Mechanism of cellular uptake of modified oligodeoxynucleotides containing methylphosphonate linkages.

The cellular uptake and intracellular distribution of methylphosphonate oligonucleotides (15 mers) has been examined using both 32P labeled and fluorescent labeled oligonucleotides. The cellular uptake process for methylphosphonate oligonucleotides is highly temperature dependent, with a major increase in uptake occurring between 15 and 20 degrees C. Most of the label which becomes cell associated at 37 degrees C cannot be removed by acid washing or trypsinization and thus seems to be within the cell. Visualization of rhodamine labeled methylphosphonate oligonucleotides using digital imaging fluorescence microscopy reveals a vesicular subcellular distribution suggestive of an endosomal localization. There was extensive co-localization of rhodamine labeled methylphosphonate oligonucleotides with fluorescein-dextran, an endosomal/lysosomal marker substance. The apparent endocytotic uptake of labeled methylphosphonate oligonucleotides could not be blocked by competition with unlabeled methylphosphonate or phosphodiester oligonucleotides, nor by ATP. This contrasts with the situation for radiolabeled phosphodiester oligonucleotides whose uptake can be completely blocked with unlabeled competitor. Uptake of phosphodiester oligonucleotides, but not of methylphosphonate oligonucleotides, could be blocked by acidification of the cytosol. These observations suggest that the pathway of cellular uptake of methylphosphonate oligonucleotides involves fluid phase or adsorbtive endocytosis, and is distinct from the uptake pathway for phosphodiester oligonucleotides.     

Infectivity of adeno-associated virus serotypes in mouse testis

Background

Recombinant adeno-associated viruses (AAVs) are emerging as favoured transgene delivery vectors for both research applications and gene therapy. In this context, a thorough investigation of the potential of various AAV serotypes to transduce specific cell types is valuable. Here, we rigorously tested the infectivity of a number of AAV serotypes in murine testis by direct testicular injection.

Results

We report the tropism of serotypes AAV2, 5, 8, 9 and AAVrh10 in mouse testis. We reveal unique infectivity of AAV2 and AAV9, which preferentially target intertubular testosterone-producing Leydig cells. Remarkably, AAV2 TM, a mutant for capsid designed to increase transduction, displayed a dramatic alteration in tropism; it infiltrated seminiferous tubules unlike wildtype AAV2 and transduced Sertoli cells. However, none of the AAVs tested infected spermatogonial cells.

Conclusions

In spite of direct testicular injection, none of the tested AAVs appeared to infect sperm progenitors as assayed by reporter expression. This lends support to the current view that AAVs are safe gene-therapy vehicles. However, testing the presence of rAAV genomic DNA in germ cells is necessary to assess the risk of individual serotypes.

     

Cloning and Characterization of the Crystal Protein-Encoding Gene of Bacillus thuringiensis subsp. yunnanensis

Molecular cloning and characterization of a novel cry gene, cry32Aa, of Bacillus thuringiensis subsp. yunnanensis was carried out. The Cry32Aa protein was predicted to have a molecular mass of 139.2 kDa and was found to have an unusual 42-amino-acid-long tail at the C terminus. The cry32Aa gene was localized on the 103-MDa plasmid of the organism. Bioassays showed no toxicity against several moths and mosquitoes. However, it exhibited weak toxicity against larvae of the diamondback moth, Plutella xylostella.     

Analysis of Fluorescence Decay by the Nonlinear Least Squares Method

Fluorescence decay deconvolution analysis to fit a multiexponential function by the nonlinear least squares method requires numerical calculation of a convolution integral. A linear approximation of the successive data of the instrument response function is proposed for the computation of the convolution integral. Deconvolution analysis of simulated fluorescence data were carried out to show that the linear approximation method is generally better when one of the lifetimes is comparable to the time interval between data.     

Analysis of sarcoplasmic reticulum Ca2+ transport and Ca2+ ATPase enzymatic properties using mouse cardiac tissue homogenates

Recent studies have focused on developing transgenic mouse models to explore the physiological roles of sarcoplasmic reticulum (SR) calcium handling proteins. The goal of this study was to develop methodology to measure SR Ca2+ transport function and enzymatic properties of SR Ca2+ ATPase (SERCA) in individual mouse hearts. We describe here the procedures to specifically measure SR Ca2+ uptake, the formation and decomposition of SERCA phosphoenzyme intermediate (E-P) in mouse cardiac homogenates. The specificity of SERCA enzymatic activity in cardiac homogenates was established by (a) the selective inhibition of SERCA enzyme by inhibitor-thapsigargin, and (b) comparison of the kinetic parameters of SERCA activity between homogenates and isolated microsomes. Here we show that the apparent affinity of SERCA for Ca2+ and ATP, the time to reach steady-state levels of E-P, and the rate of E-P decomposition (turnover rate of SERCA enzyme) are similar in homogenates and microsomes. These studies demonstrate that SERCA Ca2+ transport and enzymatic properties can be accurately measured in mouse cardiac tissue homogenates. Additionally, we show that frozen cardiac homogenates can be used without significant loss of enzymatic activity. In conclusion, we have developed and established the methods to employ tissue homogenates to study SR Ca2+ transport function in individual mouse hearts.     

Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy

Advances in molecular biology provide various methods to define the structure and function of the individual proteins that form the component parts of subcellular structures. The ability to see the dynamic behavior of a specific protein inside the living cell became possible through the application of advanced fluorescence resonance energy transfer (FRET) microscope techniques. The fluorophore molecule used for FRET imaging has a characteristic absorption and emission spectrum that should be considered for characterizing the FRET signal. In this article we describe the system development for the image acquisition for one- and two-photon excitation FRET microscopy. We also describe the precision FRET (PFRET) data analysis algorithm that we developed to remove spectral bleed-through and variation in the fluorophore expression level (or concentration) for the donor and acceptor molecules. The acquired images have been processed using a PFRET algorithm to calculate the energy transfer efficiency and the distance between donor and acceptor molecules. We implemented the software correction to study the organization of the apical endosome in epithelial polarized MDCK cells and dimerization of the CAATT/enhancer binding protein alpha (C/EBPalpha). For these proteins, the results revealed that the extent of correction affects the conventionally calculated energy transfer efficiency (E) and the distance (r) between donor and acceptor molecules by 38 and 9%, respectively.     

Characterization of a phosphoprotein phosphatase activity in ribonucleoprotein particles from HeLa cell nuclei.

Ribonucleoprotein particles containing heterogenous nuclear RNA (hnRNP) have a phosphoprotein phosphatase activity. This activity is optimal at pH 7.5, inhibited by divalent cations and by increasing ionic strength above 200 mM NaCl, stimulated by 2-mercaptoethanol and inhibited by N-ethylmaleimide. It is clearly distinct from non specific alkaline phosphatase and resembles the phosphoprotein phosphatase present in Novikoff hepatoma nucleoli (Olson et al. B.B.R.C. (1976), 70, 717–721). This enzyme may be involved in regulating the phosphorylation level of hnRNP proteins in combination with the protein kinase previously described (Blanchard et al. Eur. J. Biochem. (1977) in press).     

Relative growth before and after sexual maturity in Ocypode platytarsis (Milne Edwards) and Ocypode cordimana (Desmarest) (Crustacea: Decapoda)

The relative growth of two species of the genus Ocypode inhabiting different ecological niches was studied before and after sexual maturity. The growth coefficient of both sexes of O. cordimana, which inhabits supra-littoral zones, is higher than that of O. platytarsis found in wave-wash zones.s

Some characteristics such as eyestalk length, major chela width and obdomen width do not show any difference in the growth pattern of either species of Ocypode at the onset of sexual maturity. Other characteristics such as minor and major chela length in male O. platytarsis; carapace length, minor chela length and width in female O. platytarsis; major chela length and third walking leg length in male O. cordimana showed a difference in the growth pattern after the onset of sexual maturity.

Major chela length of males of both species is clearly a sexually dimorphic feature since it showed an increase in the growth coefficient after sexual maturity. Merus length in male O. platytarsis and minor chela length in female O. cordimana also showed such sexual dimorphism.

There are several characteristics in which the growth coefficient declines after sexual maturity. The change in growth coefficient of different characteristics may depend on the adaptive value of the characteristic in reproductive or post-reproductive activities of O. cordimana and O. platytarsis.