Bodian's silver impregnation of endocrine cells

Summary We have recently shown that a variety of proteins, including albumin and immunoglobulins conjugated to colloidal gold, strongly binds to certain basic peptide sequences, and neurohormonal peptides. Silver proteinate, used in the classical Bodian's neurohistological procedure, is now shown to bind to the same peptide sequences in cytochemical model systems. In tissue, gastrin cells and glucagon cells have been reported to show strong unspecific immunocytochemical staining and these cell types also stain in the Bodian's procedure. These results suggest that certain types of unspecific immunocytochemical staining and the Bodian's silver staining method may depend upon a common mechanism, involving binding of labelled or aggregated protein to basic and hydrophobic sequences in tissue.     

Bodian's silver impregnation of endocrine cells. A tentative explanation to the staining mechanism

We have recently shown that a variety of proteins, including albumin and immunoglobulins conjugated to colloidal gold, strongly binds to certain basic peptide sequences, and neurohormonal peptides. Silver proteinate, used in the classical Bodian's neurohistological procedure, is now shown to bind to the same peptide sequences in cytochemical model systems. In tissue, gastrin cells and glucagon cells have been reported to show strong unspecific immunocytochemical staining and these cell types also stain in the Bodian's procedure. These results suggest that certain types of unspecific immunocytochemical staining and the Bodian's silver staining method may depend upon a common mechanism, involving binding of labelled or aggregated protein to basic and hydrophobic sequences in tissue.     

Characterisation of multiple immunoreactive neurons in the central nervous system of the pond snail Lymnaea stagnalis with different fixatives and antisera adsorbed with the homologous and the heterologous antigens

Summary In the central nervous system of the pond snail Lymnaea stagnalis a large number of elements (cells and fibers) can be identified with antisera (a-FM) to the molluscan cardioactive tetrapeptide FMRFamide (Phe-Met-Arg-Phe-NH₂). Of these elements some are also reactive to antivasotocin (a-VT) and/or anti-gastrin (a-Gas). These observations suggest that the a-FM positive elements belong to more than one type. Previous results had already indicated that the immunoreactivity of many a-FM positive cells is influenced by the type of fixation. Taking into account the effects of three fixatives on the reactivity of the cells, and their staining characteristics with the two other antisera used, 8 a-FM positive types could be distinguished.Homologous and heterologous adsorptions were carried out to test the specificity of a-FM, a-VT and a-Gas. After homologous adsorptions no staining was obtained. After heterologous adsorptions only part of the multiple staining cells were identified. This indicates that in a-FM, a-VT and a-Gas in addition to (more) selective IgG molecules, less specific IgG molecules occur that can bind to other peptides than those used to raise the antisera (cross-reaction). The (more) selective IgG molecules in a-FM bind to 6 of the a-FM positive types, suggesting that in L. stagnalis a family of FMRFamide-like substances occurs. This conclusion is sustained by results obtained with a-FM adsorbed with fragments of FMRFamide. It appeared that the less selective IgG molecules in a-FM, a-VT and a-Gas cause the multiple stainings of those cells that remain unstained with an antiserum adsorbed with a heterologous antigen. Multiple staining, which can not be abolished by heterologous adsorption, probably is due to the binding of (more) selective IgG molecules to different antigenic determinants present in the cells.The results show that unexpected cross-reactions may occur in immunocytochemical staining procedures. It thus seems precarious to draw conclusions about the chemical structure of a peptide solely on the basis of immunocytochemical studies.     

Avidin- or streptavidin-biotin as a highly sensitive method to stain total protein on membranes

A sensitive method for staining proteins after transfer from polyacrylamide gels to nitrocellulose paper is described. Transferred proteins are first derivatized by reaction of the nitrocellulose replica with sulfosuccinimidobiotin and are then reacted sequentially with streptavidin, rabbit anti-streptavidin, and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. Incubation with the enzyme substrate α-chloronaphthol, produces dark protein bands against a white background. The binding of streptavidin to the proteins is dependent on biotin derivatization as demonstrated by competition with biotinylated bovine serum albumin or 10 nM biotin. The procedure detects less than 5ng of transferred protein in a single band and is thus 5–10 times more sensitive than horseradish peroxidase-conjugated avidin alone. For bovine serum albumin, the method is comparable in sensitivity to silver staining of protein in polyacrylamide gels.     

Application of polymer-embedded proteins to fabrication of DNA array

A plasma-polymerized film (PPF) of hexamethyldisiloxane [HMDS; (CH(3))(3)SiOSi(CH(3))(3)] was used to immobilize streptavidin on a glass substrate. Another layer of HMDS-PPF was also applied to the protein, which was first adsorbed to an underlayer of the same kind of film. As the result, the streptavidin was "embedded" between the two layers of HMDS, whereby biotinylated molecules could be efficiently captured. The second layer of approximately 30 to 45 A PPF was sufficient to allow the binding of biotinylated molecules, whereas thicknesses of >90 A significantly hindered the streptavidin-biotin interactions. Fluorescence analysis revealed that the absence of an HMDS plasma-polymer (HMDS-PP) layer on either side of the streptavidin film resulted in a decrease in biotin binding. This immobilization technique was used to bind biotinylated oligonucleotides in sequence-specific DNA-DNA interactions. The hydrophobic properties of the plasma-polymerized HMDS thin film acted to minimize nonspecific DNA binding to the glass substrate. A DNA array was fabricated using this procedure and showed greatly decreased nonspecific DNA binding compared with a poly-L-lysine coated substrate.     

Andrenocorticotropin and β-lipotropin in the hypothalamus

Adrenocorticotropin and β-lipotropin (β-LPH) have been localized by immunoperoxidase methods in nerve cells and fibers of the hypothalamus and brain stem of the ewe. 6-μm sections were immunostained first for either ACTH or β-LPH. The reaction products and the antibody complexes were then eluted completely from the tissue, and the same section was immunostained for the second peptide. Absorption of the primary antisera with a variety of peptide fragments of ACTH and β-LPH demonstrated, immunocytochemically as well as by radioimmunoassay, that the ACTH and β-LPH antisera were directed to the COOH- and NH(2)-termini of the peptides, respectively. Neither antiserum recognized any portion of the heterologous peptide. In the sequential staining procedure on the same tissue section, preincubation of the antisera with the homologous peptide abolished the staining, whereas preincubation with the heterologous peptide did not affect it, regardless of the order followed. Every nerve cell in the arcuate nucleus that contained ACTH also contained β-LPH, but β-LPH cells appeared, probably falsely, to be twice as numerous as ACTH cells. β-LPH-positive fibers in and beyond the hypothalamus were also more numerous and stained more intensively than ACTH fibers. The salient exception was fibers in the infundibular zona externa, where the opposite was true. Our observations establish that ACTH and β-LPH are contained in the same nerve cells They stongly favor biosynthesis in brain, probably from a common precursor molecule, as has been demonstrated in the pituitary gland. The complexity of the cytologic distribution pattern described suggests that the two peptides are not processed in the same manner by the nerve cell.     

Calcitonin is not contained within the common precursor to corticotropin and endorphin in the rat.

The suggestion that calcitonin is contained within the structure of the common precursor to ACTH and endorphin was examined. Immunohistochemical staining demonstrated calcitonin in thyroid parafollicular cells, and ACTH and 16K fragment in ACTH/endorphin cells of pituitary. No 16K fragment immunostaining was detected in thyroid parafollicular cells; no calcitonin staining was detected in pituitary. Immunoprecipitation of [³⁵S]methionine-labeled molecules synthesized by rat intermediate pituitary cells demonstrated that neither 30K precursor, 16K fragment nor any other major labeled cell product was recognized by calcitonin antiserum. Analyses of tryptic peptides of 30K precursor indicated that peptides expected from calcitonin were not present in 30K precursor.     

Does the neurotropic action of adrenocorticotrophic hormone involve a lipolytic step?

The stimulatory effect of adrenocorticotrophic hormone (ACTH)-related synthetic peptides on the hydrolysis of emulsified trioleoylglycerol by a rat brain lipase was studied. The ACTH effect was related to the net positive charge associated with the basic amino acid residues at position 15-18 in the ACTH sequence, as well as to the presence of the NH2-terminal amino acid residues at position 1-2. The ACTH effect on lipolysis was markedly reduced when lipids were partially removed from the enzyme preparation by extraction with chloroform/acetone. Full restoration of the stimulatory effect was obtained upon addition of phosphatidylcholine (2 mg/ml) to the lipolytic medium. Striking similarities between the structure-activity pattern for the stimulatory effect of ACTH on brain lipase and that described for the receptor-mediated actions of ACTH on adrenal and fat cells suggest that the ACTH effect might involve recognition of a binding site associated with the brain enzyme. Complete log concentration response curves obtained with four ACTH analogs may also be regarded as simulating hormone-receptor interaction. These findings are discussed in relation to the possibility that ACTH may have a neurohormonal role via lipase-catalyzed changes in the lipid matrix of membranes.     

Characterisation of multiple immunoreactive neurons in the central nervous system of the pond snail Lymnaea stagnalis with different fixatives and antisera adsorbed with the homologous and the heterologous antigens

In the central nervous system of the pond snail Lymnaea stagnalis a large number of elements (cells and fibers) can be identified with antisera (a-FM) to the molluscan cardioactive tetrapeptide FMRFamide (Phe-Met-Arg-Phe-NH2). Of these elements some are also reactive to antivasotocin (a-VT) and/or anti-gastrin (a-Gas). These observations suggest that the a-FM positive elements belong to more than one type. Previous results had already indicated that the immunoreactivity of many a-FM positive cells is influenced by the type of fixation. Taking into account the effects of three fixatives on the reactivity of the cells, and their staining characteristics with the two other antisera used, 8 a-FM positive types could be distinguished. Homologous and heterologous adsorptions were carried out to test the specificity of a-FM, a-VT and a-Gas. After homologous adsorptions no staining was obtained. After heterologous adsorptions only part of the multiple staining cells were identified. This indicates that in a-FM, a-VT and a-Gas in addition to (more) selective IgG molecules, less specific IgG molecules occur that can bind to other peptides than those used to raise the antisera (cross-reaction). The (more) selective IgG molecules in a-FM bind to 6 of the a-FM positive types, suggesting that in L. stagnalis a family of FMRFamide-like substances occurs. This conclusion is sustained by results obtained with a-FM adsorbed with fragments of FMRFamide.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Molar Combining Ratio of Anti-Albumin Fab' Fragments to Homologous and Heterologous Serum Albumins

The number of antibody combining sites on bovine (BSA), goat (GSA) and sheep (SSA) serum albumins was studied using rabbit and chicken antibodies. In homologous reactions, the profiles of quantitative precipitations with chicken antibody were similar to those with rabbit antibody reported previously (12), and the antigenic valence in the extreme antibody excess zone was found to be 6–7 for each albumin. The univalent Fab' fragments of rabbit and chicken antibodies were prepared. The stoichiometry of the soluble complex formed with the Fab' fragment and fluorescence labeled albumin was analyzed by gel filtration, and the number of Fab' fragment molecules capable of binding to an albumin molecule was estimated. In a homologous reaction with both rabbit and chicken Fab' fragments, the Fab' to albumin combining ratio revealed from the molecular weight of the soluble complex was 14:1. In the heterologous reactions, the combining ratio was 5:1 for rabbit Fab' fragment to BSA, and 9:1 for chicken Fab' fragment to BSA. From the heterologous reactions between GSA and SSA, it was demonstrated that the combining ratios were 10–11:1 with rabbit Fab' fragment and 11–13:1 with chicken Fab' fragment.     

The immune response in the hamster. VII. Studies on cytophilic immunoglobulin.

Hamster 7S IgG1 and IgG2 antibodies to hen-egg albumin (HEA) were tested for their capacity to bind to macrophage cytophilic Ig receptors. Both IgG1 and IgG2 were cytophilic for hamster macrophages though the membrane receptor had a predominant specificity for IgG1. Hamster IgG1 bound primarily to homologous macrophages whereas IgG2 bound to macrophages from other rodent species as well. The binding of hamster Ig to hamster macrophages was inhibited by a wide range of heterologous rodent sera. The only exception was guinea pig serum since guinea pig IgG2 was found to bind only to homologous macrophages. Sheep red blood cells (SRBC) coated with hamster IgG2 were ingested by macrophages more readily than those coated with hamster IgG1. Thus, there appeared to be a paradoxical relationship between the apparently strong affinity of IgG1 for the hamster macrophage Ig receptor and its reactivity weak ingestion promoting activity. Implications of this phenomenon are discussed.     

R-phycoerythrin as a fluorescent label for immunolocalization of bound atrazine residues

We established a highly sensitive immunofluorescence procedure for localizing bound atrazine in the aquatic macrophytes Elodea canadensis and E. densa. The technique included biotin-labeled anti-rabbit IgG as a first enhancement step and R-phycoerythrin (R-PE) coupled to streptavidin for fluorescent labeling as a second improvement on the procedure. A comparison with the conventional indirect immunofluorescence method confirmed the superior results of the R-PE approach. The use of atrazine-free plants (grown in charcoal-filtered water) and a variety of other controls excluded both contaminating atrazine and nonspecific incubation constituents as sources of tissue staining. Pre-incubations to block nonspecific binding sites proved to be unnecessary in this system. The highly sensitive procedure described here might be a useful tool for the localization of tissue-bound pesticides in general and possibly of other haptens as well.     

Defensins are mitogenic for epithelial cells and fibroblasts

Defensins are a family of structurally homologous peptides contained within phagocytic cells. Although these peptides are best known for their broad spectrum antimicrobial properties, they also inhibit ACTH (corticotropin) stimulated corticosterone production, chemoattract monocytes, and lyse mammalian cells. We now report that these peptides are potent mitogens in vitro in the same concentration range that they display potent antimicrobial activity in vitro. These concentrations are in the same range as those expected to be present in vivo during the wound healing process. All defensins tested were stimulatory for epithelial cells and fibroblasts and acted synergistically with insulin. These are the first data to disclose the strong growth-promoting effects of this unique family of peptides and point to another basic mechanism whereby the macrophage and neutrophil may participate in a variety of trophic, physiologic, and pathologic processes.     

Composite surface for blocking bacterial adsorption on protein biochips

The design and fabrication of protein biochips requires characterization of blocking agents that minimize nonspecific binding of proteins or organisms. Nonspecific adsorption of Escherichia coli, Listeria innocua, and Listeria monocytogenes is prevented by bovine serum albumin (BSA) or biotinylated BSA adsorbed on SiO(2) surfaces of a biochip that had been modified with a C(18) coating. Biotinylated BSA forms a protein-based surface that in turn binds streptavidin. Because streptavidin has multiple binding sites for biotin, it in turn anchors other biotinylated proteins, including antibodies. Hence, biotinylated BSA simultaneously serves as a blocking agent and a foundation for binding an interfacing protein, avidin or streptavidin, which in turns anchors biotinylated antibody. In our case, the antibody is C11E9, an IgG-type antibody that binds Listeria spp. Nonspecific adsorption of another bacterium, Escherichia coli, is also minimized due to the blocking action of the BSA. The blocking characteristics of BSA adsorbed on C(18)-derivatized SiO(2) surfaces for construction of a protein biochip for electronic detection of pathogenic organisms is investigated.     

Hormonal properties of avidin-biotinylinsulin and avidin-biotinylcorticotropin complexes

In connection with the development of affinity columns (based on the avidin-biotin interaction) for retrieval of peptide and protein hormone receptors, the hormonal properties of a number of avidin-biotinylinsulin and avidin-bioinylcorticotropin complexes were examined. Of particular interest was an evaluation of streptavidin as a ligand for the attachment of biotinylated hormones to solid supports and its possible advantage over SpHPP-avidin (S = succinoylated; pHPP = 3-(p-hydroxyphenyl)propionyl). As concerns binding kinetics using rat liver plasma membranes, streptavidin was found superior to avidin since it does not display apparently nonsaturable binding. Scatchard analyses of the binding of 125I-streptavidin, 125I-S-streptavidin and 125I-SpHPP-avidin to rat liver plasma membranes gave KD value of 6.7, 13.2, and 10.6 nM respectively. The binding was saturable and the unlabeled proteins competed with their labeled counterparts for the membrane binding sites. Biotinylinsulin, attached to either streptavidin or SpHPP-avidin was able to compete for 125I-insulin-binding sites on rat liver plasma membranes though somewhat larger concentrations of the complexes than of insulin were required to achieve comparable inhibition. The ID50 values for insulin and the biotinylinsulin complexes were 5 and 80 nM respectively. Biotinylcorticotropin was found to be a more effective activator of particulate rat adrenal adenylate cyclase when complexed with unmodified avidin than with streptavidin, S-streptavidin or SpHPP-avidin.     

Use of glucose-6-phosphate dehydrogenase as a new label for nucleic acid hybridization reactions

We describe here a sensitive new procedure for detecting DNA hybridization by dot blots. The method utilizes DNA or oligonucleotide probes labeled with biotin, sulfone, or haptens that can be detected by glucose-6-phosphate dehydrogenase (G6PDH) conjugates. Biotin labeling of DNA gave the best sensitivity. G6PDH activity was revealed by staining or by bioluminescence using an FMN oxidoreductase and a luciferase from Beneckea harveyi. Bioluminescent detection offered better sensitivity and faster revelation than the colorimetric assay and was found to be very useful in visualizing single mutations in human DNA after hybridization with an allele-specific biotinylated oligonucleotide probe. Revelation can be performed using a luminometer, photographic films, or a very sensitive video camera. The detection is limited by the nonspecific binding of the labeled reagent (streptavidin or antibodies). This limit is similar to that obtained with other nonisotopic labeling procedures, but our method is faster and several hybridization reactions can be performed on the same support.     

Mixed self-assembly of polydiacetylenes for highly specific and sensitive strip biosensors

Polydiacetylenes (PDAs), which possess unique properties that allow them to change color in response to environmental changes such as variations in pH, temperature, and molecular binding, have been widely investigated as signal transducers in biosensor applications. Most PDA-based sensors reported to date have been evaluated largely on the basis of their ability to detect purified samples, however, and their specificity has rarely been tested. In this study, novel PDAs fabricated on polyvinylidene fluoride (PVDF) strips by photoreaction of composite diacetylene self-assemblies were developed as biosensors, and nonspecific binding to off-target biomolecules was assessed. A mixed PDA surface containing biotin and ethanolamide bound the target, i.e., streptavidin, more specifically than did biotin alone. The optimized PDA biosensor exhibited approximately 2850-fold higher selectivity for streptavidin relative to bovine serum albumin controls. A PDA biosensor that was further prepared showed distinctive signals for the urine of diabetic patients compared to urine samples from healthy/non-diabetic person due to the concentration of microalbuminuria. To our knowledge, this is the first strip-type biosensor fabricated with PDAs and the first PDA-based biosensor that can effectively overcome the problem of nonspecific binding.     

Immunofluorescence Staining of Group B Coxsackieviruses

Studies were conducted on the sensitivity and specificity of indirect fluorescent-antibody (FA) staining for identification of group B coxsackieviruses. Antisera produced in four different species (monkeys, rabbits, horses, hamsters) and immune ascitic fluids prepared in mice were compared for suitability in FA staining. The horse antisera showed high titers of nonspecific staining, and the rabbit antisera showed relatively low homologous FA titers. Immune reagents from monkeys, hamsters, and mice were used for homologous and heterologous testing against cell cultures infected with the various group B coxsackieviruses. Antisera or immune ascitic fluids produced in these three species showed some heterotypic and nonspecific staining at low dilutions, with the monkey antisera showing the highest heterotypic titers. However, the immune reagents could be diluted to a point where they gave no heterotypic reactivity, but still showed characteristic homotypic staining. Heterotypic staining appeared as diffuse, low-level staining of the cells, whereas homotypic staining revealed characteristic, brightly staining aggregates of viral antigen in the cytoplasm of the infected cells. By using hamster immune sera, appropriately diluted to eliminate heterotypic staining and yet give strong homotypic staining, it was possible to identify correctly 79 (93%) of 85 field strains of group B coxsackieviruses at the first passage level in BS-C-1 cells; the remainder of the strains were identified after two passages in BS-CS-1 cells. No incorrect identifications were made. A limited number of field strains of group B coxsackieviruses were passed into rhesus monkey kidney and human fetal diploid kidney cells, and these were all correctly identified by FA staining, even the strains which failed to produce a cytopathic effect in the human fetal diploid kidney cells. Two human heart and brain tissues from which coxsackievirus type B4 had been isolated failed to show homotypic FA staining in excess of nonspecific or heterotypic staining.     

Screening scheme based on measurement of fluorescence lifetime in the nanosecond domain

The authors demonstrate that the fluorescence lifetime of certain fluorescent labels is a useful parameter to detect affinity binding between biotin and streptavidin, as well as between biotinylated bovine serum albumin and streptavidin. The assay is performed in a microplate format, and lifetimes are determined using dye laser-induced fluorescence. Four fluorescent labels are presented that undergo a significant change in their lifetime upon affinity binding. The scheme, referred to as the fluorescence lifetime affinity assay, has several attractive features in that it requires single labeling only, represents a homogeneous assay, allows each of the 2 binding partners to be labeled, and is compatible with the standard microwell formats used in high-throughput screening.     

Nonspecific immunoglobulin synthesis and elevated IgG levels in rabbits immunized with mucoid exopolysaccharide from cystic fibrosis isolates of Pseudomonas aeruginosa

Correlations have been observed between the presence of elevated levels of serum IgG and poor clinical status in cystic fibrosis, and between colonization with mucoid exopolysaccharide (MEP)-producing strains of Pseudomonas aeruginosa and poor clinical status. To determine if P. aeruginosa products could affect the immune system in such a way as to cause nonspecific Ig synthesis and elevated IgG levels, we immunized rabbits with whole bacterial cells and purified MEP from three strains of mucoid P. aeruginosa and with cells of a mucoid strain of E. coli. Antisera raised to whole bacterial cells reacted slightly with a panel of 12 different polysaccharide antigens from various bacteria, whereas antisera raised to purified MEP reacted moderately to strongly with these antigens. The heterologous antibodies elicited by MEP generally showed high affinities and specificities for heterologous antigens, and were functional in opsonophagocytic assays. Analysis of the kinetics of rabbit responses to MEP against homologous and heterologous antigens suggested that nonspecific Ig synthesis could be documented shortly after homologous antibody to MEP was elicited. Rabbits hyperimmunized with MEP also had elevated levels of IgG, even after removal of MEP-specific antibody. These data suggest that the change in young cystic fibrosis patients from a relatively healthy, hypogammaglobulinemic state to more progressive lung disease, associated with elevated levels of IgG and colonization with mucoid P. aeruginosa, may be mediated, in part, by the effects of MEP on mammalian immune systems.