Pseudotyped recombinant adeno-associated viral vectors mediate efficient gene transfer into primary human CD34+ peripheral blood progenitor cells

Background and aimsBecause of their pluripotency, human CD34⁺ peripheral blood progenitor cells (PBPC) are targets of interest for the treatment of many acquired and inherited disorders using gene therapeutic approaches. Unfortunately, most current vector systems lack either sufficient transduction efficiency or an appropriate safety profile. Standard single-stranded recombinant adeno-associated virus 2 (AAV2)-based vectors offer an advantageous safety profile, yet lack the required efficiency in human PBPC.MethodsA panel of pseudotyped AAV vectors (designated AAV2/x, containing the vector genome of serotype 2 and capsid of serotype x, AAV2/1–AAV2/6) was screened on primary human granulocyte–colony-stimulating factor (G-CSF)-mobilized CD34⁺ PBPC to determine their gene transfer efficacy. Additionally, double-stranded self-complementary AAV (dsAAV) were used to determine possible second-strand synthesis limitations.ResultsAAV2/6 vectors proved to be the most efficient [12.8% (1.8–25.4%) transgene-expressing PBPC after a single transduction], being significantly more efficient (all P < 0.005) than the other vectors [AAV2/2, 2.0% (0.2–7.3%); AAV2/1, 1.3% (0.1–2.9%); others, <; 1% transgene-expressing PBPC]. In addition, the relevance of the single-to-double-strand conversion block in transduction of human PBPC could be shown using pseudotyped dsAAV vectors: for dsAAV2/2 [9.3% (8.3–20.3%); P < 0.001] and dsAAV2/6 [37.7% (23.6–61.0%); P < 0.001) significantly more PBPC expressed the transgene compared with their single-stranded counterparts; for dsAAV2/1, no significant increase could be observed.ConclusionsWe have shown that clinically relevant transduction efficiency levels using AAV-based vectors in human CD34⁺ PBPC are feasible, thereby offering an efficient alternative vector system for gene transfer into this important target cell population.     

Temperature acclimation in a biochemical model of photosynthesis: a reanalysis of data from 36 species

The Farquhar et al. model of C(3) photosynthesis is frequently used to study the effect of global changes on the biosphere. Its two main parameters representing photosynthetic capacity, V(cmax) and J(max), have been observed to acclimate to plant growth temperature for single species, but a general formulation has never been derived. Here, we present a reanalysis of data from 36 plant species to quantify the temperature dependence of V(cmax) and J(max) with a focus on plant growth temperature, i.e. the plants' average ambient temperature during the preceding month. The temperature dependence of V(cmax) and J(max) within each data set was described very well by a modified Arrhenius function that accounts for a decrease of V(cmax) and J(max) at high temperatures. Three parameters were optimized: base rate, activation energy and entropy term. An effect of plant growth temperature on base rate and activation energy could not be observed, but it significantly affected the entropy term. This caused the optimum temperature of V(cmax) and J(max) to increase by 0.44 degrees C and 0.33 degrees C per 1 degrees C increase of growth temperature. While the base rate of V(cmax) and J(max) seemed not to be affected, the ratio J(max) : V(cmax) at 25 degrees C significantly decreased with increasing growth temperature. This moderate temperature acclimation is sufficient to double-modelled photosynthesis at 40 degrees C, if plants are grown at 25 degrees C instead of 17 degrees C.     

Dependence of the Q10 values on the depth of the soil temperature measuring point

The parameter Q₁₀ is commonly used to express the relationship between soil CO₂ efflux and soil temperature. One advantage of this parameter is its application in a model expression of respiration losses of different ecosystems. Correct specification of Q₁₀ in these models is indispensable. Soil surface CO₂ efflux and soil temperature at different depths were measured in a 21-year-old Norway spruce stand and a mountain grassland site located at the Experimental Ecological Study Site Bily Kriz, Beskydy Mts. (NE Czech Republic), using automated gasometric systems. A time-delay and goodness-of-fit between soil CO₂ efflux and soil temperature at different measuring depths were determined. Wide ranges of values for the time-delay of CO₂ efflux in response to temperature, Q₁₀ and the determination coefficient (R²) between CO₂ efflux and temperature were obtained at the both sites. The values of Q₁₀ and the CO₂ time-delay increased with depth, while the R² of the CO₂-temperature relationship significantly decreased. Soil temperature records obtained close to the soil surface showed the highest values of R² and the lowest value of the time-delay at both sites. Measurement of soil temperature at very shallow soil layer, preferably at the soil surface, is highly recommended to determine useable values of Q₁₀. We present a new procedure to normalize Q₁₀ values for soil temperatures measured at different depths that would facilitate comparison of different sites.     

Exendin-4, but not dipeptidyl peptidase IV inhibition, increases small intestinal mass in GK rats

Long-term treatment with dipeptidyl peptidase IV inhibitors (DPPIV-I) or glucagon-like peptide (GLP)-1 analogs may potentially affect intestinal growth by down- or upregulating the intestinotrophic hormone GLP-2. This study compared the intestinotrophic effects of 12-wk administration of vehicle, exendin-4 (Ex-4; 5 nmol/kg bid sc), or DPPIV-I (NN-7201, 10 mg/kg qd orally) in GK rats. Some animals were observed additionally for 9 wk after the end of treatment. Both treatments lowered glycated hemoglobin A1c at wk 12 vs. control (Ex-4, -0.8%; DPPIV-I, -0.4%). Body weight was reduced by Ex-4 compared with control (361 +/- 4 vs. 399 +/- 5 g; P < 0.001) because of reduced food intake, whereas neither parameter was affected by DPPIV-I. Linear bone growth was unaffected by either treatment. After treatment end, food intake in Ex-4 animals increased, and, by wk 21, body weight was identical in all groups. The small intestine of Ex-4-treated animals was larger at wk 12 compared with control (length, 135.6 +/- 1.6 vs. 124.5 +/- 2.3 cm, P < 0.001; absolute weight, 8.4 +/- 0.2 vs. 6.4 +/- 0.4 g, P < 0.001), being most pronounced proximally, where the absolute cross-sectional area related to body weight increased by 24% because of increased mucosal thickness. These effects were reversible, and 9 wk after the end of treatment, no differences between Ex-4 and control were apparent. Plasma GLP-2 concentrations were unaltered by either treatment, and Ex-4 had no agonistic or antagonistic effects on the transfected GLP-2 receptor. DPPIV-I had no intestinal effects. In conclusion, the continued presence of Ex-4 is necessary to maintain weight loss in GK rats. Effective antihyperglycemic treatment with Ex-4 increases intestinal mass reversibly, whereas DPPIV-I lacks intestinal effects.     

Interactions of Ly49 family receptors with MHC class I ligands in trans and cis

The Ly49A NK cell receptor interacts with MHC class I (MHC-I) molecules on target cells and negatively regulates NK cell-mediated target cell lysis. We have recently shown that the MHC-I ligand-binding capacity of the Ly49A NK cell receptor is controlled by the NK cells' own MHC-I. To see whether this property was unique to Ly49A, we have investigated the binding of soluble MHC-I multimers to the Ly49 family receptors expressed in MHC-I-deficient and -sufficient C57BL/6 mice. In this study, we confirm the binding of classical MHC-I to the inhibitory Ly49A, C and I receptors, and demonstrate that detectable MHC-I binding to MHC-I-deficient NK cells is exclusively mediated by these three receptors. We did not detect significant multimer binding to stably transfected or NK cell-expressed Ly49D, E, F, G, and H receptors. Yet, we identified the more distantly related Ly49B and Ly49Q, which are not expressed by NK cells, as two novel MHC-I receptors in mice. Furthermore, we show using MHC-I-sufficient mice that the NK cells' own MHC-I significantly masks the Ly49A and Ly49C, but not the Ly49I receptor. Nevertheless, Ly49I was partly masked on transfected tumor cells, suggesting that the structure of Ly49I is compatible in principal with cis binding of MHC-I. Finally, masking of Ly49Q by cis MHC-I was minor, whereas masking of Ly49B was not detected. These data significantly extend the MHC-I specificity of Ly49 family receptors and show that the accessibility of most, but not all, MHC-I-binding Ly49 receptors is modulated by the expression of MHC-I in cis.     

Expression level and activity profile of membrane bound guanylate cyclase type 2 in rod outer segments

Rod and cone cells of the mammalian retina harbor two types of a membrane bound guanylate cyclase (GC), rod outer segment guanylate cyclase type 1 (ROS-GC1) and ROS-GC2. Both enzymes are regulated by small Ca²⁺-binding proteins named GC-activating proteins that operate as Ca²⁺ sensors and enable cyclases to respond to changes of intracellular Ca²⁺after illumination. We determined the expression level of ROS-GC2 in bovine ROS preparations and compared it with the level of ROS-GC1 in ROSs. The molar ratio of a ROS-GC2 dimer to rhodopsin was 1 : 13 200. The amount of ROS-GC1 was 25-fold higher than the amount of ROS-GC2. Heterologously expressed ROS-GC2 was differentially activated by GC-activating protein 1 and 2 at low free Ca²⁺ concentrations. Mutants of GC-activating protein 2 modulated ROS-GC2 in a manner different from their action on ROS-GC1 indicating that the Ca²⁺ sensitivity of the Ca²⁺ sensor is controlled by the mode of target–sensor interaction.     

Structures of Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase provide new insights into catalysis

Isopentenyl diphosphate is the precursor of various isoprenoids that are essential to all living organisms. It is produced by the mevalonate pathway in humans but by an alternate route in plants, protozoa, and many bacteria. 1-deoxy-D-xylulose-5-phosphate reductoisomerase catalyzes the second step of this non-mevalonate pathway, which involves an NADPH-dependent rearrangement and reduction of 1-deoxy-D-xylulose 5-phosphate to form 2-C-methyl-D-erythritol 4-phosphate. The use of different pathways, combined with the reported essentiality of the enzyme makes the reductoisomerase a highly promising target for drug design. Here we present several high resolution structures of the Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase, representing both wild type and mutant enzyme in various complexes with Mn(2+), NADPH, and the known inhibitor fosmidomycin. The asymmetric unit corresponds to the biological homodimer. Although crystal contacts stabilize an open active site in the B molecule, the A molecule displays a closed conformation, with some differences depending on the ligands bound. An inhibition study with fosmidomycin resulted in an estimated IC(50) value of 80 nm. The double mutant enzyme (D151N/E222Q) has lost its ability to bind the metal and, thereby, also its activity. Our structural information complemented with molecular dynamics simulations and free energy calculations provides the framework for the design of new inhibitors and gives new insights into the reaction mechanism. The conformation of fosmidomycin bound to the metal ion is different from that reported in a previously published structure and indicates that a rearrangement of the intermediate is not required during catalysis.