The mechanics behind DNA sequence-dependent properties of the nucleosome

Chromatin organization and composition impart sophisticated regulatory features critical to eukaryotic genomic function. Although DNA sequence-dependent histone octamer binding is important for nucleosome activity, many aspects of this phenomenon have remained elusive. We studied nucleosome structure and stability with diverse DNA sequences, including Widom 601 derivatives with the highest known octamer affinities, to establish a simple model behind the mechanics of sequence dependency. This uncovers the unique but unexpected role of TA dinucleotides and a propensity for G|C-rich sequence elements to conform energetically favourably at most locations around the histone octamer, which rationalizes G|C% as the most predictive factor for nucleosome occupancy in vivo. In addition, our findings reveal dominant constraints on double helix conformation by H3-H4 relative to H2A-H2B binding and DNA sequence context-dependency underlying nucleosome structure, positioning and stability. This provides a basis for improved prediction of nucleosomal properties and the design of tailored DNA constructs for chromatin investigations.     

HIV gene expression deactivates redox-sensitive stress response program in mouse tubular cells both in vitro and in vivo

Human immunodeficiency virus (HIV)-1 has been reported to cause tubular cell injury both in in vivo and in vitro studies. In the present study, we evaluated the role of oxidative stress in the induction of apoptosis in HIV gene expressing mouse tubular cells in in vivo (Tg26, a transgenic mouse model of HIV-associated nephropathy) and in vitro (tubular cells were transduced with pNL4-3: ΔG/P-GFP, VSV.G psueudo typed virus) studies. Although Tg26 mice showed enhanced tubular cell reactive oxygen species (ROS) generation and apoptosis, renal tissue did not display a robust antioxidant response in the form of enhanced free radical scavenger (MnSOD/catalase) expression. Tg26 mice not only showed enhanced tubular cell expression of phospho-p66ShcA but also displayed nuclear Foxo3a translocation to the cytoplasm. These findings indicated deactivation of tubular cell Foxo3A-dependent redox-sensitive stress response program (RSSRP) in Tg26 mice. In in vitro studies, NL4-3 (pNL4-3: ΔG/P-GFP, VSV.G pseudotyped virus)-transduced mouse proximal tubular cells (NL4-3/MPTEC) displayed enhanced phosphorylation of p66ShcA. NL4-3/MPTECs also displayed greater (P < 0.01) ROS generation when compared with empty vector-transduced tubular cells; however, both diminution of p66ShcA and N-acetyl cysteine attenuated NL4-3-induced tubular cell ROS generation as well as apoptosis. In addition, both antioxidants and free radical scavengers partially inhibited HIV-induced tubular cell apoptosis. NL4-3/MPTEC displayed deactivation of RSSRP in the form of enhanced phosphorylation of Foxo3A and attenuated expression of superoxide dismutase (SOD) and catalase. Since both SOD and catalase were able to provide protection against HIV-1-induced tubular cell apoptosis, it suggests that HIV-1-induced proapoptotic effect may be a consequence of the deactivated RSSRP.     

Direct role of dynein motor in stable kinetochore-microtubule attachment, orientation, and alignment

Cytoplasmic dynein has been implicated in diverse mitotic functions, several involving its association with kinetochores. Much of the supporting evidence comes from inhibition of dynein regulatory factors. To obtain direct insight into kinetochore dynein function, we expressed a series of dynein tail fragments, which we find displace motor-containing dynein heavy chain (HC) from kinetochores without affecting other subunits, regulatory factors, or microtubule binding proteins. Cells with bipolar mitotic spindles progress to late prometaphase-metaphase at normal rates. However, the dynein tail, dynactin, Mad1, and BubR1 persist at the aligned kinetochores, which is consistent with a role for dynein in self-removal and spindle assembly checkpoint inactivation. Kinetochore pairs also show evidence of misorientation relative to the spindle equator and abnormal oscillatory behavior. Further, kinetochore microtubule bundles are severely destabilized at reduced temperatures. Dynein HC RNAi and injection of anti-dynein antibody in MG132-arrested metaphase cells produced similar effects. These results identify a novel function for the dynein motor in stable microtubule attachment and maintenance of kinetochore orientation during metaphase chromosome alignment.     

Radiosynthesis and evaluation of IGF1R PET ligand [11C]GSK1838705A

Radiosynthesis and evaluation of [¹¹C]GSK1838705A in mice using microPET and determination of specificity in human GBM UG87MR cells are described herein. The radioligand was synthesized by reacting desmethyl-GSK1838705A with [¹¹C]CH₃I using GE FX2MeI module in ～5% yield (EOS), >95% radiochemical purity and a specific activity of 2.5 ± 0.5 Ci/μmol. MicroPET imaging in mice indicated that [¹¹C]GSK1838705A penetrated blood brain barrier (BBB) and showed retention of radiotracer in brain. The radioligand exhibited high uptake in U87MG cells with >70% specific binding to IGF1R. Our experiments suggest that [¹¹C]GSK-1838705A can be a potential PET radiotracer for the in vivo quantification of IGF1R expression in GBM and other brain tumors.     

Spindle assembly checkpoint proteins are positioned close to core microtubule attachment sites at kinetochores

Spindle assembly checkpoint proteins have been thought to reside in the peripheral corona region of the kinetochore, distal to microtubule attachment sites at the outer plate. However, recent biochemical evidence indicates that checkpoint proteins are closely linked to the core kinetochore microtubule attachment site comprised of the Knl1–Mis12–Ndc80 (KMN) complexes/KMN network. In this paper, we show that the Knl1–Zwint1 complex is required to recruit the Rod–Zwilch–Zw10 (RZZ) and Mad1–Mad2 complexes to the outer kinetochore. Consistent with this, nanometer-scale mapping indicates that RZZ, Mad1–Mad2, and the C terminus of the dynein recruitment factor Spindly are closely juxtaposed with the KMN network in metaphase cells when their dissociation is blocked and the checkpoint is active. In contrast, the N terminus of Spindly is ∼75 nm outside the calponin homology domain of the Ndc80 complex. These results reveal how checkpoint proteins are integrated within the substructure of the kinetochore and will aid in understanding the coordination of microtubule attachment and checkpoint signaling during chromosome segregation.     

The mealybug chromosome system I: Unusual methylated bases and dinucleotides in DNA of aPlanococcus species

The methylation status of the nuclear DNA from a mealybug, aPlanococcus species, has been studied. Analysis of this DNA by High Performance Liquid Chromatography and Thin Layer Chromatography revealed the presence of significant amounts of 5-—methylcytosine. Since analysis of DNA methylation using the Msp I/Hpa II system showed only minor differences in susceptibility of the DNA to the two enzymes, it seemed possible that 5-methylcytosine (5mC) occurred adjacent to other nucleotides in addition to its usual position, next to guanosine. This was verified by dinucleotide analysis of DNA labelledin vitro by nick translation. These data show that the total amount of 5-methylcytosine in this DNA is slightly over 2.3 mol %, of which 0.61% occurs as the dinucleotide 5mCpG, 0.68% as 5mCpA, 0.59% as 5mCpT and 0.45% as 5mCpC. 5mCpG represents approximately 3.3% of all CpG dinucleotides. The experimental procedure would not have permitted the detection of 5mCp5mC, if it occurs in this system. Unusually high amounts of 6-methyladenine (approximately 4 mol %) and 7-methylguanine (approximately 2 mol %) were also detected, 6-methyladenine and 7-methylguanine occurred adjacent to all four nucleotides. The total G+C content was 33.7% as calculated from dinucleotide data and 32.9% as determined from melting profiles.     

Synthesis of 4-(5-[18F]fluoromethyl-3-phenylisoxazol-4-yl)benzenesulfonamide, a new [18F]fluorinated analogue of valdecoxib, as a potential radiotracer for imaging cyclooxygenase-2 with positron emission tomography

Fluoroalkyl and fluoroaryl analogues of valdecoxib were found to possess potent inhibitory activities against cyclooxygenase-2 comparable to that of the parent valdecoxib. Among them, the fluoromethyl analogue was chosen for 18F-labeling. Thus, 4-(5-[18F]fluoromethyl-3-phenylisoxazol-4-yl)benzenesulfonamide (approximately 2000 Ci/mmol at end of synthesis) was synthesized by [18F]fluoride-ion displacement of the corresponding tosylate in approximately 40% decay-corrected radiochemical yield within approximately 120 min from end of bombardment.     

A high molecular weight plasmid ofZymomonas mobilis harbours genes for HgCl2 resistance

Summary Plasmids fromZ. mobilis could be stably maintained inE. coli HB101 in which the expression of various drug resistance markers could be monitored. A large molecular weight plasmid (5.2 kbp) ofZ. mobilis was found to harbour the genes for mercuric chloride degradation and to confer uponE. coli, resistance to a higher mercuric chloride concentration as compared toZ. mobilis. The introduction of this plamsid madeE. coli sensitive to concentrations of cadmium acetate which were originally non-inhibitory to it.