TRIM28 interacts with EZH2 and SWI/SNF to activate genes that promote mammosphere formation.

Histone methyl transferase EZH2 (Enhancer of Zeste Homolog 2) is generally associated with H3K27 methylation and gene silencing, as a member of the polycomb repressor 2 (PRC2) complex. Immunoprecipitation and mass spectrometry of the EZH2-protein interactome in estrogen receptor positive, breast cancer-derived MCF7 cells revealed EZH2 interactions with subunits of chromatin remodeler SWI/SNF complex and TRIM28, which formed a complex with EZH2 distinct from PRC2. Unexpectedly, transcriptome profiling showed that EZH2 primarily activates, rather than represses, transcription in MCF7 cells and with TRIM28 co-regulates a set of genes associated with stem cell maintenance and poor survival of breast cancer patients. TRIM28 depletion repressed EZH2 recruitment to chromatin and expression of this gene set, in parallel with decreased CD44hi/CD24lo mammosphere formation. Mammosphere formation, inhibited by EZH2 depletion, was rescued by ectopic expression of EZH2 but not by TRIM28 expression or by EZH2 mutated at the region (pre-SET domain) of TRIM28 interaction. These results support PRC2-independent functions of EZH2 and TRIM28 in activation of gene expression that promotes mammary stem cell enrichment and maintenance.

p53 targets chromatin structure alteration to repress alpha-fetoprotein gene expression.

Many of the functions ascribed to p53 tumor suppressor protein are mediated through transcription regulation. We have shown that p53 represses hepatic-specific alpha-fetoprotein (AFP) gene expression by direct interaction with a composite HNF-3/p53 DNA binding element. Using solid-phase, chromatin-assembled AFP DNA templates and analysis of chromatin structure and transcription in vitro, we find that p53 binds DNA and alters chromatin structure at the AFP core promoter to regulate transcription. Chromatin assembled in the presence of hepatoma extracts is activated for AFP transcription with an open, accessible core promoter structure. Distal (-850) binding of p53 during chromatin assembly, but not post-assembly, reverses transcription activation concomitant with promoter inaccessibility to restriction enzyme digestion. Inhibition of histone deacetylase activity by trichostatin-A (TSA) addition, prior to and during chromatin assembly, activated chromatin transcription in parallel with increased core promoter accessibility. Chromatin immunoprecipitation analyses showed increased H3 and H4 acetylated histones at the core promoter in the presence of TSA, while histone acetylation remained unchanged at the site of distal p53 binding. Our data reveal that p53 targets chromatin structure alteration at the core promoter, independently of effects on histone acetylation, to establish repressed AFP gene expression.

Single strand DNA binding and annealing activities in the yeast recombination factor Rad59.

Saccharomyces cerevisiae RAD59 gene is required for homologous recombination processes and normal level of resistance to ionizing radiation. To study the biochemical functions of Rad59, it was overproduced in yeast and purified to near homogeneity. Rad59 binds DNA, showing much higher affinity for ssDNA than dsDNA. Rad59 also anneals complementary DNA strands, and order of addition experiments indicate that maximal annealing efficiency is achieved when both complementary DNA strands are present upon addition of Rad59. Thus, Rad59 resembles its homolog Rad52 in being able to bind ssDNA and anneal complementary DNA strands. However, unlike Rad52, DNA annealing by Rad59 is not accelerated by the ssDNA binding factor RPA. DNA binding and strand annealing are likely to be important for the biological functions of Rad59 in general recombination and in the single-strand annealing pathway of recombination.

Yeast Rad51 recombinase mediates polar DNA strand exchange in the absence of ATP hydrolysis.

Saccharomyces cerevisiae RAD51 gene is required for genetic recombination and recombinational repair of DNA strand breaks. Rad51 protein has a DNA-dependent ATPase activity, and it catalyzes ATP-dependent pairing and strand exchange between homologous DNA molecules. We show here that the rad51 Arg-191 protein, which is devoid of ATPase activity, mediates the pairing and strand exchange reaction upon binding ATP. In addition, the wild type Rad51 protein can catalyze pairing and strand exchange in the presence of the nonhydrolyzable ATP analogues adenylyl-imidodiphosphate and adenosine 5'-O-thiotriphosphate. Thus, homologous pairing and the unidirectional transfer of greater than 5 kilobases of DNA can occur efficiently without the need for nucleotide hydrolysis. Consistent with the results from the biochemical analyses, expression of the rad51 Arg-191 protein in a rad51 null mutant confers normal cellular resistance to the DNA damaging agent methylmethane sulfonate, suggesting that nucleotide binding by Rad51 is sufficient for biological function.

Spine injuries in runners: A functional approach.

Interest and participation in running has increased over the past several decades. There has also been a substantial increase in the incidence and prevalence of injuries associated with running. The etiology of running injuries is multifactorial. Of all the musculoskeletal injuries associated with running, lumbar spine injuries are among the least common - yet can be the most debilitating. The impact load generated during heelstrike travels up the lower extremity kinetic chain and converges on the lumbar spine. This impact load is minimized through a series of normal biomechanical actions of the joints and the soft tissues that support them. Dysfunction at any link in the lower extremity kinetic chain can set off a cascading series of mechanical adaptations throughout the spinal axis. Similarly, spinal pain and dysfunction can create peripheral joint adaptations. These mechanical adaptations may become painful or may create a painful dysfunction at a more distant site. There are a variety of structures in the lumbar spine that can cause pain. Mechanical, vascular, biochemical, and neurochemical factors may also be involved in the production of low back pain and radiculopathy. Running stresses the normal biomechanics of the lumbar spine. Changes in normal tissue function including strength, strength balance, flexibility, and proprioception may be caused by these stresses or be the cause of them. New adaptive functional running patterns occur. A thorough rehabilitation plan that addresses all aspects of the injury requires an accurate and complete diagnosis that is made as soon as possible. The rehabilitation program is divided into acute and subacute phases. Education, physical modalities, medication, manual therapy, traction and therapeutic exercise are used to help meet the goals of the acute phase of rehabilitation. The goals of the subacute phase are met by optimizing strength, endurance, and coordination of the neuromuscular system as it affects the lumbar spine and preventing any further injury or recurrences. Running specific rehabilitation may start early in the subacute phase and then progress as the runner's spine stabilization skills improve.

Use of he-ne laser for treatment of soft tissue trauma: evaluation by - gallium-67 citrate scanning*.

Sequential quantitative Gallium-67 Citrate (Ga-67) scanning was used to evaluate the resolution of inflammation in standardized bruises in rat thighs with and without Helium Neon (He-Ne) laser treatment. The severity of inflammation in the injured tissue was assessed as the ratio of Ga-67 localized in the bruise to the Ga-67 localized in the noninjured area of the same geometry. During the first 12 days after injury, both (He-Ne laser) treated and nontreated rats healed rapidly with no observable differences in Ga-67 localization. J Orthop Sports Phys Ther 1986;8(2):93-96.

Therapeutic Ultrasound: Its Effects on the lntegrity of a Nonpenetrating Wound*.

This study investigated the effect of ultrasound applied to traumatized muscle tissue of an adult male rat. Sixty-six rats were traumatized, treated, and sacrificed. The rats were assigned to one of 11 groups, to which experimental and control (two mock) ultrasound treatments were given at 3- and 7-minute time periods at 0.5 and 1.5 watts per square centimeter intensities (pulsed and continuous ultrasound). The rats were given a total of six treatments, once every other day three times a week. Treatments began on the third day following the receipt of a nonpenetrating wound on the right lateral thigh. The animals were subsequently analyzed histologically to determine the extent of healing cells present. The principal findings were as follows: 1) ultrasound intensities had a significant effect on healing, 2) there was no statistical difference between the overall means of the control and experimental groups, and 3) there was no difference between the pulsed and continuous modes of ultrasound or between treatment times.J Orthop Sports Phys Ther 1984;5(5):278-281.

Role of endorphins in pain modulation.

This paper describes a brief history of endorphins and the current theories on the role of endorphins in pain modulation. The use of physical therapy modalities for pain control and the interaction of endorphins may also be implicated. J Orthop Sports Phys Ther 1982;3(4):200-205.

Postoperative thoracotomy. Effect of transcutaneous electrical nerve stimulation on forced vital capacity.

The purpose of this study was to measure the effects of transcutaneous electrical nerve stimulation on patients who had undergone thoracotomies, using the objective physiological measurement of forced vital capacity. Twenty-one patients were randomly assigned to an experimental group (n = 11) or a control group (n = 10). Each group performed three trials of forced vital capacity testing over a 20-minute period. In addition, the experimental group was given a 10-minute treatment of transcutaneous electrical nerve stimulation at the sites of greatest pain. The data indicated a statistically significant increase in forced vital capacity during the stimulation (p less than .01), suggesting that transcutaneous electrical nerve stimulation during application improves chest expansion and mobility in patients who have had thoracotomies.