Identification of lactic acid bacteria associated with traditional cachaça fermentations

During the production of traditional cachaça (alembicïs cachaça), contamination of the fermented must is one of the factors leading to economic losses in the beverage manufacturing industry. The diversity of bacterial populations and the role of these microorganisms during the cachaça production process are still poorly understood in Brazil. In our work, the fermentation process was followed in two distilleries located in the state of Minas Gerais. The objective of this work was to identify the populations of lactic acid bacteria present during cachaça fermentation using physiological and molecular methods. Lactic acid bacteria were isolated in high frequencies during all of the fermentative processes, and Lactobacillus plantarum and L. casei were the most prevalent species. Other lactic acid bacteria were found in minor frequencies, such as L. ferintoshensis, L. fermentum, L. jensenii, L. murinus, Lactococcus lactis, Enterococcus sp. and Weissella confusa. These bacteria could contribute to the increase of volatile acidity levels or to the production of compounds that could influence the taste and aroma of the beverage.

Intolerancia a la lactosa: en quién y por qué / Lactose intolerance

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Comparação da eficiência dos caldos Escherichia coli e caldo verde brilhante lactose bile na enumeração de coliformes termotolerantes em leite e sorvete de massa / Efficiency of Escherichia coli liquid culture medium and brilliant greenbile salt lactose broth for counting thermotolerant coliforms in milk and ice cream samples

Foi realizada a comparação entre os caldos Escherichia coli - EC (Difco 0314-01-0) e Caldo Verde Brilhante Lactose Bile 2% - CVBLB (Difco 0007-01-2) utilizados como teste confirmativo para a detecção de coliformes termotolerantes em amostras de leite pasteurizado tipo C e sorvete. Com base nos resultados obtidos, pode-se afirmar que houve diferença significativa (p<0,05) entre os dois meios de cultura quanto à contagem do NMP de coliformes termotolerantes. Outrossim, 58,3% das amostras de leite e 19,4% das amostras de sorvetes apresentaram valores de contagem de coliformes termotolerantes em desacordo com os tolerados pela legislação (ANVISA, 2001), quando as amostras analisadas foram cultivadas em meio de CVBLB. Ao empregar o caldo EC para o cultivo das amostras, 70,8% das amostras de leite e 30,6% das amostras de sorvetes apresentaram NMP de coliformes termotolerantes em desacordo com os permitidos pela legislação.

Effecto de la diarrea inducida con lactosa sobre la disponibilidad de los macronutrientes y la función inmune en ratas nutridas y desnutridas / Effect of lactose induced diarrhea on macronutrients digestibility and immune function in well-nourished and undernourished rats

El objetivo de este trabajo fue estudiar el efecto del estado nutricional sobre la disponibilidad de los nutrientes dietarios y algunos aspectos del sistema inmunológico durante la diarrea. Para ello se utilizaron ratas a las que se les indujo una diarrea osmótica usando lactosa y desnutrición reduciendo la oferta de dieta a la mitad del consumo registrado en ratas que recibieron alimentación ad libitum. El estudio incluyó cuatro grupos de ratas con siete ratas en cada grupo. Los dos primeros grupos incluyeron ratas nutridas, a uno de ellos se les indujo diarrea y al otrono; el tercer y cuarto grupo incluyeron ratas desnutridas con y sin diarrea, respectivamente. Los resultados mostraron que la lactosa produjo diarrea tanto en las ratas nutridas como en las desnutridas; sin embargo, en las nutridas la diarrea produjo una reducción importante en el consumo de alimento y en el crecimiento, mientras que en las desnutridas no. Así mismo, en las ratas nutridas la diarrea produjo una reducción en la digestabilidad y retención de todos los macronutrientes estudiados, mientras que esta reducción fue menos aparente o no se observó en las ratas desnutridas. Una situación similar se presentó en la relación con el peso del timo y la concentración de inmunoglobina G sérica. En ambos casos la diarrea tuvo un efecto negativo en las ratas nutridas, mientras que en las nutridas no produjo un deterioro adicional al producido por la desnutrición. En general estos resultados muestran que en las ratas desnutridas no se presentan o se presentan con mucho menos intensidad los efectos negativos de la diarrea. Esta adaptación evita que la diarrea provoque un deterioro adicional en su estado nutricional. (CONICIT, S1-2265)

Hepatic lacI and cII mutation in transgenic (lambdaLIZ) rats treated with dimethylnitrosamine.

The recent introduction of a transgenic rat in vivo mutation assay is a much needed supplement to the transgenic mouse models and offers the tools necessary for collecting target tissue specific genotoxicity data in this species. The utility of the Big Blue(R) rat for the detection of in vivo mutations was investigated by studying spontaneous and dimethylnitrosamine (DMN)-induced hepatic mutations. High molecular weight DNA isolated from Big Blue(R) rat livers typically yielded good transgene rescue efficiency of up to 5x105 plaque forming units per packaging reaction. DMN, when administered by oral gavage at dose levels of 0.2, 0.6, 2.0, and 6.0 mg kg-1 day-1, induced up to a 4.5-fold increase in mutations at the highest dose level. There was no apparent difference between the lacI vs. cII target genes of the shuttle vector in either the background or DMN-induced mutant frequencies. These results suggest that the transgenic rat model is a useful tool for studying potential genotoxicity in target organs and, with further validation, the selectable cII target could be an attractive alternative to the conventional lacI color screening method for the detection of mutations in the lambdaLIZ shuttle vector.

Xanthan Production by Xanthomonas campestris in a whey-based medium

Isodados de Xanthomonas campestris foram adaptados à utilizaçäo de lactose para produçäo de xantana. Um meio mínimo de soro de leite (sem desproteinizaçäo e hidrólise) foi desenvolvido contendo 0,5 por cento K2HPO4; 0,01 por cento MgSO4 e 4 por cento leite gerando 14Kg/10 de goma. A viscosidade de soluçöes das gomas obtidas pelos isolados também foi avaliada

Removal of polymerase-produced mutant sequences from PCR products.

Heteroduplex DNA lacking d(GATC) methylation is subject to mismatch-provoked double-strand cleavage at d(GATC) sites in a reaction dependent on MutH, MutL, MutS, and ATP. We have exploited this reaction to develop a method for removal of polymerase-produced mutant sequences that arise during sequence amplification by PCR. After denaturation and reannealing, the PCR product pool is subjected to MutH, MutL, and MutS mismatch repair proteins under double-strand cleavage conditions, followed by isolation of uncleaved product by size selection. Use of an Escherichia coli lac forward mutation assay has shown that this procedure reduces the incidence of polymerase-induced mutant sequences by an order of magnitude. Twenty mutants that originated from three independent PCR amplification reactions and survived MutHLS treatment all were found to contain an infrequently occurring A.T --> T.A transversion mutation at a unique position within the product. By contrast, the majority of mutations in untreated PCR products were transitions occurring throughout the amplified region, although frameshifts and transversions also were observed. The MutHLS method thus can be used to effectively remove the majority of mutant sequences produced by polymerase errors during PCR amplification.

Intrastriatal grafts of embryonic mesencephalic rat neurons genetically modified using an adenovirus encoding human Cu/Zn superoxide dismutase.

Intrastriatal grafting of embryonic dopamine-containing neurons is a promising approach for treating clinical and experimental Parkinson's disease. However, neuropathological analyses of grafted patients and transplanted rats have demonstrated that the survival of grafted dopamine neurons is relatively poor. In the present study, we pursued a strategy of transferring a potentially neuroprotective gene into rat embryonic mesencephalic rat cells in vitro, before grafting them into the denervated striatum of 6-hydroxydopamine-lesioned rats. We performed intrastriatal grafts of embryonic day 14 mesencephalic cells infected with replication-defective adenoviruses bearing either the human copper-zinc superoxide dismutase gene or, as a control, the E. coli lac Z marker gene. The transgenes were expressed in the grafts four days after transplantation and the expression persisted for at least five weeks thereafter. After five weeks postgrafting, there was more extensive functional recovery in the superoxide dismutase group as compared to the control (uninfected cells) and beta-galactosidase groups. The functional recovery was significantly correlated with the number of tyrosine hydroxylase-positive cells in the grafts, although the clear trend to increased survival of the dopamine neurons in the superoxide dismutase grafts did not reach statistical significance. Only a moderate inflammatory reaction was revealed by OX-42 immunostaining in all groups, suggesting that ex vivo gene transfer using adenoviral vectors is a promising method for delivering functional proteins into brain grafts.

Infecçöes relacionadas ao cateter venoso central em terapia intensiva / Central venous catheter-related infections in critically-ill-patients

OBJETIVO. Determinar a incidência, a etiologia e os fatores de risco das infecçoes relacionadas ao cateter venoso central em terapia intensiva. METODOLOGIA. Estudo observacional de coorte, prospectivo, em pacientes criticamente enfermos submetidos à cateterizaçao venosa profunda por punçao percutânea. Realizadas culturas quantitativa da pele, semiquantitativa da ponta e quantitativa do canhao do cateter, e hemocultura periférica. Os possíveis fatores de risco foram submetidos à análise univariada e multivariada. RESULTADOS. Foram estudados 57 períodos de cateterizaçao em 51 pacientes. A incidência de infecçao local foi de 21,1 por cento (33,8/1.000 dias-cateter), e de bacteremia, 8,7 por cento (l4,1/1.000 dias-cateter). A pele no local de inserçao estava colonizada em 32,7 por cento dos pacientes e o canhao, em 29,1 por cento. A origem dos microrganismos causadores de infecçao foi a pele em 41,2 por cento, o canhao em 29,4 por cento, infecçao a distância em 5,9 por cento, e nao ficou esclarecida em 23,5 por cento dos casos. Estafilococos coagulase-negativa foram os agentes etiológicos predominantes. Identificou-se, como variáveis independentemente associadas à infecçao local, a purulência no orifício de inserçao e a utilizaçao de outro dispositivo intravascular. As variáveis independentemente associadas à bacteremia foram a inserçao na veia jugular interna e a colonizaçao do canhao do cateter. CONCLUSOES. A bacteremia é uma complicaçao importante do cateterismo venoso central em terapia intensiva. Os estafilococos coagulase-negativa predominam nesta modalidade de infecçao hospitalar. A inserçao do cateter na veia jugular interna e a colonizaçao do canhao aumentam o risco de bacteremia relacionada à linha venosa central.

Deamination of single-stranded DNA cytosine residues in aerobic nitric oxide solution at micromolar total NO exposures.

Deamination of cytosine to uracil is a potential source of mutations in DNA. Here we examine the deaminating ability of aerobic nitric oxide (NO) toward single-stranded DNA at very low (micromolar and below) total exposures, using a sensitive genetic method that allows us to study a single deamination event at a specific site in a 7200-nucleotide DNA molecule within a pool of ca. 100,000 other identical DNA molecules. We incubated gapped C141 M13mp2 DNA with the NO-generating compound, Et2N[N(O)NO]Na (DEA/NO), in aerobic buffer for 16 h to ensure complete autoxidation at pH 7.4 and 37 degrees C. After ultrafiltration to remove small molecules, the DNA was transformed into isogenic Escherichia coli cultures that were either deficient (NR9404, ung-) or proficient (MC1061, ung+) in uracil-DNA glycosylase activity. The gapped DNA was constructed such that the target (CCC) codon was contained in a short single-stranded segment of otherwise double-stranded circular DNA, and the incubation was performed in a closed system to prevent loss of NO to the atmosphere before the reaction was complete. An increase in the reversion frequency in the ung- strain was noted between 0 and 1 microM DEA/NO, and the reversion frequency leveled out between 3 and 30 microM. However, 30 microM "spent" DEA/NO (i.e., that which was similarly incubated for 4 h to complete the autoxidation of NO before the DNA was added) did not increase reversion frequency relative to control. Nearly all (42/43) of the mutations identified after 1 microM DEA/NO treatment were C-->T transitions, and reversion frequency in the isogenic ung+ strain was lower than in the ung- strain. The data are consistent with the hypothesis that total NO exposures in the mumol/L range can lead to C-->T mutations via a mechanism most probably involving deamination of DNA cytosine residues.

Olfactory glomeruli are innervated by more than one distinct subset of primary sensory olfactory neurons in mice.

The rodent olfactory epithelium consists of a mosaic of primary sensory olfactory neurons (PONs) which express distinct putative olfactory receptor proteins. Recent evidence suggests that individual subsets of these sensory neurons project to separate glomeruli in the olfactory bulb (Vassar et al., [1994] Cell 79:981-991). In the present study we have identified two distinct subsets of primary sensory olfactory neurons (PONs) in the H-OMP-LacZ-6 transgenic mouse. In these transgenic mice, a LacZ reporter gene under the control of a 294 base pair element from the 5' promoter region of the olfactory marker protein (OMP) gene was expressed in a subset of PONs located in a discrete band of neuroepithelium in the nasal cavity. These LacZ positive neurons were not randomly located within this band but were more concentrated within a locus between endoturbinates IIb and III. The axons of these neurons densely innervated three adjacent and bilaterally symmetrical glomeruli present in the ventromedial olfactory bulb. Labeling of tissue sections with the plant lectin Dolichos biflorus (DBA) revealed an independent subset of PONs in the transgenic mice. These neurons were present in a wide region of the nasal cavity that included the neuroepithelial band containing the LacZ expressing neurons. The DBA labeled axons terminated in glomeruli in the rostromedial and dorsolateral olfactory bulb surfaces. Although the glomeruli innervated by the LacZ and DBA positive axons were predominantly non-overlapping there were glomeruli in the ventral olfactory bulb that were labeled by both DBA and LacZ markers. Eight different types of glomeruli were characterized. Most notably, glomeruli were identified which were innervated partially by both or by either subset alone. In these cases, axon subsets were observed to terminate within discrete subregions of a glomerulus. These results support the hypothesis that phenotypically distinct subsets of PONs converge on to the same glomeruli but also indicate that some glomeruli are innervated by more than one subset of sensory neuron. These findings have implications for understanding how the olfactory projection is formed and how olfactory information is processed.

Identification and characterization of the insertion element IS1070 from Leuconostoc lactis NZ6009.

A novel insertion sequence, designated IS1070, was identified on the lactose plasmid of Leuconostoc lactis NZ6009 by nucleotide sequence analysis. The 1027-bp sequence contains partially matched (24 of 28 bp) inverted repeats and has one long open reading frame. The deduced 305-amino-acid sequence demonstrated homology to transposases of IS30 from Escherichia coli, IS4351 from Bacteroides fragilis, IS1086 from Alcaligenes eutrophus, IS1161 from Streptococcus salivarius, ISAS2 from Aeromonas salmonicida and a putative protein encoded by ORF3 of virus SpV1-R8A2 B from Spiroplasma citri. At least fifteen IS1070-like sequences were detected in the genome of the parent Lc. lactis strain and five of these were situated on plasmids. Analysis of the direct repeats of two of these copies with that of IS1070 revealed differences in the target duplication lengths.

Comparison of solution structure of free and complexed lac operator by molecular modelling with NMR constraints.

The structure difference between the free operator of the lac system d(GCTCACAAT).d(ATTGTGAGC) and the same operator complexed to the headpiece of the lac repressor has been investigated by 2-D-1H NMR spectroscopy in conjunction with molecular modelling in internal coordinates (JUMNA). The free and complexed operator adopt both a right-handed B helical conformation, but a more detailed analysis of the conformational parameters using the Curves program shows striking differences in the groove geometries, the rises, the twists and the total bending.

Highly efficient transactivation by the yeast Kluyveromyces lactis transcription factor LAC9 and its inhibition by the negative regulator GAL80 in mammalian cells.

Expression of the LAC9 gene from the yeast Kluyveromyces lactis in HepG2 human hepatoblastoma cells efficiently induced luciferase expression from reporter plasmids containing the four LAC9 binding sites from the K. lactis GAL1-GAL10 gene linked to a basal promoter. Induction was approximately 100fold and was dependent on the presence of the UAS sequence and an intact reading frame in the LAC9 gene. Additional cotransfection of constructs expressing the K. lactis GAL80 gene reduced luciferase activity by up to 98%. This inhibition was not affected by addition of 14mM galactose to the medium. No further yeast-specific factors appear necessary for efficient inhibition of LAC9 by GAL80, but additional gene products may be required for activation by galactose.

Mutants with substitutions for Glu171 in the catabolite activator protein (CAP) of Escherichia coli activate transcription from the lac promoter.

Single amino acid substitutions for residue Glu171 in helix E of the catabolite gene activator protein (CAP) of Escherichia coli have been reported to abolish activation of transcription without impairing binding to the CAP site of the lac promoter. The negative charge of Glu171 was proposed to transmit the activating signal from CAP to RNA polymerase. However, this idea has been challenged by later work. We set up a system to re-examine this issue. We analysed the ability of mutant CAP-E171L and CAP-E171K proteins to bind a near-consensus CAP site in vivo and found it to be diminished fourfold relative to wild type in each case. Activation of lac transcription by these mutant proteins remains the same as with wild-type CAP. Thus our results confirm that Glu171 in helix E of CAP is not involved directly in the activation of transcription. Yet CAP-E171K does not activate transcription as well as wild-type CAP under all circumstances. Possible reasons for this absence of activation are discussed.

Molecular rearrangement of lactose plasmid DNA associated with high-frequency transfer and cell aggregation in Lactococcus lactis 712.

High-frequency conjugation of the lactose plasmid pLP712 is associated with a constitutive cell aggregation phenotype and is facilitated by cointegration with a sex factor. Analysis of 23 independently derived enlarged lactose plasmids revealed that the sex factor DNA present in cointegrates varied in size. This suggested that more than simple cointegration with a sex factor plasmid was involved. Further analysis led to the discovery of a chromosomally located sex factor that could excise and be lost or exist as labile plasmid DNA. Cointegration with this sex factor was shown to be promoted by transposition of a copy of ISSI present on the lactose plasmid, and models are presented to account for the complex and variable structures of the resulting enlarged lactose plasmids.

In vivo interaction of Escherichia coli lac repressor N-terminal fragments with the lac operator.

Escherichia coli lac repressor is a tetrameric protein composed of 360 amino acid subunits. Considerable attention has focused on its N-terminal region which is isolated by cleavage with proteases yielding N-terminal fragments of 51 to 59 amino acid residues. Because these short peptide fragments bind operator DNA, they have been extensively examined in nuclear magnetic resonance structural studies. Longer N-terminal peptide fragments that bind DNA cannot be obtained enzymatically. To extend structural studies and simultaneously verify proper folding in vivo, the DNA sequence encoding longer N-terminal fragments were cloned into a vector system with the coliphage T7 RNA polymerase/promoter. In addition to the wild-type lacI gene sequence, single amino acid substitutions were generated at positions 3 (Pro3----Tyr) and 61 (Ser61----Leu) as well as the double substitution in a 64 amino acid N-terminal fragment. These mutations were chosen because they increase the DNA binding affinity of the intact lac repressor by a factor of 10(2) to 10(4). The expression of these lac repressor fragments in the cell was verified by radioimmunoassays. Both wild-type and mutant lac repressor N termini bound operator DNA as judged by reduced beta-galactosidase synthesis and methylation protection in vivo. These observations also resolve a contradiction in the literature as to the location of the operator-specific, inducer-dependent DNA binding domain.

Studies on the lactose character in Salmonella S:41:z10.

A number of plasmids carrying the Lac+ character have been reported. Lac+ character of salmonella S:41:z10:- studied for transfer of Lac+ character to standard Escherichia coli K12 Lac-F- Nalr and Escherichia coli K12 F- Lac- Rifr, failed to transfer in in vitro experiments. Similarly, identification and characterisation of plasmid DNA by agarose gel electrophoresis technique did not show specific plasmid DNA as compared to standard molecular weight plasmids. Plasmid DNA appeared to have been embedded with chromosomal DNA molecule.

In vitro expression of Lac-PTS and tagatose 1,6-bisphosphate aldolase genes from Lactococcus lactis subsp. cremoris plasmid pDI-21.

A 4.4-kb EcoR1-EcoR1 DNA fragment from the Lactococcus lactis subsp. cremoris plasmid pDI-21 encoded the tagatose 1,6-bisphosphate (TBP) aldolase gene and the Lac-PTS genes. In vitro transcription-translation using Escherichia coli S30 extract showed the synthesis of 41,000-, 23,000- and 12,000-dalton proteins which correspond to the TBP-aldolase, Lac-PTS enzyme II, and factor III proteins respectively.

Effects of anti-alpha monoclonal antibodies on initiation and elongation by the Escherichia coli RNA polymerase.

Anti-alpha monoclonal antibodies have been used to investigate the role of the alpha subunit of RNA polymerase in initiation and elongation. The four inhibitory monoclonal antibodies studied strongly inhibit cAMP receptor protein-dependent initiation with lac P+ and partially inhibit initiation directed by the lac UV5 promoter. The data suggest that the epitopes to which each of the monoclonal antibodies bind may be proximal to the contact domain between cAMP receptor protein and RNA polymerase. Recycling of RNA polymerase through the initiation process is slower in the presence of an inhibitory monoclonal antibody. Once a 9-nucleotide-long transcript has formed, incubation with the anti-alpha monoclonal antibody does not affect subsequent elongation. The monoclonal antibodies still bind to the elongation complex as indicated by sedimentation of the complex formed after incubation with Staphylococcus aureus cells (immunoprecipitin). These results suggest that the resistance of the elongation complex to these antibodies is not a consequence of their inability to bind to RNA polymerase. Only one of the alpha subunits may be involved in the initial process of transcription, and the antigenic domain of this subunit appears to be occluded by the nascent transcript present in the elongation complex.