An immunoblotting technique for the detection of bound and secreted bacterial Fc receptors

A rapid semiquantitative procedure that enables bacteria to be screened for surface or secreted receptors for the Fc region of human IgG is described. Surface Fc receptors were detected by direct transfer of bacterial colonies to nitrocellulose by electroblotting and then probing with ¹²⁵I-labeled human IgG in the presence of a two fold molar excess of unlabeled F(ab′)₂fragments. The blots were exposed to X-ray film and the intensity of the resulting autoradiograph was a measure of surface Fc receptors expression. This procedure reliably distinguished Staphylococcus aureus strains which expressed different levels of surface Fc receptors. When applied to the study of group A streptococci, a number of Fc receptor-positive strains were identified. Unlike the homogeneous Fc receptor expression on individual colonies of the staphylococcal strains, a wide variation in the level of Fc receptor expression was observed within a given streptococcal strain. Group A streptococcal substrains which expressed high and low levels of surface Fc receptors could be isolated from replica plates.Secreted Fc receptors were measured by a simple modification of the blotting procedure in which the nitrocellulose was placed on the opposite side of the agar from the bacterial colonies. Secreted Fc receptors was electroblotted through the agar onto nitrocellulose and probed as described above. This approach readily detected nanogram quantities of secreted type I Fc receptor (protein A) from the Staphylococcus aureus Cowan strain. None of the group A streptococcal strains tested were found to secrete detectable quantities of Fc receptors.     

Relative contribution of humoral and metastatic factors to the pathogenesis of hypercalcaemia in malignancy.

Some relations between metastatic bone disease and calcium homoeostasis were determined in a consecutive series of 81 patients with solid malignant tumours attending for radionuclide bone scans. Biochemical evaluation showed that bone resorption from metastatic disease was generally not enough to account for hypercalcaemia. While skeletal metastases were present in about half of the patients who developed hypercalcaemia, biochemical indices of bone resorption in these subjects were greatly increased and disproportionate to the extent of metastatic disease detected by the bone scans. Furthermore, a reduced renal phosphate threshold and increased tubular calcium reabsorption were generally observed in hypercalcaemic patients when compared with their normocalcaemic counterparts. These findings suggest that in most cases malignancy associated hypercalcaemia may be caused by the release of a humoral factor by tumour tissue which exhibits "parathyroid-hormone-like" activity with regard to bone resorption, renal phosphate threshold, and renal calcium handling. It may be postulated that this putative humoral mediator predisposes to hypercalcaemia both by stimulating generalised osteolysis and in most cases also by impairing the renal excretion of the resultant increase in filtered calcium load. While hypercalcaemia may arise as a result of metastatic bone disease alone, these data indicate that this may be the exception rather than the rule. Hence the term "metastatic hypercalcaemia" should probably be reserved for patients with extensive skeletal tumour disease in whom biochemical evaluation fails to yield evidence of an underlying humorally mediated cause.     

Studies on the terminal stages of immune hemolysis. VI. Osmotic blockers of differing Stokes' radii detect complement-induced transmembrane channels of differing size.

We have previously shown that 0.1 M EDTA could be used to distinguish functionally different transmembrane channels produced during complement-(C) mediated hemolysis of E. In this paper we have studied the ability of sugars of varying Stokes' radii to prevent hemoglobin release from E intermediates whose lysis was inhibitable or not inhibitable by EDTA. On the basis of these experiments we propose that the inhibition of E transformation by high molarity EDTA occurs by virtue of the size of the EDTA molecule in solution. Studies on the effect of EDTA on red cell lysis induced by polyene antibiotics that form transmembrane channels of a defined size support this conclusion. The results of these experiments were interpreted to mean: 1) The EDTA inhibitable lesion of E has a smaller effective radius than the noninhibitable lesion; 2) the effective radius of the smallest lesion that yields a lytic site was less than 3.6 A; 3) the lesions produced in the red cell membrane by C are not uniform but vary in size depending on the C9 to SACl-8 ratio used to produce E.     

Studies on the interaction between protein A and immunoglobulin G. I. Effect of protein A on the functional activity of IgG.

Staphylococcal protein (A (PA) and IgG anti-Forssman immunoglobulin formed complexes that behaved functionally like IgM in their ability to lyse sheep erythrocytes (E) in the presence of whole guinea pig complement (GPC) and to fix purified guinea pig C1. Concanavalin A, a plant lectin that inhibited IgM but not IgG hemolytic activity, inhibited the hemolytic activity of IgG-protein A complexes that behaved like IgM but had no effect on complexes that behaved functionally like IgG. Since Con A is known to bind specifically to glucose and mannose residues, our results suggested that the interaction of protein A with the Fc region of IgG led to exposure of sugar moieties that may participate in complement (C) binding. The production of IgM-like complexes depended on the ratio of protein A to IgG and the empirical formula of these IgM-like complexes was found to be [(IgG)2PA]n. As the ratio of PA to IgG was increased, the resulting complexes tended to behave functionally like IgG but with reduced hemolytic activity and C1 fixing ability. Furthermore, the binding of C1 to EIgG was inhibited by PA and the binding of PA to EIgG was inhibited by C1 indicating that the binding sites for C1 and PA were located near each other or were identical. Our results offer a reasonable explanation for the reported effects of PA or mixtures of PA and IgG in vitro and in vivo.     

Evaluation of phosphorus in forest soils: Comparison of phosphorus uptake,extraction method and soil properties

Summary Phosphorus in soils from plantation of red pine (Pinus resinosa Ait.) was determined using six extractants: 0.002N H₂SO₄ (pH 3.0); 0.025N HCl+ +0.03N NH₄F; 0.5N NaHCO₃ (pH 8.5);N NH₄OAc (pH 4.8); anion exchange resin (Dower –2, Cl-form); H₂O. Correlations of extractable P with Al- and Al-+Fe-P indicated that these fractions are the dominant forms of inorganic P in most of the soils.Uptake of P by corn and Monterey pine seedlings grown in greenhouse culture was correlated with soil P extracted by the different methods. The most successful of the extractants for predicting P uptake was resin extractable P; the simple correlation coefficients were 0.811 and 0.609 for pine and corn respectively. P uptake by pine correlated significantly with 0.002N H₂SO₄ P (r=0.679),N NH₄OAc P (r=0.443), H₂O P (r=0.549) and Al-+Fe-P (r=0.532) while P uptake by corn correlated with 0.002N H₂SO₄ P (r=0.579), H₂O P (r=0.477) and organic P (r=0.460). Per cent P in pine seedling tops correlated significantly with 0.002N H₂SO₄, resin andN NH₄ OAc extractable P. Multiple regressions which included silt+clay and organic P improved correlations of some soil tests with P uptake in corn and pine seedlings respectively.Research supported by the School of Natural Resources, College of Agricultural and Life Sciences, Univ. of Wisconsin, Madison, the Wisconsin Department of Natural Resources and FAO Fellowship to the senior author.     

The cardiac innervation of Eledone cirrhosa (Lamarck) (Mollusca: Cephalopoda)

The innervation to the cardiac organs and vessels of the octopods Eledone cirrhosa, E. moschata and Octopus vulgaris is described from vitally stained fresh material and wax-embedded sections. This innervation arises from the paired visceral nerves and includes two main peripheral ganglia (fusiform and cardiac) on each side. Several new details of the innervation are reported. Nerves supplying the lateral venae cavae arise from the ventricular nerves at the level of the ventricle. Nerve fibres run to the efferent branchial vessels from the cardiac ganglia. A small ganglion, lying on the auriculo-ventricular nerve, is described for some specimens of both species of Eledone, and is named the auricular ganglion. Commissural strands linking the right and left ventricular nerves of either side are found in Eledone, comparable to those previously described from Octopus. The detailed branching pattern of the innervation shows considerable individual variation and consistent interspecific differences. In E. cirrhosa the fine fibres innervating the inner and outer muscle layers of the auricle show distinct differences in their configuration. Innervation at the surface of the ventricular lumen and around the coronary arterial vessels shows evidence of specialization. The muscle of the branchial heart, particularly the valve leaflets at the junction of the heart and the lateral vena cava, is abundantly innervated. The observations are discussed in relation to other cephalopods and to their probable physiological significance. It is suggested that they provide evidence for a greater degree of neural influence in the control of the cardiac organs than is usually supposed and that they support the idea that the lateral venae cavae have a significant role in the generation of circulatory pressures.     

Cytolytic T lymphocyte responses to metabolically inactivated stimulator cell. I. Metabolic inactivation impairs both CD and LD antigen signals

The effects of metabolic inactivation of spleen cells on antigen presentation to precursors of alloreactive cytolytic T lymphocytes (T_c) were examined. By serological methods, populations inactivated by ultraviolet irradiation, glutaraldehyde fixation or plasma membrane isolation were found to retain normal levels of H-2K/D and Ia antigens. However, comparison of the antigen doses required to stimulate secondary T_c responses in mixed leukocyte culture showed that the inactivated preparations were approximately 10-fold less immunogenic than X-irradiated spleen cells. Their total inability to stimulate primary cytolytic responses pointed to at least a 100-fold impairment of immunogenicity for unprimed T_c precursors in the case of uv-irradiated and glutaraldehyde-treated stimulator cells, and at least a 10-fold impairment for membrane fragments. Experiments showing that the capacity of cell monolayers to absorb precursor T_c from unprimed spleen populations was reduced following uv-irradiation or glutaraldehyde treatment provided direct evidence that this loss of immunogenicity was due in part to suboptimal antigen presentation to precursor T_c. It is concluded that, in addition to the traditional view that these treatments damage the “LD” signal to helper T lymphocytes, metabolic inactivation also impairs recognition of “CD” determinants by precursor T_c.     

Evidence for functional heterogeneity in IgG Fc-binding proteins associated with group A streptococci

A number of group A streptococcal isolates have been compared for their nonimmune reactivity with each human IgG subclass, and rabbit, pig, or horse IgG. The results obtained demonstrate considerable heterogeneity in the expression of type II IgG-binding proteins among and within group A isolates. Extraction and analysis of type II IgG-binding proteins from selected strains demonstrate the existence of five functionally distinct IgG-binding proteins. The type IIo IgG binding protein displayed the greatest range of reactivities, binding to all four human IgG subclasses, and rabbit, pig, and horse IgG. A variant of this protein, designated type II'o, bound all four human subclasses and rabbit IgG, but failed to react with pig or horse IgG. A type IIa protein was recovered from certain group A strains which bound human IgG1, IgG2, IgG4, as well as reacting with rabbit, pig, and horse IgG. A functionally related type IIc activity that displayed all of the reactivities of the type IIa protein but did not bind with human IgG2 was also identified. The final functional form of group A IgG-binding protein, the type IIb protein, bound exclusively to human IgG3. Comparison of these functionally different type II IgG-binding proteins demonstrated no simple structure-function relationship. These studies underscore the heterogeneity of type II Ig-binding proteins expressed by different group A streptococci and document that a single strain can change its pattern of expression of type II IgG-binding protein both quantitatively and qualitatively.