Rapid exchange of mammalian topoisomerase II alpha at kinetochores and chromosome arms in mitosis

A stable cell line (GT2-LPk) derived from LLC-Pk was created in which endogenous DNA topoisomerase II alpha (topoII alpha) protein was downregulated and replaced by the expression of topoII alpha fused with enhanced green fluorescent protein (EGFP-topoII alpha). The EGFP-topoII alpha faithfully mimicked the distribution of the endogenous protein in both interphase and mitosis. In early stages of mitosis, EGFP-topoII alpha accumulated at kinetochores and in axial lines extending along the chromosome arms. During anaphase, EGFP-topoII alpha diminished at kinetochores and increased in the cytoplasm with a portion accumulating into large circular foci that were mobile and appeared to fuse with the reforming nuclei. These cytoplasmic foci appearing at anaphase were coincident with precursor organelles of the reforming nucleolus called nucleolus-derived foci (NDF). Photobleaching of EGFP-topoII alpha associated with kinetochores and chromosome arms showed that the majority of the protein rapidly exchanges (t1/2 of 16 s). Catalytic activity of topoII alpha was essential for rapid dynamics, as ICRF-187, an inhibitor of topoII alpha, blocked recovery after photobleaching. Although some topoII alpha may be stably associated with chromosomes, these studies indicate that the majority undergoes rapid dynamic exchange. Rapid mobility of topoII alpha in chromosomes may be essential to resolve strain imparted during chromosome condensation and segregation.     

Complete one-stage,immediate breast reconstruction with prosthetic material in patients with large or ptotic breasts

Immediate prosthetic breast reconstruction is a relatively simple, quick procedure with no donor site morbidity. This report discusses immediate one-stage breast reconstruction using prostheses in 18 patients (19 breasts) who also required a contralateral reduction or mastopexy. In all cases, an inverted-T pattern was applied to both breasts. The mean age of the patients was 49 years (range, 32 to 62 years), and the mean size of the gel implant used was 330 ml (range, 120 to 550 ml); the implant was inserted in a total submuscular pocket in seven patients and subcutaneously in 11 patients. In two patients with multiple risk factors, the prosthesis extruded, and one patient required removal for a periprosthetic infection. In 10 patients with early stage disease (T1 or T2) with tumors more than 5 cm from the nipple-areola complex, the original areola (n = 3) or nipple-areola complex (n = 7) was retained as a full-thickness skin graft.The breast shape after submuscular prosthesis insertion is different than that of the contralateral breast after a mastopexy or reduction, and nipple-areola complex symmetry was difficult to obtain; thus, this technique was abandoned in favor of the subcutaneous position (using a modified Wise keyhole pattern with a de-epithelialized portion, which still allows two-layer closure).In the subgroup of patients with large breasts or marked ptosis, a single-stage breast reconstruction procedure can be performed with symmetrical incisions. The subcutaneous position allows for symmetrical shape and nipple-areola complex symmetry to be obtained. When the tumors are small and situated in the periphery of the breast, the nipple-areola complex may be retained as a full-thickness graft.     

Inferences about human demography based on multilocus analyses of noncoding sequences

Data from 10 unlinked autosomal noncoding regions, resequenced in 15 individuals from each of three populations, were used in a multilocus analysis to test models of human demography. Each of the 10 regions consisted of approximately 2500 bp. The multilocus analysis, based on summary statistics (average and variance of Tajima's D and Fu and Li's D*), was used to test a family of models with recent population expansion. The African sample (Hausa of Cameroon) is compatible with a constant population size model and a range of models with recent expansion. For this population sample, we estimated confidence sets that showed the limited range of parameter values compatible with growth. For an exponential growth rate as low as 1 x 10(-3)/generation, population growth is unlikely to have started prior to 50,000 years ago. For higher growth rates, the onset of growth must be more recent. On the basis of the average value of Tajima's D, our sample from an Italian population was found to be incompatible with a constant population size model or any simple expansion model. In the Chinese sample, the variance of Tajima's D was too large to be compatible with the constant population size model or any simple expansion model.     

Affinities of packaging domain loops in HIV-1 RNA for the nucleocapsid protein

To design anti-nucleocapsid drugs, it is useful to know the affinities the protein has for its natural substrates under physiological conditions. Dissociation equilibrium constants are reported for seven RNA stem-loops bound to the mature HIV-1 nucleocapsid protein, NCp7. The loops include SL1, SL2, SL3, and SL4 from the major packaging domain of genomic RNA. The binding assay is based on quenching the fluorescence of tryptophan-37 in the protein by G residues in the single-stranded loops. Tightly bound RNA molecules quench nearly all the fluorescence of freshly purified NCp7 in 0.2 M NaCl. In contrast, when the GGAG-tetraloop of tight-binding SL3 is replaced with UUCG or GAUA, quenching is almost nil, indicating very low affinity. Interpreting fluorescence titrations in terms of a rapidly equilibrating 1:1 complex explains nearly all of the experimental variance for the loops. Analyzed in this way, the highest affinities are for 20mer SL3 and 19mer SL2 hairpin constructs (K(d) = 28 +/- 3 and 23 +/- 2 nM, respectively). The 20mer stem-UUCG-loop and GAUA-loop constructs have <0.5% of the affinity for NCp7 relative to SL3. Affinities relative to SL3 for the other stem-loops are the following: 10% for a 16mer construct to model SL4, 30% for a 27mer model of the 9-residue apical loop of SL1, and 20% for a 23mer model of a 1 x 3 asymmetric internal loop in SL1. A 154mer construct that includes all four stem-loops binds tightly to NCp7, with the equivalent of three NCp7 molecules bound with high affinity per RNA; it is also possible that two strong sites and several weaker ones combine to give the appearance of three strong sites.     

Pharmacokinetics and biodistribution of radioimmunoconjugates of anti-CD19 antibody and single-chain Fv for treatment of human B-cell Malignancy

The comparative advantages and disadvantages of intact antibodies and single-chain Fv as immunotoxins and radioimmunoconjugates have been widely discussed but not directly compared. In this study, the in vivo properties of anti-CD19 B43 monoclonal antibody and its derived single-chain Fv (FVS191) were studied in athymic nude mice bearing CD19-positive human lymphomas. B43 mab and FVS191 were labeled with iodine-125 using iodine-beads, and immunoreactivities were determined to be 57% and 72%, respectively. Scatchard analysis showed a similar high affinity for both. The results of pharmacokinetic studies revealed that FVS191 had a rapid biphasic clearance from the circulation (T1/2α = 2.5 min, T1/2β = 3.7 h); The T1/2α and T1/2β phases of B43 mab were determined to be 0.72 h and 57 h respectively. Biodistribution studies compared the uptake of labeled antibodies by CD19-positive and by CD19-negative tumors. The peak percentages of injected dose were 5.7% at 12 h for B43 and 2.45% at 1 h for FVS191. Radiolocalization indices (RI) demonstrated tumor-specific uptake for both, but higher uptake for B43. The optimal RI was seen at 15 min for FVS191 and 6 h for B43. FVS191 was unstable in vivo, approximately 50% of the injected dose being degraded in blood in 100 min. Radioactivity detected in the urine was present mainly as the deiodinized form of FVS191. The results suggest that B43 mab is favored over FVS191 in biodistribution properties and in vivo stability. Because B43 Mab showed early tumor-specific uptake, high RI values, and favorable tissue-to-blood ratios, it is a potential candidate for radioimmunotherapy and immunotoxin therapy of B-cell leukemia and lymphoma. Received: 17 June 1997 / Accepted: 17 June 1998     

Comparative analysis of the noncollagenous NC1 domain of type IV collagen: identification of structural features important for assembly, function, and pathogenesis.

Type IV collagen alpha1-alpha6 chains have important roles in the assembly of basement membranes and are implicated in the pathogenesis of Goodpasture syndrome, an autoimmune disorder, and Alport syndrome, a hereditary renal disease. We report comparative sequence analyses and structural predictions of the noncollagenous C-terminal globular NC1 domain (28 sequences). The inferred tree verified that type IV collagen sequences fall into two groups, alpha1-like and alpha2-like, and suggested that vertebrate alpha3/alpha4 sequences evolved before alpha1/alpha2 and alpha5/alpha6. About one fifth of NC1 residues were identified to confer either the alpha1 or alpha2 group-specificity. These residues accumulate opposite charge in subdomain B of alpha1 (positive) and alpha2 (negative) sequences and may play a role in the stoichiometric chain selection upon type IV collagen assembly. Neural network secondary structure prediction on multiple aligned sequences revealed a subdomain core structure consisting of six hydrophobic beta-strands and one short alpha-helix with a significant hydrophobic moment. The existence of opposite charges in the alpha-helices may carry implications for intersubdomain interactions. The results provide a rationale for defining the epitope that binds Goodpasture autoantibodies and a framework for understanding how certain NC1 mutations may lead to Alport syndrome. A search algorithm, based entirely on amino acid properties, yielded a possible similarity of NC1 to tissue inhibitor of metalloproteinases (TIMP) and prompted an investigation of a possible functional relationship. The results indicate that NC1 preparations decrease the activity of matrix metalloproteinases 2 and 3 (MMP-2, MMP-3) toward a peptide substrate, though not to [14C]-gelatin. We suggest that an ancestral NC1 may have been incorporated into type IV collagen as an evolutionarily mobile domain carrying proteinase inhibitor function.