Purification of bovine conglutinin using pepsin digestion

This paper describes a new method for the purification of bovine conglutinin based on the relative resistance of this protein to pepsin digestion. First, conglutinin is purified by absorption on yeast, then the preparation is treated with 2% pepsin (w/w) at 4°C for 18 hr, and finally gel filtrated on agarose A5m. The yield is 60–75% and conglutinin thus prepared appears physically, immunochemically and functionally intact. This procedure allows for a rapid production of sufficient amounts of conglutinin for immune complex detection or purification methods.     

Isolation and radiation hybrid mapping of a dinucleotide repeat polymorphism at the human calcium-sensing receptor (CASR) locus

Calcium-sensing receptor (CASR) in parathyroid gland regulates calcium homeostasis by sensing decreases in extracellular calcium levels and effecting an increase in secretion of parathyroid hormone. A polymorphic dinucleotide (CA) sequence was isolated from a genomic clone containing the human CASR gene and was mapped to 3q13.3–q21. This polymorphism will be useful in the genetic study of disorders affecting calcium metabolism, such as hypercalcemia, hypocalcemia, osteoporosis, hyperparathyroidism, and hypoparathyroidism. Received: June 2, 1998 / Accepted: June 24, 1998     

Identification of parasite proteins in a membrane preparation enriched for the surface membrane of erythrocytes infected with Plasmodium knowlesi

A subcellular fraction enriched in erythrocyte membranes has been isolated from rhesus monkey erythrocytes infected with Plasmodium knowlesi. Infected cells were lysed by centrifugation through a zone of hypotonic buffer and membranes isolated by equilibrium density gradient centrifugation in the same tube. The purified membrane fraction was shown to include the erythrocyte surface membrane by several methods: electron microscopy, identification of Coomassie Blue stained erythrocyte membrane proteins, identification of band 3 with a monoclonal antibody, and identification of radioiodinated cell surface proteins. The resulting ghosts were shown to be specifically reactive with monkey sera against the variant surface antigens of P. knowlesi by indirect immunofluorescence and membrane agglutination. No reactivity was seen with a monoclonal antibody (13C11) against the intracellular schizont surface. A number of metabolically labelled parasite proteins were enriched in this membrane function, including peptides of 277, 208, 173, 153, 134, 109, 80, 60 and 48 kDa and the variant surface antigens of variable molecular mass (180-207 kDa). These proteins were distinct from the major parasite proteins of total infected erythrocytes and isolated merozoites. The major glucosamine labelled glycoprotein of the internal schizont (230 kDa) was not found in this fraction. Moreover, no fragment of this parasite glycoprotein was found in this membrane fraction, indicating that no part of this molecule is transported to the erythrocyte surface. In contrast, the variant antigen of P. knowlesi, known to be on the erythrocyte surface, could be readily identified as peptides unique to specific cloned parasite lines. We propose that the other nine parasite proteins found within this membrane fraction represent a starting point for the identification of other parasite proteins transported to the surface membrane of the infected erythrocyte.     

Monoclonal antibodies to three distinct epitopes on human IgE: their use for determination of allergen-specific IgE

Three different monoclonal antibodies (MAb) against human immunoglobulin E have been obtained which specifically bind to human myeloma and polyclonal IgE. The antibodies showed high avidities for soluble IgE (0.7 X 10(9) to 3.3 X 10(9) M-1). These MAb defined three distinct epitopes on IgE. A mixture of these antibodies in combination with an 125I-labelled anti-mouse Kappa chain MAb has been used to measure allergen-specific IgE. This determination was performed by a solid-phase radioimmunoassay using allergen extracts coated to either chemically activated paper discs or to polyvinyl chloride wells. This method is 4-10 times more sensitive than other previously reported procedures. A similar technique has also been applied to detect individual allergens in immunoblots of allergen extracts.     

Simultaneous demonstration of phenylethanolamine N-methyltransferase immunofluorescent and catecholamine fluorescent nerve cell bodies in the rat medulla oblongata

The distribution of phenylethanolamine N-methlytransferase-immunoreactive nerve cell bodies was investigated in the rat medulla using an antiserum to bovine phenylethanolamine N-methyltransferase raised in rabbits. A procedure that combines immunohistochemistry and catecholamine fluorescence histochemistry was developed with a formaldehyde/glutaraldehyde mixture as a fixative. Three groups of immunoreactive nerve cell bodies were found in the medulla: a ventrolateral group, C1, a dorsal group, C2, in the nucleus of the tractus solitarius and a smaller medial group of cells, C3, scattered in the medial longitudinal fasciculus. Most of the phenylethanolamine N-methyltransferase positive nerve cells did not show catecholamine fluorescence and did not correspond to the catecholamine cell groups A1 and A2. Both groups C1 and C2 of immunoreactive nerve cells extended further rostrally than A1 and A2. Group C3 has not previously been described as a distinct group of catecholamine fluorescent nerve cell bodies.Inhibition of phenylethanolamineN-methyltransferase and monoamine oxidase results in the appearance of catecholamine fluorescence in the immunoreactive cell bodies suggesting that they usually store adrenaline which reacts poorly with the formaldehyde/glutaraldehyde mixture or other aldehydes which induce catecholamine fluorescence and it is for this reason that they are not normally identified in maps of catecholamine fluorescent cells.     

磷酸钙人工骨(CPC)在颈前路椎间融合的临床应用

目的探讨磷酸钙人工骨(CPC)在颈椎前路椎间融合手术中的应用效果。方法2001年4月至2003年10月颈前路手术中应用磷酸钙人工骨栓椎间融合结合钛钢板固定治疗颈椎病17例,颈椎间盘突出症5例,颈椎外伤脱位2例,共24例35个节段。采用JOA评分评价神经功能,X线片判定融合效果。结果随访18±6.5个月,术后无感染,无过敏或毒性反应。JOA评分由术前9.28±2.15分增加到14.65±2.18分(P<0.001)。术后X线片未见CPC骨栓塌陷或移位,钛板和螺钉无松动及折断。术后16.5±6.8个月均获得椎间融合。结论颈椎前路椎间融合手术应用磷酸钙人工骨替代自体骨,经济、安全、简便、效果可靠。     

Immunology of DNA. II. The effect of size and structure of the antigen on the Farr assay.

The Farr assay for the detecton of antibodies to double stranded (ds) DNA is influenced by the DNA preparations used as antigen. To elucidate this the molecular weight of the antigen preparation, contamination with proteins and presence or absence of single stranded (ss) regions were studied with the following conclusions: 1) The degree of DNA binding by antibodies is linearly dependent on the molecular weight of the DNA, provided that this does not exceed 10 X 10(6). 2) Deproteinization of E. coli DNA by chromatography on methylated albumin-kieselguhr(MAK) columns results in lower binding by most sera. 3) ds DNA preparations sometimes contain ss regions which bind antibodies to ss DNA. The difference in behaviour of different ds DNA preparations may be ascribed entirely to these factors and not to differences in antigenic determinants. We have standardized the Farr assay and enhanced its specificity by the use of circular DNA isolated from bacteriophage PM2.     

Association between HLA and Japanese patients with rheumatoid arthritis

Japanese patients with rheumatoid arthritis (RA) were observed to have a statistical association with HLA-DR4, MT3. Strong association between the clinical severity of RA and HLA was also observed. Male patients had a stronger association with HLA than female patients. Males are more resistant to RA than females. This suggested that the threshold of liability for RA is higher in males than in females. Japanese patients with RA with systemic vasculitis were negative for HLA-Bw44 and had antilymphocytotoxic autoantibody, indicating that RA with systemic vasculitis is different in etiology from RA without systemic vasculitis.     

Skeletal muscle metabolism during short duration high-intensity exercise: influence of creatine supplementation

Seven male subjects performed repeated bouts of high-intensity exercise, on a cycle ergometer, before and after 6 d of creatine supplementation (20 g Cr H₂O day^-1). The exercise protocol consisted of five 6-s exercise periods performed at a fixed exercise intensity, interspersed with 30-s recovery periods (Part I), followed (40 s later) by one 10 s exercise period (Part II) where the ability to maintain power output was evaluated. Muscle biopsies were taken from m. vastus lateralis at rest, and immediately after (i) the fifth 6 s exercise period in Part I and (ii) the 10 s exercise period in Part II. In addition, a series of counter movement (CMJ) and squat (SJ) jumps were performed before and after the administration period. As a result of the creatine supplementation, total muscle creatine [creatine (Cr) + phosphocreatine (PCr)] concentration at rest increased from (mean + SEM) 128.7 (4.3) to 151.5 (5.5)mmolkg^_1 dry wt (P < 0.05). This was accompanied by a 1.1 (0.5) kg increase in body mass (P < 0.05). After the fifth exercise bout in Part I of the exercise protocol, PCr concentration was higher [69.7 (2.3) vs. 45.6 (7.5) mmol kg“‘ dry wt, P < 0.05], and muscle lactate was lower [26.2 (5.5) vs. 44.3 (9.9) mmol kg”¹ dry wt, P < 0.05] after vs. before supplementation. In Part II, after creatine supplementation, subjects were better able to maintain power output during the 10-s exercise period (P < 0.05). There was no change in jump performance as a result of the creatine supplementation (P > 0.05). These findings show that enhanced fatigue resistance during short duration high-intensity exercise following creatine supplementation is associated with a greater availability of PCr and a lower accumulation of lactate in the muscle. The finding that jump performance was not enhanced suggests that short-term creatine feeding does not influence peak power output.     

Changes in cyclic nucleotides during the calcium paradox in the isolated rat heart

Reperfusion of hearts with a Ca²⁺-containing medium after a perfusion period in Ca²⁺-free medium results in irreversible cell damage (calcium paradox). In this investigation we have studied coronary flow and cyclic AMP and cyclic GMP levels after several periods of Ca²⁺-free perfusion in isolated rat hearts. We also investigated the effects of papaverine (Pap), noradrenaline (NA), acetylcholine (ACh) and absence of inorganic phosphate during Ca²⁺-free perfusion on coronary flow (CF) and cyclic nucleotide levels. Inability of the heart to recover contractile activity with development of contracture during the reperfusion period was accepted as indicative of the calcium paradox. Ca²⁺-free perfusion alone and NA and absence of inorganic phosphate during the Ca²⁺-free perfusion period increased CF, whereas Pap and ACh decreased it. However, only Ca²⁺-free perfusion and NA elevated cyclic AMP. On the other hand, Pap and ACh increased cyclic GMP (with a transient rise of cyclic AMP in Pap infusion), and absence of inorganic phosphate decreased both cyclic AMP and cyclic GMP. Pap, ACh and absence of phosphate prevented the calcium paradox. Our study suggests that increased cyclic AMP during the Ca²⁺-free perfusion may contribute, with the other factors, to the occurrence of the calcium paradox.