Expression patterns of anterior Hox genes in the polychaete Chaetopterus: correlation with morphological boundaries

Expression patterns for five Hox genes were examined by whole-mount in situ hybridization in larvae of Chaetopterus, a polychaete annelid with a tagmatized axial body plan. Phylogenetic analysis demonstrates that these genes are orthologs of the Drosophila genes labial, proboscipedia, zen, Deformed, and Sex combs reduced and are termed CH-Hox1, CH-Hox2, CH-Hox3, CH-Hox4, and CH-Hox5, respectively. Expression studies reveal a biphasic expression pattern. In early larval stages, well before any indications of segmental organization exist, a novel pattern of expression in bilateral posterior proliferating cell populations, corresponding to the teloblasts, was detected for each of the genes, with CH-Hox1 and CH-Hox2 expressed before the remaining three. In middle larval stages, all five genes are expressed in bilateral strips along the ventral midline, corresponding with the developing ventral nerve cord. In addition, CH-Hox1 and CH-Hox2 show strong expression at the foregut-midgut boundary. By late larval stages the expression is generally confined to the ventral CNS and ectoderm of the anterior parapodia. Anterior boundaries of expression are "colinear," at later larval stages, with CH-Hox2 expressed most rostrally, in the first segment, and anterior expression boundaries for CH-Hox1, CH-Hox3, CH-Hox4, and CH-Hox5 in segments 2, 3, 4, and 5, respectively. Like vertebrates and spiders, but unlike insects, CH-Hox3 participates in this colinear axial expression pattern. CH-Hox1 and CH-Hox2 have distinct posterior boundaries of expression in the ninth segment, which corresponds to a major morphological boundary, and may reflect a reorganization of Hox gene regulation related to the evolutionary reorganization of the Chaetopterus body plan.     

Roles for scalloped and vestigial in regulating cell affinity and interactions between the wing blade and the wing hinge

The scalloped and vestigial genes are both required for the formation of the Drosophila wing, and recent studies have indicated that they can function as a heterodimeric complex to regulate the expression of downstream target genes. We have analyzed the consequences of complete loss of scalloped function, ectopic expression of scalloped, and ectopic expression of vestigial on the development of the Drosophila wing imaginal disc. Clones of cells mutant for a strong allele of scalloped fail to proliferate within the wing pouch, but grow normally in the wing hinge and notum. Cells overexpressing scalloped fail to proliferate in both notal and wing-blade regions of the disc, and this overexpression induces apoptotic cell death. Clones of cells overexpressing vestigial grow smaller or larger than control clones, depending upon their distance from the dorsal-ventral compartment boundary. These studies highlight the importance of correct scalloped and vestigial expression levels to normal wing development. Our studies of vestigial-overexpressing clones also reveal two further aspects of wing development. First, in the hinge region vestigial exerts both a local inhibition and a long-range induction of wingless expression. These and other observations imply that vestigial-expressing cells in the wing blade organize the development of surrounding wing-hinge cells. Second, clones of cells overexpressing vestigial exhibit altered cell affinities. Our analysis of these clones, together with studies of scalloped mutant clones, implies that scalloped- and vestigial-dependent cell adhesion contributes to separation of the wing blade from the wing hinge and to a gradient of cell affinities along the dorsal-ventral axis of the wing.     

Anthracenone ABA analogue as a potential photoaffinity reagent for ABA-binding proteins

An anthracenone analogue of abscisic acid (ABA) was synthesized as a potential photoaffinity reagent and tested for biological activity. Reaction between 10,10'-dimethoxy-9-anthrone with two equivalents of the lithiated dianion of cis-3-methylpent-2-en-4-yn-1-ol afforded an acetylenic alcohol key intermediate. Subsequent reduction of the triple bond, functional group manipulation of the side chain alcohol and deprotection of the dimethoxy protected anthrone provided anthracenone ABA analogue 7 as a potential photoaffinity reagent for ABA-binding proteins. The effect of natural ABA and the potential photoaffinity anthracenone ABA 7 on corn cell growth was determined at various concentrations. The results show that anthracenone ABA 7 is perceived as ABA-like, although producing less inhibition than ABA itself. For example, 7 at 33 microM produces approximately the same inhibition as ABA at 10 microM.     

Phylogenetic utility of the nuclear gene arginine decarboxylase: an example from Brassicaceae

Arginine decarboxylase (ADC) is an important enzyme in the production of putrescine and polyamines in plants. It is encoded by a single or low-copy nuclear gene that lacks introns in sequences studied to date. The rate of Adc amino acid sequence evolution is similar to that of ndhF for the angiosperm family studied. Highly conserved regions provide several target sites for PCR priming and sequencing and aid in nucleotide and amino acid sequence alignment across a range of taxonomic levels, while a variable region provides an increased number of potentially informative characters relative to ndhF for the taxa surveyed. The utility of the Adc gene in plant molecular systematic studies is demonstrated by analysis of its partial nucleotide sequences obtained from 13 representatives of Brassicaceae and 3 outgroup taxa, 2 from the mustard oil clade (order Capparales) and 1 from the related order Malvales. Two copies of the Adc gene, Adc1 and Adc2, are found in all members of the Brassicaceae studied to data except the basal genus Aethionema. The resulting Adc gene tree provides robust phylogenetic data regarding relationships within the complex mustard family, as well as independent support for proposed tribal realignments based on other molecular data sets such as those from chloroplast DNA.      

Drug Repurposing: A Systematic Approach to Evaluate Candidate Oral Neuroprotective Interventions for Secondary Progressive Multiple Sclerosis

Objective

To develop and implement an evidence based framework to select, from drugs already licenced, candidate oral neuroprotective drugs to be tested in secondary progressive multiple sclerosis.

Design

Systematic review of clinical studies of oral putative neuroprotective therapies in MS and four other neurodegenerative diseases with shared pathological features, followed by systematic review and meta-analyses of the in vivo experimental data for those interventions. We presented summary data to an international multi-disciplinary committee, which assessed each drug in turn using pre-specified criteria including consideration of mechanism of action.

Results

We identified a short list of fifty-two candidate interventions. After review of all clinical and pre-clinical evidence we identified ibudilast, riluzole, amiloride, pirfenidone, fluoxetine, oxcarbazepine, and the polyunsaturated fatty-acid class (Linoleic Acid, Lipoic acid; Omega-3 fatty acid, Max EPA oil) as lead candidates for clinical evaluation.

Conclusions

We demonstrate a standardised and systematic approach to candidate identification for drug rescue and repurposing trials that can be applied widely to neurodegenerative disorders.     

There is no ‘Conundrum’ of InsP6

Indirect assays have claimed to quantify phytate (InsP₆) levels in human biofluids, but these have been based on the initial assumption that InsP₆ is there, an assumption that our more direct assays disprove. We have shown that InsP₆ does not and cannot (because of the presence of an active InsP₆ phosphatase in serum) exist in mammalian serum or urine. Therefore, any physiological effects of dietary InsP₆ can only be due either to its actions in the gut as a polyvalent cation chelator, or to inositol generated by its dephosphorylation by gut microflora.We are grateful to Dr Vucenik for bringing up a number of interesting points.It is true that we have not quantified the dietary intakes of our human donors any more (but also hardly any less) than has been done by those groups claiming that InsP₆ is present in bodily fluids. As a qualitative observation we should point out that in fact all our donors for ref. [1] do have a regular intake of dietary cereals and indeed, one is a strict vegetarian on a high cereal diet. But it is quantification that reveals this to be a specious issue. The limits of detection in our two relevant publications [1,2] for InsP₆ in plasma and urine were, respectively, around two and three orders of magnitude lower than the levels claimed to be present by Grases et al. [3] in the fluids of experimentally phytate-deprived human subjects. These numbers make the argument that we could not detect any InsP₆ simply because we chose donors on the ‘wrong’ diet untenable.So how have those many claims that InsP₆ is present in body fluids come about? For most of them, the simple answer appears to be that the assays used are indirect and are based entirely on the assumption that InsP₆ is present in the first place. Thus, for example, Valiente and co-workers [4,5] and Chen and co-workers [6,7] measured organic phosphate remaining after a series of fractionations of urine samples and simply assumed it was due to InsP₆, as did March et al. measuring inorganic phosphate after a similar protocol [8]. Grases co-workers [9] have used extensively a less indirect assay, which, after initial ion chromatography and dephosphorylation by a phytase, measures myo-inositol by mass spectrometry, but nevertheless the assay starts with the assumption that InsP₆ is there and that this is what they are quantifying. More recently, direct quantification of InsP₆ in plasma by mass spectrometry has been claimed [10] on the basis that there are peaks in plasma at m/z 624 running near where InsP₆ standards elute in two different HPLC separations [10,11]. But no evidence is presented to show even that these peaks are the same compound, let alone any data to establish firmly that InsP₆ is present, e.g. a minimal requirement of m/z quantified to two decimal places with allowance for C₁₃ content or a full disintegration fingerprint (see also [12]). Any quantified misidentification is likely to have a stochastic element to it, and it is noteworthy that Perelló & Grases have stated [11, p. 255]: ‘…we have found some humans and rats having undetectable [InsP₆], probably depending on their diet or other unknown factors’. In the light of the preceding discussion, we can offer a simpler explanation: the InsP₆ was never there in the first place.In contrast to these claims we have, using two entirely independent specific and sensitive assays with quantified spiking recovery, unambiguously shown that InsP₆ is not present in plasma or urine. This is crucial and central to the whole debate about the actions of dietary InsP₆, because it means that InsP₆ never enters the blood. It is only absorbed after being dephosphorylated, principally to inositol (see [1,2] for further discussion). Ironically, the most direct evidence for this lies in Dr Vucenik''s own data in experiments examining the fate of radioactive InsP₆ fed to animals, in which only inositol was detected in the blood [13]. This particular study was, as Dr Vucenik points out in her letter, conducted on mice. However, exactly the same conclusion (i.e. InsP₆ does not enter the circulation from the gut) is equally clear in her earlier study [14], which she did not cite and which was indeed on rats; does this omission ‘reflect poorly’ on Dr Vucenik''s own ‘report and the author''s credibility in culling scientific data’?In short, dietary InsP₆ can have only two fates: it can stay in the gut, ultimately to be defecated [15], and while it is there it can chelate metal ions to alter their uptake from the gut into the body. This is no ‘straw-man’ and is certainly the most likely explanation for all of the effects of InsP₆ on cultured cells, which comprise the majority of the reports cited by Dr Vucenik. Alternatively, InsP₆ can be converted to inositol (principally by the gut microflora [15]) and be taken up as such into the circulation; were any InsP₆ to get into the blood it would in any case be rapidly dephosphorylated by the phosphatase activity we have shown to be present in human plasma [1].For animal studies, we have raised the possibility [1,2] that it is the inositol so generated (Vitamin Bh, harmless as far as we know) that is the active mediator of any reported beneficial effects of dietary InsP₆. We note that most of the websites touting InsP₆ as a dietary supplement advocate inositol as an important (essential?) co-supplement; that the only human cancer study highlighted as important by Dr Vucenik that we could examine [16] did not administer InsP₆ alone, but only in conjunction with inositol; and that in the few studies where the separate contributions of inositol and InsP₆ have been considered, there are data suggesting that it may be the inositol that matters (e.g. fig. 1 of [17]). Moreover, we are not the only ones to suggest this idea. In the Discussion of their paper (on mice) in which InsP₆ was shown not to enter the blood from the gut [13], Dr Vucenik and her colleagues state: ‘Inositol may be responsible for the antitumor actions observed in both chemopreventitive and efficacy studies of IP₆ … A question remains as to whether the activity of IP₆ in animal models can be replicated by administration of inositol alone because only inositol was detected in plasma and tumor after oral gavage’. Precisely.Finally, returning to InsP₆ itself, which, incidentally, is officially classified by the FDA as a ‘fake’ cancer cure (http://www.fda.gov/drugs/guidancecomplianceregulatoryinformation/enforcementactivitiesbyfda/ucm171057.htm), our data lead inevitably to the conclusion that while InsP₆ might impact on the gut environment and thus indirectly on its microflora [2,12], its only plausible direct action on the body will be to inhibit cation uptake from the diet. Although InsP₆ binds trivalent cations with a higher affinity than divalents [18], it is nevertheless comparatively non-specific in this action. Administering chemicals to the diet to manipulate ion uptake is not unknown in modern medicine; for treatment of iron disorders such as haemochromatosis, as an alternative to injection of Desferral, oral administration of the closely related chelator Deferasirox is now sometimes recommended [19]. But Deferasirox is a highly iron-specific chelator, administered under close medical supervision for a directly iron-related pathology. Recommending unmonitored, widespread administration of InsP₆ to address a veritable multitude of different pathologies [20] seems to us to be an entirely different matter.In a well-fed human, where the cation to InsP₆ ratio in the diet is high, InsP₆ may very well do no harm (it is, after all, a natural component of our diet) and there is much evidence to support this idea, as argued by Dr Vucenik. But if InsP₆ is not impacting on cation uptake from the diet to do any harm it is difficult to understand how at exactly the same time it can impact on the same uptake to do good. (See reference [21] for the studies Dr Vucenik requested ‘unequivocally demonstrating the toxicity of pure Ca-Mg-InsP₆ as it occurs naturally’ in humans with low dietary cation uptake.) In the light of the above discussion and our rigorous data, we stand unreservedly by our original closing statement [1]: ‘…that chronically altering cation absorption from the gut by artificially loading the diet with a non-specific chelator … in the hope that it might impact indirectly on cancer or other pathologies seems highly inadvisable’.     

USE OF MIDAZOLAM/PETHIDINE AND TILETAMINE/ZOLAZEPAM COMBINATIONS FOR THE CHEMICAL RESTRAINT OF LEOPARD SEALS (HYDRURGA LEPTONYX)

We immobilized 200–550-kg leopard seals ( Hydrurga leptonyx ) on sea ice in Prydz Bay, Antarctica (68°25'S, 77°10'E) between November 1997 and February 2000. Midazolam (0.18–0.27 mg/kg)/ pethidine (1.0–1.5 mg/kg) was administered by dart to 16 leopard seals. Unpredictable immobilization, poor airway maintenance, and our inability to fully assess the suitability of flumazenil (0.003–0.01 mg/kg), naloxone (0.01–0.013 mg/kg), and naltrexone (0.05–0.12 mg/kg) as reversal agents limited suitability of midazolam/pethidine. Tiletamine/zolazepam 1:1 (0.5–1.5 mg/kg) was, therefore, administered to 19 leopard seals. It produced faster induction (19 ± 3 min), more effective and reliable response to dose (rank correlation: r _s= 0.88, n = 18), and better pulmonary ventilation and faster return of cognitive function upon recovery, in comparison to midazolam/pethidine. Best results were achieved with tiletamine/zolazepam (1.2–1.4 mg/kg) which safely immobilized seven of nine seals for 20–30 min. Entry to the water upon darting was minimized, but not eliminated, by the use of lightweight air-pressurized darts and a thorough knowledge of leopard seal behavior.