Single-strand conformational polymorphism analysis of the M(r) 170,000 isozyme of DNA topoisomerase II in human tumor cells

Five cell lines selected for resistance to the cytotoxicity of inhibitors of DNA topoisomerase II have point mutations in the gene that codes for the M(r) 170,000 form of this enzyme. In each case, the mutation results in an amino acid change in or near an ATP binding sequence of the M(r) 170,000 isozyme of topoisomerase II. We used single-strand conformational polymorphism analysis to screen for similar mutations in other drug-resistant cell lines or in leukemic cells from patients previously treated with etoposide or teniposide. We also analyzed the region of the gene that codes for amino acids adjacent to the tyrosine at position 804 of topoisomerase II which binds covalently to DNA. CEM/VM-1, CEM/VM-1-5, and HL-60/AMSA human leukemic cell lines were used as controls; 3 of 3 known mutations were detected by migration differences of polymerase chain reaction products from the RNA extracted from these three lines. A previously unknown mutation was found in the tyrosine 804 region of the M(r) 170,000 topoisomerase II expressed by CEM/VM-1 and CEM/VM-1-5 cells. Sequence analysis showed that substitution of a T for a C at nucleotide 2404 resulted in an amino acid change of a serine for a proline at amino acid 802. No mutations in any of the ATP binding sequences or in the tyrosine 804 region were detected in polymerase chain reaction products from RNA extracted from human leukemia HL-60/MX2 or CEM/MX1 cells (both cell lines selected for resistance to mitoxantrone) or in human myeloma 8226/Dox1V cells (selected for resistance by simultaneous exposure to doxorubicin and verapamil). No mutations were detected in polymerase chain reaction products from RNA extracted from blasts of 15 patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide. We conclude that: (a) single-strand conformational polymorphism analysis is useful for screening for mutations in topoisomerase II; (b) resistance to the cytotoxicity of inhibitors of DNA topoisomerase II is not always associated with mutations in ATP binding sequences or the active site tyrosine region of M(r) 170,000 topoisomerase II; and (c) mutations similar to those detected in drug resistant cells selected in culture have not been identified in blast cells from patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide.     

Protein crystal growth in microgravity-temperature induced large scale crystallization of insulin

One of the major stumbling blocks that prevents rapid structure determination using x-ray crystallography is macromolecular crystal growth. There are many examples where crystallization takes longer than structure determination. In some cases, it is impossible to grow useful crystals on earth. Recent experiments conducted in conjunction with NASA on various Space Shuttle missions have demonstrated that protein crystals often grow larger and display better internal molecular order than their earth-grown counterparts. This paper reports results from three Shuttle flights using the Protein Crystallization Facility (PCF). The PCF hardware produced large, high-quality insulin crystals by using a temperature change as the sole means to affect protein solubility and thus, crystallization. The facility consists of cylinders/containers with volumes of 500, 200, 100, and 50 ml. Data from the three Shuttle flights demonstrated that larger, higher resolution crystals (as evidenced by x-ray diffraction data) were obtained from the microgravity experiments when compared to earth-grown crystals.     

Biomimetic Formation of 2‐Tropolones by Dioxygenase‐Catalysed Ring Expansion of Substituted 2,4‐Cyclohexadienones

Substituted 2‐tropolone natural products are found in plants and fungi. Their biosynthesis is thought to occur by ring expansion from a cyclohexadienone precursor, but this reaction has not previously been demonstrated experimentally. Treatment of 6‐hydroxy‐6‐hydroxymethylcyclohexa‐2,4‐dienone with the non‐haem iron(II)‐dependent extradiol catechol dioxygenase MhpB from Escherichia coli results in the formation of the 2‐tropolone ring‐expansion product through a pinacol‐type rearrangement. Three further substituted cyclohexa‐2,4‐dienone analogues were prepared, and treatment of each analogue was found to give the substituted 2‐tropolone ring‐expansion product. This ring expansion could also be effected nonenzymatically by treatment with 1,4,7‐triazacyclononane and FeCl₂. This is a novel transformation for non‐haem iron‐dependent enzymes, and this is the first experimental demonstration of the proposed ring‐expansion reaction in tropolone biosynthesis.     

Metatarsus varus corrected by open wedge osteotomy of the first cuneiform bone

The persistence of metatarsus adductus varus has been a problem in management. We have treated a series of selected patients with this problem and believe that our results have been better than with the procedure we have used in the past. Fowler has described a procedure which seems ideal for the patients in our series. Through personal communication the procedure and its application were discussed and the series was started eight years ago. Our series is small because our patients are responding to other forms of treatment at an earlier age. The few that do not respond are now considered for the operation described. The procedure is relatively simple to perform. Full correction should be obtained at the time of surgery. Casting is utilized to hold the correction and immobilize the extremity for healing. Our unsatisfactory results occurred because of errors in technique or poor selection of patients. We believe that this procedure should be considered in the older patient with metatarsus varus.     

Structure-based design of inhibitors of purine nucleoside phosphorylase. 3. 9-Arylmethyl derivatives of 9-deazaguanine substituted on the methylene group

X-ray crystallography and computer-assisted molecular modeling (CAMM) studies aided in the design of a potent series of mammalian purine nucleoside phosphorylase (PNP) inhibitors. Enhanced potency was achieved by designing substituted 9-(arylmethyl)-9-deazaguanine analogs that interact favorably with all three of the binding subsites of the PNP active site, namely the purine binding site, the hydrophobic pocket, and the phosphate binding site. The most potent PNP inhibitor prepared during our investigation, (S)-9-[1-(3-chlorophenyl)-2-carboxyethyl]-9-deazaguanine (18b), was shown to have an IC50 of 6 nM, whereas the corresponding (R)-isomer was 30-fold less potent.     

Confinement Effects in Shallow-Water Jets

This paper documents measurements of the mean velocity field and turbulence statistics of an isothermal, round jet entering a shallow layer of water. The lower boundary of the jet was a solid wall and the upper boundary a free surface. The jet axis was midway between the solid wall and the free surface in all cases. Experiments were performed at a Reynolds number of 22,500 for water layer depths 15, 10, and 5?times the jet exit diameter (9?mm). Particle image velocimetry measurements were made on vertical and horizontal planes—both containing the axis of the jet. The measurements were taken from 10 to 80 jet diameters downstream. Results showed that, for the highly confined cases at downstream locations, the axial velocity was quite uniform over the depth, with a mild peak below the jet axis. In the horizontal plane, the velocity profiles were slightly narrower than the free jet profile, but in the vertical plane, they were wider. The mean vertical velocity profiles showed that entrainment was suppressed in the vertical direction. At the same time, the lateral velocity profiles indicate that fluid flows from the sides toward the jet centerline. For the shallow cases, the mean vertical velocity becomes negative over most of the depth at downstream locations, indicating that this inflow from the sides is directed downward toward the solid wall. The relative turbulence intensity results were suppressed in the axial and vertical directions and mildly enhanced in the lateral direction. As well, the Reynolds shear stress in the vertical plane was significantly reduced by the vertical confinement, while in the horizontal plane it was only slightly affected by the confinement.     

Revealing list-level control in the Stroop task by uncovering its benefits and a cost.

Interference is reduced in mostly incongruent relative to mostly congruent lists. Classic accounts of this list-wide proportion congruence effect assume that list-level control processes strategically modulate word reading. Contemporary accounts posit that reliance on the word is modulated poststimulus onset by item-specific information (e.g., proportion congruency of the word). To adjudicate between these accounts, we used novel designs featuring neutral trials. In two experiments, we showed that the list-wide proportion congruence effect is accompanied by a change in neutral trial color-naming performance. Because neutral words have no item-specific bias, this pattern can be attributed to list-level control. Additionally, we showed that list-level attenuation of word reading led to a cost to performance on a secondary prospective memory task but only when that task required processing of the irrelevant, neutral word. These findings indicate that the list-wide proportion congruence effect at least partially reflects list-level control and challenge purely item-specific accounts of this effect. (PsycINFO Database Record (c) 2011 APA, all rights reserved)