Requirement of ADP-ribosylation for the pertussis toxin-induced alteration in electrophoretic mobility of G-proteins.

Pertussis toxin (PTX) catalyzes the ADP-ribosylation of the alpha-subunit of GTP-binding proteins (G-proteins) in the presence of NAD+. Pertussis toxin also decreases the electrophoretic mobility of the alpha-subunit on urea SDS PAGE. This effect of PTX has been suggested to be a property of the toxin different from its ability to catalyze ADP-ribosylation. However, the present report provides evidence to the contrary; ie, this mobility shift required the ADP-ribosylation of alpha-subunits. This conclusion was based on: (1) in the presence of increasing concentrations of NAD+ (0.026-1.3 microM), there was a linear increase in the formation of the slower migrating alpha-subunit as measured by immunoblotting with selective antisera, (2) addition of NADase to the incubation mixture completely eliminated the formation of this protein, and (3) increasing concentrations of nicotinamide (50-250 mM), which inhibits ADP-ribosylation, decreased the amount of the slower migrating alpha-subunit. Thus, in addition to PTX, NAD+ was required for the mobility shift and the slower migrating alpha-subunit is likely the ADP-ribosylated form.     

Alleles of Pto and Fen occur in bacterial speck-susceptible and fenthion-insensitive tomato cultivars and encode active protein kinases.

The Pto gene was derived originally from the wild tomato species Lycopersicon pimpinellifolium and confers resistance to Pseudomonas syringae pv tomato strains expressing the avirulence gene avrPto. The Fen gene is also derived from L. pimpinellifolium and confers sensitivity to the insecticide fenthion. We have now isolated and characterized the alleles of Pto and Fen from cultivated tomato, L. esculentum, and designated them pto and fen. High conservation of genome organization between the two tomato species allowed us to identify the pto and fen alleles from among the cluster of closely related Pto gene family members. The pto and fen alleles are transcribed and have uninterrupted open reading frames that code for predicted proteins that are 87 and 98% identical to the Pto and Fen protein kinases, respectively. In vitro autophosphorylation assays revealed that both the pto and fen alleles encode active kinases. In addition, the pto kinase phosphorylates a previously characterized substrate of Pto, the Pto-interacting Pti1 serine/threonine kinase. However, the pto kinase shows impaired interaction with Pti1 and with several previously isolated Pto-interacting proteins in the yeast two-hybrid system. The observation that pto and fen are active kinases and yet do not confer bacterial speck resistance or fenthion sensitivity suggests that the amino acid substitutions distinguishing them from Pto and Fen may interfere with recognition of the corresponding signal molecule or with protein-protein interactions involved in the Pto- and Fen-mediated signal transduction pathways.     

beta-Endorphin: structure-activity relationships in the guinea pig ileum and opiate receptor binding assays.

The opiate activities of some derivatives and enzymatic digests of camel and human β-endorphin were determined in the guinea pig ileum and rat brain opiate receptor binding assays. Derivatives of β-endorphins altered within the amino-terminal five residues showed pronounced losses in activity. Anisylation of the C-terminal glutamic acid residue of β_h-endorphin produced only small reductions in activity. Chymotryptic digestion greatly weakened the opiate activities of β_h-endorphin, whereas carboxypeptidase A, tryptic and leucine aminopeptidase digests showed only small losses in potency. The C-terminus of β-endorphin appears to contribute little directly to opiate activity. Amino acid analysis and assay of the leucine aminopeptidase digests suggest that the larger potency of β-endorphin relative to Met-enkephalin may be a consequence of its greater resistance to exopeptidase attack.     

Mechanism of poliovirus inactivation by bromine chloride.

The mechanism of poliovirus inactivation by BrCl was determined by exposing poliovirus to various concentrations of BrCl and correlating the loss of virus infectivity with structural changes of the virus. Concentrations of 0.3 to 5 mg of BrCl per liter resulted in 95% to total inactivation of poliovirus. However, the inactivated virus retained structural integrity, as determined by buoyant density measurements of poliovirus labeled with radioactivity. However, at concentrations of 10 to 20 mg of BrCl per liter, total inactivation of poliovirus was associated with the degradation of the structural integrity of the virus. Since infectious ribonucleic acid at similar concentrations could be recovered from untreated poliovirus and poliovirus treated with 0.3 mg of BrCl per liter, it was concluded that BrCl as HOBr or bromamines inactivates poliovirus by reacting with the protein coat of the virus. Moreover, this inactivating reaction does not result in the degradation of the structure of the virion, nor does it affect the biological activity of the internal ribonucleic acid of the virus.     

Calibrating the Human Mutation Rate via Ancestral Recombination Density in Diploid Genomes

The human mutation rate is an essential parameter for studying the evolution of our species, interpreting present-day genetic variation, and understanding the incidence of genetic disease. Nevertheless, our current estimates of the rate are uncertain. Most notably, recent approaches based on counting de novo mutations in family pedigrees have yielded significantly smaller values than classical methods based on sequence divergence. Here, we propose a new method that uses the fine-scale human recombination map to calibrate the rate of accumulation of mutations. By comparing local heterozygosity levels in diploid genomes to the genetic distance scale over which these levels change, we are able to estimate a long-term mutation rate averaged over hundreds or thousands of generations. We infer a rate of 1.61 ± 0.13 × 10⁻⁸ mutations per base per generation, which falls in between phylogenetic and pedigree-based estimates, and we suggest possible mechanisms to reconcile our estimate with previous studies. Our results support intermediate-age divergences among human populations and between humans and other great apes.     

Unidirectional cross-activation of GRPR by MOR1D uncouples itch and analgesia induced by opioids

Spinal opioid-induced itch, a prevalent side effect of pain management, has been proposed to result from pain inhibition. We now report that the μ-opioid receptor (MOR) isoform MOR1D is essential for morphine-induced scratching (MIS), whereas the isoform MOR1 is required only for morphine-induced analgesia (MIA). MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR) in the spinal cord, relaying itch information. We show that morphine triggers internalization of both GRPR and MOR1D, whereas GRP specifically triggers GRPR internalization and morphine-independent scratching. Providing potential insight into opioid-induced itch prevention, we demonstrate that molecular and pharmacologic inhibition of PLCβ3 and IP3R3, downstream effectors of GRPR, specifically block MIS but not MIA. In addition, blocking MOR1D-GRPR association attenuates MIS but not MIA. Together, these data suggest that opioid-induced itch is an active process concomitant with but independent of opioid analgesia, occurring via the unidirectional cross-activation of GRPR signaling by MOR1D heterodimerization.     

High-Throughput Mutation Profiling Changes before and 3 Weeks after Chemotherapy in Newly Diagnosed Breast Cancer Patients

Background

Changes in tumor DNA mutation status during chemotherapy can provide insights into tumor biology and drug resistance. The purpose of this study is to analyse the presence or absence of mutations in cancer-related genes using baseline breast tumor samples and those obtained after exposure to one cycle of chemotherapy to determine if any differences exist, and to correlate these differences with clinical and pathological features.

Methods

Paired breast tumor core biopsies obtained pre- and post-first cycle doxorubicin (n = 18) or docetaxel (n = 22) in treatment-naïve breast cancer patients were analysed for 238 mutations in 19 cancer-related genes by the Sequenom Oncocarta assay.

Results

Median age of patients was 48 years (range 32–64); 55% had estrogen receptor-positive tumors, and 60% had tumor reduction ≥25% after cycle 1. Mutations were detected in 10/40 (25%) pre-treatment and 11/40 (28%) post-treatment samples. Four mutation pattern categories were identified based on tumor mutation status pre- → post-treatment: wildtype (WT)→WT, n = 24; mutant (MT)→MT, n = 5; MT→WT, n = 5; WT→MT, n = 6. Overall, the majority of tumors were WT at baseline (30/40, 75%), of which 6/30 (20%) acquired new mutations after chemotherapy. Pre-treatment mutations were predominantly in PIK3CA (8/10, 80%), while post-treatment mutations were distributed in PIK3CA, EGFR, PDGFRA, ABL1 and MET. All 6 WT→MT cases were treated with docetaxel. Higher mutant allele frequency in baseline MT tumors (n = 10; PIK3CA mutations n = 8) correlated with less tumor reduction after cycle 1 chemotherapy (R = -0.667, p = 0.035). No other associations were observed between mutation pattern category with treatment, clinicopathological features, and tumor response or survival.

Conclusion

Tumor mutational profiles can change as quickly as after one cycle of chemotherapy in breast cancer. Understanding of these changes can provide insights on potential therapeutic options in residual resistant tumors.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00212082","term_id":"NCT00212082"}}NCT00212082     

Detection and Partial Characterization of Milk vetch dwarf virus Isolates from Faba Bean (Vicia faba L.) in Yunnan Province, China

A virus disease of faba bean ( Vicia faba L.) in China, characterized by leaf yellowing and rolling and plant stunting, was shown to be caused by a virus of the genus Nanovirus based on serological reactions to nanovirus-specific monoclonal antibodies and the generation of polymerase chain reaction amplicons using nanovirus-specific primers. To identify the faba bean-infecting nanovirus, regions of the DNA components encoding the master replication initiator protein and capsid protein of two nanovirus isolates from China were cloned, sequenced and compared with those of other members of the genus Nanovirus . The two Chinese virus isolates shared nucleotide sequence identities ranging from 95 to 98% with the type isolate of Milk vetch dwarf virus (MDV) from Japan. They were thus identified as isolates of MDV, a virus so far known to cause important diseases of legumes in Japan. This is the first record of MDV-infecting faba bean in China.     

Synthesis,Structural and Antioxidant Studies of Some Novel N-Ethyl Phthalimide Esters

A series of N-ethyl phthalimide esters 4(a-n) were synthesized and characterized by spectroscopic studies. Further, the molecular structure of majority of compounds were analysed by single crystal X-ray diffraction studies. The X-ray analysis revealed the importance of substituents on the crystal stability and molecular packing. All the synthesized compounds were tested for in vitro antioxidant activity by DPPH radical scavenging, FRAP and CUPRAC methods. Few of them have shown good antioxidant activity.