Measurement of serum apolipoprotein B by radioimmunoassay

Abstract. A sensitive and specific double antibody radioimmunoassay for the major apolipoprotein (apo B) of human serum very low density lipoprotein (VLDL) and low density lipoprotein (LDL) is described. Using anti-LDL and anti-apo B antibodies the immunoreactivity of LDL and apo B were compared. Human LDL and its isolated apo B were not immunologically identical when each antiserum was used with its homologous label; a population of antibodies was selected which reacted with antigenic sites unique to the antigen itself as well as to those which were common to the closely related protein. When the heterologous label was used with either antiserum, a population of antibodies directed against antigenic sites shared by the LDL and apo B molecules was selected.
Apo B in sera samples can be measured using either anti-LDL or anti-apo B antibodies provided that intact LDL was used for preparation of the iodinated tracer and standard. Serum apo B levels in healthy normolipi-daemic males and females were 0.93 ±0.25 g/l (range 0.58–1.39) and 0.90 ± 0.15 g/l (range 0.58–1.12), respectively. The total cholesterol and apo B, and phos-pholipid and apo B concentrations for both males and females were significantly correlated (P<0.05). In another normolipidaemic population ( n = 52), total serum apo B values correlated positively with LDL cholesterol ( r= 0.92, P< 0.001).
Apo B was measured in sera from patients with abetalipoproteinaemia, familial hypercholesterolaemia and Tangiers disease. Apo B was not detected in the serum of subjects with abetalipoproteinaemia, while the apo B level in the familial hypercholesterolaemic subjects was significantly elevated (range 3.26–4.94 g/l) compared to normals (P<0.001). Serum apo B (0.80 g/l) of the subject with Tangier disease was within the normal range.     

Domain characteristics of the cyanogen bromide fragment 121–316 of thermolysin

The molecule of thermolysin was shown by X-ray crystallography to be composed of two structural domains of equal size comprising residues 1–157 and 158–316. In order to explore the possibility that these domains correspond to globular fragments able to refold autonomously, we have investigated the conformational and stability properties of fragment 121–316, which was obtained by limited chemical cleavage of thermolysin with cyanogen bromide. As judged by far-ultraviolet circular dichroism measurements, in aqueous solution under neutral conditions the fragment maintains a relative amount of helical structure which is comparable to that exhibited by the corresponding region in native thermolysin. The secondary structure attained by the fragment appears remarkably stable to the denaturing action of heat. By measuring the temperature dependence of the dichroic signal at 220 nm a Tm near 74d? was obtained. Immunodiffusion analyses indicated that the fragment recognizes and precipitates antibodies raised in rabbits using native thermolysin as immunogen. The overall conformational and immunochemical data indicate that fragment 121–316 of thermolysin is able to refold into a stable structure of native-like characteristics independently of the rest of the molecule. The results of this study complement those previously reported for fragment 206–316 (Vita, C., Fontana, A., Seeman, J.R. & Chaiken, I.M. (1979) Biochemistry 18 , 3023–3031).     

Failure of Endoscopy to Establish a Source for Upper Gastrointestinal Bleeding

In a series of 500 patients undergoing emergency endoscopic examination to detect the source(s) for upper gastrointestinal bleeding, the examination failed in its purpose in 55 cases (11%). This group was analyzed. Thiry-seven of these individuals demonstrated large and extensive esophagogastric varices which, while not observed to bleed during the examination, represented a potential bleeding source of great significance. The clinical implication of this endoscopic finding is described. Variceal bleeding frequently recurs sporadically, ceases abruptly and leaves no visible evidence of the point of rupture. Lacking this latter factor, the endoscopist is usually reluctant to assign responsibility for bleeding to these lesions. When, however, large varices are discovered as the sole potential source for bleeding, they may be assumed, with good reason, to represent the actual source and specific treatment logically may be instituted.     

THERMOLYSIN AND BACILLUS SUBTILIS NEUTRAL PROTEASE

A comparative study on thermolysin from Bacillus thermoproteolyticus and neutral protease from Bacillus subtilis involving far-and near-ultraviolet circular dichroism (CD) and immunological techniques is reported. These enzymes are homologous metalloendopeptidases, having similar size, kinetic behaviour, substrate specificity and susceptibility to inhibitors. The far-ultraviolet CD spectrum of each protein shows a minimum at 208 and a shoulder near 220 nm; differences in the extent of ellipticity, however, have been observed. Estimates of secondary structure obtained by quantitation of the far-ultraviolet CD spectra indicated a higher helicity of neutral protease relative to thermolysin. In the presence of ethylenediaminetetraacetic acid, which removes calcium and the functional zinc ion from the metalloenzymes, neutral protease is immediately denatured, whereas thermolysin maintains a globular structure, although thermolabile. On the other hand, the zinc-specific chelating agent tetraethylenepentamine does not have measurable effects on the conformation and conformational stability of either protein. Marked higher stability to temperature and guanidine hydrochloride were observed for thermolysin as compared with neutral protease, as indicated by monitoring conformational transitions with CD measurements at 220 nm. Antisera prepared in rabbits using thermolysin as immunogen do not cross-react with neutral protease, indicating differences of surface structure between the two proteins. On the basis and limitations of the techniques employed, it is proposed that the two sequentially and functionally homologous metalloendopeptidases may have similar conformations at specific regions (active and binding sites, at least) of the polypeptide chain essential for biological function, while some variability in the structure of other regions may be tolerated.     

Antibacterial and mechanical properties of restorative materials combined with chlorhexidines

Chorhexidine gluconate or chlorhexidine dihydrochloride were added to a composite resin and a glass ionomer restorative material in concentrations of 0, 1, 2, 3, 5, and 10% by weight. Antibacterial activity was measured by inhibition of growth of S. viridans, S. pyogenes, S. mutans, L. acidophilus, and E. coli, for 4 days. Compressive, tensile, and restorative material-enamel adhesive shear strength tests were performed. The addition of chlorhexidine gluconate or chlorhexidine dihydrochloride increased the antibacterial activity of the composite resin and the glass ionomer restorative material and changed the mechanical properties of the restorative materials. The addition of chlorhexidine dihydrochloride resulted in mechanical properties closest to controls.     

Enzymatically active subunits of Bacillus stearothermophilus enolase bound to Sepharose

The octameric enolase from Bacillus stearothermophilus was immobilized onto Sepharose 4B activated by the cyanogen bromide reaction under conditions for achieving essentially a single-point attachment. The immobilized enzyme was dissociated with guanidine hydrochloride to yield bound monomeric enolase. The Sepharose-bound subunit regained activity upon removal of the denaturant. It was also possible to rehybridize immobilized monomers to native octamers. Of note, the thermal stability of the immobilized enolase subunit does not appreciably differ from that of the parent soluble octameric enzyme. Thus, these results indicate that single subunits of thermophilic enolase are active and that oligomerization is not a prerequisite for the enzymic activity as well as for thermal stability.     

Catheter Ablation of Ventricular Tachycardia: Role of the Underlying Etiology and the Site of Energy Delivery

The role of DC catheter ablation (CA) to treat patients with sustained monomorphic ventricular tachycardia (VT) is still debated. To assess the efficacy of VT CA, we studied the follow-up of 49 patients with VT who underwent CA. There were 33 patients with an old myocardial infarction [MI] (group G I) and 16 patients had noncoronary VT (group G II): CA was performed at the earliest endocardial activation (EEA)(20 patients in G I, 14 patients in G II) or at the area of slow conduction (ASC) (13 patients in GI, 2 patients in GII). During the mean follow-up of 35 ± 25 (1–79) months, there were 17 patients in G I (52%) and 12 patients in G II (75%) with VT recurrences (P < 0.05). Recurrences of VT was observed in 4 of 15 patients (27%) when CA was performed at the ASC, compared to 25 of 34 patients (74%) with CA at the EEA (P < 0.01). These data show that DC CA is more successful in patients with coronary artery disease, particularly when CA is performed at the ASC.