Disruption of the non-canonical Wnt gene PRICKLE2 leads to autism-like behaviors with evidence for hippocampal synaptic dysfunction.

Autism spectrum disorders (ASDs) have been suggested to arise from abnormalities in the canonical and non-canonical Wnt signaling pathways. However, a direct connection between a human variant in a Wnt pathway gene and ASD-relevant brain pathology has not been established. Prickle2 (Pk2) is a post-synaptic non-canonical Wnt signaling protein shown to interact with post-synaptic density 95 (PSD-95). Here, we show that mice with disruption in Prickle2 display behavioral abnormalities including altered social interaction, learning abnormalities and behavioral inflexibility. Prickle2 disruption in mouse hippocampal neurons led to reductions in dendrite branching, synapse number and PSD size. Consistent with these findings, Prickle2 null neurons show decreased frequency and size of spontaneous miniature synaptic currents. These behavioral and physiological abnormalities in Prickle2 disrupted mice are consistent with ASD-like phenotypes present in other mouse models of ASDs. In 384 individuals with autism, we identified two with distinct, heterozygous, rare, non-synonymous PRICKLE2 variants (p.E8Q and p.V153I) that were shared by their affected siblings and inherited paternally. Unlike wild-type PRICKLE2, the PRICKLE2 variants found in ASD patients exhibit deficits in morphological and electrophysiological assays. These data suggest that these PRICKLE2 variants cause a critical loss of PRICKLE2 function. The data presented here provide new insight into the biological roles of Prickle2, its behavioral importance, and suggest disruptions in non-canonical Wnt genes such as PRICKLE2 may contribute to synaptic abnormalities underlying ASDs.

Efficacy comparison between intraportal and subcapsular islet transplants in a murine diabetic model.

BACKGROUND: It is important to determine the efficacy of intraportal (IP) islet transplantation in comparison with other transplant sites. In this study, we sought to determine the optimal number of islets to achieve normoglycemia following transplantation into the liver versus the kidney using a mouse model. METHODS: Streptozotocin-induced diabetic mice (Balb/C) were transplanted with syngeneic islets via the IP versus renal subcapsular (SC) routes. The transplanted islet numbers were 0 to 800 (n = 3-5). We assessed the correlation between parameters and islet numbers, comparing IP versus SC groups. The parameters were: (1) percentage of normoglycemia; (2) postoperative days to normoglycemia; (3) mean blood glucose levels at various points from pretransplantation to the end of the study (postoperative day 28); (4) mean serum insulin; and (5) area under the curve of blood glucose levels after glucose injection. RESULTS: Two hundred islets yielded normoglycemia in renal subcapsular grafts, while 800 islets were the minimum required for normoglycemia with IP transplantation. The transplant efficacy in SC transplantation was 2 to 5 times greater than that of IP transplantation. The days to normoglycemia were significantly different between IP versus renal SC islets (13.25 +/- 4.38 days vs 4.50 +/- 0.81 days; P = .007). CONCLUSION: The efficacy of islet transplantation in murine diabetic models was significantly greater under the kidney capsule. Clinical islet transplantation could benefit from trials of alternative transplant sites.

Effect of mode of birth on purine and malondialdehyde in umbilical arterial plasma in normal term newborns.

OBJECTIVE: To examine the effect of mode of birth on plasma purine and malondialdehyde levels in normal term infants. STUDY DESIGN: Umbilical arterial cord blood was obtained immediately after birth from a convenience sample of 119 normal term newborns born by vaginal delivery, with or without oxytocin augmentation or by elective cesarean delivery. Plasma was analyzed for purine and/or malondialdehyde levels. Numeric data were analyzed utilizing independent samples t-test and ordinal data were analyzed using Mann-Whitney test. Correlation coefficients were obtained using Spearman's rho. RESULT: Uric acid levels were significantly elevated (P<0.001) in neonates undergoing vaginal birth, compared to neonates born by elective cesarean delivery. When the effect of oxytocin and length of labor was analyzed, neonates born to mothers on oxytocin had lower hypoxanthine, significantly lower xanthine (P=0.05) and higher uric acid levels. In addition, malondialdehyde levels were significantly higher (P<0.006) in neonates born to mothers who received oxytocin compared to neonates born to mothers without oxytocin augmentation. We also found significant correlations between malondialdehyde (MDA) and hypoxanthine (r=-0.465, P<0.039) and between MDA and xanthine (r=-0.753, P=0.003) in neonates born via oxytocin-augmented birth. Mode of birth had no statistically significant effect on clinical outcomes, although infants born by elective cesarean had higher incidence of acute respiratory distress and transient tachypnea of the newborn compared to those born vaginally. CONCLUSION: Neonates born by elective cesarean had the lowest purine levels in cord blood compared to neonates born vaginally. Oxytocin augmentation is associated with some degree of uterine hyperstimulation which may enhance the ATP degradation pathway resulting in the rapid conversion of hypoxanthine to xanthine and xanthine to uric acid. Significantly higher MDA levels in neonates whose mothers received oxytocin as well as significant correlation between MDA and the purines hypoxanthine and xanthine, suggest free-radical production, most likely due to xanthine oxidase activation. However, despite differences in plasma purine and malondialdehyde levels, no significant differences were seen in neonatal outcome. Further studies are required to fully characterize the effect of mode of birth on purine metabolism and free-radical production.

Elevated total peripheral leukocyte count may identify risk for neurological disability in asphyxiated term neonates.

OBJECTIVE: The present study investigated the relationship between neurologic outcome and total circulating white blood cell (WBC) and absolute neutrophil counts (ANCs) in the first week of life in term infants with hypoxic-ischemic encephalopathy (HIE). STUDY DESIGN: Long-term neurologic outcome at 18 months was measured retrospectively in 30 term neonates with HIE using the Pediatric Cerebral Performance Category Scale (PCPCS) score with outcomes dichotomized as either good or poor. We then compared white blood cell and ANC levels during the first 4 days of life and magnetic resonance imaging (MRI) obtained within the first month life between the two PCPCS groups. MRI was quantified using a validated scoring system. RESULTS: Neonates with good long-term outcomes had significantly lower MRI scores (indicating lesser injury) than neonates with poor outcomes. More importantly, neonates with poor outcomes had significantly higher WBC and ANC levels as early as12 h after birth and up to 96 h after birth compared to those with good outcomes. These data suggest that elevated peripheral neutrophil counts in the first 96 h of life may signal or predict adverse long-term outcome. CONCLUSIONS: Our findings suggest that elevated peripheral neutrophil counts in the first 96 h of life in term infants with HIE may contribute to abnormal neurodevelopmental outcome.

8-oxo-7,8-dihydroguanine is removed by a nucleotide excision repair-like mechanism in Porphyromonas gingivalis W83.

A consequence of oxidative stress is DNA damage. The survival of Porphyromonas gingivalis in the inflammatory microenvironment of the periodontal pocket requires an ability to overcome oxidative stress caused by reactive oxygen species (ROS). 8-oxo-7,8-dihydroguanine (8-oxoG) is typical of oxidative damage induced by ROS. There is no information on the presence of 8-oxoG in P. gingivalis under oxidative stress conditions or on a putative mechanism for its repair. High-pressure liquid chromatography with electrochemical detection analysis of chromosomal DNA revealed higher levels of 8-oxoG in P. gingivalis FLL92, a nonpigmented isogenic mutant, than in the wild-type strain. 8-oxoG repair activity was also increased in cell extracts from P. gingivalis FLL92 compared to those from the parent strain. Enzymatic removal of 8-oxoG was catalyzed by a nucleotide excision repair (NER)-like mechanism rather than the base excision repair (BER) observed in Escherichia coli. In addition, in comparison with other anaerobic periodontal pathogens, the removal of 8-oxoG was unique to P. gingivalis. Taken together, the increased 8-oxoG levels in P. gingivalis FLL92 could further support a role for the hemin layer as a unique mechanism in oxidative stress resistance in this organism. In addition, this is the first observation of an NER-like mechanism as the major mechanism for removal of 8-oxoG in P. gingivalis.

An unexpectedly high excision capacity for mispaired 5-hydroxymethyluracil in human cell extracts.

The oxidation of thymine in DNA can generate a base pair between 5-hydroxymethyluracil (HmU) and adenine, whereas the oxidation and deamination of 5-methylcytosine (5mC) in DNA can generate a base pair between HmU and guanine. Using synthetic oligonucleotides containing HmU at a defined site, HmU-DNA glycosylase activities in HeLa cell and human fibroblast cell extracts have been observed. An HmU-DNA glycosylase activity that removes HmU mispaired with guanine has been measured. Surprisingly, the HmU:G excision activity is 60 times greater than the corresponding HmU:A activity, even though the expected rate of formation of the HmU:A base pair exceeds that of the HmU:G base pair by a factor of 10(7). The HmU:G mispair would arise from the 5mC:G base pair, and, if unrepaired, would give rise to a transition mutation. The observation of an unexpectedly high HmU:G glycosylase activity suggests that human cells may encounter the HmU:G mispair much more frequently than expected. The conversion of 5mC to HmU must be considered as a potential pathway for the generation of 5mC to T transition mutations, which are often found in human tumors.

Examination of the DNA substrate selectivity of DNA cytosine methyltransferases using mass tagging.

The biological significance of cytosine methylation is as yet incompletely understood, but substantial and growing evidence strongly suggests that perturbation of methylation patterns, resulting from the infidelity of DNA cytosine methyltransferase, is an important component of the development of human cancer. We have developed a novel in vitro assay that allows us to quantitatively determine the DNA substrate preferences of cytosine methylases. This approach, which we call mass tagging, involves the labeling of target cytosine residues in synthetic DNA duplexes with stable isotopes, such as (15)N. Methylation is then measured by the formation of 5-methylcytosine (5mC) by gas chromatography/mass spectrometry. The DNA substrate selectivity is determined from the mass spectrum of the product 5mC. With the non-symmetrical duplex DNA substrate examined in this study we find that the bacterial methyltransferase HPA:II (duplex DNA recognition sequence CCGG) methylates the one methylatable cytosine of each strand similarly. Introduction of an A-C mispair at the methylation site shifts methylation exclusively to the mispaired cytosine residue. In direct competition assays with HPA:II methylase we observe that the mispaired substrate is methylated more extensively than the fully complementary, normal substrate, although both have one HPA:II methylation site. Through the use of this approach we will be able to learn more about the mechanisms by which methylation patterns can become altered.

Multiple structures for the 2-aminopurine-cytosine mispair.

The base pair formed between 2-aminopurine (2AP) and cytosine (C) is an intermediate in transition mutations generated by 2AP. To date, several structures have been proposed for the 2AP-C mispair, including those involving a rare tautomer, a protonated base pair, and a neutral wobble structure. In this paper, we describe a series of UV, fluorescence, and NMR studies which demonstrate that an equilibrium exists between the neutral wobble and the protonated Watson-Crick structures. The apparent pK value for the transition between the structures is 5.9-6.0. Formation of a Watson-Crick base pair is accomplished predominantly by protonation of the 2AP residue as indicated by UV spectral changes, fluorescence quenching, and changes in proton chemical shifts. Rapid transfer of the shared proton between the 2AP and cytosine residues is indicated by the rapid exchange of the cytosine amino protons of the protonated Watson-Crick configuration. The relative contribution of the neutral wobble and protonated Watson-Crick configurations to 2AP-induced transition mutations is discussed.

Conformation and proton configuration of pyrimidine deoxynucleoside oxidation damage products in water.

Emerging data strongly suggest that the oxidation of DNA bases can contribute to genomic instability. Structural changes to DNA, induced by base oxidation, may reduce the fidelity of DNA replication and interfere with sequence-specific DNA-protein interactions. We have examined the structures of a series of pyrimidine deoxynucleoside oxidation damage products in aqueous solution. The modified nucleosides studied include the deoxynucleoside derivatives of 5-hydroxyuracil, 5-hydroxycytosine, 5-(hydroxymethyl)uracil, 5-(hydroxymethyl)cytosine, 5-formyluracil, and 5-formylcytosine. The influence of base oxidation on ionization constants, sugar conformation, and tautomeric configuration has been determined on the basis of UV, proton, and nitrogen NMR spectra of the (15)N-enriched derivatives. The potential biological consequences of the structural perturbations resulting from base oxidation are discussed.

15N-enriched 5-fluorocytosine as a probe for examining unusual DNA structures.

A new method is presented for the synthesis of oligonucleotides containing 15N-enriched 5-fluorocytosine (FC). Due to the reduced pK of FC, the amino protons of an unpaired FC residue may be observed at lower values of solution pH. The labeled FC residue has been placed as a template base at a model DNA replication fork. The amino protons of the FC residue have been identified in isotope-edited NMR spectra. Data is presented for a template FC residue unpaired, paired with guanine, and mispaired with adenine. These studies demonstrate the utility of labeled FC in examining unusual DNA structures.

Endogenous bile acids are ligands for the nuclear receptor FXR/BAR.

The major metabolic pathway for elimination of cholesterol is via conversion to bile acids. In addition to this metabolic function, bile acids also act as signaling molecules that negatively regulate their own biosynthesis. However, the precise nature of this signaling pathway has been elusive. We have isolated an endogenous biliary component (chenodeoxycholic acid) that selectively activates the orphan nuclear receptor, FXR. Structure-activity analysis defined a subset of related bile acid ligands that activate FXR and promote coactivator recruitment. Finally, we show that ligand-occupied FXR inhibits transactivation from the oxysterol receptor LXR alpha, a positive regulator of cholesterol degradation. We suggest that FXR (BAR) is the endogenous bile acid sensor and thus an important regulator of cholesterol homeostasis.

Identification by UV resonance Raman spectroscopy of an imino tautomer of 5-hydroxy-2'-deoxycytidine, a powerful base analog transition mutagen with a much higher unfavored tautomer frequency than that of the natural residue 2'-deoxycytidine.

UV resonance Raman spectroscopy was used to detect and estimate the frequency of the unfavored imino tautomer of the transition mutagen 5-hydroxy-2'-deoxycytidine (HO5dCyt) in its anionic form. In DNA, this 2'-deoxycytidine analog arises from the oxidation of 2'-deoxycytidine and induces C --> T transitions with 10(2) greater frequency than such spontaneous transitions. An imino tautomer marker carbonyl band (approximately 1650 cm-1) is enhanced at approximately 65 degrees C against an otherwise stable spectrum of bands associated with the favored amino tautomer. This band is similarly present in the UV resonance Raman spectra of the imino cytidine analogs N3-methylcytidine at high pH and N4-methoxy-2'-deoxycytidine at pH 7 and displays features attributable to the imino form of C residues and their derivatives. The fact that the imino tautomer of HO5dCyt occurs at a frequency consistent with its high mutagenic enhancement lends strong support to the hypothesis that unfavored base tautomers play important roles in the mispair intermediates of replication leading to substitution mutations.

Interaction of DNA polymerase I (Klenow fragment) with DNA substrates containing extrahelical bases: implications for proofreading of frameshift errors during DNA synthesis.

Frameshift mutagenesis occurs through the misalignment of primer and template strands during DNA synthesis and involves DNA intermediates that contain one or more extrahelical bases in either strand of the DNA substrate. To investigate whether these DNA structures are recognized by the proofreading apparatus of DNA polymerases, time-resolved fluorescence spectroscopy was used to examine the interaction between the Klenow fragment of DNA polymerase I and synthetic DNA primer-templates containing extrahelical bases at defined positions within the template strand. A dansyl probe attached to the DNA was used to measure the fractional occupancies of the polymerase and 3'-5' exonuclease sites of the enzyme for DNA substrates with and without the extrahelical bases. The presence of an extrahelical base at the first position from the primer 3' terminus increased the level of partitioning of the DNA substrates into the 3'-5' exonuclease site by 3-7-fold, relative to the perfectly base-paired primer-template, depending on the identity of the extrahelical base. The ability of different extrahelical bases to promote partitioning of DNA into the 3'-5' exonuclease site decreased in the following order: G > A approximately T > C. The results of partitioning measurements for DNA substrates containing a bulged adenine base at different positions within the template showed that an extrahelical base is recognized up to five bases from the primer 3' terminus. The largest effects were observed for the extrahelical base at the third or fourth positions from the primer terminus, which increased the level of partitioning of DNA into the 3'-5' exonuclease site by 8- and 18-fold, respectively, relative to that of the perfectly base-paired substrate. Steady-state fluorescence measurements of analogous primer-templates containing 2-aminopurine (AP) at the primer 3' terminus indicate that extrahelical bases increase the degree of terminus unwinding, especially when close to the terminus. In addition, steady-state kinetic measurements of removal of AP from the primer-templates indicate that the exonucleolytic cleavage activity of Klenow fragment is correlated with the increased level of partitioning of bulged DNA substrates to the 3'-5' exonuclease site relative to that of properly base-paired DNA. The results of this study indicate that misalignment of primer and template strands to generate an extrahelical base strongly promotes transfer of a DNA substrate to the 3'-5' exonuclease site, suggesting that the premutational intermediates in frameshift mutagenesis are subject to proofreading by the polymerase.

Synthesis and utilization of 13C(8)-enriched purines.

A new and more efficient method is presented for the synthesis of 13C(8)-enriched adenine. In addition, we present for the first time the synthesis of 13C(8)-enriched 2-aminopurine and purine. All three analogues have been converted to the corresponding ribonucleosides. The adenine analogue has been further converted to the 2'-deoxy-nucleoside and incorporated into a synthetic oligonucleotide. Data is presented demonstrating the utility of 13C-enrichment in heteronuclear isotope-edited NMR spectra.

Quantification of 5-(hydroxymethyl)uracil in DNA by gas chromatography/mass spectrometry: problems and solutions.

Oxidation of the thymine methyl group results in the formation of 5-(hydroxymethyl)uracil (HmU). HmU is a recognized endogenous DNA damage product, and HmU levels in DNA are increased by oxidant stress. Previous studies have reported substantially conflicting values for HmU levels in DNA. In studies utilizing postlabeling methods, HmU levels have been reported to be as high as or higher than the levels of some of the more commonly described DNA oxidation damage products such as 8-oxoguanine. In some studies utilizing GC/MS methods, however, HmU has been undetectable. In acid solution, the hydroxymethyl group of HmU can undergo condensation reactions with carboxylic acids, alcohols, and amines. While HmU can be accurately measured by GC/MS, the first step in the preparation of samples for GC/MS analysis is acid hydrolysis of the DNA. Such hydrolysis would be expected to result in substantial derivatization of HmU. We have utilized chemically synthesized oligonucleotides containing a known amount of HmU as well as an isotopically enriched standard to investigate the chemical modification of HmU during the acid hydrolysis of DNA. We conclude that HmU levels reported by GC/MS following acid hydrolysis may be up to an order of magnitude lower than the actual levels. Further, we propose modifications to the standard hydrolysis protocols which maximize recovery of HmU prior to silylation and analysis by GC/MS.

Synthesis and characterization of isotopically enriched pyrimidine deoxynucleoside oxidation damage products.

Oxidative damage to DNA is an established source of genomic instability. In this paper, we describe the synthesis and characterization of several pyrimidine deoxynucleoside oxidation damage products, enriched with stable isotopes. These products include the 2'-deoxynucleoside derivatives of 5-(hydroxymethyl)uracil, 5-formyluracil, 5-hydroxyuracil, 5-(hydroxymethyl)cytosine, 5-formylcytosine, and 5-hydroxycytosine. The common precursor is 2'-deoxy-2"-deutero[1,3-15N]uridine. Additional stable isotopes are added during functional group conversions. Characterization of these derivatives includes mass spectrometry and 1H and 15N NMR spectroscopy. Proton and nitrogen NMR studies reported here allow an examination of the influence of the modification on sugar conformation and tautomeric equilibrium, properties likely to be important in understanding the biological consequences of these DNA damage products.

Synthesis and cleavage of oligodeoxynucleotides containing a 5-hydroxyuracil residue at a defined site.

Oxidation and hydrolysis of a cytosine residue can lead to the formation of 5-hydroxyuracil in DNA. The biological consequences of this modification are not fully understood. To facilitate biochemical and biophysical studies aimed at elucidating the effects of this modification in DNA, we have developed a solid-phase synthetic method for the placement of 5-hydroxyuracil residues at defined sites in oligodeoxynucleotides. This method is based upon the enhanced acidity of the 5-hydroxyl proton which allows selective aqueous acetylation. Under standard aqueous ammonia deprotection conditions, however, we observed that 5-hydroxyuracil residues are lost substantially from synthetic oligonucleotides. Substitution of aqueous ammonia with methanolic potassium carbonate and the use of phosphoramidite derivatives with alternatively protected amino groups allow synthesis of oligonucleotides containing 5-hydroxyuracil and all normal bases in high yield. The composition of the oligodeoxynucleotides prepared by this method has been verified by enzymatic digestion followed by high-performance liquid chromatography (HPLC) analysis as well as acid hydrolysis followed by GC/MS analysis. The location of the 5-hydroxyuracil residue is demonstrated by selective permanganate oxidation of the 5-hydroxyuracil residue followed by beta-elimination. We have also probed a synthetic oligonucleotide containing a unique 5-hydroxyuracil residue with uracil DNA N-glycosylase, previously reported to remove this lesion from DNA.

Linkage between operator binding and dimer to octamer self-assembly of bacteriophage lambda cI repressor.

Cooperative binding of the bacteriophage lambda cI repressor dimer to specific sites of the phage operators OR and OL controls the developmental state of the phage. Cooperativity has long been thought to be mediated by self-assembly of repressor dimers to form tetramers which can bind simultaneously to adjacent operators. More recently, we demonstrated that when free repressor dimers self-associate in solution, tetramer is an intermediate in a concerted assembly reaction leading to octamer as the predominant higher order species [Senear, D. F., et al. (1993) Biochemistry 32, 6179-6189]. Even as a minority component in the assembly reaction, tetramer can account for pairwise cooperativity. In a similar manner, were it able to bind all three operators simultaneously, octamer could account for three-way cooperativity. In fact, based solely on repressor self-assembly, the naive prediction is that the repressor-OR interactions should be substantially more cooperative than they are. Evidently, there are unfavorable contributions to cooperativity from processes other than repressor self-assembly. Here, we focus on coupling between repressor self-association and operator binding as one possible unfavorable contribution to cooperativity. Sedimentation equilibrium analysis was used to compare the dimer-octamer association reactions of a repressor dimer-OR1 complex and free repressor dimer. Fluorescence anisotropy was used to investigate OR1 binding to free dimers and dimers assembled as higher order species. The results of these experiments indicate a significant and salt-dependent unfavorable contribution generated by such coupling. Since the oligonucleotides used in these experiments are the size of single operator sites, this coupling is mediated by the protein, not by the DNA. This mechanism does not account for an additional, salt-independent, unfavorable contribution which we presume is transmitted via the DNA. Thus, unfavorable contributions generated by structural transitions in both macromolecules serve to moderate the effect of self-association alone. We speculate that this is a general feature of cooperative protein-DNA interactions.

Spectroscopic determination of open complex formation at promoters for Escherichia coli RNA polymerase.

A considerable amount of effort has been expended studying the kinetics of association of Escherichia coli RNA polymerase with promoter DNA in forming the open complex. Strand separation occurs over about 12 base pairs and includes the transcription start site. However, these efforts have been significantly hampered by the lack of a sensitive, real time method by which formation of an open complex could be assayed. Here, we employ short (86 bp) synthetic promoters with 2-aminopurine (2-AP) substitutions in the region that becomes single-stranded to spectroscopically monitor open complex formation. We demonstrate that promoters bearing the substitutions behave in a manner similar to that of those containing only the four common bases with respect to both the region of strand separation and start site selection. Open complex formation was found to yield an increased fluorescence signal with an emission maximum characteristic of 2-aminopurine. This spectroscopic assay for open complex formation was found to be well-suited to the investigation of a strong promoter, allowing open complex formation to be followed over a time scale of seconds with a stopped flow apparatus. The introduction of two additional nonconsensus base pairs in the -35 region resulted in a promoter for which open complex formation was 100-fold slower. The same substrates were also used to monitor the promoter re-annealing that ensues upon initiation of RNA synthesis. Similar rates for this process were observed for the two promoter variants employed in this study.