Anti-cooperative interactions in single-strand oligomers of deoxyriboadenylic acid

Optical rotatory dispersion measurements were made on the deoxyribo nucleotides d(pA)₂, d(pA)₄, d(pA)₆ and poly(deoxyriboadenylic acid) at neutral pH over the temperature range 5–80°C. and were compared to similar data for the analogous oligoriboadenylic acids. The data were interpreted in terms of a temperature-dependent stacking of the bases in the single-strand deoxyribo oligomers. The thermal transition curves show an inverted chain-length dependence compared to the ribo oligomer curves. These results are explained by a theory of anti-cooperative interaction, where the nucleation parameter σ is >1. The theory, based on a one-dimensional Ising model involving both attractive nearest-neighbor and repulsive next-nearest-neighbor interactions, predicts the inverse chain length dependence and agrees rather well with the experimental data. At and above the transition temperature, the deoxyribo polymer is seen to consist of isolated stacked base pairs separated by at least one unit of random coil, there being only a very small probability for the existence of sequences of stacked residues longer than one. The partition function is seen to undergo an irregular behavior as a function of chain length because of the anti-cooperative phenomenon. It is necessary to use an enthalpy of stacking of ?5.0 kcal./mole in order to fit the experimental data with the theory. This value, 1.5 kcal./mole more positive than the ΔH found for the ribo oligomers, is reasonable, since the 2′ hydroxyl group would be expected to stabilize the stacking interaction in the ribo oligomers. Various kinds of distribution functions are calculated and plotted graphically for this theoretical model. A physical rationale is presented for the use of a repulsive next-nearest-neighbor term in this theory for the deoxyribo oligomers.     

Resistance to Gray Leaf Spot of Maize: Genetic Architecture and Mechanisms Elucidated through Nested Association Mapping and Near-Isogenic Line Analysis

Gray leaf spot (GLS), caused by Cercospora zeae-maydis and Cercospora zeina, is one of the most important diseases of maize worldwide. The pathogen has a necrotrophic lifestyle and no major genes are known for GLS. Quantitative resistance, although poorly understood, is important for GLS management. We used genetic mapping to refine understanding of the genetic architecture of GLS resistance and to develop hypotheses regarding the mechanisms underlying quantitative disease resistance (QDR) loci. Nested association mapping (NAM) was used to identify 16 quantitative trait loci (QTL) for QDR to GLS, including seven novel QTL, each of which demonstrated allelic series with significant effects above and below the magnitude of the B73 reference allele. Alleles at three QTL, qGLS1.04, qGLS2.09, and qGLS4.05, conferred disease reductions of greater than 10%. Interactions between loci were detected for three pairs of loci, including an interaction between iqGLS4.05 and qGLS7.03. Near-isogenic lines (NILs) were developed to confirm and fine-map three of the 16 QTL, and to develop hypotheses regarding mechanisms of resistance. qGLS1.04 was fine-mapped from an interval of 27.0 Mb to two intervals of 6.5 Mb and 5.2 Mb, consistent with the hypothesis that multiple genes underlie highly significant QTL identified by NAM. qGLS2.09, which was also associated with maturity (days to anthesis) and with resistance to southern leaf blight, was narrowed to a 4-Mb interval. The distance between major leaf veins was strongly associated with resistance to GLS at qGLS4.05. NILs for qGLS1.04 were treated with the C. zeae-maydis toxin cercosporin to test the role of host-specific toxin in QDR. Cercosporin exposure increased expression of a putative flavin-monooxygenase (FMO) gene, a candidate detoxification-related gene underlying qGLS1.04. This integrated approach to confirming QTL and characterizing the potential underlying mechanisms advances the understanding of QDR and will facilitate the development of resistant varieties.     

Deregulation of Rab5 and Rab4 proteins in p85R274A-expressing cells alters PDGFR trafficking

Activated receptor tyrosine kinases recruit many signaling proteins to activate downstream cell proliferation and survival pathways, including phosphatidylinositol 3-kinase (PI3K) consisting of a p85 regulatory protein and a p110 catalytic protein. We have recently shown the p85α protein also has in vitro GTPase activating protein (GAP) activity towards Rab5 and Rab4, small GTPases that regulate vesicle trafficking events for activated receptors. Expression of a GAP-defective mutant, p85R274A, resulted in sustained levels of activated platelet-derived growth factor receptors (PDGFRs) and enhanced downstream signaling. In this report we have characterized Rab5- and Rab4-mediated PDGFR trafficking in cells expressing wild type p85 and GAP-defective mutant p85R274A. Wild type p85 overexpressing cells had slower PDGFR trafficking consistent with enhanced GAP activity deactivating Rab5 and Rab4 to block their vesicle trafficking functions. Mutant p85R274A expression increased the internalization rate of PDGFRs, a Rab5-dependent process, without preventing PDGFR ubiquitination. Immunofluorescence studies further demonstrated that p85R274A-expressing cells showed Rab5 accumulation at intracellular locations. Pull-down and FRAP (fluorescence recovery after photobleaching) experiments indicate this is likely membrane-associated Rab5-GTP, sustained due to decreased p85 GAP activity for the p85R274A mutant. These cells also had substantial amounts of activated PDGFRs in Rab4-positive recycling endosomes, a compartment that usually contains primarily deactivated/dephosphorylated receptors. Our results suggest that the PDGFR-associated GAP activity of p85 regulates both Rab5 and Rab4 functions in cells to influence the movement of activated PDGFR through endosomal compartments. Disruption of this regulation by p85R274A expression impacts PDGFR phosphorylation/dephosphorylation, degradation kinetics and downstream signaling by altering the time receptors spend in specific intracellular endosomal compartments. These results demonstrate that the p85α protein is an important regulator of Rab-mediated PDGFR trafficking, which significantly impacts receptor signaling and degradation.     

Rubella vaccine-induced cellular immunity: evidence of associations with polymorphisms in the Toll-like, vitamin A and D receptors, and innate immune response genes

Toll-like, vitamin A and D receptors and other innate proteins participate in various immune functions. We determined whether innate gene-sequence variations are associated with rubella vaccine-induced cytokine immune responses. We genotyped 714 healthy children (11–19 years of age) after two doses of rubella-containing vaccine for 148 candidate SNP markers. Rubella virus-induced cytokines were measured by ELISA. Twenty-two significant associations (range of P values 0.002–0.048) were found between SNPs in the vitamin A receptor family (RARA, RARB, TOP2B and RARG), vitamin D receptor and downstream mediator of vitamin D signaling (RXRA) genes and rubella virus-specific (IFN-γ, IL-2, IL-10, TNF-α, and GM-CSF) cytokine immune responses. A TLR3 gene promoter region SNP (rs5743305, −8441A > T) was associated with rubella-specific GM-CSF secretion. Importantly, SNPs in the TRIM5 gene coding regions, rs3740996 (His43Tyr) and rs10838525 (Gln136Arg), were associated with an allele dose-related secretion of rubella virus-specific TNF-α and IL-2/GM-CSF, respectively, and have been previously shown to have functional consequences regarding the antiviral activity and susceptibility to HIV-1 infection. We identified associations between individual SNPs and haplotypes in, or involving, the RIG-I (DDX58) gene and rubella-specific TNF-α secretion. This is the first paper to present evidence that polymorphisms in the TLR, vitamin A, vitamin D receptor, and innate immunity genes can influence adaptive cytokine responses to rubella vaccination.     

Maturation of the epithelial Na+ channel involves proteolytic processing of the alpha- and gamma-subunits

The epithelial Na+ channel (ENaC) is a tetramer of two alpha-, one beta-, and one gamma-subunit, but little is known about its assembly and processing. Because co-expression of mouse ENaC subunits with three different carboxyl-terminal epitope tags produced an amiloride-sensitive sodium current in oocytes, these tagged subunits were expressed in both Chinese hamster ovary or Madin-Darby canine kidney type 1 epithelial cells for further study. When expressed alone alpha-(95 kDa), beta-(96 kDa), and gamma-subunits (93 kDa) each produced a single band on SDS gels by immunoblotting. However, co-expression of alphabetagammaENaC subunits revealed a second band for each subunit (65 kDa for alpha, 110 kDa for beta, and 75 kDa for gamma) that exhibited N-glycans that had been processed to complex type based on sensitivity to treatment with neuraminidase, resistance to cleavage by endoglycosidase H, and GalNAc-independent labeling with [3H]Gal in glycosylation-defective Chinese hamster ovary cells (ldlD). The smaller size of the processed alpha- and gamma-subunits is also consistent with proteolytic cleavage. By using alpha- and gamma-subunits with epitope tags at both the amino and carboxyl termini, proteolytic processing of the alpha- and gamma-subunits was confirmed by isolation of an additional epitope-tagged fragment from the amino terminus (30 kDa for alpha and 18 kDa for gamma) consistent with cleavage within the extracellular loop. The fragments remain stably associated with the channel as shown by immunoblotting of co-immunoprecipitates, suggesting that proteolytic cleavage represents maturation rather than degradation of the channel.     

Protein denaturant binding polynomials

We show how moments of the denaturant binding distribution function can be extracted from experimental data on the denaturation of a protein as a function of the concentration of denaturant and how in turn these moments can be used to construct the denaturant binding distribution function. This approach is similar to our recent work on using the maximum-entropy method to construct ligand-binding distributions from moments obtained from titration curves for nucleic acids and proteins. As an example we take literature data on the denaturation of ferro- and ferricytochrome c by guanidine hydrochloride and from it construct the denaturant binding polynomial and binding distribution function for the unfolded protein.     

Tomicus piniperda (Coleoptera: Scolytidae) initial flight and shoot departure along a north-south gradient

The exotic pine shoot beetle, Tomicus piniperda (L.) (Coleoptera: Scolytidae), established in the north central and northeastern United States (U.S.) and adjacent regions in Canada, is regulated by a federal quarantine that restricts movement of pine material during specific times of the year based on the beetle's life history. Although climatic variation occurs across T. piniperda's range, a single set of dates is used for timing the movement of pine logs. We monitored T. piniperda spring flight, fall shoot departure, and air and internal tree temperatures at three sites along a 300-km north-south gradient in Michigan and Indiana. We also estimated dates for initial spring flight (12 degrees C threshold) and fall shoot departure (0 degrees C threshold) across an 850-km gradient using historical temperature records (1901 to 1999). Average daily temperatures in fall 1997 (8 October to 12 December) and spring 1998 (20 February to 21 April) were 1.8 to 2.4 degrees C colder, respectively, at the northern field site than at the southern field site. Fall shoot departure began at approximately the same time (day 289 to 290) at all three field sites, but complete shoot departure was extended by 3 wk at the southern site (day 336) compared with the northern site (day 317). T. piniperda adults were first captured in funnel traps on calendar day 86 at the northern site and on day 59 at the central and southern field sites. Peak flight occurred at approximately the same time (day 86) at all three sites. Within-shoot temperatures were very similar to air temperatures in the fall and aboveground inside-bark temperatures were similar to air temperatures in the spring. Average predicted dates based on historical temperature records varied by 31 d for initial shoot departure and 84 d for initial spring flight between northern Michigan and southern Indiana. Because considerable variation can occur in T. piniperda behavior across a broad geographic range, dates specified in the U.S. Federal quarantine should be adjusted according to local temperatures.     

Free energy distributions in proteins.

Protein molecules in solution have a broad distribution of enthalpy states. A good approximation to the distribution function for enthalpy states can be calculated, using the maximum-entropy method, from the moments of the distribution that, in turn, are obtained from the experimental temperature dependence of the heat capacity. In the present paper, we show that the enthalpy probability distribution can then be formulated in terms of a free energy function that gives the free energy of the protein corresponding to a particular value of the enthalpy. By the location of the minima in this function, the free energy distribution graphically indicates the most probable values of the enthalpy for the protein. We find that the behavior of the free energy functions for proteins falls somewhere between two different cases: a two-state like function with two minima, the relative levels of the two states changing with temperature; and, a single-minimum function where the position of the minimum shifts to higher enthalpy values as the temperature is increased. We show that the temperature dependence of the free energy function can be expressed in terms of a central free energy distribution for a given, fixed temperature (which is most conveniently chosen as the temperature of the maximum in the heat capacity). The nature of this central free energy function for a given protein thus yields all of the thermodynamic behavior of that protein over the temperature range of the denaturation process.     

The persistence exponent of DNA

Using the complete genome of Thermoplasma volcanium, as an example, we have examined the distribution functions for the amount of C or G in consecutive, non-overlapping blocks of m bases in this system. We find that these distributions are very much broader (by many factors) than those expected for a random distribution of bases. If we plot the widths of the C-G distributions relative to the widths expected for random distributions, as a function of the block size used, we obtain a power law with a characteristic exponent. The broadening of the C-G distributions follows from the empirical finding that blocks containing a given C-G content tend to be followed by blocks of similar C-G content thus indicating a statistical persistence of composition. The exponent associated with the power law thus measures the strength of persistence in a given DNA. This behavior can be understood using Mandelbrot's model of a fractional Brownian walk. In this model there is a hierarchy of persistence (correlation between blocks) between all parts of the system. The model gives us a way to scale the C-G distributions such that all these functions are collapsed onto a master curve. For a fractional Brownian walk, the fractal dimension of the C-G distribution is simply related to the persistence exponent for the power law. The persistence exponent for T. volcanium is found to be gamma = 0.29 while for a 10 million base segment of the human genome we obtain gamma = 0.39, similar to but not identical with the value found for the microbe.     

Clathrin-mediated endocytosis of MUC1 is modulated by its glycosylation state

MUC1 is a mucin-like type 1 transmembrane protein associated with the apical surface of epithelial cells. In human tumors of epithelial origin MUC1 is overexpressed in an underglycosylated form with truncated O-glycans and accumulates in intracellular compartments. To understand the basis for this altered subcellular localization, we compared the synthesis and trafficking of various glycosylated forms of MUC1 in normal (Chinese hamster ovary) cells and glycosylation-defective (ldlD) cells that lack the epimerase to make UDP-Gal/GalNAc from UDP-Glc/GlcNAc. Although the MUC1 synthesized in ldlD cells was rapidly degraded, addition of GalNAc alone to the culture media resulted in stabilization and near normal surface expression of MUC1 with truncated but sialylated O-glycans. Interestingly, the initial rate of endocytosis of this underglycosylated MUC1 was stimulated by twofold compared with fully glycosylated MUC1. However, the half-lives of the two forms were not different, indicating that trafficking to lysosomes was not affected. Both the normal and stimulated internalization of MUC1 could be blocked by hypertonic media, a hallmark of clathrin-mediated endocytosis. MUC1 endocytosis was also blocked by expression of a dominant-negative mutant of dynamin-1 (K44A), and MUC1 was observed in both clathrin-coated pits and vesicles by immunoelectron microscopy of ultrathin cryosections. Our data suggest that the subcellular redistribution of MUC1 in tumor cells could be a direct result of altered endocytic trafficking induced by its aberrant glycosylation; potential models are discussed. These results also implicate a new role for O-glycans on mucin-like membrane proteins entering the endocytic pathway through clathrin-coated pits.