Effects of inspiratory muscle training on exercise responses in Paralympic athletes with cervical spinal cord injury.

We asked whether specific inspiratory muscle training (IMT) improves respiratory structure and function and peak exercise responses in highly trained athletes with cervical spinal cord injury (SCI). Ten Paralympic wheelchair rugby players with motor-complete SCI (C5-C7) were paired by functional classification then randomly assigned to an IMT or placebo group. Diaphragm thickness (B-mode ultrasonography), respiratory function [spirometry and maximum static inspiratory (PI ,max ) and expiratory (PE ,max ) pressures], chronic activity-related dyspnea (Baseline and Transition Dyspnea Indices), and physiological responses to incremental arm-crank exercise were assessed before and after 6 weeks of pressure threshold IMT or sham bronchodilator treatment. Compared to placebo, the IMT group showed significant increases in diaphragm thickness (P = 0.001) and PI ,max (P = 0.016). There was a significant increase in tidal volume at peak exercise in IMT vs placebo (P = 0.048) and a strong trend toward an increase in peak work rate (P = 0.081, partial eta-squared = 0.33) and peak oxygen uptake (P = 0.077, partial eta-squared = 0.34). No other indices changed post-intervention. In conclusion, IMT resulted in significant diaphragmatic hypertrophy and increased inspiratory muscle strength in highly trained athletes with cervical SCI. The strong trend, with large observed effect, toward an increase in peak aerobic performance suggests IMT may provide a useful adjunct to training in this population.

Mutation of the ST7 tumor suppressor gene on 7q31.1 is rare in breast, ovarian and colorectal cancers.

The gene ST7 has recently been implicated as the broad-range tumor suppressor on human chromosome 7q31.1. We did not detect somatic mutations in ST7 in any of 149 primary ovarian, breast or colon carcinomas. These data suggest that epigenetic downregulation or haploinsufficiency, rather than somatic genetic alterations, may be the primary mechanism of abrogation of ST7 function in these tumor types.

High and low mammographic density human breast tissues maintain histological differential in murine tissue engineering chambers.

Mammographic density (MD) is the area of breast tissue that appears radiologically white on mammography. Although high MD is a strong risk factor for breast cancer, independent of BRCA1/2 mutation status, the molecular basis of high MD and its associated breast cancer risk is poorly understood. MD studies will benefit from an animal model, where hormonal, gene and drug perturbations on MD can be measured in a preclinical context. High and low MD tissues were selectively sampled by stereotactic biopsy from operative specimens of high-risk women undergoing prophylactic mastectomy. The high and low MD tissues were transferred into separate vascularised biochambers in the groins of SCID mice. Chamber material was harvested after 6 weeks for histological analyses and immunohistochemistry for cytokeratins, vimentin and a human-specific mitochondrial antigen. Within-individual analysis was performed in replicate mice, eliminating confounding by age, body mass index and process-related factors, and comparisons were made to the parental human tissue. Maintenance of differential MD post-propagation was assessed radiographically. Immunohistochemical staining confirmed the preservation of human glandular and stromal components in the murine biochambers, with maintenance of radiographic MD differential. Propagated high MD regions had higher stromal (p = 0.0002) and lower adipose (p = 0.0006) composition, reflecting the findings in the original human breast tissue, although glands appeared small and non-complex in both high and low MD groups. No significant differences were observed in glandular area (p = 0.4) or count (p = 0.4) between high and low MD biochamber tissues. Human mammary glandular and stromal tissues were viably maintained in murine biochambers, with preservation of differential radiographic density and histological features. Our study provides a murine model for future studies into the biomolecular basis of MD as a risk factor for breast cancer.

ST7-mediated suppression of tumorigenicity of prostate cancer cells is characterized by remodeling of the extracellular matrix.

Multiple lines of evidence have provided compelling evidence for the existence of a tumor suppressor gene (TSG) on chromosome 7q31.1. ST7 may be the target of this genetic instability but its designation as a TSG is controversial. In this study, we show that, functionally, ST7 behaves as a tumor suppressor in human cancer. ST7 suppressed growth of PC-3 prostate cancer cells inoculated subcutaneously into severe combined immunodeficient mice, and increased the latency of tumor detection from 13 days in control tumors to 23 days. Re-expression of ST7 was also associated with suppression of colony formation under anchorage-independent conditions in MDA-MB-231 breast cancer cells and ST7 mRNA expression was downregulated in 44% of primary breast cancers. Expression profiling of PC-3 cells revealed that ST7 predominantly induces changes in genes involved in re-modeling the extracellular matrix such as SPARC, IGFBP5 and several matrix metalloproteinases. These data indicate that ST7 may mediate tumor suppression through modification of the tumor microenvironment.

Homeostatic behavior of fast Fourier transform power in very low frequency non-rapid eye movement human electroencephalogram.

Basic research shows that the physiological and molecular mechanisms of very low frequency (<1 Hz) electroencephalogram (EEG) waves of non-rapid eye movement (NREM) sleep differ from those of the higher (1-4 Hz) delta frequencies. Human studies show that the across-NREM period dynamics of very low frequency and 1-4 Hz EEG also differ. These differences and the reported failure of very low frequency EEG power to increase after a night of total sleep deprivation raise the question of whether very low frequency EEG shows the other homeostatic properties established for higher delta frequencies. Here we tested the relation of very low frequency EEG power density to prior waking duration across a normal day and whether these low frequencies meet another criterion for homeostatic sleep EEG: conservation of power across a late nap and post-nap sleep. Data from 19 young adults recorded in four separate sessions of baseline, daytime nap and post-nap sleep were analyzed. Power density in very low frequency NREM EEG increased linearly when naps were taken later in the day (i.e. were preceded by longer waking durations). In the night following an 18:00 h nap, very low frequency power was reduced by roughly the amount of power in the nap. Thus, very low frequency EEG meets two major homeostatic criteria. We hypothesize that these low frequencies reflect the executive rather than the functional processes by which NREM sleep reverses the effects of waking brain activity.

Folate-binding protein is a marker for ovarian cancer.

We describe the isolation of a complementary DNA (cDNA) sequence encoding the ovarian cancer-associated antigen recognized by monoclonal antibody MOv18 and its identification as a high-affinity folate-binding protein (FBP). Functional cDNA clones were isolated using mRNA from the ovarian carcinoma cell line SKOV3 and colon carcinoma cell line HT29, by transient expression in WOP cells and selection of expressing cells by adhesion to antibody-coated magnetic beads. The cDNAs differed in the lengths of 5'- and 3'-noncoding regions, but they encoded identical peptides. A database search clearly showed them to be adult high-affinity FBPs with amino acid sequences identical with those isolated from normal placenta and several carcinoma cell lines. Reactivity of cell lines with MOv18 was quantitatively consistent with the expression of FBP mRNA. Southern hybridizations show evidence of a family of related genes and/or pseudogenes and were mapped to chromosome 11q13.3-14.1 by fluorescent in situ hybridization using cosmid clones containing part of this region. Also identified were two PstI polymorphisms of four and three alleles, respectively, and a two-allele MspI polymorphism. The folate-binding protein locus was not amplified in any of the 16 carcinoma cell lines tested and in only 1 of 10 serous adenocarcinomas, indicating that overexpression of FBP in ovarian cancer cannot, in general, be due to gene amplification.

An ovarian tumor marker with homology to vaccinia virus contains an IgV-like region and multiple transmembrane domains.

The monoclonal antibody OVTL3 has a highly restricted reactivity with ovarian carcinomas and defines a surface glycoprotein, OA3, which has been used for immunotargeting. To understand why OA3 is found on ovarian tumors we isolated a complementary DNA by expression cloning. The clone encodes a 323-amino acid protein with 5 putative membrane spanning domains, reminiscent of a membrane receptor or channel, but of a new type with little or no sequence similarity with these families of proteins. Interestingly, the OA3 sequence is highly related to a vaccinia virus encoded protein (VA38) and its extracellular domain is a member of the immunoglobulin V region superfamily.

Refinement of two chromosome 11q regions of loss of heterozygosity in ovarian cancer.

Loss of heterozygosity on chromosome 11q23.3-qter is a frequent event in ovarian carcinoma, implying the existence of an important ovarian tumor suppressor gene(s) within the region. To refine a minimum region(s) of loss, 67 ovarian tumors were analyzed for loss of heterozygosity with eight microsatellite markers spanning 11q23.3-qter. Forty tumors (61%) demonstrated allelic losses. Twenty-seven of these had allelic losses on only part of 11q23.3, which enabled the identification of two distinct regions likely to harbor ovarian tumor suppressor genes. The proximal region, flanked by markers D11S925 and D11S1336, is less than two megabases while the second more distal region, flanked by markers D11S912 and D11S439, is approximately eight megabases. The refinement of these candidate tumor suppressor gene loci will facilitate future loss of heterozygosity studies and enable the isolation of candidate genes from these regions.

Molecular cloning of the B-CAM cell surface glycoprotein of epithelial cancers: a novel member of the immunoglobulin superfamily.

The human F8/G253 antigen, B-CAM, is a cell surface glycoprotein that is expressed with restricted distribution pattern in normal fetal and adult tissues, and is up-regulated following malignant transformation in some cell types. We have isolated a complementary DNA for B-CAM using an expression cloning technique. The complementary DNA (EMBL accession number X80026) encodes a 588-amino acid protein which is a novel member of the immunoglobulin superfamily with a characteristic V-V-C2-C2-C2 immunoglobulin domain structure. This structure has been described previously for the human MUC18 melanoma antigen (31% amino acid identity) and chicken and rat versions of a neural adhesion molecule referred to as SC1/DM-GRASP/BEN or KG-CAM, respectively (26% amino acid identity). This homology is suggestive of a role for B-CAM in cell-cell or cell-matrix adhesion. The gene for B-CAM has been mapped by fluorescence in situ hybridization to chromosome 19q13.2-13.3.

Localization of an ovarian cancer tumor suppressor gene to a 0.5-cM region between D22S284 and CYP2D, on chromosome 22q.

The detection of loss of heterozygosity, indicative of the presence of a tumor suppressor gene, has been reported to occur frequently on chromosome 22q in human ovarian cancer. In this study, 110 sporadic ovarian tumors were analyzed using 8 polymorphic loci to define a minimum region of loss. Fifty-eight (53%) tumors showed loss of heterozygosity, and of these 6 exhibited partial loss, enabling the identification of two candidate tumor suppressor gene loci. One region, of less than 0.5 cM, is flanked by D22S284 and CYP2D, and a second region lies distal to D22S276. Analysis of loss of heterozygosity with respect to grade and stage suggests that chromosome 22q loss of heterozygosity is of more relevance in tumor progression rather than initiation.

Allelotyping of endometriosis with adjacent ovarian carcinoma reveals evidence of a common lineage.

Endometriosis is a common gynecological disease in which tissue similar to the endometrium proliferates at sites outside the uterine cavity. Malignant transformation of endometriosis to endometrioid and clear cell ovarian carcinomas has been documented in histological studies, but no molecular genetic evidence exists to support that endometriosis is the clonal precursor of such malignancies. We examined 14 cases of endometriosis synchronous with ovarian cancer for loss of heterozygosity on 12 chromosome arms, X chromosome inactivation, and TP53 mutation to determine whether they shared genetic alterations. In all four of the cases where the carcinoma had arisen within endometriosis and in five of the seven cases where the carcinoma was adjacent to the endometriosis, common genetic lesions were detected, consistent with a common lineage. A TP53 mutation was also detected in one case of endometriosis adjacent to carcinoma. These findings support the numerous histological observations that endometrioid and clear cell ovarian carcinomas may arise through malignant transformation of endometriotic lesions.

The phosphatidylinositol 3'-kinase p85alpha gene is an oncogene in human ovarian and colon tumors.

Phosphatidylinositol 3'-kinases (PI3ks) are a family of lipid kinases that play a crucial role in a wide range of important cellular processes associated with malignant behavior including cell growth, migration, and survival. We have used single-strand conformational polymorphism/heteroduplex analysis to demonstrate the presence of somatic mutations in the gene for the p85alpha regulatory subunit of PI3k (PIK3R1) in primary human colon and ovarian tumors and cancer cell lines. All of the mutations lead to deletions in the inter-SH2 region of the molecule proximal to the serine608 autoregulatory site. Expression of a mutant protein with a 23 amino acid deletion leads to constitutive activation of PI3k providing the first direct evidence that p85alpha is a new oncogene involved in human tumorigenesis.

Microsatellite analysis of endometriosis reveals loss of heterozygosity at candidate ovarian tumor suppressor gene loci.

Endometriosis is a very common gynecological condition in which tissue similar to endometrium proliferates at sites outside the uterine cavity, most commonly the ovary. Although it generally remains a benign condition, malignant transformation has been documented. and it is commonly found in association with endometrioid subtype ovarian cancer. Tumor suppressor genes are commonly altered in ovarian cancers, and the development of endometriosis may involve mutations in the same class of genes. We have investigated this possibility by examining DNA from 40 cases of endometriosis for clonal status, alterations in TP53 and RASK, and allelic losses at candidate ovarian tumor suppressor loci on chromosome arms 6q, 9p, l1q, 17p, l7q, and 22q. The majority of endometriotic cysts were monoclonal, but interestingly, 8 of 10 normal endometrial glands were also monoclonal, demonstrating that both are able to develop from a single progenitor cell. No mutations were detected in TP53 or RASK. Loss of heterozygosity (LOH) was detected on chromosomes 9p (18%), 1lq (18%), and 22q (15%). In total, 11 of 40 (28%) cases demonstrated LOH at one or more of these loci. This study, which is the first to report LOH in endometriosis, supports the notion that tumor suppressor gene inactivation may play a role in the development of at least a subset of cases.

Frequent PTEN/MMAC mutations in endometrioid but not serous or mucinous epithelial ovarian tumors.

Epithelial ovarian cancer comprises three major histological subtypes (serous, mucinous, and endometrioid), and it is becoming clear that the developmental pathways for these subtypes are fundamentally different. In particular, endometrioid ovarian cancers probably arise by the malignant transformation of ectopic endometrial implants called endometriosis and not the ovarian surface epithelium. The PTEN/MMAC gene on chromosome 10q23 is a tumor suppressor implicated in the pathogenesis of a wide variety of malignancies, but to date, somatic mutations in PTEN have not been identified in studies of predominantly serous ovarian cancers. In endometrial cancers, PTEN mutations are very common in tumors of the endometrioid type but have rarely been found in serous types, and we hypothesized that a similar histological subtype bias might be occurring in ovarian cancer. We have analyzed 81 ovarian tumors, including 34 endometrioid, 29 serous, 10 mucinous, and 8 clear cell tumors, for loss of heterozygosity (LOH) on 10q23 and for mutations in all 9 coding exons of PTEN. LOH was common among the endometrioid (43%) and serous (28%) tumors but was infrequent among the other histological subtypes. Somatic PTEN mutations were detected in seven (21%) of the endometrioid tumors, and in all informative cases, the mutation was accompanied by loss of the wild-type allele. One mucinous tumor without 10q23 LOH was shown to harbor two somatic PTEN mutations. In this tumor, the histological appearance of the mucinous areas was atypical, and the mucinous areas contained foci of endometrioid differentiation. The majority of tumors with PTEN mutations were grade 1 and/or stage 1, suggesting that inactivation of PTEN is an early event in ovarian tumorigenesis. No PTEN mutations were found among the serous or clear cell tumors. The identification of frequent somatic PTEN mutations in endometrioid ovarian tumors indicates that it plays a significant role in the etiology of this subtype. The absence of mutations in other histological subtypes is consistent with the hypothesis that epithelial ovarian cancers arise through distinct developmental pathways.

Absence of PPP2R1B gene alterations in primary ovarian cancers.

The PPP2R1B gene has recently been implicated as a tumor suppressor based on the finding of somatic alterations in lung and colon cancers. PPP2R1B is located on chromosome 11q22-24 which coincides with the site of frequent loss of heterozygosity (LOH) in ovarian cancer. We investigated if the PPP2R1B gene was inactivated in ovarian cancer by single strand conformational polymorphism (SSCP) and heteroduplex (HD) analysis of 99% of the coding region. LOH at the PPP2R1B locus was detected in 32% of the malignant tumors but no somatic alterations were detected in any of 65 malignant, five borderline or six benign tumors. A germline G > A transition (GGC > GAC) in codon 90 was detected in 4/76 tumors. This alteration has previously been described as a mutation but on further investigation we found that the frequency of this variant among 167 ovarian cancers (4.2%) was not statistically significantly different from that observed in 247 non-cancer random controls (2.4%). We conclude that the PPP2R1B gene is not involved in the pathogenesis of ovarian cancer. The codon 90 Gly > Asp alteration may represent a non-pathological polymorphism and consequently the mutation frequency reported in lung cancers may have been overstated and the designation of PPP2R1B as a tumor suppressor gene should be regarded with caution.

Frequent loss of heterozygosity on chromosomes 7 and 9 in benign epithelial ovarian tumours.

It is presently unclear if ovarian cancers arise through malignant transformation of pre-existing benign tumours. The apparent rarity of loss of heterozygosity (LOH) reported for benign tumours has led to speculation that they lack malignant potential and represent a biological entity distinct from ovarian carcinoma. We reasoned that the absence of detectable LOH may be due to the masking of such losses by contamination with normal tissue present in excess in the majority of benign tumour biopsies. Therefore we utilized a microdissection technique to examine for LOH using 14 microsatellite markers on chromosome arms 6q, 7p, 7q, 9p, 11q and 17p in 31 solitary benign epithelial ovarian tumours. LOH was detected on all chromosome arms with the most frequent LOH occurring on 7p (27%) and 9p (26%). In addition, a point mutation in codon 157 of TP53 was detected in one tumour which is the first report of a TP53 mutation in a solitary benign ovarian tumour. In total 48% of tumours harboured genetic alterations which supports the idea that all benign ovarian tumours may carry a genetic predisposition to malignancy and are therefore not inherently different from their malignant counterparts.

Loss of heterozygosity on chromosomes 7p, 7q, 9p and 11q is an early event in ovarian tumorigenesis.

Forty early stage malignant and seven borderline ovarian tumours were analysed for loss of heterozygosity (LOH) on chromosomes 6, 7, 9, 11 and 17. LOH involving at least one locus was observed in 32 (80%) early stage and six (86%) borderline tumours. Frequent LOH in the early stage tumours was detected on chromosome arms 7p (31%), 7q (50%), 9p (42%) and 11q (34%) suggesting that these chromosomes harbour tumour suppressor genes which are inactivated early in tumorigenesis. Borderline tumours exhibited a similar pattern of LOH to that observed in the early stage malignant tumours, indicating that the development of both malignant and borderline forms may involve inactivation of the same set of tumour suppressor genes. Together with our previous investigation of benign ovarian tumours this data supports the theory that malignant ovarian tumours may arise from benign and borderline precursors.

High internight reliability of computer-measured NREM delta, sigma, and beta: biological implications.

BACKGROUND: Computer analysis of the sleep electroencephalogram (EEG) waveforms is widely employed, but there have been no systematic studies of its reliability. METHODS: The most commonly used computer methods are power spectral analysis with the fast-Fourier transform (FFT) and period amplitude analysis (PAA) with zero cross or zero first derivative half-wave measurement. We applied all three computer methods to the digitized EEG of 16 normal subjects who underwent 5 consecutive nights of baseline (placebo) recording. We evaluated the internight reliability of three non-rapid eye movement (NREM) frequency bands of special importance to sleep research: delta (0.3-3 Hz), sigma (12-15 Hz), and beta (15-23 Hz). RESULTS: Both FFT and the two methods of PAA gave excellent internight reliability for delta and sigma. Even a single night of recording correlated highly (r >.9) with the 5-night mean. Beta reliability was lower but still highly significant for both the PAA and the FFT measures. CONCLUSIONS: Computer-analyzed sleep EEG data are highly reliable. Period amplitude methods demonstrate that wave incidence and period as well as amplitude are reliable, indicating that the reliability of composite measures (FFT power, PAA integrated amplitude) is not solely based on individual differences in EEG amplitude. The high internight stability of NREM delta indicates that it possesses traitlike characteristics and is relatively independent of day-to-day variations in state.

Mutation analysis of RASK and the 'FLR exon' of NF1 in sporadic ovarian carcinoma.

Frequent loss of heterozygosity has been described on several chromosomes in ovarian carcinoma (OC), but few tumour suppressor genes (TSGs) have been analysed. Mutations in the GTPase-related domain (GRD) of the TSG NF1 have been described in tumours not usually associated with neurofibromatosis type 1 (NF1). We analysed 36 OCs for mutations in this domain using single-strand conformation polymorphism. The NF1-GRD can downregulate the active form of p21RAS and, therefore, we analysed the same tumours for mutations in RASK. No cases of mutations in NF1-GRD were seen, and only two cases of RASK mutations were found. Thus, activation of the RAS signalling pathway by RASK or NF1 mutations does not appear to be common in OC.

Parametric and pharmacological studies of midbrain suppression of the hind limb flexion withdrawal reflex in the rat.

These experiments quantitatively analyzed effects of electrical midbrain stimulation on a nociceptive hind limb flexion reflex in rats anesthetized with sodium pentobarbital. We recorded the force of isometric hind limb flexion withdrawal, and related flexor electromyographic (EMG) activity, elicited by noxious heat (42-54 degrees C, 10 sec) applied to the ventral hind paw. Several hind limb flexors including biceps femoris were active during the reflex. Quantified reflex responses to identical noxious heat stimuli delivered every 2 min were constant in magnitude and were reduced or abolished during stimulation (100 msec trains at 100 Hz, 3/sec, 15-325 microA) in the midbrain periaqueductal gray (PAG) or lateral reticular formation (LRF). LRF was significantly more effective than PAG stimulation in suppressing reflex responses. The magnitude of the reflex responses increased with graded increases in the temperature of the noxious heat stimulus. The slope of the temperature-response relationship was significantly reduced during PAG stimulation, whereas it was shifted toward higher temperatures with significantly increased threshold during LRF stimulation. To investigate possible transmitters involved, we tested if PAG- or LRF-evoked reflex suppression was affected following systemic administration of the opiate antagonist naloxone, the serotonin antagonist methysergide, the noradrenergic antagonist phentolamine, or the cholinergic antagonist scopolamine. Naloxone had little effect, while methysergide and phentolamine reduced PAG- and LRF-evoked reflex suppression in about one-half of the cases. Scopolamine largely reduced PAG- and LRF-evoked reflex suppression (in 8/9 and 4/6 rats, respectively). These results indicate that the flexion reflex is under parametrically but not pharmacologically distinct inhibitory midbrain controls.