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Metaphase spindles assemble to a steady state in length by mechanisms that involve microtubule dynamics and motor proteins, but they are incompletely understood. We found that Xenopusextract spindles recapitulate the length of egg meiosis II spindles, by using mechanisms intrinsic to the spindle. To probe these mechanisms, we perturbed microtubule polymerization dynamics and opposed motor proteins and measured effects on spindle morphology and dynamics. Microtubules were stabilized by hexylene glycol and inhibition of the catastrophe factor mitotic centromere-associated kinesin (MCAK) (a kinesin 13, previously called XKCM) and destabilized by depolymerizing drugs. The opposed motors Eg5 and dynein were inhibited separately and together. Our results are consistent with important roles for polymerization dynamics in regulating spindle length, and for opposed motors in regulating the relative stability of bipolar versus monopolar organization. The response to microtubule destabilization suggests that an unidentified tensile element acts in parallel with these conventional factors, generating spindle shortening force.

Sciatic nerves were excised from 3 beagle dogs about 5 h after their sacrifice, treated three times by freezing and thawing, and stored in physiological saline for 3 months at −20°C until used. Nerve segments 5 cm in length prepared from these stored nerves were transplanted to the common peroneal nerve in the right hindlimb of beagle dogs. Sixteen beagle dogs in total were used, in four treatment groups of two pairs each studied at 1 and 3 months. Five-hundred microliters basic fibroblast growth factor (bFGF) of two different concentrations (10 μg/300 μl and 100 μg/300 μl) which were impregnated in 0.5 ml gelatin hydrogels was applied around the sutured allografts. Autografting was also done in 4 beagle dogs, with no bFGF application. One month after the grafting, no regenerating nerves extended beyond the middle of the transplant in any of the allografts, except in the autografts in which a number of regenerated (myelinated) axons were present. Three months after the grafting, an abundance of myelinated axons was found at the middle of the graft: the numbers of axons per 104μm2were 22.6 in the autografts and 10.6, 10.4 and 19.2 in the allografts treated with no bFGF, low-dose bFGF, and high-dose bFGF, respectively. Regenerating axons extended into the host nerve: the numbers of myelinated axons at the level 1.5 cm distal to the distal suture were 35.7, 0.9, 3.8, and 12.1 per 104μm2in the above respective order. Although it was inferior in quality to the autograft, peripheral nerve regeneration was extensive in the distal nerve using freeze–thawed and bFGF-treated allografts at 3 months. Electromyography showed that the peroneus longus muscle responded to the electrical stimuli given at the site proximal to the transplant in all four groups. These data indicate that a 5-cm acellular nerve segment containing Schwann cell basal laminae can be used successfully as an allograft without any immunosuppressants and that exogenously applied bFGF can improve nerve regeneration by enhancing the growth of regenerating axons.

To investigate the site-specific implantation of cancer cells in peritoneal dissemination, we inoculated CDF1 mice intraperitoneally with mouse P388 leukaemia cells labelled with bromodeoxyuridine (BrdU) and then observed immunohistochemically the distribution of the cells in the greater omentum taken from the mice using an anti-BrdU antibody. We found the BrdU-labelled cells infiltrating selectively into the milky spots in the omentum. Furthermore, we intraperitoneally inoculated the BrdU-labelled P388 cells at 10(5), 10(6) and 10(7) cells per mouse into three groups of ten CDF1 mice and then quantified the distribution of the BrdU-labelled cells by counting the number of the labelled cells per unit area at each milky spot and non-milky spot site in the omentum. Inoculations of 10(5), 10(6) and 10(7) BrdU-labelled P388 cells per mouse resulted in 15.8 +/- 13.3, 120 +/- 46.5 and 504 +/- 208 cells mm-2 respectively in the milky spot sites and 9.14 x 10(-3) +/- 1.58 x 10(-2), 1.14 x 10(-1) +/- 7.82 x 10(-2) and 7.07 x 10(-1) +/- 5.98 x 10(-1) cells mm-2 respectively in the non-milky spot sites. The ratios of the mean labelled cell numbers in the milky spot sites vs those in the non-milky spot sites were 1728:1, 1049:1 and 713:1 respectively. In all cases, there were statistically significant differences in the number of BrdU-labelled cells mm-2 between milky spot sites and non-milky spot sites. However, the ratios decreased as the numbers of inoculated cells increased. In addition, we inoculated C57/BL mice intraperitoneally with B-16 PC melanoma cells, which were easily differentiated from the other cells by the intrinsic black melanin, and examined the distribution of the cells macro- and microscopically. The B-16 PC melanoma cells were also found to be infiltrating preferentially into the milky spots in the omentum. These results suggest that cancer cells seeded intraperitoneally specifically infiltrate the milky spots in the early stages of peritoneal dissemination.

The tumour-suppressor gene p53 encodes a transcription factor that plays a critical role in the induction of G1 cell cycle arrest and apoptosis after DNA damage. To clarify the role of the p53 gene and apoptosis in combined hyperthermia, chemotherapy and radiation (hyperthermochemoradiotherapy, HCR therapy) for rectal cancer, we examined the histological response, rate of apoptosis, DNA fragmentation and p53 status in tumours from 28 patients undergoing HCR therapy before surgery and from 22 patients who did not have preoperative treatment. The therapeutic effect of HCR therapy was closely correlated with the rate of apoptosis; the correlation was statistically significant, suggesting that this effect occurs through apoptosis. The incidence of p53 mutations in the treated group were as follows: in tumours resistant to HCR therapy, four of seven (57.1%); intermediately sensitive, 7 of 13 (53.9%); or sensitive, three of eight (37.5%), suggesting that the therapeutic effect and apoptosis rate were related to the p53 status of the tumours to some extent, but the relation was not statistically significant. In the 22 control tumours (non-treated group), the apoptosis rate was 2.0 +/- 1.1%, and there was no significant difference in p53 status compared with the HCR group. Our study indicates that the pathological response to HCR therapy correlates with the rate of apoptosis with statistical significance and that it induces the therapeutic effect more significantly in rectal cancer cells with wild-type p53, although HCR therapy-induced apoptosis also occurs in some rectal cancers with mutated p53. Therefore, this combination therapy can induce an additive or synergistic anti-tumour effect in rectal cancers with wild-type p53 as well as in those with mutated p53 through apoptosis, offering new therapeutic opportunities and a better prognosis.