Frequent epigenetic inactivation of Wnt antagonist genes in breast cancer.

Although mutation of APC or CTNNB1 (beta-catenin) is rare in breast cancer, activation of Wnt signalling is nonetheless thought to play an important role in breast tumorigenesis, and epigenetic silencing of Wnt antagonist genes, including the secreted frizzled-related protein (SFRP) and Dickkopf (DKK) families, has been observed in various tumours. In breast cancer, frequent methylation and silencing of SFRP1 was recently documented; however, altered expression of other Wnt antagonist genes is largely unknown. In the present study, we found frequent methylation of SFRP family genes in breast cancer cell lines (SFRP1, 7 out of 11, 64%; SFRP2, 11 out of 11, 100%; SFRP5, 10 out of 11, 91%) and primary breast tumours (SFRP1, 31 out of 78, 40%; SFRP2, 60 out of 78, 77%; SFRP5, 55 out of 78, 71%). We also observed methylation of DKK1, although less frequently, in cell lines (3 out of 11, 27%) and primary tumours (15 out of 78, 19%). Breast cancer cell lines express various Wnt ligands, and overexpression of SFRPs inhibited cancer cell growth. In addition, overexpression of a beta-catenin mutant and depletion of SFRP1 using small interfering RNA synergistically upregulated transcriptional activity of T-cell factor/lymphocyte enhancer factor. Our results confirm the frequent methylation and silencing of Wnt antagonist genes in breast cancer, and suggest that their loss of function contributes to activation of Wnt signalling in breast carcinogenesis.

Cardiac and peripheral vascular responses to head-up tilt during whole body thermal stress.

During acute orthostatic stress, neurally mediated control of cardiac output (CO) and total peripheral vascular resistance (TPR) play an important role in the maintenance of systemic blood pressure. To examine the influence of thermal stress on the CO and TPR responses to orthostatic stress, 10 healthy male volunteers were exposed to normothermic control conditions followed by whole-body thermal stress produced by a cold or hot water-perfused suit during 5 min-70 degrees head-up tilt (HUT). HUT increased mean arterial pressure (MAP) by 3% of the pre-tilt value during normothermic control and cooling, whereas it decreased MAP by 4% of the pre-tilt value during heating. HUT decreased CO by 16-17% of the pre-tilt value under each thermal condition. The increase of TPR during HUT was exaggerated during cooling and inhibited during heating compared to normothermic control. Tilt-induced decrease of skin blood flow was greater during heating than cooling. These results suggest that the smaller increase of TPR rather than the CO change is responsible for the decreased MAP during acute orthostatic stress in hyperthermic humans. The contribution of skin vascular constriction to TPR changes during HUT is increased during heating and decreased during cooling.

Overproduction of elongation factor 1alpha, an essential translational component, causes aberrant cell morphology by affecting the control of growth polarity in fission yeast.

BACKGROUND: Elongation factor 1alpha (EF1alpha), an essential component of the eukaryotic translational machinery, has been shown to possess various biochemical and biological activities, including F-actin-binding and -bundling, microtubule- severing, and the activity of making fibroblasts highly susceptible to transformation. However, our understanding of the biological significance of EF1alpha with respect to these various biochemical or biological activities remains limited. Here we report the identification of EF1alpha-encoding genes as genes whose over-expression causes aberrant cell morphology in fission yeast. RESULTS: Overproduction of EF1alpha caused aberrant cell morphology-elliptic, curved or branched-and growth defects in yeast cells at high temperatures. EF1alpha-overproducing cells showed a supersensitivity to the actin inhibitor cytochalasin D and to the tubulin inhibitor thiabendazole. Genetic analyses using cdc mutants suggested that excess EF1alpha disturbed the establishment and the maintenance of growth polarity in the G1 phase by pre- venting the localization of F-actin to the polarized growing site and the organization of microtubules. Results from DNase I column chromatography indicated that EF1alpha was bound to G-actin. Indeed, the fission yeast actin was immunoprecipitated along with EF1alpha. Moreover, the temperature sensitivity caused by the overproduction of EF1alpha was restored by co-overproduction of actin. CONCLUSIONS: Fission yeast EF1alpha has the ability to alter the cell morphology of yeast by affecting the control of actin and microtubule cytoskeletons.

Somatolactin in the white sturgeon and African lungfish and its evolutionary significance.

Somatolactin (SL) is a newly characterized pituitary hormone belonging to the growth hormone-prolactin family. Until now SL has been identified only in teleosts, the most highly derived ray-finned fishes. We report here the cloning of SL cDNAs from two species of bony fish, the white sturgeon (Acipenser transmontanus) and the African lungfish (Protopterus annectens). Overlapping partial cDNA clones corresponding to teleost SLs were amplified by polymerase chain reaction (PCR) from either single-strand or double-strand cDNA from pituitary glands. Excluding the poly(A) tail, the sturgeon SL cDNA is 881 base pairs (bp). This is comparable to 1.0 kb estimated by Northern blot analysis. It contains a 696-bp open reading frame encoding a prehormone of 232 amino acids (aa) with a signal peptide of 24 aa and a mature protein of 208 aa. Excluding the poly(A) tail, the lungfish SL cDNA is 938 bp. This is comparable to 1.1 kb estimated by Northern blot analysis. It contains a 696-bp open reading frame encoding a prehormone of 232 aa with a signal peptide of 26 aa and a mature protein of 206 aa. The deduced aa sequences of sturgeon and lungfish SLs show 76-60% and 65-54% identity with teleost SLs, respectively. These values are significantly higher than the 30% identity with nonteleostean growth hormones and prolactins. Immunostaining of sturgeon pituitary with anti-salmon SL serum demonstrated that the SL cells were localized in the pars intermedia, as in teleosts. The present results demonstrate that the SL gene is present in two divergent lineages, the Actinopterygii (Chondrostei: white sturgeon) and the Sarcopterygii (Dipnoi: African lungfish).

Purification, molecular cloning, and expression of lipase from Pseudomonas aeruginosa.

An extracellular lipase secreted by Pseudomonas aeruginosa TE3285 was purified. A genomic library of this strain was constructed in lambda EMBL3, and a DNA fragment 2.7 kb long containing the lipase gene, lipA, was isolated with an oligonucleotide probe synthesized on the basis of the partial amino acid sequence of a purified preparation of the enzyme. Nucleotide sequence analysis showed an open reading frame of 933 bases, and the deduced amino acid sequence agreed well with the molecular mass and partial amino acid sequences of mature lipase. The results of alignment of the amino acid sequences of five lipases from Pseudomonas species considered together with the published crystal structure studied with human pancreatic lipase showed that Ser82, His251, and Asp209 were catalytic residues and that a surface loop from residues 172 to 204 was responsible for the substrate specificity. About 50 bases downstream of lipA, there was another gene, lipB. The sequence of lipB was highly homologous to that of putative modulators of the production of active lipases in other Pseudomonas species. Expression plasmids encoding lipA followed by the complete or incomplete lipB gene downstream of the lac promoter of pUC18 were constructed. lipA was expressed in Escherichia coli 1100 only in the presence of the complete lipB gene.

Changes in insulation of wetsuits during repetitive exposure to pressure.

This investigation was intended to a) establish a relationship between pressure and thickness (and hence insulation) of wetsuits during acute changes in pressure from 0.5 to 5 atmospheres absolute (ATA); b) determine the effect of repetitive compression-decompression (C-D) procedures on the insulation properties of wetsuits; and c) assess the possible recovery of insulation after cessation of repetitive C-D. Various wetsuits with different thicknesses and types of linings were selected. In all wetsuits, insulation and thickness were both reduced as similar, curvilinear functions of acutely increasing pressure in the range of 0.5-5.0 ATA. Effects of repetitive C-D (3 ATA) were studied in 5-mm-thick suit samples with various linings. The insulation of suits (0.100-0.120 degrees C.W-1.m-2) rapidly decreased after the first 5000 C-D. In suits without linings (skin suits), the insulation was reduced to 0.080 degrees C.W-1.m-2 at 5000 C-D and revealed no further reduction during the continuation of C-D cycles up to 30,000. Suits lined with single- or double-sided jersey showed a continuous decrease in the insulation and thickness as the number of cycles increased up to 12,000 and remained at a lowered value (0.060 degrees C.W-1.m-2) until the termination of C-D (30,000 times). A nearly complete recovery of thickness was seen 10 days after cessation of C-D treatment in the skin suit when C-D cycles were less than 3500; however, no recovery was observed in suits with linings (either single- or double-sided) in 25 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Vertical abnormal retinal correspondence in three patients with congenital absence of the superior oblique muscle.

Three patients with large vertical deviations diagnosed as palsy of the superior oblique muscle were found to have a congenital absence of the muscle during surgery. They also showed large, vertical abnormal retinal correspondence, which caused paradoxical vertical diplopia when the deviations were corrected with Fresnel membrane prisms or surgery. Each patient showed large, long-standing, vertical deviations for which head tilting could not have compensated.

Modification of a conventional bicycle ergometer for underwater use.

With a few simple modifications, a conventional row-bicycle ergometer could be made suitable for underwater use. By attaching three sizes of fins to the pedal cranks of the ergometer, work load in water could be achieved to an oxygen consumption (VO2) level range of 400 to 2000 ml/min. The preferable range of pedaling speeds was 30-40 rpm to maintain a constant speed for a prolonged time. When using three different fins VO2 was found to be: VO2 = 254.3 + 4.50n - 0.12n2 + 0.0030n3 for no fins, VO2 = 250.1 + 8.30n - 0.32n2 + 0.0062n3 for small fins, VO2 = 254.0 + 6.10n - 0.24n2 + 0.0068n3 for medium fins, and VO2 = 256.4 + 13.77n - 0.72n2 + 0.0131n3 for large fins, when n is the number of pedaling rate. Major advantages of this modified ergometer for underwater exercise are 1) the modification is simple and the size of the device is small enough to place in a tank of a climatic chamber, 2) the ergometer can be biologically calibrated and used for a wide range of exercise oxygen uptakes, and 3) a subject can exercise in a comfortable upright position.