Faecal avoidance and the risk of infection by nematodes in a natural population of reindeer

We tested whether Svalbard reindeer (Rangifer tarandus platyrhynchus) minimise the risk of gastro- intestinal nematode infection by avoiding patches with a high density of faeces. This experiment was performed in preferred summer foraging habitat. The possibility that reindeer assess infection risk on the basis of faecal contamination levels across plant communities was determined by measuring the distribution of faeces in seven plant communities, and nematode developmental success in two plant communities with contrasting soil moisture content. We explored whether variation within individual reindeer in the levels of infection by gastro-intestinal nematodes was related to their diet. Reindeer avoided pastures where faecal contamination was increased, and thereby potentially reduced the risk of becoming infected by Trichostrongyle nematodes. Dung density was inversely related to soil moisture content, with high densities of faeces in dry plant communities and low densities in wet communities. However, nematode developmental success was positively related to soil moisture content, and was highest in the wetter sites. Thus, by avoiding dry areas with high dropping densities, reindeer would tend to feed in wetter areas where nematodes thrive. Therefore, dung density may be an unreliable predictor of the risk of infection. The absence of a strong relationship between an individual’s infection level and its diet might be due to the unpredictability of pasture infection level. Received: 8 July 1999 / Accepted: 20 January 2000     

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’.     

The Oral History and Literature of the Wolof People of Waalo, Northern Senegal

The Oral History and Literature of the Wolof People of Waalo, Northern Senegal. Samba Diop. Lewiston, Canada; Edwin Mellen Press, 1995. 389 pp.     

Immunoreactive Corticotrophin Levels in Adrenocortical Insufficiency

Plasma concentrations of immunoreactive corticotrophin (ACTH) have been determined in 14 patients with untreated Addison''s disease and in 42 patients with secondary adrenocortical insufficiency. Basal morning plasma ACTH levels were markedly raised in those with Addison''s disease but were either in the normal range or undetectable in the group with secondary adrenocortical insufficiency. In the group with Addison''s disease circulating ACTH values showed a definite nyctohemeral rhythm, a pronounced rise in response to insulin-induced hypoglycaemia, and an immediate fall following the intravenous injection of corticosteroids, with a half-life of between 13·5 and 44·2 minutes. When assays were performed with antisera directed against the portion of the ACTH molecule responsible for corticosteroidogenesis (the N-terminal portion) the apparent ACTH concentrations were lower than with antisera directed against the non-steroidogenic (C-terminal) portion of the molecule. This emphasizes that different antisera may give different apparent hormone concentrations, and that the ranges of values obtained in normal and abnormal states must be established for each antiserum.     

Purification of cysteine proteinases from trichomonads using bacitracin-sepharose

Abstract Bacitracin affinity chromatography has been used to purify proteinases of the parasitic protozoon Tritrichomonas foetus . It proved superior to other affinity chromatography methods we have tested for the purification of trichomonad proteinases and should prove a useful procedure for purifying cysteine proteines from these parasites and other parasitic protozoa. The main cysteine proteinases of T. foetus were purified over 100-fold to be free from the majority of other cell proteins. About 90 μg of protein containing 1.56-fold more proteinase activity than was detectable in the original cell lysate was obtained from 10⁹ cells (7.2 mg protein). SDS-PAGE revealed that the eluate contained two main Coomassie blue-staining bands. N-terminal amino acid sequence analysis of these proteins confirmed that one of them was a cysteine proteinase with unusuall features. Cysteine proteinases were also purified from cell lysates of Trichomonas vaginalis and a N-terminal sequence determined. This is the first amino acid sequence information that has been obtained for trichomonad cysteine proteinases. The method was also used to purify proteinases from the medium of T. foetus cultures. Some selectivity in binding of the proteinases to the affinity column was found.     

Differential release of LH and FSH in cyclic mares in response to synthetic Gn-RH.

Mares at different stages of the oestrous cycle were given a single intravenous injection of 0.5 mg synthetic Gn-RH. The mean area of the induced LH peak was significantly less at mid-cycle (Day 10-11) than at any other time. The mean height of the LH peak above preinjection concentration was greater at late oestrus and early cycle (Day 5-6) than at mid-cycle and early oestrus. There were no significant different in mean FSH responses. The LH:FSH ratio for both height and area of induced peaks was significantly less at mid-cycle than at other times of injection. These results suggest that one releasing hormone could cause the release of both FSH and LH in the normal cyclic mare.     

Electrophoretic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates: application to proenkephalin processing enzymes

A novel method is described for the zymographic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates such as [35S]methionine-labeled proenkephalin or 125I-labeled proinsulin. After electrophoresis the enzyme is reactivated and cleaves the radiolabeled in situ substrate into smaller peptides. These small peptides are able to diffuse out of the gel, leaving clear areas against a dark background when visualized by autoradiography. The technique can be used to detect as little as 200 fg of trypsin using only 50 ng (1.25 microCi) of [35S]proenkephalin. Soluble- and membrane-bound adrenal trypsin-like enzyme were isolated from bovine adrenal chromaffin granules. Both proteinases cleaved [35S]methionine-labeled proenkephalin but not 125I-labeled proinsulin. Moreover, both had a Mr of approximately 30,000. The potential of this technique for general use is discussed. An additional method using the synthetic fluorogenic substrate t-butoxycarbonyl Glu-Lys-Lys aminomethylcoumarin is also described.     

Diversity and dynamics of the DNA and cDNA-derived bacterial compost communities throughout the <Emphasis Type="Italic">Agaricus bisporus</Emphasis> mushroom cropping process

The cultivation of Agaricus bisporus involves the conversion of agricultural materials via fermentation into utilisable simple sugars as a nutrient source for the fungal crop during mushroom cropping. However, little is currently known about the role of the bacterial community contributing to the fermentation process. In this investigation we characterised the composition and dynamics of the DNA and cDNA-derived bacterial populations throughout a commercial mushroom cropping process using MiSeq sequencing. Both methods indicated substantial changes in the bacterial community structure after the first flush of the mushroom crop. However, differences were observed between the composition of the bacterial community determined by each of the two methods. The cDNA-derived community indicated that thermotolerant genera with known sulphur-reducing characteristics were highly active up to the first flush. Activity of the phyla Actinobacteria and Firmicutes was observed to increase as fermentation progressed, indicating that the members of these phyla played prominent roles in the conversion of wheat straw into utilisable sugars. The cDNA-derived community comprised genera with roles in the nitrification process that became highly active at post flush 1. Subsequent chemical analysis of extractable nitrate indicated that substantial nitrification occurred up until the termination of the cropping process. This study has demonstrated that a highly dynamic bacterial community is present throughout the mushroom cropping process.