Chromatographic fractionation of Penicillium polonicum fermentation metabolites in search of the nephrotoxin(s) for rats

Complex renal histopathological changes in rats, in silent response to dietary contamination with wheat moulded by a common Penicillium from the Balkans, have long eluded attribution of a causal toxin. So far, water-soluble amphoteric glyco-peptides seem responsible, at least for the nuclear pyknoses in nephron epithelia after several days of dietary exposure. Recently, refined histology analysis has diagnosed pyknosis as apoptosis, and followed the finding through application of medium-pressure liquid chromatography, anion exchange and silica layer chromatography to fractionate a water/alcohol-soluble extract of a fungal fermentation on wheat. Proline was revealed, with other amino acids, in acid hydrolysate of the fermentation extract. Application of mass spectrometry has recognized prominent ions (m/z 550 and 564) correlated with fragmentations consistent with a terminal proline moiety for the putative toxins, coupled with other structural fragments and correlated with apoptosis. Use of 14C-proline in probing Penicillium polonicum fermentation to aid isolation of the new potential toxins, along with application of gel electrophoresis, may further aid characterization of the apoptosis toxin(s). The present focus on proline peptides in mycotoxicosis fits easily with their increasingly recognised pharmacological activity associated with proline’s rigid secondary amine structure, which causes conformational contortion in peptides. Nevertheless, there remains the striking rat renal karyocytomegaly by P. polonicum, for which there is yet no causative mycotoxin

1H NMR Spectroscopy-Based Metabolomic Assessment of Uremic Toxicity, with Toxicological Outcomes, in Male Rats Following an Acute, Mid-Life Insult from Ochratoxin A

Overt response to a single 6.25 mg dose of ochratoxin A (OTA) by oral gavage to 15 months male rats was progressive loss of weight during the following four days. Lost weight was restored within one month and animals had a normal life-span without OTA-related terminal disease. Decline in plasma OTA concentration only commenced four days after dosing, while urinary excretion of OTA and ochratoxin alpha was ongoing. During a temporary period of acute polyuria, a linear relationship between urine output and creatinine concentration persisted. Elimination of other common urinary solutes relative to creatinine was generally maintained during the polyuria phase, except that phosphate excretion increased temporarily. 1H NMR metabolomic analysis of urine revealed a progressive cyclic shift in the group principal components data cluster from before dosing, throughout the acute insult phase, and returning almost completely to normality when tested six months later. Renal insult by OTA was detected by 1H NMR within a day of dosing, as the most sensitive early indicator. Notable biomarkers were trimethylamine N-oxide and an aromatic urinary profile dominated by phenylacetylglycine. Tolerance of such a large acute insult by OTA, assessed by rat natural lifetime outcomes, adds a new dimension to toxicology of this xenobiotic

Changes in male rat urinary protein profile during puberty: a pilot study

BACKGROUND: Androgen-dependent proteins (lipocalins) circulate in blood of male rats and mice and, being small (~ 18 kDa), pass freely into glomerular filtrate. Some are salvaged in proximal nephrons but some escape in urine. Several organic molecules can bind to these proteins causing, where salvage occurs, nephropathy including malignancy in renal cortex. In urine, both free lipocalins and ligands contribute to an increasingly-recognised vital biological role in social communication between adults, especially in the dark where reliance is on smell and taste. Crystal structure of the first-characterised lipocalin of male rats, α2u-globulin, has been determined and peptide sequences for others are available, but no study of occurrence during early puberty has been made. We have followed temporal occurrence in urine of juveniles (n = 3) for non-invasive pilot study by high resolution gradient mini-gel electrophoresis, tryptic digest of excised protein bands, and LC-MS/MS of digest to identify peptide fragments and assign to specific lipocalins. Study objective refers directly to external availability for social communication but also indirectly to indicate kinetics of circulating lipocalins to which some xenobiotics may bind and constitute determinants of renal disease. RESULTS: Mini-gels revealed greater lipocalin complexity than hitherto recognised, possibly reflecting post-translational modifications. Earliest patterns comprised rat urinary protein 1, already evident in Sprague-Dawley and Wistar strains at 36 and 52 days, respectively. By 44 and 57 days major rat protein (α2u-globulin) occurred as the progressively more dominant protein, though as two forms with different electrophoretic mobility, characterised by seven peptide sequences. No significant change in urinary testosterone had occurred in Wistars when major rat protein became evident, but testosterone surged by 107 days concomitant with the marked abundance of excreted lipocalins. CONCLUSIONS: Qualitative temporal changes in the composition of excreted lipocalins early in puberty, and apparent increase in major urinary protein as two resolvable forms, should catalyse systematic non-invasive study of urinary lipocalin and testosterone dynamics from early age, to illuminate this aspect of laboratory rodent social physiology. It could also define the potential temporal onset of nephrotoxic ligand risk, applicable to young animals used as toxicological models

Pearl Millet as an Alternate Host of the Sorghum Ergot Pathogen, Claviceps africana

The infectivity of C. africana on pearl millet (Pennisetum glaucum) lines in Africa was studied. C. africana consistently gave 100% disease incidence on male-sterile sorghum in experiments undertaken in Zimbabwe. C. fusiformis established disease with moderate incidence on most but not all lines tested. In contrast, C. africana established a parasitic association with all the pearl millet lines tested, with incidence as high as 23% in ICMSR 260, the genotype that also supported the highest incidence with C. fusiformis. However, the disease severities were always low, between 1 and 5%. All infections on pearl millet were verified as the sphacelial stage by virtue of their conidial characteristics. After 1 passage through a pearl millet host, C. africana did not apparently become more infectious on this host

Quantitative characterization of machining-induced white layers in Ti–6Al–4V

Machining-induced white layers can affect the functional performance of engineered components, due to the resulting mechanical and microstructural properties. Destructive inspection methods such as cross-sectional microscopy are typically used to identify white layers, however, these methods are inherently costly and time-consuming. It is, therefore, desirable to detect this anomalous surface feature using non-destructive methods which requires improved knowledge around the characteristics of white layers. The present paper reports on the characterization of white layers formed during machining of Ti–6Al–4V, to aid future development of a reliable non-destructive assessment method. The microstructure of the material in the white layer was found to have a basal α-hexagonal close packed texture and there was no evidence of an α→β phase transformation during white layer formation. The white layer has a highly refined grain structure with an increased nanohardness of up to 15% compared with the bulk material. It is proposed that white layers in Ti–6Al–4V are formed by continuous dynamic recrystallization driven by severe plastic deformation during machining. According to the measured micro-mechanical properties of the white layer, suitable non-destructive testing methods are suggested for the detection of this surface feature

Spring salmon enhancement on the Delphi Fishery, Ireland

This paper summarises the results of an Atlantic salmon (Salmo salar) enhancement programme on the Delphi Fishery in the west of Ireland between 1991 and 1995. The aim of the programme was to increase salmon rod catches in the wake of a sea trout stock collapse. Smolts from two other Irish rivers, Burrishoole and Corrib, were released alongside Delphi fish and differentially tagged. Record angling catches were subsequently recorded. Catch rates varied from 0.6 to 12.7 per 1,000 smolts released. The comparative performance of the different stocks is assessed, with significant differences emerging between the stocks and between year classes In terms of survival/exploitation rates, run limes, sex ratios and homing patterns. The Delphi fish produced consistently lower overall returns than the Burrishoole groups, but consistently much higher numbers of early-running multi-sea-winter (MSW) salmon. These MSW salmon were predominantly female, while Delphi grilse were predominantly male. The Corrib fish performed relatively poorly. The smaller MSW salmon component of the non-indigenous groups ran later in the season than their Delphi counterparts. Important size differences in adult returns were noted and related to stock, sex, husbandry and selection by interceptory fisheries. It is suggested that the MSW salmon component of Delphi stocks is attributable to genetic factors, possibly linked to low freshwater temperature regimes. The programme has contributed to an increase in estuarine droll netting. Exploitation of grilse by all forms of coastal net ranged from 56% to 87%. Exploitation of MSW salmon by nets was lower, ranging from 0% to 54%, the lowest rates being achieved by the early-running Delphi MSW salmon. The programme is expensive and cannot be justified in terms of direct angling revenue. But when related accommodation income is taken into account, the financial results and the sustainment of the fishery's capital value broadly justify the programme to date