Effects of electron mediator charge properties on the reaction kinetics of hydrogenase from Chlamydomonas

Anions modulate hydrogenase activity in cell-free preparations of Chlamydomonas reinhardtii, and this modulation is greatly influenced by the charge properties of the redox agent included to mediate electron transfer to hydrogenase. With cationic methyl viologen as the electron mediator, anions stimulate the maximum velocity of H2 production (e.g., a 320% increase in the presence of 1 M NaCl) but have little effect on the Km for methyl viologen. Conversely, when hydrogenase activity is mediated by polyanionic metatungstate or ferredoxin, H2 production is strongly inhibited by anions (e.g., 70-77% inhibition by 0.2 M NaCl). This inhibition is primarily due to a reduced affinity of hydrogenase for these mediators (as evidenced by a large increase in Km values), rather than a change in the maximum velocity of the reaction. Anions have little effect on the kinetics of hydrogenase activity mediated by zwitterionic sulfonatopropyl viologen, a redox agent with a nearly neutral net charge. These results suggest the presence of a cationic region near the active site of hydrogenase. This cationic region, probably due to lysine and/or arginine residues, may serve in vivo to facilitate the interaction between hydrogenase and ferredoxin, the polyanionic, physiological electron mediator.     

Effects of iron deficiency on heme biosynthesis in Rhizobium japonicum.

The effects of iron deficiency on heme biosynthesis in Rhizobium japonicum were examined. Iron-deficient cells had a decreased maximum cell yield and a decreased cytochrome content and excreted protoporphyrin into the growth medium. The activities of the first two enzymes of heme biosynthesis, delta-aminolevulinic acid synthase (EC 2.3.1.37) and delta-aminolevulinic acid dehydrase (EC 4.2.1.24), were diminished in iron-deficient cells, but were returned to normal levels upon addition of iron to the cultures. The addition of iron salts, iron chelators, hemin, or protoporphyrin to cell-free extracts did not affect the activity of these enzymes. The addition of levulinic acid to iron-deficient cultures blocked protoporphyrin excretion and also resulted in high delta-aminolevulinic acid synthase and delta-aminolevulinic acid dehydrase activities. These results suggest the possibility that rhizobial heme biosynthesis in the legume root nodule may be affected by the release of iron from the host plant to the bacteroids.     

A Novel Effect in Phycomyces Phototropism : Positive Bending and Compensation Spectrum in Far UV

A novel effect—positive phototropic bending under far UV irradiation (between 260 and 305 nanometers) at low intensities—is reported. Natural compensation points (intensities which cause no bending under unilateral irradiation) have been determined for different wavelengths. The curve connecting these points, the compensation spectrum, divides the intensity-wavelength plane into areas of negative and positive tropism. It is further shown that a highly asymmetrical pattern of light stimulus within the sporangiophore underlies the symmetrical growth response at each compensation point. This suggests that some unknown additional factor is involved in perceiving a UV stimulus at the level of the photoreceptor. It is also demonstrated here that positive tropism in the UV range is due to a lens effect. We conclude that the hypothesis of optical attenuation of the stimulus (considered until now as the most plausible explanation of negative tropism in the UV spectral range) must be dismissed. The results presented here represent the first application of our quantitative theoretical consideration of spatial factors in phototropism heretofore neglected by others.     

Choline acetyltransferase activity of spinal cord cell cultures increased by co-culture with muscle and by muscle-conditioned medium

Activity of the enzyme choline acetyltransferase (CAT), which mediates the synthesis of the neurotransmitter, acetylcholine, was increased up to 20- fold in spinal cord (SC) cells grown in culture with muscle cells for 2 wk. This increase was directly related to the duration of co-culture as well as to the cell density of both the SC and muscle involved and was not affected by the presence of the acetylcholine receptor blocking agent, α-bungarotoxin. Glutamic acid decarboxylase (GAD) activity was often markedly decreased in SC-muscle cultures while the activities of acetylcholinesterase and several other enzymes were little changed. Increased CAT activity was also observed when SC cultures were maintained in medium which had been conditioned by muscle cells or by undifferentiated cells from embryonic muscle. Muscle-conditioned medium (CM) did not affect the activities of SC cell GAD or acetylcholinesterase. Dilution or concentration of the CM directly affected its ability to increase SC CAT activity , as did the duration and timing of exposure of the SC cells to the CM. The medium could be conditioned by muscle cells in the presence or absence of serum, and remained effective after dialysis or heating to 58 degrees C. Membrane filtration data were consistent with the conclusion that the active material(s) in CM had a molecular weight in excess of 50,000 daltons. We conclude that large molecular weight material that is released by muscle cells is capable of producing a specific increase in CAT activity of SC cells.     

<Emphasis Type="Italic">SONIC HEDGEHOG</Emphasis> mutations causing human holoprosencephaly impair neural patterning activity

Holoprosencephaly (HPE) is a common forebrain malformation associated with mental retardation and craniofacial anomalies. Multiple lines of evidence indicate that loss of ventral neurons is associated with HPE. The condition is etiologically heterogeneous, and abnormalities in any of several genes can cause human HPE. Among these genes, mutations in SONIC HEDGEHOG ( SHH) are the most commonly identified single gene defect causing human HPE. SHH mediates a number of processes in central nervous system development and is required for the normal induction of ventral cell types in the brain and spinal cord. Although a number of missense mutations in SHH have been identified in patients with HPE, the functional significance of these mutations has not yet been determined. We demonstrate that two SHH mutations that cause human HPE result in decreased in vivo activity of SHH in the developing nervous system. These mutant forms of SHH fail to regulate genes properly that are normally responsive to SHH signaling and do not induce ventrally expressed genes. In addition, the immunoreactivity of the mutant proteins is altered, suggesting that the conformation of the SHH protein has been disrupted. These studies are the first demonstration that mutations in SHH associated with human HPE perturb the in vivo patterning function of SHH in the developing nervous system.     

Substituted beta-cyclodextrins interact with PAMAM dendrimer-DNA complexes and modify transfection efficiency

The efficiency of PAMAM dendrimer-mediated DNA transfer can be improved by the addition of substituted beta-cyclodextrins (beta-CDs) as formulation excipients. In vitro CAT expression increased approximately 200-fold when dendrimer/DNA/beta-CD formulations were applied on the surface of collagen membranes. The inclusion of beta-CD into the formulations resulted in particles that were smaller and more evenly distributed on the surface of the solid support. The average size of the complex formed at 50 microg/ml and at charge ratio of 1 decreased from 156 nm to 5.8 nm and 21.2 nm in 0.025-0.1% w/vol beta-CDs. Sulfonated beta-CDs bind to dendrimer and in the increased concentration may displace DNA in the dendrimer/DNA complex. High concentrations of amphoteric beta-CD do not dissociate dendrimer/DNA complexes; however, they may decrease their ability to transfect cells. At the optimized formulations the surface-modified beta-CDs may enhance solid support-based transfection in vitro, through modification of dendrimer/DNA complex composition and improved surface distribution.     

iSubgraph: Integrative Genomics for Subgroup Discovery in Hepatocellular Carcinoma Using Graph Mining and Mixture Models

The high tumor heterogeneity makes it very challenging to identify key tumorigenic pathways as therapeutic targets. The integration of multiple omics data is a promising approach to identify driving regulatory networks in patient subgroups. Here, we propose a novel conceptual framework to discover patterns of miRNA-gene networks, observed frequently up- or down-regulated in a group of patients and to use such networks for patient stratification in hepatocellular carcinoma (HCC). We developed an integrative subgraph mining approach, called iSubgraph, and identified altered regulatory networks frequently observed in HCC patients. The miRNA and gene expression profiles were jointly analyzed in a graph structure. We defined a method to transform microarray data into graph representation that encodes miRNA and gene expression levels and the interactions between them as well. The iSubgraph algorithm was capable to detect cooperative regulation of miRNAs and genes even if it occurred only in some patients. Next, the miRNA-mRNA modules were used in an unsupervised class prediction model to discover HCC subgroups via patient clustering by mixture models. The robustness analysis of the mixture model showed that the class predictions are highly stable. Moreover, the Kaplan-Meier survival analysis revealed that the HCC subgroups identified by the algorithm have different survival characteristics. The pathway analyses of the miRNA-mRNA co-modules identified by the algorithm demonstrate key roles of Myc, E2F1, let-7, TGFB1, TNF and EGFR in HCC subgroups. Thus, our method can integrate various omics data derived from different platforms and with different dynamic scales to better define molecular tumor subtypes. iSubgraph is available as MATLAB code at http://www.cs.umd.edu/~ozdemir/isubgraph/.     

A Linear Relationship between Crystal Size and Fragment Binding Time Observed Crystallographically: Implications for Fragment Library Screening Using Acoustic Droplet Ejection

High throughput screening technologies such as acoustic droplet ejection (ADE) greatly increase the rate at which X-ray diffraction data can be acquired from crystals. One promising high throughput screening application of ADE is to rapidly combine protein crystals with fragment libraries. In this approach, each fragment soaks into a protein crystal either directly on data collection media or on a moving conveyor belt which then delivers the crystals to the X-ray beam. By simultaneously handling multiple crystals combined with fragment specimens, these techniques relax the automounter duty-cycle bottleneck that currently prevents optimal exploitation of third generation synchrotrons. Two factors limit the speed and scope of projects that are suitable for fragment screening using techniques such as ADE. Firstly, in applications where the high throughput screening apparatus is located inside the X-ray station (such as the conveyor belt system described above), the speed of data acquisition is limited by the time required for each fragment to soak into its protein crystal. Secondly, in applications where crystals are combined with fragments directly on data acquisition media (including both of the ADE methods described above), the maximum time that fragments have to soak into crystals is limited by evaporative dehydration of the protein crystals during the fragment soak. Here we demonstrate that both of these problems can be minimized by using small crystals, because the soak time required for a fragment hit to attain high occupancy depends approximately linearly on crystal size.     

Genome Resilience and Prevalence of Segmental Duplications Following Fast Neutron Irradiation of Soybean

Fast neutron radiation has been used as a mutagen to develop extensive mutant collections. However, the genome-wide structural consequences of fast neutron radiation are not well understood. Here, we examine the genome-wide structural variants observed among 264 soybean [Glycine max (L.) Merrill] plants sampled from a large fast neutron-mutagenized population. While deletion rates were similar to previous reports, surprisingly high rates of segmental duplication were also found throughout the genome. Duplication coverage extended across entire chromosomes and often prevailed at chromosome ends. High-throughput resequencing analysis of selected mutants resolved specific chromosomal events, including the rearrangement junctions for a large deletion, a tandem duplication, and a translocation. Genetic mapping associated a large deletion on chromosome 10 with a quantitative change in seed composition for one mutant. A tandem duplication event, located on chromosome 17 in a second mutant, was found to cosegregate with a short petiole mutant phenotype, and thus may serve as an example of a morphological change attributable to a DNA copy number gain. Overall, this study provides insight into the resilience of the soybean genome, the patterns of structural variation resulting from fast neutron mutagenesis, and the utility of fast neutron-irradiated mutants as a source of novel genetic losses and gains.     

The Determination of Axis Lengths of Dunaliella from the Image Analysis of TEM Sections through Randomly Orientated Cells

We present a method which improves the determination of geometric parameters of unicellular organisms with prolate ellipsoid geometry, such as Dunaliella. Approximately determined axis lenghts were obtained from transmission electron micrographs of ultrathin sections of microalgae. The density functions generated by these ‘axis lengths’ have poles which can be used to determine the short half axis length and ratio of axis lengths of the ellipsoid in order to determine the true cell volume.