A modified walking rhythm employed during righting behavior in the cockroachGromphadorhina portentosa

Summary Electromyograms were recorded from leg muscles of the cockroachGromphadorhina during walking and righting under free-ranging and tethered conditions. Two muscles which are essentially synergistic during walking become antagonistic during righting (Fig. 3, 4). This explains in part the difference in the direction of the leg stroke in the two behaviors (Fig. 2). Other properties of the muscle activity are very similar during the two rhythms: the same motoneurons appear to be active (Fig. 5, 6); cycle frequencies are the same; the burst length of one motoneuron studied varies with burst frequency in a generally similar manner in both behaviors (Fig. 7); inter-leg coordination is the same (Fig. 9); and transganglionic coupling characteristic of walking can occur while a leg on one side is engaged in walking, and its contralateral homologue is engaged in righting (Fig. 10). Although other properties of the leg rhythms are different in walking and righting, these differences appear to result from dissimilarities in sensory feedback. It is concluded that although the two leg rhythms are superficially quite different, the underlying central neuronal rhythms are very similar, and possibly result from activity in the same central oscillatory cell or circuit.We thank Carol Smith for technical assistance. This work was supported by NIH grant #NS09083-05. Computation was done at the New York State Veterinary College Computer Facility which is supported by NIH grant RR 326.     

Myeloid-derived suppressor cells are associated with disease progression and decreased overall survival in advanced-stage melanoma patients

Myeloid-derived suppressor cells are increased in the peripheral blood of advanced-stage cancer patients; however, no studies have shown a correlation of these immunosuppressive cells with clinical outcomes in melanoma patients. We characterized the frequency and suppressive function of multiple subsets of myeloid-derived suppressor cells in the peripheral blood of 34 patients with Stage IV melanoma, 20 patients with Stage I melanoma, and 15 healthy donors. The frequency of CD14⁺ MDSCs (Lin^? CD11b⁺ HLA-DR^? CD14⁺ CD33⁺) and CD14^? MDSCs (Lin^? CD11b⁺ HLA-DR^? CD14^? CD33⁺) was increased in the peripheral blood of Stage IV melanoma patients relative to healthy donors. The frequency of CD14⁺ and CD14^? MDSCs correlated with each other and with the increased frequency of regulatory T cells, but not with classically defined monocytes. CD14^? MDSCs isolated from the peripheral blood of Stage IV melanoma patients suppressed T cell activation more than those isolated from healthy donors, and the frequency of these cells correlated with disease progression and decreased overall survival. Our study provides the first evidence that the frequency of CD14^? MDSCs negatively correlates with clinical outcomes in advanced-stage melanoma patients. These data indicate that suppressive MDSCs should be considered as targets for future immunotherapies.     

Stop codon recognition in ciliates: Euplotes release factor does not respond to reassigned UGA codon

In eukaryotes, the polypeptide release factor 1 (eRF1) is involved in translation termination at all three stop codons. However, the mechanism for decoding stop codons remains unknown. A direct interaction of eRF1 with the stop codons has been postulated. Recent studies focus on eRF1 from ciliates in which some stop codons are reassigned to sense codons. Using an in vitro assay based on mammalian ribosomes, we show that eRF1 from the ciliate Euplotes aediculatus responds to UAA and UAG as stop codons and lacks the capacity to decipher the UGA codon, which encodes cysteine in this organism. This result strongly suggests that in ciliates with variant genetic codes eRF1 does not recognize the reassigned codons. Recent hypotheses describing stop codon discrimination by eRF1 are not fully consistent with the set of eRF1 sequences available so far and require direct experimental testing.     

Characterization of an antigenically related family of cell-type specific proteins implicated in slug migration in Dictyostelium discoideum

The monoclonal antibody MUD50 recognizes a group of developmentally regulated proteins, which are almost exclusively expressed by prespore cells in developing aggregates of Dictyostelium discoideum. Some of these antigens are integrally associated with the cell membrane, as assessed by physical and detergent-fractionation procedures. The MUD50-reactive proteins are glycosylated and some are phosphorylated. Post-translational modification is the common antigenic feature that is recognized by the MUD50 antibody in these cell-type-specific proteins. A glycosylation-defective mutant, DL118, (modB) does not express the MUD50 epitope, but does express the MUD52 epitope, which is found on a different group of glycoproteins. Therefore, we conclude that MUD50 recognizes a particular carbohydrate epitope on a restricted group of proteins. These proteins are structurally diverse, but are apparently involved in the maintenance of structure and movement of the multicellular D. discoideum slug.     

Cyclic AMP suppresses expression of v-rasH oncogene linked to the mouse mammary tumor virus promoter

Clone 433.3 of NIH 3T3 cells is a stable carrier of the MMTV LTR:v-rasH chimeric DNA. Only in the presence of dexamethasone (a synthetic glucocorticoid), 433.3 cells exhibit an induced level of p21 transforming protein and phenotypic transformation. N6,O2'-dibutyryl cAMP (DBcAMP) antagonized the effect of dexamethasone in a time - and concentration - dependent manner. DBcAMP (5 X 10(-4)M) added 18 hr prior to the addition of dexamethasone (10(-7)M) almost completely blocked the hormone effect: cells contained levels of p21 20% of that in the cells treated with dexamethasone alone, and formed flat, contact inhibited monolayers. On the basis of these results together with our previous data on mammary carcinomas in vivo, we postulate that cAMP may be an intracellular suppressor acting at a regulatory locus of both cellular and viral ras genes.     

Nonflowering plants possess a unique folate-dependent phenylalanine hydroxylase that is localized in chloroplasts

Tetrahydropterin-dependent aromatic amino acid hydroxylases (AAHs) are known from animals and microbes but not plants. A survey of genomes and ESTs revealed AAH-like sequences in gymnosperms, mosses, and algae. Analysis of full-length AAH cDNAs from Pinus taeda, Physcomitrella patens, and Chlamydomonas reinhardtii indicated that the encoded proteins form a distinct clade within the AAH family. These proteins were shown to have Phe hydroxylase activity by functional complementation of an Escherichia coli Tyr auxotroph and by enzyme assays. The P. taeda and P. patens AAHs were specific for Phe, required iron, showed Michaelian kinetics, and were active as monomers. Uniquely, they preferred 10-formyltetrahydrofolate to any physiological tetrahydropterin as cofactor and, consistent with preferring a folate cofactor, retained activity in complementation tests with tetrahydropterin-depleted E. coli host strains. Targeting assays in Arabidopsis thaliana mesophyll protoplasts using green fluorescent protein fusions, and import assays with purified Pisum sativum chloroplasts, indicated chloroplastic localization. Targeting assays further indicated that pterin-4a-carbinolamine dehydratase, which regenerates the AAH cofactor, is also chloroplastic. Ablating the single AAH gene in P. patens caused accumulation of Phe and caffeic acid esters. These data show that nonflowering plants have functional plastidial AAHs, establish an unprecedented electron donor role for a folate, and uncover a novel link between folate and aromatic metabolism.     

The Susceptibility of Pseudomonas aeruginosa Strains from Cystic Fibrosis Patients to Bacteriophages

Phage therapy may become a complement to antibiotics in the treatment of chronic Pseudomonas aeruginosa infection. To design efficient therapeutic cocktails, the genetic diversity of the species and the spectrum of susceptibility to bacteriophages must be investigated. Bacterial strains showing high levels of phage resistance need to be identified in order to decipher the underlying mechanisms. Here we have selected genetically diverse P. aeruginosa strains from cystic fibrosis patients and tested their susceptibility to a large collection of phages. Based on plaque morphology and restriction profiles, six different phages were purified from “pyophage”, a commercial cocktail directed against five different bacterial species, including P. aeruginosa. Characterization of these phages by electron microscopy and sequencing of genome fragments showed that they belong to 4 different genera. Among 47 P. aeruginosa strains, 13 were not lysed by any of the isolated phages individually or by pyophage. We isolated two new phages that could lyse some of these strains, and their genomes were sequenced. The presence/absence of a CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and Crisper associated genes) was investigated to evaluate the role of the system in phage resistance. Altogether, the results show that some P. aeruginosa strains cannot support the growth of any of the tested phages belonging to 5 different genera, and suggest that the CRISPR-Cas system is not a major defence mechanism against these lytic phages.