An isothermal primer extension method for whole genome amplification of fresh and degraded DNA: applications in comparative genomic hybridization, genotyping and mutation screening

We describe a protocol that uses a bioinformatically optimized primer in an isothermal whole genome amplification (WGA) reaction. Overnight incubation at 37 degrees C efficiently generates several hundred- to several thousand-fold increases in input DNA. The amplified product retains reasonably faithful quantitative representation of unamplified whole genomic DNA (gDNA). We provide protocols for applying this isothermal primer extension WGA protocol in three different techniques of genomic analysis: comparative genomic hybridization (CGH), genotyping at simple tandem repeat (STR) loci and screening for single base mutations in a common monogenic disorder, beta-thalassemia. gDNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues can also be amplified with this protocol.     

Ex vivo hypothermic recirculatory adenoviral gene transfer to the transplanted pig heart

BACKGROUND: To facilitate the application of adenoviral gene therapy in clinical heart transplantation, we developed an ex vivo hypothermic recirculatory adenoviral gene transfer method to the transplanted pig heart. METHODS: Experimental animals were assigned into three groups; controls, 1x10(8) plaque-forming units (pfu)/ml group and 1x10(9) pfu/ml group. During the 30 min gene transfer perfusion, 200 ml of University of Wisconsin solution containing the adenoviral vector was recirculated through the coronary vessels. The myocardial temperature was maintained below 4 degrees C and the perfusion pressure was adjusted at 50 mmHg. RESULTS: Cardiac myocyte transduction efficiencies in the 1x10(8) pfu/ml group were 0.04% and 0.07%, whereas transduction efficiencies in the 1x10(9) pfu/ml group were widely distributed from 0.45% to 22.62%. The gene transduction efficiency increased with the virus titer. Additionally, no difference in the transduction efficiency was observed between different segments of the left ventricle. The current gene transfer method at 1x10(9) pfu/ml of adenovirus titer enabled homogeneous gene transduction into the transplanted pig heart up to a maximum of 22.62%. CONCLUSIONS: This model can be applied to a large isolated heart and will greatly facilitate the investigation of gene therapy in large animal models of heart transplantation.     

An isothermal method for whole genome amplification of fresh and degraded DNA for comparative genomic hybridization, genotyping and mutation detection.

Molecular genotyping has important biomedical and forensic applications. However, limiting amounts of human biological material often yield genomic DNA (gDNA) in insufficient quantity and of poor quality for a reliable analysis. This motivated the development of an efficient whole genome amplification method with quantitatively unbiased representation usable on fresh and degraded gDNA. Amplification of fresh frozen, formalin-fixed paraffin-embedded (FFPE) and DNase-degraded DNA using degenerate oligonucleotide-primed PCR or primer extension amplification using a short primer sequence bioinformatically optimized for coverage of the human genome was compared with amplification using current primers by chromosome-based and BAC-array comparative genomic hybridization (CGH), genotyping at short tandem repeats (STRs) and single base mutation detection. Compared with current primers, genome amplification using the bioinformatically optimized primer was significantly less biased on CGH in self-self hybridizations, and replicated tumour genome copy number aberrations, even from FFPE tissue. STR genotyping could be performed on degraded gDNA amplified using our technique but failed with multiple displacement amplification. Of the 18 different single base mutations 16 (89.5%) were correctly identified by sequencing gDNA amplified from clinical samples using our technique. This simple and efficient isothermal method should be helpful for genetic research and clinical and forensic applications.     

Interaction between the N-terminal and middle regions is essential for the in vivo function of HSP90 molecular chaperone

At the primary structure level, the 90-kDa heat shock protein (HSP90) is composed of three regions: the N-terminal (Met(1)-Arg(400)), middle (Glu(401)-Lys(615)), and C-terminal (Asp(621)-Asp(732)) regions. In the present study, we investigated potential subregion structures of these three regions and their roles. Limited proteolysis revealed that the N-terminal region could be split into two fragments carrying residues Met(1) to Lys(281) (or Lys(283)) and Glu(282) (or Tyr(284)) to Arg(400). The former is known to carry the ATP-binding domain. The fragments carrying the N-terminal two-thirds (Glu(401)-Lys(546)) and C-terminal one-third of the middle region were sufficient for the interactions with the N- and C-terminal regions, respectively. Yeast HSC82 that carried point mutations in the middle region causing deficient binding to the N-terminal region could not support the growth of HSP82-depleted cells at an elevated temperature. Taken together, our data show that the N-terminal and middle regions of the HSP90 family protein are structurally divided into two respective subregions. Moreover, the interaction between the N-terminal and middle regions is essential for the in vivo function of HSP90 in yeast.     

Acoustic detection of termite infestations in urban trees

A portable, low-frequency acoustic system was used to detect termite infestations in urban trees. The likelihood of infestation was rated independently by a computer program and an experienced listener that distinguished insect sounds from background noises. Because soil is a good insulator, termite sounds could be detected easily underneath infested trees, despite the presence of high urban background noise. Termite sounds could be detected also in trunks, but background noise often made it difficult to identify termite signals unambiguously. High likelihoods of termite infestation were predicted at four live oak (Quercus virginiana Mill, Fagacae), two loblolly pine (Pinus taeda L., Pinacae), and two baldcypress (Taxodium distichum Rich. Pinacae) trees that wood-baited traps had identified as infested with Coptotermes formosanus Shiraki. Infestations also were predicted at two pine trees with confirmed recoveries of Reticulitermes flavipes (Kollar). Low likelihoods of infestation were predicted in four oak trees where no termites were found. Additional tests were conducted in anechoic environments to determine the range of acoustic detectability and the feasibility of acoustically estimating termite population levels. There was a significant regression between the activity rate and the number of termites present in a wood trap block, with a minimum detectable number of approximately 50 workers per liter of wood. The success of these field tests suggests that currently available acoustic systems have considerable potential to detect and monitor hidden infestations of termites in urban trees and around building perimeters in addition to their present uses to detect and monitor termite infestations in buildings.     

Purification and characterization of trans-permethrin metabolizing microsomal esterases from workers of the eastern subterranean termite, Reticulitermes flavipes (Kollar)

Three alpha-naphthyl acetate hydrolyzing esterase isozymes were purified from microsomes prepared from Reticulitermes flavipes workers. The two step process involved sequential preparative IEF followed by continuous elution preparative electrophoresis on a 5% non-denaturing polyacrylamide gel. The first IEF run resulted in 5.4-fold purification with a yield of 46.1%. Subsequent IEF further purified the esterases 14.3-fold and 12% yield. Preparative electrophoresis of the pooled IEF fractions produced three major peaks of alpha-naphthyl acetate hydrolyzing activity. The esterases were correspondingly designated microsomal esterase (ME) 1, ME 2, and ME 3 based on increasing molecular retention on a native PAGE gel. ME 1, ME 2, and ME 3 were acidic proteins with pI values of 4.61, 4.70, and 4.77, respectively. Molecular mass as determined by gel filtration chromatography of ME 1, ME 2, and ME 3 was 69, 64, and 62 kDa, respectively. SDS-PAGE gels produced a single band for each of the isozymes with a molecular mass of 63 kDa indicating that the esterases were monomers. Specific activities of ME 1, ME 2, and ME 3 increased with increasing pH and the enzymes were active over a broad temperature range (25-55 degrees C). The three purified isozymes were inhibited at low concentration by paraoxon (10(-10) M), chlorpyrifos (10(-6) M), DEF (10(-6) M), and PMSF (10(-6) M) indicating that they were "B" type serine esterases. Conversely, inhibition was not observed at 10(-4) M eserine, PHMB, or CaCl(2), further supporting the conclusion that the microsomal esterases were of the "B" type. None of the isozymes was inhibited by 10(-4) M imidacloprid, fipronil, or PBO. Quantitatively, ME 1, ME 2 and ME 3 metabolized t-permethrin at 21.8, 21.0, and 38.8 nmol/h/mg protein, representing a purification factor of 333-, 318-, and 591-fold over microsomes, respectively. The three isozymes produced the same type and number of t-permethrin metabolites.     

Purification and characterization of 31-kDa palm pollen glycoprotein (Ela g Bd 31 K), which is recognized by IgE from palm pollinosis patients

A basic glycoprotein, which was recognized by IgE from oil palm pollinosis patients, has been purified from oil palm pollen (Elaeis guineensis Jacq.), which is a strong allergen and causes severe pollinosis in Malaysia and Singapore. Soluble proteins were extracted from defatted palm pollen with both Tris-HCl buffer (pH 7.8) and Na-acetate buffer (pH 4.0). The allergenic glycoprotein was purified from the total extract to homogeneity with 0.4% yield by a combination of DEAE- and CM-cellulose, SP-HPLC, and gel filtration. The purified oil palm pollen glycoprotein with molecular mass of 31 kDa was recognized by the beta1-2 xylose specific antibody, suggesting this basic glycoprotein bears plant complex type N-glycan(s). The palm pollen basic glycoprotein, designated Ela g Bd 31 K, was recognized by IgE of palm pollinosis patients, suggesting Ela g Bd 31 K should be one of the palm pollen allergens. The preliminary structural analysis of N-glycans linked to glycoproteins of palm pollens showed that the antigenic N-glycans having alpha1-3 fucose and alpha1-2 xylose residues (GlcNAc(2 to approximately 0)Man3Xyl1Fuc(1 to approximately 0)GlcNAc2) actually occur on the palm pollen glycoproteins, in addition to the high-mannose type structures (Man(9 to approximately 5)GlcNAc2).     

Comparison of Toxoplasma gondii Seroprevalence in Shelter Cats and Dogs during 1999–2001 and 2009–2011 in Tokyo,Japan

Toxoplasma gondii is an important human health concern with respect to abortion, congenital hydrocephalus, and encephalitis in immunocompromised people. Cats and dogs both are potential sources of T. gondii because they have close contact with humans. However, no epidemiological surveys have been conducted in Tokyo over the past decade. Therefore, the present study investigated and compared the seroprevalence of T. gondii infection in shelter cats and dogs during 1999–2001 and 2009–2011 in Tokyo, Japan. Serum samples were collected from 337 shelter cats and 325 shelter dogs in urban and suburban areas of Tokyo, during 1999–2001 (233 cats and 219 dogs) and 2009–2011 (104 cats and 106 dogs). T. gondii antibodies were measured in the serum samples using a commercial latex agglutination test. Data were compared using the Fisher’s exact test, and significance was indicated at P < 0.05. The overall seroprevalence of T. gondii infection in cats was 5.6% (13 of 233) in 1999–2001 and 6.7% (7 of 104) in 2009–2011, and that in dogs was 1.8% (4 of 219) and 1.9% (2 of 106), respectively. Significantly higher seroprevalence was observed in cats from suburban areas compared with cats in urban areas during both periods (P < 0.05). These results reveal that there has been little change in the feline and canine seroprevalence over the past decade, indicating that the risk of T. gondii exposure for cats and dogs in Tokyo is considerably low as the seroprevalence has reached a steady state.     

A Chrysin Derivative Suppresses Skin Cancer Growth by Inhibiting Cyclin-dependent Kinases

Chrysin (5,7-dihydroxyflavone), a natural flavonoid widely distributed in plants, reportedly has chemopreventive properties against various cancers. However, the anticancer activity of chrysin observed in in vivo studies has been disappointing. Here, we report that a chrysin derivative, referred to as compound 69407, more strongly inhibited EGF-induced neoplastic transformation of JB6 P⁺ cells compared with chrysin. It attenuated cell cycle progression of EGF-stimulated cells at the G₁ phase and inhibited the G₁/S transition. It caused loss of retinoblastoma phosphorylation at both Ser-795 and Ser-807/811, the preferred sites phosphorylated by Cdk4/6 and Cdk2, respectively. It also suppressed anchorage-dependent and -independent growth of A431 human epidermoid carcinoma cells. Compound 69407 reduced tumor growth in the A431 mouse xenograft model and retinoblastoma phosphorylation at Ser-795 and Ser-807/811. Immunoprecipitation kinase assay results showed that compound 69407 attenuated endogenous Cdk4 and Cdk2 kinase activities in EGF-stimulated JB6 P⁺ cells. Pulldown and in vitro kinase assay results indicated that compound 69407 directly binds with Cdk2 and Cdk4 in an ATP-independent manner and inhibited their kinase activities. A binding model between compound 69407 and a crystal structure of Cdk2 predicted that compound 69407 was located inside the Cdk2 allosteric binding site. The binding was further verified by a point mutation binding assay. Overall results indicated that compound 69407 is an ATP-noncompetitive cyclin-dependent kinase inhibitor with anti-tumor effects, which acts by binding inside the Cdk2 allosteric pocket. This study provides new insights for creating a general pharmacophore model to design and develop novel ATP-noncompetitive agents with chemopreventive or chemotherapeutic potency.