Bio-inspired compensatory strategies for damage to flapping robotic propulsors.

Natural swimmers and flyers can fully recover from catastrophic propulsor damage by altering stroke mechanics: some fish can lose even 76% of their propulsive surface without loss of thrust. We consider applying these principles to enable robotic flapping propulsors to autonomously repair functionality. However, direct transference of these alterations from an organism to a robotic flapping propulsor may be suboptimal owing to irrelevant evolutionary pressures. Instead, we use machine learning techniques to compare these alterations with those optimal for a robotic system. We implement an online artificial evolution with hardware-in-the-loop, performing experimental evaluations with a flexible plate. To recoup thrust, the learned strategy increased amplitude, frequency and angle of attack (AOA) amplitude, and phase-shifted AOA by approximately 110°. Only amplitude increase is reported by most fish literature. When recovering side force, we find that force direction is correlated with AOA. No clear amplitude or frequency trend is found, whereas frequency increases in most insect literature. These results suggest that how mechanical flapping propulsors most efficiently adjust to damage may not align with natural swimmers and flyers.

K-ras 4A and 4B are co-expressed widely in human tissues, and their ratio is altered in sporadic colorectal cancer.

Ras activating mutations result in constitutive activation of Ras signalling pathways and occur in 30% of human malignancies. K-ras encodes two splice variants, K-ras 4A and 4B, and K-ras activating mutations which jointly affect both isoforms are prevalent in lung, pancreatic and colorectal cancers. Using RT-PCR we examined their expression in normal adult human tissues and addressed whether K-ras splicing is altered in sporadic colorectal cancer by comparing normal colon with colon carcinoma cell lines, and 'matched' tumour and tumour-free colon tissues from the same patient. K-ras 4B was expressed ubiquitously and was the predominant splice variant. K-ras 4A was expressed differentially, with detection in colorectal tumours and cell lines, and normal colon, pancreas and lung--sites where tumours with K-ras activating mutations arise. Both K-ras splice variants were co-expressed by single colon carcinoma cells. The K-ras 4A/4B ratio was significantly reduced in all 6 cell lines examined, including two that lacked K-ras activating mutations, and in 4/9 primary adenocarcinomas. We conclude that K-ras activating mutations do not affect K-ras splicing per se, both isoforms may play a role in neoplastic progression, and altered splicing of either the K-ras proto-oncogene or oncogene, in favour of K-ras 4B, may modulate tumour development.

Effects of heterozygosity for the Rb-1t19neo allele in the mouse.

We describe the pathology of a cohort of 80 mice heterozygous for an inactive allele of the Rb-1 tumour suppressor gene. The majority of these mice developed locally invasive tumours, arising from the pituitary gland. The time of onset of overt signs of disease in mice known to have inherited their mutant allele paternally shows a small but statistically significant shift to the lower end of the spectrum, suggesting that tumorigenesis is influenced by gametic imprinting. In situ hybridisation analysis demonstrates the presence in the tumours of pro-opiomelanocortin mRNA, which is normally found both in corticotroph and melanotroph cells. Mice within this cohort also develop systemic defects. Most notably, there is increased siderosis in the spleen indicating the possibility of an abnormality in red blood cell turnover. This is consistent with the abnormalities of erythropoiesis described previously in homozygous Rb-1-deficient mice. In addition, a proportion of mice developed liver steatosis, probably representing the end organ effects of hormonal imbalance as a direct consequence of tumour presence. A significant proportion showed C cell hyperplasia in the thyroid. The spectrum of pathology in mice differs from that in the human but does provide a useful model of site-specific tumour predisposition.

p53 dependence of early apoptotic and proliferative responses within the mouse intestinal epithelium following gamma-irradiation.

p53 is now well characterized as a tumour suppressor gene, with loss of normal p53 function being recorded as the commonest genetic event associated with human malignancy. In particular, its involvement with tumorigenesis within the intestine is well established. Normal p53 function has been shown to be crucial for the induction of apoptosis in tumour cell lines, murine thymocytes and murine haematopoietic cells following DNA damage. To elucidate further the role of p53 in the cellular response to DNA damage we have investigated the response to gamma-irradiation of crypt cells in vivo from the small and large intestine of mice bearing a constitutive p53 deletion. Four hours after gamma-irradiation, a time point at which wild type crypt cells show abundant apoptosis, crypt cells from p53-deficient mice differed in that they were completely resistant to the induction of apoptosis. The p53 dose dependence of this phenomenon was clearly shown by the intermediate level of apoptosis observed in p53 heterozygotes. Analysis of the mitotic index and the bromodeoxyuridine labelling index showed that two other responses of wild type crypts to gamma-irradiation, namely the G2 block and the reduction in bromodeoxyuridine incorporation, were both largely intact in p53 deficient animals. These observations demonstrate that p53 function is essential for a major component of the normal response to gamma-irradiation induced DNA damage in intestinal mucosal cells, and suggest that p53 deficiency permits a population of cells bearing DNA damage to escape the normal process of deletion.

Ganciclovir-induced ablation non-proliferating thyrocytes expressing herpesvirus thymidine kinase occurs by p53-independent apoptosis.

In adult mice of the transgenic strain TG66.19, in which expression of herpes simplex type 1 virus thymidine kinase (HSVI-TK) is driven in thyrocytes from the thyroglobulin promoter, the drug Ganciclovir causes the death (ablation) of thyrocytes. Ablation occurred in the absence of thyrocyte proliferation or nuclear DNA synthesis, but was accompanied by transient expression of proliferating cell nuclear antigen and the dying thyrocytes exhibited the ultrastructural features of apoptosis. Control experiments show that the apoptosis is a result of the production of Ganciclovir phosphates in thyrocytes that express HSV1-TK. However, cell death was not dependent upon the presence of a functional copy of the oncosuppressor gene p53. We conclude that the apoptosis is probably not mediated by induction of DNA damage and occurs via a pathway that is independent of p53. The fact that Ganciclovir phosphate can kill cells by a p53-independent apoptotic pathway is encouraging in relation to tumour ablation by methods based on transfection with HSV1-tk genes and administration of Ganciclovir.

Tumour incidence, spectrum and ploidy in mice with a large deletion in the p53 gene.

In human tumourigenesis the tumour suppressor gene most commonly affected by mutation, inactivation or allele loss is p53. Loss of p53 function is associated both with failure to maintain a normal diploid status and inability to delete cells by apoptosis following DNA damage. To investigate further the role of p53 we have generated mice carrying a large deletion within the gene. All animals homozygous for this deletion develop spontaneous tumours, predominantly lymphomas, by the age of 6 months. 10% of heterozygotes develop a range of neoplasms, with a lower predisposition towards lymphoma, by 9 months. Both tumour incidence and spectrum in heterozygotes differ from those previously reported in another p53 mutant stock, suggesting either difference in exposure to carcinogens between the two stocks, or a role for modulating genes within different genetic backgrounds. Tumours showed frequent loss of diploid status, and the majority of those arising in heterozygotes showed loss of the wild type allele. These findings are consistent with the concept that p53 acts as a tumour suppressor by preventing the propagation of DNA damage to daughter cells.

Absence of p53 permits propagation of mutant cells following genotoxic damage.

Much evidence has been gathered in support of a critical role for p53 in the cellular response to DNA damage. p53 dysfunction is associated with progression and poor prognosis of many human cancers and with a high incidence of tumours in p53 knockout mice. The absence of a p53-dependent G1 arrest that facilitates DNA repair or apoptosis might impact critically on clinical cancer in two ways. First, by abrogating the impact on therapy that operates via genotoxic damage and apoptosis; and second, by encouraging progression either by inducing genomic instability and DNA mis-repair or by permitting survival of mutants. However, experiments examining the relationship between p53 deficiency and mutation frequency have so far failed to confirm these predictions. The precise role played by p53 is therefore unclear. We now report use of a short term in vitro approach to assess the influence of p53 on radiation-induced mutations at the hprt locus in murine B cell precursors that are normally radiation ultrasensitive. We find a high number of hprt mutants among X-irradiated p53 null cells, which results from preferential survival as clonogenic mutants rather than from a p53-dependent increase in mutation rate. This result has important implications for genotoxic cancer therapy.

Developmentally-regulated expression of murine K-ras isoforms.

The products (p21) of the three mammalian H-, N- and K-ras genes play important roles in intracellular signal transduction, linking membrane receptor kinases to the nuclear pathway through raf and mitogen activated protein kinase. They are involved in the regulation of proliferation and differentiation, and activating mutations of these genes are commonly associated with human cancers. Two p21 proteins are encoded by the K-ras gene (p21K-rasA and p21K-rasB) due to alternative splicing of the last exon. While the four p21ras proteins are highly homologous, their sequences diverge significantly at the C-termini, to which distinct biochemical and perhaps even functional differences may be ascribed. However, H-, N- and K-rasB appear to be ubiquitously expressed, with little evidence of tissue-specific or developmental regulation. In contrast, we now demonstrate that the expression of K-rasA is strikingly different. K-rasA is induced during differentiation of pluripotent embryonal stem cells in vitro. Its expression during early embryogenesis is limited temporally and spatially in a tissue-specific distribution which is largely maintained as an adult. This suggests a distinct biological role for p21K-rasA.

129/Ola mice carrying a null mutation in PrP that abolishes mRNA production are developmentally normal.

The neural membrane glycoprotein PrP is implicated in the pathogenesis of the transmissible spongiform encephalopathies; however, the normal function of PrP and its precise role in disease are not understood. Recently, gene targeting has been used to produce mice with neo/PrP fusion transcripts, but no detectable PrP protein in the brain (1). Here we report the use of a different targeting strategy, to produce inbred mice with a complete absence of both PrP protein and mRNA sequences. At 7 mo of age, these mice show no overt phenotypic abnormalities despite the normal high levels of expression of PrP during mouse development. The mice are being used in experiments designed to address the role of PrP in the pathogenesis of scrapie and the replication of infectivity.

Infusion into the brain of an antisense oligonucleotide to the immediate-early gene c-fos suppresses production of fos and produces a behavioral effect.

While many studies have examined the numerous physiological and pharmacological factors which can induce the expression of c-fos and other immediate-early genes, few have examined the physiological/biochemical consequences of altering their expression pattern. Using antisense oligonucleotides to c-fos, we demonstrate that D-amphetamine-induced c-fos expression can be attenuated in specific brain regions in vivo. This unilateral attenuation of c-fos expression in D-amphetamine-stimulated animals results in a directed rotational behavior. We show that animals rotate only when they express a difference in Fos-like immunoreactivity between hemispheres. The attenuation of Fos-like immunoreactivity by the antisense oligonucleotides appears to be dependent on the c-fos messenger RNA site that these antisense oligonucleotides target and the degree of chemical protection of the oligonucleotide against degradation. The attenuation of Fos-like immunoreactivity and the increase in unilaterally directed rotation are both time- and dose-dependent. These results demonstrate that manipulating immediate-early gene expression by the direct infusion of antisense oligonucleotides in specific brain regions can have behavioral consequences.

Antisense oligonucleotide eliminates in vivo expression of c-fos in mammalian brain.

Immediate-early genes such as c-fos and NGFI-A are rapidly and transiently expressed in the striatum following amphetamine administration in vivo. Here we show that direct infusion of an antisense oligodeoxynucleotide to c-fos into striatum will reduce amphetamine-induced production of Fos-like immunoreactivity without affecting NGFI-A expression. These results suggest that it is possible to use antisense technology to study the role of immediate-early genes in specific sites in the brain in vivo.

Tolerance to self-administration of cocaine in rats: time course and dose-response determination using a multi-dose method.

To assess tolerance to cocaine in a self-administration paradigm, rats were trained to self-administer cocaine (0.25 mg/injection) on a fixed-ratio 2 (FR2) schedule of reinforcement. The development of tolerance was studied during chronic administration of cocaine (20 mg/kg per 8 h for 10 days), given either contingently (self-administered by the rats) or non-contingently (infused by the experimenter). Both contingent and non-contingent administration of cocaine produced comparable tolerance, as indicated by a faster rate of cocaine self-administration (the average inter-reinforcer time, ISRT, decreased significantly). Tolerance developed by day 2 of the chronic regimen and reached a floor value (60% of baseline) from day 4 through day 10. Termination of chronic cocaine then resulted in recovery from tolerance, with ISRTs returning to baseline within 6 days of termination. A second set of experiments determined whether tolerance could be studied using a multi-dose method to obtain dose-response data in a single session. A system of multiple pumps allowed testing of three doses of cocaine during a single experimental session. Cocaine dose-response curves obtained from the multi-dose method: (i) did not differ from that obtained from a single-dose method; (ii) were reproducible; and (iii) were shifted to the right by Schering 23390. Rats were then subjected to a 7-day chronic regimen of infused cocaine (20 mg/kg per 8 h) or infused saline. At the end of this chronic cocaine period, they were tested with the multi-dose method. Chronic cocaine, as compared to chronic saline, shifted the cocaine dose-response curve to the right, indicating that the multi-dose method can be successfully applied to demonstrate tolerance to the effects of cocaine in a self-administration paradigm.

Failure of ondansetron to block the discriminative or reinforcing stimulus effects of cocaine in the rat.

Ondansetron (GR38032F), a serotonin 5HT3 antagonist, is active in numerous behavioral paradigms and neurochemical systems. Since 5HT3 antagonists have been suggested as therapeutic agents for the treatment of drug abuse, the action of ondansetron on cocaine drug discrimination and self-administration paradigms in rats was investigated. Doses of ondansetron (0.001 - 1.0 mg/kg) had no effect on the discriminative stimulus properties of 10 mg/kg cocaine. In contrast SCH23390, a dopamine D1 antagonist known to block cocaine discrimination, acted as previously reported. Ondansetron did not augment the effects of SCH23390, but at higher doses, combinations of ondansetron and SCH23390 produced disruption of lever pressing in the presence of cocaine. Ondansetron (0.001-1.0 mg/kg) had no effect on the self-administration of various doses of cocaine, nor did it have any effect on reacquisition of cocaine self-administration in animals with a history of active administration followed by a period of abstinence. As before, SCH23390, known to block cocaine self-administration, acted as previously reported. Although other 5HT antagonists may prove to be efficacious in cocaine abuse, ondansetron appears unlikely to alter the subjective or rewarding stimulus properties of cocaine.

Antisense Therapeutics in the Central Nervous System : The Induction of c-fos.

Immediate-early genes (IEGs) are members of a class of genes that respond, in many cell types, to a variety of stimuli by rapid, but transient expression (1). Several of these IEGs code for transcription factors and include the widely studied activator protein-1 (AP-1) transcription factor complex believed to be homo- and heterodimeric assemblies of the Fos and Jun families (1-3). IEGs are induced in the central nervous system (CNS) by diverse physiological and pharmacological stimuli, many of which, when presented once or on multiple occasions, can alter the "normal" functioning of the brain in a permanent or semipermanent fashion. Examples of pharmacological stimuli that lead to long-term changes are the highly addictive psychostimulant drugs, amphetamine and cocaine These drugs produce a robust activation of IEGs (e.g., c-fos, jun-B, egr-1) in areas of the brain that are believed to be part of the neural substrates of addiction (4-8) In animal models of epileptogenesis or memory, such as kindling and long-term potentiation (LTP), respectively, electrical stimuli produce activation of IEGs within the brain structures thought to underlie the long-lasting changes associated with these experimental procedures (9-16). IEGs can also be induced by noninvasive stimuli, such as a simple light pulse given to animals in a dark room. The circadian rhythms of animals that are housed in darkened conditions can be shifted by exposing them to a light during then subjective night. Activation of IEGs in such experiments are restricted to the suprachiasmatic nucleus (SCN), which is believed to be the seat of the biological clock (17).

Patterns of sustained attentional demands in elementary classrooms: an observational study.

This study investigates the patterns of sustained attentional demands of events in Kindergarten through Grade 5. The durations and variability of sustained attention for events occurring in 12 elementary classrooms were observed. The data show a progression of sustained attentional demands across the grades; however, a substantial increase in students' sustained attention is required between Kindergarten and Grade 1. The implications of these findings on curriculum development are discussed.

Comparison of demands of sustained attentional events between public and private children's television programs.

The durations and variability of changing events were analyzed for 20 min. each of 13 children's television programs. These programs included selections from both publically and privately produced shows. Significantly different patterns of attentional demands were found between the programs. Public television programming is characterized by longer and more variable durations of sustained attentional events, while private television programming is best described as having fast-paced shorter events. The implications of this finding for difficulties in learning by school-age children to attend for longer periods are discussed.

Gonadal effects of a mouse Denys-Drash syndrome mutation.

Gonadal effects of the Denys-Drash syndrome (DDS) mutation Wt1(tmT396 )were examined in chimaeric and heterozygous mice. Since the only heterozygote was 41,XXY, Sertoli cell function was assessed by comparison with age-matched control XXY testes. Control XXY Sertoli cells showed immuno-expression of WT1 and androgen receptor (AR) indistinguishable from wild-type (40,XY), but expressed anti-Mullerian hormone (AMH). In contrast, DDS Sertoli cells showed only faint immuno-expression of WT1 and did not express AR or AMH. While XY<-->XY DDS chimaeras were male, XX<-->XY chimaeras were predominantly female. In the rare XX<-->XY DDS males the Sertoli cell lineage was largely derived from Wt1 mutant XY cells. We conclude that DDS mutant cells can form Sertoli cells, that the dominant mutation does not cause male sex reversal in mice but distorts the sex ratio of XX<-->XY chimaeras, and that there may be a link between WT1, AMH and AR expression by Sertoli cells in vivo.