo dimorphic transition. However, small but repeatable differences were found between the wild-type and the null mutant with respect to susceptibility to chitin and glucan synthesis inhibitors. Using a murine model of experimental infection, no significant differences in virulence were observed. The xog1 null strain is thus a suitable recipient for studying Candida gene expression using the exoglucanase as a reporter gene.

The ARG5,6 gene from the dimorphic fungus Candida albicans was cloned by functional complementation of the arginine auxotrophy present in strain EL2 (Arg-) using a gene library constructed in the double autonomously replicating sequence vector pRM1. Sequence analysis revealed a putative 857 amino acid polypeptide (95 kDa) which showed high homology (63% protein identity) to the Saccharomyces cerevisiae ARG5,6 gene. Similarly to the S. cerevisiae gene, the C. albicans ARG5,6 gene is responsible for both the acetylglutamate kinase and acetylglutamyl-phosphate reductase activities, the second and third steps of arginine biosynthesis at the mitochondria. The C. albicans ARG5,6 gene complemented the arg6 mutation present in S. cerevisiae (strain D160-4D) on a yeast episomal plasmid using its own regulatory signals. A set of non-integrative high-efficiency plasmid vectors based on this gene marker was constructed and a null C. albicans arg5,6 delta strain was obtained using the common URA3-blaster strategy. In addition, we generated an arg5,6 delta null mutant in a single transformation event, thus improving the basic strategy for generating gene deletions in C. albicans.

The yeast SEC14 gene product is required for the transport of proteins from the Golgi complex. We have cloned the homologous Candida albicans SEC14 gene (CaSEC14) by functional complementation of a Saccharomyces cerevisiae thermosensitive mutant, sec14ts. Some putative TATA boxes have been identified in CaSEC14 and, contrary to S. cerevisiae SEC14, no introns were found in the Candida homologue. Sequence analysis revealed that CaSec14p is a 301 amino acid protein, 67% identical to S. cerevisiae and Kluyveromyces iactis Sec14p, and 61% identical to the 300 amino-terminal residues of Yarrowia lipolytica Sec14p. Hydrophatic profile analysis of CaSec14p suggests a soluble protein without transmembrane domains as has been described for the S. cerevisiae counterpart. While it was easy to disrupt one allele of SEC14 in C. albicans, repeated attempts to disrupt the second allele were unsuccessful, thus suggesting that the gene could be essential for vegetative growth in C. albicans.

Unlabelled: A multicentric clinical trial was performed to evaluate two routes of access for implantable subcutaneous central venous devices: by way of the subclavian vein (group A) and peripheral access by way of the veins of the flexion side of the elbow (group B). The indications for implantation were: antineoplastic treatment of solid tumors, myelo- and lymphoproliferative syndromes, antiviral treatment, and parenteral nutrition at home. The study was composed of 87 patients, 48 men and 39 women, with a mean age of 57.1 years (SD = 12.6). Group A was made up of 48 patients, and group B of 39., Results: Implantation failure was 8.3% in group A and 5.1% in group B (p = n.s.). Complications of implantation were 23.4% in group A and 15.4% in group B (p = n.s.). The complications of permanence were diagnosed at 27.6% in group A and at 15.4% in group B (p = n.s.)., Conclusions: We have not been able to find significant differences between the two groups, probably due to the fact that the series is still short. Nevertheless, the manageability, comfort for the nursing staff and for the patients, appears to be greater with the devices implanted in the infraclavicular region, by means of a subclavian vein puncture.

Objective: To analyse the attitudes of Pediatrics interns to their Primary Care training by comparing it to their preferences and professional expectations., Design: Descriptive study using a questionnaire sent out by post. SITE. Those hospitals in the Valencian Community with Pediatrics interns., Participants: All the Pediatrics interns (n = 87) in the Valencian Community who were being trained in 1991., Measurements and Results: 72 interns (82.7%) answered the questionnaire. Only three interns (4.2%) considered it essential to carry through the placements set out in the Primary Care Programme within the specialist course; and 46 (63.9%) considered it useful. The proposed period of these placements (6 months) was considered far too long by 28 interns (38.6%). Ten (13.9%) thought it should be dropped altogether., Conclusions: Pediatrics interns still only show scant interest in doing part of their training in Health Centres or in subsequently working in Primary Care. Nevertheless there is some encouragement in that it is the fourth-year doctors who show greater interest both in training and working in Primary Care. Even so, we considered this training is essential for doctors to acquire the specific knowledge, techniques, attitudes and skills required in Primary Care; especially as a high percentage think, as is the reality of the situation, that they are going to be working in Primary Care on finishing their specialist training.

Objective: To assess user-satisfaction on the introduction of an Appointments Scheme., Design: Crossover and observational, with a random sample. SITE. 47?97 Health Centres and 72 Clinics in the Community of Valencia., Patients and Others Participants: A random sample of 400 users., Main Measurements and Results: A month after the introduction of the scheme, a survey was administered by staff of the centres themselves to a sample of users coming out from consulting rooms. The percentage of users who waited less than 15 minutes to see their doctor increased from 18% to 75.5% after the introduction of the Scheme. The average number of people in the waiting-rooms decreased from 61 to 14. 72.5% of users considered that the quality of health care received had improved., Conclusions: The users' evaluation of the programme is satisfactory. There was moreover a significant difference in favour of Health Centres as to fewer crowds in waiting-rooms, greater access to telephones, the evaluation of the quality of care and the average time of a consultation.

Separate renal arteries and renal portal veins supply the three divisions of domestic fowl kidneys, leading to uncertainty concerning the structural and functional homology of the different divisions. To assess the degree of anatomical similarity, separate glomerular size and volume distribution profiles were constructed for cranial, medial, and caudal divisions from the left and right kidneys of male and female domestic fowl. There were no significant differences between left and right divisions (intradivision comparisons) or between cranial, medial, and caudal divisions (interdivision comparisons) when the total number of glomeruli or total glomerular volumes were compared on a per gram kidney weight basis. Glomerular size distribution profiles were unimodal in shape, with the peaks for all divisions falling in the 0.15-0.22 mm circumference range in males and in the 0.19-0.26 mm circumference range in females. There were no significant differences in glomerular size distribution profiles for intradivision (left vs. right) comparisons. Interdivisional comparisons revealed that medial divisions had significantly lower values than caudal divisions in one of 11 glomerular circumference categories. For all remaining comparisons, there were no significant differences in glomerular size distribution profiles, nor were there any significant interdivisional differences when glomerular volume distribution profiles were compared. These data indicate that the three divisions of domestic fowl kidneys are structurally homologous on a per gram kidney weight basis, suggesting the separate divisions also are functionally homologous.

The location of FtsQ, an Escherichia coli protein essential for cell division, is, under physiological conditions, in the cytoplasmic membrane facing towards the periplasmic space. An amino-proximal hydrophobic domain is required for FtsQ to reach its location and for its activity in the cell. Overexpression of modified forms of FtsQ is deleterious for the cell.