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’.     

Interleukin-6 Serum Levels in Patients with Parkinson’s Disease

Several lines of evidence suggest that neuroimmune mechanisms may be involved in the neurodegenerative process of Parkinson’s disease (PD). Interleukin-6 (IL-6) is increased in the nigrostriatal region and in the cerebrospinal fluid of patients with PD. IL-6 serum level was evaluated in PD patients. The effects of levodopa treatment and disease severity on IL-6 were also studied. The IL-6 levels were similar between PD patients (treated and not treated) and controls. However, there was a negative correlation of IL-6 levels and the activities of daily living scale (P < 0.05), indicating that patients with more severe disease have higher levels of this cytokine. No correlation involving levodopa treatment and IL-6 serum level was found. The results suggest that only marginal effects of IL-6 occur on the peripheral immune system, and that the role of IL-6 and others neuroimmune factors needs to be well elucidated on PD.     

Antimicrobial efficacy of some N,N’-Dialkyl-N,N’-dimethyl-1, 6-hexanediamine dioxides

A total of 17 N,N'-dialkyl-N,N'-dimethyl-1,6-hexanediamine dioxides were tested for activity against three microorganisms. A relationship was found between the length of the alkyl substituent and antimicrobial activity.     

Molecular Profiling of a 6-Hydroxydopamine Model of Parkinson’s Disease

Convection enhanced delivery of 6-hydroxydopamine (6-OHDA) to the rat striatum results in a model of Parkinson’s disease. An important feature of this unilateral model is the progressive loss of dopaminergic (DA) neurons over the course of several weeks. To improve the understanding of this model, gene expression changes in the substantia nigra, which contains the DA neuron cell bodies, and the striatum, which contains the DA neuron synaptic terminals, were examined using DNA microarrays. Samples were collected and behavior was analyzed from vehicle and toxin treated animals at 3 days, 1 week, 2 weeks and 4 weeks following 6-OHDA treatment. Tissue DA content was determined and samples from animals which exhibited a substantial depletion of striatal DA were included in the subsequent gene expression analysis. The results of the gene expression analysis indicated that 6-OHDA elicits a vigorous inflammatory response, comprised of several distinct pathways, in the striatum at the earliest time point tested. In contrast, relatively few gene expression changes were observed in the SN at the 3-day time point. In both tissues examined there was evidence for a vigorous inflammatory response at the 1- and 2-week time points, which was substantially diminished by the 4-week time point. Inflammation plays a prominent role in the 6-OHDA model of Parkinson’s disease.     

2,4-Diamino-6-methylpyrimidines for the potential treatment of Chagas’ disease

Chagas’ disease, caused by the protozoan parasite Trypanosoma cruzi, affects 8–10 million people across the Latin American population and is responsible for around 12,500 deaths per annum. The current frontline treatments, benznidazole and nifurtimox, are associated with side effects and lack efficacy in the chronic stage of the disease, leading to an urgent need for new treatments. A high throughput screening campaign against the physiologically relevant intracellular form of the parasite identified a series of 2,4-diamino-6-methylpyrimidines. Demonstrating the series did not work through the anti-target TcCYP51, and was generally cytocidal, confirmed its suitability for further development. This study reports the optimisation of selectivity and metabolic stability of the series and identification of a suitable lead for further optimisation.     

L’intégrine α6β1 ovocytaire et spermatique dans l’interaction gamétique

Based on inhibition tests, the α6β1 integrin was suggested to be a sperm receptor, but further experiments using gene deletion techniques have shown that neither oocyte α6, nor β1 integrin subunits were essential for mouse fertilization. Using Western blot analysis and immunofluorescence (flow cytometry and microscopy), we have shown that mouse sperm expresses the α6β1 integrin. As for oocytes, binding of GoH3 anti-alpha6 antibody to sperm induces specific inhibition of sperm fertilizing ability. Comparing zona-intact and zona-free eggs in fusion tests, we have shown that removal of the zona pellucida bypasses the α6β1 integrin role in the adhesion/fusion process of oocyte fertilization. The α6β1 integrin is expressed by both gametes and is functional during their membrane interactions. Our results, previous reports on fertilization of α6 or β1 integrin subunit-deleted oocytes by wild-type sperm and the fusion ability of β1 mutant myoblasts when they were co-cultured with wild-type myoblasts suggest that the presence of α6βl integrin on one of the two gamete membranes can rescue the fertilization process. This hypothesis is further supported by the recently reported exchange of membrane fragments occurring between gametes prior to fusion.     

Synthesis of biflavones having a 6-O-7’’ linkage and effects on cyclooxygenase-2 and inducible nitric oxide synthase

In order to establish anti-inflammatory potential of biflavonoids, 17 biflavone derivatives having a 6-O-7″ linkage were synthesized and their effects on cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were evaluated. The basic molecule (6-O-7″ biflavone) potently inhibited COX-2-mediated PGE₂ production (IC₅₀: < 2 μM), being less active on iNOS-mediated NO production (IC₅₀: > 50 μM) from lipopolysaccharide-treated RAW 264.7 cells, a mouse macrophage cell line. Generally, the hydroxyl/methoxyl substitution(s) on the basic biflavone (6-O-7″) reduced the inhibitory activity of PGE₂ production, while the effects on NO production were varied. It is suggested that the basic biflavone (6-O-7″) may have a potential for new anti-inflammatory agent.     

A reciprocal translocation between ’Garfi’ almond and ’Nemared’ peach

A map with 51 markers (46 RFLPs and five isozymes) was constructed using an interspecific F₂ population between ’Garfi’ almond (Prunus amygdalus Batsch.) and ’Nemared’ peach [Prunus persica (L.) Batsch.]. This map was developed by selecting markers covering most of the distance of the eight linkage groups from previously constructed Prunus maps. The markers studied in this population mapped to seven linkage groups instead of the eight expected in Prunus. Markers belonging to groups 6 and 8 in previous maps formed a single group in the ’Garfi’×’Nemared’ F₂ and several marker pairs placed in different groups in other maps exhibited tight linkages. The study of pollen fertility and chromosome behavior during meiosis in the F₁ generation allowed us to confirm the hypothesis that a reciprocal translocation exists between ’Garfi’ and ’Nemared’. Based on independent evidence of linkage between markers and pollen fertility data in the F₂ population, we concluded that the breakpoint of the reciprocal translocation was placed between markers AC50 and AG26A in group 6 and between markers AG112A and FG230A in group 8. Received: 28 June 2000 / Accepted: 17 October 2000     

Analysis of 6 VNTR loci by ‘multiplex’ PCR and automated fluorescent detection

The polymerase chain reaction was used to amplify six small variable number of tandem repeat loci in two reactions (D19S20 co-amplifying with D17S5 and D1S80; D17S766 co-amplifying with D16S83 and D17S24). When coupled with fluorescent detection of the products, this provides a rapid, highly discriminating automated test. Preferential amplification of small alleles, leading to allelic dropout was found to occur in D19S20 and D16S83. Population databases are presented for Caucasians and Afro-Caribbeans at loci D19S20, D16S83 and D17S24, and for Asians at D19S20.     

Defense Responses of Cherry Rootstock ‘Gisela 6’ Elicited by Agrobacterium tumefaciens Infection

Journal of Plant Growth Regulation - Agrobacterium tumefaciens causes crown gall disease in plants by transferring a portion of the tumor-inducing plasmid, transfer DNA, into the plant genome. To...     

Probucol Affords Neuroprotection in a 6-OHDA Mouse Model of Parkinson’s Disease

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the degeneration of dopaminergic nigrostriatal neurons. Although the etiology of the majority of human PD cases is unknown, experimental evidence points to oxidative stress as an early and causal event. Probucol is a lipid-lowering phenolic compound with anti-inflammatory and antioxidant properties that has been recently reported as protective in neurotoxicity and neurodegeneration models. This study was designed to investigate the effects of probucol on the vulnerability of striatal dopaminergic neurons to oxidative stress in a PD in vivo model. Swiss mice were treated with probucol during 21 days (11.8 mg/kg; oral route). Two weeks after the beginning of treatment, mice received a single intracerebroventricular (i.c.v.) infusion of 6-hydroxydopamine (6-OHDA). On the 21st day, locomotor performance, striatal oxidative stress-related parameters, and striatal tyrosine hydroxylase and synaptophysin levels, were measured as outcomes of toxicity. 6-OHDA-infused mice showed hyperlocomotion and a significant decrease in striatal tyrosine hydroxylase (TH) and synaptophysin levels. In addition, 6-OHDA-infused mice showed reduced superoxide dismutase activity and increased lipid peroxidation and catalase activity in the striatum. Notably, probucol protected against 6-OHDA-induced hyperlocomotion and striatal lipid peroxidation, catalase upregulation and decrease of TH levels. Overall, the present results show that probucol protects against 6-OHDA-induced toxicity in mice. These findings may render probucol as a promising molecule for further pharmacological studies on the search for disease-modifying treatment in PD.     

Genetic Analysis of ‘PAX6-Negative’ Individuals with Aniridia or Gillespie Syndrome

We report molecular genetic analysis of 42 affected individuals referred with a diagnosis of aniridia who previously screened as negative for intragenic PAX6 mutations. Of these 42, the diagnoses were 31 individuals with aniridia and 11 individuals referred with a diagnosis of Gillespie syndrome (iris hypoplasia, ataxia and mild to moderate developmental delay). Array-based comparative genomic hybridization identified six whole gene deletions: four encompassing PAX6 and two encompassing FOXC1. Six deletions with plausible cis-regulatory effects were identified: five that were 3ʹ (telomeric) to PAX6 and one within a gene desert 5ʹ (telomeric) to PITX2. Sequence analysis of the FOXC1 and PITX2 coding regions identified two plausibly pathogenic de novo FOXC1 missense mutations (p.Pro79Thr and p.Leu101Pro). No intragenic mutations were detected in PITX2. FISH mapping in an individual with Gillespie-like syndrome with an apparently balanced X;11 reciprocal translocation revealed disruption of a gene at each breakpoint: ARHGAP6 on the X chromosome and PHF21A on chromosome 11. In the other individuals with Gillespie syndrome no mutations were identified in either of these genes, or in HCCS which lies close to the Xp breakpoint. Disruption of PHF21A has previously been implicated in the causation of intellectual disability (but not aniridia). Plausibly causative mutations were identified in 15 out of 42 individuals (12/32 aniridia; 3/11 Gillespie syndrome). Fourteen of these mutations presented in the known aniridia genes; PAX6, FOXC1 and PITX2. The large number of individuals in the cohort with no mutation identified suggests greater locus heterogeneity may exist in both isolated and syndromic aniridia than was previously appreciated.     

Classic toxin-induced animal models of Parkinson’s disease: 6-OHDA and MPTP

Neurological disorders in humans can be modeled in animals using standardized procedures that recreate specific pathogenic events and their behavioral outcomes. The development of animal models of Parkinsons disease (PD) is important to test new neuroprotective agents and strategies. Such animal models of PD have to mimic, at least partially, a Parkinson-like pathology and should reproduce specific features of the human disease. PD is characterized by massive degeneration of dopaminergic neurons in the substantia nigra, the loss of striatal dopaminergic fibers and a dramatic reduction of the striatal dopamine levels. The formation of cytoplasmic inclusion bodies (Lewy bodies) in surviving dopaminergic neurons represents the most important neuropathological feature of PD. Furthermore, the massive striatal dopamine deficiency causes easily detectable motor deficits in PD patients, including bradykinesia, rigidity, and resting tremor, which are the cardinal symptoms of PD. Over the years, a broad variety of experimental models of PD were developed and applied in diverse species. This review focuses on the two most common classical toxin-induced PD models, the 6-hydroxy-dopamine (6-OHDA model) and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model. Both neurotoxins selectively and rapidly destroy catecholaminergic neurons, whereas in humans the PD pathogenesis follows a progressive course over decades. This discrepancy reflects one important and principal point of weakness related to most animal models. This review discusses the most important properties of 6-OHDA and MPTP, their modes of administration, and critically examines advantages and limitations of selected animal models. The new genetic and environmental toxin models of PD (e.g. rotenone, paraquat, maneb) are discussed elsewhere in this special issue.This work was supported by grants from the Deutsche Forschungsgemeinschaft.     

Neuroprotective Effects of Paeoniflorin on 6-OHDA-Lesioned Rat Model of Parkinson’s Disease

Paeoniflorin (PF) is the main active component extracted from the roots of Paeonialactiflora, a traditional Chinese medicine used for the treatment of neurodegenerative disorders, especially Parkinson’s disease (PD). The degeneration of dopaminergic (DA-) neurons in PD may be caused by pathological activation of acid-sensing ion channels (ASICs). Thus, we designed a series of experiments to evaluate the therapeutic effects of PF and to test whether its effects are related to its inhibitory effect on ASIC1a. We found that systemic administration of PF or ASICs blockers (psalmotoxin-1 and amiloride) improved behavioral symptoms, delayed DA-neuronal loss and attenuated the reduction of dopamine (DA) and its metabolites in a rat model of 6-hydroxydopamine (6-OHDA)-induced PD. In addition, our data showed that PF, like ASICs blockers, regulated the expression of ASIC1a, decreased the level of α-synuclein (α-SYN), and improved autophagic dysfunction. Further experiments showed that ASIC1a knockdown down-regulated the α-SYN level and alleviated the autophagic injury in the 6-OHDA-treated ASIC1a-silenced PC12 cells. In summary, these findings indicate that PF enhanced the autophagic degradation of α-SYN and, thus, protected DA-neurons against the neurotoxicity caused by 6-OHDA. These findings also provide experimental evidence that PF may be a neuroprotectant for PD by acting on ASIC1a and that ASIC1a may be involved in the pathogenesis of PD.     

Chromatoid Body Protein TDRD6 Supports Long 3’ UTR Triggered Nonsense Mediated mRNA Decay

Chromatoid bodies (CBs) are spermiogenesis-specific organelles of largely unknown function. CBs harbor various RNA species, RNA-associated proteins and proteins of the tudor domain family like TDRD6, which is required for a proper CB architecture. Proteome analysis of purified CBs revealed components of the nonsense-mediated mRNA decay (NMD) machinery including UPF1. TDRD6 is essential for UPF1 localization to CBs, for UPF1-UPF2 and UPF1-MVH interactions. Upon removal of TDRD6, the association of several mRNAs with UPF1 and UPF2 is disturbed, and the long 3’ UTR-stimulated but not the downstream exon-exon junction triggered pathway of NMD is impaired. Reduced association of the long 3’ UTR mRNAs with UPF1 and UPF2 correlates with increased stability and enhanced translational activity. Thus, we identified TDRD6 within CBs as required for mRNA degradation, specifically the extended 3’ UTR-triggered NMD pathway, and provide evidence for the requirement of NMD in spermiogenesis. This function depends on TDRD6-promoted assembly of mRNA and decay enzymes in CBs.     

不同细胞对ECHO 6 D’Amorl毒株的血凝能力改变的影响

(一)ECHO 6 D’Amori毒株10-0及10-3稀释的病毒在原代人腎细胞上传至第四代就出现血凝,即可以使无血凝能力的毒株变为有血凝能力的毒株。用10-5,及终稀释度的病毒传代,刖不能产生血凝。 (二)D’Amori毒株无论是10-0、10-3或10-5,在原代人羊膜细胞上传代均无血凝出现,但已获得血疑能力的D’Amori毒株传于原代人羊膜细胞,则能保持其原有的血凝能力。 (三)已获得血凝能力的D’Amori毒株在KB细胞上传1一20代均无血凝出现,但一旦传回至原代人肾细胞,就能产生血凝。 (四)本文就不同细胞对D’Amori毒株的血凝能力影响的机制进行了讨论。     

The ‘president’s drug’

Netherlands Heart Journal -