A BASIC microcomputer program to calculate the secondary structure of proteins from their circular dichroism spectrum

A BASIC program (CDPROT) has been developed to calculate thesecondary structure of proteins from their far UV circular dichroismspectrum. This implementation can use different reference spectra,calculated either from model polypeptides or proteins of knowntertiary structure. Apart from obtaining the a-helical, ß-structure,ß-turns or random percentages which would generatethe spectrum of best fit with respect to the experimental measures,CDPROT represents on screen both theoretical and experimentalspectra indicating the root-mean-square error. The provisionof additional reference spectra by the user is also considered,and another program (STOREREF) performs the editing in an adequateformat for CDPROT. Received on March 8, 1988; accepted on June 3, 1988     

DNA surveillance: web-based molecular identification of whales,dolphins, and porpoises

DNA Surveillance is a Web-based application that assists in the identification of the species and population of unknown specimens by aligning user-submitted DNA sequences with a validated and curated data set of reference sequences. Phylogenetic analyses are performed and results are returned in tree and table format summarizing the evolutionary distances between the query and reference sequences. DNA Surveillance is implemented with mitochondrial DNA (mtDNA) control region sequences representing the majority of recognized cetacean species. Extensions of the system to include other gene loci and taxa are planned. The service, including instructions and sample data, is available at http://www.dna-surveillance.auckland.ac.nz.     

Cardiac sodium channel Na(v)1.5 interacts with and is regulated by the protein tyrosine phosphatase PTPH1

In order to identify proteins interacting with the cardiac voltage-gated sodium channel Na(v)1.5, we used the last 66 amino acids of the C-terminus of the channel as bait to screen a human cardiac cDNA library. We identified the protein tyrosine phosphatase PTPH1 as an interacting protein. Pull-down experiments confirmed the interaction, and indicated that it depends on the PDZ-domain binding motif of Na(v)1.5. Co-expression experiments in HEK293 cells showed that PTPH1 shifts the Na(v)1.5 availability relationship toward hyperpolarized potentials, whereas an inactive PTPH1 or the tyrosine kinase Fyn does the opposite. The results of this study suggest that tyrosine phosphorylation destabilizes the inactivated state of Na(v)1.5.     

Poliovirus induces autophagic signaling independent of the ULK1 complex

Poliovirus (PV), like many positive-strand RNA viruses, subverts the macroautophagy/autophagy pathway to promote its own replication. Here, we investigate whether the virus uses the canonical autophagic signaling complex, consisting of the ULK1/2 kinases, ATG13, RB1CC1, and ATG101, to activate autophagy. We find that the virus sends autophagic signals independent of the ULK1 complex, and that the members of the autophagic complex are not required for normal levels of viral replication. We also show that the SQSTM1/p62 receptor protein is not degraded in a conventional manner during infection, but is likely cleaved in a manner similar to that shown for coxsackievirus B3. This means that SQSTM1, normally used to monitor autophagic degradation, cannot be used to accurately monitor degradation during poliovirus infection. In fact, autophagic degradation may be affected by the loss of SQSTM1 at the same time as autophagic signals are being sent. Finally, we demonstrate that ULK1 and ULK2 protein levels are greatly reduced during PV infection, and ATG13, RB1CC1, and ATG101 protein levels are reduced as well. Surprisingly, autophagic signaling appears to increase as ULK1 levels decrease. Overexpression of wild-type or dominant-negative ULK1 constructs does not affect virus replication, indicating that ULK1 degradation may be a side effect of the ULK1-independent signaling mechanism used by PV, inducing complex instability. This demonstration of ULK1-independent autophagic signaling is novel and leads to a model by which the virus is signaling to generate autophagosomes downstream of ULK1, while at the same time, cleaving cargo receptors, which may affect cargo loading and autophagic degradative flux. Our data suggest that PV has a finely-tuned relationship with the autophagic machinery, generating autophagosomes without using the primary autophagy signaling pathway.

Abbreviations: ACTB - actin beta; ATG13 - autophagy related 13; ATG14 - autophagy related 14; ATG101 - autophagy related 101; BECN1 - beclin 1; CVB3 - coxsackievirus B3; DMV - double-membraned vesicles; EM - electron microscopy; EMCV - encephalomyocarditis virus; EV-71 - enterovirus 71; FMDV - foot and mouth disease virus; GFP - green fluorescent protein; MAP1LC3B/LC3B - microtubule associated protein 1 light chain 3 beta; MOI - multiplicity of infection; MTOR - mechanistic target of rapamycin kinase; PIK3C3 - phosphatidylinositol 3-kinase catalytic subunit type 3; PRKAA2 - protein kinase AMP-activated catalytic subunit alpha 2; PSMG1 - proteasome assembly chaperone 1; PSMG2 - proteasome assembly chaperone 2PV - poliovirus; RB1CC1 - RB1 inducible coiled-coil 1; SQSTM1 - sequestosome 1; ULK1 - unc-51 like autophagy activating kinase 1; ULK2 - unc-51 like autophagy activating kinase 2; WIPI1 - WD repeat domain, phosphoinositide interacting 1     

High temperature effects on photosynthetic activity of two tomato cultivars with different heat susceptibility

The functional activities of the photosynthetic apparatus of two tomato cultivars of different thermotolerance were investigated after a short period of high temperature treatment. Seedlings of two tomato genotypes, Lycopersicon esculentum var. Campbell-28 and the wild thermotolerant Nagcarlang, were grown under a photoperiod of 16h at 25 degrees C and dark period of 8h at 20 degrees C. At the fourth true leaf stage, a group of plants was exposed to heat stress of 45 degrees C for 2 h. The heat shock treatment caused important reductions of the net photosynthetic rate (Pn) of Campbell-28 plants due to non-stomatal components. These non-stomatal effects were not evident in Nagcarlang-treated plants. This reduction in the CO2 assimilation rate observed in Campbell-28 was generated by affections in the Calvin cycle and also in the PSII functioning. No changes in these parameters were observed in the thermotolerant genotype after the stress. Injury to the plasma membrane because of the heat stress was evident only in the Campbell-28 genotype. Heat led to a sun-type adaptation response of the photosynthesis pigment apparatus for the Nagcarlang genotype, but not for Campbell-28, and thus an increase in chlorophyll a/b ratio and a decrease in chlorophyll/carotenoid ratio were shown in Nagcarlang stressed plants.     

Detection of antigens on nitrocellulose paper immunoblots with monoclonal antibodies

A very sensitive method for the detection of antigen-antibody complexes on nitrocellulose paper immunoblots is described. The protein antigens are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by their electrophoretic transfer onto a nitrocellulose sheet (“Western blot”). The protein antigens bound to the nitrocellulose paper are exposed to the monoclonal antibody and the antibody-antigen complexes are detected on the paper by an immunoenzymatic reaction. The improved sensitivity of this method is the result of (i) the use of the detergent Tween 20 in blocking the nonspecific binding of the antibodies to the nitrocellulose paper, (ii) the use of a peroxidase-antiperoxidase (PAP) reaction, and (iii) the intensification of the diaminobenzidine reaction product with nickel and cobalt ions in phosphate buffer.     

Selected factors limiting the microbial degradation of recalcitrant compounds

Summary The focus of this review is to examine some of the reasons biodegradation may not take place in the environment even though its occurrence in the laboratory has been demonstrated. Some approaches for dealing with chemical persistence will be discussed. In addition, the potential of bioremediation as an in situ clean-up technology will be considered.