Identification of the Severe Acute Respiratory Syndrome Coronavirus by Simultaneous Multigene DNA Sequencing

The recent severe acute respiratory syndrome (SARS) outbreak resulted in calls for an accurate diagnostic test that can be used not only for routine testing but also for generating nucleotide sequences to monitor the epidemic. Although the identity of the SARS coronavirus (SARS-CoV) genome was confirmed by DNA sequencing, it is impractical to sequence the entire 29-kb SARS-CoV genome on a routine basis. Therefore, alternative assay methods such as the enzyme-linked immunosorbent assay and PCR have been pursued for routine testing, primarily to resolve probable cases. We report here a modification of standard DNA sequencing technology for accurate identification of SARS-CoV in routine testing. Instead of requiring the sequencing of the whole SARS-CoV genome, our modification enables the simultaneous sequencing of three regions of the SARS-CoV genome, the spike protein-encoding gene (35 nucleotides), gene M (43 nucleotides), and gene N (45 nucleotides), in a single electropherogram. Comparing these nucleotide sequences to DNA databank entries (National Institutes of Health) conclusively identified them as SARS-CoV sequences

A PDZ-3 mediated physical and functional interaction between the CaV3.2 T-type calcium channel and neuronal nitric oxide synthase

T-type voltage-gated calcium channels are expressed throughout the central and peripheral nervous systems as well as in several non-neuronal tissues and contribute to variety of functions such as neuronal excitability, intracellular calcium influx, shaping action potentials, pace-making activity, hormone secretion, and neurotransmitter release. Of the three T-type channel isoforms, Cav3.2 is uniquely sensitive to redox modulation with oxidizing reagents inhibiting and reducing compounds enhancing channel activity. This modulation has been shown to alter firing patterns of reticular thalamic neurons and to affect the nociceptive threshold in vivo suggesting that redox modulation of Cav3.2 may play an important role in regulating neuronal activity. A potential source of oxidizing molecules in vivo is neuronal nitric oxide synthase (nNOS), a calcium dependent enzyme which synthesizes nitric oxide (NO) from arginine. Interestingly, the carboxyl terminus of Cav3.2 possesses a putative PDZ-3 binding ligand which is compatible with the PDZ-3 domain of nNOS. I hypothesize that Cav3.2 and nNOS physically interact via the PDZ-3 binding ligand of Cav3.2 and that this physical interaction mediates a functional interaction whereby Cav3.2 activity stimulates nNOS to produce NO which, in turn, inhibits Cav3.2 activity. Cav3.2 and nNOS were expressed in a heterologous system which allowed us to examine the putative PDZ-3 mediated interactions between the two proteins. Immunoprecipitation experiments using Cav3.2 specific antibodies demonstrate that Cav3.2 and nNOS can interact via the carboxyl PDZ-3 ligand of Cav3.2 and that this interaction is disrupted when the PDZ-3 ligand is mutated. Utilizing a NO sensitive fluorometric assay we show that Cav3.2 activity can stimulate nNOS to produce NO and that disruption the PDZ-3 interaction precludes nNOS activation. We also demonstrate that the PDZ-3 mediated physical interaction facilitates the inhibition of Cav3.2 by nNOS derived NO. Disruption of the Cav3.2/nNOS interaction in vivo using intraperitoneal injection of membrane permeable peptides designed to competitively disrupt the PDZ-3 interaction produces an exaggerated respiratory response to changes in available oxygen and a blunted response in the hyperoxic response test. These results indicate that Cav3.2 and nNOS physically and functionally interact to contribute to normal physiological processes.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat

Nucleotide Sequence-Based Multitarget Identification

MULTIGEN technology (T. Vinayagamoorthy, U.S. patent 6,197,510, March 2001) is a modification of conventional sequencing technology that generates a single electropherogram consisting of short nucleotide sequences from a mixture of known DNA targets. The target sequences may be present on the same or different nucleic acid molecules. For example, when two DNA targets are sequenced, the first and second sequencing primers are annealed to their respective target sequences, and then a polymerase causes chain extension by the addition of new deoxyribose nucleotides. Since the electrophoretic separation depends on the relative molecular weights of the truncated molecules, the molecular weight of the second sequencing primer was specifically designed to be higher than the combined molecular weight of the first sequencing primer plus the molecular weight of the largest truncated molecule generated from the first target sequence. Thus, the series of truncated molecules produced by the second sequencing primer will have higher molecular weights than those produced by the first sequencing primer. Hence, the truncated molecules produced by these two sequencing primers can be effectively separated in a single lane by standard gel electrophoresis in a single electropherogram without any overlapping of the nucleotide sequences. By using sequencing primers with progressively higher molecular weights, multiple short DNA sequences from a variety of targets can be determined simultaneously. We describe here the basic concept of MULTIGEN technology and three applications: detection of sexually transmitted pathogens (Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum), detection of contaminants in meat samples (coliforms, fecal coliforms, and Escherichia coli O157:H7), and detection of single-nucleotide polymorphisms in the human N-acetyltransferase (NAT1) gene (S. Fronhoffs et al., Carcinogenesis 22:1405-1412, 2001)

Regulation of Macropinocytosis by Diacylglycerol Kinase ζ.

Macropinosomes arise from the closure of plasma membrane ruffles to bring about the non-selective uptake of nutrients and solutes into cells. The morphological changes underlying ruffle formation and macropinosome biogenesis are driven by actin cytoskeleton rearrangements under the control of the Rho GTPase Rac1. We showed previously that Rac1 is activated by diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show DGKζ is required for optimal macropinocytosis induced by growth factor stimulation of mouse embryonic fibroblasts. Time-lapse imaging of live cells and quantitative analysis revealed DGKζ was associated with membrane ruffles and nascent macropinosomes. Macropinocytosis was attenuated in DGKζ-null cells, as determined by live imaging and vaccinia virus uptake experiments. Moreover, macropinosomes that did form in DGKζ-null cells were smaller than those found in wild type cells. Rescue of this defect required DGKζ catalytic activity, consistent with it also being required for Rac1 activation. A constitutively membrane bound DGKζ mutant substantially increased the size of macropinosomes and potentiated the effect of a constitutively active Rac1 mutant on macropinocytosis. Collectively, our results suggest DGKζ functions in concert with Rac1 to regulate macropinocytosis

Selective inhibition of Cav3.3 T-type calcium channels by Galphaq/11-coupled muscarinic acetylcholine receptors

T-type calcium channels play critical roles in controlling neuronal excitability, including the generation of complex spiking patterns and the modulation of synaptic plasticity, although the mechanisms and extent to which T-type Ca(2+) channels are modulated by G-protein-coupled receptors (GPCRs) remain largely unexplored. To examine specific interactions between T-type Ca(2+) channel subtypes and muscarinic acetylcholine receptors (mAChRS), the Cav3.1 (alpha(1G)), Cav3.2 (alpha(1H)), and Cav3.3 (alpha) T-type Ca(2+)(1I)channels were co-expressed with the M1 Galpha(q/11)-coupled mAChR. Perforated patch recordings demonstrate that activation of M1 receptors has a strong inhibitory effect on Cav3.3 T-type Ca(2+) currents but either no effect or a moderate stimulating effect on Cav3.1 and Cav3.2 peak current amplitudes. This differential modulation was observed for both rat and human T-type Ca(2+) channel variants. The inhibition of Cav3.3 channels by M1 receptors is reversible, use-independent, and associated with a concomitant increase in inactivation kinetics. Loss-of-function experiments with genetically encoded antagonists of Galpha and Gbetagamma proteins and gain-of-function experiments with genetically encoded Galpha subtypes indicate that M1 receptor-mediated inhibition of Cav3.3 occurs through Galpha(q/11). This is supported by experiments showing that activation of the M3 and M5 Galpha(q/11)-coupled mAChRs also causes inhibition of Cav3.3 currents, although Galpha(i)-coupled mAChRs (M2 and M4) have no effect. Examining Cav3.1-Cav3.3 chimeric channels demonstrates that two distinct regions of the Cav3.3 channel are necessary and sufficient for complete M1 receptor-mediated channel inhibition and represent novel sites not previously implicated in T-type channel modulation

Amyotrophic lateral sclerosis-immunoglobulins selectively interact with neuromuscular junctions expressing P/Q-type calcium channels

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a gradual loss of motoneurons. The majority of ALS cases are associated with a sporadic form whose etiology is unknown. Several pieces of evidence favor autoimmunity as a potential contributor to sporadic ALS pathology. To gain understanding concerning possible antigens interacting with IgGs from sporadic ALS patients (ALS-IgGs), we studied immunoreactivity against neuromuscular junction (NMJ), spinal cord and cerebellum of mice with and without the Ca V2.1 pore-forming subunit of the P/Q-type voltage-gated calcium (Ca 2+) channel. ALS-IgGs showed a strong reactivity against NMJs of wild-type diaphragms. ALS-IgGs also increased muscle miniature end-plate potential frequency, suggesting a functional role for ALS-IgGs on synaptic signaling. In support, in mice lacking the Ca V2.1 subunit ALS-IgGs showed significantly reduced NMJ immunoreactivity and did not alter spontaneous acetylcholine release. This difference in reactivity was absent when comparing N-type Ca 2+ channel wild-type or null mice. These results are particularly relevant because motoneurons are known to be early pathogenic targets in ALS. Our findings add further evidence supporting autoimmunity as one of the possible mechanisms contributing to ALS pathology. They also suggest that serum autoantibodies in a subset of ALS patients would interact with NMJ proteins down-regulated when P/Q-type channels are absent. © 2011 International Society for Neurochemistry.Fil: Gonzalez, Laura Elisabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Kotler, Monica Lidia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Vattino, Lucas Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Conti, Eugenia. Hospital Británico de Buenos Aires; ArgentinaFil: Reisin, Ricardo C.. Hospital Británico de Buenos Aires; ArgentinaFil: Mulatz, Kirk J.. University of British Columbia; CanadáFil: Snutch, Terrance P.. University of British Columbia; CanadáFil: Uchitel, Osvaldo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin