Tackling a Curious Case: Generation of Charge-Tagged Guanosine Radicals by Gas-Phase Electron Transfer and Their Characterization by UV-vis Photodissociation Action Spectroscopy and Theory.

We report the generation of gas-phase riboguanosine radicals that were tagged at ribose with a fixed-charge 6-(trimethylammonium)hexane-1-aminocarbonyl group. The radical generation relied on electron transfer from fluoranthene anion to noncovalent dibenzocrown-ether dication complexes which formed nucleoside cation radicals upon one-electron reduction and crown-ether ligand loss. The cation radicals were characterized by collision-induced dissociation (CID), photodissociation (UVPD), and UV-vis action spectroscopy. Identification of charge-tagged guanosine radicals was challenging because of spontaneous dissociations by loss of a hydrogen atom and guanine that occurred upon storing the ions in the ion trap without further excitation. The loss of H proceeded from an exchangeable position on N-7 in guanine that was established by deuterium labeling and was the lowest energy dissociation of the guanosine radicals according to transition-state energy calculations. Rate constant measurements revealed an inverse isotope effect on the loss of either hydrogen or deuterium with rate constants kH = 0.25-0.26 s-1 and kD = 0.39-0.54 s-1. We used time-dependent density functional theory calculations, including thermal vibronic effects, to predict the absorption spectra of several protomeric radical isomers. The calculated spectra of low-energy N-7-H guanine-radical tautomers closely matched the action spectra. Transition-state-theory calculations of the rate constants for the loss of H-7 and guanine agreed with the experimental rate constants for a narrow range of ion effective temperatures. Our calculations suggest that the observed inverse isotope effect does not arise from the isotope-dependent differences in the transition-state energies. Instead, it may be caused by the dynamics of post-transition-state complexes preceding the product separation.

Nucleosome Binding by the Lysine Specific Demethylase 1 (LSD1) Enzyme Enables Histone H3 Demethylation.

The essential human enzyme lysine specific demethylase 1 (LSD1) silences genes by demethylating mono- and dimethylated lysine 4 in histone H3 (H3K4me1/2). Studies of the minimal requirements for LSD1 activity are complicated by the heterogeneity of histone modification states in cells. We overcame this challenge by generating homogeneous mononucleosome substrates containing semisynthetic H3K4me2. Biophysical and biochemical assays with full-length LSD1 revealed its ability to bind and demethylate nucleosomes. Consistent with a requirement for nucleosome binding prior to demethylation, a competing nucleosome-binding peptide from the high-mobility group protein effectively inhibited LSD1 activity. Thus, our studies provide the first glimpse of nucleosome demethylation by LSD1 in the absence of other scaffolding proteins.

Trimethylation of the R5 Silica-Precipitating Peptide Increases Silica Particle Size by Redirecting Orthosilicate Binding.

The unmodified R5 peptide from silaffin in the diatom Cylindrotheca fusiformis rapidly precipitates silica particles from neutral aqueous solutions of orthosilicic acid. A range of post-translational modifications found in R5 contribute toward tailoring silica morphologies in a species-specific manner. We investigated the specific effect of R5 lysine side-chain trimethylation, which adds permanent positive charges, on silica particle formation. Our studies revealed that a doubly trimethylated R5K3,4me3 peptide has reduced maximum activity yet, surprisingly, generates larger silica particles. Molecular dynamics simulations of R5K3,4me3 binding by the precursor orthosilicate anion revealed that orthosilicate preferentially associates with unmodified lysine side-chain amines and the peptide N terminus. Thus, larger silica particles arise from reduced orthosilicate association with trimethylated lysine side chains and their redirection to the N terminus of the R5 peptide.

Valproic acid-induced alterations in growth and neurotrophic factor gene expression in murine embryos [corrected].

Although the teratogenicity of valproic acid (VPA) has been well established, the mechanism(s) by which this anticonvulsant drug induces malformations remains controversial. Using the combined molecular techniques of in situ-transcription (IST) and antisense RNA (aRNA) amplification we analyzed VPA-induced alterations in the gene expression for 10 genes within the neural tubes of embryos from two murine strains that have been shown to differ in their susceptibility to VPA-induce neural tube defects (NTD). Pregnant dams from both SWV (susceptible) and LM/Bc (resistant) strains were either treated with saline (control) or VPA (600 mg/kg) on gestational day (GD) 8:12 (day:hour). Neural tubes were isolated from control or VPA exposed embryos at three gestational time points, which represented the beginning (GD 8:18), middle (GD 9:00), and end (GD 9:12) of neural tube closure (NTC) in both of these murine strains. Using univariant statistics we demonstrated that in LM/Bc embryos with NTDs, the expression of bdnf, ngf, and trk, ngf-R were significantly elevated at all three time points, and the cytokine, cntf was significantly decreased at GD 9:00. In contrast, the major gene alterations observed in SWV embryos were a significant increase in tfgalpha and tgfbeta1-3 at GD 9:00. In an effort to better define the more intricate interactions between VPA exposure and the expression of these genes, we analyzed our data using Principal Component Analysis. The results from this analysis demonstrated that embryos from these two stains behaved differently, not only in response to a VPA exposure, but also under control conditions, which may explain the multifactorial nature of NTDs in these mice.

The effects of differently weighting interview scores on the admission of underrepresented minority medical students.

PURPOSE: Seeking to admit medical students who will later practice medicine in underserved areas, but faced with the national debate over affirmative action programs, the authors evaluate the effects that giving different weightings to academic and interview scores have upon the acceptance or rejection of certain applicants. METHOD: The authors reviewed the admission records of 439 applicants to Texas A&M University College of Medicine in 1996-97. They compared the applicants actually admitted (accepted under a formula that equally weighted the two scores) with applicants who would have been admitted if the formula had weighted the interview scores at either 60% or 70% and the academic scores at either 40% or 30%. RESULTS: Weighting the academic score at 40% and the interview score at 60% produced little change in the make-up of the admissions. Weighting the academic score at 30% and the interview score at 70%, however, would have resulted in offers of acceptance to three additional underrepresented minority applicants, two of whom were disadvantaged students. CONCLUSION: Readjusting the weights of the criteria by which applicants are offered admission to medical schools may help meet the goal of educating doctors who will practice in underserved communities. More research must be done to explore other adjustments to admission criteria.

Effects of environmental management on seasonal decrease in milk production in dairy cattle.

OBJECTIVE: To describe effects of season on milk production in Holstein dairy cows and to determine the location and effectiveness of fans and sprinklers in the management of stress attributable to season. DESIGN: Longitudinal observational study. ANIMALS: 141 dairy herds for which owners used the Dairy Herd Improvement Association's database for production and reproduction record keeping. PROCEDURE: Owners were interviewed to identify location of fans, shade structures, and sprinklers. Production and reproduction data were retrieved from the database, and a mixed model ANOVA was used to estimate effects of season, parity, and use of sprinklers, and fans on milk production. RESULTS: Daily peak milk production decreased for all parity groups in the summer, but the effect decreased with increasing days in lactation. Use of sprinklers increased peak milk production in parity-1 and -3 or higher cows, but use of fans did not significantly alter effects of season. After calving in the summer, 305-day milk production decreased in parity-2 and -3 cows. This decrease was not significantly modified by the presence of sprinklers or fans. CLINICAL IMPLICATIONS: Use of sprinklers may increase peak milk production in high-producing cows and could be recommended for reducing heat and total stress during this time. Production-oriented veterinarians should be cautious when recommending use of sprinklers and fans to increase production because of the wide confidence intervals describing their effectiveness. Management of parity-2 or higher cows so that they calve from October to June could increase 305-day milk production.

Levels of maternal serum alpha-fetoprotein (AFP) in pregnant women and subsequent breast cancer risk.

High maternal serum alpha-fetoprotein (AFP) levels during pregnancy may be instrumental in reducing the subsequent risk of breast cancer. This hypothesis was tested in a nested case-control study using stored frozen sera accrued between 1959 and 1966 by the University of California at Berkeley Child Health and Development Studies (CHDS) group from a cohort of pregnant women. Cases with histologically confirmed breast cancer were identified from California Cancer Registry files covering their date of enrollment in the CHDS until 1994. Controls were selected from the CHDS cohort by using randomized recruitment. Third-trimester maternal serum AFP levels were analyzed by using both a radioimmunoassay and an immunoenzymatic method. After controlling for multiple confounders in logistic regression models, the authors found an inverse association between high levels of maternal serum AFP (top quartile) during the index pregnancy and the risk of breast cancer. The protective effect of high levels of maternal serum AFP varied by age at first full-term pregnancy (age 20 years or less: odds ratio (OR) = 0.43, 95% confidence interval (CI) 0.28-0.65; age 21-23 years: OR = 0.62, 95% CI 0.41-0.92). After age 27 years, the estimated risk exceeded unity (OR = 1.67, 95% CI 1.14-2.45). These study findings suggest that some of the protection against breast cancer conferred by early first full-term pregnancy may result from high levels of maternal serum AFP. After age 27 years, a high maternal serum AFP level is not protective and may increase risk.

Neurulation abnormalities secondary to altered gene expression in neural tube defect susceptible Splotch embryos.

The murine mutant Splotch (Sp) is a well-established model for studying neural tube closure defects. In the current investigation, the progression through neural tube closure (NTC) as well as the expression patterns of 12 developmentally regulated genes were examined in the neural tissue of wildtype (+/+), Splotch heterozygous (Sp/+), and Splotch homozygous (Sp/Sp) embryos during neurulation. The overall growth of the embryos, as measured by the number of somite pairs, did not differ significantly between the three genotypes at any of the collection time-points. There was, however, a significant delay in the progression through NTC for both the Sp/+ and Sp/Sp embryos. A univariate analysis on the expression of the 12 candidate genes (bcl-2, FBP-2, Hmx-2, Msx-3, N-cam, N-cad, noggin, p53, Pax-3, Shh, Wee-1, wnt-1) revealed that although 11 were statistically altered, across time or by genotype, there were no significant interactions between gestation age and genotype for any of these genes during NTC. However, a multivariate statistical analysis on the simultaneous expression of these genes revealed interactions at both gestation day (GD) 8:12 (day:hour) and 9:00 among Pax-3, N-cam, N-cad, bcl-2, p53, and Wee-1 that could potentially explain the aberrant NTC. The data from these studies suggest that a disruption in the genes that govern the cell cycle or extracellular matrices of the developing neural tube might play a critical role in the occurrence of the NTDs observed in Splotch embryos.

Developmental expression of morphoregulatory genes in the mouse embryo: an analytical approach using a novel technology.

The molecular techniques of in situ transcription and antisense RNA amplification (IST/aRNA) have allowed for the monitoring of coordinate changes in the expression of multiple genes simultaneously. However, the analysis of their concurrent behavior during murine embryogenesis has been problematic. Studies involving the investigation of temporal and spatial gene expression during embryogenesis have focused solely on the analysis of isolated, single gene events. Such an approach has failed to provide an integrative picture of genetic control over the varied and complicated cellular processes governing embryogenesis. In order to interpret the enormous amount of gene expression data generated by these procedures, we have attempted to develop an analytical framework by employing the statistical concepts of principal components analysis (PCA). For the current study, we performed IST/aRNA on neural tubes dissected from the highly inbred LM/Bc murine strain collected during four gestational time periods. A subset of these genes, representing a partial signaling pathway in the developing neuroepithelium, was then subjected to PCA. Here, we report that PCA highlighted the transcriptional interplay among the genes p53, wee-1, Tgf beta-2, and bcl-2 such that the combined reciprocal regulation of their gene products is suggestive of a predominant proliferative state for the developing neuroepithelium. The application of PCA to the gene expression data has elucidated previously unknown interrelationships among cell cycle genes, growth, and transcription factors on a transcriptional level during critical stages of neurulation. The information gleaned from this analysis, while not definitive, suggests distinct hypotheses to guide future research.

Phenytoin-induced teratogenesis: a molecular basis for the observed developmental delay during neurulation.

PURPOSE: We wished to determine whether chronic phenytoin (PHT) exposure could impair neural development and if any morphological alterations could be linked to changes in gene expression. METHODS: Pregnant SWV mice were chronically administered PHT 40 mg/kg/day from gestational day (GD) 0:12 (day:h) until they were killed at various timepoints throughout neural tube closure (NTC). At each timepoint, embryos from both treated and control dams were collected and scored for their progression through NTC. The neural tubes were then isolated and subjected to in situ transcription (IST) and antisense RNA amplification procedures. Using these techniques, we examined the expression of 10 genes: N-cadherin (Ncad), collagen type IV (col-IV), bcl-2, c-jun, PAX-3, collular retinol binding protein-2 (CRBP-2), retinoic acid receptor alpha (RAR alpha), transforming growth factor(beta2) (TGF(beta2)), wee-1, and EMX-2. RESULTS: Chronic PHT exposure not only caused a delay in NTC whereby exposed embryos lagged behind the controls at each collection timepoint, but also significantly altered the expression of specific genes at distinct times during NTC. Early in NTC, PHT induced a significant reduction in the expression of N-cad, col-IV, and c-jun in exposed embryos as compared with controls. In contrast, during the midstages of NTC, the only significant molecular alterations observed in the PHT-exposed embryos was the continued decreased expression of col-IV and an increase in CRBP-2 expression. Finally, in the latter stages of NTC, PHT caused a significant reduction in the expression of bcl-2, RAR alpha, TGF(beta2), EMX-2, and PAX-3. CONCLUSIONS: These results show that although the effects of PHT are morphologically subtle, causing a delay in the development of the neural tube, this delay is accompanied by alterations in critical genes at crucial times of neural development that may account for the observed neurological deficits often associated with PHT exposure.

Valproic acid-induced changes in gene expression during neurulation in a mouse model.

The teratogenic potential of valproic acid has been well established both in experimental models and in human clinical studies. As with all human teratogens, there are genetically determined differences in individual susceptibility to the induction of congenital defects. Using a mouse model of valproate-induced neural tube defects, a study was undertaken to examine differential changes in gene expression for selected transcription factor (Pax-3, Emx-1, Emx-2, c-fos, c-jun, creb) and cell cycle checkpoint genes (bcl-2, p53, wee-1) during neural tube closure. In general, exposure to teratogenic concentrations of valproic acid elicited GD 9:12 control levels of transcription factor mRNA expression in GD 9:0 embryos of both strains. This accelerated developmental profile is marked by significant elevation of Emx-1, Emx-2, c-fos, c-jun, and creb expression. There was also a significant over expression of the cell cycle genes p53 and bcl-2 in the LM/Bc embryos in response to the teratogenic insult. Examination of the ratio of expression of these genes clearly favored bcl-2, which supports the hypothesis that altered neuroepithelial cell proliferation rates, rather than increased apoptosis, is the underlying mechanism by which valproic acid alters normal neural tube morphogenesis. An investigation into interactive effects of these genes on the molecular profile of GD 9:0 embryos further validated this observation. That is, the overall proliferative state among the control embryos was prematurely modified into a more differentiated state following teratogenic insult. These results suggest that alterations in the expression of multiple genes are most likely responsible for valproic acid-induced neural tube defects.

Arsenic-induced neural tube defects in mice: alterations in cell cycle gene expression.

The potential of arsenic to cause neural tube defects (NTD) in the human population remains a topic of controversy. While clearly toxic, the lack of well-defined human epidemiologic studies on this subject has made it difficult to fully understand the effects arsenic may have on the developing human neural tube. In the absence of good clinical data, we have tried to develop a murine model where hypotheses about the reproductive toxicity of arsenate can be tested. For these studies a murine strain (LM/Bc) that has proven to be susceptible to arsenic-induced NTD was use. Because cellular proliferation is vital for normal neural tube closure (NTC) to occur, in the present study we investigated whether an acute arsenate treatment could alter the expression of several cell cycle genes during murine neurulation. Pregnant LM/Bc dams were injected intraperitoneally on gestation day (GD) 7:12 (day:hour) and 8:12 with 40 mg/kg of arsenate, a treatment that causes exencephaly in 90 to 100% of the exposed fetuses. Neural tubes were then isolated from both control and arsenic treated embryos at GD 9:00, 9:12, 10:00, and 10:12, which encompasses all the stages of neurulation for this murine strain. Using the molecular techniques of in situ transcription and antisense RNA amplification (RT/aRNA) the expression pattern for bc1-2, p53, wee-1, and wnt-1 was analyzed at each of these time points. In the neural tubes isolated from control embryos, the expression of all four genes was significantly altered as neurulation progressed, demonstrating their developmental regulation. Following arsenate treatment, however, there was a significant upregulation in the expression of bc1-2 and p53 at gestational day 9:0, compared to their control values. The heightened expression of both of these genes suggests that arsenic inhibits cell proliferation, rather than inducing apoptosis, which delayed NTC and ultimately led to the neural tube defects observed in exposed embryos.

Arsenic-induced alterations in embryonic transcription factor gene expression: implications for abnormal neural development.

We examined the morphological and molecular consequences of acute in utero exposure to teratogenic concentrations of arsenate. The treatment produced a dose-related increase in neural tube defects, along with a significant alteration in the pattern of gene expression for several transcription factors (creb, Hox 3.1, Pax3, and Emx-1) that were examined using in situ transcription and antisense RNA amplification procedures. On gestational day 9:0, there was a significant delay in the embryos progression through neural tube closure, accompanied by a significant downregulation of Hox 3.1 expression and a significant upregulation of Pax3, Emx-1, and creb. As both Hox 3.1 and Pax3 serve to regulate N-CAM expression, it is possible that abnormalities associated with N-CAM may compromise neural crest cell migration and normal neural tube closure.

Pregnancy attrition associated with pregnancy testing by rectal palpation.

The objective of this study was to estimate the effect on calving risk of interval from AI date until scheduled date of pregnancy examination. First AI (n = 7105) from 65 dairy herds in the United States and Canada were followed for 294 d to determine whether cows calved. Calving was modeled as a function of the number of days in the interval, herd, season, and breeding at PGF2 alpha-induced estrus by multivariate logistic regression. The main effects of herd and AI following PGF2 alpha-induced estrus were significantly associated with calving rate from first AI. The main effects of interval and season were not significantly associated with calving.

Antipyrine and caffeine dispositions in clinically normal dogs and dogs with progressive liver disease.

Dispositions of caffeine and antipyrine were compared as indicators of decreasing hepatic function in dogs with experimentally induced progressive liver disease. Dimethylnitrosamine, a hepatospecific toxin, was administered orally to 16 dogs; 6 dogs served as controls (group 1). Three classes of liver disease were defined by histologic features: mild (group 2; n = 5), moderate (group 3; n = 6), and severe (group 4; n = 5). Disposition of antipyrine, and 24 hours later, caffeine was studied 3 weeks after the last dose of toxin in each dog. For both drugs, rapid IV administration of 20 mg/kg of body weight was administered and serum samples were obtained at intervals for determination of at least 5 terminal-phase drug half-lives. For both drugs, clearance and mean residence time differed among groups (P < or = 0.01). Clearance of antipyrine and caffeine was decreased in groups 3 and 4, compared with groups 1 and 2. Antipyrine and caffeine mean residence times were longer in group-3 dogs, compared with dogs of groups 1 and 2. Correction of caffeine and antipyrine clearances for hepatic weight increased discrimination between groups 3 and 4. The clearance and mean residence time ratios of antipyrine to caffeine were calculated for each group and, when compared with values for group-1 dogs, were used to test for differences between the 2 drugs in response to disease. Ratios did not differ among groups. These results indicate that the disposition of antipyrine and caffeine may change similarly with progression of dimethylnitrosamine-induced liver disease.

Effects of vena caval banding in experimentally induced multiple portosystemic shunts in dogs.

Effects of vena caval banding on portal venous and vena caval hemodynamics were examined in 6 control dogs and in 10 dogs that had undergone attenuation (banding) of the abdominal part of the caudal vena cava and had dimethylnitrosamine-induced multiple portosystemic shunts (PSS). Additionally, indocyanine green (ICG) extraction and clearance after infusion to steady state were used to calculate hepatic plasma flow in these dogs. Sixteen dogs were randomly assigned to 2 groups: control (n = 6) or diseased (n = 10). Diseased dogs were administered dimethylnitrosamine (2 mg/kg, PO, twice weekly) until multiple PSS developed, as assessed by results of clinical laboratory tests, ultrasonography, and hepatic scintigraphy. Shunts were confirmed visually at celiotomy and by contrast portography. Venous pressures (caudal vena caval, portal, and hepatic) were recorded before and after vena caval banding for up to 7 days in dogs from both groups. Peritoneal cavity pressures were recorded in all dogs after closure of the body wall. To determine ICG extraction and clearance, a bolus injection of ICG (0.5 mg/kg, i.v.) was administered, followed by steady-state infusion of 0.097 mg/min. Extractions and clearances of ICG were measured, and from these, hepatic plasma flow rates were determined immediately before and after banding and at 6 hours, 48 hours, and 7 days after banding. The gradient (caudal vena caval pressure within 1 to 2 mm of Hg of portal pressure) between caudal vena cava and portal venous pressures established at banding was maintained after the first hour in both groups. Caudal vena cava pressures established at banding were maintained throughout the study, with the exception of the first hour in diseased dogs. Extraction ratios were higher in control dogs at all times, except at 48 hours. Clearance was higher in control dogs at all times. Hepatic plasma flow did not differ between groups, except immediately after banding, when flow was greater in diseased dogs, and differences were not found over time in either group. This study indicated that vena caval banding in this model of experimentally induced multiple PSS increases and maintains caudal vena cava pressure, relative to portal venous pressure (after the first hour) for 7 days, and that calculated hepatic plasma flow is not persistently improved by vena caval banding.

Morphological alterations of neurons and astrocytes in guinea pigs exposed to low levels of inorganic lead.

Both astrocytes and neurons potentially undergo structural and functional alterations in the brains of animals exposed to low levels of lead (Pb). No morphometric studies of astrocytes have been reported to date in animals in low Pb exposure. In the present study, morphometric measurements of astrocytes and pyramidal neurons in the frontoparietal cortex were made in guinea pigs exposed postnatally (5 or 10 days) or prenatally (gestational day 22 to birth) to low Pb levels. Although few significant effects of Pb treatment were detected by the rigorous statistical model applied, a recurring trend was noted for postnatal Pb treatment to increase astrocyte maximum diameter (dmax). In addition, prenatal Pb treatment was associated with increased apical and basal dendritic length, increased total apical dendrites per cell and an increased basal branching complexity in neurons.