Preimplantation development of in vitro-produced bovine embryos treated with hydroxychloroquine.

Hydroxychloroquine (HCQ) is a safe antimalarial drug but its overdosage or inappropriate use, such as during the pandemic, may cause adverse effects once this drug is considered a potent inhibitor of autophagy. Information about HCQ's effects on the reproductive field, including gametes and initial embryos, is limited. In this study, we evaluated the effect of HCQ (1, 6, 12, and 24 µM) on pre-implantation embryo development, autophagy, and apoptosis of bovine embryos produced in vitro. A dose-response experiment showed a reduction (p < 0.05) in cleavage only at the highest concentration. Blastocyst rate was gradually reduced (p < 0.05) with the increase of HCQ dosage starting at 6 µM, with no embryo formation occurring at 24 µM. Further analysis showed that embryos treated with 12 µM of HCQ had a higher (p < 0.05) accumulation of acidic autophagic vesicles on Days 5 and 7 of development and a higher (p < 0.01) apoptotic index on Day 7. To our knowledge, this is the first study to evaluate the effects of HCQ on embryo pre-implantation development in mammals. The results contribute with more information related to the study of autophagy in embryology as well as add some discussion on HCQ toxicology and its effects on reproductive cells.

Septin localization in the dimorphic fungus Paracoccidioides brasiliensis.

Septins are evolutionarily conserved proteins that contain a GTPase domain and are capable of forming filaments at the cell periphery. Septins are involved in many essential cellular processes, such as cytokinesis and cell polarization, and are used as markers of morphogenesis in several fungi. Dimorphism in fungi enables cells to switch between morphologies (yeast or filament forms), due to changes in the temperature of the environment. We analysed the localization of septin proteins in yeast and filamentous cells of the dimorphic fungus Paracoccidioides brasiliensis, a common cause of granulomatous mycosis. In order to determine septin localization, we first cloned Cdc12p, a septin homolog from P. brasiliensis, and expressed it in Escherichia coli. Following PbCdc12p purification, specific serum against PbCdc12p were raised for use in immunofluorescence assays. We observed the hourglass and ring forms of septin filaments during cell division in yeast. Septin filaments were also simultaneously localized in the necks of multiple budding cells. A distinctive pattern of punctuate and/or diffuse localization was also seen in the periphery of multinucleate yeast cells and at the tips and septa of filamentous cells. A more diffuse and punctuate pattern of localization observed in P. brasiliensis cells seems to be unique to filamentous and dimorphic fungi and may be related to their specialization in cell wall deposition, morphogenesis and cell cycle control.

The control of BhB10-1 gene expression in the salivary gland of Bradysia hygida (Diptera, Sciaridae) is disrupted in vivo by a delayed effect of cycloheximide.

BhB10-1 is an amplified gene present in DNA puff B10. This gene is very active in the salivary gland regions S1 and S3 at the end of the larval development. Two transcripts of this gene, 1.3 and 1.1 kb in size, were detected. A secretory protein, SP23, is the product of BhB10-1. In this work, we present evidence supporting the hypothesis that a biphasic process of mRNA degradation is an important component in the control of BhB10-1 gene expression. The 1.3 kb transcript, by a process of poly(A) tail shortening, is converted to the inactive transcript of 1.1 kb which is detected during and after the period of SP23 expression. Cycloheximide in very low concentration, if applied at a proper time, can disrupt this process leading to extended periods of 1.3 kb RNA detection and SP23 synthesis. A tentative model is proposed to explain this phenomenon.

The induction of DNA puff BhC4-1 gene is a late response to the increase in 20-hydroxyecdysone titers in last instar dipteran larvae.

The characterization of DNA puff BhC4-1 expression was extended and its response to 20-hydroxyecdysone investigated in Bradysia hygida and in transgenic Drosophila carrying the BhC4-1 gene. In both organisms the activation of BhC4-1 in salivary glands occurs at the end of the larval stage coinciding with the peak in ecdysone titers which induces metamorphosis. Injections of 20-hydroxyecdysone into mid-fourth instar larvae of B. hygida show that the induction of BhC4-1 expression, as well as amplification and puff C4 expansion, are late events induced by the hormone. This late response of BhC4-1 expression was also observed in transgenic salivary glands cultivated in the presence of 20-hydroxyecdysone. In vitro studies using transgenic Drosophila indicate that both repressor and activator factors regulate the timing of BhC4-1 expression in salivary glands.

Avaliaçäo da qualidade microbiológica do leite UAT integral comercializado em Belo Horizonte / Microbiological aspects of UHT whole milk commercialized in Belo Horizonte, Brazil

Foram analisadas 80 amostras de leite UAT integral de oito marcas comercializadas em Belo Horizonte, entre janeiro e abril de 1999. Foram realizadas contagens de bactérias mesófilas aeróbicas em meio APC e ágar BHI enriquecido com vitamina B12, sendo os resultados comparados aos padröes estabelecidos pelo Regulamento Técnico de Identidade e Qualidade (RTIQ) do Serviço Federal de Inspeçäo do Ministério da Agricultura e Abastecimento do Brasil. Das 80 amostras analisadas, 33 (41,2(porcento)) apresentaram contagem de bactérias mesófilas aeróbicas entre 1,3x10 elevado a quarta potência e 1,4x10 elevado a quinta potência UFC/ml (4,1 e 5,1 log UFC/ml). De 174 amostras de microrganismos isoladas, 162 (93,1(porcento)) säo do gênero Bacillus, 3 cocos (1,7(porcento)) pertencentes ao gênero Micrococcus, 9 (5,2(porcento)) bastonetes Gram positivos pleomórficos, sugestivos de pertencerem ao gênero Corynebacterium. Cinco marcas de leite UAT foram consideradas como fora dos padröes microbiológicos estabelecidos pelo RTIQ. Bactérias do gênero Bacillus foram as mais isoladas

A dual role of 20-hydroxyecdysone in the control of protein synthesis related to DNA puff activity in the anterior region of Bradysia hygida (Diptera, Sciaridae) salivary gland.

During the last 30 h of the larval stage, the salivary glands of Bradysia hygida show the amplification of some genes, resulting in the formation of two successive groups of DNA puffs, which direct the synthesis of two different sets of polypeptides. Incubation of anterior (S1) salivary gland regions, at age E7, beginning of first group of DNA puffs activity, in culture medium for 2 to 10 h results in a decrease in the synthesis of the polypeptides characteristic of this period. However, during subsequent incubation (from E7 to E7+12 h-24 h), when the second group of DNA puffs is active, S1 regions were able to synthesize some polypeptides characteristic of this period. The role of 20-OH ecdysone was studied, in vitro and in vivo, during these two periods of protein synthesis in S1 regions. The presence of the hormone was shown to be necessary to maintain, in vitro, the synthesis of the first set of polypeptides and was strongly inhibitory, in vitro and in vivo, to the synthesis of the second set of polypeptides. Thus, it is likely that the activity of the two distinct groups of DNA puffs is under opposite 20-OH-ecdysone control mechanisms.

Genome-wide mutant collections: toolboxes for functional genomics.

The sequencing of entire genomes has led to the identification of many genes. A future challenge will be to determine the function of all of the genes of an organism. One of the best ways to ascertain function is to disrupt genes and determine the phenotype of the resulting organism. Novel large-scale approaches for generating gene disruptions and analyzing the resulting phenotype are underway in the budding yeast Saccharomyces cerevisiae and other organisms including flies, Mycoplasma, worms, plants and mice. These approaches and mutant collections will be extremely valuable to the scientific community and will dramatically alter the manner in which science is performed in the future.

TRIPLES: a database of gene function in Saccharomyces cerevisiae.

Using a novel multipurpose mini-transposon, we have generated a collection of defined mutant alleles for the analysis of disruption phenotypes, protein localization, and gene expression in Saccharomyces cerevisiae. To catalog this unique data set, we have developed TRIPLES, a Web-accessible database of TRansposon-Insertion Phenotypes, Localization and Expression in Saccharomyces. Encompassing over 250 000 data points, TRIPLES provides convenient access to information from nearly 7800 transposon-mutagenized yeast strains; within TRIPLES, complete data reports of each strain may be viewed in table format, or if desired, downloaded as tab-delimited text files. Each report contains external links to corresponding entries within the Saccharomyces Genome Database and International Nucleic Acid Sequence Data Library (GenBank). Unlike other yeast databases, TRIPLES also provides on-line order forms linked to each clone report; users may immediately request any desired strain free-of-charge by submitting a completed form. In addition to presenting a wealth of information for over 2300 open reading frames, TRIPLES constitutes an important medium for the distribution of useful reagents throughout the yeast scientific community. Maintained by the Yale Genome Analysis Center, TRIPLES may be accessed at http://ycmi.med.yale.edu/ygac/triples.htm

Large-scale analysis of the yeast genome by transposon tagging and gene disruption.

Economical methods by which gene function may be analysed on a genomic scale are relatively scarce. To fill this need, we have developed a transposon-tagging strategy for the genome-wide analysis of disruption phenotypes, gene expression and protein localization, and have applied this method to the large-scale analysis of gene function in the budding yeast Saccharomyces cerevisiae. Here we present the largest collection of defined yeast mutants ever generated within a single genetic background--a collection of over 11,000 strains, each carrying a transposon inserted within a region of the genome expressed during vegetative growth and/or sporulation. These insertions affect nearly 2,000 annotated genes, representing about one-third of the 6,200 predicted genes in the yeast genome. We have used this collection to determine disruption phenotypes for nearly 8,000 strains using 20 different growth conditions; the resulting data sets were clustered to identify groups of functionally related genes. We have also identified over 300 previously non-annotated open reading frames and analysed by indirect immunofluorescence over 1,300 transposon-tagged proteins. In total, our study encompasses over 260,000 data points, constituting the largest functional analysis of the yeast genome ever undertaken.

DNA puff C4 of Bradysia hygida (Diptera: Sciaridae) contains genes unequally amplified and differentially expressed during development.

We report here the isolation and characterization of a 2.3 kb genomic EcoRI fragment that co-localizes in the DNA puff C4 of Bradysia hygida with a 4 kb EcoRI fragment previously characterized as containing part of a gene amplified and expressed in the salivary gland at the time when puff C4 expands. Verification of the relative amount of DNA complementary to these two genomic fragments shows that they are unequally amplified in the salivary gland. The fragment containing part of the gene expressed when puff C4 expands amplifies about eight times more than the 2.3 kb fragment. This 2.3 kb fragment also carries sequences complementary to RNA species present in the gland in a period when puff C4 has already receded. Based on these data we discuss the nature of the DNA puff and the possible way in which amplification is occurring at these sites.