Lactate detection in inducible and orthotopic Her2/neu mammary gland tumours in mouse models.

This study compared the steady state concentration of lactate in an inducible Her2/nue transgenic breast cancer mouse model and in tumours from the same Her2/neu model grown orthotopically. In vivo lactate was detected by MRS using the Hadamard encoded selective multiple quantum coherence pulse sequence (HadSelMQC) recently developed by our laboratory. A lower lactate signal was observed in the inducible tumours compared to orthotopic tumours in vivo, while ex vivo analysis of perchloric acid extracts revealed similar amounts of this metabolite in both models. Histological staining of mammary tumour specimens showed a much higher level of fat tissue in inducible tumours compared to the orthotopic model. Phantom studies with [3-(13) C] lactate indicated that a lipid environment could significantly reduce the T2 of lactate and impede its detection. The transgenic inducible model for breast cancer not only better recapitulated the biological aspects of the human disease but also provided additional characteristics related to in vivo detection of lactate that are not available in orthotopic or xenograft models. This study suggests that the level of lactate measured by the HadSelMQC pulse sequence may be underestimated in human patients in the presence of high lipid levels that are typically encountered in the breast.

Dynamics of long-period gratings tuned with internal fiber electrodes.

The physics of electrically switched long-period grating in a twin-hole fiber with internal electrodes is studied. Dynamic measurements for the two polarizations show how the grating spectra shift in time due to the mechanical stress and heat transfer in the core and the cladding.

Photonic scanning receiver using an electrically tuned fiber Bragg grating.

A 5-cm-long electrically tuned fiber Bragg grating is used to filter a microwave signal on an optical carrier at 1.55 mum. A chirped distributed-feedback structure is employed, with a transmission bandwidth of 54 MHz and relative optical carrier rejection of >30 dB for rf frequencies >2 GHz. The rapid monotonic sweep of the Bragg wavelength is translated into a fast-frequency sweep for rf analysis.

Mutational analysis of CDKN2 (MTS1/p16ink4) in human breast carcinomas.

The CDKN2 gene that encodes the cell cycle regulatory protein cyclin-dependent kinase-4 inhibitor (p16) has recently been mapped to chromosome 9p21. Frequent homozygous deletions of this gene have been documented in cell lines derived from different types of tumors, including breast tumors, suggesting that CDKN2 is a tumor suppressor gene involved in a wide variety of human cancers. To determine the frequency of CDKN2 mutations in breast carcinomas, we screened 37 primary tumors and 5 established breast tumor cell lines by single-strand conformation polymorphism analysis. In addition, Southern blot analysis was performed on a set of five primary breast carcinoma samples and five breast tumor cell lines. Two of the five tumor cell lines revealed a homozygous deletion of the CDKN2 gene, but no mutations were observed in any of the primary breast carcinomas. These results suggest that the mutation of the CDKN2 gene may not be a critical genetic change in the formation of primary breast carcinoma.

MUC1-specific cytotoxic T lymphocytes eradicate tumors when adoptively transferred in vivo.

We have reported previously that MUC1 transgenic mice with spontaneous tumors of the pancreas (designated MET) naturally develop MHC class I-restricted, MUC1-specific CTLs as tumors progress (P. Mukherjee et al., J. Immunol., 165: 3451-3460, 2000). From these MET mice, we have isolated, expanded, and cloned naturally occurring MUC1-specific CTLs in vitro. In this report, we show that the CTL line is predominantly CD8+ T cells and expresses T-cell receptor Vbeta chains 5.1/5.2, 11, 13, and 2 and Valpha chains 2, 8.3, 3.2, and 11.1/11.2. These CTLs recognize several epitopes on the MUC1 tandem repeat with highest affinity to APGSTAPPA. The CTL clone, on the other hand, is 100% CD8+ cells and expresses a single Vbeta chain of 5.1/5.2 and Valpha2. It recognizes only the H-2Db class I-restricted epitope of MUC1, APGSTAPPA. When adoptively transferred, the CTLs were effective in eradicating MUC1-expressing injected tumor cells including mammary gland cells (C57mg) and B16 melanomas. These results suggest that MUC1-specific CTLs are capable of possibly preventing, or at least substantially delaying, MUC1-expressing tumor formation. To our knowledge, this is the first evidence that demonstrates that the naturally occurring MUC1-specific CTLs isolated from one tumor model has antitumor effects on other MUC1-expressing tumors in vivo. Therefore, our data confirm that MUC1 is an important tumor rejection antigen and can serve as a target for immunotherapy.

Pathogenesis of Adrenal Aldosterone-Producing Adenomas Carrying Mutations of the Na(+)/K(+)-ATPase.

Aldosterone-producing adenoma (APA) is a major cause of primary aldosteronism, leading to secondary hypertension. Somatic mutations in the gene for the α1 subunit of the Na(+)/K(+)-ATPase were found in about 6% of APAs. APA-related α1 subunit of the Na(+)/K(+)-ATPase mutations lead to a loss of the pump function of the Na(+)/K(+)-ATPase, which is believed to result in membrane depolarization and Ca(2+)-dependent stimulation of aldosterone synthesis in adrenal cells. In addition, H(+) and Na(+) leak currents via the mutant Na(+)/K(+)-ATPase were suggested to contribute to the phenotype. The aim of this study was to investigate the cellular pathophysiology of adenoma-associated Na(+)/K(+)-ATPase mutants (L104R, V332G, G99R) in adrenocortical NCI-H295R cells. The expression of these Na(+)/K(+)-ATPase mutants depolarized adrenal cells and stimulated aldosterone secretion. However, an increase of basal cytosolic Ca(2+) levels in Na(+)/K(+)-ATPase mutant cells was not detectable, and stimulation with high extracellular K(+) hardly increased Ca(2+) levels in cells expressing L104R and V332G mutant Na(+)/K(+)-ATPase. Cytosolic pH measurements revealed an acidification of L104R and V332G mutant cells, despite an increased activity of the Na(+)/H(+) exchanger. The possible contribution of cellular acidification to the hypersecretion of aldosterone was supported by the observation that aldosterone secretion of normal adrenocortical cells was stimulated by acetate-induced acidification. Taken together, mutations of the Na(+)/K(+)-ATPase depolarize adrenocortical cells, disturb the K(+) sensitivity, and lower intracellular pH but, surprisingly, do not induce an overt increase of intracellular Ca(2+). Probably, the autonomous aldosterone secretion is caused by the concerted action of several pathological signaling pathways and incomplete cellular compensation.

Zebrafish beta tubulin 1 expression is limited to the nervous system throughout development, and in the adult brain is restricted to a subset of proliferative regions.

Tubulin, the building block of microtubules, consists of an alpha and beta subunit, each in itself a family of several highly homologous isotypes. Abundance, tissue specificity, developmental regulation, and possibly function vary between isotypes. Six isotypes of beta tubulin (class I to class VI) have been cloned from several vertebrate species. Class I beta tubulin is believed to be widely expressed, but has not been studied by in situ hybridization in any vertebrate species so far. We have cloned a beta tubulin from zebrafish that appears most similar to other vertebrate class I tubulins and name it zbeta1 tubulin, accordingly. We report a distinct expression pattern of zbeta1 tubulin in the zebrafish embryo in restricted regions of the peripheral and central nervous system that comprise early-differentiating neurons. The expression pattern changes during development and in the adult zebrafish expression mostly is confined to a subset of proliferative zones that include the subependymal zone around the telencephalic ventricle, zones in the preoptic and hypothalamic area and in the olfactory epithelium. Thus, zbeta1 tubulin is expressed with remarkable selectivity during neuronal differentiation and neurogenesis in the embryonic and adult nervous system, respectively.

Neurofibromatosis 2 gene in human colorectal cancer.

Colon cancers commonly have allelic losses of chromosome 22q, which suggests the presence of a tumor suppressor gene on 22q. The candidate tumor suppressor gene on 22q is the neurofibromatosis 2 (NF2) gene. Using single strand conformation polymorphism (SSCP) analysis, we screened 24 pairs of colorectal cancer and adjacent normal mucosa, as well as 10 colon cancer cell lines from non-NF2 patients, for mutations in the coding sequence of the NF2 gene. Two SSCP variants, one in exon 14 and another one in exon 16, were detected in two of the sporadic colorectal cancers, but not in adjacent normal mucosa samples. Sequencing of these variants in one tumor detected an A-to-G transition in bp 1459 of the NF2 cDNA, resulting in the change of Ile to Val at codon 487 of merlin, the NF2 protein product. The other tumor showed a 2-bp (CT) deletion in the intronic sequence of the alternatively spliced exon 16. These results suggest that the NF2 gene is probably involved in some colorectal tumors, but is not the critical chromosome 22q tumor suppressor gene involved in colon tumorigenesis.

Pharmacology and pathophysiology of mutated KCNJ5 found in adrenal aldosterone-producing adenomas.

Somatic mutations of the potassium channel KCNJ5 are found in 40% of aldosterone producing adenomas (APAs). APA-related mutations of KCNJ5 lead to a pathological Na(+) permeability and a rise in cytosolic Ca(2+), the latter presumably by depolarizing the membrane and activating voltage-gated Ca(2+) channels. The aim of this study was to further investigate the effects of mutated KCNJ5 channels on intracellular Na(+) and Ca(2+) homeostasis in human adrenocortical NCI-H295R cells. Expression of mutant KCNJ5 led to a 2-fold increase in intracellular Na(+) and, in parallel, to a substantial rise in intracellular Ca(2+). The increase in Ca(2+) appeared to be caused by activation of voltage-gated Ca(2+) channels and by an impairment of Ca(2+) extrusion by Na(+)/Ca(2+) exchangers. The mutated KCNJ5 exhibited a pharmacological profile that differed from the one of wild-type channels. Mutated KCNJ5 was less Ba(2+) and tertiapin-Q sensitive but was inhibited by blockers of Na(+) and Ca(2+)-transporting proteins, such as verapamil and amiloride. The clinical use of these drugs might influence aldosterone levels in APA patients with KCNJ5 mutations. This might implicate diagnostic testing of APAs and could offer new therapeutic strategies.

Analysis of molecular domains of epitope-tagged merlin isoforms in Cos-7 cells and primary rat Schwann cells.

The Neurofibromatosis 2 gene product, merlin, has striking similarity to ezrin, radixin, and moesin (ERM), members of the protein 4.1 family which have been demonstrated to connect proteins in the plasma membrane to the cytoskeletal components. The recent localization of merlin to the motile regions in cultured cells such as membrane ruffles further supports the notion that merlin represents a new class of tumor suppressors. Here we describe the localization of full-length and truncated polypeptides of merlin expressed as Flag-tagged proteins in transfected cells. Similar to endogenous merlin, the epitope-tagged full-length merlin localizes to the membrane ruffles in transfected Cos-7 cells and rat Schwann cells. In addition, the over-expressed merlin localizes to other actin-rich cortical structures, such as microvilli and filopodia. The amino-terminal half of merlin is seen dispersed throughout the cells and in membrane ruffles. Compared to the amino-terminal half of merlin, its carboxy-terminal half localizes more distinctly to membrane ruffles. The full-length and the carboxy-terminal portion of merlin co-localize with F-actin at the membrane ruffles. However, distinct from the ERM proteins, the carboxy-terminal-truncated merlin and F-actin do not co-localize with each other at the stress fibers. Our results suggest that both the amino- and the carboxy-terminal domains of merlin contribute to its membrane ruffle localization.

Mice with spontaneous pancreatic cancer naturally develop MUC-1-specific CTLs that eradicate tumors when adoptively transferred.

Pancreatic cancer is a highly aggressive, treatment refractory cancer and is the fourth leading cause of death in the United States. In humans, 90% of pancreatic adenocarcinomas overexpress altered forms of a tumor-specific Ag, mucin 1 (MUC1; an epithelial mucin glycoprotein), which is a potential target for immunotherapy. We have established a clinically relevant animal model for pancreatic cancer by developing a double transgenic mouse model (called MET) that expresses human MUC1 as self molecule and develops spontaneous tumors of the pancreas. These mice exhibit acinar cell dysplasia at birth, which progresses to microadenomas and acinar cell carcinomas. The tumors express large amounts of underglycosylated MUC1 similar to humans. Tumor-bearing MET mice develop low affinity MUC1-specific CTLs that have no effect on the spontaneously occurring pancreatic tumors in vivo. However, adoptive transfer of these CTLs was able to completely eradicate MUC1-expressing injectable tumors in MUC1 transgenic mice, and these mice developed long-term immunity. These CTLs were MHC class I restricted and recognized peptide epitopes in the immunodominant tandem repeat region of MUC1. The MET mice appropriately mimic the human condition and are an excellent model with which to elucidate the native immune responses that develop during tumor progression and to develop effective antitumor vaccine strategies.

Alternative splicing of the tuberous sclerosis 2 (TSC2) gene in human and mouse tissues.

The recently isolated gene for tuberous sclerosis 2 (TSC2) encodes a 5.5-kb transcript that is widely expressed. The TSC2 gene product, named tuberin, is a 1784-amino-acid protein that shows a small stretch of homology to the GTPase activating protein rap1GAP. We have detected a novel variant of the TSC2 mRNA lacking 129 nucleotides, predicting an in-frame deletion of 43 amino acids spanning codons 946-988 of tuberin. This 129-bp deletion precisely corresponds to exon 25 of the TSC2 gene suggesting that alternative splicing leads to production of two forms of transcripts designated isoforms 1 and 2. Further molecular analysis revealed a third isoform exhibiting a deletion of 44 amino acids spanning codons 946-989 of tuberin. Amino acid 989 is a Ser residue encoded by the first codon of exon 26. The two isoforms also exist in newborn and adult mouse tissues, reinforcing the potential functional importance of these alternatively spliced products. These alternative isoforms should have implications for efforts aimed at identifying mutations in TSC patients. The distinct polypeptides encoded by the TSC2 gene may have different targets as well as functions involved in the regulation of cell growth.

Evidence for an oscillatory signature in atmospheric neutrino oscillations.

Muon neutrino disappearance probability as a function of neutrino flight length L over neutrino energy E was studied. A dip in the L/E distribution was observed in the data, as predicted from the sinusoidal flavor transition probability of neutrino oscillation. The observed L/E distribution constrained nu(micro)<-->nu(tau) neutrino oscillation parameters; 1.9x10(-3)0.90 at 90% confidence level.

Limits on the neutrino magnetic moment using 1496 days of Super-Kamiokande-I solar neutrino data.

A search for a nonzero neutrino magnetic moment has been conducted using 1496 live days of solar neutrino data from Super-Kamiokande-I. Specifically, we searched for distortions to the energy spectrum of recoil electrons arising from magnetic scattering due to a nonzero neutrino magnetic moment. In the absence of a clear signal, we found micro(nu)