Cell proliferation, drug distribution and therapeutic effects in relation to the vascular system of solid tumours.

In this perspective, the authors summarise some properties of the solid tumour micro-environment that have been explored during the last 55 years. It is well established that the concentrations of nutrients, including oxygen, decrease with increasing distance from tumour blood vessels, and that low extracellular pH is found in nutrient-poor regions. Cell proliferation is dependent on nutrient metabolites and decreases in regions distal from patent blood vessels. Proliferating cells cause migration of neighbouring cells further from blood vessels where they may die, and their breakdown products pass into regions of necrosis. Anticancer drugs reach solid tumours via the vascular system and establish concentration gradients such that drug concentration within tumours may be quite variable. Treatment with chemotherapy such as doxorubicin or docetaxel can kill well-nourished proliferating cells close to blood vessels, thereby interrupting migration toward necrotic regions and lead to re-oxygenation and renewed proliferation of distal cells, as can occur with radiotherapy. This effect leads to the paradox that cancer treatment can rescue cells that were destined to die in the untreated tumour. Renewed and sometimes accelerated repopulation of surviving tumour cells can counter the effects of cell killing from repeated treatments, leading to tumour shrinkage and regrowth without changes in the intrinsic sensitivity of cells to the administered treatment. Strategies to prevent these effects include the combined use of chemotherapy with agents that selectively kill hypoxic tumour cells, including inhibitors of autophagy, since this is a process that may allow recycling of cellular macromolecules from dying cells and improve their survival.

Biotic interactions drive ecosystem responses to exotic plant invaders.

Ecosystem process rates typically increase after plant invasion, but the extent to which this is driven by (i) changes in productivity, (ii) exotic species' traits, or (iii) novel (non-coevolved) biotic interactions has never been quantified. We created communities varying in exotic plant dominance, plant traits, soil biota, and invertebrate herbivores and measured indicators of carbon cycling. Interactions with soil biota and herbivores were the strongest drivers of exotic plant effects, particularly on measures of soil carbon turnover. Moreover, plant traits related to growth and nutrient acquisition explained differences in the ways that exotic plants interacted with novel biota compared with natives. We conclude that novel biological interactions with exotic species are a more important driver of ecosystem transformation than was previously recognized.

Strengthening pharmacovigilance in South Africa.

This report outlines findings and recommendations of a national pharmacovigilance workshop held in August 2012 in South Africa (SA). A survey of current pharmacovigilance activities, conducted in preparation for the meeting, identified multiple programmes collecting drug safety data in SA, with limited co-ordination at national level. The meeting resolved that existing pharmacovigilance programmes need to be strengthened and consolidated to ensure that important local safety issues are addressed, data can be pooled and compared and outputs shared more widely. Pharmacovigilance activities should inform treatment guidelines with the goal of improving patient care. A variety of pharmaco-epidemiological approaches should be employed, including nesting drug safety studies within existing sentinel cohorts and the creation of a pregnancy exposure registry. The attendees agreed on key principles that will inform a national pharmacovigilance plan and compiled a list of priority pharmacovigilance issues facing public health programmes in SA.

Designing an adaptive phase II/III trial to evaluate efficacy, safety and immune correlates of new TB vaccines in young adults and adolescents.

This article summarises the consensus arrived at a meeting of South African and international stakeholders on specific late phase clinical trial design issues integrating the investigation of immune correlates as an integral part of a phase III protocol for a preventative TB vaccine in an adolescent/adult population. The challenge ahead is to optimize the planning for phase 3 TB vaccine preventative trials, under resource constraints, given that there are no known correlates of protection to shorten and increase the efficiencies of efficacy trials. An adaptive, multi-arm, group sequentially designed trial protocol is proposed incorporating design features that address uncertainties arising from both advances in the field and dynamic study populations and disease states. Such a design allows modifications that protect research subjects, save time, and maximize the impact of scarce financial resources. Further, the protocol underwent joint review by regulators from several African nations at a meeting of the African Vaccine Regulatory Forum (AVAREF), a regional regulatory harmonization initiative, and recommendations are included.

Key issues in the clinical development and implementation of TB vaccines in South Africa.

Significant progress has been made in advancing the development pipeline for a new and more effective TB vaccine with some candidate vaccines now in late stage clinical evaluation. However, progress has been hampered by an incomplete understanding of the components of a protective immune response and limited animal models, rendering the field unable to reliably predict vaccine efficacy earlier in preclinical development, including by evaluation in animal models, and limiting the predictive utility of comparing immunogenic effects across vaccine candidates in phase I/II studies. Consequently, new candidate vaccines have to be evaluated for efficacy in large-scale phase II/III trials using clinical endpoints. Apart from the technical challenges of characterising TB incidence in target populations at high risk of acquiring TB disease and standardising case definitions in order to improve both the sensitivity and more importantly the specificity of trial endpoints, there is an urgency in expanding and supporting the considerable trial infrastructure that will be required to evaluate and ultimately license a new TB vaccine. In the longer term, implementation strategies are dependent on what policy makers most value. Economic analyses will be essential to guide policy and implementation. This paper outlines the gaps and challenges and identifies solutions for effectively developing and efficiently introducing a new TB vaccine.

Validation of the New Zealand Physical Activity Questionnaire (NZPAQ-LF) and the International Physical Activity Questionnaire (IPAQ-LF) with accelerometry.

BACKGROUND: Validation of instruments used to measure physical activity patterns is essential when attempting to assess the effectiveness of physical activity interventions. OBJECTIVES: To assess the validity of two self-report physical activity questionnaires on a representative sample of New Zealand adults. METHODS: 70 adults aged 18-65 years from around Christchurch, New Zealand were required to wear an ActiGraph GT1M accelerometer during all waking hours for 7 consecutive days. Immediately following the 7 day accelerometer period participants were required to complete the long forms of both the New Zealand Physical Activity Questionnaire (NZPAQ-LF) and the International Physical Activity Questionnaire (IPAQ-LF). RESULTS: Both the NZPAQ-LF and the IPAQ-LF questionnaires showed small to moderate correlations with ActiGraph data for time spent in moderate-intensity physical activity (r=0.19-0.30) and total physical activity (sum of moderate and vigorous-intensity physical activity, r=0.30-0.32). In comparison with the ActiGraph data, both self-report questionnaires tended to overestimate activity levels by approximately 165%. Total physical activity levels gathered from both questionnaires were strongly correlated with each other (r=0.79) and showed good levels of agreement in the Bland-Altman plots. CONCLUSIONS: The long forms of the NZPAQ and IPAQ were found to have acceptable validity when detecting participants' ability to meet activity guidelines based on exercise duration, but a significant amount of overestimation was evident. This presents a need for both instruments to be further developed and tested in order to increase validity.

Characterisation of the mouse vasoactive intestinal peptide receptor type 2 gene, Vipr2, and identification of a polymorphic LINE-1-like sequence that confers altered promoter activity.

The VPAC(2) receptor is a seven transmembrane spanning G protein-coupled receptor for two neuropeptides, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). It has a distinct tissue-specific, developmental and inducible expression that underlies an important neuroendocrine role. Here, we report the characterisation of the gene that encodes the mouse VPAC(2) receptor (Vipr2), localisation of the transcriptional start site and functional analysis of the promoter region. The Vipr2 gene contains 12 introns within its protein-coding region and spans 68.6 kb. Comparison of the 5' untranslated region sequences for cloned 5'-RACE products amplified from different tissues showed they all were contained within the same exon, with the longest extending 111 bp upstream of the ATG start site. Functional analysis of the 3.2-kb 5'-flanking region using sequentially deleted sequences cloned into a luciferase gene reporter vector revealed that this region is active as a promoter in mouse AtT20 D16:16 and rat GH4C1 cell lines. The core promoter is located within a 180-bp GC-rich region proximal to the ATG start codon and contains potential binding sites for Sp1 and AP2, but no TATA-box. Further upstream, in two out of three mice strains examined, we have discovered a 496-bp polymorphic DNA sequence that bears a significant identity to mouse LINE-1 DNA. Comparison of the promoter activity between luciferase reporter gene constructs derived from the BALB/c (which contains this sequence) and C57BL/6J (which lacks this sequence) Vipr2 promoter regions has shown three-fold difference in luciferase gene activity when expressed in mouse AtT20 D16:16 and alphaT3-1 cells, but not when expressed in the rat GH4C1 cells or in COS 7 cells. Our results suggest that the mouse Vipr2 gene may be differentially active in different mouse strains, depending on the presence of this LINE-1-like sequence in the promoter region.

Whole lot of parts: stress in extreme environments.

Stress has been a central interest for researchers of human behavior in extreme and unusual environments and also for those who are responsible for planning and carrying out expeditions involving such environments. This paper compares the actuarial and case study methods for predicting reactions to stress. Actuarial studies are useful, but do not tap enough variables to allow us to predict how a specific individual will cope with the rigors of an individual mission. Case histories provide a wealth of detail, but few investigators understand the challenges of properly applying this method. This study reviews some of the strengths and weaknesses of the actuarial and case history methods, and presents a four celled taxonomy of stress based on method (actuarial and case history) and effects (distress and eustress). For both research and operational purposes, the person, the setting, and time should not be considered independently; rather, it is an amalgam of these variables that provides the proper basis of analysis.

Death from anorexia nervosa: age span and sex differences.

The purpose of this study was to assess characteristics of individuals who died from anorexia nervosa by assessing the frequency with which anorexia nervosa is listed as a causal factor related to the death of individuals in the USA. Data from over 10 million death records (all National Center for Health Statistic registered deaths in the USA for 1986-90) were examined for mention of anorexia nervosa as a primary or contributing cause of death. Only 724 were found, which equals an average of 145 annual deaths, and a rate of 6.73 per 100,000 deaths. The age and sex distribution suggests two fatal forms of anorexia nervosa, an early-onset form comprising 89% women and a later form comprising 24% men. The findings suggest that the mortality risk from current anorexia nervosa may be lower than formerly supposed and that it is not confined to young adults and adolescents.

The case against apoptosis.

Apoptosis is not as dominant a process in cell loss from normal tissues and tumours as has sometimes been claimed. The term 'programmed cell death', which many authors regard as synonymous with apoptosis, is an unsatisfactory term that is best avoided. In studies on the response of tumours to drug and radiation treatment, the use of apoptosis assays concentrates attention on the first decade of cell killing (0-90%), whereas the outcome of treatment depends on multi-log cell kill; an assay of clonogenic cell survival is the appropriate method for this purpose. Loss of colony-forming ability is the key event in treated tumour cells, and the appearance of morphological and molecular evidence of apoptosis is probably downstream from this event. Published studies that have compared apoptosis and cell survival responses in tumour cells have generally failed to find a causal relationship.

Hyperammonemia with reduced ornithine, citrulline, arginine and proline: a new inborn error caused by a mutation in the gene encoding delta(1)-pyrroline-5-carboxylate synthase.

delta(1)-pyrroline-5-carboxylate synthase (P5CS), a bifunctional ATP- and NADPH-dependent mitochondrial enzyme, catalyzes the reduction of glutamate to delta(1)-pyrroline-5-carboxylate, a critical step in the biosynthesis of proline, ornithine and arginine. Recently, we reported the cloning and expression of human and murine P5CS cDNAs. Previously, we showed that mammalian P5CS undergoes alternative splicing to generate two isoforms differing only by a 2 amino acid insert at the N-terminus of the gamma-glutamyl kinase active site. The short isoform has high activity in the gut, where it participates in arginine biosynthesis and is inhibited by ornithine. The long isoform, expressed in multiple tissues, is necessary for the synthesis of proline from glutamate and is insensitive to ornithine. Here, we describe a newly recognized inborn error due to the deficiency of P5CS in two siblings with progressive neurodegeneration, joint laxity, skin hyperelasticity and bilateral subcapsular cataracts. Their metabolic phenotype includes hyperammonemia, hypoornithinemia, hypocitrullinemia, hypoargininemia and hypoprolinemia. Both are homozygous for the missense mutation, R84Q, which alters a conserved residue in the P5CS gamma-glutamyl kinase domain. R84Q is not present in 194 control chromosomes and dramatically reduces the activity of both P5CS isoforms when expressed in mammalian cells. Additionally, R84Q appears to destabilize the long isoform. This is the first documented report of an inborn error of P5CS and suggests that this disorder should be considered in the differential diagnosis in patients with neurodegeneration and/or cataracts and connective tissue disease.

A screen for dominant negative mutants of SEC18 reveals a role for the AAA protein consensus sequence in ATP hydrolysis.

An evolutionarily ancient mechanism is used for intracellular membrane fusion events ranging from endoplasmic reticulum-Golgi traffic in yeast to synaptic vesicle exocytosis in the human brain. At the heart of this mechanism is the core complex of N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). Although these proteins are accepted as key players in vesicular traffic, their molecular mechanisms of action remain unclear. To illuminate important structure-function relationships in NSF, a screen for dominant negative mutants of yeast NSF (Sec18p) was undertaken. This involved random mutagenesis of a GAL1-regulated SEC18 yeast expression plasmid. Several dominant negative alleles were identified on the basis of galactose-inducible growth arrest, of which one, sec18-109, was characterized in detail. The sec18-109 phenotype (abnormal membrane trafficking through the biosynthetic pathway, accumulation of a membranous tubular network, growth suppression, increased cell density) is due to a single A-G substitution in SEC18 resulting in a missense mutation in Sec18p (Thr(394)-->Pro). Thr(394) is conserved in most AAA proteins and indeed forms part of the minimal AAA consensus sequence that serves as a signature of this large protein family. Analysis of recombinant Sec18-109p indicates that the mutation does not prevent hexamerization or interaction with yeast alpha-SNAP (Sec17p), but instead results in undetectable ATPase activity that cannot be stimulated by Sec17p. This suggests a role for the AAA protein consensus sequence in regulating ATP hydrolysis. Furthermore, this approach of screening for dominant negative mutants in yeast can be applied to other conserved proteins so as to highlight important functional domains in their mammalian counterparts.

PEX7 gene structure, alternative transcripts, and evidence for a founder haplotype for the frequent RCDP allele, L292ter.

We recently reported cloning a cDNA encoding Pex7p, the peroxisomal PTS2 receptor. PEX7 mutations cause the peroxisome biogenesis disorder (PBD) rhizomelic chondrodysplasia punctata (RCDP). In a survey of 44 RCDP probands, we found that one PEX7 allele, L292ter, accounted for 50% of mutant PEX7 genes. Here we report the characterization of the PEX7 structural gene, which spans 102 kb on chromosome 6q21-q22.2 and contains at least 10 exons. In addition to the predominant full-length transcript, we identified eight smaller PEX7 transcripts generated by alternative exon splicing in several tissues. However, none of these splice forms was able to restore PTS2 protein import into peroxisomes when expressed in RCDP fibroblasts nor did they inhibit PTS2 protein import when expressed in normal fibroblasts. To determine whether the high frequency of the L292ter allele is due to a founder effect, we identified five polymorphic markers (four diallelic markers and one CA repeat) spanning the PEX7 gene. We show that all 12 L292ter homozygotes in our patient sample have an identical haplotype at these five sites, consistent with the hypothesis that the L292ter mutation arose once on an ancestral chromosome in the Caucasian population.

Correction of ornithine accumulation prevents retinal degeneration in a mouse model of gyrate atrophy of the choroid and retina.

Deficiency of ornithine-delta-aminotransferase (OAT) in humans results in gyrate atrophy of the choroid and retina (GA), an autosomal recessive disorder characterized by ornithine accumulation and a progressive chorioretinal degeneration of unknown pathogenesis. To determine whether chronic, systemic reduction of ornithine can prevent this form of retinal degeneration, we used an arginine-restricted diet to maintain long term reduction of ornithine in a mouse model of OAT-deficiency (Oat(-/-)) produced by gene targeting. We evaluated the mice over a 12-month period by measurement of plasma amino acids, electroretinograms, and retinal histologic and ultrastructural studies. We found that an arginine-restricted diet substantially reduces plasma ornithine levels and completely prevents retinal degeneration in Oat(-/-). This result indicates that ornithine accumulation is a necessary factor in the pathophysiology of the retinal degeneration in GA and that restoration of OAT activity in retina is not required for effective treatment of GA.

Psychological adjustment during three Japanese Antarctic research expeditions.

This study investigates the individual characteristics of Japanese polar team members. Four sets of measures from the Polar Psychology Project Battery have been used in Asuka Station. These measures are thought to reflect adjustment and well-being. Data have been collected during 3 consecutive years. The psychological profile of the subjects was relatively stable, and comparable to the standard means of Western sojourners. The results show the subjects were generally high in stress resistance. However, an increase in the Planning Orientation score and a decrease in the Hardiness score appear at the end of the wintering-over. These modifications correspond to psychological changes linked to the approaching end of wintering-over.

The environmental psychology of capsule habitats.

Capsule habitats make it possible for human beings to survive and function in environments that would otherwise be lethal, such as space, the ocean depths, and the polar regions. The number of people entering capsules in the course of their work or for purposes of recreation is constantly increasing. However, long-term living in such habitats imposes physical and psychological risks as well as offering opportunities and benefits. This paper reviews what is known about the environmental, social, and personality aspects of adaptation to capsules, including sources of stress, selection criteria, obstacles to and facilitators of adequate coping, changes in group interaction, the role of temporal factors, and post-mission consequences.

Biochemical analysis of the Saccharomyces cerevisiae SEC18 gene product: implications for the molecular mechanism of membrane fusion.

The SEC18 gene product is 48% identical to mammalian NSF (N-ethylmaleimide-sensitive fusion protein), and both proteins encode cytoplasmic ATPases which are essential for membrane traffic in yeast and mammalian cells, respectively. A wealth of biochemical analysis has led to the description of a model for the action of NSF; through its interaction with SNAPs (soluble NSF attachment proteins), NSF can associate with SNAP receptors (SNAREs) on intracellular membranes, forming 20S complexes. SNAPs then stimulate the intrinsic ATPase activity of NSF, leading to the disassembly of the 20S complex, which is essential for subsequent membrane fusion. Although this model is based almost entirely on in vitro studies of the original clones of NSF and alpha-SNAP, it is nevertheless widely assumed that this mechanism of membrane fusion is conserved in all eukaryotic cells. If so, the crucial biochemical properties of NSF and SNAPs should be shared by their yeast homologues, Sec18p and Sec17p. Using purified recombinant proteins, we report here that Sec18p can specifically interact not only with Sec17p but also with its mammalian homologue, alpha-SNAP. This interaction leads to a stimulation of Sec18p D1 domain ATPase activity, with kinetics similar to those of alpha-SNAP stimulation of NSF, although differences in temperature and N-ethylmaleimide sensitivity were observed between NSF and Sec18p. Furthermore, Sec18p can interact with synaptic SNARE proteins and can synergize with alpha-SNAP to stimulate regulated exocytosis in mammalian cells. We conclude that the mechanistic properties of NSF and SNAPs are shared by Sec18p and Sec17p, thus demonstrating that the biochemistry of membrane fusion is conserved from yeast to mammals.

Hyperornithinaemia-hyperammonaemia-homocitrullinuria syndrome is caused by mutations in a gene encoding a mitochondrial ornithine transporter.

Neurospora crassa ARG13 and Saccharomyces cerevisiae ARG11 encode mitochondrial carrier family (MCF) proteins that transport ornithine across the mitochondrial inner membrane. We used their sequences to identify EST candidates that partially encode orthologous mammalian transporters. We thereby identified such a gene (ORNT1) that maps to 13q14 and whose expression, similar to that of other urea cycle (UC) components, was high in liver and varied with changes in dietary protein. ORNT1 expression restores ornithine metabolism in fibroblasts from patients with hyperammonaemia-hyperornithinaemia-homocitrullinuria (HHH) syndrome. In a survey of 11 HHH probands, we identified 3 ORNT1 mutant alleles that account for 21 of 22 possible mutant ORNT1 genes in our patients: F188delta, which is common in French-Canadian HHH patients and encodes an unstable protein; E180K, which encodes a stable, properly targeted protein that is inactive; and a 13q14 microdeletion. Our results show that ORNT1 encodes the mitochondrial ornithine transporter involved in UC function and is defective in HHH syndrome.

Differences between a human bladder carcinoma cell line and its radiosensitive clone in the formation of radiation-induced DNA double-strand breaks in different chromatin substrates.

It is well established that DNA-associated proteins, as well as soluble free-radical scavengers, can significantly influence the amount of damage inflicted in DNA by ionising radiation. It is not known, however, to what degree there is variation between cell lines in the effectiveness of these cellular components to protect DNA. In this study we have examined the level of strand break induction in a human bladder carcinoma cell line, MGH-U1, and its radiosensitive mutant, U1-S40b, when soluble scavengers and DNA-associated proteins were progressively removed. DNA double-strand breaks were measured using pulsed-field gel electrophoresis when cells were irradiated after lysis in solutions containing various salt concentrations. The two cell lines showed only a small, non-significant difference in damage induced in intact cells but isolated nuclei and chromatin devoid of non-histone proteins showed significantly more damage in the U1-S40b cells. Once the histone H1 was removed again there was no difference between the cell lines in the damage induced. We conclude that the different components of the cellular defences against free radical attack can have different influences in different cells. It is not clear whether this has an influence on the cellular sensitivity to the killing effects of radiation but it does suggest that artificial manipulation of the different components of the system may not affect overall damage induction to the same degree in all cells.

Comparison between pulsed-field gel electrophoresis and the comet assay as predictive assays for radiosensitivity in fibroblasts.

The radiosensitivity of skin fibroblasts derived from patients as measured in vitro by a clonogenic survival assay appears to correlate with the risk of developing severe late reactions to radiation. Unfortunately, these assays are clinically impractical as a predictive test for radiosensitivity. The purpose of this study was to assess the utility of two possible surrogate assays for radiosensitivity, pulsed-field gel electrophoresis (PFGE) and single-cell gel electrophoresis (comet assay), both of which can be used to measure DNA double-strand breaks. Twenty-three nontransformed human fibroblast cell lines exhibiting a range of radiosensitivities were studied with both of these assays. The results were correlated with measurements of radiosensitivity obtained as part of a larger study examining the correlation between cellular radiosensitivity and clinical response. [2-(14)C]Thymidine-labeled confluent cultures were irradiated at 1.0 Gy/min with doses of 0 to 150 Gy. After allowing 4 h for repair at 37 degrees C, cells were trypsinized and aliquots were used for preparing slides for the comet assay. After neutral lysis and electrophoresis, the slides were stained with ethidium bromide and 50 comet moments were measured for each dose. The remainder of the cells were formed into agarose plugs and, after neutral lysis, were subjected to PFGE. The fraction of activity released (FAR) from the well was measured by scintillation counting of appropriate segments of each gel lane. Cellular radiosensitivity was measured with a standard clonogenic assay at a low dose rate of 1.2 cGy/min, and the dose that resulted in a surviving fraction of 0.01 (D0.01) was calculated. The slope of the plot of comet moment as a function of dose for each cell line did not correlate with D0.01 (R = 0.36, P > 0.1). In contrast, the slope of the FAR as a function of dose had a weak inverse correlation with D0.01 (R = 0.43 and P = 0.05) such that the more radiosensitive cell lines exhibited a steeper dose response for FAR. Although the correlation between the slope of the dose response for FAR and D0.01 was weak, refinement of the PFGE technique may provide a potentially useful predictive assay for radiosensitivity.