Magnetized boxes for housing polarized spins in homogeneous fields.

We present novel types of permanently magnetized as well as current powered boxes built from soft-ferromagnetic materials. They provide shielded magnetic fields which are homogeneous within a large fraction of the enclosed volume, thus minimizing size, weight, and costs. For the permanently magnetized solutions, homogenization is achieved either by an optimized distribution of the permanent field sources or by jacketing the field with a soft-ferromagnetic cylindrical shell which is magnetized in parallel to the enclosed field. The latter principle may be applied up to fields of about 0.1T. With fields of about 1mT, such boxes are being used for shipping spin-polarized (3)He worldwide for MRI purposes. For current powered boxes, we present concepts and realizations of uniaxial and tri-axial shielded magnetic fields which are homogeneous on the level of 10(-4) within the entire shielded volume. This is achieved by inserting tightly fitting solenoids into a box from soft-magnetic material. The flexible tri-axial solution suits in particular laboratory applications, e.g. for establishing a spin quantization axis.

Expression of pp32 gene family members in breast cancer.

The pp32 gene family consists of at least three closely related members, pp32, pp32r1 and pp32r2. In spite of a high degree of identity at the nucleotide level, pp32 functionally behaves as a tumor suppressor where as pp32r1 and pp32r2 are pro-oncogenic. The purpose of this pilot study was to determine pp32-related expression and whether alternative gene use among the pp32 family members occurred in human breast cancer. As a first step, in situ hybridization with a riboprobe capable of hybridizing with all the three members showed abundant pp32-related mRNA in benign ducts and acini and in infiltrating ductal carcinomas. A total of 100/102 cases were positive. Further, a detailed molecular analysis by RT-PCR, cloning, and sequencing was performed in five frozen infiltrating breast carcinomas and matched benign breast tissues. Oncogenic pp32r1 (5/5) and pp32r2 (3/5) expression was observed in carcinomas where as benign breast tissues expressed pp32. 4/5 carcinomas continued to express pp32 but one was devoid of pp32 expression. These results suggest that alternative expression of pp32 family members may be common in human breast cancer and the analysis of the profile of pp32-related expression might be helpful in understanding the role of these genes in breast cancer pathogenesis.

Rolling circle amplification: a new approach to increase sensitivity for immunohistochemistry and flow cytometry.

Immunohistochemistry is a method that can provide complementary diagnostic and prognostic information to morphological observations and soluble assays. Sensitivity, specificity, or requirements for arduous sample preparation or signal amplification procedures often limit the application of this approach to routine clinical specimens. Rolling circle amplification (RCA) generates a localized signal via an isothermal amplification of an oligonucleotide circle. The application of this approach to immunohistochemistry could extend the utility of these methods to include a more complete set of immunological and molecular probes. RCA-mediated signal amplification was successfully applied to the sensitive and specific detection of a variety of cell surface antigens (CD3, CD20, and epithelial membrane antigen) and intracellular molecules (vimentin and prostate-specific antigen) within a variety of routinely fixed specimens, as well as samples prepared for flow cytometry. RCA technology, which has an intrinsically wide dynamic range, is a robust and simple procedure that can provide a universal platform for the localization of a wide variety of molecules as a function of either antigenicity or nucleic acid sequence. The use of RCA in this way could enhance the use of markers of current interest as well as permit the integration of emerging information from genomics and proteomics into cell- and tissue-based analyses.

Association between extent of axillary lymph node dissection and survival in patients with stage I breast cancer.

BACKGROUND: The role of axillary lymph node dissection for stage I (T1N0) breast cancer remains controversial because patients can receive adjuvant chemotherapy regardless of their nodal status and because its therapeutic benefit is in question. The purpose of this study was to determine whether extent of axillary dissection in patients with T1N0 disease is associated with survival. METHODS: Data from 464 patients with T1N0 breast cancer who underwent axillary dissection from 1973 to 1994 were examined retrospectively. Kaplan-Meier estimates of overall survival, disease-free survival, and recurrence were calculated for patients according to the number of lymph nodes removed (<10 or > or = 10; <15 or > or = 15), and survival curves compared using the Wilcoxon-Gehan statistic. Cox proportional hazards regression modelling was used to adjust for confounding prognostic variables. RESULTS: Median follow-up time was 6.4 years. Patient groups were similar in age, menopausal status, tumor size, hormonal receptor status, type of surgery, and adjuvant therapy. There was a statistically significant improvement in disease-free survival in the > or = 10 versus <10 nodal groups (P <.01). Five-year estimates of survival were 75.7% and 86.2% for <10 nodes and > or = 10 nodes, respectively; 10-year estimates were 66.1% and 74.3%. There also was a notable improvement in the survival comparison of patients with <15 versus > or = 15 nodes (P < or = .05). These findings were confirmed in the multivariate analysis. CONCLUSIONS: These results may reflect a potential for misclassification of tumor stage among patients who had fewer nodes removed. The data, however, suggest that in patients with Stage I breast cancer, improved survival is associated with a more complete axillary lymph node dissection.

Thyroid-specific expression of cholera toxin A1 subunit causes thyroid hyperplasia and hyperthyroidism in transgenic mice.

Thyroid cell growth and function are regulated by hormones and growth factors binding to cell surface receptors that are coupled via G proteins, Gs and Gq, to the adenylyl cyclase and phospholipase C signal transduction systems, respectively. Activating mutations of the TSH receptor and G alpha s have been documented in subsets of thyroid neoplasms. To test the oncogenic potential of activated G alpha s in transgenic mice, we used the cholera toxin A1 subunit that constitutively activates G alpha s and used the rat thyroglobulin gene promoter for targeting this transgene (TGCT) to thyroid follicular cells. Three (M1392, F1358, and F1286) of six founders identified were able to transmit the transgene to their offspring and thyroid glands from these mice contained elevated levels of cAMP. Concentrations of serum thyroxine were elevated as early as 2 months of age (M 1392 and F 1286). F1358 mice were euthyroid until 8 months of age, at which time they developed hyperthyroidism. All three TGCT lines developed thyroid hyperplasia independent of their thyroxine levels. DNA image analysis of thyroid follicular cells from both the hyper and euthyroid mice showed that DNA index and "S+G2/M" phase were increased compared with normal, changes similar to that seen in poor prognosis human carcinomas. These data suggest that the G alpha s-adenylyl cyclase-cAMP pathway has an important role in thyroid hyperplasia and the transgenic mouse models reported herein will allow further examination of the role of this pathway in thyroid oncogenesis.

Ras oncogene-transformed and nontransformed cell populations are each heterogeneous but respond differently to the chemotherapeutic drug cytosine arabinoside (Ara-C).

In order to determine whether the growth of ras oncogene-transformed cells and nontransformed cells was inhibited differently by the chemotherapuetic drug cytosine arabinoside (Ara-C) their growth was analyzed by a novel colony-based assay that is sensitive and appropriate for heterogeneous cell populations. Colonies of nontransformed NIH3T3 cells, or ras oncogene-transformed NIH(ras) cells, were grown in the absence of drug and then divided into subclones. Subclones were allowed to continue to grow in the absence or presence of drug. Growth inhibition was determined by comparing the growth of drug-treated subclones. Colonies of nontransformed cells grown in the absence of the drug displayed a large variation in growth, and when grown in the presence of the drug displayed a large variation in growth inhibition. Colonies of transformed cells also displayed a large variation in the absence and presence of the drug. For each cell line, related subclones were more similar to each other than to unrelated subclones, implying inheritance of growth rates and drug response. For NIH3T3 cells, the growth of subclones in the presence of drug was highly correlated with the growth of related subclones in the absence of drug. However, for NIH3T3(ras) cells the growth of subclones in the presence of drug was not correlated with the growth of related subclones in the absence of drug. Therefore, ras oncogene-transformed and nontransformed cell populations differ in their response to Ara-C.

pp32 overexpression induces nuclear pleomorphism in rat prostatic carcinoma cells.

Nuclear pleomorphism is an important diagnostic factor in tumour pathology. Traditionally, nuclear pleomorphism is evaluated qualitatively or semiquantitatively, often as a component of tumour grade; the molecular basis of nuclear pleomorphism, however, remains unclear. In this study, we investigated the quantitative effects on nuclear morphology of overexpressing pp32, a recently described nuclear phosphoprotein highly expressed in self-renewing and neoplastic cell populations. Assessment of Feulgen-stained transfected and control lines of AT3.1, a rat prostatic carcinoma cell line, using a computerized Cellular Image Analysis System (BD CAS-200) showed that stable overexpression of human pp32 in AT3.1 cells is accompanied by marked increases in the coefficient of variation of nuclear shape, nuclear size and chromatin textures but not in DNA content. In contrast, stable transfection with control vector, with ras, or with bcl-2 failed to affect nuclear morphology. Cell cycle analysis further showed that pp32-related increases in variation of nuclear structure manifested principally in G1. These studies suggest that pp32 plays a role either directly or indirectly in the control of nuclear shape of G1 cells.

Persistence of cell cycle times over many generations as determined by heritability of colony sizes of ras oncogene-transformed and non-transformed cells.

The persistence of cell lifetimes during about 10 successive cell generations was investigated by comparing the number of cells in primary colonies and in secondary colonies derived from primary colonies. Primary colonies were grown from single cells for 3 or 4 days (a time equivalent to an average of five cell generations) and the number of cells in each primary colony determined. Cells in each primary colony were dispersed to initiate secondary colonies, grown for the same time, and the number of cells in secondary colonies determined. Several criteria were used to compare primary and related secondary colonies, the most informative was found to be regression and correlation coefficients between number of cells in primary colonies and mean numbers of cells in related secondary colonies. For two non-transformed mouse fibroblast cell lines, NIH 3T3 and BALB 3T3, the regression and correlation coefficients of cell number in primary and secondary colonies were positive. This suggests inheritance of cell lifetimes over many cell generations. After the addition of an activated ras oncogene (human cellular Harvey ras, or viral Kirsten ras) some regression and correlation coefficients changed in magnitude but all remained positive. Comparison of experimental data and the results of computer simulations suggest that several models of inheritance of cell lifetimes are not adequate to explain the results, including a model of independence between lifetimes of mother and daughter cells and the common model that describes daughter cells as inheriting the lifetime of their mother with deviation. Simulations do suggest that cell lifetimes are inherited within clones as deviation from the lifetime of the initial cell, and that the ras oncogene does not destroy persistence within clones but does increase heterogeneity of cell lifetimes.