Stable versus unstable orientations of sex chromosomes in two grasshopper species

The structural basis of orientation stability was investigated. The stable unipolar orientation of the Melanoplus sanguinipes X-chromosome univalent is unique in that it is stable without tension created by forces towards opposite poles; tension is thought to be the principle component in stabilizing kinetochore orientations to a pole. Stable orientation of the X chromosome in Melanoplus sanguinipes was compared with unstable X orientation in Melanoplus differentialis. Ten cells (five of each species) were studied, firstly in living cultures where chromosome behavior was followed, then by serial-section electron microscopy where the structural basis for chromosome behavior was examined. Microtubules other than kinetochore microtubules were observed impinging on the X chromosomes. One end of these microtubules was buried in chromatin, while the other ran towards a pole. The X chromosomes of M. sanguinipes had more of these microtubules than did M. differentialis X chromosomes. It is suggested that M. sanguinipes X chromosomes are less condensed than M. differentialis X chromosomes and so allow more microtubules to penetrate the chromosome. The extra microtubules impinging on the M. sanguinipes X chromosome probably prevent reorientation by inhibiting the turning of the chromosome towards the opposite pole, i.e., more force is needed to turn a kinetochore towards the opposite pole than can be generated and attempts at reorientation fail. This may be analogous to the effect that tension has on the orientation stability of bivalents.     

The effect of natural killer cells on the development of syngeneic hematopoietic progenitors

To determine whether natural killer (NK) cells are involved in the regulation of hematopoiesis, well-characterized, cell sorter-purified NK cells were incubated with syngeneic bone marrow, and the effect of this interaction on the development of various hematopoietic progenitors was assessed. NK cells were obtained from the peritoneal exudates of CBA/J mice after i.p. infection with live Listeria monocytogenes (LM). These NK cells were nylon wool-nonadherent and were purified by using M1/70, a rat anti-murine macrophage monoclonal antibody, and a fluorescence-activated cell sorter (FACS). Syngeneic bone marrow was incubated overnight with these M1/70-purified NK cells. The cells were then assayed in vitro to determine the effect on the colony formation of the following hematopoietic progenitor cells: the myeloid progenitor that produces mixed granulocyte/macrophage colonies (CFU-G/M), the myeloid progenitor that is committed to macrophage differentiation (CFU-M), and the early erythroid progenitor that is known as the burst-forming unit-erythroid (BFU-E). The marrow cells, after incubation with NK cells, were also injected into lethally irradiated syngeneic recipients to assay for the splenic colony formation capacity of the trilineage myeloid stem cell (CFU-S). Although the formation of BFU-E-, CFU-G/M-, and CFU-M-derived colonies was not adversely affected by the exposure of syngeneic bone marrow to purified NK cells, there was a dramatic decrease in the number of CFU-S-derived colonies. Incubation with NK-depleted cells did not result in an inhibition of colony formation by the CFU-S. Mixing experiments showed that the M1/70-labeled NK cells exerted their effect directly on the CFU-S and not on any accessory cells. The effect of the NK cells on colony formation by the CFU-S could be blocked competitively and selectively by the addition, before incubation, of a classic murine NK tumor target, Yac-1. Another tumor line (WTS) that is poorly recognized by NK cells was less effective in blocking the inhibitory effect of NK cells on CFU-S. The demonstration that purified NK cells can selectively inhibit the development of the tripotential CFU-S may point to the importance of NK cells in the regulation of hematopoiesis, in the development of some types of marrow dysfunction, and in the failure of engraftment of transplanted bone marrow.     

Characterization of natural killer cells induced in the peritoneal exudates of mice infected with Listeria monocytogenes: a study of their tumor target specificity and their expression of murine differentiation antigens and human NK-associated antigens

That M1/70, a monoclonal anti-murine macrophage antibody, recognizes murine natural killer cells (NK) and that there is an increase in NK following intraperitoneal infection with live Listeria monocytogenes (LM) was previously reported. Here, LM-induced NK cells were further characterized with respect to tumor target specificity and the expression of murine mast cell, mononuclear phagocyte, and lymphocyte differentiation antigens plus human NK-associated antigens. The M1/70-selected NK (Mac 1 NK) lysed Yac 1, RLmale 1, and WEHI 164.1, but not EL 4 or WTS cells. Immunoprecipitation with M1/70 demonstrated that Mac 1, the antigen recognized by M1/70, was present on NK and thioglycollate-elicited macrophages. Contaminating macrophages in the NK-enriched population did not account for the immunoprecipitated Mac 1. Mac 1 NK that lysed Yac 1 displayed Qa 5, LFA 1, asialo GM 1, Ly 5.1, and NK 1.2, but not Lyt 1, Lyt 2, Mac 2, Mac 3, or Mac 4. Thirty percent of these Mac 1 NK bore Thy 1.2. The presence of Thy 1.2 did not correlate with individual lytic efficiency or cell cycle. Antibodies to human NK antigens Leu 7, Leu 11a, and Leu 15 did not recognize LM-induced NK cells.     

A transmissible dicentric chromosome in Drosophila melanogaster

A transmissible dicentric chromosome was recovered in Drosophila melanogaster. The radiation-induced secondary chromosome rearrangement consists essentially of the entire Y and fourth chromosomes joined by 2R heterochromatin. The Y ^S · Y ^L 2Rh4 · chromosome pairs with the X and the free fourth chromosome to form a trivalent in meiosis that is unusual because it forms few chromosome bridges in primary spermatocytes and is transmitted at high frequency. We suggest that the orientation of the weaker fourth chromosome kinetochore eventually fails when opposing the stronger Y kinetochore so that the Y ^S · Y ^L 2Rh4 · moves to the pole to which the Y kinetochore is oriented. There is however an increased frequency of sex chromosome nondisjunction (14%) and of chromosome laggards (6%) in primary spermatocytes; the frequency of exceptional progeny of males containing the Y ^S · Y ^L 2Rh4 · was 7.44% compared with 0.25% in the controls. Disruption of normal sex chromosome disjunction also occurs in females containing the Y ^S · Y ^L 2Rh4 · and a compound X chromosome; the frequency of exceptional progeny was 2.55% versus 0.91% in the controls. Chromosome nondisjunction appears to occur when orientation of the X and Y kinetochores to the same pole is stabilized through tension by the orientation of one or both fourth chromosome kinetochores to the opposite pole. During anaphase, the orientation of the fourth chromosome kinetochore of the Y ^S · Y ^L 2Rh4 · appears to fail and the X and Y ^S · Y ^L 2Rh4 · chromosomes move to the same pole. Y ^S · Y ^L 2Rh4 · chromosome laggards occur with both the Y and fourth chromosome kinetochores amphitelically oriented. This orientation appears to be stable as a result of equal opposing forces toward opposite poles.     

Meiosis in Drosophila males

The conjunctive mechanism of the XY bivalent is believed to differ from that of the autosomal bivalents in the achiasmate Drosophila melanogaster male. It has been proposed that hypothetical cohesive elements, termed collochores, hold the X and Y chromosomes together at or near their nucleolar organizing regions (NORs) and that collochores are not exhibited by autosomal bivalents. In electron micrographs, unique fibrillar material is observed between the X and Y chromosomes at the synaptic site. Recently, the 240 bp nontranscribed spacer associated with rRNA genes at the NOR has been implicated as the essential DNA sequence for XY pairing. To test whether this DNA sequence is always associated with XY pairing and to determine its relationship to the unique fibrillar material, we studied the XY bivalent in Drosophila simulans. The D. simulans Y chromosome has few, if any, rRNA genes, but does have a large block (3,000 kb or 12,500 copies) of the nontranscribed spacer repeat located at the distal end of its long arm. This is in contrast to the D. melanogaster Y, which has the repeat located among rRNA genes on its short arm. Using light and electron microscopy, we show that the X does indeed pair with the distal end of the long arm of the D. simulans Y. However, no fibrillar material is evident in serial thin sections of the D. simulans XY bivalent, suggesting that this material (in D. melanogaster) may be remnants of the NOR rather than a morphological manifestation of the hypothetical collochores. Indeed, in electron micrographs, the synaptic regions of the XY and autosomal bivalents appear similar with no obvious pairing structures, suggesting that the conjunctive mechanism holding homologous chromosomes together is the same for the XY and autosomal bivalents.     

Young patients with colorectal cancer have poor survival in the first twenty months after operation and predictable survival in the medium and long-term: Analysis of survival and prognostic markers

Introduction

Male breast cancer (MBC) is a rare, yet potentially aggressive disease. Although literature regarding female breast cancer (FBC) is extensive, little is known about the etiopathogenesis of male breast cancer. Studies from our laboratory show that MBCs have a distinct immunophenotypic profile, suggesting that the etiopathogenesis of MBC is different from FBCs. The aim of this study was to evaluate and correlate the immunohistochemical expression of cell cycle proteins in male breast carcinoma to significant clinico-biological endpoints.

Methods

75 cases of MBC were identified using the records of the Saskatchewan Cancer Agency over 26 years (1970-1996). Cases were reviewed and analyzed for the immunohistochemical expression of PCNA, Ki67, p27, p16, p57, p21, cyclin-D1 and c-myc and correlated to clinico-biological endpoints of tumor size, node status, stage of the disease, and disease free survival (DFS).

Results

Decreased DFS was observed in the majority of tumors that overexpressed PCNA (98%, p = 0.004). The overexpression of PCNA was inversely correlated to the expression of Ki67 which was predominantly negative (78.3%). Cyclin D1 was overexpressed in 83.7% of cases. Cyclin D1 positive tumors were smaller than 2 cm (55.6%, p = 0.005), had a low incidence of lymph node metastasis (38.2%, p = 0.04) and were associated with increased DFS of >150 months (p = 0.04). Overexpression of c-myc (90%) was linked with a higher incidence of node negativity (58.3%, p = 0.006) and increased DFS (p = 0.04). p27 over expression was associated with decreased lymph node metastasis (p = 0.04). P21 and p57 positive tumors were related to decreased DFS (p = 0.04). Though p16 was overexpressed in 76.6%, this did not reach statistical significance with DFS (p = 0.06) or nodal status (p = 0.07).

Conclusion

Aberrant cell cycle protein expression supports our view that these are important pathways involved in the etiopathogenesis of MBC. Tumors with overexpression of Cyclin D1 and c-myc had better outcomes, in contrast to tumors with overexpression of p21, p57, and PCNA with significantly worse outcomes. P27 appears to be a predictive marker for lymph nodal status. Such observation strongly suggests that dysregulation of cell cycle proteins may play a unique role in the initiation and progression of disease in male breast cancer. Such findings open up new avenues for the treatment of MBC as a suitable candidate for novel CDK-based anticancer therapies in the future.     

Localization of pyruvate:NADP+ oxidoreductase in sporozoites of Cryptosporidium parvum

Cryptosporidium parvum contains a unique fusion protein pyruvate:NADP+ oxidoreductase (CpPNO) that is composed of two distinct, conserved domains, an N-terminal pyruvate:ferredoxin oxidoreductase (PFO) and a C-terminal cytochrome P450 reductase (CPR). Unlike a similar fusion protein that localizes to the mitochondrion of the photosynthetic protist Euglena gracilis, CpPNO lacks an N-terminal mitochondrial targeting sequence. Using two distinct polyclonal antibodies raised against CpPFO and one polyclonal antibody against CpCPR, Western blot analysis has shown that sporozoites of C. parvum express the entire CpPNO fusion protein. Furthermore, confocal immunofluorescence and transmission electron microscopy confirm that CpPNO is localized within the cytosol rather than the relict mitochondrion of C. parvum. The distribution of this protein is not, however, strictly confined to the cytosol. CpPNO also appears to localize posteriorly within the crystalloid body.     

Characterization of Cu,Zn superoxide dismutase (SOD1) gene knock-out mutant of Cryptococcus neoformans var. gattii: role in biology and virulence

The pathogenic yeast Cryptococcus neoformans (Cn) var. gattii causes meningoencephalitis in healthy individuals, unlike the better known Cn varieties grubii and neoformans, which are common in immunocompromised individuals. The virulence determinants and mechanisms of host predilection are poorly defined for var. gattii. The present study focused on the characterization of a Cu,Zn superoxide dismutase (SOD1) gene knock-out mutant constructed by developing a DNA transformation system. The sod1 mutant was highly sensitive to the redox cycling agent menadione, and showed fragmentation of the large vacuole in the cytoplasm, but no other defects were seen in growth, capsule synthesis, mating, sporulation, stationary phase survival or auxotrophies for sulphur-containing amino acids. The sod1 mutant was markedly attenuated in virulence in a mouse model, and it was significantly susceptible to in vitro killing by human neutrophils (PMNs). The deletion of SOD1 also resulted in defects in the expression of a number of virulence factors, i.e. laccase, urease and phospholipase. Complementation of the sod1 mutant with SOD1 resulted in recovery of virulence factor expression and menadione resistance, and in restoration of virulence. Overall, these results suggest that the antioxidant function of Cu,Zn SOD is critical for the pathogenesis of the fungus, but is dispensable in its saprobic life. This report constitutes the first instance in which superoxide dismutase has been directly implicated in the virulence of a fungal pathogen.     

Evaluation of healing following frenectomy

Aberrant frenum attachment would cause plaque accumulation and malalignment of teeth. It can be managed by frenotomy or frenectomy methods, through a conventional surgical technique or laser technique. Therefore, it is of interest to compare frenectomy healing surgical and laser techniques. Data from 51 outpatients and post-operative healing of frenectomy was assessed by Landry’s healing score index using 3 weeks postoperative photographs followed by statistical analysis. Based on the healing score index, the laser technique showed better outcomes than the surgical technique. Moreover, the association between the management of high frenal attachment and the healing score index was found to be statistically significant.