Aglycosylation of human IgG1 and IgG3 monoclonal antibodies can eliminate recognition by human cells expressing Fc gamma RI and/or Fc gamma RII receptors.

Aglycosylated human IgG1 and IgG3 monoclonal anti-D (Rh) and human IgG1 and IgG3 chimaeric anti-5-iodo-4-hydroxy-3-nitrophenacetyl (anti-NIP) monoclonal antibodies produced in the presence of tunicamycin have been compared with the native glycosylated proteins with respect to recognition by human Fc gamma RI and/or Fc gamma RII receptors on U937, Daudi or K562 cells. Human red cells sensitized with glycosylated IgG3 form rosettes via Fc gamma RI with 60% of U937 cells. Inhibition of rosette formation required greater than 35-fold concentrated more aglycosylated than glycosylated human monoclonal anti-D (Rh) antibody. Unlabelled polyclonal human IgG and glycosylated monoclonal IgG1 and anti-D (Rh) antibody inhibited the binding of 125I-labelled monomeric human IgG binding by U937 Fc gamma RI at concentrations greater than 50-fold lower than the aglycosylated monoclonal IgG1 anti-D (Rh) (K50 approximately 3 x 10(-9) M and approximately 6 x 10(-7) M respectively). Similar results were obtained using glycosylated and aglycosylated monoclonal human IgG1 or IgG3 chimaeric anti-NIP antibody-sensitized red cells rosetting with Fc gamma RI-/Fc gamma RII+ Daudi and K562 cells. Rosette formation could be inhibited by the glycosylated form (at greater than 10(-6) M) but not by the aglycosylated form. Haemagglutination analysis using a panel of murine monoclonal antibodies specific for epitopes located on C gamma 2, C gamma 3 or C gamma 2/C gamma 3 interface regions did not demonstrate differences in Fc conformation between the glycosylated or aglycosylated human monoclonal antibodies. These data suggest that the Fc gamma RI and Fc gamma RII sites on human IgG are highly conformation-dependent and that the carbohydrate moiety serves to stabilize the Fc structure rather than interacting directly with Fc receptors.     

Central noradrenergic activity in intact and sinoaortic denervated Dahl rats

Lesions in forebrain areas richly innervated by noradrenergic terminals and involved in cardiovascular function reduce or prevent hypertension in the Dahl salt-sensitive (S) rats fed a high (H) salt diet. This led us to examine two questions. (1) Is the noradrenergic activity altered in discrete forebrain and brainstem areas of SH rats? (2) Are these changes in noradrenergic activity eliminated by sinoaortic denervation (SAD)? Studies were done in 10-week-old female SH and Dahl salt-resistant (RH) rats. Half of the rats in each group had SAD surgery 1 week prior to study. An index of norepinephrine (NE) turnover was determined by measuring the decline in tissue NE concentration 8 h after administering alpha-methyl-p-tyrosine, a NE synthesis blocker, to animals from each of four groups: sham-RH, SAD-RH, sham-SH, and SAD-SH (n = 18-20 per group). Various discrete brain areas were obtained using the "punch technique." In SH rats the index of NE turnover was increased in the median preoptic nucleus and decreased in the paraventricular nucleus compared with RH rats regardless of SAD. In contrast, in SH rats the index of NE turnover was increased in the supraoptic nucleus and locus ceruleus compared with RH rats; however, SAD-RH had greater turnover of NE at these sites than SAD-SH. In summary, changes in noradrenergic activity in the median preoptic nucleus and the paraventricular nucleus may be related to genetic predisposition to hypertension in SH rats. In contrast, changes in the locus ceruleus and the supraoptic nucleus of SH rats may be related to impaired baroreflexes and thereby contribute to hypertension.     

Membrane solubilization by detergent: use of brominated phospholipids to evaluate the detergent-induced changes in Ca2+-ATPase/lipid interaction

The solubilization and delipidation of sarcoplasmic reticulum Ca2+-ATPase by different nonionic detergents were measured from changes in turbidity and recovery of intrinsic fluorescence of reconstituted ATPase in which tryptophan residues had been quenched by replacement of endogenous phospholipids with brominated phospholipids. It was found that incorporation of C12E8 or dodecyl maltoside (DM) at low concentrations in the membrane, resulting in membrane "perturbation" without solubilization, displaced a few of the phospholipids in contact with the protein; perturbation was evidenced by a parallel drop in ATPase activity. As a result of further detergent addition leading to solubilization, the tendency toward delipidation of the immediate environment of the protein was stopped, and recovery of enzyme activity was observed, suggesting reorganization of phospholipid and detergent molecules in the solubilized ternary complex, as compared to the perturbed membrane. After further additions of C12E8 or DM to the already solubilized membrane, the protein again experienced progressive delipidation which was only completed at a detergent concentration about 100-fold higher than that necessary for solubilization. Delipidation was correlated with a decrease in enzyme activity toward a level similar to that observed during perturbation. On the other hand, Tween 80, Tween 20, and Lubrol WX failed to solubilize SR membranes and to induce further ATPase delipidation when added after preliminary SR solubilization by C12E8 or dodecyl maltoside. For Tween 80, this can be related to an inability to solubilize pure lipid membrane; in contrast, Tween 20 and Lubrol WX were able to solubilize liposomes but not efficiently to solubilize SR membranes. In all three cases, insertion of the detergent in SR membranes is, however, demonstrated by perturbation of enzyme activity. Correlation between detergent structure and ability to solubilize and delipidate the ATPase suggests that one parameter impeding ATPase solubilization might be the presence of a bulky detergent polar headgroup, which could not fit close to the protein surface. We also conclude that in the active protein/detergent/lipid ternary complexes, solubilized by C12E8 or dodecyl maltoside, most phospholipids remain closely associated with the ATPase hydrophobic surface as in the membranous form. Binding of only a few detergent molecules on this hydrophobic surface may be sufficient for inhibition of ATPase activity observed at high ATP concentration, both during perturbation and in the completely delipidated, solubilized protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

The perception of painful and nonpainful stimuli during voluntary motor activity in man

Previous studies have shown that voluntary movement diminishes the transmission of cutaneous afferent input through the dorsal column-medial lemniscal system, and also raises the threshold for detecting nonpainful, cutaneous stimuli (electrical shocks). Although there is some evidence that pain elicited by electrical stimulation is diminished during movement, no studies have tested the effect of movement on the perception of pain produced by natural stimulation. For this reason, we tested the effects of voluntary motor activity on the perception of noxious thermal stimuli in human volunteers. We first developed a motor paradigm in which the thermal stimulation could be applied to the immobile limb (isometric elbow flexion-extension). Both isometric and isotonic muscle contractions about the elbow increased the threshold for detecting weak cutaneous stimuli (electrical shocks) applied to the forearm, and to a lesser extent the detection of stimuli applied to the dorsum of the hand. Afterwards, noxious and innocuous heat stimuli were applied to the forearm during isometric contractions and at rest. Magnitude estimates for the intensity of the pain, as well as latency measures of the onset of pain, were recorded. We found no evidence that isometric motor activity diminished either the threshold for pain or the subjective intensity of the noxious and innocuous thermal stimuli. Thus, motor activity decreases the ability to detect weak low-threshold cutaneous inputs, but has no effect on the perception of warmth and heat pain.     

Synergism between thrombin and adrenaline (epinephrine) in human platelets. Marked potentiation of inositol phospholipid metabolism.

We have studied synergism between adrenaline (epinephrine) and low concentrations of thrombin in gel-filtered human platelets prelabelled with [32P]Pi. Suspensions of platelets, which did not contain added fibrinogen, were incubated at 37 degrees C to measure changes in the levels of 32P-labelled phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and phosphatidate (PA), aggregation and dense-granule secretion after stimulation. Adrenaline alone (3.5-4.0 microM) did not cause a change in any parameter (phosphoinositide metabolism, aggregation and dense-granule secretion), but markedly enhanced the thrombin-induced responses over a narrow range of thrombin concentrations (0.03-0.08 units/ml). The thrombin-induced hydrolysis of inositol phospholipids by phospholipase C, which was measured as the formation of [32P]PA, was potentiated by adrenaline, as was the increase in the levels of [32P]PIP2 and [32P]PIP. The presence of adrenaline caused a shift to the left for the thrombin-induced changes in the phosphoinositide metabolism, without affecting the maximal levels of 32P-labelled compounds obtained. A similar shift by adrenaline in the dose-response relationship was previously demonstrated for thrombin-induced aggregation and dense-granule secretion. Also, the narrow range of concentrations of thrombin over which adrenaline potentiates thrombin-induced platelet responses is the same for changes in phosphoinositide metabolism and physiological responses (aggregation and dense-granule secretion). Our observations clearly indicate that adrenaline directly or indirectly influences thrombin-induced changes in phosphoinositide metabolism.     

Assessment of methods for covalent binding of nucleic acids to magnetic beads, Dynabeads, and the characteristics of the bound nucleic acids in hybridization reactions.

Dynabeads are magnetic monosized beads with high stability, high uniformity, unique paramagnetic properties, low particle-particle interaction, and high dispersibility. Different reactive groups; hydroxyl, carboxyl and amino groups can be attached to the surface. Several methods for covalent attachment of DNA or oligonucleotides to the beads were investigated. Best coupling yields were obtained by carbodiimide-mediated end-attachment of 5'-phosphate and 5'-NH2 modified nucleic acids to respectively amino and carboxyl beads. The carboxyl beads showed a low degree of non-specific binding, while a better yield of end-attached nucleic acids was obtained using the amino beads. The DNA-beads worked efficiently in hybridization experiments, and the kinetics of hybridization approach those of solution hybridization.     

Linkage relationships in the bovine MHC region. High recombination frequency between class II subregions

Class II genes of the bovine major histocompatibility complex (MHC) have been investigated by Southern blot analysis using human DNA probes. Previous studies revealed the presence of bovine DO , DQ , DQ , DR and DR genes, and restriction fragment length polymorphisms for each of these genes were documented. In the present study, the presence of three additional class II genes, designated DZ , DY , and DY , are reported. DZ was assumed to correspond to the human DZ gene while the other two were designated DY because their relationship to human class II genes could not be firmly established. The linkage relationships among bovine class II genes and two additional loci, TCP1B and C4, were investigated by family segregation analysis and analysis of linkage disequilibrium. The results clearly indicated that all these loci belong to the same linkage group. This linkage group is divided into two subregions separated by a fairly high recombination frequency. One region includes the C4, DQ , DQ , DR and DR loci and the other one is composed of the DO DY , DY , and TCPIB loci. No recombinant was observed within any of these subregions and there was a strong or fairly strong linkage disequilibrium between loci within groups. In contrast, as many as five recombinants among three different families were detected in the interval between these subregions giving a recombination frequency estimate of 0.17 ± 0.07. The fairly high recombination frequency observed between class 11 genes in cattle is strikingly different from the corresponding recombination estimates in man and mouse. The finding implies either a much larger molecular distance between some of the bovine class II genes or alternatively the presence of a recombinational hot spot in the bovine class II region.     

Developmental expression of bovine adrenocortical steroid hydroxylases. Regulation of P-450(17 alpha) expression leads to episodic fetal cortisol production

The developmental expression of adrenocortical steroid hydroxylases was studied in bovine fetuses from 40 to 280 days gestational age. The expression of P-450(17 alpha) is first detected at a gestational age of 50 days and reaches a maximum at 60-70 days. The expression of P-450(17 alpha) then declines and is nondetectable at a gestational age of 100 days. P-450(17 alpha) is not expressed again until about 240 days, i.e. shortly before birth (approximately 280 days). P-450scc, P-450c21, P-450(11 beta) and adrenodoxin were present in fetal adrenals throughout gestation. This "on-off-on" pattern of P-450(17 alpha) expression during fetal development was associated with a corresponding episodic production of cortisol. Immunoreactive corticotropin (ACTH) levels in fetal plasma were elevated in small fetuses (corresponding to less than or equal to 100 days) and in near-term fetuses (corresponding to greater than 250 days) compared with those in mid-gestation fetuses. In primary culture, adrenal cells from mid-gestation fetuses contained no detectable P-450(17 alpha) but rapidly responded to ACTH with an increase in P-450(17 alpha) protein and mRNA. The tissue specificity of the developmental patterns is emphasized by the fact that both P-450(17 alpha) and P-450scc were detectable throughout the development of the fetal testes, whereas only P-450scc was detectable in fetal bovine ovary prior to 200 days. Thus, in fetal bovine adrenal it appears that ACTH is the major regulatory factor effecting the intermittent presence of P-450(17 alpha), whereas the presence of the other steroid hydroxylases is either regulated by additional factors or shows a much different sensitivity to ACTH.     

In situ hybridization using 32P labelled oligodeoxyribonucleotides for the cellular localisation of mRNA in neuronal and endocrine tissue

Summary Methodological variables for in situ hybridization using ³²P labelled oligodeoxyribonucleotides (oligomers) have been examined. Four different oligomers directed against proglucagon messenger RNA (mRNA) and two different oligomers against prosomatostatin mRNA have been used. Specific hybridization was obtained in adult rat brain, stomach and pancreas and in neonatal rat ileum. Tissue was perfusion fixed with 4% paraformaldehyde 0.2% glutaraldehyde and hybridization was carried out in 50% formamide for 72 h at 42° C. Using hybridization conditions of lower stringency (33% formamide) labelling was also obtained in guinea pig tissue. Other variables which affected hybridization signal intensity were the inclusion of a prehybridization dehydration stage, the probe concentration, the inclusion of ammonium acetate in the posthybridization dehydrating ethanols and in the autoradiographic emulsion, and the exposure time. The localisation of proglucagon mRNA in rat pancreas using a 20mer was used as a model tissue for testing these methodological variables and the results were found generally also to apply to the other probes and tissues tested. The methods described provide single cell resolution and show that ³²P labelled oligomers may be used to localise neuropeptide and endocrine mRNAs in different types of tissue and in different mammalian species.