DNA crosslinking and biological activity of a hairpin polyamide–chlorambucil conjugate

A prototype of a novel class of DNA alkylating agents, which combines the DNA crosslinking moiety chlorambucil (Chl) with a sequence-selective hairpin pyrrole-imidazole polyamide ImPy-beta-ImPy-gamma-ImPy-beta-Dp (polyamide 1), was evaluated for its ability to damage DNA and induce biological responses. Polyamide 1-Chl conjugate (1-Chl) alkylates and interstrand crosslinks DNA in cell-free systems. The alkylation occurs predominantly at 5'-AGCTGCA-3' sequence, which represents the polyamide binding site. Conjugate-induced lesions were first detected on DNA treated for 1 h with 0.1 muM 1-Chl, indicating that the conjugate is at least 100-fold more potent than Chl. Prolonged incubation allowed for DNA damage detection even at 0.01 muM concentration. Treatment with 1-Chl decreased DNA template activity in simian virus 40 (SV40) in vitro replication assays. 1-Chl inhibited mammalian cell growth, genomic DNA replication and cell cycle progression, and arrested cells in the G(2)/M phase. Moreover, cellular effects were observed at 1-Chl concentrations similar to those needed for DNA damage in cell-free systems. Neither of the parent compounds, unconjugated Chl or polyamide 1, demonstrated any cellular activity in the same concentration range. The conjugate molecule 1-Chl possesses the sequence-selectivity of a polyamide and the enhanced DNA reactivity of Chl

Brain barriers and functional interfaces with sequential appearance of ABC efflux transporters during human development

Abstract Adult brain is protected from entry of drugs and toxins by specific mechanisms such as ABC (ATP-binding Cassette) efflux transporters. Little is known when these appear in human brain during development. Cellular distribution of three main ABC transporters (ABCC1, ABCG2, ABCB1) was determined at blood-brain barriers and interfaces in human embryos and fetuses in first half of gestation. Antibodies against claudin-5 and -11 and antibodies to α-fetoprotein were used to describe morphological and functional aspects of brain barriers. First exchange interfaces to be established, probably at 4–5 weeks post conception, are between brain and embryonic cerebrospinal fluid (eCSF) and between outer surface of brain anlage and primary meninx. They already exclude α-fetoprotein and are immunopositive for both claudins, ABCC1 and ABCG2. ABCB1 is detectable within a week of blood vessels first penetrating into brain parenchyma (6–7 weeks post conception). ABCC1, ABCB1 and ABCG2 are present at blood-CSF barrier in all choroid plexuses from first appearance (7 weeks post conception). Outer CSF-brain interfaces are established between 9–11 weeks post conception exhibiting immunoreactivity for all three transporters. Results provide evidence for sequential establishment of brain exchange interfaces and spatial and temporal timetable for three main ABC transporters in early human brain

Reduced ventricular proliferation in the foetal cortex following maternal inflammation in the mouse

It has been well established that maternal inflammation during pregnancy alters neurological function in the offspring, but its impact on cortical development and long-term consequences on the cytoarchitecture is largely unstudied. Here we report that lipopolysaccharide-induced systemic maternal inflammation in C57Bl/6 mice at embryonic Day 13.5 of pregnancy, as early as 8 h after challenge, caused a significant reduction in cell proliferation in the ventricular zone of the developing cerebral cortex, as revealed by quantification of anti-phospho-Histone H3 immunoreactivity and bromodeoxyuridine pulse labelling. The angle of mitotic cleavage, determined from analysis of haematoxylin and eosin staining, cyclin E1 gene expression and the pattern of β-catenin immunoreactivity were also altered by the challenge, which suggests a change from symmetric to asymmetric division in the radial progenitor cells. Modifications of cortical lamination and gene expression patterns were detected at post-natal Day 8 suggesting prolonged consequences of these alterations during embryonic development. Cellular uptake of proteins from the cerebrospinal fluid was observed in brains from lipopolysaccharide-treated animals in radial progenitor cells. However, the foetal blood–brain barrier to plasma proteins remained intact. Together, these results indicate that maternal inflammation can disrupt the ventricular surface and lead to decreased cellular proliferation. Changes in cell density in Layers IV and V at post-natal Day 8 show that these initial changes have prolonged effects on cortical organization. The possible shift in the fate of progeny and the resulting alterations in the relative cell numbers in the cerebral cortex following a maternal inflammatory response shown here will require further investigation to determine the long-term consequences of inflammation on the development of neuronal circuitry and behaviour

The nucleotide and partial amino acid sequences of rat fetuin

Fetuins are among the major plasma proteins, yet their biological role has remained elusive. Here we report the molecular cloning of rat fetuin and the sequence analysis of a full-length clone, RF619 of 1456 bp with an open reading frame of 1056 bp encoding 352 amino acid residues. The coding part of RF619 was identical with the cDNA sequence of the natural inhibitor of the insulin receptor tyrosine kinase from rat (pp63) except for four substitutions and a single base insertion causing divergence of the predicted protein sequences. Partial amino acid sequences of rat plasma fetuin were in agreement with the predictions based on the RF619 cDNA. Purified rat fetuin inhibited the insulin receptor tyrosine kinase in vitro. Therefore, we conclude that RF619 and pp63 cDNA encode the same protein, i.e. authentic rat fetuin which is a functional tyrosine kinase inhibitor

Prognostic relevance of a T-type calcium channels gene signature in solid tumours: A correlation ready for clinical validation

BackgroundT-type calcium channels (TTCCs) mediate calcium influx across the cell membrane. TTCCs regulate numerous physiological processes including cardiac pacemaking and neuronal activity. In addition, they have been implicated in the proliferation, migration and differentiation of tumour tissues. Although the signalling events downstream of TTCC-mediated calcium influx are not fully elucidated, it is clear that variations in the expression of TTCCs promote tumour formation and hinder response to treatment.MethodsWe examined the expression of TTCC genes (all three subtypes; CACNA-1G, CACNA-1H and CACNA-1I) and their prognostic value in three major solid tumours (i.e. gastric, lung and ovarian cancers) via a publicly accessible database.ResultsIn gastric cancer, expression of all the CACNA genes was associated with overall survival (OS) among stage I-IV patients (all pConclusionsAlterations in CACNA gene expression are linked to tumour prognosis. Gastric cancer represents the most promising setting for further evaluation

Heated indoor swimming pools, infants, and the pathogenesis of adolescent idiopathic scoliosis: a neurogenic hypothesis

<p>Abstract</p> <p>Background</p> <p>In a case-control study a statistically significant association was recorded between the introduction of infants to heated indoor swimming pools and the development of adolescent idiopathic scoliosis (AIS). In this paper, a neurogenic hypothesis is formulated to explain how toxins produced by chlorine in such pools may act deleteriously on the infant's immature central nervous system, comprising brain and spinal cord, to produce the deformity of AIS.</p> <p>Presentation of the hypothesis</p> <p>Through vulnerability of the developing central nervous system to circulating toxins, and because of delayed epigenetic effects, the trunk deformity of AIS does not become evident until adolescence. In mature healthy swimmers using such pools, the circulating neurotoxins detected are chloroform, bromodichloromethane, dibromochloromethane, and bromoform. Cyanogen chloride and dichloroacetonitrile have also been detected.</p> <p>Testing the hypothesis</p> <p>In infants, the putative portals of entry to the blood could be dermal, oral, or respiratory; and entry of such circulating small molecules to the brain are via the blood-brain barrier, blood-cerebrospinal fluid barrier, and circumventricular organs. Barrier mechanisms of the developing brain differ from those of adult brain and have been linked to brain development. During the first 6 months of life cerebrospinal fluid contains higher concentrations of specific proteins relative to plasma, attributed to mechanisms continued from fetal brain development rather than immaturity.</p> <p>Implications of the hypothesis</p> <p>The hypothesis can be tested. If confirmed, there is potential to prevent some children from developing AIS.</p

A Hepatic Protein, Fetuin-A, Occupies a Protective Role in Lethal Systemic Inflammation

A liver-derived protein, fetuin-A, was first purified from calf fetal serum in 1944, but its potential role in lethal systemic inflammation was previously unknown. This study aims to delineate the molecular mechanisms underlying the regulation of hepatic fetuin-A expression during lethal systemic inflammation (LSI), and investigated whether alterations of fetuin-A levels affect animal survival, and influence systemic accumulation of a late mediator, HMGB1.LSI was induced by endotoxemia or cecal ligation and puncture (CLP) in fetuin-A knock-out or wild-type mice, and animal survival rates were compared. Murine peritoneal macrophages were challenged with exogenous (endotoxin) or endogenous (IFN-γ) stimuli in the absence or presence of fetuin-A, and HMGB1 expression and release was assessed. Circulating fetuin-A levels were decreased in a time-dependent manner, starting between 26 h, reaching a nadir around 24-48 h, and returning towards base-line approximately 72 h post onset of endotoxemia or sepsis. These dynamic changes were mirrored by an early cytokine IFN-γ-mediated inhibition (up to 50-70%) of hepatic fetuin-A expression. Disruption of fetuin-A expression rendered animals more susceptible to LSI, whereas supplementation of fetuin-A (20-100 mg/kg) dose-dependently increased animal survival rates. The protection was associated with a significant reduction in systemic HMGB1 accumulation in vivo, and parallel inhibition of IFN-γ- or LPS-induced HMGB1 release in vitro.These experimental data suggest that fetuin-A is protective against lethal systemic inflammation partly by inhibiting active HMGB1 release

Building more effective partnerships for innovation in urban water management 1

Abstract This paper discusses experiences within the Sustainable Water Improves Tomorrow&apos;s Cities&apos; Health (SWITCH) consortium -a research partnership focused on long-term improvements in urban water management in developed and developing countries -to apply innovative research methodologies that may lead to more effective urban water science and wider and more integrated use of research findings. It introduces learning alliances as an attempt to build multi-stakeholder partnerships for demand-led research and the scaling-up of research impacts, and several related tools used to date to underpin an action research process: visioning and scenario-based planning with stakeholders, scoring ladders to monitor outcomes, process documentation to record change and matrix management to guide a diverse consortium. Examples drawn from the SWITCH project illustrate successes and failures from which the project aims to learn and improve its own effectiveness

Building more effective partnerships for innovation in urban water management 1

Abstract This paper discusses experiences within the Sustainable Water Improves Tomorrow&apos;s Cities&apos; Health (SWITCH) consortium -a research partnership focused on long-term improvements in urban water management in developed and developing countries -to apply innovative research methodologies that may lead to more effective urban water science and wider and more integrated use of research findings. It introduces learning alliances as an attempt to build multi-stakeholder partnerships for demand-led research and the scaling-up of research impacts, and several related tools used to date to underpin an action research process: visioning and scenario-based planning with stakeholders, scoring ladders to monitor outcomes, process documentation to record change and matrix management to guide a diverse consortium. Examples drawn from the SWITCH project illustrate successes and failures from which the project aims to learn and improve its own effectiveness