New surface contacts formed upon reductive lysine methylation: Improving the probability of protein crystallization

Surface lysine methylation (SLM) is a technique for improving the rate of success of protein crystallization by chemically methylating lysine residues. The exact mechanism by which SLM enhances crystallization is still not clear. To study these mechanisms, and to analyze the conditions where SLM will provide the optimal benefits for rescuing failed crystallization experiments, we compared 40 protein structures containing N,N-dimethyl-lysine (dmLys) to a nonredundant set of 18,972 nonmethylated structures from the PDB. By measuring the relative frequency of intermolecular contacts (where contacts are defined as interactions between the residues in proximity with a distance of 3.5 Å or less) of basic residues in the methylated versus nonmethylated sets, dmLys-Glu contacts are seen more frequently than Lys-Glu contacts. Based on observation of the 10 proteins with both native and methylated structures, we propose that the increased rate of contact for dmLys-Glu is due to both a slight increase in the number of amine-carboxyl H-bonds and to the formation of methyl C–H···O interactions. By comparing the relative contact frequencies of dmLys with other residues, the mechanism by which methylation of lysines improves the formation of crystal contacts appears to be similar to that of Lys to Arg mutation. Moreover, analysis of methylated structures with the surface entropy reduction (SER) prediction server suggests that in many cases SLM of predicted SER sites may contribute to improved crystallization. Thus, tools that analyze protein sequences and mark residues for SER mutation may identify proteins with good candidate sites for SLM.     

Small marker chromosomes in two patients with segmental aneusomy for proximal 17p

We report a nine-year-old girl (patient 1934) and a five-year-old boy (patient 2170) with small, de novo supernumerary marker chromosomes (SMCs) derived from proximal 17p. The clinical features of patient 1934 include developmental delay, triangular face, prominent forehead, low set ears, dental abnormalities, a high arched palate, long, flexible fingers, and joint laxity. Patient 2170 is affected with developmental delay, oral-motor dyspraxia/verbal apraxia, thick upper and lower lips, bilateral fifth finger clinodactyly, joint laxity and mild hypotonia. G-banded chromosome analysis of patient 1934 revealed mosaicism for a SMC in 72% of peripheral lymphocytes analyzed, whereas analysis of patient 2170 identified a smaller SMC present in 100% of cells analyzed. Fluorescence in situ hybridization (FISH) studies demonstrated that both of the SMCs derived from 17p10-p11.2. Using FISH and array-CGH analysis, the proximal breakpoints mapped within the centromere and the distal breakpoints were both located within the Smith-Magenis syndrome (SMS) common deletion region. We compare the clinical characteristics of our patients with those previously reported to have either SMC including 17p or duplications of proximal 17p in an effort to further delineate the phenotype of trisomy 17p10-p11.2 and to elucidate genotype-phenotype correlations.     

Changing excitation and inhibition in simulated neural networks: effects on induced bursting behavior

 The development of synchronous bursting in neuronal ensembles represents an important change in network behavior. To determine the influences on development of such synchronous bursting behavior we study the dynamics of small networks of sparsely connected excitatory and inhibitory neurons using numerical simulations. The synchronized bursting activities in networks evoked by background spikes are investigated. Specifically, patterns of bursting activity are examined when the balance between excitation and inhibition on neuronal inputs is varied and the fraction of inhibitory neurons in the network is changed. For quantitative comparison of bursting activities in networks, measures of the degree of synchrony are used. We demonstrate how changes in the strength of excitation on inputs of neurons can be compensated by changes in the strength of inhibition without changing the degree of synchrony in the network. The effects of changing several network parameters on the network activity are analyzed and discussed. These changes may underlie the transition of network activity from normal to potentially pathologic (e.g., epileptic) states. Received: 21 May 2002 / Accepted in revised form: 3 December 2002 / Published online: 7 March 2003 Correspondence to: P. Kudela (e-mail: pkudela@jhmi.edu) Acknowledgements. This research was supported by NIH grant NS 38958.     

Arabidopsis thaliana Ogg1 protein excises 8-hydroxyguanine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine from oxidatively damaged DNA containing multiple lesions

A functional homologue of eukaryotic Ogg1 proteins in the model plant Arabidopsis thalianahas recently been cloned, isolated, and characterized [Garcia-Ortiz, M. V., Ariza, R. R., and Roldan-Arjona, T. (2001) Plant Mol. Biol. 47, 795-804]. This enzyme (AtOgg1) exhibits a high degree of sequence similarity in several highly conserved regions with Saccharomyces cerevisiae, Drosophila melanogaster, and human Ogg1 proteins. We investigated the substrate specificity and kinetics of AtOgg1 for excision of modified bases from oxidatively damaged DNA that contained multiple pyrimidine- and purine-derived lesions. Two different DNA substrates prepared by exposure to ionizing radiation in aqueous solution under N2O or air were used for this purpose. Gas chromatography/isotope-dilution mass spectrometry was applied to identify and quantify modified bases in DNA samples. Of the 17 modified bases identified in DNA samples, only 8-hydroxyguanine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine were significantly excised from both DNA substrates. This is in agreement with the substrate specificities of other eukaryotic Ogg1 proteins that had previously been studied under identical conditions. Excision depended on incubation time, enzyme concentration, and substrate concentration and followed Michaelis-Menten kinetics. A significant dependence of excision on the nature of DNA substrate was observed in accord with previous studies on other DNA glycosylases. A comparison of excision kinetics pointed to significant differences between AtOgg1 and other Ogg1 proteins. We also investigated the effect of base-pairing on the excision using double-stranded oligodeoxynucleotides that contained 8-OH-Gua paired with each of the four DNA bases. The activity of AtOgg1 was most effective on the 8-OH-Gua:C pair with some or very low activity on other pairs in agreement with the activity of other Ogg1 proteins. The results unequivocally show that AtOgg1 possesses common substrates with other eukaryotic Ogg1 proteins albeit significant differences between their excision kinetics.     

Strain fidelity of chronic wasting disease upon murine adaptation

Chronic wasting disease (CWD), a prion disease of deer and elk, is highly prevalent in some regions of North America. The establishment of mouse-adapted CWD prions has proven difficult due to the strong species barrier between mice and deer. Here we report the efficient transmission of CWD to transgenic mice overexpressing murine PrP. All mice developed disease 500 +/- 62 days after intracerebral CWD challenge. The incubation period decreased to 228 +/- 103 days on secondary passage and to 162 +/- 6 days on tertiary passage. Mice developed very large, radially structured cerebral amyloid plaques similar to those of CWD-infected deer and elk. PrP(Sc) was detected in spleen, indicating that murine CWD was lymphotropic. PrP(Sc) glycoform profiles maintained a predominantly diglycosylated PrP pattern, as seen with CWD in deer and elk, across all passages. Therefore, all pathological, biochemical, and histological strain characteristics of CWD appear to persist upon repetitive serial passage through mice. These findings indicate that the salient strain-specific properties of CWD are encoded by agent-intrinsic components rather than by host factors.     

Type-1 polarized dendritic cells primed for high IL-12 production show enhanced activity as cancer vaccines

While multiple pathways of dendritic cell (DC) maturation result in transient production of IL-12, fully mature DCs show reduced ability to produce IL-12p70 upon a subsequent interaction with Ag-specific T cells, limiting their in vivo performance as vaccines. Such “DC exhaustion” can be prevented by the presence of IFNγ during the maturation of human DCs (type-1-polarization), resulting in improved induction of tumor-specific Th1 and CTL responses in vitro. Here, we show that type-1 polarization of mouse DCs strongly enhances their ability to induce CTL responses against a model tumor antigen, OVA, in vivo, promoting the induction of protective immunity against OVA-expressing EG7 lymphoma. Interestingly, in contrast to the human system, the induction of mouse DC1s requires the participation of IL-4, a nominal Th2-inducing cytokine. The current data help to explain the previously reported Th1-driving and anti-tumor activities of IL-4, and demonstrate that type-1 polarization increases in vivo activity of DC-based vaccines. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Adam S. Giermasz and Julie A. Urban contributed equally to this work.     

Novel purine and pyrazolo[3,4-d]pyrimidine inhibitors of PI3 kinase-α: Hit to lead studies

Series of purine and pyrazolo[3,4-d]pyrimidine inhibitors of phosphatidylinositol-3-kinases (PI3K) have been prepared. The optimized purine inhibitors show good potency in a PI3K p110α (PI3K-α) fluorescence polarization assay with good selectivity versus PI3K p110γ (PI3K-γ) and the mammalian target of rapamycin (mTOR). The related pyrazolo[3,4-d]pyrimidines show potent PI3K-α and mTOR inhibition with good selectivity versus PI3K-γ. Representative compounds showed activity in a cellular proliferation assay against Caco-2 colorectal, LoVo colorectal and PC3MM2 prostate adenocarcinoma cancer cells. Signaling through the PI3K pathway was confirmed via inhibition of phospho-AKT in MDA-361 cells.