Effects of high-oil corn on feedlot performance, carcass characteristics, fatty acid profiles, beef palatability, and retail case life traits of beef top loin steaks.

Our objective was to compare the effects of feeding steam-flaked, high-oil corn with normal steam-flaked corn to which yellow grease was added to equalize dietary fat on performance and carcass characteristics of finishing beef steers, and palatability, retail case life, and fatty acid composition of strip loins. Angus steers (n = 120; initial BW = 288 kg) were allotted to dietary treatments consisting of 1) normal mill-run, steam-flaked corn plus added fat (NMR) or 2) high-oil, steam-flaked corn (HOC) and assigned randomly to pens (12 pens/treatment with 5 steers/pen). Performance (ADG, DMI, and G:F) was measured over time, and cattle were shipped to a commercial abattoir for collection of carcass data after 165 d on feed. Carcass data were collected at 48 h postmortem on all carcasses, and 2 carcasses from each pen were selected randomly for collection of strip loins (IMPS #180A). At 14 d postmortem, 4 steaks (2.54 cm thick) were removed for retail display, trained sensory panel analysis, Warner-Bratzler shear force determination, and fatty acid analysis. Daily BW gain was greater (P = 0.03) and G:F was increased 8.4% (P = 0.01) for steers fed NMR compared with HOC, but DMI was not affected (P > 0.10) by treatment. No treatment differences were observed (P > 0.10) for HCW, 12th-rib fat, KPH, and yield grade. Marbling scores were greater (P = 0.01) for NMR than for HOC, and LM area tended (P = 0.07) to be greater in NMR than in HOC carcasses. The proportion of carcasses grading USDA Choice did not differ (P = 0.77) between treatments, but a greater (P = 0.04) proportion of carcasses graded in the upper two-thirds of Choice for NMR vs. HOC. Trained sensory panel traits and Warner-Bratzler shear force values did not differ between treatments (P > 0.10), and no differences (P > 0.10) were detected for purge loss or fatty acid composition. Overall, ADG and G:F were less and marbling score was decreased, but there were no differences between treatments in beef palatability, retail case life, or concentrations of fatty acids in strip loins.

An elastically compressible phantom material with mechanical and x-ray attenuation properties equivalent to breast tissue.

We have developed a novel phantom material: a solution of polyvinyl alcohol (PVAL) in ethanol and water, freeze-thawed to produce a solid yet elastically compressible gel. The x-ray attenuation and mechanical properties of these gels are compared with published measurements of breast tissue. Gels with PVAL concentrations from 5 to 20% w/v were produced. The linear x-ray attenuation coefficients of these gels range from 0.76 to 0.86 cm(-1) at 17.5 keV, increasing with PVAL concentration. These values are very similar to the published values of breast tissue at this energy, 0.8-0.9 cm(-1). Under compression cancerous breast tissue is approximately ten times stiffer than healthy breast tissue. The Young's moduli of the gels increase with PVAL concentration. Varying the PVAL concentration from 7.5 to 20% w/v produces gels with Young's moduli from 20 to 220 kPa at 15% strain. These values are characteristic of normal and cancerous breast tissue, respectively.

Assessing the validity of modulation transfer function evaluation techniques with application to small area and scanned digital detectors.

A novel prototype step and shoot x-ray system, the intelligent imaging system (I-Imas), has been developed in the UCL Radiation Physics laboratories. The system uses collimators to split the beam into two: the first, "scout" beam, strongly attenuated, identifies regions of interest in the image. This information is then fed back to the system so that the intensity of the second beam is modulated to optimize the dose distribution--i.e., "interesting" regions of the sample receive a higher dose, whereas other regions receive a lower one. Such systems may be capable of improved diagnostic capability at the same overall patient dose levels as typical when using large area digital plates. This acquisition mode means that large overall images are obtained by aligning many smaller images. This paper investigates the effects that this acquisition modality has on the overall spatial resolution of the system. We review different modulation transfer function (MTF) evaluation techniques and those shown to be optimal are used in the investigation of two considerations key to such a system: (i) whether there is a minimum size sensor whose MTF can accurately be determined using these techniques and (ii) whether the MTF of the large overall image differs significantly from those of the many constituent images. As the use of step and shoot systems is becoming more and more widespread, both are important considerations. We found that, for a fixed pixel pitch, the MTF is determined marginally less accurately the smaller the sensor area, with the perceived resolution varying by up to 0.1 lp/mm. It was also found that use of such a step and shoot technique does cause a very small overall degradation in resolution. The resolution of overall images was calculated to be 0.1 lp/mm lower than that of the individual images acquired.

DNA-directed expression of an animal virus RNA for replication-dependent colony formation in Saccharomyces cerevisiae.

To date, the insect nodavirus flock house virus (FHV) is the only virus of a higher eukaryote that has been shown to undergo a full replicative cycle and produce infectious progeny in the yeast Saccharomyces cerevisiae. The genome of FHV is composed of two positive-sense RNA segments: RNA1, encoding the RNA replicase, and RNA2, encoding the capsid protein precursor. When yeast cells expressing FHV RNA replicase were transfected with a chimeric RNA composed of a selectable gene flanked by the termini of RNA2, the chimeric RNA was replicated and transmitted to daughter cells indefinitely. In the work reported here, we developed a system in which a selectable chimeric RNA replicon was transcribed from an inducible RNA polymerase II (polII) promoter in vivo in yeast. To render marker gene expression absolutely dependent on RNA replication, the primary polII transcript was made negative in sense and contained an intron that blocked the translation of cryptic transcripts from the opposite DNA strand. The RNA products of DNA-templated transcription, processing, and RNA replication were characterized by Northern blot hybridization and primer extension analysis. Marker gene expression and colony growth under selective conditions depended strictly on FHV RNA replication, with background colonies arising at a frequency of fewer than 1 in 10(8) plated cells. The utility of the system was demonstrated by introducing a second chimeric replicon and showing that at least two different selectable markers could be simultaneously expressed by means of RNA replication. This is the first example of FHV RNA1-dependent selectable marker expression initiated in vivo and will greatly facilitate the identification and characterization of the requirements and inhibitors of RNA replication.

Characterization and template properties of RNA dimers generated during flock house virus RNA replication.

Flock house virus (FHV) is the best studied member of the Nodaviridae, a family of small, nonenveloped, isometric RNA viruses of insects and fish. Nodavirus genomes comprise two single-stranded positive-sense RNA segments (RNAs 1 and 2) that encode the viral RNA-dependent RNA polymerase (RdRp) and capsid protein precursor, respectively. The RdRp replicates both genomic RNAs and also generates a subgenomic RNA (RNA3) that is not encapsidated. Although genomic RNAs replicate through negative-sense intermediates, little is known about these RNAs or the details of the replication mechanism. Negative-sense RNAs 1, 2, and 3, as well as putative dimers of RNAs 2 and 3, have been detected in previous studies. In this study we detected dimers of RNAs 1, 2, and 3 by Northern blot analyses of RNA samples from FHV-infected Drosophila cells, as well as from mammalian and yeast cells supporting FHV RNA replication. Characterization of these RNA species by RT-PCR and sequence determination showed that they contained head-to-tail junctions of FHV RNAs. RNAs containing the complete sequence of RNA2 joined to RNA3 were also detected during replication. To examine the template properties of these dimeric RNAs, we made corresponding cDNAs and transcribed them from a T7 promoter in mammalian cells constitutively expressing T7 RNA polymerase, together with RNA1 to provide the RdRp. Although heterologous terminal extensions inhibit FHV RNA replication, monomeric RNA2 was resolved and replicated from complete or partial homodimer templates and from an RNA2-RNA3 heterodimer.

Activation of p53 transcriptional activity requires ATM's kinase domain and multiple N-terminal serine residues of p53.

The ATM protein kinase regulates the cell's response to DNA damage by regulating cell cycle checkpoints and DNA repair. ATM phosphorylates several proteins involved in the DNA-damage response, including p53. We have examined the mechanism by which ATM regulates p53's transcriptional activity. Here, we demonstrate that reintroduction of ATM into AT cells restores the activation of p53 by the radio-mimetic agent bleomycin. Further, p53 activation is lost when a kinase inactive ATM is used, or if the N-terminal of ATM is deleted. In addition, AT cells stably expressing ATM showed decreased sensitivity to Ionizing Radiation-induced cell killing, whereas cells expressing kinase inactive ATM or N-terminally deleted ATM were indistinguishable from AT cells. Finally, single point-mutations of serines 15, 20, 33 or 37 did not individually block the ATM-dependent activation of p53 transcriptional activity by bleomycin. However, double mutations of either serines 15 and 20 or serines 33 and 37 blocked the ability of ATM to activate p53. Our results indicate that the N-terminal of ATM and ATM's kinase activity are required for activation of p53's transcriptional activity and restoration of normal sensitivity to DNA damage. In addition, activation of p53 by ATM requires multiple serine residues in p53's transactivation domain.

Glycogen synthase kinase3 beta phosphorylates serine 33 of p53 and activates p53's transcriptional activity.

BACKGROUND: The p53 protein is activated by genotoxic stress, oncogene expression and during senescence, p53 transcriptionally activates genes involved in growth arrest and apoptosis. p53 activation is regulated by post-translational modification, including phosphorylation of the N-terminal transactivation domain. Here, we have examined how Glycogen Synthase Kinase (GSK3), a protein kinase involved in tumorigenesis, differentiation and apoptosis, phosphorylates and regulates p53. RESULTS: The 2 isoforms of GSK3, GSK3alpha and GSK3beta, phosphorylate the sequence Ser-X-X-X-Ser(P) when the C-terminal serine residue is already phosphorylated. Several p53 kinases were examined for their ability to create GSK3 phosphorylation sites on the p53 protein. Our results demonstrate that phosphorylation of serine 37 of p53 by DNA-PK creates a site for GSK3beta phosphorylation at serine 33 in vitro. GSK3alpha did not phosphorylate p53 under any condition. GSK3beta increased the transcriptional activity of the p53 protein in vivo. Mutation of either serine 33 or serine 37 of p53 to alanine blocked the ability of GSK3beta to regulate p53 transcriptional activity. GSK3beta is therefore able to regulate p53 function in vivo. p53's transcriptional activity is commonly increased by DNA damage. However, GSK3beta kinase activity was inhibited in response to DNA damage, suggesting that GSK3beta regulation of p53 is not involved in the p53-DNA damage response. CONCLUSIONS: GSK3beta can regulate p53's transcriptional activity by phosphorylating serine 33. However, GSK3beta does not appear to be part of the p53-DNA damage response pathway. Instead, GSK3beta may provide the link between p53 and non-DNA damage mechanisms for p53 activation.

Reproducible and inexpensive probe preparation for oligonucleotide arrays.

We present a new protocol for the preparation of nucleic acids for microarray hybridization. DNA is fragmented quantitatively and reproducibly by using a hydroxyl radical-based reaction, which is initiated by hydrogen peroxide, iron(II)-EDTA and ascorbic acid. Following fragmentation, the nucleic acid fragments are densely biotinylated using a biotinylated psoralen analog plus UVA light and hybridized on microarrays. This non-enzymatic protocol circumvents several practical difficulties associated with DNA preparation for microarrays: the lack of reproducible fragmentation patterns associated with enzymatic methods; the large amount of labeled nucleic acids required by some array designs, which is often combined with a limited amount of starting material; and the high cost associated with currently used biotinylation methods. The method is applicable to any form of nucleic acid, but is particularly useful when applying double-stranded DNA on oligonucleotide arrays. Validation of this protocol is demonstrated by hybridizing PCR products with oligonucleotide-coated microspheres and PCR amplified cDNA with Affymetrix Cancer GeneChip microarrays.

Life history attributes of Indian meal moth (Lepidoptera: Pyralidae) and Angoumois grain moth (Lepidoptera: Gelechiidae) reared on transgenic corn kernels.

The Indian meal moth, Plodia interpunctella (Hübner), and Angoumois grain moth, Sitotroga cerealella (Olivier), are two globally distributed stored-grain pests. Laboratory experiments were conducted to examine the impact that corn (Zea mays L.) kernels (i.e., grain) of some Bacillus thuringiensis Berliner (Bt) corn hybrids containing CrylAb Bt delta-endotoxin have on life history attributes of Indian meal moth and Angoumois grain moth. Stored grain is at risk to damage from Indian meal moth and Angoumois grain moth; therefore, Bt corn may provide a means of protecting this commodity from damage. Thus, the objective of this research was to quantify the effects of transgenic corn seed containing CrylAb delta-endotoxin on Indian meal moth and Angoumois grain moth survival, fecundity, and duration of development. Experiments with Bt grain, non-Bt isolines, and non-Bt grain were conducted in environmental chambers at 27 +/- 1 degrees C and > or = 60% RH in continuous dark. Fifty eggs were placed in ventilated pint jars containing 170 g of cracked or whole corn for the Indian meal moth and Angoumois grain moth, respectively. Emergence and fecundity were observed for 5 wk. Emergence and fecundity of Indian meal moth and emergence of Angoumois grain moth were significantly lower for individuals reared on P33V08 and N6800Bt, MON 810 and Bt-11 transformed hybrids, respectively, than on their non-Bt transformed isolines. Longer developmental times were observed for Indian meal moth reared on P33V08 and N6800Bt than their non-Bt-transformed isolines. These results indicate that MON 810 and Bt-11 CrylAb delta-endotoxin-containing kernels reduce laboratory populations of Indian meal moth and Angoumois grain moth. Thus, storing Bt-transformed grain is a management tactic that warrants bin scale testing and may effectively reduce Indian meal moth and Angoumois grain moth populations in grain without application of synthetic chemicals or pesticides.

DNA-Directed expression of functional flock house virus RNA1 derivatives in Saccharomyces cerevisiae, heterologous gene expression, and selective effects on subgenomic mRNA synthesis.

Flock house virus (FHV), a positive-strand RNA animal virus, is the only higher eukaryotic virus shown to undergo complete replication in yeast, culminating in production of infectious virions. To facilitate studies of viral and host functions in FHV replication in Saccharomyces cerevisiae, yeast DNA plasmids were constructed to inducibly express wild-type FHV RNA1 in vivo. Subsequent translation of FHV replicase protein A initiated robust RNA1 replication, amplifying RNA1 to levels approaching those of rRNA, as in FHV-infected animal cells. The RNA1-derived subgenomic mRNA, RNA3, accumulated to even higher levels of >100,000 copies per yeast cell, compared to 10 copies or less per cell for 95% of yeast mRNAs. The time course of RNA1 replication and RNA3 synthesis in induced yeast paralleled that in yeast transfected with natural FHV virion RNA. As in animal cells, RNA1 replication and RNA3 synthesis depended on FHV RNA replicase protein A and 3'-terminal RNA1 sequences but not viral protein B2. Additional plasmids were engineered to inducibly express RNA1 derivatives with insertions of the green fluorescent protein (GFP) gene in subgenomic RNA3. These RNA1 derivatives were replicated, synthesized RNA3, and expressed GFP when provided FHV polymerase in either cis or trans, providing the first demonstration of reporter gene expression from FHV subgenomic RNA. Unexpectedly, fusing GFP to the protein A C terminus selectively inhibited production of positive- and negative-strand subgenomic RNA3 but not genomic RNA1 replication. Moreover, changing the first nucleotide of the subgenomic mRNA from G to T selectively inhibited production of positive-strand but not negative-strand RNA3, suggesting that synthesis of negative-strand subgenomic RNA3 may precede synthesis of positive-strand RNA3.

Highly selective isolation of unknown mutations in diverse DNA fragments: toward new multiplex screening in cancer.

Cancer research would greatly benefit from technologies that allow simultaneous screening of several unknown gene mutations. Lack of such methods currently hampers the large-scale detection of genetic alterations in complex DNA samples. We present a novel mismatch-capture methodology for the highly efficient isolation and amplification of mutation-containing DNA from diverse nucleic acid fragments of unknown sequence. To demonstrate the potential of this method, heteroduplexes with a single A/G mismatch are formed via cross-hybridization of mutant (T-->G) and wild-type DNA-fragment populations. Aldehydes are uniquely introduced at the position of mismatched adenines via the Escherichia coli glycosylase, MutY. Subsequent treatment with a biotinylated hydroxylamine results in highly specific and covalent biotinylation of the site of mismatch. For PCR amplification, synthetic linkers are then ligated to the DNA fragments. Biotinylated DNA is then isolated and PCR amplified. Mutation-containing DNA fragments can subsequently be sequenced to identify type and position of mutation. This method correctly detects a single T-->G transversion introduced into a 7-kb plasmid containing full-length cDNA from the p53 gene. In the presence of a high excess wild-type DNA (1:1000 mutant:normal plasmids) or in the presence of diverse DNA fragment sizes, the DNA fragments containing the mutation are readily detectable and can be isolated and amplified. The present Aldehyde-Linker-Based Ultrasensitive Mismatch Scanning has a current limit of detection of one base substitution in 7 Mb of DNA and increases the limit for unknown mutation scanning by two to three orders of magnitude. Homozygous and heterozygous p53 regions (G-->T, exon 4) from genomic DNA are also examined, and correct identification of mutations is demonstrated. This method should allow large-scale detection of genetic alterations in cancer samples without any assumption as to the genes of interest.

Constitutive activation of IkappaB kinase alpha and NF-kappaB in prostate cancer cells is inhibited by ibuprofen.

Apoptotic pathways controlled by the Rel/NF-kappaB family of transcription factors may regulate the response of cells to DNA damage. Here, we have examined the NF-kappaB status of several prostate tumor cell lines. In the androgen-independent prostate tumor cells PC-3 and DU-145, the DNA-binding activity of NF-kappaB was constitutively activated and IkappaB-alpha levels were decreased. In contrast, the androgen-sensitive prostate tumor cell line LNCaP had low levels of NF-kappaB which were upregulated following exposure to cytokines or DNA damage. The activity of the IkappaB-alpha kinase, IKKalpha, which mediates NF-kappaB activation, was also measured. In PC-3 cells, IKKalpha activity was constitutively active, whereas LNCaP cells had minimal IKKalpha activity that was activated by cytokines. The anti-inflammatory agent ibuprofen inhibited the constitutive activation of NF-kappaB and IKKalpha in PC-3 and DU-145 cells, and blocked stimulated activation of NF-kappaB in LNCaP cells. However, ibuprofen did not directly inhibit IkappaB-alpha kinase. The results demonstrate that NF-kappaB is constitutively activated in the hormone-insensitive prostate tumor cell lines PC-3 and DU-145, but not in the hormone responsive LNCaP cell line. The constitutive activation of NF-kappaB in prostate tumor cells may increase expression of anti-apoptotic proteins, thereby decreasing the effectiveness of anti-tumor therapy and contributing to the development of the malignant phenotype.

Caffeine inhibits the checkpoint kinase ATM.

The basis of many anti-cancer therapies is the use of genotoxic agents that damage DNA and thus kill dividing cells. Agents that cause cells to override the DNA-damage checkpoint are predicted to sensitize cells to killing by genotoxic agents. They have therefore been sought as adjuncts in radiation therapy and chemotherapy. One such compound, caffeine, uncouples cell-cycle progression from the replication and repair of DNA [1] [2]. Caffeine therefore servers as a model compound in establishing the principle that agents that override DNA-damage checkpoints can be used to sensitize cells to the killing effects of genotoxic drugs [3]. But despite more than 20 years of use, the molecular mechanisms by which caffeine affects the cell cycle and checkpoint responses have not been identified. We investigated the effects of caffeine on the G2/M DNA-damage checkpoint in human cells. We report that the radiation-induced activation of the kinase Cds1 [4] (also known as Chk2 [5]) is inhibited by caffeine in vivo and that ATM kinase activity is directly inhibited by caffeine in vitro. Inhibition of ATM provides a molecular explanation of the attenuation of DNA-damage checkpoint responses and for the increased radiosensitivity of caffeine-treated cells [6] [7] [8].

An essential role of NFkappaB in tyrosine kinase signaling of p38 MAP kinase regulation of myocardial adaptation to ischemia.

We have recently demonstrated that myocardial adaptation to ischemia triggers a tyrosine kinase regulated signaling pathway leading to the translocation and activation of p38 MAP kinase and MAPKAP kinase 2. Since oxidative stress is developed during ischemic adaptation and since free radicals have recently been shown to function as an intracellular signaling agent leading to the activation of nuclear transcription factor, NFkappaB, we examined whether NFkappaB was involved in the ischemic adaptation process. Isolated perfused rat hearts were adapted to ischemic stress by repeated ischemia and reperfusion. Hearts were pretreated with genistein to block tyrosine kinase while SB 203580 was used to inhibit p38 MAP kinases. Ischemic adaptation was associated with the nuclear translocation and activation of NFkappaB which was significantly blocked by both genistein and SB 203580. The ischemically adapted hearts were more resistant to ischemic reperfusion injury as evidenced by better function recovery and less tissue injury during post-ischemic reperfusion. Ischemic adaptation developed oxidative stress which was reflected by increased malonaldehyde formation. A synthetic peptide containing a cell membrane-permeable motif and nuclear sequence, SN 50, which blocked nuclear translocation of NFkappaB during ischemic adaptation, significantly inhibited the beneficial effects of adaptation on functional recovery and tissue injury. In concert, SN 50 reduced the oxidative stress developed in the adapted myocardium. These results demonstrate that p38 MAP kinase might be upstream of NFkappaB which plays a role in ischemic preconditioning of heart.

The DNA-dependent protein kinase participates in the activation of NF kappa B following DNA damage.

The NFkB transcription factor is activated by diverse stimuli, including Ionizing Radiation (IR) and the cytokine TNF alpha. The role of DNA-PK, a protein kinase involved in the response to DNA damage, in the activation of NF kappa B by IR and TNF alpha was examined. In M059K cells, which express DNA-PK, NF kappa B was activated by both TNF alpha and IR. In M059J cells, which do not express DNA-PK, IR did not activate NF kappa B, whereas TNF alpha induction of NF kappa B was still observed. In HeLa cells, wortmannin, an inhibitor of DNA-PK, blocked the induction of NF kappa B by IR but not by TNF alpha. DNA-PK also phosphorylated the NF kappa B inhibitory proteins IkB-alpha and IkB-beta in vitro, and deletion analysis demonstrated that DNA-PK phosphorylates 2 distinct regions of IkB-beta. These results indicate that DNA-PK participates in the activation of NF kappa B by IR but not by TNF alpha.

Regulation of the p53 protein by protein kinase C alpha and protein kinase C zeta.

The C-terminal of p53 (amino-acids 368-383) represses the DNA binding activity of p53. In vitro, phosphorylation of this region by Protein Kinase C (PKC) is associated with increased DNA binding activity. However, whether PKC can directly modulate p53 function in vivo is not known. Here, we demonstrate that cotransfection of p53 with either PKC alpha or PKC zeta increases p53's transcriptional activity. Mutagenesis of p53 indicates that serine 371 is the major site for phosphorylation by PKC alpha in vitro. Mutation of serine 371 caused a small decline in p53 activation by PKC alpha and PKC zeta. However, the alternatively spliced murine p53, which lacks the PKC phosphorylation sites, still demonstrated increased transcriptional activation when cotransfected with either PKC alpha or PKC zeta. The results indicate that phosphorylation of p53 by PKC in vitro does not correlate with the ability of PKC to upregulate p53's transcriptional activity in vivo.

Radiosensitization of human tumor cells by the phosphatidylinositol3-kinase inhibitors wortmannin and LY294002 correlates with inhibition of DNA-dependent protein kinase and prolonged G2-M delay.

Members of the phosphatidylinositol (PI) 3-kinase gene family, including the ataxia telangiectasia gene and the DNA-dependent protein kinase (DNA-PK), are involved in regulating cellular radiosensitivity. We have investigated two structurally unrelated PI 3-kinase inhibitors, wortmannin and LY294002, to determine whether they inhibit DNA-PK and increase cellular radiosensitivity. The PI 3-kinase inhibitors wortmannin and LY294002 were effective radiosensitizers of human tumor cells, with sensitizer enhancement ratios (at 10% survival) of 2.8 and 1.9, respectively, in SW480 cells. Wortmannin and LY294002 inhibited the kinase activity of purified DNA-PK and inactivated cellular DNA-PK kinase activity. Inhibition of cellular DNA-PK activity occurred at the same concentrations of wortmannin that caused radiosensitization, and this correlation was found in a range of tumor cell lines. However, cells deficient in either DNA-PK (scid cells) or the ataxia telangiectasia protein were also partly sensitized to radiation by wortmannin, indicating the involvement of more than one protein kinase in the mechanism of action of wortmannin. Wortmannin also affected the G2-M checkpoint. SW480 cells had a reversible G2-M delay of 20 h following irradiation. However, wortmannin-treated SW480 cells had a prolonged G2-M delay; more than 75% of cells were arrested in G2 at 50 h postirradiation. This suggests the accumulation of significant unrepaired DNA damage following inhibition of PI 3-kinase family members. Therefore, PI 3-kinase inhibitors may represent a new class of radiosensitizers that inhibit the repair of DNA damage.

A simple demonstration of hindfoot flexibility in the cavovarus foot.

A simple prone examination for demonstrating both hindfoot flexibility and the "tripod" effect in the cavovarus foot has been implemented. This clinical evaluation can be performed without the use of any special equipment. Prone positioning also allows easy demonstration of deformity to family members.

Sequential phosphorylation by mitogen-activated protein kinase and glycogen synthase kinase 3 represses transcriptional activation by heat shock factor-1.

Mammalian heat shock genes are regulated at the transcriptional level by heat shock factor-1 (HSF-1), a sequence-specific transcription factor. We have examined the role of serine phosphorylation of HSF-1 in the regulation of heat shock gene transcription. Our experiments show that mitogen-activated protein kinases (MAPKs) of the ERK-1 family phosphorylate HSF-1 on serine residues and repress the transcriptional activation of the heat shock protein 70B (HSP70B) promoter by HSF-1 in vivo. These effects of MAPK are transmitted through a specific serine residue (Ser-303) located in a proline-rich sequence within the transcriptional regulatory domain of human HSF-1. However, despite the importance of Ser-303 in transmitting the signal from the MAPK cascade to HSP70 transcription, there was no evidence that Ser-303 could be phosphorylated by MAPK in vitro, although an adjacent residue (Ser-307) was avidly phosphorylated by MAPK. Further studies revealed that Ser-303 is phosphorylated by glycogen synthase kinase 3 (GSK3) through a mechanism dependent on primary phosphorylation of Ser-307 by MAPK. Secondary phosphorylation of Ser-303 by GSK3 may thus repress the activity of HSF-1, and its requirement for priming by MAPK phosphorylation of Ser-307 provides a potential link between the MAPK cascade and HSF-1. Our experiments thus indicate that MAPK is a potent inhibitor of HSF-1 function and may be involved in repressing the heat shock response during normal growth and development and deactivating the heat shock response during recovery from stress.