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BACKGROUND: Pathogenic BRCA1 mutations are usually inherited. Constitutional low-level BRCA1 mosaicism has never been reported. METHODS: Next-generation sequencing (NGS) of cancer gene panel of germline and tumour DNA in a patient with early onset, triple-negative breast cancer. RESULTS: Constitutional de novo mosaicism (5%) for a pathogenic (c.1953dupG; p.Lys652Glufs*21) BRCA1mutation was detected in leukocytes, buccal tissue and normal breast tissue DNA, with ∼50% mutation in tumorous breast tissue. CONCLUSION: This is the first reported case of low-level, multiple tissue, constitutional mosaicism in BRCA1, and highlights the need to consider deep sequencing in affected individuals clinically suspected of having cancer predisposition whose tumours display a BRCA mutation.

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We examined the effects of naturally occurring mastitis on bovine oocyte developmental competence in vitro. Specifically, we investigated the effects of intramammary infection on the ovarian pool of oocytes (i.e., follicle-enclosed oocytes) and their ability to undergo in vitro maturation, fertilization, and further development to the blastocyst stage. Culled Holstein cows (n=50) from 9 commercial dairy farms in Israel were allotted to 3 groups according to somatic cell count (SCC) records of the last 3 monthly milk tests as well as of quarter samples collected before slaughter: (1) low SCC (n=7), (2) medium SCC (n=16), or (3) high SCC (n=27). Means of SCC values differed among low-, medium-, and high-SCC groups: 148,000, 311,000 and 1,813,000 cell/mL milk, respectively. Milk yield and days in milk did not differ among the 3 groups. Bacterial isolates included coagulase-negative staphylococci, Escherichia coli, Streptococcus dysgalactiae, or no bacteria found. Ovaries were collected at the abattoir and brought to the laboratory. Cumulus oocyte complexes were recovered separately from each cow and subjected individually to in vitro maturation and fertilization, followed by 8d in culture. The number of aspirated oocytes did not differ among groups, with a range of 17 to 21 oocytes per cow. The proportion of oocytes that cleaved into 2- to 4-cell-stage embryos (86.1 ± 3.4%) did not differ among groups. In contrast, mean percentages of embryos developed to the blastocyst stage on d 7 and 8 after fertilization were less in both medium- and-high SCC groups than in the low-SCC group (5.6 ± 2.3 and 4.1 ± 1.8 vs. 18.1 ± 4.6%, respectively). Additional analysis indicated that cleavage and blastocyst-formation rates did not differ among the bacterial types in the low-, medium-, and high-SCC groups. These are the first results to demonstrate that naturally occurring mastitis disrupts the developmental competence of the ovarian pool of oocytes, (i.e., oocytes at the germinal vesicle stage). The disruption was associated with elevation of SCC rather than bacterial type. The results may provide a partial explanation for the low fertility of cows that have contracted mastitic pathogens before insemination.

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BACKGROUND: The administration of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists to low-density lipoprotein (LDL)-receptor-deficient mice resulted in a reduction in the atherosclerotic lesion area in male mice, but not in female mice. The male mice also exhibited reduction in insulin resistance while the female mice did not. To further examine the relationship between PPARgamma agonists, insulin resistance and atherosclerosis, we used the model of accelerated atherosclerosis in male apolipoprotein E (apoE)-deficient mice rendered diabetic by low-dose streptozotocin (STZ). METHODS: Male, apoE-deficient mice (n = 48) were randomly divided into four groups. To induce diabetes, two groups received low-dose STZ and two groups served as controls. After diabetes induction, rosiglitazone (a PPARgamma agonist) was administered by oral gavage to one of the diabetic and one of the non-diabetic groups. RESULTS: Rosiglitazone reduced significantly the atherosclerotic aortic plaque area in both diabetic and non-diabetic apoE-deficient mice: 340 +/- 54 vs. 201 +/- 27 micromol2 (p = 0.001) in diabetic mice; 243 +/- 22 vs. 158 +/- 27 micromol2 (p = 0.001) in non-diabetic mice. Also, rosiglitazone reduced the correlation coefficient between plasma glucose and the degree of atherosclerosis (p < 0.0025) without affecting plasma glucose levels. The rosiglitazone-treated mice, both diabetic and non-diabetic, had higher lipid levels. CONCLUSIONS: Rosiglitazone-treated animals showed less atherosclerosis despite higher lipid levels and similar glucose levels. These data suggest a direct anti-atherogenic effect of rosiglitazone on the arterial wall.

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OBJECTIVE: To determine whether known historical risk factors of brachial plexus injury differ between affected neonates and healthy controls. METHODS: The files of all 62 children with Erb's palsy who were diagnosed after birth were reviewed. The control group consisted of 124 randomly selected uninjured infants born within the same period. RESULTS: Compared with the control group, the mothers of the neonates with brachial plexus injury were found to be significantly older (32.1+/-5.2 years vs. 28.9+/-5.8 years, P = 0.01), and had a significantly higher incidence of diabetic pregnancy (69% vs. 14.5%, P = 0.001); the infants had a significantly higher mean birth weight (3846+/-576 g vs. 3220+/-582 g, P = 0.0001) and higher incidence of birth weight > or = 4000 g (27% vs. 4.8%, P = 0.0001). Two of the infants in the study group (3.2%) were born by elective cesarean section. CONCLUSIONS: Brachial plexus injury is associated with several non-predictable or preventable risk factors.

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TFIIH is a multifunctional RNA polymerase II general initiation factor that includes two DNA helicases encoded by the Xeroderma pigmentosum complementation group B (XPB) and D (XPD) genes and a cyclin-dependent protein kinase encoded by the CDK7 gene. Previous studies have shown that the TFIIH XPB DNA helicase plays critical roles not only in transcription initiation, where it catalyzes ATP-dependent formation of the open complex, but also in efficient promoter escape, where it suppresses arrest of very early RNA polymerase II elongation intermediates. In this report, we present evidence that ATP-dependent TFIIH action in transcription initiation and promoter escape requires distinct regions of the DNA template; these regions are well separated from the promoter region unwound by the XPB DNA helicase and extend, respectively, approximately 23-39 and approximately 39-50 bp downstream from the transcriptional start site. Taken together, our findings bring to light a role for promoter DNA in TFIIH action and are consistent with the model that TFIIH translocates along promoter DNA ahead of the RNA polymerase II elongation complex until polymerase has escaped the promoter.

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Recently, key advances in biochemical and structural studies of RNA polymerase II (pol II) and the basal transcriptional machinery have shed considerable light on the basic mechanisms underlying the initiation stage of eukaryotic mRNA synthesis. The development of methods for obtaining crystal structures of pol II and its complexes has revolutionized transcriptional studies and holds promise that aspects of initiation will soon be understood at atomic resolution; crosslinking studies have revealed intriguing features of the topology of the pol II initiation complex and provided working models for dynamic steps of initiation; and mechanistic studies have identified promoter escape as a critical step during initiation and brought to light novel roles for the general initiation factors TFIIE, TFIIF, and TFIIH in this process.

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The elongation stage of eukaryotic mRNA synthesis can be regulated by transcription factors that interact directly with the RNA polymerase II (pol II) elongation complex and by activities that modulate the structure of its chromatin template. Recent studies have revealed new elongation factors and have implicated the general initiation factors TFIIE, TFIIF and TFIIH, as well as the C-terminal domain (CTD) of the largest subunit of pol II, in elongation. The recently reported high-resolution crystal structure of RNA polymerase II, which provides insight into the architecture of the elongation complex, marks a new era of investigation into transcription elongation.

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Shortly after initiating promoter-specific transcription in vitro, mammalian RNA polymerase II becomes highly susceptible to arrest in a promoter-proximal region 9-13 base pairs downstream of the transcriptional start site (Dvir, A., Conaway, R. C., and Conaway, J. W. (1996) J. Biol. Chem. 271, 23352-23356). Arrest by polymerase in this region is suppressed by TFIIH in an ATP-dependent reaction (Dvir, A., Conaway, R. C., and Conaway, J. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9006-9010). In this report, we present evidence that, in addition to TFIIH and an ATP cofactor, efficient transcription by RNA polymerase II through this promoter-proximal region requires formation of an "escape-competent" transcriptional intermediate. Formation of this intermediate requires template DNA 40-50 base pairs downstream of the transcriptional start site. This requirement for downstream DNA is transient, since template DNA downstream of +40 is dispensable for assembly of the preinitiation complex, for initiation and synthesis of the first 10-12 phosphodiester bonds of nascent transcripts and for further extension of transcripts longer than approximately 14 nucleotides. Thus, promoter escape requires that the RNA polymerase II transcription complex undergoes a critical structural transition, likely driven by interaction of one or more components of the transcriptional machinery with template DNA 40-50 base pairs downstream of the transcriptional start site.

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TFIIH is a multifunctional RNA polymerase II transcription factor that possesses DNA-dependent ATPase, DNA helicase, and protein kinase activities. Previous studies have established that TFIIH enters the preinitiation complex and fulfills a critical role in initiation by catalyzing ATP-dependent formation of the open complex prior to synthesis of the first phosphodiester bond of nascent transcripts. In this report, we present direct evidence that TFIIH also controls RNA polymerase II activity at a postinitiation stage of transcription, by preventing premature arrest by very early elongation complexes just prior to their transition to stably elongating complexes. Unexpectedly, we observe that TFIIH is capable of entering the transcription cycle not only during assembly of the preinitiation complex but also after initiation and synthesis of as many as four to six phosphodiester bonds. These findings shed new light on the role of TFIIH in initiation and promoter escape and reveal an unanticipated flexibility in the ability of TFIIH to interact with RNA polymerase II transcription intermediates prior to, during, and immediately after initiation.

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With the availability of the general initiation factors (TFIIB, TFIID, TFIIE, TFIIF, and TFIIH), it is now possible to investigate aspects of the mechanism of eukaryotic messenger RNA synthesis in purified, reconstituted RNA polymerase II transcription systems. Rapid progress in these investigations has been spurred by use of a growing number of assays that are proving valuable not only for dissecting the molecular mechanisms of transcription initiation and elongation by RNA polymerase II, but also for identifying and purifying novel transcription factors that regulate polymerase activity. Here we describe a variety of these assays and discuss their utility in the analysis of transcription by RNA polymerase II.

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It is well established that TFIIH-dependent transcription by RNA polymerase II requires a hydrolyzable ATP cofactor for synthesis of the first phosphodiester bond of nascent transcripts. Whether an ATP cofactor is also required after initiation for escape of RNA polymerase II from the promoter has, however, been controversial. We have now addressed this question directly by investigating the ability of RNA polymerase II transcription complexes containing short, approximately 5-8-nucleotide transcripts synthesized in the presence of limiting nucleotides to escape the promoter in the absence of an ATP cofactor in a basal transcription system reconstituted with purified RNA polymerase II and general initiation factors. Depletion of ATP had a profound effect on the ability of initiated complexes to progress into the elongation phase: whereas in the presence of ATP, the majority of transcription complexes could be chased away from the promoter-proximal region, most complexes deprived of ATP catalyzed synthesis of only a few phosphodiester bonds and then ceased elongation after synthesizing transcripts less than 10-14 nucleotides in length. A significant fraction of these transcripts could be extended following addition of ATP, indicating that they were contained in arrested, but potentially active elongation complexes. Like the ATP-requiring step in initiation, ATP-dependent suppression of arrest by RNA polymerase II at promoter-proximal sites is inhibited by adenosine 5'-O-(thio)triphosphate. Transcription complexes containing transcripts longer than 9-10 nucleotides are insensitive to inhibition by ATPgammaS, indicating that susceptibility to ATP-sensitive arrest is a property of very early elongation complexes. Taken together, our findings reveal a novel role for an ATP cofactor in transcription by RNA polymerase II.

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A requirement for an ATP cofactor in synthesis of the first 8-10 bonds of promoter-specific transcripts by RNA polymerase II is well established. Whether ATP is required for synthesis of the first phosphodiester bond or at a slightly later stage in synthesis of nascent transcripts, however, remains controversial. Goodrich and Tjian (Goodrich, J.A., and Tjian, R. (1994) Cell 77, 145-156) recently proposed that synthesis of the first phosphodiester bond of promoter-specific transcripts by RNA polymerase II is independent of ATP and general transcription factors TFIIE and TFIIH. Here we investigate this model. Taken together, our findings indicate that ATP, TFIIE, and TFIIH can have a profound effect on the efficiency of transcription initiation. First, we observe that synthesis of the first phosphodiester bond of transcripts initiated at the adenovirus 2 major late promoter depends strongly on ATP, TFIIE, and TFIIH in a transcription system reconstituted with RNA polymerase II, TFIIH, and recombinant TBP, TFIIB, TFIIE, and TFIIF. Second, we demonstrate that, in this enzyme system, ATP-dependent activation of transcription initiation can occur immediately prior to synthesis of the first phosphodiester bond of nascent transcripts. Finally, we demonstrate that the activated initiation complex is unstable and decays rapidly to an inactive state in the presence of the inhibitor ATP-gammaS (adenosine 5'-O-(thio)triphosphate), even during reiterative synthesis of abortive transcripts.

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The DNA-dependent protein kinase (DNA-PK) phosphorylates RNA polymerase II and a number of transcription factors. We now show that the activity of DNA-PK is directly stimulated by certain transcriptional activator proteins, including the human heat shock transcription factor 1 (HSF1) and a transcriptionally active N-terminal 147 amino acid GAL4 derivative. Stimulation of DNA-PK activity required specific sequences in the activator proteins outside the minimal DNA binding domains. The stimulation of DNA-PK activity also required DNA and was greater with DNA containing relevant activator binding sites. Comparison of different HSF binding fragments showed that optimal stimulation occurred when two HSF binding sites were present. Stimulation with HSF and GAL4 was synergistic with Ku protein, another regulator of DNA-PK activity. DNA-PK is tightly associated with the transcriptional template, and an increase in its activity could potentially influence transcription through the phosphorylation of proteins associated with the transcription complex.

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A two-stage study was carried out to characterize the bile and plasma lipid composition in normolipidemic non-obese patients with and without cholesterol gallstones. The first stage involved 11 patients with cholesterol gallstones admitted for elective cholecystectomy and a control group of 16 patients without cholesterol gallstones undergoing elective laparotomy. Bile samples were obtained intraoperatively by aspiration from the gallbladder. The bile of all the gallstone patients was supersaturated with cholesterol and its nucleation time was much shorter than that of bile in the control group (2.5 days vs 22.5 days, respectively, P < 0.001). The biliary fatty acid profile of phosphatidylcholine (PC) and free fatty acids (FFA) of gallstone patients was similar to that of the control group. C-22 fatty acids were found in a higher concentration in the FFA than in the PC fatty acids (P < 0.05) in both groups of patients. Plasma triglyceride levels in the gallstone patients were significantly higher than those in the control group and the biliary cholesterol level correlated with that of plasma triglycerides. In the second stage of the study, plasma lipid profiles were obtained in two additional groups of patients, 20 patients with and 24 patients without cholesterol gallstones, for an in-depth characterization of the differences in plasma lipid profiles. The gallstone patients were found to have not only significantly higher concentrations of plasma triglycerides but increased cholesterol and phospholipid level as well. These differences were essentially due to a higher lipid content of the plasma VLDL fraction, similar to the pattern of patients with type IV hyperlipoproteinemia.

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We have recently shown that a template-associated protein kinase, which phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II, is a two-component system. We describe here the purification of these two components to apparent homogeneity from human (HeLa) cell nuclear extract. Kinase component A has a 340-kDa native molecular mass, consists of a single large polypeptide, and contains the kinase active site. Kinase component B, which is identical to the Ku autoantigen, has a 180-kDa native molecular mass, and consists of apparently equimolar 67- and 83-kDa polypeptides. Component B stimulates the activity of component A, and under some conditions, confers DNA dependence on the reaction. The purified kinase converts the CTD to the multiply phosphorylated CTD0 form. Conversion occurs processively, and this processivity is an inherent property of component A. The in vitro phosphorylated CTD0 form contains approximately equimolar phosphoserine and phosphothreonine, but no detectable phosphotyrosine.

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The carboxyl-terminal domain of RNA polymerase II contains a tandemly repeated heptapeptide sequence. Previous work has shown that this sequence is phosphorylated at multiple sites by a template-associated protein kinase, in a reaction that is closely associated with the initiation of RNA synthesis. We have purified this kinase to apparent homogeneity from human (HeLa) cells. The purified kinase phosphorylates native RNA polymerase II only in the presence of DNA and the general transcription factors TFIID (TBP), TFIIB, and TFIIF. Two kinase components are required for full activity: a catalytic component and a DNA-binding regulatory component. The regulatory component has been identified as Ku autoantigen, based on the molecular weights of its component polypeptides, its DNA-binding properties, and its reactivity with anti-Ku monoclonal antibodies. The Ku autoantigen recruits the catalytic component of the kinase to the template. Ku autoantigen has been previously proposed to interact with DNA by a characteristic bind-and-slide mechanism. This mode of interaction may provide a mechanism for targeting the kinase to the transcription complex and other DNA-bound substrates.

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A prospective study was performed to investigate the effect of short term lipid infusion on bile composition and its lithogenicity in humans. Thirty five patients shown to be free of cholesterol gall stones participated in the study. Starting 48 hours before surgery they were infused randomly with a lipid emulsion of either long chain triglycerides (LCT) or a mixture of medium and long chain triglycerides (MCT/LCT) (50%/50%) for six hours each 24 hours. A group of patients infused with a solution of 5% glucose in NaCl 0.9% served as a control. Bile samples were obtained by puncture of the gall bladder during operation. Both lipids caused an increase in biliary cholesterol and phospholipids but this effect was more pronounced and significant (p < 0.001) only with the MCT/LCT emulsion. The fatty acid composition of biliary phospholipids was not affected by either lipid infusion. The cholesterol saturation index increased significantly (p < 0.005) with the MCT/LCT emulsion and there was shortening in the nucleation time but this was not significant. There was no effect on the distribution of cholesterol between micelles and vesicles. This study shows that infusion of MCT/LCT lipid emulsion can cause lithogenic changes in bile composition in humans and may thus contribute to sludge formation and cholelithiasis during long term parenteral nutrition.

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Isolated transcription complexes contain a protein kinase that phosphorylates the heptapeptide repeats of the carboxy-terminal domain (CTD) of the RNA polymerase II (RNAP II) large subunit in an apparently promoter-dependent manner. We now show that the essential features of this reaction can be reproduced in a reconstituted system containing three macromolecular components: a fusion protein consisting of the CTD of RNAP II fused to a heterologous DNA-binding domain, an activating DNA fragment containing the recognition sequence for the fusion protein, and a protein kinase that binds nonspecifically to DNA. This kinase closely resembles a previously known DNA-dependent protein kinase. Evidently, the association of the CTD with DNA provides a key signal for phosphorylation. There appears to be no absolute requirement for specific contacts with other DNA-bound transcription factors.

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Protein tyrosine kinase blockers of the tyrphostin family inhibited the EGF-dependent proliferation of human and guinea pig keratinocytes grown in culture and induced their growth arrest. These blockers also significantly inhibited the growth of epidermal keratinocytes, but not of dermal cells, in whole skin organ culture from both guinea pig and human origin. The antiproliferative activity of these tyrphostins correlated quantitatively with their potency as inhibitors of EGF receptor autophosphorylation and the EGF-dependent protein phosphorylation of intracellular target proteins in the keratinocyte. Furthermore, no significant cell cytotoxicity or reduction in serine and threonine phosphorylation of many intracellular polypeptides were observed upon incubation of the cells with tyrphostins like AG213. The complete growth arrest induced by the tyrphostins is fully reversible and upon their removal the keratinocytes resumed their growth with the original growth rate. Because of the nontoxic nature of these compounds and their growth-arresting properties, we suggest their use as agents to treat hyperproliferative conditions of human skin.

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