N-glycolylneuraminic acid deficiency in mice: implications for human biology and evolution.
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Humans and chimpanzees share >99% identity in most proteins. One rare difference is a human-specific inactivating deletion in the CMAH gene, which determines biosynthesis of the sialic acid N-glycolylneuraminic acid (Neu5Gc). Since Neu5Gc is prominent on most chimpanzee cell surfaces, this mutation could have affected multiple systems. However, Neu5Gc is found in human cancers and fetuses and in trace amounts in normal human tissues, suggesting an alternate biosynthetic pathway. We inactivated the mouse Cmah gene and studied the in vivo consequences. There was no evidence for an alternate pathway in normal, fetal, or malignant tissue. Rather, null fetuses accumulated Neu5Gc from heterozygous mothers and dietary Neu5Gc was incorporated into oncogene-induced tumors. As with humans, there were accumulation of the precursor N-acetylneuraminic acid and increases in sialic acid O acetylation. Null mice showed other abnormalities reminiscent of the human condition. Adult mice showed a diminished acoustic startle response and required higher acoustic stimuli to increase responses above the baseline level. In this regard, histological abnormalities of the inner ear occurred in older mice, which had impaired hearing. Adult animals also showed delayed skin wound healing. Loss of Neu5Gc in hominid ancestors approximately 2 to 3 million years ago likely had immediate and long-term consequences for human biology. (+info)
A simple and sensitive assay for ascorbate using a plate reader.
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We have developed a rapid, inexpensive, and reliable assay for the determination of ascorbate using a plate reader. In this assay, ascorbic acid is oxidized to dehydroascorbic acid using Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy) and then reacted with o-phenylenediamine to form the condensation product, 3-(dihydroxyethyl)furo[3,4-b]quinoxaline-1-one. The rate of appearance of this product is monitored over time using fluorescence. With this method, it is possible to analyze 96 wells in less than 10min. This permits the analysis of 20 samples with a full set of standards and blanks, all in triplicate. The assay is robust for a variety of samples, including orange juice, swine plasma, dog plasma, and cultured cells. To demonstrate the usefulness of the assay for the rapid determination of experimental parameters, we investigated the uptake of ascorbate and two different ascorbate derivatives in U937 cells. We found similar plateau levels of intracellular ascorbate at 24h for ascorbate and ascorbate phosphate. However, the intracellular accumulation of ascorbate via the phosphate ester had an initial rate that was three to five times slower than that via the palmitate ester. Only lower concentrations of the palmitate ester could be examined because the ethanol needed as solvent decreased cell viability; it behaved similarly to the other two compounds at lower concentrations. To come to these conclusions, only nine plates needed to be analyzed to provide us with the end result after only 7h of analysis. This clearly demonstrates the strength of the plate reader assay, which allows the analysis of large-sample sets in a fraction of the time required for the methods that are most commonly used today. The assay is quick, is very economical, and provides results with uncertainties on the order of only 5%. (+info)
Patterns of "severe acute renal failure" in a referral center in Sudan: excluding intensive care and major surgery patients.
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Acute renal failure (ARF) is a common health problem worldwide. There is limited data on the pattern of ARF in Sudan. Moreover, glomerular diseases, which are a well-known cause of ARF, have not been accurately and adequately diagnosed previously. A retrospective study on the patterns of ARF was carried out in a general nephrology referral center in Sudan during the period from February 2003-February 2004. Patients from intensive care units with ARF and those who developed ARF after massive surgery were excluded from the study. Renal biopsy was performed when indicated and studied with light and immunofluorescent microscopy. Eighty-nine patients (57 (64%) cases were males and mean age was 39+/-19.4 years) fulfilled the criteria for the diagnosis of advanced renal failure requiring renal function replacement therapy. Acute tubular necrosis (ATN) was diagnosed in 50 (56%) patients; 33 (66%) ATN patients had renal failure as a complication of volume depletion, fulminant infections (particularly malaria and typhoid fever) or snakebites and 12 (13.4%) patients ingested paraphenylene-diamine (PPD) (hair/Henna dye) in suicidal attempts. Eight (9%) patients of the total study group had glomerular diseases and 11 (12.3%) had obstructive uropathy associated with ARF; the cause of ARF could not be determined in 17 (19%) patients. Fifty-three (60%) patients recovered their renal function, six (6.7%) patients progressed to chronic kidney disease (CKD), 16 (18%) died and 14 (16%) were lost to follow-up. In conclusion, patients with ARF associated with ATN had a favorable prognosis except when ATN was associated with PPD poisoning. (+info)
Molecular expression and pharmacological identification of a role for K(v)7 channels in murine vascular reactivity.
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BACKGROUND AND PURPOSE: This study represents a novel characterisation of KCNQ-encoded potassium channels in the vasculature using a variety of pharmacological and molecular tools to determine their role in contractility. EXPERIMENTAL APPROACH: Reverse transcriptase polymerase chain reaction (RT-PCR) experiments were undertaken on RNA isolated from mouse aorta, carotid artery, femoral artery and mesenteric artery using primers specific for all known KCNQ genes. RNA isolated from mouse heart and brain were used as positive controls. Pharmacological experiments were undertaken on segments from the same blood vessels to determine channel functionality. Immunocytochemical experiments were performed on isolated myocytes from thoracic aorta. KEY RESULTS: All blood vessels expressed KCNQ1, 4 and 5 with hitherto 'neuronal' KCNQ4 being, surprisingly, the most abundant. The correlated proteins K(v)7.1, K(v)7.4 and K(v)7.5 were identified in the cell membranes of aortic myocytes by immunocytochemistry. Application of three compounds known to activate K(v)7 channels, retigabine (2 -20 microM), flupirtine (20 microM) and meclofenamic acid (20 microM), relaxed vessels precontracted by phenylephrine or 1 mM 4-aminopyridine but had no effect on contractions produced by 60 mM KCl or the K(v)7 channel blocker XE991 (10 microM). All vessels tested contracted upon application of the K(v)7 channel blockers XE991 and linopirdine (0.1-10 microM). CONCLUSIONS AND IMPLICATIONS: Murine blood vessels exhibit a distinctive KCNQ expression profile with 'neuronal' KCNQ4 dominating. The ion channels encoded by KCNQ genes have a crucial role in defining vascular reactivity as K(v)7 channel blockers produced marked contractions whereas K(v)7 channel activators were effective vasorelaxants. (+info)
Permeability of hair dye compounds p-phenylenediamine, toluene-2,5-diaminesulfate and resorcinol through protective gloves in hairdressing.
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Dermal exposure to skin irritants and contact allergens is frequent in hairdressing. Hair dyeing is popular today and involves exposure to highly potent contact allergens, such as p-phenylenediamine (PPD). Use of protective gloves to prevent contact with skin-damaging substances is essential. The aim of the present study was to determine the resistance to permeation by PPD, toluene-2,5-diaminesulfate (TDS) and resorcinol (RES) through protective gloves used in hairdressing in Sweden. The permeation of PPD, TDS and RES through four types of protective gloves made of natural rubber latex (NRL), polyvinylchloride (PVC), nitrile rubber (NR) and polyethene (PE) was tested using the American Society for Testing and Materials (1-inch) test cell. Exposure solutions were 5% PPD (w/v), 0.75% TDS and 10% RES in borate buffer with 0.2 M ascorbic acid. The cumulative breakthrough, the so-called 'time-lag breakthrough' (Lag-BT), and permeation rate were determined for each substance and glove. For the NRL glove, the permeated amounts were below the analytical detection levels for all the tested substances. The NR glove was permeated only by RES, with a Lag-BT of 183 min. The PE glove was the thinnest glove and had a Lag-BT of 32 min for PPD; however, the steady-state permeation rate was only 0.031 nmol cm(-2) min(-1). The PVC glove gave the lowest protection against PPD and RES. TDS did not permeate any of the tested gloves. All the tested gloves were disposable, and all need to be changed often and disposed of after use. In conclusion, if properly used, all the tested gloves give considerable protection against permeation of PPD, TDS and RES. (+info)
Paraphenylene diamine ingestion: an uncommon cause of acute renal failure.
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Paraphenylene diamine (PPD) is a major component of hair dyes. The aim is to study the renal manifestations and outcome of PPD consumption. During a four-year period from 2002 to February 2006, 10 persons were admitted to our Institute after consuming a hair dye in a suicidal bid. The percentage of ARF due to PPD at our Institute was 0.95%. Seven patients out of 10 (70%) who consumed PPD developed ARF. All 10 patients, including the patients who had normal renal function had features of rhabdomyolysis. Two patients required ventilator support for respiratory distress and two more required tracheostomy due to upper airway tract edema. One patient has expired after two sessions of dialysis. Renal biopsy in two patients (one, postmortem) showed acute tubular necrosis along with presence of casts in tubules due to myoglobin. (+info)
Biological monitoring of TDI-derived amines in polyurethane foam production.
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BACKGROUND: Toluene diisocyanate (TDI) is used in industry in the production of flexible polyurethane foam, commonly a mixture of the 2,4- and 2,6- isomers. The production process may lead to exposure to diisocyanates which are associated with respiratory disease. A method has been available for the determination of TDI biomarkers in urine for some years. AIMS: To explore the usefulness of urinary toluenediamine (uTDA) in assessing whether dermal absorption of diisocyanates makes a significant contribution to a worker's total exposure. METHODS: Twenty-six workers took part in the study. Thirteen workers whose duties brought them into physical contact with uncured polyurethane foam during their shift (handlers) were compared to a control group of 13 workers in the same block plant environment had no physical contact with uncured foam on the day that sampling took place (non-handlers). Creatinine-adjusted uTDA levels in the two groups were compared across a work shift. RESULTS: Both groups of workers were exposed to similar levels of airborne TDI. Ten handlers were found to have TDA in post-shift urine samples above detection limits compared with two non-handlers (P < 0.05). No clear relationship was found between the level of airborne TDI exposure and post-shift uTDA. CONCLUSIONS: uTDA provides a useful indication of the contribution which skin absorption makes to total TDI exposure. The results suggest that skin protection when handling uncured polyurethane foam may not receive sufficient consideration. (+info)
Activation of T-cells from allergic patients and volunteers by p-phenylenediamine and Bandrowski's base.
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Allergic contact dermatitis is commonly associated with exposure to p-phenylenediamine. The aim of this study was to determine whether p-phenylenediamine (PPD) and/or Bandrowski's base (BB) stimulate T cells from allergic patients and volunteers, and to explore the relationship between T-cell immunogenicity and allergy. Lymphocytes from allergic patients proliferated with PPD and BB (n=8). Lymphocytes from 14/16 non-allergic individuals also proliferated following stimulation, but only with BB; cord blood lymphocytes failed to respond (n=6). Glutathione, which prevented BB formation, but not binding of PPD to cells and serum, did not prevent p-phenylenediamine-specific stimulation of patient lymphocytes. T-cell clones generated from allergic patients were stimulated separately with PPD and BB, while clones from volunteers proliferated with BB alone. Patient and volunteer clones secreted IL-4, IL-5, IL-13, TNF-alpha, MIP-1alpha, MIP-1beta, and RANTES. These data show that activation of T lymphocytes from allergic individuals alone with PPD represents an important discrimination between allergic and non-allergic groups. BB-specific T cells are found in both allergic patients and volunteers, but not in cord blood. Their presence seems to reflect an acquired immune response, which is not translated into an allergic reaction. (+info)