Bone marrow cell therapy after acute myocardial infarction: the HEBE trial in perspective, first results

During the last decennium, the role of bone marrow mononuclear cells (BMMC) has been underscored in the healing process after acute myocardial infarction (AMI). Although these cells improve left ventricular recovery after AMI in experimental studies, results from large-scale randomised trials investigating BMMC therapy in patients with AMI have shown contradictory results. To address this issue the HEBE study was designed, a multicentre, randomised trial, evaluating the effects of intracoronary infusion of BMMCs and the effects of intracoronary infusion of peripheral blood mononuclear cells after primary percutaneous coronary intervention. The primary endpoint of the HEBE trial is the change in regional myocardial function in dysfunctional segments at four months relative to baseline, based on segmental analysis as measured by magnetic resonance imaging. The results from the HEBE trial will provide detailed information about the effects of intracoronary BMMC therapy on post-infarct left ventricular recovery. In addition, further analysis of the data and material obtained may provide important mechanistic insights into the contribution of BMMCs to natural recovery from AMI as well as the response to cell therapy. This may significantly contribute to the development of improved cell-based therapies, aiming at optimising post-infarct recovery and preventing heart failure. (Neth Heart J 2008;16:436-9.)     

Population Shift Underlies Ca2+-induced Regulatory Transitions in the Sodium-Calcium Exchanger (NCX)

In eukaryotic Na⁺/Ca²⁺ exchangers (NCX) the Ca²⁺ binding CBD1 and CBD2 domains form a two-domain regulatory tandem (CBD12). An allosteric Ca²⁺ sensor (Ca3–Ca4 sites) is located on CBD1, whereas CBD2 contains a splice-variant segment. Recently, a Ca²⁺-driven interdomain switch has been described, albeit how it couples Ca²⁺ binding with signal propagation remains unclear. To resolve the dynamic features of Ca²⁺-induced conformational transitions we analyze here distinct splice variants and mutants of isolated CBD12 at varying temperatures by using small angle x-ray scattering (SAXS) and equilibrium ⁴⁵Ca²⁺ binding assays. The ensemble optimization method SAXS analysis demonstrates that the apo and Mg²⁺-bound forms of CBD12 are highly flexible, whereas Ca²⁺ binding to the Ca3–Ca4 sites results in a population shift of conformational landscape to more rigidified states. Population shift occurs even under conditions in which no effect of Ca²⁺ is observed on the globally derived D_max (maximal interatomic distance), although under comparable conditions a normal [Ca²⁺]-dependent allosteric regulation occurs. Low affinity sites (Ca1–Ca2) of CBD1 do not contribute to Ca²⁺-induced population shift, but the occupancy of these sites by 1 mm Mg²⁺ shifts the Ca²⁺ affinity (K_d) at the neighboring Ca3–Ca4 sites from ∼ 50 nm to ∼ 200 nm and thus, keeps the primary Ca²⁺ sensor (Ca3–Ca4 sites) within a physiological range. Thus, Ca²⁺ binding to the Ca3–Ca4 sites results in a population shift, where more constraint conformational states become highly populated at dynamic equilibrium in the absence of global conformational transitions in CBD alignment.     

Selective inhibition of thymidine transport at low doses of the alkylating agents triethyleneiminobenzoquinone (Trenimon)

The alkylating antitumor agent triethyleneiminobenzoquinone (Trenimon) causes a rapid decrease in the incorporation of labeled thymidine into the DNA of Yoshida or Ehrlich ascites tumor cells. The effect is expressed 4 h after administration of 6 × 10⁻⁸ moles/kg of the drug to mice bearing Yoshida ascites tumors or of 6 × 10⁻⁷ moles/kg to Ehrlich ascites tumor-bearing animals, respectively. The reduced incorporation of labeled thymidine which is observed under these conditions is not due to an inhibition of DNA synthesis. DNA synthesis was measured by an isotope dilution assay after pulse-labeling with ³H-thymidine and by monitoring the increase in the total amount of DNA of the cell populations. The data demonstrate that DNA synthesis is not affected during the first 8 h after exposure to the drug. This conclusion is supported by cell kinetic measurements which indicate that the alkylating agent does not interfere with the progression of cells into the S phase, but exerts a block at the G 2 stage of the cell cycle. The reduced incorporation of thymidine into DNA is explained by a decreased transport of the nucleoside into the cells.     

Import of a new chloroplast inner envelope protein is greatly stimulated by potassium phosphate

A cDNA clone encoding a major chloroplast inner envelope membrane protein of 96 kDa (IEP96) was isolated and characterized. The protein is synthesized as a larger-molecular-weight precursor (pIEP96) which contains a cleavable N-terminal transit sequence of 50 amino acids. The transit peptide exhibits typical stromal targeting information. It is cleaved in vitro by the stromal processing peptidase, though the mature protein is clearly localized in the inner envelope membrane. Translocation of pIEP96 into chloroplasts is greatly stimulated in the presence of 80 mM potassium phosphate which results in an import efficiency of about 90%. This effect is specific for potassium and phosphate, but cannot be ascribed to a membrane potential across the inner envelope membrane. Protein sequence analysis reveals five stretches of repeats of 26 amino acids in length. The N-terminal 300 amino acids are 45% identical (76% similarity) to the 35 kDa -subunit of acetyl-CoA carboxyl-transferase from Escherichia coli. The C-terminal 500 amino acids share significant similarity (69%) with USOI, a component of the cytoskeleton in yeast.Abbreviations P_i phosphate - IEP inner envelope membrane protein - pIEP precursor form of IEP - SSU small subunit of ribulose-1,5-bisphosphate carboxylase oxygenase - IEP96_pep peptide specific antiserum to IEP96 - IEP96_pol polyspecific antiserum to IEP96     

Indirect trophic interactions with an invasive species affect phenotypic divergence in a top consumer

While phenotypic responses to direct species interactions are well studied, we know little about the consequences of indirect interactions for phenotypic divergence. In this study we used lakes with and without the zebra mussel to investigate effects of indirect trophic interactions on phenotypic divergence between littoral and pelagic perch. We found a greater phenotypic divergence between littoral and pelagic individuals in lakes with zebra mussels and propose a mussel-mediated increase in pelagic and benthic resource availability as a major factor underlying this divergence. Lakes with zebra mussels contained higher densities of large plankton taxa and large invertebrates. We suggest that this augmented resource availability improved perch foraging opportunities in both the littoral and pelagic zones. Perch in both habitats could hence express a more specialized foraging morphology, leading to an increased divergence of perch forms in lakes with zebra mussels. As perch do not prey on mussels directly, we conclude that the increased divergence results from indirect interactions with the mussels. Our results hence suggest that species at lower food web levels can indirectly affect phenotypic divergence in species at the top of the food chain.     

Synthesis of a series of fully methylated aldobiouronic acids,and β-elimination reactions of their synthetic precursors

Treatment of methyl 2,3,4-tri-O-acetyl-l-bromo-l-deoxy-α-d-glucopyranuronate severally with 2,4,6-, 2,3,6-, and 2,3,4-tri-O-methyl derivatives of methyl α-d-glucopyranoside and with methyl 4,6-O-benzylidene-3-O-methyl-α-d-glucopyranoside, in the presence of silver carbonate, afforded crystalline aldobiouronic acid derivatives in high yield. Deacetylation followed by methylation gave a series of fully methylated derivatives of laminaribiouronic, cellobiouronic, and gentiobiouronic acids, and the (1 → 2)-linked analogue. Methylation with methyl iodide and silver oxide in N,N-dimethylformamide was invariably accompanied by a small amount ofβ-elimination, with the formation of olefinic disaccharides which were also obtained by β-elimination reactions of the precursor acetates followed by methylation. Methyl 4,5-unsaturated 4-deoxyhexopyranosyluronate derivatives were the main products of the reaction, but these underwent further degradation with cleavage of the interglycosidic linkage and formation of 6-methoxycarbonyl-4-pyrone.     

Raman-based dynamic feeding strategies using real-time glucose concentration monitoring system during adalimumab producing CHO cell cultivation

The use of Process Analytical Technology tools coupled with chemometrics has been shown great potential for better understanding and control of mammalian cell cultivations through real-time process monitoring. In-line Raman spectroscopy was utilized to determine the glucose concentration of the complex bioreactor culture medium ensuring real-time information for our process control system. This work demonstrates a simple and fast method to achieve a robust partial least squares calibration model under laboratory conditions in an early phase of the development utilizing shake flask and bioreactor cultures. Two types of dynamic feeding strategies were accomplished where the multi-component feed medium additions were controlled manually and automatically based on the Raman monitored glucose concentration. The impact of these dynamic feedings was also investigated and compared to the traditional bolus feeding strategy on cellular metabolism, cell growth, productivity, and binding activity of the antibody product. Both manual and automated dynamic feeding strategies were successfully applied to maintain the glucose concentration within a narrower and lower concentration range. Thus, besides glucose, the glutamate was also limited at low level leading to reduced production of inhibitory metabolites, such as lactate and ammonia. Consequently, these feeding control strategies enabled to provide beneficial cultivation environment for the cells. In both experiments, higher cell growth and prolonged viable cell cultivation were achieved which in turn led to increased antibody product concentration compared to the reference bolus feeding cultivation.     

Genetic and Morphometric Analysis of Cob Architecture and Biomass-Related Traits in the Intermated B73 × Mo17 Recombinant Inbred Lines of Maize

Expected future cellulosic ethanol production increases the demand for biomass in the US Corn Belt. With low nutritious value, low nitrogen content, and compact biomass, maize cobs can provide a significant amount of cellulosic materials. The value of maize cobs depends on cob architecture, chemical composition, and their relation to grain yield as primary trait. Eight traits including cob volume, fractional diameters, length, weight, tissue density, and grain yield have been analyzed in this quantitative trait locus (QTL) mapping experiment to evaluate their inheritance and inter-relations. One hundred eighty-four recombinant inbred lines of the intermated B73?×?Mo17 (IBM) Syn 4 population were evaluated from an experiment carried out at three locations and analyzed using genotypic information of 1,339 public SNP markers. QTL detection was performed using (1) comparison-wise thresholds with reselection of cofactors (α?=?0.001) and (2) empirical logarithm of odds score thresholds (P?=?0.05). Several QTL with small genetic effects (R ²?=?2.9–13.4 %) were found, suggesting a complex quantitative inheritance of all traits. Increased cob tissue density was found to add value to the residual without a commensurate negative impact on grain yield and therefore enables for simultaneous selection for cob biomass and grain yield.