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D Bergstrom

Publications and source records attributed to D Bergstrom.

11 recordsLinked to original sources
PubMed1999

Nucleotide analogs facilitate base conversion with 3' mismatch primers.

We compared the efficiency of PCR amplification using primers containing either a nucleotide analog or a mismatch at the 3' base. To determine the distribution of bases inserted opposite eight different analogs, 3' analog primers were used to amplify four different templates. The products from the reactions with the highest amplification efficiency were sequenced. Analogs allowing efficient amplification followed by insertion of a new base at that position are herein termed 'convertides'. The three convertides with the highest amplification efficiency were used to convert sequences containing C, T, G and A bases into products containing the respective three remaining bases. Nine templates were used to generate conversion products, as well as non-conversion control products with no base change. We compared the ability of natural bases to convert specific sites with and without a preconversion step using nucleotide analog primers. Conversion products were identified by a ligation detection reaction using primers specific for the converted sequence. We found that conversions resulting in transitions were easier to accomplish than transversions and that sequence context influences conversion. Specifically, primer slippage appears to be an important mechanism for producing artifacts via polymerase extension of a 3' base or analog transiently base paired to neighboring bases of the template. Nucleotide analogs could often reduce conversion artifacts and increase the yield of the expected product. While new analogs are needed to reliably achieve transversions, the current set have proven effective for creating transition conversions.

Base Pair Mismatch↗
PubMed1999

Nucleotide analogs and new buffers improve a generalized method to enrich for low abundance mutations.

A high sensitivity method for detecting low level mutations is under development. A PCR reaction is performed in which a restriction site is introduced in wild-type DNA by alteration of specific bases. Digestion of wild-type DNA by the cognate restriction endonuclease (RE) enriches for products with mutations within the recognition site. After reamplification, mutations are identified by a ligation detection reaction (LDR). This PCR/RE/LDR assay was initially used to detect PCR error in known wild-type samples. PCR error was measured in low |Deltap K a| buffers containing tricine, EPPS and citrate, as well as otherwise identical buffers containing Tris. PCR conditions were optimized to minimize PCR error using perfect match primers at the Msp I site in the p53 tumor suppressor gene at codon 248. However, since mutations do not always occur within pre-existing restriction sites, a generalized PCR/RE/LDR method requires the introduction of a new restriction site. In principle, PCR with mismatch primers can alter specific bases in a sequence and generate a new restriction site. However, extension from 3' mismatch primers may generate misextension products. We tested conversion of the Msp I (CCGG) site to a Taq I site (TCGA). Conversion was unsuccessful using a natural base T mismatch primer set. Conversion was successful when modified primers containing the 6 H,8 H -3, 4-dihydropyrimido[4,5- c ][1,2]oxazine-7-one (Q6) base at 3'-ends were used in three cycles of preconversion PCR prior to conversion PCR using the 3' natural base T primers. The ability of the pyrimidine analog Q6 to access both a T-like and C-like tautomer appears to greatly facilitate the conversion.

Base Sequence↗
PubMed1996

NeuroD2 and neuroD3: distinct expression patterns and transcriptional activation potentials within the neuroD gene family.

We have identified two new genes, neuroD2 and neuroD3, on the basis of their similarity to the neurogenic basic-helix-loop-helix (bHLH) gene neuroD. The predicted amino acid sequence of neuroD2 shows a high degree of homology to neuroD and MATH-2/NEX-1 in the bHLH region, whereas neuroD3 is a more distantly related family member. neuroD3 is expressed transiently during embryonic development, with the highest levels of expression between days 10 and 12. neuroD2 is initially expressed at embryonic day 11, with persistent expression in the adult nervous system. In situ and Northern (RNA) analyses demonstrate that different regions of the adult nervous system have different relative amounts of neuroD and neuroD2 RNA. Similar to neuroD, expression of neuroD2 in developing Xenopus laevis embryos results in ectopic neurogenesis, indicating that neuroD2 mediates neuronal differentiation. Transfection of vectors expressing neuroD and neuroD2 into P19 cells shows that both can activate expression through simple E-box-driven reporter constructs and can activate a reporter driven by the neuroD2 promoter region, but the GAP-43 promoter is preferentially activated by neuroD2. The noncongruent expression pattern and target gene specificity of these highly related neurogenic bHLH proteins make them candidates for conferring specific aspects of the neuronal phenotype.

Adult↗
PubMed1989

Clinical management using direct and derived parameters.

In the past, left heart function was estimated from the level of CVP, clinical information and chest x-ray; cardiac output was estimated from mixed venous oxygen saturation or content. These indirect assessments frequently led to incorrect conclusions and irrationale therapy. Currently, direct and derived hemodynamic parameters have been invaluable in the diagnosis, management, and prognosis in critically ill patients. Continuous mixed venous oxygen saturation monitoring affords yet another valuable parameter to facilitate optimal patient care outcomes.

Adolescent↗
PubMed1986

In vitro and in vivo studies of a promising antileukemic thymidine analogue, 5-hydroxymethyl-2' deoxyuridine.

The toxicity and metabolism of a thymidine analogue, 5-hydroxymethyl-2'-deoxyuridine (5HmdUrd) were studied with human leukemia cells (HL-60) and with human platelets. 3 X 10(-5) M 5HmdUrd caused a 50% inhibition in the proliferation of HL-60 cells. The compound was hydrolyzed to 5-hydroxymethyluracil (5HmUra) by the enzyme thymidine phosphorylase (EC 2.4.2.4) present in leukemia cells; this catabolic product was non-toxic. The catabolism of 5HmdUrd by human platelet thymidine phosphorylase could be inhibited by 6-aminothymine. The toxicity of 5HmdUrd was effectively reversed by deoxycytidine and 5HmdUrd increased the incorporation of deoxycytidine into dCTP and DNA several fold. The two latter phenomena are explicable in terms of a feedback action to ribonucleotide reductase, resulting in deoxycytidylate starvation, which is a known effect of excess thymidine. We report here also our preliminary observations that 5HmdUrd is active against mouse leukemia in vivo.

Animals↗
PubMed1985

5-Ethyl-2'-deoxyuridine. Cytotoxicity and DNA incorporation demonstrated with human leukemic cells and PHA-stimulated lymphocytes in vitro.

5-Ethyl-2'-deoxyuridine (5EtdUrd) is a biologically active thymidine analogue. The cytotoxicity of 5EtdUrd was investigated with seven established human leukemia cell lines as well as with human peripheral blood PHA-stimulated lymphocytes. All types of leukemia cells were susceptible to the toxicity of 5EtdUrd as assayed with a [U-14C]-L-leucine incorporation system developed for this study. A 50% inhibition of leucine incorporation in 3-day cultures was induced by 1.3-3.8 microM 5EtdUrd with leukemic cells, but the concentration required to induce similar inhibition with PHA-stimulated lymphocytes was approximately was approximately 100-fold. The toxicity of 5EtdUrd seemed to require active DNA synthesis, since the inhibition of leucine incorporation became obvious only after the first 24 hours of culture. The DNA incorporation studies were based on a new isotopically labeled 5EtdUrd derivative, [2-14C]5EtdUrd, synthesized for this study in our laboratory. It was demonstrated for the first time that most of the radioactivity derived from [2-14C]5EtdUrd in DNA was in 5-ethyluracil. 5EtdUrd has a powerful antileukemic potency in vitro. Its effects against human leukemia in vivo remain to be tested.

Burkitt Lymphoma↗
PubMed1981

Mutagens in feces from vegetarians and non-vegetarians.

Mutagens in water extracts from feces of persons in 3 different diet groups were measured with the fluctuation test for weak mutagens using Salmonella typhimurium TA100 and TA98 as tester strains. The 3 diet groups were ovo-lacto vegetarians (N = 6), strict vegetarians (N = 11) and non-vegetarians (N = 12). All subjects were from the urban area of Vancouver, British Columbia, Canada. On TA100 ovo-lacto vegetarians and strict vegetarians had significantly lower levels of fecal mutagens than non-vegetarians (P less than or equal to 0.025 and P less than 0.010, resp.). The same pattern, although less significant, was obtained with TA98. Correlation studies between mutagenicity on TA100 and TA98 and between the pH of the fecal homogenate and mutagenicity indicate the presence of 2 or more major fecal mutagens.

Diet, Vegetarian↗