Drosophila projectin: relatedness to titin and twitchin and correlation with lethal(4) 102 CDa and bent-dominant mutants.

We have investigated projectin, a large protein of insect muscles, in Drosophila melanogaster. The 5.3 kilobases of coding sequence reported here contains Class I and Class II motifs characteristic of titin and twitchin, arranged in a three domain ... [II-I-I] [II-I-I] ... pattern. Two mutants mapped to the location of the projectin gene in the 102C subdivision of chromosome 4, lethal(4) 102 CDa and bent-Dominant, have DNA rearrangements within their projectin gene. The lethal(4) 102 CDa mutant has a 141 nucleotide insertion containing stop codons in all three reading frames within an exon sequence, showing that it cannot synthesize normal projectin. Both bent-Dominant and lethal(4) 102 CDa homozygotes die at the completion of embryogenesis because they are unable to escape the egg vitelline membrane. We propose that this hatching failure is due to muscle weakness caused by projectin defects.     

Phosphorylation switches specific for the cardiac isoform of myosin binding protein-C: a modulator of cardiac contraction?

Cardiac myosin binding protein-C (cardiac MyBP-C, cardiac C protein) belongs to a family of proteins implicated in both regulatory and structural functions of striated muscle. For the cardiac isoform, regulatory phosphorylation in vivo by cAMP-dependent protein kinase (PKA) upon adrenergic stimulation is linked to modulation of cardiac contraction. The sequence of human cardiac MyBP-C now reveals regulatory motifs specific for this isoform. Site-directed mutagenesis identifies a LAGGGRRIS loop in the N-terminal region of cardiac MyBP-C as the key substrate site for phosphorylation by both PKA and a calmodulin-dependent protein kinase associated with the native protein. Phosphorylation of two further sites by PKA is induced by phosphorylation of this isoform-specific site. This phosphorylation switch can be mimicked by aspartic acid instead of phosphoserine. Cardiac MyBP-C is therefore specifically equipped with sensors for adrenergic regulation of cardiac contraction, possibly implicating cardiac MyBP-C in cardiac disease. The gene coding for cardiac MyBP-C has been assigned to the chromosomal location 11p11.2 in humans, and is therefore in a region of physical linkage to subsets of familial hypertrophic cardiomyopathy (FHC). This makes cardiac MyBP-C a candidate gene for chromosome 11-associated FHC.     

Oligonucleotide Probes That Hybridize with rRNA as a Tool To Study Frankia Strains in Root Nodules

Oligonucleotide probes that hybridize with specific sequences in variable regions of the 16S rRNA of the nitrogen-fixing actinomycete Frankia were used for the identification of Frankia strains in nodules. Frankia cells were released from plant tissue by grinding glutaraldehyde-fixed root nodules in guanidine hydrochloride solution. rRNA was obtained after sonication, precipitation with ethanol, and purification by phenolchloroform extraction. Degradation of rRNA, evident in Northern blots, did not affect hybridization with the oligonucleotides. Nodules of about 1 mg (fresh weight) provided sufficient rRNA for reliable detection of the Frankia strain. The utility of this rRNA extraction method was tested in a competition experiment between two effective Frankia strains on cloned Alnus glutinosa plants.     

SorCS2 Controls Functional Expression of Amino Acid Transporter EAAT3 and Protects Neurons from Oxidative Stress and Epilepsy-Induced Pathology

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Eukaryotic expression plasmid transfer from the intracellular bacterium Listeria monocytogenes to host cells

The facultative intracellular, Gram-positive bacterium Listeria monocytogenes invades phagocytic and non-phagocytic cells from the tissues and organs of a wide variety of animals and humans. Here, we report the use of these bacteria as vehicles for gene transfer. Eukaryotic expression plasmids were introduced into the nucleus of host cells following lysis of the intracytosolic, plasmid-carrying bacteria with antibiotics. Cell lines of different tissues and species could be transfected in this way. We examined bacterial properties required for delivery of the expression plasmids and found that this was strictly dependent on the ability of these bacteria to both invade eukaryotic cells and egress from the vacuole into the cytosol of the infected host cells. Macrophage-like cell lines or primary, peritoneal macrophages proved to be almost refractory to Listeria ‐mediated gene transfer. Thus, attenuated L. monocytogenes represents a serious candidate for consideration as a DNA-transfer vehicle for in vivo somatic gene therapy. The potential for oral administration of L. monocytogenes and the ease in producing and cultivating recombinant strains are further attributes that make its use as a gene transfer vehicle attractive.     

Molecular imaging of gliomas

Gliomas are the most common types of brain tumors. Although sophisticated regimens of conventional therapies are being carried out to treat patients with gliomas, the disease invariably leads to death over months or years. Before new and potentially more effective treatment strategies, such as gene- and cell-based therapies, can be effectively implemented in the clinical application, certain prerequisites have to be established. First of all, the exact localization, extent, and metabolic activity of the glioma must be determined to identify the biologically active target tissue for a biological treatment regimen; this is usually performed by imaging the expression of up-regulated endogenous genes coding for glucose or amino acid transporters and cellular hexokinase and thymidine kinase genes, respectively. Second, neuronal function and functional changes within the surrounding brain tissue have to be assessed in order to save this tissue from therapy-induced damage. Third, pathognomonic genetic changes leading to disease have to be explored on the molecular level to serve as specific targets for patient-tailored therapies. Last, a concerted noninvasive analysis of both endogenous and exogenous gene expression in animal models as well as the clinical setting is desirable to effectively translate new treatment strategies from experimental into clinical application. All of these issues can be addressed by multi-modal radionuclide and magnetic resonance imaging techniques and fall into the exciting and fast growing field of molecular and functional imaging. Noninvasive imaging of endogenous gene expression by means of positron emission tomography (PET) may reveal insight into the molecular basis of pathogenesis and metabolic activity of the glioma and the extent of treatment response. When exogenous genes are introduced to serve for a therapeutic function, PET imaging may reveal the assessment of the "location," "magnitude," and "duration" of therapeutic gene expression and its relation to the therapeutic effect. Detailed reviews on molecular imaging have been published from the perspective of radionuclide imaging (Gambhir et al., 2000; Blasberg and Tjuvajev, 2002) as well as magnetic resonance and optical imaging (Weissleder, 2002). The present review focuses on molecular imaging of gliomas with special reference on the status and perspectives of imaging of endogenous and exogenously introduced gene expression in order to develop improved diagnostics and more effective treatment strategies of gliomas and, in that, to eventually improve the grim prognosis of this devastating disease.     

Evolution after gene duplication: models, mechanisms, sequences, systems, and organisms

Gene duplication is postulated to have played a major role in the evolution of biological novelty. Here, gene duplication is examined across levels of biological organization in an attempt to create a unified picture of the mechanistic process by which gene duplication can have played a role in generating biodiversity. Neofunctionalization and subfunctionalization have been proposed as important processes driving the retention of duplicate genes. These models have foundations in population genetic theory, which is now being refined by explicit consideration of the structural constraints placed upon genes encoding proteins through physical chemistry. Further, such models can be examined in the context of comparative genomics, where an integration of gene-level evolution and species-level evolution allows an assessment of the frequency of duplication and the fate of duplicate genes. This process, of course, is dependent upon the biochemical role that duplicated genes play in biological systems, which is in turn dependent upon the mechanism of duplication: whole genome duplication involving a co-duplication of interacting partners vs. single gene duplication. Lastly, the role that these processes may have played in driving speciation is examined.     

Targeted proteomics on its way to discovery

For a long time, targeted and discovery proteomics covered different corners of the detection spectrum, with targeted proteomics focused on small target sets. This changed with the recent advances in highly multiplexed analysis. While discovery proteomics still pushes higher numbers of identified and quantified proteins, the advances in targeted proteomics rose to cover large parts of less complex proteomes or proteomes with low protein detection counts due to dynamic range restrictions, like the blood proteome. These new developments will impact, especially on the field of biomarker discovery and the possibility of using targeted proteomics for diagnostic purposes.     

<Emphasis Type="Italic">ScreenMill</Emphasis>: A freely available software suite for growth measurement,analysis and visualization of high-throughput screen data

Background  

Many high-throughput genomic experiments, such as Synthetic Genetic Array and yeast two-hybrid, use colony growth on solid media as a screen metric. These experiments routinely generate over 100,000 data points, making data analysis a time consuming and painstaking process. Here we describe ScreenMill, a new software suite that automates image analysis and simplifies data review and analysis for high-throughput biological experiments.     

Effect of hyperbaric air on endotoxin from <Emphasis Type="Italic">Bacteroides fragilis</Emphasis> strains

The aim of the project was to determine any effect of hyperbaric air on Bacteroides fragilis strains cultivated under hyperbaric conditions. Previously, it was hypothesized that there was a correlation between the presence of Bacteroides bacteria in patients preferring a meaty diet and cancer of the small intestine, and particularly of the large intestine and rectum. With respect to the fact that Bacteroides fragilis (BAFR) group are important producers of endotoxins, measurement and statistical evaluation of endotoxin production by individual strains of isolated Bacteroides species were used to compare bacteria isolated from various clinical samples from patients with colon and rectum cancer in comparison with strains isolated from other non-cancer diagnoses. Endotoxin production was proven by quantitative detection using the limulus amebocyte lysate (LAL) test in EU/mL. Production of endotoxins in these bacteria cultured under hyperbaric air conditions was higher than those strains cultured under normobaric anaerobic conditions. But these differences in endotoxin production were not statistically significant (t test with log-transformed data, p value = 0.0910). Based on a two-tier t test for lognormal data, it is possible to cautiously conclude that a statistically significant difference was found between endotoxin production by Bacteroides fragilis strains isolated from non-carcinoma diagnoses (strains (1–6) and strains isolated from colorectal carcinoma diagnoses (strains 7–8; Wilcoxon non-parametric test p = 0.0132; t test = 0.1110; t test with log-transformed data, p value = 0.0294).