Modulation of the classical multidrug resistance (MDR) phenotype by RNA interference (RNAi)

For reversal of MDR1 gene-dependent multidrug resistance (MDR), two small interfering RNA (siRNA) constructs were designed to inhibit MDR1 expression by RNA interference. SiRNA duplexes were used to treat human pancreatic carcinoma (EPP85-181RDB) and gastric carcinoma (EPG85-257RDB) cells. In both cellular systems, siRNAs could specifically inhibit MDR1 expression up to 91% at the mRNA and protein levels. Resistance against daunorubicin was decreased to 89% (EPP85-181RDB) or 58% (EPG85-257RDB). The data indicate that this approach may be applicable to cancer patients as a specific means to reverse tumors with a P-glycoprotein-dependent MDR phenotype back to a drug-sensitive one.     

NSF and p97/VCP: similar at first, different at last

N-Ethylmaleimide sensitive factor (NSF) and p97/valosin-containing protein (VCP) are distantly related members of the ATPases associated with a variety of cellular activities (AAA) family of proteins. While both proteins have been implied in cellular morphology changes involving membrane compartments or vesicles, more recent evidence seems to imply that NSF is primarily involved in the soluble NSF attachment receptor (SNARE)-mediated vesicle fusion by disassembling the SNARE complex whereas p97/VCP is primarily involved in the extraction of membrane proteins. These functional differences are now corroborated by major structural differences based on recent crystallographic and cryo-electron microscopy studies. This review discusses these recent findings.     

Mapping of synergistic components of weakly interacting protein-protein motifs using arrays of paired peptides

Protein-protein recognition usually involves multiple interactions among different motifs that are scattered over protein surfaces. To identify such weak interactions, we have developed a novel double peptide synthesis (DS) method. This method allows us to map protein-protein interactions that involve two linear dis- continuous components from a polypeptide by the use of spatially addressable synergistic pairs of synthetic peptides. The DS procedure is based on the "SPOT" membrane-bound peptide synthesis technique, but to synthesize a mixture of two peptides, it uses both Fmoc (N-(9-fluorenyl)methoxycarbonyl))-alanine and Alloc-alanine at the first cycle. This allows their selective deprotection by either piperidine or tributyltin/palladium treatment, respectively. Using SPOT DS, we confirmed as a proof of principle that Elk-1 Ser(383) phosphorylation by ERK-2 kinase is stimulated by the presence of the Elk-1-docking domain. SPOT DS can also be used to dissect protein-protein motifs that define phosphatase substrate affinity. Using this technique, we identified three new regions in the insulin receptor that stimulate the dephosphorylation of the receptor by protein-tyrosine phosphatase (PTP) 1B and presumably increase the selectivity of PTP for this substrate. These data demonstrate that the SPOT DS technique allows the identification of non-linear weakly interacting protein motifs, which are an important determinant of protein kinase and phosphatase substrate specificity and of protein-protein interactions in general.     

A chemosensory gene family encoding candidate gustatory and olfactory receptors in Drosophila

A novel family of candidate gustatory receptors (GRs) was recently identified in searches of the Drosophila genome. We have performed in situ hybridization and transgene experiments that reveal expression of these genes in both gustatory and olfactory neurons in adult flies and larvae. This gene family is likely to encode both odorant and taste receptors. We have visualized the projections of chemosensory neurons in the larval brain and observe that neurons expressing different GRs project to discrete loci in the antennal lobe and subesophageal ganglion. These data provide insight into the diversity of chemosensory recognition and an initial view of the representation of gustatory information in the fly brain.     

Analysis of right ventricular function in healthy mice and a murine model of heart failure by in vivo MRI

Because of its complex geometry, assessment of right ventricular (RV) function is more difficult than it is for the left ventricle (LV). Because gene-targeted mouse models of cardiomyopathy may involve remodeling of the right heart, the purpose of this study was to develop high-resolution functional magnetic resonance imaging (MRI) for in vivo quantification of RV volumes and global function in mice. Thirty-three mice of various age were studied under isoflurane anesthesia by electrocardiogram-triggered cine-MRI at 7 T. MRI revealed close correlations between RV and LV stroke volume and cardiac output (r = 0.97, P < 0.0001 each). Consistent with human physiology, murine RV end-diastolic and end-systolic volumes were significantly higher compared with LV volumes (P < 0.05 each). MRI in mice with LV heart failure due to myocardial infarction revealed significant structural and functional changes of the RV, indicating RV dysfunction. Hence, MRI allows for the quantification of RV volumes and global systolic function with high accuracy and bears the potential to evaluate mechanisms of RV remodeling in mouse models of heart failure.     

The mitochondrial genome of the pufferfish,Fugu rubripes,and ordinal teleostean relationships

The small nuclear genome of the pufferfish, Fugu rubripes (order Tetraodontiformes), makes this species highly interesting for genome research. In order to establish the phylogenetic position of the Tetraodontiformes relative to other teleostean orders that might also have a reduced nuclear genome size, we have sequenced the mitochondrial (mt) genome of the pufferfish. The gene order, nucleotide composition and evolutionary rate of the mt genome of the fugu correspond to those of other teleosts. This suggests that the evolution of this genome has not been affected by the processes that led to the dramatic reduction of the size of the nuclear genome of the fugu. The phylogenetic analyses, which were based on the concatenated amino acid sequences of twelve protein-coding mt genes, placed the fugu among the percomorphs. The affinities between the Tetraodontiformes and either the Perciformes or the Zeiformes were limited, however. The common notion of a separate euteleostean clade remained unsupported. The analyses did not support the traditional systematic understanding that the Clupeiformes constitute a basal teleostean lineage. In addition the findings strongly suggest that three teleostean orders, the Perciformes, Zeiformes and Scorpaeniformes, are paraphyletic.     

Discovering lactic acid bacteria by genomics

This review summarizes a collection of lactic acid bacteria that are now undergoing genomic sequencing and analysis. Summaries are presented on twenty different species, with each overview discussing the organisms fundamental and practical significance, nvironmental habitat, and its role in fermentation, bioprocessing, or probiotics. For those projects where genome sequence data were available by March 2002, summaries include a listing of key statistics and interesting genomic features. These efforts will revolutionize our molecular view of Gram–positive bacteria, as up to 15 genomes from the low GC content lactic acid bacteria are expected to be available in the public domain by the end of 2003. Our collective view of the lactic acid bacteria will be fundamentally changed as we rediscover the relationships and capabilities of these organisms through genomics.     

Correct conduct of full-committee and designated-member protocol reviews

Only two protocol review methods fulfill USDA and PHS requirements-full committee and designated member. The author attempts to lessen the confusion surrounding this issue by describing these methods, along with examples of faulty hybrids and convenient strategies to bring unacceptable methods into compliance.