Development of an enzyme‐linked immunosorbent assay (ELISA) for the detection of porcine pepsin as a milk clotting enzyme

An enzyme‐linked immunosorbent assay (ELISA) for the detection of porcine pepsin in milk‐clotting enzyme preparations has been developed. The assay is capable of detecting porcine pepsin in the range 1 μgto 1 mg ml^‐1 without enhancement or modification. The specificity of the technique was studied by inhibition assay. Slight cross‐reactions with bovine rennet and Mucor miehei rennet occurred at high concentrations (1.0 mg ml^‐1). The ELISA used in this investigation appears to provide a quick, sensitive and specific method for the detection of porcine pepsin and has potential applications in the dairy industry.     

Analysis of the four heart sounds statistical study and spectro-temporal characteristics

Abstract

Heart auscultation has been recognised for a long time as an important tool for the diagnosis of heart disease; it is the most common and widely recommended method to screen for structural abnormalities of the cardiovascular system. Detecting relevant characteristics and forming a diagnosis based on the sounds heard through a stethoscope, however, is a skill that can take years to be acquired and refine. The efficiency and accuracy of diagnosis based on heart sound auscultation can be improved considerably by using digital signal processing techniques to analyse phonocardiographic (PCG) signals. The study of the functioning of the heart is very important for the diagnosis of different cardiac pathologies. The phonocardiogram signal (PCG) is the signal generated after conversion of the sound noises coming from the heart into an electrical signal, it groups together a set of four cardiac noises (S1, S2, S3, S4) which are in direct correlation with cardiac activity. The short-term Fourier Transform (STFT) is an analytical technique that describes the evolution of the time and frequency behaviour of these four heart sounds. A statistical study has been carried out in this direction in order to better highlight the characteristics of the PCG signal. A fairly high number of cycles (twenty) was used to further refine the expected results. The objective of this paper is to use a statistical analysis based on the results obtained by the use of The STFT technic this in order to find statistical parameters (mean, standard deviation, etc.) which can give us a clear vision of the electrophysiological behaviour of the phonocardiogram signal. This aspect has not been done so far and which however can give appreciable practical results.     

Mechanotransduction of bone cells<Emphasis Type="Italic">in vitro</Emphasis>: Mechanobiology of bone tissue

Mechanical force plays an important role in the regulation of bone remodelling in intact bone and bone repair. In vitro, bone cells demonstrate a high responsiveness to mechanical stimuli. Much debate exists regarding the critical components in the load profile and whether different components, such as fluid shear, tension or compression, can influence cells in differing ways. During dynamic loading of intact bone, fluid is pressed through the osteocyte canaliculi, and it has been demonstrated that fluid shear stress stimulates osteocytes to produce signalling molecules. It is less clear how mechanical loads act on mature osteoblasts present on the surface of cancellous or trabecular bone. Although tissue strain and fluid shear stress both cause cell deformation, these stimuli could excite different signalling pathways. This is confirmed by our experimental findings, in human bone cells, that strain applied through the substrate and fluid flow stimulate the release of signalling molecules to varying extents. Nitric oxide and prostaglandin E₂ values increased by between two- and nine-fold after treatment with pulsating fluid flow (0.6±0.3 Pa). Cyclic strain (1000 μstrain) stimulated the release of nitric oxide two-fold, but had no effect on prostaglandin E₂. Furthermore, substrate strains enhanced the bone matrix protein collagen I two-fold, whereas fluid shear caused a 50% reduction in collagen I. The relevance of these variations is discussed in relation to bone growth and remodelling. In applications such as tissue engineering, both stimuli offer possibilities for enhancing bone cell growth in vitro.     

Increases in renal epsilon-(gamma-glutamyl)-lysine crosslinks result from compartment-specific changes in tissue transglutaminase in early experimental diabetic nephropathy: pathologic implications

Diabetic nephropathy (DN) is characterized by an early, progressive expansion and sclerosis of the glomerular mesangium leading to glomerulosclerosis. This is associated with parallel fibrosis of the renal interstitium. In experimental renal scarring, the protein cross-linking enzyme, tissue transglutaminase (tTg), is up-regulated and externalized causing an increase in its crosslink product, epsilon-(gamma-glutamyl)-lysine, in the extracellular space. This potentially contributes to the extracellular matrix (ECM) accumulation central to tissue fibrosis by increasing deposition and inhibiting breakdown. We investigated if a similar mechanism may contribute to the ECM expansion characteristic of DN using the rat streptozotocin model over 120 days. Whole kidney epsilon-(gamma-glutamyl)-lysine (HPLC analysis) was significantly increased from Day 90 (+337%) and peaked at Day 120 (+650%) (p < 0.05). Immunofluorescence showed this increase to be predominantly extracellular in the peritubular interstitial space, but also in individual glomeruli. Total kidney transglutaminase (Tg) was not elevated. However, using a Tg in situ activity assay, increased Tg was detected in both the extracellular interstitial space and glomeruli by Day 60, with a maximal 53% increase at Day 120 (p < 0.05). Using a specific anti-tTg antibody, immunohistochemistry showed a similar increase in extracellular enzyme in the interstitium and glomeruli. To biochemically characterize glomerular changes, glomeruli were isolated by selective sieving. In line with whole kidney measurement, there was an increase in glomerular epsilon-(gamma-glutamyl) lysine (+361%); however, in the glomeruli this was associated with increases in Tg activity (+228%) and tTg antigen by Western blotting (+215%). Importantly, the ratio of glomerular epsilon-(gamma-glutamyl) lysine to hydroxyproline increased by 2.2-fold. In DN, changes in the kidney result in increased translocation of tTg to the extracellular environment where high Ca(2+) and low GTP levels allow its activation. In the tubulointerstitium this is independent of increased tTg production, but dependent in the glomerulus. This leads to excessive ECM cross-linking, contributing to the renal fibrosis characteristic of progressive DN.     

Study of β‐casein denaturation by microparticle‐enhanced nephelometric immunoassay

Rabbit anti‐native bovine ß‐casein antiserum reacted with native ß‐casein and fragments f( 1–105/7) and f( 106–209) formed during ß‐casein proteolysis by plasmin. Agglutination of ß‐casein‐coated microparticles by anti‐native ß‐casein antiserum was weakly inhibited by ß‐casein f(1–105/7) and ß‐casein f( 106–209) (0·04 and 1·4%, respectively, compared with native ß‐casein). Immunoreactivity of these ß‐casein peptides in microparticle‐enhanced nephelometric immunoassay was more preserved in the whole ß‐casein than in its isolated fragments. The protein concentration producing 50% inhibition of the ß‐casein‐coated microparticle agglutination with anti‐native ß‐casein antiserum increased during ß‐casein denaturation. A microparticle‐enhanced nephelometric immunoassay, quantifying changes of this inhibiting protein concentration, permitted detection of alteration of the immunoreactivity of ß‐casein during its plasmin proteolysis and heat denaturation, providing an adequate test for the integrity of the whole molecule.     

Widespread IgE-mediated hypersensitivity in the Sudan to the 'green nimitti' midge, Cladotanytarsus lewisi (Diptera: Chironomidae) II. Identification of a major allergen.

A major allergen has been identified in an aqueous extract of the 'green nimitti' midge, Cladotanytarsus lewisi (Diptera: Chironomidae). Following chromatography on Sephadex G-100 allergenic activity, as assessed by skin ('prick') testing, eluted as two closely related peaks (pools I and II) at about 50% bed volume. When these pools were applied separately to columns of CM-cellulose, activity in each eluted with 0 . 05 M NaCl. Isoelectric focusing of the unfractionated allergen gave a single peak of activity at pI 4 . 3. By SDS--PAGE, biological activity in the whole 'green nimitti' extract and the material eluting from both pools I and II of the Sephadex G-100 column migrated to the same positions and were associated with a molecular size of 15,000--20,000 daltons. Skin test reactivity of the unfractionated material and the Sephadex G-100 pool I and II eluates were all destroyed following incubation with trypsin, chymotrypsin, thermolysin and neuraminidase. These experiments indicate that a major allergen derived from the 'green nimitti' midge, a cause of widespread and severe immediate-type allergy in the Sudan, is an acidic glycoprotein of 15,000--20,000 molecular weight.     

Neurosurgical anatomy of the anterior interhemispheric approach for aneurysms of the anterior communicating artery (26.6.92)

Summary The anterior interhemispheric approach for aneurysms of the anterior communicating artery was studied in ten cadavers. This approach presents several advantages over the pterional approach widely used in neurosurgery. It allows direct access to the region of the anterior communicating artery complex with minimal retraction of the brain and preservatioin of the olfactory tract and the gyrus rectus.

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Bases anatomiques de l'abord inter-hémisphérique antérieur lors de la chirurgie des anévrysmes de l'artère communicante antérieure

Résumé Ce travail concerne l'abord neurochirurgical des anévrysmes de l'artére communicante antérieure par voie frontale interhémisphérique. L'étude anatomique a été réalisée sur dix sujets. Cette exposition possède de nombreux avantages comparée à la voie ptérionale habituelle : voie d'abord reduite médiane permettant une visualisation directe et symétrique du complexe artériel de l'artére communicante antérieure ; avec le moindre manipulation et retraction du cerveau en respectant les voies olfactives et le gyrus rectus.

     

Follicular thyroid lesions coexisting with Hashimoto's thyroiditis: incidence and possible sources of diagnostic errors

Our objectives were to study the types and incidence of thyroid follicular lesions coexisting with Hashimoto's thyroiditis (HT), the pitfalls in their cytodiagnosis, and the effect on management. All cases of HT diagnosed by fine-needle aspiration (FNA) and/or histology over a 7-yr period were retrospectively studied. HT coexisted with follicular adenoma (FA) in 6 cases, follicular variant of papillary carcinoma (FVPC) in 1 case, and goitrous nodule (GN) in 2 cases. The overall incidence rates of thyroid neoplasm and goitrous nodules coexistent with HT were 15% and 3.5%, respectively. A preoperative FNA diagnosis was available in 10 histologically proven cases of HT. A false-positive diagnosis of follicular neoplasm (FN) that led to unnecessary thyroidectomies was given in 3 cases. In 2 of these, the cytological diagnosis was HT with the possibility of coexisting FN, and in the third case, the cytological finding of HT was misinterpreted as FN. The main causes of these diagnostic pitfalls were the presence of hyperplastic follicular cells with nuclear pleomorphism, a paucity of lymphoid cells in burned-out HT, and lack of ones exposure. Nuclear pleomorphism was observed in none of the follicular adenomas. FNA diagnosed accurately the coexisting lesions in 6 cases; 3 FA, 1 FVPC, and 2 GN, but it did not sample HT. In one case, FNA diagnosed correctly both HT and the coexisting FA. Therefore, the presence of a coexistent neoplasm or goitrous nodule reduced the chances of sampling HT by 85.7%, with no false-negative results. Indeed, aspiration on and around the thyroid nodule helps in sampling HT. However, HT may dominate the smear and obscure neoplasia. This can be avoided if the procedure is performed by the pathologist and the aspiration is done on the nodule only. The overlapping cytological features of FN and HT were the main causes of false-positive results. This can be reduced by avoiding the diagnosis of FN in the presence of follicular-cell pleomorphism and/or moderate to excessive numbers of lymphoid cells, provided proper aspiration technique is maintained.     

Correlation of oxacillin MIC with mecA gene carriage in coagulase-negative staphylococci

The National Committee for Clinical Laboratory Standards has recently changed the oxacillin breakpoint from >/=4 mg/liter to >/=0. 5 mg/liter to detect methicillin-resistant coagulase-negative staphylococci (CoNS) because the previous breakpoint lacked sensitivity. To determine the correlation between the new oxacillin breakpoint and the presence of the mecA gene, 493 CoNS of 11 species were tested. The presence of the mecA gene was determined by PCR, and oxacillin susceptibility was determined by the agar dilution method with Mueller-Hinton agar containing 2% NaCl and oxacillin (0. 125 to 4.0 mg/liter). The new breakpoint correctly classified all CoNS strains with mecA as methicillin resistant and strains of Staphylococcus epidermidis, S. haemolyticus, and S. hominis without mecA as methicillin susceptible. The breakpoint of >/=0.5 mg/liter was not specific for S. cohnii, S. lugdunensis, S. saprophyticus, S. warneri, and S. xylosus, in that it categorized 70 of 74 strains of these species without mecA (94.6%) as methicillin resistant. The results of this study indicate that the new oxacillin breakpoint accurately identifies strains of CoNS with mecA but is not specific for strains of certain species of CoNS without mecA.     

Quantitation of the reconstruction quality of a four-dimensional computed tomography process for lung cancer patients

We have developed a four-dimensional computed tomography (4D CT) technique for mapping breathing motion in radiotherapy treatment planning. A multislice CT scanner (1.5 mm slices) operated in ciné mode was used to acquire 12 contiguous slices in each couch position for 15 consecutive scans (0.5 s rotation, 0.25 s between scans) while the patient underwent simultaneous quantitative spirometry measurements to provide a sorting metric. The spirometry-sorted scans were used to reconstruct a 4D data set. A critical factor for 4D CT is quantifying the reconstructed data set quality which we measure by correlating the metric used relative to internal-object motion. For this study, the internal air content within the lung was used as a surrogate for internal motion measurements. Thresholding and image morphological operations were applied to delineate the air-containing tissues (lungs, trachea) from each CT slice. The Hounsfield values were converted to the internal air content (V). The relationship between the air content and spirometer-measured tidal volume (v) was found to be quite linear throughout the lungs and was used to estimate the overall accuracy and precision of tidal volume-sorted 4D CT. Inspection of the CT-scan air content as a function of tidal volume showed excellent correlations (typically r>0.99) throughout the lung volume. Because of the discovered linear relationship, the ratio of internal air content to tidal volume was indicative of the fraction of air change in each couch position. Theoretically, due to air density differences within the lung and in room, the sum of these ratios would equal 1.11. For 12 patients, the mean value was 1.08 +/- 0.06, indicating the high quality of spirometry-based image sorting. The residual of a first-order fit between v and V was used to estimate the process precision. For all patients, the precision was better than 8%, with a mean value of 5.1% +/- 1.9%. This quantitative analysis highlights the value of using spirometry as the metric in sorting CT scans. The 4D reconstruction provides the CT data required to measure the three-dimensional trajectory of tumor and lung tissue during free breathing.