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PubMed2002

Species-specific identification of commercial probiotic strains.

Products containing probiotic bacteria are gaining popularity, increasing the importance of their accurate speciation. Unfortunately, studies have suggested that improper labeling of probiotic species is common in commercial products. Species identification of a bank of commercial probiotic strains was attempted using partial 16S rDNA sequencing, carbohydrate fermentation analysis, and cellular fatty acid methyl ester analysis. Results from partial 16S rDNA sequencing indicated discrepancies between species designations for 26 out of 58 strains tested, including two ATCC Lactobacillus strains. When considering only the commercial strains obtained directly from the manufacturers, 14 of 29 strains carried species designations different from those obtained by partial 16S rDNA sequencing. Strains from six commercial products were species not listed on the label. The discrepancies mainly occurred in Lactobacillus acidophilus and Lactobacillus casei groups. Carbohydrate fermentation analysis was not sensitive enough to identify species within the L. acidophilus group. Fatty acid methyl ester analysis was found to be variable and inaccurate and is not recommended to identify probiotic lactobacilli.

Carbohydrate Metabolism↗

PubMed2005

A model of bidirectional synaptic plasticity: from signaling network to channel conductance.

In many regions of the brain, including the mammalian cortex, the strength of synaptic transmission can be bidirectionally regulated by cortical activity (synaptic plasticity). One line of evidence indicates that long-term synaptic potentiation (LTP) and long-term synaptic depression (LTD), correlate with the phosphorylation/dephosphorylation of sites on the alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit protein GluR1. Bidirectional synaptic plasticity can be induced by different frequencies of presynaptic stimulation, but there is considerable evidence indicating that the key variable is calcium influx through postsynaptic N-methyl-d-aspartate (NMDA) receptors. Here, we present a biophysical model of bidirectional synaptic plasticity based on [Ca2+]-dependent phospho/dephosphorylation of the GluR1 subunit of the AMPA receptor. The primary assumption of the model, for which there is wide experimental support, is that the postsynaptic calcium concentration, and consequent activation of calcium-dependent protein kinases and phosphatases, is the trigger for phosphorylation/dephosphorylation at GluR1 and consequent induction of LTP/LTD. We explore several different mathematical approaches, all of them based on mass-action assumptions. First, we use a first order approach, in which transition rates are functions of an activator, in this case calcium. Second, we adopt the Michaelis-Menten approach with different assumptions about the signal transduction cascades, ranging from abstract to more detailed and biologically plausible models. Despite the different assumptions made in each model, in each case, LTD is induced by a moderate increase in postsynaptic calcium and LTP is induced by high Ca2+ concentration.

Animals↗

PubMed1984

DNA methylation versus gene expression.

Vertebrate DNA is methylated at a high proportion of cytosine residues in the sequence CpG, and it has been suggested that the distribution of methylated and non-methylated CpGs in a given cell type influences the pattern of gene expression in those cells. Since a DNA methylation pattern is normally transmitted faithfully to daughter cells via cell division, this idea suggests an origin for stable, clonally inherited patterns of gene expression. This article discusses some of the current evidence for a relationship between DNA methylation and gene expression. Although the evidence is incomplete, it appears already that the relationship is variable: transcription of some genes is repressed by the presence of 5-methylcytosine at certain CpGs, and may be controlled by methylation, while transcription of other genes is indifferent to methylation. In attempting to explain this variability it is helpful to adopt an evolutionary perspective.

5-Methylcytosine↗

PubMed1995

Elementary forms of synaptic plasticity in the visual cortex.

The neocortex is an important site of memory storage, and memories are believed to be formed in the cortex by the activity-dependent modification of synaptic connections. However, in contrast to the hippocampus where there has been an increasingly sophisticated analysis of synaptic plasticity, relatively little is known about the mechanisms of synaptic modification in neocortex. Here we summarize the results of a series of experiments conducted on slices of visual cortex in vitro, aimed at elucidating the elementary mechanisms of synaptic plasticity in the superficial layers of neocortex. We show that long-term potentiation (LTP) and depression (LTD) result from high- and low-frequency conditioning stimulation, respectively, of the middle layers of cortex. Both forms of synaptic plasticity are input-specific and dependent on activation of postsynaptic N-methyl-D-aspartate (NMDA) receptors. The critical variable in determining the sign of the synaptic modification appears to be the level of postsynaptic depolarization during conditioning stimulation. The data support a model in which the state of correlation of pre- and post-synaptic activity is converted by the voltage-dependent NMDA receptor channel into a graded postsynaptic Ca2+ signal. LTD is triggered by a modest but sustained elevation in postsynaptic Ca2+, while LTP is elicited by larger changes in Ca2+. An important variable that regulates synaptic plasticity in the neocortex is intracortical inhibition, which constrains the patterns of activity that can reach the modifiable synapses in layer III.

Calcium↗

PubMed2005

Global assessment of promoter methylation in a mouse model of cancer identifies ID4 as a putative tumor-suppressor gene in human leukemia.

DNA methylation is associated with malignant transformation, but limitations imposed by genetic variability, tumor heterogeneity, availability of paired normal tissues and methodologies for global assessment of DNA methylation have limited progress in understanding the extent of epigenetic events in the initiation and progression of human cancer and in identifying genes that undergo methylation during cancer. We developed a mouse model of T/natural killer acute lymphoblastic leukemia that is always preceded by polyclonal lymphocyte expansion to determine how aberrant promoter DNA methylation and consequent gene silencing might be contributing to leukemic transformation. We used restriction landmark genomic scanning with this mouse model of preleukemia reproducibly progressing to leukemia to show that specific genomic methylation is associated with only the leukemic phase and is not random. We also identified Idb4 as a putative tumor-suppressor gene that is methylated in most mouse and human leukemias but in only a minority of other human cancers.

Animals↗

PubMed1995

Temporal and spatial regulation of H19 imprinting in normal and uniparental mouse embryos.

The mouse H19 gene is imprinted so that the paternal copy is both methylated and repressed during fetal development. However, the CpG-rich promoter region encompassing the transcription start is not methylated in sperm; this region must therefore become methylated postzygotically. We first examined the timing of DNA methylation of this region and the corresponding expression of H19. Both parental copies are initially undermethylated in blastocysts and the paternal copy then becomes fully methylated in the embryo around implantation; this methylation is more protracted in the extraembryonic lineages, especially in the trophoblast. By contrast to the lineage-dependent methylation, we observed exclusive expression of the maternal copy in preimplantation embryos and in all the lineages of early postimplantation embryos although variability may exist in cultured embryos. This indicates that methylation of the CpG-rich promoter is not a prerequisite for the paternal repression. We then examined whether methylation and expression occurs appropriately in the absence of a maternal or a paternal genome. Both H19 copies in androgenetic embryos are fully methylated while they are unmethylated in parthenogenetic embryos. This correlates with the lack of expression in androgenetic embryos but expression in parthenogenetic embryos. However, the androgenetic trophoblast was exceptional as it shows reduced methylation and expresses H19. These results suggest that promoter methylation is not the primary inactivation mechanism but is a stabilizing factor. Differential methylation in the more upstream region, which is established in the gametes, is a likely candidate for the gametic signal and may directly control H19 activity.

Animals↗

PubMed1991

The sequence specificity domain of cytosine-C5 methylases.

Prokaryotic DNA[cytosine-C5]methyltransferases (m5C-methylases) share a common architectural arrangement of ten conserved sequence motifs. A series of eleven hybrids have been constructed between the HpaII (recognition sequence: Cm5CGG) and HhaI (recognition sequence: Gm5CGC) DNA-methylases. The hybrids were over-expressed in E.coli and their in vivo methylation phenotypes investigated. Six were inactive by our assay while five of them retained partial methylation activity and full specificity. In all five cases the specificity matched that of the parent methylase which contributed the so-called variable region, located between conserved motifs VIII and IX. This was the only sequence held in common between the active hybrids and for the first time provides unequivocal evidence that the specificity determinants of the mono-specific m5C-methylases are located within the variable region. Correlation of the hybrid methylase structure with the efficiency of methylation suggests that conserved motif IX may interact with the variable region whereas motif X most probably interacts with the N-terminal half of the molecule.

Amino Acid Sequence↗

PubMedDate not supplied

The hair-organ relationship in mercury concentration in contemporary Japanese.

The hair-organ relationship of mercury concentration was investigated in 46 autopsy samples in Tokyo, Japan. Hair mercury levels were highly significantly correlated with organ Hg levels in the cerebrum, cerebellum, heart, spleen, liver, kidney cortex, and kidney medulla, when the total mercury or methyl mercury value in the organ was compared with the hair total mercury or organic mercury, respectively. When the inorganic mercury value was tested, significant correlations remained, with weaker coefficients in all the organs but the spleen. Stepwise multiple regression analysis evidenced that the hair organic mercury value was the major explanatory variable for the organ total mercury or organ methyl mercury value in all the organs. To explain the organ inorganic mercury value, the hair organic mercury value was the major variable for the cerebrum and kidney (both cortex and medulla), the hair inorganic mercury value was the major variable for the cerebellum and heart, and the hair phosphorous and hair organic mercury were the major variables for the liver; no explanatory variable existed for the spleen. Auxiliary explanatory variables accounted for the organ total mercury and inorganic mercury levels, among which the hair selenium value was conspicuous with negative regression coefficients.

Adolescent↗

PubMed2004

Field capture of northern and western corn rootworm beetles relative to attractant structure and volatility.

We used field assays to study attraction of feral northern and western corn rootworm beetles (Diabrotica barberi and D. virgifera virgifera) to a series of mostly nitrogenous and benzenoid synthetic compounds allied with host plant and floral aromas. Vaporization rates were obtained for most field-tested compounds and selected additional lures under both ideal and field-representative, but constant, conditions. Although many test compounds showed at least trace activity for one or both species, methyl benzoate and some of its derivatives, notably methyl anthranilate and methyl 4-methoxybenzoate, merited emphasis as effective new lures for females. Structural alteration of methyl benzoate had consistently negative effects on northern corn rootworm captures despite variable effects on release rate, whereas western corn rootworm was more strongly attracted to methyl anthranilate and methyl 4-methoxybenzoate than to the considerably more volatile parent compound. Phenylacetaldoxime was attractive to females of both species, but no more so than syn-benzaldoxime, included as reference. Release rate was disproportionately low for benzaldoxime, as well as other nitrogenous lures, under field compared with ideal conditions. The attractiveness of salicylaldoxime to northern corn rootworm, despite its low field release rate, and the unattractiveness of methyl salicylate, having a methyl ester in place of the oxime group, similarly highlighted importance of the oxime moiety for reactivity of this species.

Animals↗

PubMed2004

Nickel oxidation states of F(430) cofactor in methyl-coenzyme M reductase.

Magnetic circular dichroism (MCD) spectroscopy and variable-temperature variable-field MCD are used in combination with density functional theory (DFT) and time-dependent DFT (TD-DFT) calculations to characterize the so-called ox1-silent, red1, and ox1 forms of the Ni-containing cofactor F430 in methyl-coenzyme M reductase (MCR). Previous studies concluded that the ox1 state, which is the precursor of the key reactive red1 state of MCR, is a Ni(I) species that derives from one-electron reduction of the Ni(II)-containing ox1-silent state. However, our absorption and MCD data provide compelling evidence that ox1 is actually a Ni(II) species. In support of this proposal, our DFT and TD-DFT calculations indicate that addition of an electron to the ox1-silent state leads to formation of a hydrocorphin anion radical rather than a Ni(I) center. These results and biochemical evidence suggest that ox1 is more oxidized than red1, which prompted us to test a new model for ox1 in which the ox1-silent species is oxidized by one electron to form a thiyl radical derived from coenzyme M that couples antiferromagnetically to the Ni(II) ion. This alternative ox1 model, formally corresponding to a Ni(III)/thiolate resonance form but with predicted S = 1/2 EPR parameters reminiscent of a Ni(I) (3dx2-y2)1 species, rationalizes the requirement for reduction of ox1 to yield the red1 species and the seemingly incongruent EPR and electronic spectra of the ox1 state.

Archaea↗

PubMed2006

Methyl group metabolism gene polymorphisms as modifier of breast cancer risk in Italian BRCA1/2 carriers.

BRCA1 and 2 are major cancer susceptibility genes but their penetrance is highly variable. The folate metabolism plays an important role in DNA methylation and its alterated metabolism is associated with cancer risk. The role of allele variants 677T and 1298C (MTHFR gene) and 2756G (MS gene) has been investigated as potentially modifying factors of BRCA gene penetrance, evaluated as age at first diagnosis of cancer, in 484 BRCA1/BRCA2 carriers and in 108 sporadic breast cancer cases as a control group. The genotype analysis has been performed by means of PCR/RFLP's. The analysis of association between a particular genotype and disease risk was performed using Cox Regression with time to breast or ovarian cancer onset as the end-point. The presence of 677T allele confers an increased risk of breast cancer in BRCA1 carriers (P = 0.007) and the presence of 1298C allele confers an increased risk of breast cancer in sporadic cases (P = 0.015).

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1997

Abnormal c-myc oncogene DNA methylation in human bladder cancer: possible role in tumor progression.

OBJECTIVE: It has been suggested that the hypermethylation of normally unmethylated DNA sequences plays a critical role in the genesis and progression of human tumors. Although the molecular bases of this mechanism have not been completely explained, the altered methylation pattern of the c-myc oncogene is supposed to represent an important step in tumor development. METHODS: We have analyzed tissue samples from 47 urinary bladder tumors (43 primary transitional and 4 squamous cell carcinomas) and the respective blood with HpaII methyl-sensitive endonuclease digestion and the Southern blotting technique to detect the methylation pattern in a widespread area in and around the c-myc oncogene. RESULTS: Data presented in this study showed significant differences between the c-myc methylation pattern and pathological grade (p < 0.05). On the other hand, we did not find a significant correlation between the c-myc methylation pattern and clinical stage. However, a variable covalent alteration of c-myc DNA existed in bladder cancer as compared to normal tissue. CONCLUSION: Although the correlation between superficial and infiltrating forms was not statistically significant, we did, however, find differences in aggressive neoplastic behavior. This suggested that local hypermethylation may be considered as one potential mechanism for increasing genetic alterations in bladder cancer formation.

Aged↗

PubMed2005

DNMT3B mutations and DNA methylation defect define two types of ICF syndrome.

ICF syndrome is a rare autosomal recessive disease characterized by variable immunodeficiency, centromeric instability, and facial abnormalities. Mutations in the catalytic domain of DNMT3B, a gene encoding a de novo DNA methyltransferase, have been recognized in a subset of patients. ICF syndrome is a genetic disease directly related to a genomic methylation defect that mainly affects classical satellites 2 and 3, both components of constitutive heterochromatin. The variable incidence of DNMT3B mutations and the differential methylation defect of alpha satellites allow the identification of two types of patients, both showing an undermethylation of classical satellite DNA. This classification illustrates the specificity of the methylation process and raises questions about the genetic heterogeneity of the ICF syndrome.

Centromere↗

PubMed1990

Intestinal absorption of menhaden and rapeseed oils and their fatty acid methyl and ethyl esters in the rat.

The relative cellular uptake and incorporation into prechylomicrons and chylomicrons was investigated for the menhaden and rapeseed oil fatty acids, when given by stomach tube as the original oils or the corresponding methyl and ethyl esters. The intermediates and final products of cellular acylation were determined by chromatographic methods at various times over a period of 1-24 h. There was little selectivity in the uptake among the oligo- and poly-unsaturated fatty acids of menhaden oil, when either oil or esters were fed. In contrast, the long-chain saturated and monounsaturated fatty acids of rapeseed oil were discriminated against during both cellular uptake and reacylation (60% overall reduction in utilization). Also, there was detectable discrimination against the long-chain polyunsaturated monoacylglycerols of menhaden oil and against the long-chain saturated and monounsatured monoacylglycerols of rapeseed oil during both cellular uptake and reacylation (30% overall reduction in utilization). Evidence was obtained for an indiscriminate cellular uptake of variable amounts (4-22%) of intact dietary methyl and ethyl esters of fatty acids, which, however, appeared in the chylomicrons only to a very limited extent (0.1-1.0% of total lipid). During peak absorption the cellular and lymphatic appearance of fatty acids from the digestion and absorption of the alkyl esters was nearly 50% lower than that from the corresponding triacylglycerols. The slower absorption of the fatty acids from the alkyl ester feeding is hypothetically attributed to a lower efficiency of the phosphatidic acid pathway, which is required in the absence of dietary 2-monoacylglycerols, but other mechanisms cannot be excluded.

Animals↗

PubMed1977

Temperature-sensitive variants for saturation density and anchorage dependencey of a simian virus 40-transformed human X mouse hybrid cell line.

Several variant clones temperature sensitive for some parameters of transformation from a hybrid cell line containing a stable diploid mouse genome and two human chromosomes 7 carrying an integrated defective simian virus 40 have been isolated. Like the wild-type (wt) cells, at the permissive temperature the temperature-sensitive (ts) clones grew to high saturation densities and readily formed colonies in methyl cellulose. In contrast to the wt cells, at the nonpermissive temperature they had variable but always lower saturation densities and were unable to form colonies in methyl cellulose. The fraction of cells that synthesized DNA decreased at both temperatures when the cells reached saturation density, but it represented always at least 20% of the total population. All of the ts clones so far tested had a near triploid chromosome number, contained from one to three human chromosomes 7, and were T-antigen-positive both at the permissive and nonpermissive temperatures. The ts clones maintained low saturation density at the nonpermissive temperature because of a decrease in the number of cells that were actively proliferating and because of the shedding of cells into the medium. Temperature downshifts and refeeding allowed for expression of the permissive phenotype. In most of the isolated clones anchorage independency was not correlated with unrestricted cell proliferation. These variant clones may provide useful systems for a better understanding of the number of interdependent pathways involved in the expression of the phenotype of a transformed cell and elucidate the molecular mechanism required for the maintenance of a normal state in a cell population.

Antigens, Viral↗

PubMed1996

Arsenic methylation patterns before and after changing from high to lower concentrations of arsenic in drinking water.

Inorganic arsenic (In-As), an occupational and environmental human carcinogen, undergoes biomethylation to monomethylarsonate (MMA) and dimethylarsinate (DMA). It has been proposed that saturation of methylation capacity at high exposure levels may lead to a threshold for the carcinogenicity of In-As. The relative distribution of urinary In-As, MMA, and DMA is used as a measure of human methylation capacity. The most common pathway for elevated environmental exposure to In-As worldwide is through drinking water. We conducted a biomarker study in northern Chile of a population chronically exposed to water naturally contaminated with high arsenic content (600 micrograms/l). In this paper we present the results of a prospective follow-up of 73 exposed individuals, who were provided with water of lower arsenic content (45 micrograms/l) for 2 months. The proportions of In-As, MMA, and DMA in urine were compared before and after intervention, and the effect of other factors on the distribution of arsenic metabolites was also analyzed. The findings of this study indicate that the decrease in arsenic exposure was associated with a small decrease in the percent In-As in urine (from 17.8% to 14.6%) and in the MMA/DMA ratio (from 0.23 to 0.18). Other factors such as smoking, gender, age, years of residence, and ethnicity were associated mainly with changes in the MMA/DMA ratio, with smoking having the strongest effect. Nevertheless, the factors investigated accounted for only about 20% of the large interindividual variability observed. Genetic polymorphisms in As-methylating enzymes and other co-factors are likely to contribute to some of the unexplained variation. The changes observed in the percent In-As and in the MMA/DMA ratio do not support an exposure-based threshold for arsenic methylation in humans.

Adult↗

PubMed1978

Inhibition of morphological transformation induced with N-methyl-N'-nitro-N-nitrosoguanidine in cultures of hamster embryo cells by 5'-bromo-2'-deoxyuridine-photolysis.

The present study was performed in order to determine whether type III transformed foci induced by N-methyl-N'-nitro-N-nitrosoguanidine originate from the small subpopulation of cells stimulated by the carcinogen to enter DNA synthesis. During the last 30 min of variable treatment periods using different doses of N-methyl-N'-nitro-N-nitrosoguanidine, administered alone or in association with the thymidine analogue, 5'-bromo-2'-deoxyuridine (0.98 x 10(-5)M), the density-inhibited monolayers of hamster embryo cells were exposed to fluorescent light and then assayed for abnormal growth patterns by the focus formation method. Mock-irradiated cultures as well as monolayers whose medium lacked N-methyl-N'-nitro-N-nitro-soguanidine, 5'-bromo-2'-deoxyuridine, or both, served as controls. The cytotoxicity of 5'-bromo-2'- deoxyuridine + N-methyl-N'-nitro-N-nitrosoguanidine + photolysis (BMP) protocol on confluent as well as logarithmically growing hamster embryo cells was estimated in single-cell survival experiments. Plating efficiency determinations have demonstrated that, unlike their actively growing counterparts, confluent hamster embryo cell monolayers are extremely resistant to the cytotoxic effects of the BMP protocol. The quantitative transformation assays indicated that: (1) in non-illuminated cultures addition of 5'-bromo-2'-deoxyuridine to carcinogen-containing medium does affect transformation frequency of hamster embryo cells in the sense that the incidence of type III foci did not subside at later intervals during the post-carcinogen administration period as it did in the absence of the analogue; (2) irradiation of N-methyl-N'-nitro-N-nitrosoguanidine and halogenated pyrimidine analogue-treated cultures with fluorescent light practically suppressed transformation; (3) analogue-added and analogue-removed experiments pointed out that the event(s) on which 5'-bromo-2'-deoxyuridine fluorescent light sensitization of morphological transformation largely depends, takes place between 5 and 15 h after N-methyl-N'-nitro-N-nitrosoguanidine administration, i.e., during the period of maximal carcinogen-stimulated DNA synthesis; and (4) neither fluorescent light nor 5'-bromo-2'-deoxyuridine, singly or in combination, were able to transform cultures of hamster embryo cells. These findings are strong indirect arguments for the concept that carcinogen-induced DNA synthesis and the initiation of transformed clones are causally related.

Animals↗

PubMed2001

Decreased expression of catenins (alpha and beta), p120 CTN, and E-cadherin cell adhesion proteins and E-cadherin gene promoter methylation in prostatic adenocarcinomas.

BACKGROUND: Catenin/E-cadherin complex proteins play an important role in cell-cell adhesion with decreased expression correlating with adverse prognostic variables in several human malignancies. METHODS: Archival formalin fixed, paraffin embedded (FFPE) sections from 118 prostatic adenocarcinomas (PACs) were immunostained by an automated method (Ventana Medical Systems, Tuscon, AZ) using monoclonal antibodies to catenins alpha and beta, p120 CTN, and E-cadherin proteins. Immunoreactivity was semiquantitatively graded, and results correlated with traditional prognostic parameters. In a subset of 10 randomly selected cases, E-cadherin gene promoter methylation status was determined on FFPE tissues using sodium bisulfite/hydroquinone DNA modification and polymerase chain reaction (PCR) with methylation specific primers (CpG wiz E-cadherin methylation assay; Intergen Co., Purchase, NY). RESULTS: Decreased expression of alpha-catenin (17%), beta catenin (4%), p120 CTN (45%), and E-cadherin (25%) proteins was noted in PACs with downregulation of each protein correlating with high tumor grade (P = 0.01-0.0001). In addition, p120 CTN and E-cadherin expression levels correlated with pathologic stage (P = 0.05; P = 0.02), aneuploidy (P = 0.001; P = 0.0001), and alpha-catenin with aneuploidy (P = 0.0001). p120 CTN loss also correlated with preoperative serum prostate specific antigen (P = 0.05). Two of 10 cases featured no evidence of E-cadherin gene promoter methylation by PCR and both cases retained expression of E-cadherin protein on immunohistochemistry. Of the 8 cases that showed E-cadherin methylation, 5 (68%) featured loss of expression of the protein on immunohistochemistry (P = 0.11). There was no correlation between E-cadherin methylation and adverse prognostic variables. CONCLUSIONS: Decreased expression of catenin/E-cadherin complex cell adhesion proteins is associated with aggressive phenotype in prostatic adenocarcinoma. E-cadherin gene promote methylation is a common event in prostate carcinoma but does not appear to bear prognostic significance in the subset of cases analyzed.

Adenocarcinoma↗