Zinc mediates assembly of the T1 domain of the voltage-gated K channel 4.2

An intermolecular Zn(2+)-binding site was identified in the structure of the T1 domain of the Shaw-type potassium channels (aKv3.1). T1 is a BTB/POZ-type domain responsible for the ordered assembly of voltage-gated potassium channels and interactions with other macromolecules. In this structure, a Zn(2+) ion was found to be coordinated between each of the four assembly interfaces of the T1 tetramer by three Cys and one His encoded in the sequence motif (HX(5)CX(20)CC) of the T1 domain. This sequence motif is conserved among all non-Shaker-type voltage-dependent potassium (Kv) channels, but not in Shaker-type channels. The presence of this conserved Zn(2+)-binding site is a primary molecular determinant that distinguishes the tetrameric assembly of non-Shaker Kv channel subunits from that of Shaker channels. We report here that tetramerization of the Shal (rKv4.2) T1 in solution requires the presence of Zn(2+), and the addition/removal of Zn(2+) reversibly switches the protein between a stable tetrameric or monomeric state. We further show that the conversion from tetramers to monomers is profoundly pH-dependent: as the solution pH gets lower, the dissociation rate increases significantly. The unfolding energy of the T1 tetramer as a measure of the conformational stability of the structure is also pH-dependent. Surprisingly, at a lower pH we observe a distinctly altered conformational state of the T1 tetramer trapped during the process of unfolding of the T1 tetramer in the presence of Zn(2+). The conformational alteration may be responsible for increased rate of dissociation at lower pH by allowing Zn(2+) to be removed more effectively by EDTA. The ability of the T1 domain to adopt stable alternative conformations may be essential to its function as a protein-protein interaction/signaling domain to modulate the ion conduction properties of intact full-length Kv channels.     

Antiviral and immunological benefits in HIV patients receiving intranasal peptide T (DAPTA)

D-Ala-Peptide T-amide (DAPTA), the first viral entry inhibitor, blocks chemokine (CCR5) receptors, not CD4. Early investigators could not "replicate" DAPTAs potent in vitro antiviral effect using the lab-adapted, X4, peptide T-insensitive strain, IIIB, delaying clinical virological studies. We now report that DAPTA, administered to eleven long-term infected (mean=17 years) patients with stable persistent plasma "virus" for up to 32 weeks did not change this level. Infectious virus could not be isolated from their plasma suggesting HIV RNA was devoid of replicative capacity. Progressively less actual virus (P<0.01) could be isolated from white blood cells (PBMCs). DAPTA flushed the monocyte reservoir to undetectable viral levels in most patients. Five of eleven had a mean CD4 increase of 33%. Immune benefits also included a four-fold increase in gamma-interferon-secreting T-cells (antiviral cytotoxic T-cells) in the absence of drug-related toxicity. All five CD4 responders had increases in antiviral T cells and decreases in infected monocytes, an argument for initiating further studies promptly.     

Toward development of neem-based repellents against the Scottish Highland biting midge Culicoides impunctatus

Oil of neem, from the tree Azadirachta indica A. Juss (Meliaceae), was evaluated for repellent and antifeedant activity against Culicoides biting midges (Diptera: Ceratopogonidae), by three complementary methods with serial dilutions. Electroantennograms revealed the sensitivity of Culicoides nubeculosus (Meigen) females to neem > or = 0.10%. Culicoides impunctatus Goetghebuer females were repelled by > or = 1% in a Y-tube olfactometer, Using a membrane feeder for wild-caught parous females of C. impunctatus, the proportion blood-feeding was significantly reduced by topical applications of neem oil > or = 0.10% concentrations, with blood-feeding completely prevented by > or =1%. On the basis of these response data, we developed 2% neem-based formulations for personal protection against biting midges.     

Glucose-stimulated insulin response in pregnant sheep following acute suppression of plasma non-esterified fatty acid concentrations

Background  

Elevated non-esterified fatty acids (NEFA) concentrations in non-pregnant animals have been reported to decrease pancreatic responsiveness. As ovine gestation advances, maternal insulin concentrations fall and NEFA concentrations increase. Experiments were designed to examine if the pregnancy-associated rise in NEFA concentration is associated with a reduced pancreatic sensitivity to glucose in vivo. We investigated the possible relationship of NEFA concentrations in regulating maternal insulin concentrations during ovine pregnancy at three physiological states, non-pregnant, non-lactating (NPNL), 105 and 135 days gestational age (dGA, term 147+/- 3 days).     

Inhibition of transferrin recycling and endosome tubulation by phospholipase A2 antagonists

We report here that a broad spectrum of phospholipase A(2) (PLA(2)) antagonists produce a concentration-dependent, differential block in the endocytic recycling pathway of transferrin (Tf) and Tf receptors (TfRs) but have no acute affect on Tf uptake from the cell surface. At low concentrations of antagonists (approximately 1 microm), Tf and TfR accumulated in centrally located recycling endosomes, whereas at higher concentrations (approximately 10 microm), Tf-TfR accumulated in peripheral sorting endosomes. Several independent lines of evidence suggest that this inhibition of recycling may result from the inhibition of tubule formation. First, BFA-stimulated endosome tubule formation was similarly inhibited by PLA(2) antagonists. Second, endocytosed tracers were found in larger spherical endosomes in the presence of PLA(2) antagonists. And third, endosome tubule formation in a cell-free, cytosol-dependent reconstitution system was equally sensitive PLA(2) antagonists. These results are consistent with the conclusion that endosome membrane tubules are formed by the action of a cytoplasmic PLA(2) and that PLA(2)-dependent tubules are involved in intracellular recycling of Tf and TfR. When taken together with previous studies on the Golgi complex, these results also indicate that an intracellular PLA(2) activity provides a novel molecular mechanism for inducing tubule formation from multiple organelles.     

Noninvasive magnetic resonance imaging of the development of individual colon cancer tumors in rat liver

Monitoring tumor development is essential for the understanding of mechanisms involved in tumor progression and to determine efficacy of therapy. One of the evolving approaches is longitudinal noninvasive magnetic resonance imaging (MRI) of tumors in experimental models. We applied high-resolution MRI at 7 Tesla to study the development of colon cancer tumors in rat liver. MRI acquisition was triggered to the respiratory cycle to minimize motion artifacts. A special radio frequency (RF) coil was designed to acquire detailed T1-weighted and T2-weighted images of the liver. T2-weighted images identified hyperintense lesions representing tumors with a minimum diameter of 2 mm, enabling the determination of growth rates and morphological aspects of individual tumors. It is concluded that high-resolution MRI using a dedicated RF coil and triggering to the respiratory cycle is an excellent tool for quantitative and morphological analysis of individual diffusely distributed tumors throughout the liver. However, at present, MRI requires expensive equipment and expertise and is a time-consuming methodology. Therefore, it should preferably be used for dedicated applications rather than for high-throughput assessment of total tumor load in animals.     

Kinetic investigations in single muscle fibres using luminescent and fluorescent Ca2+ probes

The Ca2+-sensitive photoprotein aequorin and the Ca2+-dependent fluorescent indicators quin 2 and TnCDANZ have been used to investigate contractile processes in single crustacean muscle fibres. The investigations with quin 2 indicate that the free Ca2+ rises to a maximum value before peak force as with aequorin light (approximately 200 msec delay at 12 degrees C) and subsequently decays more slowly, unlike the majority of the aequorin signal, although an aequorin 'tail' signal remains. The resting quin 2 fluorescence from the cell suggests an upper limit of 348 nM for the resting calcium concentration. Experiments with TnCDANZ indicate that this fluorescence response rises rapidly but then the rate of rise slows to reach a maximum value at a time when peak force is achieved and then the fluorescence signal decays more slowly than force. The latter result implies that Ca2+ is attached to the Ca2+-specific sites of TnC when externally recorded force is small.