Polymorphisms in the MLL breakpoint cluster region (BCR)

The MLL gene is involved in many chromosomal translocations leading to both acute myeloid and lymphoid leukemia. Some patients treated for primary malignancies with chemotherapeutic agents that inhibit DNA topoisomerase II (topo II) develop treatment-related leukemia (t-AML) caused by MLL gene rearrangement. Whether these patients are unusually susceptible to anti-topo II drugs, or whether this is a random adverse event is unknown. To discover genetic polymorphisms that may predispose patients to t-AML development, we sequenced the 8.3-kb MLL breakpoint cluster region (BCR) from 22 patients who had been treated with topo II inhibitors and who developed t-AML and from 37 patients who did not, and from eight infants and 20 normal individuals. Four polymorphic sites within Alu repetitive elements were identified; three affected the length of poly-A tracts and one altered the size of a trinucleotide repeat. The three poly-A tract polymorphisms occurred with equal frequency in leukemic patients and controls and hence are not predictors of risk. The trinucleotide GAA repeat has three alleles: (GAA)4, (GAA)5, and (GAA)6. The (GAA)6 allele is very rare. The adult t-AML patients are almost exclusively (GAA)4/5 heterozygotes (83%), whereas the normal population is only 55% (GAA)4/5 heterozygotic and is represented equally by (GAA)4 and (GAA)5 homozygotes (20% each). Only certain trends could be established because of the small sample size of these leukemic groups. Whereas adult t-AML patients are more likely to be (GAA)4/5 heterozygotes, this is not statistically significant, and this polymorphism within the MLL BCR has only a suggestive association with t-AML development.     

Vav-1 and the IKK alpha subunit of I kappa B kinase functionally associate to induce NF-kappa B activation in response to CD28 engagement

We have recently observed that CD28 engagement initiates a signaling pathway leading to the activation of I kappa B kinase (IKK) complex and, consequently, to NF-kappa B activation, and we identified Vav-1 as an important mediator of this function. Here we report for the first time that Vav-1 constitutively associates with IKK alpha in both Jurkat and primary CD4(+) T cells. Vav-1/IKK alpha association is mediated by their helix-loop-helix domains, does not involve IKK beta, and is functionally relevant in that Vav-1-associated IKK alpha kinase activity is increased following CD28 engagement by B7. Moreover, we demonstrate that CD28-induced NF-kappa B activation is augmented by both IKK alpha and Vav-1, but not IKK beta. Confocal microscopy showed that endogenous Vav-1 and IKK alpha, but not IKK beta, were recruited to the membrane and colocalized in response to CD28 stimulation. Taken together, these data evidence that Vav-1 plays a key role in the control of NF-kappa B pathway by targeting IKK alpha in the T cell membrane and favoring its activation in response to CD28 stimulation.     

Viscosity and potentiometric measurements of poly(L-histidyl-L-alanyl-α-L-glutamic acid) and poly(L-lysyl-L-alanyl-α-L-glutamic acid)

Poly(His-Ala-Glu) and poly(Lys-Ala-Glu) were examined by viscosity and potentiometric titration. These measurements were interpreted in terms of the hydrodynamic size of the above sequential polypeptides. Effects of polymer, size and concentration, and solution-salt concentration were demonstrated. Although the sequential polypeptides generally behave like polyampholytes, they do demonstrate some differences. These differences my be attributed to the ability of ionized side chains three residues apart to repel themselves, in the order His < Glu < Lys.     

Effect of Ethylenediaminetetraacetate upon the Surface of Escherichia coli

The effect of ethylenediaminetetraacetate (EDTA) on the envelope of two strains of Escherichia coli (B and Cla) was studied with freeze-fracturing methods. Untreated cells showed the outer membrane's outer surface with a fine texture of randomly spaced depressions of about 4.5-nm diameter; small areas with symmetrical arrangements of structural surface elements were also observed. The outer membrane's fracture plane revealed a random distribution of particles on its "concave" plane, only occasionally interrupted by particle-free areas. The "convex" aspect of the outer membrane's fracture plane showed only a few scattered particles. The cleavage plane of the inner membrane was often interrupted by many localized elevated plateaus, at which the cleaving process had, for short distances, switched to the outer membrane. The effects of EDTA treatment were mainly seen in the structure of the freeze-etched outer membrane: (i) the pits as well as the symmetrical surface elements of the outer membrane's outer surface had disappeared; (ii) a number of plateaus (about 20 to 50/cell) were seen at which a cleavage plane within the inner membrane had switched to the hydrophobic portion of the outer membrane (outer membrane's fracture plane). These plateaus were also visible in untreated cells; however, EDTA treatment apparently caused an increased exposure of plateaus. Surface areas, exposed by freeze-etching, revealed the underlying plateaus as elevations in the surface contour of the cell, suggesting a slower etching rate in the zones of the plateaus relative to the rest of the outer membrane. Well-defined, particle-free patches in the outer membrane's fracture plane, concave, were more frequent and larger in size after EDTA treatment than in the controls. In the presence of glycerol, the cells often cleaved in the outer membrane's fracture plane, but isolated plateaus were rarely observed. After metabolic poisoning of cells for 15 to 25 min at 37 degrees C, the plateaus had widened. These data suggest that the material of the plateaus has a slow rate of lateral diffusion. Placement of EDTA-treated cells in fresh medium at 37 degrees C caused, after 3 to 5 min, the reoccurrence of the pitted surface structure. We propose that the plateaus represent localized zones, at which newly synthesized lipopolysaccharide has been inserted.     

Reproductive system of the genus <Emphasis Type=Italic>Crasiella</Emphasis> (Gastrotricha,Macrodasyida)

Crasiella diplura from Sweden and Crasiella sp. from Italy were studied alive and with transmission electron microscopy (TEM). The two species are simultaneous hermaphrodites and share the same reproductive system lay-out: paired ovaries extend along the posterior part of the intestine and join mid-dorsally, while bilateral, club-shaped testes lie at the sides of the anterior gut, extending as deferentia that fuse on the mid-ventral plane and open into a single pore; gametes mature in a caudocephalic and centripetal direction. The bulky, sac-like, frontal organ is lined by a simple epithelium and lies dorsolaterally to the intestine, on the left side of the body. The spindle-shaped caudal organ is musculo-glandular and is located ventrolaterally to the gut on the right side. It is characterized by the presence of a roughly Y-shaped internal channel that opens into two pores close to each other, which function for the intake and outlet of the (auto)sperm, respectively. The spermatozoa, which are peculiar and similar in the two species, are characterized by a long and complex acrosome consisting of four ultrastructurally distinct regions, three of which find equivalence in other gastrotrich species. The flagellum lacks a striated cylinder. Anatomy and ultrastructure enable us to hypothesize a modality of sperm transfer in Crasiella that is similar to that observed in Macrodasys.     

Characterization of quinolinate synthases from Escherichia coli, Mycobacterium tuberculosis, and Pyrococcus horikoshii indicates that [4Fe-4S] clusters are common cofactors throughout this class of enzymes

Quinolinate synthase (NadA) catalyzes a unique condensation reaction between iminoaspartate and dihydroxyacetone phosphate, affording quinolinic acid, a central intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD). Iminoaspartate is generated via the action of l-aspartate oxidase (NadB), which catalyzes the first step in the biosynthesis of NAD in most prokaryotes. NadA from Escherichia coli was hypothesized to contain an iron-sulfur cluster as early as 1991, because of its observed labile activity, especially in the presence of hyperbaric oxygen, and because its primary structure contained a CXXCXXC motif, which is commonly found in the [4Fe-4S] ferredoxin class of iron-sulfur (Fe/S) proteins. Indeed, using analytical methods in concert with Mossbauer and electron paramagnetic resonance spectroscopies, the protein was later shown to harbor a [4Fe-4S] cluster. Recently, the X-ray structure of NadA from Pyrococcus horikoshii was solved to 2.0 A resolution [Sakuraba, H., Tsuge, H.,Yoneda, K., Katunuma, N., and Ohshima, T. (2005) J. Biol. Chem. 280, 26645-26648]. This protein does not contain a CXXCXXC motif, and no Fe/S cluster was observed in the structure or even mentioned in the report. Moreover, rates of quinolinic acid production were reported to be 2.2 micromol min (-1) mg (-1), significantly greater than that of E. coli NadA containing an Fe/S cluster (0.10 micromol min (-1) mg (-1)), suggesting that the [4Fe-4S] cluster of E. coli NadA may not be necessary for catalysis. In the study described herein, nadA genes from both Mycobacterium tuberculosis and Pyrococcus horikoshii were cloned, and their protein products shown to contain [4Fe-4S] clusters that are absolutely required for activity despite the absence of a CXXCXXC motif in their primary structures. Moreover, E. coli NadA, which contains nine cysteine residues, is shown to require only three for turnover (C113, C200, and C297), of which only C297 resides in the CXXCXXC motif. These results are consistent with a bioinformatics analysis of NadA sequences, which indicates that three cysteines are strictly conserved across all species. This study concludes that all currently annotated quinolinate synthases harbor a [4Fe-4S] cluster, that the crystal structure reported by Sakuraba et al. does not accurately represent the active site of the protein, and that the "activity" reported does not correspond to quinolinate formation.     

Searching for new NO-donor aspirin-like molecules: Furoxanylacyl derivatives of salicylic acid and related furazans

A new group of derivatives of salicylic acid containing NO-donor furoxans, and the related des-NO-furazans, were synthesized and evaluated as new aspirin-like molecules. Their stability was assessed in acid (pH 1) and physiological solutions (pH 7.4), and in human serum. No compound exhibited COX-inhibitory activity against COX-1 and COX-2 isoforms, when tested up to 100μM, respectively, on isolated platelets and on monocytes. Phenylsulfonyl- and cyano-substituted furoxans inhibited platelet aggregation induced by collagen in human platelet-rich plasma, through a cGMP dependent mechanism. Furoxan derivatives displayed cGMP-dependent vasodilator activities, tested on rat aorta strips precontracted with phenylephrine. All products showed anti-inflammatory activity similar to that of ASA, tested on rats by the carrageenan-induced paw edema assay. Unlike ASA, all products showed markedly reduced gastrotoxicity in a rat lesion model.