N-cadherin-mediated cell adhesion restricts cell proliferation in the dorsal neural tube

Neural progenitors are organized as a pseudostratified epithelium held together by adherens junctions (AJs), multiprotein complexes composed of cadherins and α- and β-catenin. Catenins are known to control neural progenitor division; however, it is not known whether they function in this capacity as cadherin binding partners, as there is little evidence that cadherins themselves regulate neural proliferation. We show here that zebrafish N-cadherin (N-cad) restricts cell proliferation in the dorsal region of the neural tube by regulating cell-cycle length. We further reveal that N-cad couples cell-cycle exit and differentiation, as a fraction of neurons are mitotic in N-cad mutants. Enhanced proliferation in N-cad mutants is mediated by ligand-independent activation of Hedgehog (Hh) signaling, possibly caused by defective ciliogenesis. Furthermore, depletion of Hh signaling results in the loss of junctional markers. We therefore propose that N-cad restricts the response of dorsal neural progenitors to Hh and that Hh signaling limits the range of its own activity by promoting AJ assembly. Taken together, these observations emphasize a key role for N-cad-mediated adhesion in controlling neural progenitor proliferation. In addition, these findings are the first to demonstrate a requirement for cadherins in synchronizing cell-cycle exit and differentiation and a reciprocal interaction between AJs and Hh signaling.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding

Background

The mini-chromosome maintenance protein (MCM) complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7), the archaeon Sulfolobus solfataricus has only one MCM protein (ssoMCM), six subunits of which form a homohexamer. We have recently reported a 4.35Å crystal structure of the near full-length ssoMCM. The structure reveals a total of four β-hairpins per subunit, three of which are located within the main channel or side channels of the ssoMCM hexamer model generated based on the symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM) structure. The fourth β-hairpin, however, is located on the exterior of the hexamer, near the exit of the putative side channels and next to the ATP binding pocket.

Results

In order to better understand this hairpin's role in DNA binding and helicase activity, we performed a detailed mutational and biochemical analysis of nine residues on this exterior β-hairpin (EXT-hp). We examined the activities of the mutants related to their helicase function, including hexamerization, ATPase, DNA binding and helicase activities. The assays showed that some of the residues on this EXT-hp play a role for DNA binding as well as for helicase activity.

Conclusions

These results implicate several current theories regarding helicase activity by this critical hexameric enzyme. As the data suggest that EXT-hp is involved in DNA binding, the results reported here imply that the EXT-hp located near the exterior exit of the side channels may play a role in contacting DNA substrate in a manner that affects DNA unwinding.

     

Assessment of the potential for hybridisation between Laricobius nigrinus (Coleoptera: Derodontidae) and Laricobius osakensis,predators of the hemlock woolly adelgid (Hemiptera: Adelgidae)

In 2003, Laricobius nigrinus Fender was introduced into the eastern United States as a biological control agent of the hemlock woolly adelgid (Adelges tsugae Annand). Following its release, it was discovered that L. nigrinus was hybridising and producing viable progeny with Laricobius rubidus LeConte, a species native to eastern North America. Recently, Laricobius osakensis Montgomery and Shiyake was imported from Japan into the USA as a potential biological control agent of hemlock woolly adelgid. Hybridisation between L. nigrinus and L. rubidus led to interest in the outcome of interactions between L. osakensis and the other two Laricobius spp. The purpose of this study was to determine if L. osakensis could mate with L. nigrinus, if they could produce hybrid progeny, and whether mating interferes with reproductive output. Laricobius spp. were observed mating directly following emergence and found to be capable of producing sterile eggs in the absence of a mating event. Laboratory and confined field studies found no evidence that L. osakensis and L. nigrinus could produce hybrid progeny and the interaction between the two species did not result in a lower reproduction associated with interspecific mating attempts. Interbreeding should therefore not have an impact on biological control using these species. Fecundity experiments showed that L. osakensis produced eggs earlier in the season and at a higher rate than L. nigrinus, suggesting that L. osakensis may have the potential to be an even more successful biological control agent than L. nigrinus.     

Host effects of glandular-haired alfalfa on alfalfa weevil (Coleoptera: Curculionidae) and potato leafhopper (Homoptera: Cicadellidae) populations in Virginia

Cultivars of glandular-haired alfalfa, Medicago sativa L., such as '54H69', are currently available and marketed as being resistant to potato leafhopper, Empoasca fabae (Harris). Between 2000 and 2002, studies were conducted to compare the effects of '54H69' and a standard, nonglandular-haired alfalfa cultivar, 'Choice', on alfalfa weevil, Hypera postica (Gyllenhal), and potato leafhopper populations at Campbell and Montgomery counties, Virginia. '54H69' had no effect on alfalfa weevil populations. At each location, densities of alfalfa weevil in '54H69' and 'Choice' were similar, but pest pressure was higher at Campbell Co. than at Montgomery Co. and always exceeded the economic threshold before insecticide was applied. Densities of potato leafhopper also did not differ between '54H69' and 'Choice' in any year at the two locations. Insecticide treatment effectively reduced potato leafhopper densities in the two cultivars, although populations were below the economic threshold at both locations when the insecticides were applied. Overall, postinsecticide treatment comparisons showed that the densities of alfalfa weevil and potato leafhoppers were similar or higher in untreated '54H69' compared with insecticide-treated 'Choice'. In addition, there were no differences in seasonal dry yields between '54H69' and 'Choice' in any year at either location. Our results indicate that the glandular-haired alfalfa '54H69' does not provide acceptable resistance to potato leafhopper and also does not offer a yield advantage to growers in Virginia.     

Yield and forage quality of glandular-haired alfalfa under alfalfa weevil (Coleoptera: Curculionidae) and potato leafhopper (Hemiptera: Cicadellidae) pest pressure in Virginia

Cultivars of glandular-haired alfalfa, Medicago sativa L., such as '54H69', are currently available and marketed as being resistant to potato leafhopper, Empoasca fabae (Harris) (Hemiptera: Cicadellidae). 54H69 and a standard, nonglandular-haired alfalfa 'Choice' were evaluated at two locations in Virginia over a 3-yr period. Dry matter yields and concentrations of crude protein and acid detergent fiber were compared at the first, second, and third harvests. Overall, the two cultivars produced similar dry matter yields of comparable forage quality in the absence of insecticides at both locations in each year. Untreated 54H69 did not produce greater dry matter yields than untreated Choice under either light or heavier potato leafhopper pest pressure. Concentrations of crude protein did not vary between the two cultivars at any harvest. Some differences in concentrations of acid detergent fiber were detected between cultivars, but these differences were not consistent among years, harvests, or between locations. Further comparisons between untreated 54H69 and treated Choice were made, but few significant differences were detected in dry matter yields or forage quality. An economic analysis for the study indicated that a grower planting 54H69 would realize less net revenue than a grower planting Choice, largely because of the seed premium for the glandular-haired cultivar and the evident need to treat 54H69 with insecticide for control of alfalfa weevil, Hypera postica (Gyllenhal) (Coleoptera: Curculionidae), and potato leafhopper.