Molecular mechanisms that distinguish TFIID housekeeping from regulatable SAGA promoters

An important distinction is frequently made between constitutively expressed housekeeping genes versus regulated genes. Although generally characterized by different DNA elements, chromatin architecture and cofactors, it is not known to what degree promoter classes strictly follow regulatability rules and which molecular mechanisms dictate such differences. We show that SAGA‐dominated/TATA‐box promoters are more responsive to changes in the amount of activator, even compared to TFIID/TATA‐like promoters that depend on the same activator Hsf1. Regulatability is therefore an inherent property of promoter class. Further analyses show that SAGA/TATA‐box promoters are more dynamic because TATA‐binding protein recruitment through SAGA is susceptible to removal by Mot1. In addition, the nucleosome configuration upon activator depletion shifts on SAGA/TATA‐box promoters and seems less amenable to preinitiation complex formation. The results explain the fundamental difference between housekeeping and regulatable genes, revealing an additional facet of combinatorial control: an activator can elicit a different response dependent on core promoter class.     

Fine needle aspiration cytology diagnosis of renal medullary carcinoma: a case report

BACKGROUND: Renal medullary carcinoma is a recently described, highly aggressive neoplasm that affects predominantly young African American males with a history of sickle cell trait. To the best of our knowledge, this is the first report of fine needle aspirate cytology (FNAC) findings of renal medullary carcinoma. CASE: A 14-year-old, African American male with a history of sickle cell trait presented with the sudden onset of third cranial nerve palsy. Radiographic examination demonstrated possible tumor masses in the brain, thorax and left kidney. Ultrasound-guided fine needle aspiration was performed on the left kidney, and a cytologic diagnosis of "suspect renal medullary carcinoma" was rendered. The cytologic diagnosis was confirmed by tissue examination. CONCLUSION: The cytologic features of renal medullary carcinoma include loosely cohesive clusters and single epithelioid cells with cytologic atypia, including high nuclear/cytoplasmic ratios, hyperchromasia, prominent nucleoli and cytoplasmic vacuolation. These cytologic findings, coupled with clinical findings (young black male with sickle cell trait), allow recognition of this rare renal neoplasm.     

Effect of humic acids from Leonardite on the stability of soil aggregates and melon roots under greenhouse conditions

Leonardite is an oxidized form of lignite carbon, which is obtained from fossilized organic materials. Such materials are used for the extraction of humic acids (HA). The result of the addition of HA of organic origin on soil structure is known; however, the effects of adding HA of Leonardite on soil structure have been scarcely investigated. The objectives of this research were (1) to determine the influence of humic acids derived from Leonardite in increasing the aggregate stability of an Aridisol under greenhouse conditions, and (2) evaluate the morphology of the root xylem during the phenological development of melon plants (Cucumis melo L.). Three treatments of HA solution application to the soil were used: soil without solution application (HA0), and application of HA solution to the soil with pH 6 (HA6) or (HA7). Aggregate stability (As) and bulk density (Da) were evaluated as soil variables. Development and quantification of xylem area were studied on plants. There were significant differences in aggregate stability. Also, there was an increase in the root xylem area, and the best treatment was when AH7 solution was applied. Humic acids derived from Leonardite increased the stability of soil aggregates when plants grew under greenhouse conditions, and fostered the development of xylem conduits during the fruiting stage.     

Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies

Introduction

Early degeneration of the intervertebral disc (IVD) involves a change in cellular differentiation from notochordal cells (NCs) in the nucleus pulposus (NP) to chondrocyte-like cells (CLCs). The purpose of this study was to investigate the gene expression profiles involved in this process using NP tissue from non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occurring IVD degeneration.

Methods

Dual channel DNA microarrays were used to compare 1) healthy NP tissue containing only NCs (NC-rich), 2) NP tissue with a mixed population of NCs and CLCs (Mixed), and 3) NP tissue containing solely CLCs (CLC-rich) in both non-chondrodystrophic and chondrodystrophic dogs. Based on previous reports and the findings of the microarray analyses, canonical Wnt signaling was further evaluated using qPCR of relevant Wnt target genes. We hypothesized that caveolin-1, a regulator of Wnt signaling that showed significant changes in gene expression in the microarray analyses, played a significant role in early IVD degeneration. Caveolin-1 expression was investigated in IVD tissue sections and in cultured NCs. To investigate the significance of Caveolin-1 in IVD health and degeneration, the NP of 3-month-old Caveolin-1 knock-out mice was histopathologically evaluated and compared with the NP of wild-type mice of the same age.

Results

Early IVD degeneration involved significant changes in numerous pathways, including Wnt/β-catenin signaling. With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant down-regulation of axin2 gene expression. IVD degeneration involved significant down-regulation in Caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of wild-type mice was rich in viable NCs, whereas the NP of Caveolin-1 knock-out mice contained chondroid-like matrix with mainly apoptotic, small, rounded cells.

Conclusions

Early IVD degeneration involves down-regulation of canonical Wnt signaling and Caveolin-1 expression, which appears to be essential to the physiology and preservation of NCs. Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration.     

A new method for surface staining large slices of fixed brain using a copper phthalocyanine dye

Here we describe a method for gross staining of gray matter in slices of formaldehyde-fixed human brain. After protection of white matter with 4% phenol at 60°C for 5 min followed by a cold water wash, the gray matter was stained for 10-15 min at 20-25°C with 1% aqueous copper(II) phthalocyanine tetrasulfonic acid tetrasodium salt (CPTS). The staining resisted all attempts to be washed from the gray matter. Stained slices can be stored indefinitely in slightly acidified water, or plastinated as permanent dry specimens.     

Estimating loss to follow-up in HIV-infected patients on antiretroviral therapy: the effect of the competing risk of death in Zambia and Switzerland

Background

Loss to follow-up (LTFU) is common in antiretroviral therapy (ART) programmes. Mortality is a competing risk (CR) for LTFU; however, it is often overlooked in cohort analyses. We examined how the CR of death affected LTFU estimates in Zambia and Switzerland.

Methods and Findings

HIV-infected patients aged ≥18 years who started ART 2004–2008 in observational cohorts in Zambia and Switzerland were included. We compared standard Kaplan-Meier curves with CR cumulative incidence. We calculated hazard ratios for LTFU across CD4 cell count strata using cause-specific Cox models, or Fine and Gray subdistribution models, adjusting for age, gender, body mass index and clinical stage. 89,339 patients from Zambia and 1,860 patients from Switzerland were included. 12,237 patients (13.7%) in Zambia and 129 patients (6.9%) in Switzerland were LTFU and 8,498 (9.5%) and 29 patients (1.6%), respectively, died. In Zambia, the probability of LTFU was overestimated in Kaplan-Meier curves: estimates at 3.5 years were 29.3% for patients starting ART with CD4 cells <100 cells/µl and 15.4% among patients starting with ≥350 cells/µL. The estimates from CR cumulative incidence were 22.9% and 13.6%, respectively. Little difference was found between naïve and CR analyses in Switzerland since only few patients died. The results from Cox and Fine and Gray models were similar: in Zambia the risk of loss to follow-up and death increased with decreasing CD4 counts at the start of ART, whereas in Switzerland there was a trend in the opposite direction, with patients with higher CD4 cell counts more likely to be lost to follow-up.

Conclusions

In ART programmes in low-income settings the competing risk of death can substantially bias standard analyses of LTFU. The CD4 cell count and other prognostic factors may be differentially associated with LTFU in low-income and high-income settings.     

Spontaneous coronary artery dissection: long stenting in a patient with polycythemia vera

Spontaneous coronary artery dissection is a rare cause of myocardial ischemia or sudden cardiac death. We describe a patient with polycythemia vera and a chronic spontaneous coronary artery dissection who was treated with successful angioplasty and long stenting.     

Dissociation between adenosine receptors and adenylate cyclase in the smooth muscle of guinea pig myometrium

We have previously demonstrated that adenosine causes contraction of guinea-pig myometrium in a fashion consistent with the presence of a purinergic receptor of the A1 subtype. Incubation of guinea-pig uterine smooth muscle membranes with the stable adenosine analogue [3H]cyclohexyladenosine [( 3H]CHA) resulted in rapid, reversible association of radioligand to saturable sites. The affinity (KD) of the receptor for [3H]CHA determined from kinetic experiments (3.14 nM) is in good agreement with that determined in saturation experiments (KD = 4.5 nM). Scatchard analysis of specific [3H]CHA binding (Bmax = 79 fmol/mg protein) is consistent with a single class of binding sites for [3H]CHA. Computer analysis of competition of [3H]CHA binding by the stereoisomers of phenylisopropyl adenosine, R-PIA (KI = 5.3 nM) and S-PIA (KI = 69 nM), as well as the 5'-substituted analogue, ethylcarboxamide adenosine (NECA; KI = 4.2 nM) suggest that [3H]CHA binding occurs to a single class of receptors of the AI subtype. Contractile studies employing these agents reveal that the relative order of potency, based on ED50 values, correlates well with the relative order of competition of agonist binding, based on equilibrium binding constants. Direct assay of myometrial adenylate cyclase failed to show that adenosine receptors in this smooth muscle are coupled to adenylate cyclase. We conclude here that a smooth muscle adenosine receptor is not coupled to adenylate cyclase, yet subserves muscle contraction. These data are important in light of recent attempts to classify adenosine receptors as dual regulators of adenylate cyclase.     

Studies on the mechanism for entry of vesicular stomatitis virus glycoprotein g mRNA into membrane-bound polyribosome complexes

Glycoprotein mRNA (G mRNA) of vesicular stomatitis virus is synthesized in the cytosol fraction of infected HeLa cells. Shortly after synthesis, this mRNA associates with 40S ribosomal subunits and subsequently forms 80S monosomes in the cytosol fraction. The bulk of labeled G mRNA is then found in polysomes associated with the membrane, without first appearing in the subunit or monomer pool of the membrane-bound fraction. Inhibition of the initiation of protein synthesis by pactamycin or muconomycin A blocks entry of newly synthesized G m RNA into membrane-bound polysomes. Under these circumstances, labeled G mRNA accumulates into the cytosol. Inhibition of the elongation of protein synthesis by cucloheximide, however, allows entry of 60 percent of newly synthesized G mRNA into membrane-bound polysomes. Furthermore, prelabeled G mRNA associated with membrane-bound polysomes is released from the membrane fraction in vivo by pactamycin or mucomycon A and in vitro by 1mM puromycin - 0.5 M KCI. This release is not due to nonspecific effects of the drugs. These results demonstrate that association of G mRNA with membrane-bound polysomes is dependent upon polysome formation and initiation of protein synthesis. Therefore, direct association of the 3' end of G mRNA with the membrane does not appear to be the initial event in the formation of membrane-bound polysomes.     

Gene transfer into mouse embryonic stem cell-derived cardiac myocytes mediated by recombinant adenovirus

Summary The main purpose of this study was to examine, for the first time, the ability of recombinant adenovirus to mediate gene transfer into cardiac myocytes derived from mouse embryonic stem (ES) cells differentiating in vitro. In addition, observations were made on the effect of adenovirus infection on cardiac myocyte differentiation and contractility in this in vitro system of cardiogenesis. ES cell cultures were infected at various times of differentiation with a recombinant adenovirus vector (AdCMVlacZ) containing the bacterial lacZ gene under the control of the cytomegalovirus (CMV) promoter. Expression of the lacZ reporter gene was determined by histochemical staining for β-galactosidase activity. LacZ expression was not detected in undifferentiated ES cells infected with AdCMVlacZ. In contrast, infection of differentiating ES cell cultures showed increasing transgene expression with continued time in culture. Expression in ES-cell-derived cardiac myocytes was demonstrated by codetection of β-galactosidase activity and troponin T with indirect immunofluorescence. At 24 h postinfection, approximately 27% of the cardiac myocytes were β-galactosidase positive, and lacZ gene expression appeared to be stable for up to 21 postinfection. Adenovirus infection had no apparent effect on the onset, extent, or duration of spontaneously contracting ES-cell-derived cardiomyocytes, indicating that cardiac differentiation and contractile function were not significantly altered in the infected cultures. The demonstration of adenovirus-mediated gene transfer into ES-cell-derived cardiac myocytes will aid studies of gene expression with this in vitro model of cardiogenesis and may facilitate future studies involving the use of these myocytes for grafting experiments in vivo.