Safety and feasibility pilot study of continuous low-dose maternal supplemental oxygen in fetal single ventricle heart disease.

OBJECTIVES: Fetuses with single ventricle physiology (SVP) exhibit reductions in fetal cerebral oxygenation with associated delays in fetal brain growth and neurodevelopmental outcomes. Maternal supplemental oxygen (MSO) has been proposed to improve fetal brain growth but current evidence on dosing, candidacy, and outcomes are limited. In this pilot study, we evaluated the safety and feasibility of continuous low-dose MSO in the setting of SVP. METHODS: This single-centre, open-label, pilot phase 1 safety and feasibility clinical trial included 25 pregnant individuals with a fetal diagnosis of SVP. Participants self-administered continuous supplemental oxygen using medical-grade oxygen concentrators for up to 24 hours per day from the second half of gestation until delivery. The primary aim was the evaluation of the safety profile and feasibility of MSO. A secondary preliminary analysis was performed to assess the impact of MSO on the fetal circulation by echocardiography and late-gestational cardiovascular magnetic resonance, early outcomes including brain growth and pre-operative brain injury, and 18-month neurodevelopmental outcomes by the Bayley Scales of Infant and Toddler Development 3rd Edition compared to a contemporary fetal SVP cohort that received standard of care (SOC). RESULTS: Among 25 participants, the average maternal age at conception was 35 years, and fetal SVP diagnoses included 16 right ventricle dominant, 8 left ventricle dominant, and 1 indeterminant ventricular morphology. Participants started the trial at approximately 29.3 gestational weeks and took MSO for a median 16.1 hours per day for 63 days, cumulating a median 1029 hours of oxygen intake from enrollment until delivery. The only treatment-associated adverse events were nasal complications that were typically resolved by attaching a humidifier unit to the oxygen concentrator. No premature closure of the ductus arteriosus or unexpected fetal demise was observed. In the secondary analysis, MSO was not associated with any changes in fetal growth, middle cerebral artery pulsatility index, cerebroplacental ratio, nor head circumference to abdominal circumference ratio Z-scores over gestation compared to SOC. Although MSO was associated with changes in umbilical artery pulsatility index Z-score over gestation compared to SOC (p=0.02), this was likely due to initial baseline differences in placental resistance. At late-gestational cardiovascular magnetic resonance, MSO was not associated with any significant increase in umbilical vein oxygen saturation, fetal oxygen delivery, or fetal cerebral oxygen delivery. Similarly, we observed no differences in newborn outcomes including brain volume and pre-operative brain injury, nor mortality by 18 months of age, nor neurodevelopmental outcomes at 18 months of age. CONCLUSIONS: This pilot phase 1 clinical trial indicates low-dose maternal supplemental oxygen therapy is safe and well tolerated in pregnancies diagnosed with fetal SVP. However, our protocol was not associated with any significant changes in fetal circulatory physiology or improvements in early neurologic or neurodevelopmental outcomes. This article is protected by copyright. All rights reserved.

MRI characterization of hemodynamic patterns of human fetuses with cyanotic congenital heart disease.

OBJECTIVES: To characterize, using magnetic resonance imaging (MRI), the distribution of blood flow and oxygen transport in human fetuses with subtypes of congenital heart disease (CHD) that present with neonatal cyanosis. METHODS: Blood flow was measured in the major vessels of 152 late-gestation human fetuses with CHD and 40 gestational-age-matched normal fetuses, using cine phase-contrast MRI. Oxygen saturation (SaO2 ) was measured in the major vessels of 57 fetuses with CHD and 40 controls. RESULTS: Compared with controls, we found lower combined ventricular output in fetuses with single-ventricle physiology, with the lowest being observed in fetuses with severe forms of Ebstein's anomaly. Obstructive lesions of the left or right heart were associated with increased flow across the contralateral side. Pulmonary blood flow was reduced in fetuses with Ebstein's anomaly, while those with Ebstein's anomaly and tricuspid atresia had reduced umbilical flow. Flow in the superior vena cava was elevated in fetuses with transposition of the great arteries, normal in fetuses with hypoplastic left heart, tetralogy of Fallot or tricuspid atresia and reduced in fetuses with Ebstein's anomaly. Umbilical vein SaO2 was reduced in fetuses with hypoplastic left heart or tetralogy of Fallot. Ascending aorta and superior vena cava SaO2 were reduced in nearly all CHD subtypes. CONCLUSIONS: Fetuses with cyanotic CHD exhibit profound changes in the distribution of blood flow and oxygen transport, which result in changes in cerebral, pulmonary and placental blood flow and oxygenation. These alterations of fetal circulatory physiology may influence the neonatal course and help account for abnormalities of prenatal growth and development that have been described in newborns with cyanotic CHD. © 2021 International Society of Ultrasound in Obstetrics and Gynecology.

Long-term postnatal outcome of fetuses with prenatally suspected septo-optic dysplasia.

OBJECTIVES: Septo-optic dysplasia (SOD) is a clinical syndrome characterized by varying combinations of optic nerve hypoplasia, pituitary gland hypoplasia and abnormal cavum septi pellucidi. It is suspected on prenatal imaging when there is non-visualization or hypoplasia of the septal leaflets. Long-term postnatal outcomes of fetuses with prenatally suspected SOD have been documented poorly. The aims of this study were to describe the natural history of deficient septal leaflets, to quantify the incidence of postnatally confirmed SOD and to document the visual, endocrine and long-term neurodevelopmental outcomes of these infants. METHODS: This was an observational retrospective study of all fetuses with prenatal imaging showing isolated septal agenesis, assessed at a single tertiary center over an 11-year period. Pregnancy, delivery and neonatal outcomes and pre- and postnatal imaging findings were reviewed. Neonatal evaluations or fetal autopsy reports were assessed for confirmation of SOD. Ophthalmologic, endocrine, genetic and long-term developmental evaluations were assessed. Imaging findings and outcome were compared between infants with and those without postnatally confirmed SOD. RESULTS: Of 214 fetuses presenting with septal absence on prenatal ultrasound and magnetic resonance imaging (MRI), 18 (8.4%) were classified as having suspected isolated septal agenesis suspicious for SOD. Uniform prenatal MRI findings in cases with suspected SOD included remnants of the leaflets of the cavum septi pellucidi, fused forniceal columns, normal olfactory bulbs and tracts and a normal optic chiasm. Twelve fetuses were liveborn and five (27.8%) had postnatally confirmed SOD. Only two of these five fetuses had additional prenatal imaging features (pituitary cyst, microphthalmia and optic nerve hypoplasia) supporting a diagnosis of SOD. The other three confirmed SOD cases had no predictive prenatal or postnatal imaging findings that reliably differentiated them from cases without confirmed SOD. Visual and endocrine impairments were present in two (40%) and four (80%) cases with confirmed SOD, respectively. In those with visual and/or endocrine impairment, developmental delay (median age at follow-up, 2.5 (interquartile range, 2.5-7.0) years) was common (80%) and mostly severe. Neonates with isolated septal agenesis and a lack of visual or endocrine abnormalities to confirm SOD had normal development. CONCLUSIONS: Only a quarter of fetuses with isolated septal agenesis suggestive of SOD will have postnatal confirmation of the diagnosis. Clinical manifestations of SOD are variable, but neurodevelopmental delay may be more prevalent than thought formerly. © 2020 Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Recruitment of PP1 to the centrosomal scaffold protein CEP192.

Centrosomal protein of 192 kDa (CEP192) is a scaffolding protein that recruits the mitotic protein kinases Aurora A and PLK1 to the centrosome. Here we demonstrate that CEP192 also recruits the type one protein phosphatase (PP1) via a highly conserved KHVTF docking motif. The threonine of the KHVTF motif is phosphorylated during mitosis and protein kinase inhibition studies suggest this to be a PLK1-dependent process.

Expression of DNA damage response proteins in cervical cancer patients treated with radical chemoradiotherapy.

OBJECTIVE: The management of locally advanced cervical cancer has improved significantly with the advent of cisplatin-based chemoradiotherapy (CRT) as the primary treatment regimen. Nevertheless, a significant proportion of patients fail to respond or relapse on this treatment and have a very poor prognosis. Our goal was to determine the prognostic value of a panel of proteins involved in detection and repair of DNA damage. METHODS: We performed fluorescence immunohistochemistry, and used software analysis to assess expression of DNA damage response proteins ATM, DNA-PKcs, PARP-1, Ku70 and Ku86 in 117 pre-treatment specimens from patients with locally advanced cervical cancer. We compared expression to clinicopathologic correlates to determine prognostic significance. RESULTS: Five-year progression-free survival was significantly lower in the low expressors than in high expressors of ATM (35% vs. 58%, p=0.044) and PARP-1 (24% vs. 61%, p=0.003), and showed a trend to significance for DNA-PKcs (30% vs. 60%, p=0.050). Low expression of the same proteins also correlated significantly with lower overall survival. In multivariable analysis, adjusted for FIGO stage and tumor size, low ATM and PARP-1 expression was significantly associated with both poorer progression-free and overall survival. Pairwise analyses indicated that expression levels of these proteins were correlated. CONCLUSIONS: Expression of DNA damage response proteins in cervical cancer is associated with outcome in patients treated with CRT. Immunohistochemical analysis of these proteins may be useful in guiding treatment decisions in such patients.

Utilization of macrominerals and trace elements in pregnant heifers with distinct feed efficiencies.

The objective of the study was to evaluate utilization of dietary minerals and trace elements in pregnant heifers with distinct residual feed intakes (RFI). Feed intake, body weight (BW), and body composition traits were recorded in 36 crossbred heifers over a period of 37 wk, starting shortly after weaning at 8.3 (0.10; standard deviation) mo of age with an average BW of 276 (7.8) kg. Both BW and body composition were monitored regularly throughout the study, whereas individual feed intake was assessed during the last 84 d of the trial. Data recorded were used to calculate RFI for each heifer. Heifers were ranked based on RFI and assigned to high (n=14) or low (n=10) RFI groups. After the RFI study, 24 selected heifers [age 18.2 (0.14) mo; 87.5 (4.74) d in gestation; 497 (8.5) kg of BW] were used in an indirect digestibility trial (lignin as internal marker). Heifers were fed a ration containing corn silage, haylage, and a mineral premix in which Ca, P, K, Na, Mg, S, Cu, Fe, Mn, Mo, Se, Zn, and Co were provided in the diet according to National Research Council requirements of pregnant replacement heifers. The digestibility trial lasted 1 wk, during which samples of feces were gathered twice daily, and blood and liver biopsy samples were collected on the last day. We noted no significant differences between low- and high-RFI heifers in dry matter digestibility. Apparent absorption of Cu, Zn, and Mn was increased in heifers with low RFI, and apparent absorption of Co tended to be greater for these animals. Concentrations of macrominerals and trace elements in serum of pregnant heifers were similar for both groups except for Se, which was increased in the serum of low-RFI heifers. Liver concentrations of Cu, Fe, Mn, Mo, Se, and Zn did not differ between low- and high-RFI heifers. In conclusion, whereas improved absorption of some trace elements (Cu, Zn, Mn, and Co) and increased Se serum concentration appear to be associated with superior feed efficiency in pregnant heifers, further studies are needed to investigate the causality of such relationships.

ATM associates with and phosphorylates p53: mapping the region of interaction.

The human genetic disorder ataxia-telangiectasia (AT) is characterized by immunodeficiency, progressive cerebellar ataxia, radiosensitivity, cell cycle checkpoint defects and cancer predisposition. The gene mutated in this syndrome, ATM (for AT mutated), encodes a protein containing a phosphatidyl-inositol 3-kinase (PI-3 kinase)-like domain. ATM also contains a proline-rich region and a leucine zipper, both of which implicate this protein in signal transduction. The proline-rich region has been shown to bind to the SH3 domain of c-Abl, which facilitates its phosphorylation and activation by ATM. Previous results have demonstrated that AT cells are defective in the G1/S checkpoint activated after radiation damage and that this defect is attributable to a defective p53 signal transduction pathway. We report here direct interaction between ATM and p53 involving two regions in ATM, one at the amino terminus and the other at the carboxy terminus, corresponding to the PI-3 kinase domain. Recombinant ATM protein phosphorylates p53 on serine 15 near the N terminus. Furthermore, ectopic expression of ATM in AT cells restores normal ionizing radiation (IR)-induced phosphorylation of p53, whereas expression of ATM antisense RNA in control cells abrogates the rapid IR-induced phosphorylation of p53 on serine 15. These results demonstrate that ATM can bind p53 directly and is responsible for its serine 15 phosphorylation, thereby contributing to the activation and stabilization of p53 during the IR-induced DNA damage response.

Sildenafil citrate therapy for severe early-onset intrauterine growth restriction.

Sildenafil citrate therapy for severe early-onset intrauterine growth restriction. BJOG 2011;118:624-628. Currently, there is no effective therapy for severe early-onset intrauterine growth restriction (IUGR). Sildenafil citrate vasodilates the myometrial arteries isolated from women with IUGR-complicated pregnancies. Women were offered Sildenafil (25 mg three times daily until delivery) if their pregnancy was complicated by early-onset IUGR [abdominal circumference (AC)< 5th percentile] and either the gestational age was <25(+0) weeks or an estimate of the fetal weight was <600 g (excluding known fetal anomaly/syndrome and/or planned termination). Sildenafil treatment was associated with increased fetal AC growth [odds ratio, 12.9; 95% confidence interval (CI), 1.3, 126; compared with institutional Sildenafil-naive early-onset IUGR controls]. Randomised controlled trial data are required to determine whether Sildenafil improves perinatal outcomes for early-onset IUGR-complicated pregnancies.

Genetic analysis of survival and fitness in turkeys with multiple-trait animal models.

Genetic parameters for production, survival, and structural fitness traits recorded in pedigreed turkey sire and dam parental lines from a nucleus breeding program were estimated with multiple-trait animal models. Survival and conformation traits were scored in binary terms of health, where 0 = died or affected, and 1 = survived or healthy. Walking ability at 20 wk was subjectively scored from 1 (poor) to 6 (excellent). Body weights and egg production displayed moderate heritability (h(2) = 0.18 to 0.35). Early survival (to 3 wk) displayed low heritability (h(2) = 0.02 and 0.04 for the dam and sire lines, respectively). Late survival (3 to 23 wk) and longevity (age at death or cull) had low to moderate heritability (h(2) = 0.12 to 0.14). Walking ability had moderate heritability (h(2) = 0.26, 0.25). Leg structure health displayed low heritability (h(2) = 0.08), as did hip structure, foot, and skin health (h(2) ≤ 0.02). Crop health displayed moderate heritability (h(2) = 0.12). Walking ability, hip and leg structures, footpad, and breast skin health had negative genetic correlations with BW (r(G) = -0.50 to -0.23). Egg production had moderate positive genetic correlation with late survival (r(G) = 0.61). Genetic correlations between early and late survival were close to zero (r(G) = 0.10 and 0.03 for the dam and sire lines, respectively). Walking ability had high positive genetic correlations with late survival, longevity, hip structure, and leg structure in both lines (r(G) = 0.51 to 0.91). These genetic parameters indicate that unchecked selection for growth could decrease survival, walking ability, and hip, leg, footpad, and skin health in turkeys. However, index selection should be effective at improving fitness, survival, and growth simultaneously in commercial turkey lines. Walking ability should be a good indicator trait for selection to improve overall late survival and hip and leg health in turkeys.

Interleukin-1 beta-converting enzyme-like protease cleaves DNA-dependent protein kinase in cytotoxic T cell killing.

Cytotoxic T cells (CTL) represent the major defense mechanism against the spread of virus infection. It is believed that the pore-forming protein, perforin, facilitates the entry of a series of serine proteases (particularly granzyme B) into the target cell which ultimately leads to DNA fragmentation and apoptosis. We demonstrate here that during CTL-mediated cytolysis the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), an enzyme implicated in the repair of double strand breaks in DNA, is specifically cleaved by an interleukin (IL)-1 beta-converting enzyme (ICE)-like protease. A serine protease inhibitor, 3,4-dichloroisocoumarin (DCl), which is known to block granzyme B activity, inhibited CTL-induced apoptosis and prevented the degradation of DNA-PKcs in cells but failed to prevent the degradation of purified DNA-PKcs by CTL extracts. However, Tyr-Val-Ala-Asp-CH2Cl (YVAD-CMK) and other cysteine protease inhibitors prevented the degradation of purified DNA-PKcs by CTL extracts. Furthermore, incubation of DNA-PKcs with granzyme B did not produce the same cleavage pattern observed in cells undergoing apoptosis and when this substrate was incubated with either CTL extracts or the ICE-like protease, CPP32. Sequence analysis revealed that the cleavage site in DNA-PKcs during CTL killing was the same as that when this substrate was exposed to CPP32. This study demonstrates for the first time that the cleavage of DNA-PKcs in this intact cell system is exclusively due to an ICE-like protease.

Absence of p350 subunit of DNA-activated protein kinase from a radiosensitive human cell line.

The radiosensitive rodent mutant cell line xrs-5 is defective in DNA double-strand break repair and lacks the Ku component of the DNA-activated protein kinase, DNA-PK. Here radiosensitive human cell lines were analyzed for DNA-PK activity and for the presence of related proteins. The radiosensitive human malignant glioma M059J cell line was found to be defective in DNA double-strand break repair, but fails to express the p350 subunit of DNA-PK. These results suggest that DNA-PK kinase activity is involved in DNA double-strand break repair.

Relationships of nutritional plane and feed efficiency with sexual development and fertility related measures in young beef bulls.

Sexual development in beef bulls appears to be influenced by nutritional plane and feed efficiency. Yearling bulls fed high (GRAIN = 49) and moderate (ROUGHAGE = 109) nutritional planes, were submitted to a performance test where scrotal circumference (SC), scrotum and testis ultrasonograms and hormones were monitored throughout. Scrotal thermographs, blood cell counts and semen were evaluated at the end of the test. Residual feed intake (RFI) was the measure of feed efficiency, and bulls within each population were characterized as efficient and inefficient. During the test period, the GRAIN group had greater triiodothyronine (T3), leptin and scrotal skin thickness (SST) while having greater T3, leptin, % motile sperm, % normal sperm, scrotal temperature and values for red blood cell variables at the end of the test when compared to the ROUGHAGE group. During the test, the efficient GRAIN group had lesser testis pixel intensity while at the end of the performance test the bulls of this group had greater % normal sperm and lower testis pixel intensity compared to bulls in inefficient GRAIN group. In comparison, the efficient ROUGHAGE group had less T3, SC and SST while at the end of the test these bulls had less T3, leptin, SC, SST and scrotal temperature compared to bulls in the inefficient ROUGHAGE group. Complete blood cell variables, semen quality, scrotal biometry and thermography explained most of the variation in RFI. Results of the present study support the hypothesis that there is an antagonistic relationship between feed efficiency and sexual development in young bulls, which seems partially offset when there is a greater plane of nutrition. Thus, feed efficiency should be factored in the reproductive evaluation of sires.

Associations of rumen parameters with feed efficiency and sampling routine in beef cattle.

Characterizing ruminal parameters in the context of sampling routine and feed efficiency is fundamental to understand the efficiency of feed utilization in the bovine. Therefore, we evaluated microbial and volatile fatty acid (VFA) profiles, rumen papillae epithelial and stratum corneum thickness and rumen pH (RpH) and temperature (RT) in feedlot cattle. In all, 48 cattle (32 steers plus 16 bulls), fed a high moisture corn and haylage-based ration, underwent a productive performance test to determine residual feed intake (RFI) using feed intake, growth, BW and composition traits. Rumen fluid was collected, then RpH and RT logger were inserted 5.5±1 days before slaughter. At slaughter, the logger was recovered and rumen fluid and rumen tissue were sampled. The relative daily time spent in specific RpH and RT ranges were determined. Polynomial regression analysis was used to characterize RpH and RT circadian patterns. Animals were divided into efficient and inefficient groups based on RFI to compare productive performance and ruminal parameters. Efficient animals consumed 1.8 kg/day less dry matter than inefficient cattle (P⩽0.05) while achieving the same productive performance (P⩾0.10). Ruminal bacteria population was higher (P⩽0.05) (7.6×1011 v. 4.3×1011 copy number of 16S rRNA gene/ml rumen fluid) and methanogen population was lower (P⩽0.05) (2.3×109 v. 4.9×109 copy number of 16S rRNA gene/ml rumen fluid) in efficient compared with inefficient cattle at slaughter with no differences (P⩾0.10) between samples collected on-farm. No differences (P⩾0.10) in rumen fluid VFA were also observed between feed efficiency groups either on-farm or at slaughter. However, increased (P⩽0.05) acetate, and decreased (P⩽0.05) propionate, butyrate, valerate and caproate concentrations were observed at slaughter compared with on-farm. Efficient had increased (P⩽0.05) rumen epithelium thickness (136 v. 126 µm) compared with inefficient cattle. Efficient animals also spent 318% and 93.2% more time (P⩽0.05) in acidotic (4.14% v. 1.30%) (pH⩽5.6) and optimal (5.6

The normal neonatal brain: MR imaging, diffusion tensor imaging, and 3D MR spectroscopy in healthy term neonates.

BACKGROUND AND PURPOSE: There is a lack of normative diffusion tensor imaging (DTI) and 3D MR spectroscopy (MRS) data in the early neonatal period. We report quantitative values from a cohort of healthy term neonates to serve as baseline data for studies assessing brain development and injury. MATERIALS AND METHODS: Sixteen healthy term neonates (median age, 7 days) were studied with spin-echo T1- and T2-weighted MR imaging, DTI, and 3D point-resolved spectroscopy sequence (PRESS) MRS without sedation on a 1.5 T scanner. Average diffusivity (D(av)), fractional anisotropy (FA), eigenvalues (EV), and metabolite ratios (N-acetylaspartate [NAA]/choline, lactate/choline) were calculated by automated processing in 7 brain regions. Neurodevelopment was assessed by blinded and validated neuromotor examinations and the Bayley II test at 3 and 14 months. RESULTS: Two neonates were excluded from the cohort: one had brain injury on T2-weighted imaging, and the other, who had normal MR imaging, showed mildly delayed cognition at 14 months. The mean DTI values of the remaining 14 neonates were between these ranges: D(av)=0.98-1.48 10(-3) mm(2)/s, FA=0.14-0.30, EV1=1.21-1.88, EV2=0.95-1.46, and EV3=0.77-1.24 (all x 10(-3) mm(2)/s). The NAA/choline ratio ranged between 0.58 and 0.73, and minimal lactate/choline (<0.15) could be detected in each neonate. All neonates exhibited clinically normal neuromotor status. CONCLUSIONS: Our study demonstrates the feasibility of obtaining high-quality quantifiable MR data in nonsedated healthy term neonates that can be used to study normal early brain development and as control data in studies of perinatal brain injury.

Three yeast proteins related to the human candidate tumor suppressor p33(ING1) are associated with histone acetyltransferase activities.

Three Saccharomyces cerevisiae proteins (Yng1/YOR064c, Yng2/YHR090c, and Pho23) and two Schizosaccharomyces pombe proteins (Png1/CAA15917 and Png2/CAA21250) share significant sequence identity with the human candidate tumor suppressor p33(ING1) in their C-terminal regions. The homologous regions contain PHD finger domains which have been implicated in chromatin-mediated transcriptional regulation. We show that GFP-Yng2, like human Ing1, is localized in the nucleus. Deletion of YNG2 results in several phenotypes, including an abnormal multibudded morphology, an inability to utilize nonfermentable carbon sources, heat shock sensitivity, slow growth, temperature sensitivity, and sensitivity to caffeine. These phenotypes are suppressed by expression of either human Ing1 or S. pombe Png1, suggesting that the yeast and human proteins are functionally conserved. Yng1- and Pho23-deficient cells also share some of these phenotypes. We demonstrated by yeast two-hybrid and coimmunoprecipitation tests that Yng2 interacts with Tra1, a component of histone acetyltransferase (HAT) complexes. We further demonstrated by coimmunoprecipitation that HA-Yng1, HA-Yng2, HA-Pho23, and HA-Ing1 are associated with HAT activities in yeast. Genetic and biochemical evidence indicate that the Yng2-associated HAT is Esa1, suggesting that Yng2 is a component of the NuA4 HAT complex. These studies suggest that the yeast Ing1-related proteins are involved in chromatin remodeling. They further suggest that these functions may be conserved in mammals and provide a possible mechanism for the human Ing1 candidate tumor suppressor.

Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen.

HeLa cells contain a serine/threonine protein kinase (DNA-PK) that is strongly activated in vitro by low concentrations of double-stranded DNA (dsDNA). Activation was specific for dsDNA; both natural DNAs and synthetic oligonucleotides functioned as kinase activators. The fact that DNA-PK activity was rapidly inhibited by incubation with dsDNA and ATP suggests that DNA-PK activity also may be regulated by autophosphorylation. During gel filtration, DNA-PK activity behaved as a 350-kDa protein, and highly purified DNA-PK contained a dsDNA-binding, 350-kDa polypeptide that was phosphorylated in a dsDNA-dependent manner. We conclude that this 350-kDa polypeptide is likely to be DNA-PK. Previously we showed that the dsDNA-activated kinase phosphorylates two threonines at the N terminus of hsp90 alpha (S. P. Lees-Miller and C. W. Anderson, J. Biol. Chem. 264:17275-17280, 1989). Here we show that DNA-PK also phosphorylates the simian virus 40 large tumor antigen, the mouse tumor-suppressor protein p53, the human Ku autoantigen, and two unidentified HeLa DNA-associated polypeptides of 52 and 110 kDa. Identification of these and other newly identified DNA-binding substrates suggest that the dsDNA-activated kinase may regulate transcription, DNA replication, or cell growth.

Human DNA-activated protein kinase phosphorylates serines 15 and 37 in the amino-terminal transactivation domain of human p53.

Human DNA-PK is a nuclear, serine/threonine protein kinase that, when activated by DNA, phosphorylates several DNA-binding substrates, including the tumor suppressor protein p53. To identify which p53 residues are phosphorylated, we examined DNA-PK's ability to phosphorylate synthetic peptides corresponding to human p53 sequences. Serines 15 and 37 in the amino-terminal transactivation domain of human p53, and serines 7 and 18 of mouse p53, were phosphorylated by DNA-PK in the context of synthetic peptides. Other serines in these p53 peptides, and serines in other p53 peptides, including peptides containing the serine 315 p34cdc2 site and the serine 392 casein kinase II site, were not recognized by DNA-PK or were phosphorylated less efficiently. Phosphorylation of the conserved serine 15 in human p53 peptides depended on the presence of an adjacent glutamine, and phosphorylation was inhibited by the presence of a nearby lysine. Phosphorylation of recombinant wild-type mouse p53 was inhibited at high DNA concentrations, suggesting that DNA-PK may phosphorylate p53 only when both are bound to DNA at nearby sites. Our study suggests that DNA-PK may have a role in regulating cell growth and indicates how phosphorylation of serine 15 in DNA-bound p53 could alter p53 function.

Inactivation of DNA-dependent protein kinase by protein kinase Cdelta: implications for apoptosis.

Protein kinase Cdelta (PKCdelta) is proteolytically cleaved and activated at the onset of apoptosis induced by DNA-damaging agents, tumor necrosis factor, and anti-Fas antibody. A role for PKCdelta in apoptosis is supported by the finding that overexpression of the catalytic fragment of PKCdelta (PKCdelta CF) in cells is associated with the appearance of certain characteristics of apoptosis. However, the functional relationship between PKCdelta cleavage and induction of apoptosis is unknown. The present studies demonstrate that PKCdelta associates constitutively with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The results show that PKCdelta CF phosphorylates DNA-PKcs in vitro. Interaction of DNA-PKcs with PKCdelta CF inhibits the function of DNA-PKcs to form complexes with DNA and to phosphorylate its downstream target, p53. The results also demonstrate that cells deficient in DNA-PK are resistant to apoptosis induced by overexpressing PKCdelta CF. These findings support the hypothesis that functional interactions between PKCdelta and DNA-PK contribute to DNA damage-induced apoptosis.

Associations of acute stress and overnight heart rate with feed efficiency in beef heifers.

Proxies have the potential to accelerate feed efficiency (residual feed intake (RFI); kg dry matter/day) improvement, assisting with the reduction of beef cattle feed costs and environmental impact. Heart rate (HR) (beats per minute (BPM)) is associated with feed efficiency and influenced by autonomic activity and peripheral metabolism, suggesting HR could be used as a proxy for feed efficiency. Objectives were to assess associations between overnight HR, lying patterns and RFI, and between acute stress HR and RFI. Heifer calves (n=107; 408±28 days of age, 341±42.2 kg) and yearling heifers (n=36; 604±92 days of age, 539±52.2 kg) were exposed to a performance test to determine productive performance. Overnight HR (electrode based) and lying patterns (accelerometer based) were monitored on a subgroup of heifer calves (n=40; 20 lowest RFI; 20 highest RFI). In the 10-min acute stress assessment, all heifers were individually exposed to the opening and closing of an umbrella and HR before (HRBEF), in response to (HRMAX), after (HRAFT) and change (HRCHG; HRAFT-HRBEF) as a result of exposure were determined. Using polynomial regression, rate of HR decrease pre-exposure (ß 1) and rates of HR increase (ß 2) and decrease (ß 3, ß 4) post-exposure were determined. Heifer calves in the overnight assessment were classified into equal RFI groups (low RFI; high RFI) and HR means were treated as repeated measures and compared using multiple regression. In the acute stress assessment, heifers were classified within cattle category into equal RFI groups (low RFI; high RFI) and means and polynomial regression parameters were compared using multiple regression. Low-RFI heifer calves had a lower overnight HR (69.2 v. 72.6 BPM), similar HR change from lying to standing intervals (8.9 v. 9.2 BPM) and similar time lying (61.1% v. 64.5%) compared with high-RFI heifer calves. Low-RFI heifer calves had a higher absolute HRMAX (162.9 v. 145.7 BPM) and ß 2 (-0.34 v. -0.20) than high-RFI heifer calves. Low-RFI yearling heifers had similar acute stress HR means and a lower ß 1 (0.003 v. 0.006) than high-RFI yearling heifers. Overnight HR and acute stress HR are potential indicators of RFI in heifer calves. However, acute stress HR results varied in yearling heifers, suggesting previous handling experience and/or age influence stress response. Pending further development (predictive ability, repeatability), the acute stress assessment could have potential for on-farm application as a feed efficiency proxy in young heifers with minimal handling experience.

MR imaging, MR spectroscopy, and diffusion tensor imaging of sequential studies in neonates with encephalopathy.

BACKGROUND: Although the imaging, spectroscopic, and diffusion characteristics of brains of infants with neonatal encephalopathy have been described, the time course during which these changes evolve is not clear. The results of sequential MR imaging studies--including anatomic MR imaging, proton MR spectroscopy, and diffusion tensor imaging (DTI)--of 10 patients enrolled prospectively in a study of neonatal encephalopathy are reported to help to clarify the time course of changes in different brain regions during the first 2 weeks of life. METHODS: Ten neonates were prospectively enrolled in a study of the evolution of MR findings in neonatal encephalopathy and were studied 2 (8 patients) or 3 (2 patients) times within the first 2 weeks of life. The MR examination included spin-echo T1 and T2-weighted images, DTI, and long echo time (288 milliseconds) proton MR spectroscopy. Diffusion parameters (diffusivity [D(av)], fractional anisotropy [FA], and individual eigenvalues) were calculated for 10 1-cm2 regions of interest in each hemisphere that were placed based on anatomic landmarks. D(av) and FA were then measured manually in the same areas on a workstation. Metabolite ratios (NAA/Ch, Cr/Ch, Cr/NAA, Lac/Ch, and Lac/NAA) were calculated in 7 regions of interest. Imaging appearance, diffusion parameters, and metabolite ratios were then evaluated longitudinally (comparing with other studies on the same patient at different times) and cross-sectionally (comparing all studies performed on the same postnatal day). RESULTS: In most of the patients a characteristic evolution of DTI and MR spectroscopy parameters was seen during the first 2 weeks after birth. Although the anatomic images were normal or nearly normal on the first 2 days after birth in most patients, abnormalities were detected on DTI (both visually and by quantitative interrogation of D(av) maps) and proton MR spectroscopy (abnormal metabolite ratios). These parameters tended to worsen until about day 5 and then normalize, though in several patients abnormal metabolite ratios persisted. Of interest, as areas of abnormal diffusivity pseudonormalized within one region of the brain they would develop in other areas. Therefore, the pattern of injury looked very different when imaging was performed at different times during this evolution. CONCLUSION: Patterns of injury detected by standard anatomic imaging sequences, DTI sequences, and proton MR spectroscopy varied considerably during the first 2 weeks after injury. The appearance of new areas of reduced diffusion simultaneous with the pseudonormalization of areas that had reduced diffusion at earlier times can result in an entirely different pattern of injury on diffusivity maps acquired at different time points. Awareness of these evolving patterns is essential if studies are performed and interpreted during this critical period of time.