Wnt7a is a novel inducer of ß-catenin-independent tumor-suppressive cellular senescence in lung cancer.

Cellular senescence is an initial barrier for carcinogenesis. However, the signaling mechanisms that trigger cellular senescence are incompletely understood, particularly in vivo. Here we identify Wnt7a as a novel upstream inducer of cellular senescence. In two different mouse strains (C57Bl/6J and FVB/NJ), we show that the loss of Wnt7a is a major contributing factor for increased lung tumorigenesis owing to reduced cellular senescence, and not reduced apoptosis, or autophagy. Wnt7a-null mice under de novo conditions and in both the strains display E-cadherin-to-N-cadherin switch, reduced expression of cellular senescence markers and reduced expression of senescence-associated secretory phenotype, indicating a genetic predisposition of these mice to increased carcinogen-induced lung tumorigenesis. Interestingly, Wnt7a induced an alternate senescence pathway, which was independent of ß-catenin, and distinct from that of classical oncogene-induced senescence mediated by the well-known p16(INK4a) and p19(ARF) pathways. Mechanistically, Wnt7a induced cellular senescence via inactivation of S-phase kinase-associated protein 2, an important alternate regulator of cellular senescence. Additionally, we identified Iloprost, a prostacyclin analog, which initiates downstream signaling cascades similar to that of Wnt7a, as a novel inducer of cellular senescence, presenting potential future clinical translational strategies. Thus pro-senescence therapies using either Wnt7a or its mimic, Iloprost, might represent a new class of therapeutic treatments for lung cancer.

A kinetic comparison of back-loading and head-loading in Xhosa women.

The purpose of this study was to compare the kinetic responses associated with ground reaction force measurements to both head-loading and back-loading in a group of Xhosa women. Altogether, 16 women were divided into two groups based on their experience of head-loading. They walked over a force plate in three conditions: unloaded or carrying 20 kg in either a backpack or on their head. The most striking finding was that there was no difference in kinetic response to head-loading as a consequence of previous experience. Considering the differences between the load carriage methods, most changes were consistent with increasing load. Head-loading was, however, associated with a shorter contact time, smaller thrust maximum and greater vertical force minimum than back-loading. Both loading conditions differed from unloaded walking for a number of temporal variables associated with the ground contact phase, e.g. vertical impact peak was delayed whilst vertical thrust maximum occurred earlier. STATEMENT OF RELEVANCE: Consideration of the kinetics of head and back load carriage in African women is important from a health and safety perspective, providing an understanding of the mechanical adaptations associated with both forms of load carriage for a group of people for whom such load carriage is a daily necessity.

A comparison of the physiological consequences of head-loading and back-loading for African and European women.

The aim is to quantify the physiological cost of head-load carriage and to examine the 'free ride' hypothesis for head-load carriage in groups of women differing in their experience of head-loading. Twenty-four Xhosa women [13 experienced head-loaders (EXP), 11 with no experience of head-loading (NON)] attempted to carry loads of up to 70% of body mass on both their heads and backs whilst walking on a treadmill at a self-selected walking speed. Expired air was collected throughout. In a second study nine women, members of the British Territorial Army, carried similar loads, again at a self-selected speed. Maximum load carried was greater for the back than the head (54.7 +/- 15.1 vs. 40.8 +/- 13.2% BM, P < 0.0005). Considering study one, head-loading required a greater oxygen rate than back-loading (10.1 +/- 2.6 vs. 8.8 +/- 2.3 ml kg bodymass(-1) min(-1), P = 0.043, for loads 10-25% BM) regardless of previous head-loading experience (P = 0.333). Percentage changes in oxygen consumption associated with head-loading were greater than the proportional load added in both studies but were smaller than the added load for the lighter loads carried on the back in study 1. All other physiological variables were consistent with changes in oxygen consumption. The data provides no support for the 'free ride' hypothesis for head-loading although there is some evidence of energy saving mechanisms for back-loading at low speed/load combinations. Investigating the large individual variation in response may help in identifying combinations of factors that contribute to improved economy.

Subjective perceptions of load carriage on the head and back in Xhosa women.

The purpose of this study was to compare the subjective perceptual responses to both head-loading and back-loading in a group of Xhosa women. Thirty two women were divided into three groups based on their experience of head-loading and walked on a treadmill on two occasions, head-loading and back-loading, at a self selected walking speed for 4 min with a variety of loads until pain or discomfort caused the test to be terminated or a load of 70% body mass was successfully carried. After each workload there was a 1 min rest period during which the women indicated feelings of pain or discomfort in particular areas of the body via visual analogue scales. At the end of each test the women were asked to complete further questionnaires relating to pain and discomfort and on completion of the second test were also asked to compare the two loading conditions. Finally the women were interviewed to establish their history of load carriage and associated pain and discomfort. The data indicate that whilst back-loading was generally associated with more areas of discomfort than head-loading, the pain and discomfort in the neck associated with head-loading was the predominant factor in the termination of tests and that this was independent of head-loading experience. This early termination meant that, on average, the women could carry greater loads on their backs than on their heads. The study suggests that further work needs to be carried out to establish viable alternatives to head-loading for rural dwellers in Africa.

Peripheral arterial disease and intermittent claudication: efficacy of short-term upper body strength training, dynamic exercise training, and advice to exercise at home.

OBJECTIVE: To compare the effect of two training programmes and advice to exercise at home on physiological adaptations in patients with peripheral arterial disease (PAD). DESIGN: 30 patients with a typical history of PAD and intermittent claudication were randomised to either an upper body strength training programme (UBST), a dynamic (walking, cycling, circuit) conventional exercise rehabilitation programme (CER), or advice to 'walk as much as possible at home' (CONT). Before and after intervention groups performed a standard graded treadmill exercise test (GTET) and a 6-minute walk test (SMWT) to determine peak physiological parameters and walking distances. Maximal walking distance (MWD), pain-free walking distance (PFWD), peak oxygen uptake (VO2) , heart rate and perceived pain were measured. RESULTS: MWD on the GTET increased significantly in the CER group compared with the CONT and UBST groups (93.9 +/- 79% v. 7.0 +/- 19.8% v. 7.3 +/- 46%; CER v. UBST v. CONT p = 0.003). Similarly, peak VO2 increased with CER compared with the CONT and UBST groups (28.4 +/- 20 v. -6.2 +/- 15 v. -1.0 +/- 21%; CER v. UBST v. CONT p = 0.004). During the SMWT the CER and UBST groups improved in PFWD compared with the CONT group (37 +/- 47% v. 27 +/- 71% v. -30 +/- 29%; CER v. UBST v. CONT p = 0.03), and perceived pain decreased in the CER group compared with the UBST group (-24 +/- 39% v. 27 +/- 48%; CER v. UBST p = 0.01). CONCLUSION: CER improves physiological parameters and walking distances more than UBST does. CER is effective within 6 weeks. Verbal encouragement to exercise is an ineffective form of management.

Factors predicting walking intolerance in patients with peripheral arterial disease and intermittent claudication.

OBJECTIVE: To determine which physiological variables conduce to walking intolerance in patients with peripheral arterial disease (PAD). DESIGN: The physiological response to a graded treadmill exercise test (GTT) in patients with PAD was characterised. SETTING: Patients were recruited from the Department of Vascular Surgery, Groote Schuur Hospital, Cape Town. SUBJECTS: Thirty-one patients diagnosed with PAD were included in the study. OUTCOME MEASURES: During a GTT, peak oxygen consumption (VO(2peak)), peak minute ventilation (VE(peak)), peak heart rate and peak venous lactate concentrations were measured and compared with those from a comparison group. Ankle-brachial index (ABI) was measured at rest and after exercise. During the GTT, maximum walking distance (MWD) and pain-free walking distance (PFWD) were measured to determine walking tolerance. RESULTS: Peak venous lactate concentrations did not correlate significantly with either PFWD (r = -0.08; p = 0.3) or MWD (r = -0.03; p = 0.4). Resting ABI did not correlate with either MWD (r = 0.09; p = 0.64) or PFWD (r = -0.19; p = 0.29). Subjects terminated exercise at significantly (p < 0.05) lower levels of cardiorespiratory effort and venous lactate concentrations than did a sedentary but otherwise healthy comparison group: peak heart rate 156 +/- 11 v. 114 +/- 22 beats per minute (BPM); p = 0.001; and peak venous lactate concentration 9.7 +/- 2.7 mmol/l v. 3.28 +/- 1.39 mmol/1; p = 0.001. CONCLUSION: Perceived discomfort in these patients is not caused by elevated blood lactate concentrations, a low ABI or limiting cardiorespiratory effort but by other factors not measured in this study.

Wnt7b regulates placental development in mice.

Secreted Wnt proteins regulate many developmental processes in multicellular organisms. We have generated a targeted mutation in the mouse Wnt7b gene. Homozygous Wnt7b mutant mice die at midgestation stages as a result of placental abnormalities. Wnt7b expression in the chorion is required for fusion of the chorion and allantois during placental development. The alpha4 integrin protein, required for chorioallantoic fusion, is not expressed by cells in the mutant chorion. Wnt7b also is required for normal organization of cells in the chorionic plate. Thus, Wnt7b signaling is central to the early stages of placental development in mammals.

Validity of inspiratory and expiratory methods of measuring gas exchange with a computerized system.

The accuracy of a computerized metabolic system, using inspiratory and expiratory methods of measuring ventilation, was assessed in eight male subjects. Gas exchange was measured at rest and during five stages on a cycle ergometer. Pneumotachometers were placed on the inspired and expired side to measure inspired (VI) and expired ventilation (VE). The devices were connected to two systems sampling expired O(2) and CO(2) from a single mixing chamber. Simultaneously, the criterion (Douglas bag, or DB) method assessed VE and fractions of O(2) and CO(2) in expired gas (FE(O(2)) and FE(CO(2))) for subsequent calculation of O(2) uptake (VO(2)), CO(2) production (VCO(2)), and respiratory exchange ratio. Both systems accurately measured metabolic variables over a wide range of intensities. Though differences were found between the DB and computerized systems for FE(O(2)) (both inspired and expired systems), FE(CO(2)) (expired system only), and VO(2) (inspired system only), the differences were extremely small (FE(O(2)) = 0.0004, FE(CO(2)) = -0.0003, VO(2) = -0.018 l/min). Thus a computerized system, using inspiratory or expiratory configurations, permits extremely precise measurements to be made in a less time-consuming manner than the DB technique.

Retinoic acid receptor-beta as a prognostic indicator in stage I non-small-cell lung cancer.

PURPOSE: Retinoids are pivotal in the growth and differentiation of certain epithelial tissues, interacting with nuclear retinoid receptors (the retinoic acid receptors [RARs] and retinoid X receptors [RXRs]), which function as transcription factors. RAR-beta mRNA is undetectable by in situ hybridization (ISH) in 50% of non-small-cell lung cancers (NSCLC). RAR-beta may suppress tumorigenicity. Therefore, we hypothesized that loss of expression of RAR-beta gene in stage I NSCLC is a prognostic factor of a poor clinical outcome. PATIENTS AND METHODS: We retrospectively analyzed RAR-beta mRNA levels (by ISH using a digoxigenin-labeled antisense riboprobe) in specimens from 185 consecutive patients with completely resected clinical/radiographic stage I NSCLC for whom clinical follow-up data were available. RESULTS: One hundred fifty-six patients who met the criteria of pathologic stage I NSCLC and positivity for RXR-alpha mRNA (used as a control to assess RNA degradation) and who had adequate follow-up could be evaluated. RAR-beta mRNA expression was undetectable in 51 patients, weakly positive in 64 patients, and strongly positive in 41 patients. Overall survival of the 41 patients with strongly positive RAR-beta was significantly worse than for the 115 patients with weak or absent RAR-beta (P =.045). CONCLUSION: Unexpectedly, strong RAR-beta expression was associated with a significantly worse outcome of early-stage NSCLC. The mechanisms underlying this clinically and biologically important finding should be further explored.

Tbx12, a novel T-box gene, is expressed during early stages of heart and retinal development.

T-box genes encode transcription factors that regulate many developmental processes. We have cloned a novel mouse T-box gene, Tbx12. Tbx12 is the vertebrate homologue of the Drosophila H15 gene and the Caenorhabditis elegans tbx-12 gene. Tbx12 is expressed in extraembryonic tissues such as the amnion and allantois. In the embryo, Tbx12 is strongly expressed in the neural retina and the heart.

Sexually dimorphic development of the mammalian reproductive tract requires Wnt-7a.

An important feature of mammalian development is the generation of sexually dimorphic reproductive tracts from the Müllerian and Wolffian ducts. In females, Müllerian ducts develop into the oviduct, uterus, cervix and upper vagina, whereas Wolffian ducts regress. In males, testosterone promotes differentiation of Wolffian ducts into the epididymis, vas deferens and seminal vesicle. The Sertoli cells of the testes produce Müllerian-inhibiting substance, which stimulates Müllerian duct regression in males. The receptor for Müllerian-inhibiting substance is expressed by mesenchymal cells underlying the Müllerian duct that are thought to mediate regression of the duct. Mutations that inactivate either Müllerian-inhibiting substance or its receptor allow development of the female reproductive tract in males. These pseudohermaphrodites are frequently infertile because sperm passage is blocked by the presence of the female reproductive system. Here we show that male mice lacking the signalling molecule Wnt-7a fail to undergo regression of the Müllerian duct as a result of the absence of the receptor for Müllerian-inhibiting substance. Wnt7a-deficient females are infertile because of abnormal development of the oviduct and uterus, both of which are Müllerian duct derivatives. Therefore, we propose that signalling by Wnt-7a allows sexually dimorphic development of the Müllerian ducts.

The classical mouse mutant postaxial hemimelia results from a mutation in the Wnt 7a gene.

The study of spontaneous mutations has aided the understanding of developmental processes. A large collection of spontaneous or "classical" mouse mutations has been accumulated over many decades. One of the mutations causes the postaxial hemimelia (px) phenotype, which consists of limb patterning defects accompanied by Müllerian duct-associated sterility in both sexes. We were intrigued that both the limb and the Müllerian duct px phenotypes are similar to those caused by mutations in the gene encoding the Wnt 7a signaling molecule. In this paper, we investigate the nature of the px mutation. Morphological analysis and breeding experiments demonstrate that the px phenotype indeed results from a mutation in the Wnt 7a gene. Molecular analysis demonstrates that px results from a 515-bp deletion in the Wnt 7a gene. This generates an abnormal splicing event, which ultimately produces a truncated Wnt 7a protein of half the normal size. Thus, the px mutation is predicted to be a likely null allele of the Wnt 7a gene. Our results provide another interesting example of a classical mutation that disrupts an important patterning gene in development.

Dorsalizing signal Wnt-7a required for normal polarity of D-V and A-P axes of mouse limb.

Formation of the vertebrate limb requires specification of cell position along three axes. Proximal-distal identity is regulated by the apical ectodermal ridge (AER) at the distal tip of the growing limb. Anterior-posterior identity is controlled by signals from the zone of polarizing activity (ZPA) within the posterior limb mesenchyme. Dorsal-ventral identity is regulated by ectodermally derived signals. Recent studies have begun to identify signalling molecules that may mediate these patterning activities. Members of the fibroblast growth factor (FGF) family are expressed in the AER and can mimic its proximal-distal signalling activity. Similarly, the gene Sonic hedgehog (Shh) is expressed in the ZPA, and Shh-expressing cells, like ZPA cells, can cause digit duplications when transplanted to the anterior limb margin. In contrast, no signal has yet been identified for the dorsal-ventral axis, although Wnt-7a is expressed in the dorsal ectoderm, suggesting that it may play such a role. To test this possibility, we have generated mice lacking Wnt-7a activity. The limb mesoderm of these mice shows dorsal-to-ventral transformations of cell fate, indicating that Wnt-7a is a dorsalizing signal. Many mutant mice also lack posterior digits, demonstrating that Wnt-7a is also required for anterior-posterior patterning. We propose that normal limb development requires interactions between the signalling systems for these two axes.

Wnt genes and vertebrate development.

A variety of experimental approaches have underscored the critical role played by secreted polypeptide factors, such as those encoded by members of the Wnt gene family, in many aspects of vertebrate embryogenesis. Recent papers have revealed restricted patterns of Wnt gene expression that delineate important subdivisions within the early forebrain and spinal cord, demonstrated that Wnt gene products can regulate mesoderm formation and gastrulation, and investigated how Wnt protein signaling may affect cell adhesion.

Independent regulatory elements in the nestin gene direct transgene expression to neural stem cells or muscle precursors.

Changes in intermediate filament gene expression occur at key steps in the differentiation of cell types in the mammalian CNS. Neuroepithelial stem cells express the intermediate filament protein nestin and down-regulate it sharply at the transition from proliferating stem cell to postmitotic neuron. Nestin is also expressed in muscle precursors but not in mature muscle cells. We show here that in transgenic mice, independent cell type-specific elements in the first and second introns of the nestin gene consistently direct reporter gene expression to developing muscle and neural precursors, respectively. The second intron contains an enhancer that functions in CNS stem cells, suggesting that there may be a single transcriptional mechanism regulating the CNS stem cell state. This enhancer is much less active in the PNS. The identification of these elements facilitates analysis of mechanisms controlling the switch in gene expression that occurs when muscle and brain precursors terminally differentiate.

Mouse Wnt genes exhibit discrete domains of expression in the early embryonic CNS and limb buds.

Mutation and expression studies have implicated the Wnt gene family in early developmental decision making in vertebrates and flies. In a detailed comparative analysis, we have used in situ hybridization of 8.0- to 9.5-day mouse embryos to characterize expression of all ten published Wnt genes in the central nervous system (CNS) and limb buds. Seven of the family members show restricted expression patterns in the brain. At least three genes (Wnt-3, Wnt-3a, and Wnt-7b) exhibit sharp boundaries of expression in the forebrain that may predict subdivisions of the region later in development. In the spinal cord, Wnt-1, Wnt-3, and Wnt-3a are expressed dorsally, Wnt-5a, Wnt-7a, and Wnt-7b more ventrally, and Wnt-4 both dorsally and in the floor plate. In the forelimb primordia, Wnt-3, Wnt-4, Wnt-6 and Wnt-7b are expressed fairly uniformly throughout the limb ectoderm. Wnt-5a RNA is distributed in a proximal to distal gradient through the limb mesenchyme and ectoderm. Along the limb's dorsal-ventral axis, Wnt-5a is expressed in the ventral ectoderm and Wnt-7a in the dorsal ectoderm. We discuss the significance of these patterns of restricted and partially overlapping domains of expression with respect to the putative function of Wnt signalling in early CNS and limb development.

The Wnt family of cell signalling molecules in postimplantation development of the mouse.

The mammalian Wnt gene family consists of at least ten members, all of which share a common structure. The N-terminus encodes a putative signal peptide sequence, suggesting that Wnt proteins are secreted. A number of absolutely conserved cysteine residues imply that inter- or intramolecular disulphide bonding is important to Wnt protein function. Wnt RNAs are localized to discrete regions of the postimplantation embryo and fetus, particularly within the developing central nervous system. Studies on Wnt gene expression strongly suggest that Wnt-mediated signalling is likely to be an important aspect of mouse development. One member of the family, Wnt-1, has been studied in some detail. By generating mutant alleles, we have demonstrated that Wnt-1 regulates regional development of the central nervous system at early somite stages. There is circumstantial evidence that some aspects of the pathway through which Wnt-1 action is mediated may be evolutionarily conserved. We propose that the Wnt family plays a major role in cell-cell interactions in the mouse.

Promoter structure and protein sequence of msp130, a lipid-anchored sea urchin glycoprotein.

The early fate specification of primary mesenchyme cells in sea urchin embryos makes them an attractive system for studying alterations in gene expression and protein synthesis during cell lineage determination and differentiation. To analyze the developmental regulation of gene expression in Strongylocentrotus purpuratus, we have isolated and sequenced genomic and cDNA clones encoding msp 130, a mesenchyme-specific cell surface glycoprotein. We have located the transcription initiation site of the msp130 gene and sequenced several kilobases of the promoter region. The region of the gene that encodes the protein is divided into numerous small (less than 500 base pairs) exons. The msp130 protein possesses two novel glycine-rich domains and a signal peptide, but apparently lacks a transmembrane domain. The carboxyl-terminal sequence suggests that msp130 may be phosphatidylinositol-linked to the cell membrane, and experiments with phospholipases support this conclusion. The implications of the msp130 sequence for its possible functions are discussed.