Mesophilic Lactic Acid Bacteria Diversity Encountered in Brazilian Farms Producing Milk with Particular Interest in Lactococcus lactis Strains.

The milk produced in regions with different traditions in Brazil is used for artisanal product production, which is characterized by different sensorial characteristics. This study aimed to identify the bacterial ecosystem of farms located in a traditional dairy region in the state of Minas Gerais and to characterize Lactococcus lactis strains, the species of interest in this study, using a multilocus sequence typing (MLST) protocol and pulsed-field gel electrophoresis (PFGE) technique. Samples were collected from raw milk and dairy environment from six farms. A total of 50 isolates were analyzed using 16S rRNA sequencing and species-specific PCR. Five genera were identified: Lactobacillus, Leuconostoc, Lactococcus, Enterococcus, and Staphylococcus, from ten different species. MLST (with six housekeeping genes) and PFGE (with SmaI endonuclease) were used for the characterization of 20 isolates of Lactococcus lactis from a dairy collection in this study. Both methods revealed a high clonal diversity of strains with a higher discriminatory level for PFGE (15 pulsotypes), compared to MLST (12 ST). This study contributes to the preservation of the Brazilian dairy heritage and provides insights into a part of the LAB population found in raw milk and dairy environment.

Efficacy of fish embryo vitrification protocols in terms of embryo morphology - a systematic review.

BACKGROUND: Embryo cryopreservation has been used for the creation of genetic banks with diploid resources, and among different techniques, vitrification is considered as the most promising method. OBJECTIVE: The goal is to evaluate the major aspects of the existing vitrification techniques and to evaluate their efficacy in terms of embryo morphology. METHODS: Electronic searches in the PubMed and ScienceDirect databases were performed with the keyword combination: fish, embryo and vitrification. Pubmed retrieved 26 articles and Science Direct resulted in 464 articles. For this review, only studies that developed and tested vitrification protocols in fish embryos were included. Research regarding cryoprotectant toxicity and permeability were excluded. There were no restrictions on publication date or language. With these criteria, a total of ten articles were evaluated. RESULTS: In these articles, the major aspects to be considered for the development of new vitrification protocols are: the cryoprotectants' toxicity, the embryos' development stage, the exposure to and the permeability of the cryoprotectants, vitrification devices and vitrification-warning cycle. CONCLUSION: The survival were limited, however, the preservation of embryonic morphology after thawing indicates the possibility of preserving fish embryos via the vitrification technique.

Molecular characterization of a new lytic bacteriophage isolated from cheese whey.

In this study, we isolated and characterized a lytic Lactococcus lactis bacteriophage from the sera of a failed fermentation. The phage was isolated and cultured in L. lactis subsp. cremoris in M17 medium. The isolated bacteriophage was characterized by multiplex PCR, pulsed-field electrophoresis, DNA restriction digestion, analysis of the N-terminal sequence of the phage major structural protein, transmission electron microscopy and sequencing and analysis of a conserved fragment of its genome. Analysis of the viral genome indicates that its genome is composed of a DNA strand of approximately 48 kb in length, and PCR and microscopy confirmed that IL-P1 belongs to the group of 936-type phages in the family Siphoviridae, which is the most abundant type of lactococcal virus in dairy products worldwide. To our knowledge, this is the first report of a virus within this family that has a presumptive genome larger than 40 kb.

Biological activities of a human amniotic membrane interferon.

In order to characterize further the human amniotic membrane interferon (IFN-AM), an interferon antigenically unrelated to human IFN-alpha, -beta, and -gamma or TNF, we analysed its biological activities. Here, we present direct evidence of its ability to affect cell growth and to induce the IFN-stimulated genes (ISGs) 6-16 and 2'-5' oligoadenylate synthetase (OAS), in addition to its crossed anti-viral activity. The cellular growth arrest effect of IFN-AM was dose-dependent and paralleled that of IFN-beta. IFN-AM was also able to inhibit thymidine incorporation into DNA, similar to IFN-beta. The mRNA induction of 6-16 gene with IFN-AM treatment reached its highest level at 500 IU/ml and remained constant up to 2000 IU/ml. Conversely, 2'-5' OAS mRNA induction was dose-dependent, with the maximum level detected at 2000 IU/ml of IFN-AM treatment. The time course of mRNA accumulation by ISGs with IFN-AM (500 IU/ml) stimulation was also investigated. Gene induction reached a maximum at 16 h after IFN treatment for 2'-5' OAS and at 48 h for the 6-16 gene. IFN-AM and human IFN-alpha induced similar levels of the OAS enzyme. IFN-AM also showed small but significant activity in bovine cells. In conclusion, the amniotic membrane IFN here studied showed both anti-cellular activity and the ability to stimulate ISG-transcriptional activation in a similar manner to IFN-beta. In addition, IFN-AM was also as able to induce the expression of the enzyme 2'-5' OAS, as did IFN-alpha. Lastly, amniotic IFN showed a significant cross-species anti-viral activity, which was different from both human IFN-alpha and -beta. Taken together, these data strongly suggest that IFN-AM is a novel sub-type I IFN.

Identification of diary Propionibacterium species by rRNA gene restriction patterns.

A total of 78 strains of dairy propionibacteria, 4 reference strains of Propionibacterium and 8 related bacteria were characterized by ribosomal ribonucleic acid (rRNA) gene restriction patterns (ribotyping). The patterns were obtained after cleavage of total DNA with either BamHI or ClaI restriction endonucleases and hybridization of fragments with acetylaminofluorene-labelled 16 + 23S rRNA from Escherichia coli. The four different species of dairy propionibacteria, P. freudenreichii, P. jensenii, P. thoenii and P. acidipropionici, gave different restriction patterns with species-specific fragments. Moreover, ribotyping allowed the differentiation of P. freudenreichii subsp. freudenreichii from P. freudenreichii subsp. shermanii. The patterns of dairy propionibacteria were different from those of closely related bacteria and other bacteria used in the dairy industry.

Reclassification of "Propionibacterium rubrum" as P. jensenii.

The taxonomic relationship of strains previously designated as "Propionibacterium rubrum" to P. thoenii and P. jensenii was investigated by use of 16S ribosomal RNA sequence comparison, biochemical characteristics and DNA hybridization. A total of 46 strains representing the species P. jensenii and P. thoenii and the former species "P. rubrum" and also including 21 reference strains and 25 strains isolated from dairy sources were studied. The 16S rRNA sequence of strain "P. rubrum" CNRZ 85 (= ATCC 4871) was found to be almost identical to that of the type strain of P. jensenii. DNA hybridization data indicated that "P. rubrum" should belong to the species P. jensenii rather than P. thoenii, as formerly proposed. The "P. rubrum" strains should then be reclassified as a beta-haemolytic biovar of P. jensenii. The genomic species P. jensenii and P. thoenii could be differentiated by biochemical characteristics such as the production of acid from myo-inositol and starch.

Pixel clustering by adaptive pixel moving and chaotic synchronization.

In this paper, a network of coupled chaotic maps for pixel clustering is proposed. Time evolutions of chaotic maps in the network corresponding to a pixel cluster are synchronized with each other. Those synchronized trajectories are desynchronized with respect to the time evolutions of chaotic maps corresponding to other pixel clusters in the same image. A pixel motion mechanism is also introduced, which makes each group of pixels more compact and, consequently, makes the model robust enough to classify ambiguous pixels. Another feature of the proposed model is that the number of pixel clusters does not need to be previously known.

Present status of approval procedures in the EU member states and Switzerland.

The European Union Council Directive 96/29/EURATOM requires that 'individual monitoring shall be ... based on individual measurements which are established by an approved dosimetric service' and that 'Each Member State shall make arrangements to recognise, as appropriate, the capacity of ... approved dosimetric services'. At present, approval of dosimetric services does not have the same meaning within EU Member States and Switzerland. In some countries, service and dosemeter approval is clearly separated, in some others only one of the two is supposed to be tested, and in others no approval is required. Dosimetric requirements and criteria are based on different international documents (e.g. IEC, ISO, ANSI, CEC report) or national specific rules. Approval frequency can be once, every 2 or more years. Approval can be based on either evaluation of technical and management reports, irradiation tests, inspection on-site or the three steps together. In most cases, approval involves photon dosimetry while beta and neutron dosimetry test procedures are not as well established. However, comparisons may lead to some convergent evolution of procedures and to a greater degree of harmonisation and quality consolidation.

Harmonisation and dosimetric quality assurance in individual monitoring for external radiation.

The current situation amongst Member States is that there are widely differing national requirements for dosimetric services and for dosemeter performance. It is clear that with the free movement of workers within the European Union (EU) and the requirements for individual dosimetry given in Council Directive 96/29 EURATOM, a degree of harmonisation of requirements and procedures of EU Member States would be desirable. A EURADOS action group, made up of members from each of the EU Member States plus Switzerland, was set up with the overall objectives of consolidating within the EU the quality of individual monitoring using personal dosemeters and assisting movement towards harmonised procedures. An outline of the work of the action group is given and the term 'harmonisation' is discussed.

Genes involved in lactose catabolism and organic acid production during growth of Lactobacillus delbrueckii UFV H2b20 in skimmed milk.

There are three main reasons for using lactic acid bacteria (LAB) as starter cultures in industrial food fermentation processes: food preservation due to lactic acid production; flavour formation due to a range of organic molecules derived from sugar, lipid and protein catabolism; and probiotic properties attributed to some strains of LAB, mainly of lactobacilli. The aim of this study was to identify some genes involved in lactose metabolism of the probiotic Lactobacillus delbrueckii UFV H2b20, and analyse its organic acid production during growth in skimmed milk. The following genes were identified, encoding the respective enzymes: ldh - lactate dehydrogenase, adhE - Ldb1707 acetaldehyde dehydrogenase, and ccpA-pepR1 - catabolite control protein A. It was observed that L. delbrueckii UFV H2b20 cultivated in different media has the unexpected ability to catabolyse galactose, and to produce high amounts of succinic acid, which was absent in the beginning, raising doubts about the subspecies in question. The phylogenetic analyses showed that this strain can be compared physiologically to L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis, which are able to degrade lactose and can grow in milk. L. delbrueckii UFV H2b20 sequences have grouped with L. delbrueckii subsp. bulgaricus ATCC 11842 and L. delbrueckii subsp. bulgaricus ATCC BAA-365, strengthening the classification of this probiotic strain in the NCFM group proposed by a previous study. Additionally, L. delbrueckii UFV H2b20 presented an evolutionary pattern closer to that of probiotic Lactobacillus acidophilus NCFM, corroborating the suggestion that this strain might be considered as a new and unusual subspecies among L. delbrueckii subspecies, the first one identified as a probiotic. In addition, its unusual ability to metabolise galactose, which was significantly consumed in the fermentation medium, might be exploited to produce low-browning probiotic Mozzarella cheeses, a desirable property for pizza cheeses.

Genes involved in protein metabolism of the probiotic lactic acid bacterium Lactobacillus delbrueckii UFV H2b20.

A basic requirement for the prediction of the potential use of lactic acid bacteria (LAB) in the dairy industry is the identification of specific genes involved in flavour-forming pathways. The probiotic Lactobacillus delbrueckii UFV H2b20 was submitted to a genetic characterisation and phylogenetic analysis of genes involved in protein catabolism. Eight genes belonging to this system were identified, which possess a closely phylogenetic relationship to NCFM strains representative, as it was demonstrated for oppC and oppBII, encoding oligopeptide transport system components. PepC, PepN, and PepX might be essential for growth of LAB, probiotic or not, since the correspondent genes are always present, including in L. delbrueckii UFV H2b20 genome. For pepX gene, a probable link between carbohydrate catabolism and PepX expression may exists, where it is regulated by PepR1/CcpA-like, a common feature between Lactobacillus strains and also in L. delbrueckii UFV H2b20. The well conserved evolutionary history of the ilvE gene is evidence that the pathways leading to branched-chain amino acid degradation, such as isoleucine and valine, are similar among L. delbrueckii subsp. bulgaricus strains and L. delbrueckii UFV H2b20. Thus, the involvement of succinate in flavour formation can be attributed to IlvE activity. The presence of aminopeptidase G in L. delbrueckii UFV H2b20 genome, which is absent in several strains, might improve the proteolytic activity and effectiveness. The nucleotide sequence encoding PepG revealed that it is a cysteine endopeptidase, belonging to Peptidase C1 superfamily; sequence analysis showed 99% identity with L. delbrueckii subsp. bulgaricus ATCC 11842 pepG, whereas protein sequence analysis revealed 100% similarity with PepG from the same organism. The present study proposes a schematic model to explain how the proteolytic system of the probiotic L. delbrueckii UFV H2b20 works, based on the components identified so far.

Gross and microscopic anatomy of the pineal gland in Nasua nasua--coati (Linnaeus, 1766).

Nasua nasua, coati, is a mammal of the Carnivora order and Procyonidae family. It lives in bands composed of females and young males. The pineal gland or epiphysis of brain is endocrine, producing the melatonin. Its function is the control of the cycle of light environment, characteristic of day and night. For this research, five adult coatis were used, originating from CECRIMPAS-UNIfeob (Proc. IBAMA 02027.003731/04-76), Brazil. The animals were killed and perfusion-fixed in 10% formaldehyde. Pineals were measured and a medium size was found to be 2.3-mm-long and 1.3-mm-wide. Pineal gland was located in the habenular commissure in the most caudal portion of the third ventricular roof, lying in a dorso-caudal position from the base to the apex. Pinealocytes were predominantly found in the glandular parenchyma. Distinct and heterogeneous arrangements of these cells throughout the three pineal portions were observed as follows: linear cords at the apex, circular cords at the base of the gland, whereas at the body a transition arrangement was found. Calcareous concretions could be observed in the apex. The pineal gland was classified as subcallosal type [Rec. Méd. Vét.1, 36 (1956)] and as AB type [Prog. Brain Res. 42, 25 (1979); The Pineal Organ, Berlin/Heidelberg: Springer-Verlag (1981)].

Alterations in the level of insulin receptor and GLUT-4 mRNA in skeletal muscle from rats fed a kidney bean (Phaseolus vulgaris) diet.

1. A decline in the level of circulating insulin was observed in rats fed a diet containing kidney bean. 2. Consumption of a diet containing kidney bean caused an increase in the level of mRNAs for the insulin receptor (327%) and GLUT-4 (185%) in the gastrocnemius muscle. In contrast there was only a small increase in the amount of actin mRNA (125%). Since the kidney bean-fed rats are euglycaemic the results suggest that insulin receptor and GLUT-4 mRNA levels are regulated in response to circulating insulin concentrations rather than glucose. 3. No increases in the level of insulin receptor and actin mRNA were evident in the soleus muscle of rats fed the diet containing kidney bean; however a decline was observed in the level of GLUT-4 mRNA. 4. It is proposed that a component of kidney beans, most likely the lectin phytohaemagglutinin, has systemic effects which lead to changes in expression of the insulin receptor and GLUT-4 genes and to the sensitivity of muscle to insulin.

NABC1 (BCAS1): alternative splicing and downregulation in colorectal tumors.

We have identified a new splicing variant of the gene "novel amplified in breast cancer 1," NABC1 (HGMW-approved symbol BCAS1). This variant, which we call NABC1_5B, uses a previously unidentified 135-bp exon. Also in this report, we confirm that NABC1 is overexpressed in breast tumors and show that both NABC1 and NABC1_5B are downregulated in colorectal tumors.

Lipid accumulation in obese Zucker rats is reduced by inclusion of raw kidney bean (Phaseolus vulgaris) in the diet.

The effects of inclusion of different levels of raw kidney bean (Phaseolus vulgaris) of high lectin content (27 g/kg meal) in a high-quality (lactalbumin) control diet were tested in nutritional trials on the growth and metabolism of obese Zucker (fafa) rats and their lean littermates in comparison with pair-fed controls. All diets contained 100 g total protein/kg and either 50 g lipids/kg (low fat) or 150 g lipids/kg (moderate fat). The growth of both obese and lean rats on bean diets was retarded by the daily bean intake in a dose-dependent manner. However, most of this was because bean-fed rats contained less body fat than the controls after 10 d. Thus, after feeding low-fat diets containing up to 130 g kidney bean/kg (lectin intake < or = 0.2 g/kg body weight (BW) per d) in both 10 d and 70 d trials, the bodies of obese rats contained less fat but not protein than their pair-fed controls. Moreover, by increasing the lipid content of the diet to 150 g/kg, the level of bean inclusion could be increased to 280 g/kg (lectin intake > or = 0.4 g/kg BW per d) without loss of body protein and skeletal muscle. Although these rats contained more body fat than those which were fed on low-fat diets, their weight reduction could be accounted for exclusively by reduced lipid content. In contrast, significant body protein loss occurred when the same diet of high lectin content was fed to lean littermates. Plasma insulin levels were significantly depressed in the obese Zucker rats on bean diets but the pancreas was not significantly enlarged nor its insulin content changed in 10 d trials. However, significant pancreatic growth occurred on long-term (70 d) bean feeding compared with pair-fed controls. The results suggest that, in addition to animal nutrition, it may also be possible to use the bean lectin as a dietary adjunct or therapeutic agent to stimulate gut function and ameliorate obesity if a safe and effective dose-range can be established for human subjects.