DNA typing forHLA-DPB1*02 and−DPB1*04 in multiple sclerosis and juvenile rheumatoid arthritis

DP gene typing using in vitro DNA amplification combined with sequence-specific oligonucleotide probes (SSOP) has recently been reported. The amplification step may be specific for theHLA-DPB locus, or it may be specific for one or a group ofHLA-DPB alleles, thus increasing the discriminatory power of the system. We report the combined use of group-specific DNA in vitro amplification followed by SSOP in typing forDPB1*02 andDPB1*04 variants. The method was used to type for these variants in 96 randomly selected, healthy Danes, in 37 patients with pauciarticular juvenile rheumatoid arthritis (PJRA); and in 38 patients with multiple sclerosis (MS). Increased frequencies of the cellularly defined HLA-DPw2 in PJRA and of HLA-DPw4 in MS have previously been reported. In the patient groups, the frequencies of theDPB1*02 andDPB1*04 variants did not differ significantly from those expected based on the cellularly defined HLA-DP types of the patients and the frequencies of theDPB1*02 andDPB1*04 variants among healthy Danes.     

Detection of β-thalassemia and hemoglobin E genes in Thai by a DNA amplification technique

Summary Enzymatic DNA amplification and polyacrylamide gel electrophoresis, which demonstrate different sizes of DNA fragments, were used to detect the common mutations causing -thalassemia and hemoglobin (Hb) E in Thai people. The 4-bp deletion at codons 41 and 42 can be detected directly by polyacrylamide gel electrophoresis and ethidium bromide staining. Whereas the nonsense mutations at codon 17 (AAG TAG) and Hb E (GAGAAG at codon 26) were detected after digestion of the amplified DNA with the enzymes MaeI and MnlI, respectively.     

Visual detection of Hg²⁺ in aqueous solution using gold nanoparticles and thymine-rich hairpin DNA probes

We report a sensitive method for visual detection of mercury ions (II) (Hg2?) in aqueous solution by using gold nanoparticles (Au-NPs) and thymine (T)-rich hairpin DNA probes. The thiolated hairpin DNA probe was immobilized on the Au-NP surface through a self-assembling method. Another thymine-rich, digoxin-labeled DNA probe was introduced to form DNA duplexes on the Au-NP surface with thymine-Hg2?-thymine (T-Hg2?-T) coordination in the presence of Hg2?. The Au-NPs associated with the formed duplexes were captured on the test zone of a lateral flow strip biocomponent (LFSB) by immunoreaction events between the digoxin on the duplexes and anti-digoxin antibodies on the LFSB. The accumulation of Au-NPs produced a characteristic red band on the test zone, enabling visual detection of Hg2? without instrumentation. A detection limit of 0.1 nM was obtained under optimal experimental conditions. This method provides a simple, rapid, sensitive approach for the detection of Hg2? and shows great promise for point-of-care and in-field detection of environmentally toxic mercury.     

PI (α1) polymorphism in the Japanese: Confirmation of PI*M4 and description of new PI variants

PI phenotyping by separator isoelectric focusing (SIEF) was performed on a total of 1000 unrelated Japanese individuals from two different areas of Western Japan. The PI M1M4 subtype was observed together with the six common PI M subtypes. PI*M4 was confirmed to be present but rare in the Japanese. Several new PI variants were identified by comparison runs of each variant with previously reported genetic variants. The significance of treatment of serum with dithiothreitol (DTT) followed by iodacetic acid (IAC) in determination of PI variants is also described.     

Detection of RASSF1A and RAR? Hypermethylation in Serum DNA from Breast Cancer Patients

Breast cancer is fast emerging as the leading cancer amongst females, especially in young females in metropolitan cities in India. The epigenetic alterations involved in the onset and progression of breast cancer may serve as biomarkers for early detection and prognosis of the disease. Furthermore, using body fluids such as serum offers a non-invasive method to procure multiple samples for such analyses. In this study, we examined methylation status of two normally unmethylated but biologically significant cancer genes, RAS association domain family protein 1A (RASSF1A) and Retionic acid receptor ? (RAR?) by Methylation Specific PCR (MSP) in invasive ductal carcinomas of the breast and paired serum DNA. RASSF1A was found to be methylated in 17 of 20 (85%) breast tumors; while sera from 15 of 20 (75%) of the patients showed concordant methylated RASSF1A, with a sensitivity of 88%. RAR? was methylated in 2/20 (10%) breast tumors. A gene unmethylated in the tumor DNA was always found to be unmethylated in the matched serum DNA for both RASSF1A and RAR? genes; hence specificity was 100%. Immunohistochemical analysis of RAR? protein in 15 breast carcinoma patients harboring unmethylated RAR? in tumors and serum DNA showed the expression of RAR? protein in tumors and paired normal breast tissues, confirming the MSP findings, suggesting that RAR? promoter is functional in these cases. This study underscores the potential utility of DNA methylation based screening of serum, a readily accessible body fluid, as a surrogate marker for early detection of breast cancer.      

Identification and classification of cut-flower “Huangyinghua”using DNA marker techniques

rch Laboratory, Nanjing Agricultural University, Nanjing 210095, China) Abstract “Huangyinghua” is a popular cut-flower in China, but it is unclear as to whether “Huangyinghua” is an invasive Solidago canadensis or not. The genetic relationship of a total of 45 samples of “Huangyinghua” with S. canadensis, and S. decurrens were investigated using AFLP technique so as to determine the identity of “Huangyinghua”. Genomic DNA was digested with EcoRI and MseI enzymes and amplified with six E+3 and M+3 primer combinations. AFLP analysis produced 661 endonucleotide discernable bands, of which 639 (96.61%) were polymorphic. Cluster analysis through using UPGMA method indicated that “Huangyinghua” and S. canadensis were clustered into the same group that was different from S. decurrens. Sequence analysis based on the ITS regions showed that their sequences of 5.8S rDNA were the same, and the differences were found only in the ITS1 and ITS2 regions. ITS phylogenetic trees of the tested samples and closely-related species were reconstructed based on our sequence data in combination with those from GenBank. Based on the trees, “Huangyinghua” was found to belong to S. canadensis complex, but not to S. decurrens. Moreover, it was found that there were considerable genetic variation in both “Huangyinghua” and S. canadensis. Therefore, the cut-flower “Huangyinghua” may be invasive, and proper measures should be taken to control the further spread of its propagules.     

Designing a new biosensor “DNA ELISA” to detect Escherichia coli using genomic DNA and comparison of this method to PCR-ELISA

In this experiment, DNA-ELISA biosensor was introduced, bearing the ability to detect specific bacteria in about 4?h. This is a more rapid system in comparison to conventional methods, like colony counting method. Moreover, this method does not require any amplification and directly detects genomic DNA of bacteria, giving a lower limit to the sensitivity of 40,000 bacteria. In this study, two specific probes capture (biotin labelled) and detector (dig labelled), were used against special regions of 16s rRNA gene of Escherichia coli ATCC 25922. The capture probe has the ability to trap the target bacterial DNA from a pool of other kinds of bacteria under specific conditions. The detector probe then was used to hybridize to the genome of trapped bacteria. The detection proceeds by adding HRP-anti dig enzyme and its substrate, ABTS to emit light. Light absorbance is measured for verifying the detection.     

Commentary on “Prevalence and evolution of drug resistance HIV-1 variants in Henan, China”

Critical to the implementation and scale-up of successful antiretroviral therapy worldwide is an understanding of the efficacy, toxicities and durability of antiretroviral regimens in the specific populations, which they are utilized. In order to obtain this information monitoring and evaluation or operational research must be done in each country or population. The article by Li et al in this issue represents an outstanding example of the importance of data obtained in the populations treated . The authors proactively evaluated the outcomes at 3 and 6 months of 118 and 124 HIV infected individuals, respectively, treated with zidovudine, didanosine and nevirapine in one region in China.     

“Self-tanning”—a new and important source of stoichiometric error in cytophotometric determination of nuclear DNA content in plants

A Feulgen-densitometric comparison of nuclear DNA contents (C-values) was performed in various plant species (a fern, four gymnosperms, 16 woody and herbaceous angiosperms) after two types of fixation, additive (neutral formaldehyde) and non-additive (methanol-acetic acid, 3:1, MAA). Nuclei from tissues containing a significant amount of polyphenols (of the hydrolysable and non-hydrolysable tannin type) always showed reduced stainability and distorted spectral absorbance curves after MAA-fixation, while after formaldehyde-fixation no evidence for distorted staining was found. No fixation-dependent differences in Feulgen-DNA contents were stated in nuclei from tissues having no polyphenols. Distorted Feulgen-staining is a consequence of cellular self-tanning during fixation. Tanning is impaired by formaldehyde which binds to tannins and inactivates them. The rationale for using formaldehyde as a fixative in Feulgen-cytophotometry can be mainly seen in its capability of eliminating the self-tanning error. Standardization in plant DNA cytophotometry, and recent reports on unorthodox nuclear DNA variation in conifers are critically discussed.The author dedicates this paper, with emotions of respect and gratitude, to emer. O. Prof. DrElisabeth Tschermak-Woess on the occasion of the 70th anniversary of her birthday. She guided his Ph.D. Thesis in the years 1968 to 1972.     

Positioning of the Alzheimer Aβ(1–40) peptide in SDS micelles using NMR and paramagnetic probes

NMR spectroscopy combined with paramagnetic relaxation agents was used to study the positioning of the 40-residue Alzheimer Amyloid β-peptide Aβ(1–40) in SDS micelles. 5-Doxyl stearic acid incorporated into the micelle or Mn²⁺ ions in the aqueous solvent were used to determine the position of the peptide relative to the micelle geometry. In SDS solvent, the two α-helices induced in Aβ(1–40), comprising residues 15–24, and 29–35, respectively, are surrounded by flexible unstructured regions. NMR signals from these unstructured regions are strongly attenuated in the presence of Mn²⁺ showing that these regions are positioned mostly outside the micelle. The central helix (residues 15–24) is significantly affected by 5-doxyl stearic acid however somewhat less for residues 16, 20, 22 and 23. This α-helix therefore resides in the SDS headgroup region with the face with residues 16, 20, 22 and 23 directed away from the hydrophobic interior of the micelle. The C-terminal helix is protected both from 5-doxyl stearic acid and Mn²⁺, and should be buried in the hydrophobic interior of the micelle. The SDS micelles were characterized by diffusion and ¹⁵N-relaxation measurements. Comparison of experimentally determined translational diffusion coefficients for SDS and Aβ(1–40) show that the size of SDS micelle is not significantly changed by interaction with Aβ(1–40). Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Increased detectability of somatic changes in the DNA from human tumours after probing with “synthetic” and “genome-derived” hypervariable multilocus probes

Summary DNA fingerprinting with two minisatellite (33.15, M13) and two simple repeat probes [(GACA)₄, (CAC)₄/ (GTG)₅] was performed to screen for somatic changes in the DNA from various solid human tumours in comparison with constitutional DNA from the same patient. Loss of bands or changes in band intensitities were observed. Together the probes 33.15 and (CAC)₅/(GTG)₅ detected deviating fingerprint patterns in 63% of the colorectal carcinomas investigated. In mammary and stomach carcinomas, only 1/11 and 2/11 tumours, respectively, showed differences with either of the three probes, 33.15, (GACA)₄ and (CAC)₅/(GTG)₅.     

Phenomenon of “Siamese embryos” in cereals in vivo and in vitro: Cleavage polyembryony and fasciations

The genesis of wheat microsporial polyembryoids in vitro was analyzed in detail. The nature of different phenotypes of cereal polymeric embryos was identified. They represent the class “multiple shoot meristems,” which results from a cleavage polyembryony and is accompanied by organ fasciations of all known types (radial, flat, or ring). The morphological nature of cereal embryonic organs has been clarified: shoot meristem—axial organ; scutellum—lateral outgrowth of this axis; coleoptile—derivative of shoot meristem but fused with scutellum; terminality of scutellum—the result of linear fasciation that occurred historically. An explanation is given on how the structural model of an auxin polar transport works during the establishment of bilateral symmetry in a cereal embryo that is associated with the inverted polarization of the carrier protein PIN1 on cell membranes and, correspondingly, with the inverted auxin transport performed by this carrier (Fischer-Iglesias et al., 2001; Forestan et al., 2010).     

Turnover of “new” and “old” carbon in soil microbial biomass

The contributions of “new” carbon coming from plants with the C4-type of photosynthesis (maize) and “old” carbon from soil organic matter (SOM) formed under C3 vegetation as carbon sources for microorganisms was determined. Soil samples were taken from the plots of field experiments on Chernozem and Phaeozem. The values of δ¹³C were determined in evolved CO₂, SOM, total microbial biomass (C_mic), and phospholipid fatty acids (PLFA), assuming that the PLFA markers for certain taxonomic groups of microorganisms enriched in C4 carbon indicated a more significant role of these microorganisms in the transformation of root exudates and plant residues. Carbon pools were arranged in the following order by the degree of their enrichment with “new” C: SOM < C_mic < CO₂. Consequently, the “new” carbon proved to be a more preferable substrate for microbial growth than the “old” one. The share of C4 in the markers varied from 18 to 60% (on average 38%) in Phaeozem and from 15 to 40% in Chernozem (on average 28%). The groups of microorganisms in Phaeozem were arranged in the following order by the degree of their enrichment with “new” carbon: protozoa < saprotrophic fungi < actinomycetes < gram-positive bacteria < gramnegative bacteria < mycorrhizal fungi. In Chernozem, the contribution of C4 to the carbon composition of PLFA did not differ significantly for various groups of microorganisms. The C4 content within the PLFA markers of fungi and gram-negative bacteria did not demonstrate any crucial contribution of these groups of organisms to the transformation of “new” C. The long-term C3–C4 transition probably results in formation of a broad range of carbon pools similar in their C4 content but different in resistance to mineralization; therefore, gram-positive bacteria could assimilate C4 from resistant C pools. The low content of “new” carbon in the PLFA markers of fungi may be explained by a considerable portion of dormant forms.     

Detection of designer steroid methylstenbolone in “nutritional supplement” using gas chromatography and tandem mass spectrometry: Elucidation of its urinary metabolites

The use of “nutritional supplements” containing unapproved substances has become a regular practice in amateur and professional athletes. This represents a dangerous habit for their health once no data about toxicological or pharmacological effects of these supplements are available. Most of them are freely commercialized online and any person can buy them without medical surveillance. Usually, the steroids intentionally added to the “nutritional supplements” are testosterone analogues with some structural modifications.In this study, the analyzed product was bought online and a new anabolic steroid known as methylstenbolone (2,17α-dimethyl-17β-hydroxy-5α-androst-1-en-3-one) was detected, as described on label. Generally, anabolic steroids are extensively metabolized, thus in-depth knowledge of their metabolism is mandatory for doping control purposes. For this reason, a human excretion study was carried out with four volunteers after a single oral dose to determine the urinary metabolites of the steroid. Urine samples were submitted to enzymatic hydrolysis of glucuconjugated metabolites followed by liquid–liquid extraction and analysis of the trimethylsilyl derivatives by gas chromatography coupled to tandem mass spectrometry. Mass spectrometric data allowed the proposal of two plausible metabolites: 2,17α-dimethyl-16ξ,17β-dihydroxy-5α-androst-1-en-3-one (S1), 2,17α-dimethyl-3α,16ξ,17β-trihydroxy-5α-androst-1-ene (S2). Their electron impact mass spectra are compatible with 16-hydroxylated steroids O-TMS derivatives presenting diagnostic ions such as m/z 231 and m/z 218. These metabolites were detectable after one week post administration while unchanged methylstenbolone was only detectable in a brief period of 45 h.     

Definition of the binding mode of a new class of phosphoinositide 3-kinase α-selective inhibitors using in vitro mutagenesis of non-conserved amino acids and kinetic analysis

The binding mechanism of a new class of lipid-competitive, ATP non-competitive, p110α isoform-selective PI3K (phosphoinositide 3-kinase) inhibitors has been elucidated. Using the novel technique of isoform reciprocal mutagenesis of non-conserved amino acids in the p110α and p110β isoforms, we have identified three unique binding mechanisms for the p110α-selective inhibitors PIK-75, A-66S and J-32. Each of the inhibitor's p110α-isoform-selective binding was found to be due to interactions with different amino acids within p110. The PIK-75 interaction bound the non-conserved region 2 amino acid p110α Ser(773), A-66S bound the region 1 non-conserved amino acid p110α Gln(859), and J-32 binding had an indirect interaction with Lys(776) and Ile(771). The isoform reciprocal mutagenesis technique is shown to be an important analytical tool for the rational design of isoform-selective inhibitors.     

Structure determination of DNA methylation lesions N1-meA and N3-meC in duplex DNA using a cross-linked protein–DNA system

N¹-meA and N³-meC are cytotoxic DNA base methylation lesions that can accumulate in the genomes of various organisms in the presence of S_N2 type methylating agents. We report here the structural characterization of these base lesions in duplex DNA using a cross-linked protein–DNA crystallization system. The crystal structure of N¹-meA:T pair shows an unambiguous Hoogsteen base pair with a syn conformation adopted by N¹-meA, which exhibits significant changes in the opening, roll and twist angles as compared to the normal A:T base pair. Unlike N¹-meA, N³-meC does not establish any interaction with the opposite G, but remains partially intrahelical. Also, structurally characterized is the N⁶-meA base modification that forms a normal base pair with the opposite T in duplex DNA. Structural characterization of these base methylation modifications provides molecular level information on how they affect the overall structure of duplex DNA. In addition, the base pairs containing N¹-meA or N³-meC do not share any specific characteristic properties except that both lesions create thermodynamically unstable regions in a duplex DNA, a property that may be explored by the repair proteins to locate these lesions.