Lectins from tunicates: structure-function relationships in innate immunity.
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Biomedical subjects
Publications and source records attributed to N O'Leary.
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To assess the affect of haemolysis on routine photometric methods used by participants in the Irish External Quality Assessment Scheme for Clinical Biochemistry (IEQAS) a sample was distributed with a supplied diluent containing haemoglobin. A sample of the same batch had previously been circulated as a normal distribution. Many analytes/methods were affected by the haemolysis. The range of values reported for each analyte was as follows. Albumin 34-48 g/L (baseline 37 g/L). Calcium 2.16-2.68 mmol/L (baseline 2.34 mmol/L). Urate 90-470 mumol/L (baseline 195 mumol/L). Creatinine 55-161 mumol/L (baseline 140 mumol/L). Glucose 4.1-5.6 mmol/L (baseline 4.8 mmol/L). Bilirubin 14-60 mumol/L (baseline 36 mumol/L). Urea 3.4-7.3 mmol/L (baseline 3.8 mmol/L). Iron 17.9-55.0 mumol/L (baseline 26.8 mumol/L). Inorganic phosphorus 0.22-2.41 mmol/L (baseline 1.59 mmol/L). Cholesterol 3.0-5.9 mmol/L (baseline 4.1 mmol/L). In some situations the use of an appropriate blank may solve the problem, but it must be remembered that interference caused by haemoglobin may be more complex than merely spectral.
Two hundred consecutive female patients, who were referred to a university-based facial pain clinic, were asked to mark all painful sites on sketches showing the contours of a human body in the frontal and rear views. The drawings were analyzed with transparent templates containing 1875 (frontal view) and 1929 (rear view) square cells of equal size. The average patient scored 71.8 cells in the frontal and 99.7 cells in the rear view (corresponding to 3.8% and 5.2% of the maximum possible scores). In individual patient drawings, however, up to 42.7% and 44.9% of all cells were marked. Only 37 cases (18.5%) exhibited pain that was limited to the trigeminal system. An analysis of the pain distribution according to the arrangements of dermatomes revealed three distinct clusters of patients: (1) pain restricted to the region innervated by the trigeminal nerves (n = 37); (2) pain in the trigeminal dermatomes and any combination involving the spinal dermatomes C2, C3, and C4, but no other dermatomes (n = 32); and (3) pain sites involving dermatomes in addition to the ones listed above (n = 131). Mean ages in the three clusters were 38.7, 35.5, and 37.5 years, respectively (p = 0.62, n.s.). Widespread pain existed for longer durations (median, 48 months) than conditions involving local and regional pain (median, 24 months) (p = 0.02, s.). Our findings showed that among a great percentage of persistent facial pain patients the pain distribution is more widespread than commonly assumed, and that the persistence of pain in the regional and widespread pain presentations is significantly greater than in cases with pain limited to the trigeminal system.
Measurement of the affinity of a protein for a promoter sequence is critical when assessing its potential to regulate transcription. Here we report that the DNA protein crosslinking (DPC) assay can be used to measure affinity, amount and molecular weight of DNA binding proteins to specific and non-specific DNA sequences. By applying a theoretical analysis to evaluate the binding data, it was shown that the affinity constants of two proteins (named DPC80 and DPC107) to the MT3 region of the mouse thymidine kinase promoter were 2 x 10(-9) M, which is 10(4) times higher than to non-specific DNA. Similar affinity constants were found when the purified proteins corresponding to DPC80 and DPC107 instead of nuclear extracts were used to assess the reliability of the DPC assay. A value for crosslinking efficiency was determined as 0.07, however, it is not needed for computation of the DNA-protein affinity, but with it the abundance of a binding protein can be estimated. In summary, the DPC assay is useful for quantifying DNA binding proteins and thereby judging their influence on transcription.
A fundamental cause of cancer is changed properties of genetic material, which may deregulate normal development of the tissue or provide selective growth advantage to the tumor cell. This deregulation of cell proliferation results from altered production of a handful of proteins that play key roles in progression through the eukaryotic cell cycle. Some of these proteins include tumor suppressor genes or oncogenes. However, no one general change or alteration of a critical gene has yet been found in all cancers. Using surgical material obtained from patients with various malignancies, we show that breast cancers and other solid tumors, as well as malignant lymphocytes from patients with lymphatic leukemia, show severe quantitative and qualitative alterations in cyclin E protein production independent of the S-phase fraction of the samples. Hence, these alterations represent a true difference between normal versus tumor tissue. In addition, in breast cancer, the alterations in cyclin E expression become progressively worse with increasing stage and grade of the tumor, suggesting its potential use as a new prognostic marker.
Traditionally external quality assessment schemes (EQAS) distribute samples spectrally similar to a normal serum. This does not allow laboratories to assess the performance of their methods at measuring lipaemic samples. The Irish EQAS distributed a sample with a supplied lipid diluent. A sample of the same batch had previously been circulated as a normal distribution to obtain baseline values. Most analytes were affected by the lipaemia. The degree of interference was instrument/method dependent and a wide range of values was reported for each analyte: total protein 51-134 g/L (baseline 65 g/L); albumin 33-56 g/L (baseline 42 g/L); calcium 2.12-6.10 mmol/L (baseline 2.34 mmol/L); urate 38-1100 mumol/L (baseline 195 mumol/L); creatinine 6-199 mumol/L (baseline 140 mumol/L); glucose 4.2-9.5 mmol/L (baseline 4.8 mmol/L); total bilirubin 1-192 mumol/L (baseline 36 mumol/L); urea 1.1-8.1 mmol/L (baseline 3.8 mmol/L); aspartate aminotransferase (AST) 12-105 iu/L (baseline 44 iu/L); iron 4.3-107 mumol/L (baseline 26.8 mumol/L); inorganic phosphorus 1.12-4.96 mmol/L (baseline 1.59 mmol/L); and cholesterol 2.5-17.1 mmol/L (baseline 4.1 mmol/L). In situations where no clearing of the lipid occurs in the reagent, interference can be minimized by taking a sample blank reading or using a kinetic procedure. If clearing of the lipid does occur, time must be allowed for this to be completed before analytical readings are taken.
Bilirubin is oxidized to biliverdin by 1.0 mmol/L potassium ferricyanide in the presence of caffeine. The absorbance decline at 480 nm with blanking at 660 nm is proportional to bilirubin concentration. The assay is linear up to 1000 mumol/L and is suitable for the measurement of bilirubin in adult and neonatal patients. The assay is accurate and precise and is not significantly affected by haemolysis up to 5.0 g/L haemoglobin. Reagents and calibration are stable for at least 3 months.
The negative interference by bilirubin in the measurement of creatinine by the Jaffé procedure is well documented. We describe a procedure for the elimination of interfering bilirubin by oxidation to biliverdin through the addition of potassium ferricyanide before the alkaline picrate reagent. We detected no negative interference with the addition of as much as 600 mumol/L bilirubin. The procedure shows good agreement with a recently described blank rate method for analyzing icteric samples.
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The Technicon SMAC system was adapted to function with much smaller volumes of plasma than those used in standard procedures. With only minimal operator-entered changes to the 7.6 computer program, and simple modifications of the glucose and alkaline phosphatase channels, the SMAC analyser performed equally well when the plasma volume was decreased from 235 microliter sequentially down through 176 microliter, 142 microliter, and 105 microliter. The basic principle employed was to maintain a constant flow rate of diluted sample plus diluent with decreasing sample size. The instrument proved to have the capability of operating successfully on all channels with lower signals than those encountered with the unmodified system.
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In recent years, a 'new' pathway for complement activation mediated by the mannose-binding lectin (MBL) has been described as a key mechanism for the mammalian acute phase response to infection. This complement activation pathway is initiated by a non-self recognition step: the binding of a humoral C-type lectin [mannose-binding lectin (MBL)] to microbial surfaces bearing 'foreign' carbohydrate determinants. The recognition factor, MBL, is associated with a serine protease [MBL-associated serine protease (MASP)] which, upon MBL binding to the microbial ligand, activates the complement component C3, leading to either (a) phagocytosis of the opsonized target via the complement receptor, or (b) humoral cell killing via assembly of the membrane attack complex. Galectins (formerly known as S-type lectins) modulate activity of the complement receptor 3 (CR3), the macrophage membrane receptor for complement components C3b and iC3b, downstream products of the MBL pathway which are covalently bound to 'target cells. Galectins also mediate macrophage- and dendrocyte-adhesion to lymphocytes activated by signaling through another C-type lectin, the L-selectin, leading to immunoglobulin-mediated responses. Thus, the functional interplay of MBL, galectins and L-selectin in the acute phase response neutralizes the microbial challenge, and lead to further adaptive immunity. Although the observation of various components of the lectin pathway in different invertebrate species demonstrates the high conservation and ancient roots of the components of innate immunity, there has previously been no evidence supporting the possibility that the integral lectin-mediated complement activation pathway is present in invertebrates. We now have evidence for the coexistence of homologs of all the pathway's key components (MBL, MASP, C3, and galectin) in the protochordate Clavelina picta, suggesting the lectin-mediated pathway of complement activation preceded the immunoglobulin pathway in evolution. Therefore, despite being 'new' to the textbooks, experimental evidence indicates that this pathway is ancient, and has been conserved intact throughout its evolution.