The cucumber mosaic virus 1a protein regulates interactions between the 2b protein and ARGONAUTE 1 while maintaining the silencing suppressor activity of the 2b protein

The cucumber mosaic virus (CMV) 2b viral suppressor of RNA silencing (VSR) is a potent counter-defense and pathogenicity factor that inhibits antiviral silencing by titration of short double-stranded RNAs. It also disrupts microRNA-mediated regulation of host gene expression by binding ARGONAUTE 1 (AGO1). But in Arabidopsis thaliana complete inhibition of AGO1 is counterproductive to CMV since this triggers another layer of antiviral silencing mediated by AGO2, de-represses strong resistance against aphids (the insect vectors of CMV), and exacerbates symptoms. Using confocal laser scanning microscopy, bimolecular fluorescence complementation, and co-immunoprecipitation assays we found that the CMV 1a protein, a component of the viral replicase complex, regulates the 2b-AGO1 interaction. By binding 2b protein molecules and sequestering them in P-bodies, the 1a protein limits the proportion of 2b protein molecules available to bind AGO1, which ameliorates 2b-induced disease symptoms, and moderates induction of resistance to CMV and to its aphid vector. However, the 1a protein-2b protein interaction does not inhibit the ability of the 2b protein to inhibit silencing of reporter gene expression in agroinfiltration assays. The interaction between the CMV 1a and 2b proteins represents a novel regulatory system in which specific functions of a VSR are selectively modulated by another viral protein. The finding also provides a mechanism that explains how CMV, and possibly other viruses, modulates symptom induction and manipulates host-vector interactions.     

The isolation of spirochaetes from the rat caecum

Spirochaetes indigenous to the conventional rat caecum were cultivated on agar medium using selective isolation techniques. The 27 axenic isolates represented four morphological types of spirochaetes differing in cell size, cell coiling pattern and number of protoplasmic flagella.     

Mutations in the tobacco mosaic virus 30-kD protein gene overcome Tm-2 resistance in tomato.

A resistance-breaking strain of tobacco mosaic virus (TMV), Ltb1, is able to multiply in tomatoes with the Tm-2 gene, unlike its parent strain, L. Nucleotide sequence analysis of Ltb1 RNA revealed two amino acid changes in the 30-kD protein: from Cys68 to Phe and from Glu133 to Lys (from L to Ltb1). Strains with these two changes generated in vitro multiplied in tomatoes with the Tm-2 gene and induced essentially the same symptoms as those caused by Ltb1. Strains with either one of the two changes did not overcome the resistance as efficiently as Ltb1, although increased levels of multiplication were observed compared with the L strain. Results showed that both mutations are involved in the resistance-breaking property of Ltb1. Sequence analysis indicated that another resistance-breaking strain and its parent strain had two amino acid changes in the 30-kD protein: from Glu52 to Lys and from Glu133 to Lys. The fact that the amino acid changes occurred in or near the well conserved regions in the 30-kD protein suggests that the mechanism of Tm-2 resistance may be closely related to the fundamental function of the 30-kD protein, presumably in cell-to-cell movement.     

In vivo cellular responses to electrophoretically applied dynorphin in the rat hippocampus

Recent immunohistochemical and radioimmunochemical observations have demonstrated a differential distribution of immunoreactive dynorphin (DYN) in rat brain. The presence of DYN immunoreactivity in a major intrinsic fiber pathway within the rat hippocampus (the mossy fiber system) has led us to evaluate the possible role of DYN and other closely related peptides in this structure. Single cell activity and hippocampal field potentials have been recorded from the CA1-CA3 cellular fields in halothane or urethane anesthetized rats. DYN, DYN1-13, DYN1-8, and alpha-neo-endorphin had an excitatory effect on most CA1-CA3 neurons encountered as has been previously observed for opiates and other opioid peptides. This response could be blocked by naloxone or by co-administration of Mg++ ion suggesting an indirect (synaptic) mechanism of excitation similar to that hypothetized for enkephalin. A significant number of CA3 neurons, however, exhibited a non-naloxone sensitive inhibitory response to DYN, related opioid peptides, and the kappa agonist WIN 35-197 (ethylketocyclazocine). Field potential analysis of CA1-CA3 neuronal responses to mossy fiber activation also indicated an excitatory, Mg++ reversible, action of iontophoretically applied DYN. These observations support our cytochemical and assay studies indicating diverse opioid systems within the rat hippocampus. In addition, these functional studies are congruent with other evidence suggesting multiple opioid mechanisms in this structure.     

Susceptibility of neuroactive peptides to aminopeptidase digestion is related to molecular size.

Peptide fragments derived from the NH₂-terminus of corticotropin were found to exhibit widely differing degrees of stability to degradation by aminopeptidase M. Corticotropin itself was 135 times more stable than its NH₂-terminal pentapeptide, and similar differences in stability were observed with peptides derived from the B-chain of bovine insulin. Enkephalin linked covalently to the A-chain of bovine insulin was at least 100 times more stable than the pentapeptide. The results demonstrate that the molecular size of a peptide is one factor that determines its NH₂-terminal stability.     

Cytochrome b, the var 1 protein, and subunits I and III of cytochrome c oxidase are synthesized without transient presequences in Saccharomyces cerevisiae

The N-termini of four mitochondrial translation products, the var 1 protein, cytochrome b, and subunits I and III of cytochrome c oxidase have been characterized in Saccharomyces cerevisiae and compared with the known DNA sequences of the respective structural genes. The four mature proteins correspond to the predicted primary translation products and retain the formylated methionine residue. Thus, subunit II of cytochrome c oxidase studied previously [Pratje et al. (1983) EMBO J.2, 1049-1054] is so far the only mitochondrial translation product carrying a N-terminal-extended transient presequence in S. cerevisiae.     

Atrial natriuretic factor alters autonomic interactions in the control of heart rate in conscious rats

Regulation of heart rate was studied in rats receiving either i.v. saline at 64 microL/min or synthetic 28-residue rat atrial natriuretic peptide (ANF) at a dose sufficient to decrease mean arterial blood pressure by 10%. Autonomic influences were deduced from steady-state heart rate responses of each group to propranolol, atropine, or propranolol and atropine combined. A multiplicative model of heart rate control was used to derive quantitatively from the data the modulation of intrinsic heart rate by sympathetic and parasympathetic mechanisms. Animals receiving ANF showed a lower heart rate than control animals. This relative bradycardia was abolished by atropine. Blocking of sympathetic effects with propranolol had no effect on basal heart rate in either group, and atropinization led to significant increases in heart rate in both groups of rats. Mathematical analysis of the results showed that the bradycardia produced by ANF was due predominantly to a reduced intrinsic heart rate and to enhanced vagal inhibition of postganglionic sympathetic activity. Parasympathetic contribution to heart rate in the absence of sympathetic activity was negligible in control rats and small during ANF. We conclude that the major influences of ANF on heart rate control are a decrease of intrinsic heart rate and enhanced parasympathetic inhibition of postganglionic presynaptic sympathetic activity.     

The specificity of the syngeneic mixed leukocyte response, a primary anti-I region T cell proliferative response, is determined intrathymically

In previous studies, the syngeneic MLR of peripheral T cells was shown to be predominantly an I region-restricted function. In this report we show that adult thymocytes are also capable of responding to syngeneic irradiated stimulator cells in a syngeneic MLR, provided that TCGF is added to the culture system. Using this assay, it was possible for the first time to examine the pattern of I region restriction within the thymus itself. Analysis of the thymocyte syngeneic MLR in thymuses from radiation-induced bone marrow chimeras demonstrated that the MHC preference seen in the peripheral T cell population also existed in cells resident within the thymus. Experiments utilizing congenitally athymic mice transplanted with allogeneic thymic grafts demonstrated that both peripheral T cells and thymocytes from such animals displayed a strong preferential proliferation toward stimulator cells bearing thymic-type MHC determinants. The results in the nude model thus demonstrate that the thymus by itself is sufficient to impart such restriction specificity on a developing T cell repertoire. These results are consistent with the notion that the thymus exerts selective pressure on maturing T cell populations that results in a skewing of the T cell repertoire toward the recognition of thymic-type I region products, and that this MHC preference exists before expansion of T cells in the periphery.     

Modulation by dopamine of population spikes in area CA1 hippocampal neurons elicited by paired stimulus pulses

Extracellular recording techniques were used to study the effects of dopamine on postactivation excitability of rat area CA1 hippocampal neurons maintained in vitro. Population spikes were elicited by delivery of conditioning and test stimulus pulses to afferent fibers. The interval between the conditioning and test volley was set to separate delivery of stimuli by 10 to 80 msec. The effect of superfusion or microtopical application of dopamine (DA) on population responses to test stimulus pulses was studied. When paired stimulus volleys, separated by brief intervals (up to 40 msec), were delivered to afferent fibers, paired-pulse suppression (PPS) was indicated by the amplitude of the population spike elicited by the test volley being smaller than that elicited by the conditioning volley. When paired volleys were separated by longer intervals (40 to 80 msec), the response elicited by the test volley was larger in amplitude than that elicited by the conditioning volley, indicating paired-pulse facilitation (PPF). Following exposure to DA, the amplitude of the population response elicited by the conditioning volley was larger than the amplitude before exposure to DA. This effect was long-lasting, enduring for tens of minutes. However, when the amplitude of the conditioning population response was held constant, the PPS was decreased, indicating disinhibition. It is suggested that dopamine produces a long-lasting attenuation of an intervening inhibitory influence onto CA1 pyramidal neurons.