Tumor attenuation by 2(6-hydroxynaphthyl)-beta-D-xylopyranoside requires priming of heparan sulfate and nuclear targeting of the products

We have previously reported that the heparan sulfate-priming glycoside 2-(6-hydroxynaphthyl)-beta-D-xylopyranoside selectively inhibits growth of transformed or tumor-derived cells. To investigate the specificity of this xyloside various analogs were synthesized and tested in vitro. Selective growth inhibition was dependent on the presence of a free 6-hydroxyl in the aglycon. Because cells deficient in heparan sulfate synthesis were insensitive to the xyloside, we conclude that priming of heparan sulfate synthesis was required for growth inhibition. In growth-inhibited cells, heparan sulfate chains primed by the active xyloside were degraded to products that contained anhydromannose and appeared in the nuclei. Hence the degradation products were generated by nitric oxide-dependent cleavage. Accordingly, nitric oxide depletion reduced nuclear localization of the degradation products and counteracted the growth-inhibitory effect of the xyloside. We propose that 2-(6-hydroxynaphthyl)-beta-D-xylopyranoside entered cells and primed synthesis of heparan sulfate chains that were subsequently degraded by nitric oxide into products that accumulated in the nucleus. In vivo experiments demonstrated that the xyloside administered subcutaneously, perorally, or intraperitoneally was adsorbed and made available to tumor cells located subcutaneously. Treatment with the xyloside reduced the average tumor load by 70-97% in SCID mice. The present xyloside may serve as a lead compound for the development of novel antitumor strategies.     

Identification of DNA-PKcs phosphorylation sites in XRCC4 and effects of mutations at these sites on DNA end joining in a cell-free system

Nonhomologous end joining (NHEJ) is the principal mechanism for repairing DNA double-strand breaks in mammalian cells. NHEJ requires at least three protein components: the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku protein, and the DNA ligase IV/XRCC4 (DNL IV/XRCC4) complex. Although DNA-PKcs phosphorylates several sites within itself and these other proteins, the significance of phosphorylation at individual sites is not yet understood. Here we investigate the effects of DNA-PKcs-mediated phosphorylation at two sites in XRCC4. One is a previously described site at serine 260; the other is a newly mapped site at serine 318. XRCC4 bearing mutations at these sites was co-expressed with DNL IV, the resulting complexes were purified, and activity was tested in a cell-free end-joining system reconstituted from recombinant and purified proteins. Substitution of alanine for serine 260 or 318, which prevents phosphorylation at these positions, or aspartate for serine 260, which mimics constitutive phosphorylation, had no significant effect on overall end-joining activity. In the assay system used, DNA-PKcs is not essential, but when present, arrests the reaction until phosphorylation occurs, in effect establishing a reaction checkpoint. Mutations at serines 260 and 318 did not affect establishment or release from the checkpoint. Results demonstrate that DNA-PKcs-mediated phosphorylation of XRCC4 serine 260 and serine 318 does not directly control end-joining under the conditions tested.     

V(D)J recombination frequencies can be profoundly affected by changes in the spacer sequence

Each V, D, and J gene segment is flanked by a recombination signal sequence (RSS), composed of a conserved heptamer and nonamer separated by a 12- or 23-bp spacer. Variations from consensus in the heptamer or nonamer at specific positions can dramatically affect recombination frequency, but until recently, it had been generally held that only the length of the spacer, but not its sequence, affects the efficacy of V(D)J recombination. In this study, we show several examples in which the spacer sequence can significantly affect recombination frequencies. We show that the difference in spacer sequence alone of two V(H)S107 genes affects recombination frequency in recombination substrates to a similar extent as the bias observed in vivo. We show that individual positions in the spacer can affect recombination frequency, and those positions can often be predicted by their frequency in a database of RSS. Importantly, we further show that a spacer sequence that has an infrequently observed nucleotide at each position is essentially unable to support recombination in an extrachromosmal substrate assay, despite being flanked by a consensus heptamer and nonamer. This infrequent spacer sequence RSS shows only a 2-fold reduction of binding of RAG proteins, but the in vitro cleavage of this RSS is approximately 9-fold reduced compared with a good RSS. These data demonstrate that the spacer sequence should be considered to play an important role in the recombination efficacy of an RSS, and that the effect of the spacer occurs primarily subsequent to RAG binding.     

Sodium channel modulating activity in a delta-conotoxin from an Indian marine snail

A 26 residue peptide (Am 2766) with the sequence CKQAGESCDIFSQNCCVG-TCAFICIE-NH(2) has been isolated and purified from the venom of the molluscivorous snail, Conus amadis, collected off the southeastern coast of India. Chemical modification and mass spectrometric studies establish that Am 2766 has three disulfide bridges. C-terminal amidation has been demonstrated by mass measurements on the C-terminal fragments obtained by proteolysis. Sequence alignments establish that Am 2766 belongs to the delta-conotoxin family. Am 2766 inhibits the decay of the sodium current in brain rNav1.2a voltage-gated Na(+) channel, stably expressed in Chinese hamster ovary cells. Unlike delta-conotoxins have previously been isolated from molluscivorous snails, Am 2766 inhibits inactivation of mammalian sodium channels.     

Radiotherapy: effects on expanded skin

Although the combination of radiation and tissue expansion has been associated with a significant rate of complications, the specific pathophysiology has yet to be clearly elucidated. The objective of this study was to develop a model to identify and examine specific histologic changes associated with tissue expansion and irradiation. Rectangular 50-cc silicone tissue expanders were placed subcutaneously over the midline dorsum of 18 adult New Zealand white rabbits. Preoperative radiographic dosimetry demonstrated that the radiation portal was away from vital intraabdominal structures. The expanders were inflated with 10 cc of saline every other day for a total of 80 cc. Expanders were left in place for 2 to 3 weeks to allow fibrovascular capsule formation. The rabbits were then divided into three groups (six rabbits per group), each receiving one of three nonfractionated doses of radiation (20, 25, or 35 Gy). Half of the expanded skin was irradiated using a single dose, and the other half served as a nonirradiated control. Capsules and skin were harvested 6 weeks after the delivery of radiation, allowing the beginning of chronic radiation changes to occur. Using hematoxylin and eosin staining, histomorphometric analysis was performed. The data were analyzed using Student's test. Although irradiation did not affect dermal thickness, it did cause a statistically significant increase in epidermal thickness. At 20, 25, and 35 Gy the increase in epidermal thickness was 43, 90, and 130 percent, respectively. Although significant epidermal changes could be identified, capsular and dermal alterations were not evident. Further studies evaluating the long-term effects of alterations in capsular formation caused by radiation may be required.