Potential use of ellagic acid for endometriosis treatment: its effect on a human endometrial cell cycle, adhesion and migration.

Endometriosis is a common and challenging condition of reproductive-aged women that is defined as the presence of endometrial-like tissue outside the uterine cavity. Despite its prevalence, there is still no effective therapeutics; so we aim to evaluate the ellagic acid (EA) effect on the most relevant aspects that are known to be altered in endometriosis. Endometrial primary cultures from women with and without endometriosis and endometrial cell lines were incubated with EA (50 and 100 µM) for 24 and 48 h. The results demonstrated that EA arrests an endometrial stromal cell cycle on the G2/M phase, after 48 h. In addition, 100 µM EA treatment significantly decreased ECC-1 cell migration at 20 h and T-HESC cell migration at 10 h and 20 h, while 50 µM EA significantly decreased T-HESC cell migration at 20 h. On the other hand, we proved that the treatment with EA for 24 h reduces T-HESC and ECC-1 adhesion to plastic. However, we did not find an effect of EA on cell proliferation. EA has an inhibitory effect on endometrial cell adhesion, migration and cell cycle progression in vitro. These highlight the idea to investigate natural compounds as novel and promising candidates for therapeutic treatment of endometriosis.

Do recommended protein intakes improve neurodevelopment in extremely preterm babies?

OBJECTIVE: To determine whether achieving recommended protein intakes for extremely low birthweight (ELBW; birth weight <1000 g) babies, resulting in better growth, improves neurodevelopmental outcomes. DESIGN: A prospective cohort study of ELBW babies before and after the introduction of a new nutritional policy designed to meet international consensus protein recommendations. Forty-five children born 'before' and 42 born 'after' the policy change were assessed at 2 years' corrected age (CA). Associations between nutritional intakes, growth and neurodevelopmental outcome (Bayley Scales of Infant and Toddler Development, Third edition (Bayley-III), motor and sensory impairment) were assessed using univariate and multivariate analyses. RESULTS: Bayley-III cognitive (mean (SD) 96 (12) vs 96 (15)), motor (96 (13) vs 95 (15)) or language scores (89 (11) vs 91 (17)) were not different between the 'before' and 'after' cohorts. In the 'before' cohort, motor scores were positively associated with enteral nutrition intakes and growth velocity. Neither were sensory impairments different between groups (visual impairment 4 vs 2, hearing impairment 2 vs 0) nor was the gross motor function classification score (any cerebral palsy 2 vs 1). CONCLUSIONS: In this prospective cohort study, increasing intravenous and enteral protein intakes to recommended levels in the first month after birth was not associated with improved cognitive, language or motor scores or decreased sensory impairments at 2 years' CA despite significantly improved early growth and reduced postnatal faltering growth. Appropriate randomised controlled trials are needed to answer definitively whether higher early protein intakes improve neurodevelopmental outcome in this population.

Optimum feeding and growth in preterm neonates.

Approximately 10% of all babies worldwide are born preterm, and preterm birth is the leading cause of perinatal mortality in developed countries. Although preterm birth is associated with adverse short- and long-term health outcomes, it is not yet clear whether this relationship is causal. Rather, there is evidence that reduced foetal growth, preterm birth and the long-term health effects of both of these may all arise from a suboptimal intrauterine environment. Further, most infants born preterm also experience suboptimal postnatal growth, with potential adverse effects on long-term health and development. A number of interventions are used widely in the neonatal period to optimise postnatal growth and development. These commonly include supplementation with macronutrients and/or micronutrients, all of which have potential short-term risks and benefits for the preterm infant, whereas the long-term health consequences are largely unknown. Importantly, more rapid postnatal growth trajectory (and the interventions required to achieve this) may result in improved neurological outcomes at the expense of increased cardiovascular risk in later life.

Pregnancy and neonatal outcomes following hyperemesis gravidarum.

Undernutrition during pregnancy is associated with detrimental pregnancy and neonatal outcomes, which can have long-term implications for the infant. Hyperemesis gravidarum may severely limit nutritional intake. The aim of this study was to investigate the effect of hyperemesis on pregnancy and neonatal outcome, particularly gestation length and infant size at birth. Seventy-five prospectively recruited women admitted to a tertiary level hospital in Auckland, with hyperemesis gravidarum between March 2003 and October 2005, were compared to 142 controls matched for age, parity, ethnicity and expected date of delivery. Data were obtained from electronic records and analysed by Student's t-test, χ2, Wilcoxon, Fisher's exact tests and linear regression. Length of gestation, birth weight and crown-heel length were not different between participants and controls. Infants born to women with hyperemesis gravidarum had smaller head circumferences (Z-score mean (s.d.) 0.02 (0.16) v. 0.43 (0.11), P = 0.04 in all infants and -0.02 (1.24) v. 0.48 (1.29), P = 0.01 in-term infants). This study found hyperemesis gravidarum to be associated with smaller head circumferences in offspring. Given the reported associations between smaller head circumference at birth and lower cognitive ability and higher risk of cardiovascular disease in later life, further study is necessary to confirm these results and to determine whether there are any long-term implications for the offspring.

Aquaporin in Candida: characterization of a functional water channel protein.

The Candida albicans genome database contains one ORF with homology to aquaporins, AQY1. Xenopus oocytes injected with cRNA encoding C. albicans Aqy1p displayed a coefficient of water permeability (P(f)) that was equivalent to the P(f) for oocytes injected with the cRNA of S. cerevisiae Aqy1p. In addition, as seen in Saccharomyces for Aqy1p and Aqy2p, deletion of AQY1 in C. albicans resulted in cells that were less sensitive than wild-type to osmotic shock. In Saccharomyces, aquaporin null cells also have a cell surface that is more hydrophobic. However, unlike Saccharomyces, there was no effect on the cell surface hydrophobicity, flocculation or cell aggregation in aqy1 null C. albicans cells. Perhaps as a result, there was no difference between the virulence of C. albicans wild-type and aqy1 null strains in a murine model for systemic candidiasis.

Directed mutagenesis using the polymerase chain reaction.

The polymerase chain reaction (PCR) is most often used for the enzymatic amplification and direct sequencing of small quantities of nucleic acids. This technology can also be used as a quick and efficient method for introducing any desired sequence change into the DNA of interest. This unit contains two basic protocols for introducing base changes into specific DNA sequences. The first describes the incorporation of a restriction site and the second details the generation of specific point mutations. An alternate protocol describes generating point mutations by sequential PCR steps. Although the general procedure is the same in all three protocols, there are differences in the design of the synthetic oligonucleotide primers and in the subsequent cloning and analyses of the amplified fragments.

Directed mutagenesis using the polymerase chain reaction.

This unit contains two basic protocols for introducing base changes into specific DNA sequences. The first describes the incorporation of a restriction site and the second details the generation of specific point mutations. An Alternate Protocol describes generating point mutations by sequential PCR steps. Although the general procedure is the same in all three protocols, there are differences in the design of the synthetic oligonucleotide primers and in the subsequent cloning and analyses of the amplified fragments.

An adhesin of the yeast pathogen Candida glabrata mediating adherence to human epithelial cells.

Candida glabrata is an important fungal pathogen of humans that is responsible for about 15 percent of mucosal and systemic candidiasis. Candida glabrata adhered avidly to human epithelial cells in culture. By means of a genetic approach and a strategy allowing parallel screening of mutants, it was possible to clone a lectin from a Candida species. Deletion of this adhesin reduced adherence of C. glabrata to human epithelial cells by 95 percent. The adhesin, encoded by the EPA1 gene, is likely a glucan-cross-linked cell-wall protein and binds to host-cell carbohydrate, specifically recognizing asialo-lactosyl-containing carbohydrates.

Efficient homologous and illegitimate recombination in the opportunistic yeast pathogen Candida glabrata.

The opportunistic pathogen Candida glabrata causes significant disease in humans. To develop genetic tools to investigate the pathogenicity of this organism, we have constructed ura3 and his3 auxotrophic strains by deleting the relevant coding regions in a C. glabrata clinical isolate. Linearized plasmids carrying a Saccharomyces cerevisiae URA3 gene efficiently transformed the ura3 auxotroph to prototrophy. Homologous recombination events were observed when the linearized plasmid carried short terminal regions homologous with the chromosome. In contrast, in the absence of any chromosomal homology, the plasmid integrated by illegitimate recombination into random sites in the genome. Sequence analysis of the target sites revealed that for the majority of illegitimate transformants there was no microhomology with the integration site. Approximately 0.25% of the insertions resulted in amino acid auxotrophy, suggesting that insertion was random at a gross level. Sequence analysis suggested that illegitimate recombination is nonrandom at the single-gene level and that the integrating plasmid has a preference for inserting into noncoding regions of the genome. Analysis of the relative numbers of homologous and illegitimate recombination events suggests that C. glabrata possesses efficient systems for both homologous and nonhomologous recombination.

Advances in molecular genetics of Candida albicans and Candida glabrata.

Ten years ago, when molecular genetic methods were being applied vigorously to viruses, bacterial pathogens and eukaryotic parasites, there seemed to be a partial paralysis in applying them to infectious fungi; this state of affairs was more than apparent in the composition of the symposia at the ISHAM conference in 1987. Since then, however, things have changed. The ISHAM conference held in Italy in 1997 was replete with studies utilizing molecular genetic techniques to answer questions related to epidemiology, pathogenesis, drug development and typing. In the symposium Advances in Molecular Genetics of Fungal Pathogens, several new applications of molecular biology to fungal pathogenesis were reviewed. Although the presentations in this symposium covered only a fraction of the molecular methods now being applied to Candida pathogenesis, they nevertheless provided an intriguing view of what is in store for us in the coming years.

Green fluorescent protein as a reporter of transcription and protein localization in fungi.

Green fluorescent protein (GFP) is a versatile and powerful tool for analysis of diverse biological processes. The recent development of GFP variants with altered spectral properties and altered codon composition has allowed efficient expression of GFP in a number of fungal species. GFP has been successfully used to analyze transcription regulation as well as protein and organelle localization, and promises to give an unprecedented view into the dynamic subcellular processes that shape the fungal cell.

FACS-optimized mutants of the green fluorescent protein (GFP).

We have constructed a library in Escherichia coli of mutant gfp genes (encoding green fluorescent protein, GFP) expressed from a tightly regulated inducible promoter. We introduced random amino acid (aa) substitutions in the twenty aa flanking the chromophore Ser-Tyr-Gly sequence at aa 65-67. We then used fluorescence-activated cell sorting (FACS) to select variants of GFP that fluoresce between 20-and 35-fold more intensely than wild type (wt), when excited at 488 nm. Sequence analysis reveals three classes of aa substitutions in GFP. All three classes of mutant proteins have highly shifted excitation maxima. In addition, when produced in E. coli, the folding of the mutant proteins is more efficient than folding of wt GFP. These two properties contribute to a greatly increased (100-fold) fluorescence intensity, making the mutants useful for a number of applications.

Conserved and nonconserved functions of the yeast and human TATA-binding proteins.

Although the TATA-binding protein (TBP) is highly conserved throughout the eukaryotic kingdom, human TBP cannot functionally replace yeast TBP for cell viability. To investigate the basis of this species specificity, we examine the in vivo transcriptional activity of human TBP at different classes of yeast promoters. Consistent with previous results, analysis of yeast/human hybrid TBPs indicates that growth defects are not correlated with the ability to promote TATA-dependent polymerase II (Pol II) transcription or to respond to acidic activator proteins. Human TBP partially complements the growth defects of a yeast TBP mutant with altered TATA element-binding specificity, suggesting that it carries out sufficient Pol II function to support viability. However, human TBP does not complement the defects of yeast TBP mutants that are specifically defective in transcription by RNA polymerase III. Three independently isolated derivatives of human TBP that permit yeast cell growth replace arginine 231 with lysine; the corresponding amino acid in yeast TBP (lysine 133) has been implicated in RNA polymerase III transcription. Transcriptional analysis indicates that human TBP functions poorly at promoters recognized by RNA polymerases I and III and at RNA Pol II promoters lacking a conventional TATA element. These observations suggest that species specificity of TBP primarily reflects evolutionarily diverged interactions with TBP-associated factors (TAFs) that are necessary for recruitment to promoters lacking TATA elements.

Regional codon randomization: defining a TATA-binding protein surface required for RNA polymerase III transcription.

The TATA-binding protein (TBP) is required for transcription by all three nuclear RNA polymerases. TBP was subjected to regional codon randomization, a codon-based mutagenesis method that generates complex yet compact protein libraries. Analysis of 186 temperature-sensitive TBP mutants yielded 65 specifically defective in transcription by RNA polymerase III (Pol III). These mutants map to a limited TBP surface that may interact with Tds4, a component of the Pol III transcription factor TFIIIB. Strains that contain the Pol III-defective derivatives have increased amounts of messenger RNA, which suggests that competition among TBP-interacting factors for limiting quantities of TBP determines the ratio of Pol II and Pol III transcription in vivo.

The TATA-binding protein is required for transcription by all three nuclear RNA polymerases in yeast cells.

Using temperature- and proteolytically sensitive derivatives to inactivate the function of the yeast TATA-binding protein (TBP) in vivo, we investigated the requirement of TBP for transcription by the three nuclear RNA polymerases in yeast cells. TBP is required for RNA polymerase II (pol II) transcription from promoters containing conventional TATA elements as well as functionally distinct promoters that lack TATA-like sequences. TBP is also required for transcription of the U6 snRNA and two different tRNA genes mediated by RNA pol III as well as transcription of ribosomal RNA mediated by RNA pol I. For all promoters tested, transcription decreases rapidly and specifically upon inactivation of TBP, strongly suggesting that TBP is directly involved in the transcription process. These observations suggest that TBP is required for transcription of all nuclearly encoded genes in yeast, although distinct molecular mechanisms are probably involved for the three RNA polymerase transcription machineries.

Contribution of a risk factor clinic to lipid management in patients with coronary artery disease.

The progress of a cohort of 145 patients seen between June 1986 and June 1989 was reviewed. These patients had treatment prescribed by the clinic and had data recorded over serial visits; they allowed us to determine the contribution of the risk factor clinic. Eighty-six percent had coronary artery disease. Patients were given nutritional advice, partly in groups. In addition 61% were treated with drug therapy. Seventy-four percent had modified their diet before the clinic visit but only 32% received less than 30% of energy from fat; the number rose to 67% by discharge. Sixty-four percent had a body mass index of 25 or greater, falling to 53% at discharge. Mean total cholesterol of the 145 patients was 7.9, HDL cholesterol 1.06, and total:HDL cholesterol ratio 7.7 mmol/L. Changes with clinic management were: total cholesterol -19%, HDL cholesterol +11%, total:HDL cholesterol ratio -25%, LDL cholesterol -21%. Despite these changes, levels were less than optimal for patients with coronary arterial disease in at least 50% of patients at the time of discharge. Improved results can be achieved only with a more aggressive approach to drug therapy. Recent studies in patients with coronary disease provide strong support for such a change in management.

Functional differences between yeast and human TFIID are localized to the highly conserved region.

TFIID, the general transcription factor that binds TATA promoter elements, is highly conserved throughout the eukaryotic kingdom. TFIIDs from different organisms contain C-terminal core domains that are at least 80% identical and display similar biochemical properties. Despite these similarities, yeast cells containing human TFIID instead of the endogenous yeast protein grow extremely poorly. Surprisingly, this functional distinction reflects differences in the core domains, not the divergent N-terminal regions. The N-terminal region is unimportant for the essential function(s) of yeast TFIID because expression of the core domain permits efficient cell growth. Analysis of yeast-human hybrid TFIIDs indicates that several regions within the conserved core account for the phenotypic difference, with some regions being more important than others. This species specificity might reflect differences in DNA-binding properties and/or interactions with activator proteins or other components of the RNA polymerase II transcription machinery.

RNA structure, not sequence, determines the 5' splice-site specificity of a group I intron.

The group I self-splicing introns act at exon-intron junctions without recognizing a particular sequence. In order to understand splice-site selection, we have developed an assay system based on the Tetrahymena ribozyme to allow the study of numerous 5'-splice-site variants. Cleavage at the correct site requires formation of the correct secondary structure and occurs most efficiently within a 3-base-pair window centered on base pair 5 from the bottom of the P1 stem. Within this window the ribozyme recognizes and cleaves at a "wobble" base pair; the base pair above the cleavage site also influences splicing efficiency. The recognition of RNA structure rather than sequence explains the ability of these transposable introns to splice out of a variety of sequence contexts.