Biochemical Binding Assays Research Articles

Liquid-phase peptide synthesis on polyethylene glycol (PEG) supports using strategies based on the 9-fluorenylmethoxycarbonyl amino protecting group: application of PEGylated peptides in biochemical assays.

Stepwise synthetic assembly of polypeptide chains reversibly linked to polyethylene glycol represents a hybrid between traditional solution and solid-phase chemistries and combines the inherent advantages of both approaches. The technical simplicity and scalability of the liquid-phase peptide synthesis method renders it particularly attractive for multiple parallel syntheses, combinatorial approaches and the large-scale preparation of peptides. The versatile protection strategy based on the N alpha-fluorenylmethoxycarbonyl group commonly used in solid-phase peptide synthesis was adapted to the liquid-phase approach. Fluoride ions were used rather than the conventional organic base piperidine for the repetitive amino-deprotection step. Using a range of acid- and base-labile linkers between the polymer and the peptide, it was shown that free and fully side-chain protected peptides can be obtained using our version of the liquid-phase peptide synthesis method. Protocols for simultaneous multiple syntheses requiring a minimum of equipment are presented and the use of polyethylene glycol-bound peptides in biochemical binding and functional assay systems is demonstrated.

Down-regulation of protease-activated receptor-1 is regulated by sorting nexin 1.

Degradation or "down-regulation" of protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin, is critical for termination of receptor signaling. Toward understanding the molecular mechanisms by which activated PAR1 is internalized, sorted to lysosomes, and degraded, we investigated whether PAR1 interacted with sorting nexin 1 (SNX1). SNX1 is a membrane-associated protein that functions in lysosomal sorting of the epidermal growth factor receptor. In vitro biochemical binding assays revealed a specific interaction between a glutathione S-transferase fusion of SNX1 and PAR1. In HeLa cells, activated PAR1 colocalized with endogenous SNX1 and coimmunoprecipitated SNX1. SNX1 contains a phox homology domain predicted to bind phosphatidylinositol-3-phosphate and a C-terminal coiled-coil region. To assess SNX1 function, we examined the effects of SNX1 deletion mutants on PAR1 trafficking. Neither the N terminus nor phox homology domain of SNX1 affected PAR1 trafficking. By contrast, overexpression of SNX1 C-terminal domain markedly inhibited agonist-induced degradation of PAR1, whereas internalization remained virtually intact. Immunofluorescence microscopy studies revealed substantial PAR1 accumulation in an early endosome antigen-1-positive compartment in agonist-treated cells expressing SNX1 C terminus. By contrast, lysosome-associated membrane protein-1 distribution was unperturbed. Together, these findings strongly suggest a role for SNX1 in sorting of PAR1 from early endosomes to lysosomes. Moreover, this study provides the first example of a protein involved in lysosomal sorting of a G protein-coupled receptor in mammalian cells.

Gβ Residues That Do Not Interact with Gα Underlie Agonist-independent Activity of K+ Channels

Gbetagamma subunits interact directly and activate G protein-gated Inwardly Rectifying K(+) (GIRK) channels. Little is known about the identity of functionally important interactions between Gbetagamma and GIRK channels. We tested the effects of all mammalian Gbeta subunits on channel activity and showed that whereas Gbeta1-4 subunits activate heteromeric GIRK channels independently of receptor activation, Gbeta5 does not. Gbeta1 and Gbeta5 both bind the N and C termini of the GIRK1 and GIRK4 channel subunits. Chimeric analysis between the Gbeta1 and Gbeta5 proteins revealed a 90-amino acid stretch that spans blades two and three of the seven-propeller structure and is required for channel activation. Within this region, eight non-conserved amino acids were critical for the activity of Gbeta1, as mutation of each residue to its counterpart in Gbeta5 significantly reduced the ability of Gbeta1 to stimulate channel activity. In particular, mutation of residues Ser-67 and Thr-128 to the corresponding Gbeta5 residues completely abolished Gbeta1 stimulation of GIRK channel activity. Mapping these functionally important residues on the three-dimensional structure of Gbeta1 shows that Ser-67, Ser-98, and Thr-128 are the only surface accessible residues. Galpha(i)1 interacts with Ser-98 but not with Ser-67 and Thr-128 in the heterotrimeric Galphabetagamma structure. Further characterization of the three mutant proteins showed that they fold properly and interact with Ggamma2. Of the three identified functionally important residues, the Ser-67 and Thr-128 Gbeta mutants significantly inhibited basal currents of a channel point mutant that displays Gbetagamma-mediated basal but not agonist-induced currents. Our findings indicate that the presence of Gbeta residues that do not interact with Galpha are involved in Gbetagamma interactions in the absence of agonist stimulation.

Spectroscopic characterization of insulin and small molecule ligand binding to the insulin receptor

We have applied spectroscopic techniques to study two kinds of ligand binding to the insulin receptor. First, a fluorescently labelled insulin analogue is used to characterize the mechanism of reversible 1 :1 complex formation with a fragment of the insulin receptor ectodomain. The receptor induced fluorescence enhancement of the labelled insulin analogue provides the basis for stopped flow kinetic experiments. The kinetic data are consistent with a bimolecular binding event followed by a conformational change. This emphasizes the importance of insulin induced conformational changes in the activation of the insulin receptor. Second, the binding of fluorescein derivatives to the insulin receptor is studied. These small molecule ligands displace insulin from its receptor with micromolar affinity. The binding is verified by transferred NOESY NMR experiments. Their chromophoric properties are used to measure the affinity by UV‐vis and fluorescence difference spectroscopies and the resulting Kd values are similar to those observed in the displacement receptor binding assay. However, these experiments and a stoichiometry determination indicate multiple binding sites, of which one overlaps with the insulin binding site. These two examples illustrate how spectroscopy complements biochemical receptor binding assays and provides information on ligand–insulin receptor interactions in the absence of three dimensional structures.

Inhalation of Low Concentrations of Toluene Induces Persistent Effects on a Learning Retention Task, Beam-Walk Performance, and Cerebrocortical Size in the Rat

The organic solvent toluene is widely used in industry. The threshold limit value for extended occupational exposure to toluene is presently set to 200 ppm in the United States. We have investigated the effect of an inhalation exposure of 80 ppm for 4 weeks (6 h/day, 5 days/week), followed by a postexposure period of at least 4 weeks, on behavior and brain features in the rat. Toluene exposure appeared to affect spatial memory, since toluene-exposed rats showed a longer time in the correct quadrant in a Morris swim maze. This effect may indicate that the exposed rats used their praxis strategy longer before they started to look for the platform elsewhere. Toluene-exposed rats showed trends for increases in both locomotion and rearing behaviors and a significantly reduced beam-walk performance. The area of the cerebral cortex, especially the parietal cortex, was decreased by 6–10% in toluene-exposed rats, as shown by magnetic resonance imaging of living rats and autoradiograms of frozen brain sections. The KD and Bmax values of the dopamine D3 agonist [3H]PD 128907 were not affected by toluene, as measured in caudate-putamen and subcortical limbic area using biochemical receptor binding assays and in caudate-putamen and islands of Calleja using quantitative receptor autoradiography. Hence, previously demonstrated persistent effects by toluene on the binding characteristics of radioligands binding to both D2 and D3 receptors seem to indicate a persistent effect of toluene selectively on dopamine D2 receptors. Taken together, the present results indicate that exposure to low concentrations of toluene leads to persistent effects on cognitive, neurological, and brain-structural properties in the rat.

Immunohistochemical Biomarkers in Breast Cancer

Most treatment decisions for breast cancer patients are based on an assessment of prognostic and predictive factors. The traditional staging variables (tumor size, axillary nodal status, and distant metastasis) remain the most important and powerful prognostic factors. Estrogen and progesterone receptor status as assessed by biochemical ligand binding assays are the only predictive factors recommended for routine clinical use. However, most laboratories today are evaluating hormone receptors, and often many other factors (eg, Ki67, c-erbB-2, p53, angiogenesis, micrometastasis), almost exclusively by immunohistochemistry (IHC) on fixed archival tissue. None of these tests currently meets the guidelines for prognostic and predictive factors agreed upon by expert panels of pathologists and oncologists. Although many studies suggest that these factors as assessed by IHC may indeed be helpful in making treatment decisions, overall their clinical utility is still controversial and there are important unresolved technical issues in how to measure them. A few laboratories have developed sensitive, specific, and reproducible IHC assays for evaluating most of these factors and demonstrated their usefulness in clinical studies. However, most laboratories offering these tests have not validated them and may not even be aware of the issues needing attention. Unless laboratories validate their tests, or follow the procedures of others who have, they run the risk of reporting wasteful, meaningless and potentially harmful results. (The J Histotechnol 22:249, 1999)

Different domains of the M-band protein myomesin are involved in myosin binding and M-band targeting.

Myomesin is a 185-kDa protein located in the M-band of striated muscle where it interacts with myosin and titin, possibly connecting thick filaments with the third filament system. By using expression of epitope-tagged myomesin fragments in cultured cardiomyocytes and biochemical binding assays, we could demonstrate that the M-band targeting activity and the myosin-binding site are located in different domains of the molecule. An N-terminal immunoglobulin-like domain is sufficient for targeting to the M-band, but solid-phase overlay assays between individual N-terminal domains and the thick filament protein myosin revealed that the unique head domain contains the myosin-binding site. When expressed in cardiomyocytes, the head domains of rat and chicken myomesin showed species-specific differences in their incorporation pattern. The head domain of rat myomesin localized to a central area within the A-band, whereas the head domain of chicken myomesin was diffusely distributed in the cytoplasm. We therefore conclude that the head domain of myomesin binds to myosin but that this affinity is not sufficient for the restriction of the domain to the M-band in vivo. Instead, the neighboring immunoglobulin-like domain is essential for the precise incorporation of myomesin into the M-band, possibly because of interaction with a yet unknown protein of the sarcomere.

Agonist-independent internalization and activity of a C-terminally truncated somatostatin receptor subtype 2 (delta349).

The rat somatostatin receptor subtype 2 (SSTR2) is rapidly internalized and phosphorylated in the presence of somatostatin 14 (SST14). Several C-terminal deletion constructs of SSTR2 have been investigated for their ability to undergo agonist-dependent internalization by using biochemical ligand binding assays and confocal microscopic analysis. Whereas mutant receptors lacking either 10 (delta359), 30 (delta339), or 44 (delta325) amino acid residues at the C terminus required SST14 for internalization, a construct lacking the last 20 amino acids (delta349) was detected mostly intracellularly and independently of the presence of the agonist. When internalization was blocked by sucrose, the delta349 receptor remained at the cell surface, strongly indicating that this mutant is internalized in an agonist-independent fashion. An increased affinity for agonists as measured in membrane binding assays and a reduced level of forskolin-stimulated cyclic AMP accumulation in human embryonic kidney cells expressing delta349 are properties that are characteristic of agonist-independent receptor activity. Delta349 is not phosphorylated detectably in the absence of agonist, demonstrating that phosphorylation per se is not a prerequisite for internalization of SSTR2. This observation is in line with data obtained for the delta325 mutant, which was internalized in an agonist-dependent manner, but not phosphorylated in either the presence or absence of SST14. We conclude that truncation of the SSTR2 C terminus at position 349 leads to agonist-independent, constitutive activity and internalization.

Complex binding interactions between multicomponent mixtures and odorant receptors in the olfactory organ of the Caribbean spiny lobster Panulirus argus.

Our study was designed to examine how components of complex mixtures can inhibit the binding of other components to receptor sites in the olfactory system of the spiny lobster Panulirus argus. Biochemical binding assays were used to study how two- to six-component mixtures inhibit binding of the radiolabeled odorants taurine, L-glutamate and adenosine-5'-monophosphate to a tissue fraction rich in dendritic membrane of olfactory receptor neurons. Our results indicate that binding inhibition by mixtures can be large and is dependent on the nature of the odorant ligand and on the concentration and composition of the mixture. The binding inhibition by mixtures of structurally related components was generally predicted using a competitive binding model and binding inhibition data for the individual components. This was not the case for binding inhibition by most mixtures of structurally unrelated odorants. The binding inhibition for these mixtures was generally smaller than that for one or more of their components, indicating that complex binding interactions between components can reduce their ability to inhibit binding. The magnitude of binding inhibition was influenced more by the mixture's precise composition than by the number of components in it, since mixtures with few components were sometimes more inhibitory than mixtures with more components. These findings raise the possibility that complex binding interactions between components of a mixture and their receptors may shape the output of olfactory receptor neurons to complex mixtures.

Selective decline of 5-HT 1A receptor binding sites in rat cortex, hippocampus and cholinergic basal forebrain nuclei during aging

The effect of aging on 5-HT 1A receptor binding in several forebrain areas associated with the basal forebrain cholinergic system was investigated in rats of 3-, 24- and 30-months-old by receptor autoradiography and biochemical binding assay using [ 3H]8-OH-DPAT as a ligand. Autoradiographic measurements demonstrated a marked region-specific decline of ligand binding in: (i) regions of the basal forebrain cholinergic cell groups, i.e. the medial septum, diagonal band nuclei and magnocellular nucleus basalis, (ii) the frontal and parietal neocortex and (iii) the dentate gyrus of the hippocampus. No change or only a slight decrease of the 5-HT 1A receptor density was found in other areas investigated: the CA1 and CA3 sectors of hippocampus, the cingular and perirhinal cerebral cortex and the lateral septum. The autoradiographic findings were substantiated by the biochemical binding assay, which revealed a comparable loss of 5-HT 1A receptor in the hippocampus and neocortex at the age of 30 months. The results clearly show that with increasing age the decrement of 5-HT 1A receptor binding in the rat forebrain is remarkably region-selective and particularly affects the cholinergic cell groups that innervate cortex and hippocampus. This phenomenon appears to be especially significant in relation to the neuronal substrates underlying the age-related alterations of mood and cognition.

Endocytosis of the rat somatostatin receptors: subtype discrimination, ligand specificity, and delineation of carboxy-terminal positive and negative sequence motifs.

Endocytosis of the five rat somatostatin receptor subtypes (SSTR1-5) was investigated in transfected HEK cells by biochemical ligand binding assays and confocal microscopic analysis. Phenylarsine oxide-sensitive internalization of SSTR1-3 is dependent on SST-14 or SST-28, whereas only the octacosapeptide triggers this reaction with SSTR5. SSTR4 is not internalized with either SST. Internalized SSTR3 is cycled back to the plasma membrane while endocytosed rho-Ala1-SST-14 remains inside the cell. Delineation of sequence motifs responsible for internalization of SSTR3 revealed multiple serines and a threonine (Ser-341, Ser-346, Ser-351, and Thr-357) within the carboxy-terminal tail of which Ser-351 and Thr-357 were the most effective ones. Chimeras in which various segments of the carboxyl terminus of SSTR4 were replaced by the corresponding regions of SSTR3 were internalized as long as they contain the Ser/Thr motif. However, this internalization reaction was suppressed when the chimeras were extended by the carboxyl terminus of SSTR4 (residues 320-384), suggesting the presence of a negative control element in that region. Step-wise truncation of the carboxyl terminus of wild-type SSTR4 revealed a motif of three amino acid residues Glu-Thr-Thr (SSTR4-330-332) that is responsible for preventing internalization and may be important in regulating endocytosis of this receptor subtype.

Felodipine inhibits intimal lesion formation in the hypercholesterolemic rabbit: differential effects on endothelial and monocyte determinants of atherogenesis.

The purpose of this study was to determine if the calcium entry antagonist felodipine inhibited intimal lesion formation in hypercholesterolemic rabbits, and to determine if this was due to an effect upon monocyte and/or endothelial determinants of this interaction. Twenty-three male New Zealand White rabbits received the following treatment regimen for 10 weeks: normal chow (NP, n = 3); normal chow with felodipine infusion (NF, n = 6); 0.5% cholesterol chow (CP, n = 7); or 0.5% cholesterol chow and felodipine infusion (CF, n = 7). After 10 weeks blood was collected for biochemical measurements and mononuclear cell binding assays, and thoracic aortae were harvested for vascular reactivity studies and histomorphometry. In the animals receiving normal chow, felodipine did not significantly affect blood pressure, plasma cholesterol levels, binding studies, vascular reactivity, or structure; therefore these animals were analyzed as one group (N). Plasma cholesterol levels were significantly elevated in groups receiving the 0.5% cholesterol diet (N, 29 +/- 3 mg/dl; CP, 1221 +/- 73 mg/dl; CF, 979 +/- 108 mg/dl). High-density lipoprotein cholesterol was not different between the groups (25 +/- 4 vs 23 +/- 4 vs 27 +/- 4 mg/dl; N vs CF vs CP respectively; p = NS). Cholesterol feeding markedly augmented the adhesiveness of mononuclear cells, as demonstrated by a 250% increase in cell binding. Felodipine did not alter the adhesiveness of mononuclear cells in hypercholesterolemic animals. Cholesterol feeding significantly impaired endothelium-dependent relaxations. Endothelium-dependent relaxations were restored by felodipine treatment as reflected by the maximal responses to acetylcholine (40 +/- 7% vs 58 +/- 4% vs 67 +/- 5%; CP vs CF vs N respectively). The improvement in endothelium-dependent relaxation in the felodipine-treated animals was associated with a 2.2-fold reduction in lesion surface area of the thoracic aorta (8.2 +/- 6.3% vs 18.2 +/- 9.5%; CF vs CP; p < 0.01). Moreover, the intima/media ratio reflecting lesion thickness was substantially reduced by felodipine treatment (0.05 +/- 0.02 vs 0.20 +/- 0.07; CF vs CP; p = 0.006). Ex vivo studies revealed that felodipine inhibited the adhesiveness of vascular endothelium, but not mononuclear cells, derived from hypercholesterolemic animals. Low-dose felodipine appears to inhibit monocyte-endothelial interaction, as indicated by a reduction in the formation of lesions in hypercholesterolemic animals. This effect is not due to an alteration in adhesiveness of mononuclear cells. The salutary effect of felodipine is associated with an increase in vascular nitric oxide activity which may reduce endothelial adhesiveness.

Binding of Peptides from the N-Terminal Region of α-Gliadin to the Celiac Disease-Associated HLA-DQ2 Molecule Assessed in Biochemical and T Cell Assays

Celiac disease (CD) is most probably an immunological disease, precipitated in susceptible individuals by ingestion of wheat gliadin and related proteins from other cereals. The disease shows a strong HLA association predominantly to thecis- ortrans-encoded HLA-DQ(α1*0501, β1*02) (i.e., DQ2) heterodimer. T cell recognition of gliadin peptides presented by DQ2 in the intestinal mucosa is central in the immunopathogenesis of CD. Here we describe a study where overlapping peptides from the N-terminal region of α-gliadin have been tested in biochemical assays for binding to affinity-purified DQ2 and DR3 (i.e., DR(α, β1*0301)) molecules. The peptides were also tested for binding to DQ2 in a functional binding assay, where binding was measured as the capacity to inhibit the stimulation of a gliadin-specific, DQ2-restricted T lymphocyte clone RNnTα33. In both assay systems the overlapping gliadin peptides were found to bind with weak or intermediate affinity to DQ2. No or only very weak binding was found to DR3 in the biochemical binding assay. Overall, the results question the role of these peptides in the T-cell-mediated immunopathogenesis of CD. Thein vitroassays described here provide new methods for the screening of potentially toxic peptides.

Developmental profile of estrogen receptor mRNA in the preoptic area of male and female neonatal rats

Exposure to estrogen or estrogenic metabolites of testosterone during the early postnatal period has permanent effects on rodent brain development. Differential sensitivity to estrogen, as reflected by transcription of the estrogen receptor gene, might determine the period of maximal sensitivity to the masculinizing effects of estrogen. We used an 35S-labeled riboprobe and in situ hybridization to chart the development of estrogen receptor (ER) mRNA expression in the rat preoptic area, a brain region for which sexual dimorphisms and the effects of estrogen on development are particularly well documented. Neonatal male and female rats were sacrificed by perfusion fixation on postnatal days 0, 2, 4, 7 or 10 (PND; day of birth is PND 0). Many ER mRNA-containing, cells were detected in the periventricular preoptic area and medial preoptic nucleus and the distribution of ER-synthesizing cells was similar in both sexes. Analysis of film autoradiograms showed that the relative steady state level of ER mRNA was significantly higher in females than in males at all ages except PND 0 and 10. The temporal profile of ER mRNA expression was different in males and females. ER mRNA did not change with age in males, whereas in females, ER mRNA was significantly higher on PND 2 compared with PND 0 and 10. These results demonstrate that the pattern of ER mRNA expression is quantitatively and qualitatively different between the sexes during the neonatal period. The pattern of ER mRNA expression contrasts markedly with previous reports of estrogen binding based on biochemical and autoradiographie steroid binding assays. The profile of ER mRNA expression is not consistent with the hypothesis that a decrease in estrogen receptor availability terminates the critical period for estrogen-dependent masculinization of the preoptic area.

Binding of Ras Oncogene Peptides to Purified HLA‐DQ(αl*0102, ß1*0602) and ‐DR(α, ß1*0101) Molecules

Mutated oncogene peptides may be presented to T cells by HLA molecules. To be able to design the optimal peptides for stimulation of T cells in individuals with different HLA molecules, it is important to analyse the binding characteristics of oncogene peptides to HLA. HLA‐DQ6 (DQ(α1*0102, ß1*0602)) and HLA‐DRI (DR(α, ßl*0101)) molecules were purified from lysates of homozygous EBV‐transformed cell lines. Purified HLA molecules were then tested for their ability to bind synthetic peptides in gel filtration assays. A p21 ras oncogene peptide (previously found to stimulate DQ6‐restricted T‐cell clones) and an influenza matrix peptide were labelled with 125I and served as indicator peptides for binding to DQ6 and DR1 respectively. Binding of homologous truncated and mutated p21 ras peptides and unrelated peptides was then evaluated by their capacity to inhibit binding of the indicator peptides. p21 ras‐derived peptides were found to bind to both DQ6 and DR1 molecules indicating the existence of a promiscuous binding motif in these peptides. The binding affinities seemed to vary between the different peptides, but the amino acid substitutions resulting from natural mutations were not critical for binding. Notably, the results obtained for DQ6 in the biochemical peptide binding assay correlated well with results obtained in a functional assay using T‐cell clones as probes.

Direct binding of autoimmune disease related T cell epitopes to purified Lewis rat MHC class II molecules.

New strategies applied in the treatment of experimental autoimmune disease models involve blocking or modulation of MHC–peptide–TCR interactions either at the level of peptide–MHC interaction or, alternatively, at the level of T cell recognition. In order to identify useful competitor peptldes one must be able to assess peptide–MHC interactions. Several well described autoimmune disease models exist in the Lewis rat and thus this particular rat strain provides a good model system to study the effect of competitor peptldes. So far no information has been available on the peptide binding characteristics of the Lewis rat MHC class II RT1.BI molecule. We have now developed a biochemical binding assay which enables competition studies in which the relative MHC binding affinity of a set of non-labelled peptldes can be assessed while employing detection of blotlnylated marker peptides by chemllumlnescence. The assay is sensitive and specific. We have used this assay to determine the binding characteristics of several disease associated T cell determinants and their sequence analogues in the Lewis rat. Notably, most of the autoimmune disease associated peptide sequences tested were found to be intermediate to poor binders. Single amino acid substitutions at defined positions were sufficient to turn certain peptldes into good binders. These results are relevant to the design of competitor peptldes in the treatment of experimental autoimmune diseases.

Binding of [ 3H]MK-801, NMDA-displaceable [ 3H]glutamate, [ 3H]glycine, [ 3H]spermidine, [ 3H]kainate and [ 3H]AMPA to regionally discrete brain membranes of the gerbil: A biochemical study

Bindings of glutamate receptor agonists and related modulators were investigated in 10 discrete tissues from gerbil brain using a biochemical technique. There appeared considerable discrepancies, in respect of intrahippocampal profiles, from reported data by autoradiography on rat brain. In the gerbil, an almost equivalent level of N- methyl- d-aspartate (NMDA)-displaceable [ 3H]glutamate binding was found in field CA1 and the dentate gyrus, while approx 30% less in field CA3, a profile which was strikingly similar to that of (+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]-cycloheptene-5,10-imine maleate (MK-801) or of [ 3H]glycine. [ 3H]Kainate binding was highest in the dentate gyrus followed by field CA3 and then field CA1, the ratio of the highest to the lowest being 3 to 2. Binding of [ 3H] dl-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) differed, to a certain extent, from that of [ 3H]kainate and showed the dentate gyrus followed by field CA1 and then field CA3 in the rank order of decreasing binding. Taking together, intrahippocampal localization of glutamate receptor subtypes in the gerbil, when analyzed with a biochemical binding assay, looks to be less region selective than the distribution obtained on autoradiography in the rat. Thus, it is likely that these different distribution profiles show different status of receptor function respectively, or are due merely to species difference.

PH dependence of the interaction between immunogenic peptides and MHC class II molecules. Evidence for an acidic intracellular compartment being the organelle of interaction.

The pH dependence of the interaction between immunogenic peptide and MHC class II was studied both in a direct biochemical binding assay and in a functional Ag presentation assay. The two approaches yielded similar results. All of the peptides tested bound optimally to their relevant MHC class II restriction element at around pH 4.5. Indeed, several of the peptides did not bind at neutral pH. These results demonstrate that Ag under physiologic conditions meet MHC class II in a quite acidic environment. The very acidic pH optimal for peptide-MHC class II interaction is only found intracellularly and most notably in the endosome-lysosome compartment in which Ag processing is thought to occur. Thus, Ag processing and interaction with MHC class II molecules can potentially happen in the very same compartment. This yet undefined acidic compartment would have to contain proteolytic enzymes and MHC class II molecules.

Relationship between epidermal growth factor receptor status and various prognostic factors in human breast cancer

Relationship between epidermal growth factor receptor (EGFR) status and various prognostic factors was investigated in 91 human breast cancer tissues. Epidermal growth factor receptor was measured by biochemical competitive binding assay using iodine 125 epidermal growth factor (125I)-EGF. The EGFR status was not correlated with axillary lymph node involvement, tumor size, stage, and histologic type, but significantly correlated with histologic grading (P less than 0.05) and lymphatic invasion (P less than 0.01). Between EGFR and estrogen receptor (ER) status, a clear inverse relationship was observed (P less than 0.01). The Ki-67-positive stained cell rate, which reveals the proportion of cycling cells, was significantly higher in EGFR-positive tumor tissues than in EGFR-negative cases. Furthermore, preliminary postoperative survey demonstrated a high tendency of recurrence rate of patients with EGFR-positive tumors as compared with those with EGFR-negative tumors. These data suggest that EGFR status may be important for the prediction of biologically high malignant potential.

Assessment of oestrogen receptor content of breast carcinoma by immunohistochemical techniques on fixed and frozen tissue and by biochemical ligand binding assay.

The oestrogen receptor content of 61 breast carcinomas was assessed by biochemical ligand binding assay and three immunohistochemical techniques--a frozen section method (Abbott ER-ICA) and on paraffin wax sections after fixation by two methods. The two fixatives used were Carson's buffered formalin and methacarn, and a DNAse pretreatment of sections was used. Overall agreement for the immunohistochemical methods with the ligand binding technique were 95%, 85%, and 86% for the frozen, formalin, and methacarn methods, respectively. A semiquantitative staining score was performed and all three methods gave significant correlations of staining scores with biochemical ligand binding values. The frozen section method was best (r = 0.88) with the fixed tissue methods yielding poorer correlation coefficients. Several factors affected staining, including the nature of the fixative and variable activity of DNAse. It is concluded that immunohistochemical assessment of oestrogen receptor content on fixed tissue provides acceptable qualitative information but that standardisation of protocols for tissue processing will be necessary for optimal utility and especially for quantitative assessments.