Adeno-associated virus of a single-polarity DNA genome is capable of transduction in vivo.

The adeno-associated virus (AAV) is a promising vector for gene therapy. Further improvement of the virus for clinical application depends on better understanding of the molecular structure and fate of the vector genome. AAV vectors with wild-type inverted terminal repeats package either the plus- or the minus-strand DNA genomes with equal frequency. By creating a series of deletions within the, we have developed a genetic approach that can generate an AAV vector that packages its single-stranded DNA genome predominantly in a single polarity (99.4%). This novel reagent efficiently transduced muscle, brain and liver in whole animals. The transduction efficiencies were similar to those of the control mixed-polarity vectors. Our results showed that reannealing of plus- and minus-strand DNA was not required for AAV-mediated transduction in vivo, supporting the hypothesis that second-strand DNA synthesis is a primary pathway in converting the single-stranded AAV genome into double-stranded forms. The availability of the single-polarity AAV vector would aid further studies on the mechanism of AAV transduction as well as the application of AAV vector for gene replacement therapy.

[Study on the relationship between some genetic factors and peak bone mineral density in Beijing young women].

OBJECTIVE: To evaluate the relationship between the peak bone mineral density (PBMD) and vitamin D receptor (VDR), estrogen receptor (ER) allelic variants in Beijing young women. METHODS: From March, 2000 to July, 2001, one hundred and fifty-nine young healthy women (25 - 37 years old) in Beijing were voluntarily enrolled in the study. (1) BMD were measured by dual energy X-ray absorptiometry (DXEA) at lumbar and hip. (2) The polymorphism of VDR and ER genes were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). (3) The relationship between BMD and polymorphism of VDR and ER genes were examined. RESULTS: (1) Lumber BMD was positively correlated to height, weight and body mass index (BMI), whereas, the femoral neck BMD only to weight, and the other sites of hip BMD to BMI. (2) Although subjects with the VDR bb genotype had higher BMD than those with Bb genotype at lumber, femoral neck, inter and troch, no significant difference was found (P > 0.05). (3) In Ward triangle, subjects with ER PP genotype had significantly lower BMD than those in ER Pp and pp genotypes (P < 0.05). (4) Women with BbPP genotype combination had lower BMD levels at lumber and hip, and with bbPP and Bbpp genotypes combination significantly higher lumber BMD levels than BbPP genotype (P < 0.05). However, the differences of BMD among subjects with different VDR and ER genotypes became not significant after adjusting the confounder of body weight. CONCLUSIONS: (1) Body weight and BMI play important roles to PBMD of Beijing women. (2) There was no significant difference of BMD levels between VDR genotypes at any site. (3) PvuII polymorphism of ER gene was associated with low Ward triangle BMD. (4) There was significant relationship between the combination of ER and VDR polymorphisms at lumbar and hip BMD. Our data suggest that genetic variation at the ER locus, singly and in relation to the VDR locus, may influence the attainment and maintenance of peak bone mass in young women.

Molecular mechanisms involved in adenosine-induced endothelial cell barrier enhancement.

Extracellular adenosine is a physiologically relevant agonist released by various sources, including endothelial cells (EC) and activated platelets, with complex effects mediated via activation of P1 purinergic receptors. Adenosine-induced EC production of glutathione peroxidase1 and nitric oxide is recognized, and an anti-inflammatory mechanism has been described. Effects of extracellular adenosine on the pulmonary EC barrier function and vascular permeability, however, remain poorly characterized. In this study, we demonstrated the adenosine-induced rapid dose-dependent barrier enhancement in human pulmonary artery EC (HPAEC) as measured by an increase in transendothelial electrical resistance (TER). We have shown that HPAEC express only A2A and A2B adenosine receptors. Pharmacological and siRNA depletion studies indicate that A2A, but not A2B receptor activation is required for the adenosine-induced TER increase. Depletion of Galphas with a specific siRNA significantly attenuated the adenosine-induced TER response in HPAEC. In contrast, depletion of either Galphaq or Galphai2 did not affect the adenosine-induced TER increase. This suggests that the adenosine-induced TER increase is cAMP-dependent. The adenosine-induced barrier enhancement effects were associated with a rearrangement of the EC F-actin component of the cytoskeleton, enhanced cell-surface presentation of cell-cell junctional protein VE-cadherin and an involvement of Myosin-light-chain phosphatase (MLCP). Our results suggest, for the first time, that adenosine regulates the EC barrier function via A2A receptors followed by Galphas engagement and is associated with cytoskeletal activation.

[Characterization of bubble size distribution in ES-DAF unit].

ES-DAF unit was introduced and studied in this paper. Without a costly air saturator, ES-DAF consists of an ejector and a static mixer between the pressure side and suction side of the recycle rotary pump. The bubble size distribution in this novel unit was studied by using a CCD imagination through a microscope. The bubble size decreased with the increase of cycle ratio or the decrease of superficial air-water ratio. These results suggest that smaller bubbles would be formed when the initial number of nucleation sites increase by enhancing the turbulence intensity in the saturation system. The bubble size distribution became lower and wider with the increase of saturation pressure because of more frequent bubble collision and coalescence.

Truncation of herpes simplex virus type 2 glycoprotein B increases its cell surface expression and activity in cell-cell fusion, but these properties are unrelated.

Formation of small polykaryons by cell-cell fusion is characteristic of herpes simplex virus (HSV) lesions, but the great majority of viruses isolated from such lesions produce only limited cell fusion in tissue culture. Because of this, HSV laboratory strains that produce extensive cell fusion (syncytium formation) in culture are regarded as variants or mutants. Furthermore, the rarity of clinical isolates able to produce syncytia in culture suggests that extensive cell fusion is deleterious in vivo. Mutations that confer a syncytial phenotype can then be regarded as bypassing a mechanism that normally limits cell fusion. Determination of how these mutations, some of which are in the cytoplasmic tail of glycoprotein B (gB), lead to syncytium formation will likely reveal how fusion is controlled. Here we show the following. (i) Truncation of the cytoplasmic tail of HSV type 2 gB (gB-2) by a minimum of 25 residues or a maximum of 49 residues produces a syncytial phenotype. (ii) Truncation by 20 to 49 residues increases cell fusion when gB-2 is coexpressed with only gD-2, gH-2, and gL-2. (iii) Truncation by 25 or more residues removes a potential endocytosis motif and increases gB-2 cell surface expression. (iv) Mutation of this motif increases gB-2 cell surface expression but does not increase fusogenic activity, whereas mutation of another potential endocytosis motif does not increase surface expression but does increase fusogenic activity. Therefore, syncytial mutations in the cytoplasmic tail of gB-2 do not act by increasing cell surface levels of the protein.