Dohn G. Glitz

Localization of eosinophil-derived neurotoxin and eosinophil cationic protein in neutrophilic leukocytes.

Eosinophil‐derived neurotoxin (EDN) and eosinophil cationic protein (ECP) are generally regarded as eosinophil‐specific proteins. We tested whether EDN and ECP are present in mature neutrophils. By indirect immunofluorescence, both eosinophils and neutrophils stained with antibodies to EDN and ECP. Lysates of purified (<0.1% eosinophil contamination) neutrophils contained EDN, 112 ± 4 ng/106 cells, and ECP, 163 ± 2 ng/106 cells, whereas eosinophil major basic protein (MBP) was not detectable. Electron microscopic examination of immunogold‐labeled buffy coat cells stained with EDN antibody showed that EDN is localized to neutrophil granules. Finally, EDN mRNA was detected in lysates of highly purified neutrophils (0.001% eosinophil contamination) by the reverse transcription‐polymerase chain reaction. We conclude that proteins that are either identical to or immunologically cross‐reactive with EDN and ECP are present in neutrophils and that EDN is synthesized and localized to neutrophil granules. Thus, caution must be exercised in interpreting the presence of EDN and ECP as specific markers of eosinophil‐associated inflammation in human disease. J. Leukoc. Biol. 63: 715–722; 1998.

The amino acid sequence of human pancreatic ribonuclease

The primary structure of human (Homo sapiens) pancreatic ribonuclease has been determined by automatic sequencing of the native protein and by analysis of peptides obtained by cleavage with proteolytic enzymes, cyanogen bromide, and hydroxylamine. The following sequence was deduced: (sequence in text). Human pancreatic ribonuclease differs at 37 positions from bovine pancreatic ribonuclease. In addition the human enzyme has three more residues at the C-terminus. About half of the enzyme molecules contain carbohydrate attached to the sequence Asn-Met-Thr (34-36). Two other Asn-X-Ser/Thr sequences are carbohydrate free. Human pancreatic ribonuclease contains many positively charged residues, especially near the N-terminus, while negatively charged residues are more concentrated near the C-terminus.

Ribonuclease activity and substrate preference of human eosinophil cationic protein (ECP)

The eosinophil cationic protein (ECP), a potent helminthotoxin with considerable neurotoxic activity, was recently shown to also have ribonucleolytic activity. In this work the substrate preference of ECP ribonuclease action was studied in detail. With single‐stranded RNA or synthetic polyribonucleotide substrates ECP showed significant but low activity, 70‐ to 200‐fold less than that of bovine RNase A. ECP hydrolyzed RNA more rapidly than it did any synthetic polynucleotide. Poly(U) was degraded more rapidly than poly(C), and poly(A) and double‐stranded substrates were extremely resistant. Defined low molecular weight substrates in the form of the 16 dinucleoside phosphates (NpN′) and uridine and cytidine 2′, 3′‐cyclic phosphates were tested, and none showed hydrolysis by ECP at a significant rate. The results link ECP ribonucleolytic activity to the ‘non‐secretory’ liver‐type enzymes rather than to the ‘secretory’ pancreatic‐type RNases.

Nucleotide-specific antibodies as potential blocking agents in the structural analysis of nucleic acids

Abstract Anti-nucleotide antibodies might be useful blocking agents in directing the nuclease catalyzed hydrolysis of RNA for sequence analysis, but only if the antibodies recognized a nucleotide or sequence with sufficient specificity and affinity to compete with a nuclease at only specific sites in an RNA. As a test, antibodies directed against mononucleotide haptens were induced with 5′-nucleotide (oxidized with periodate) coupled to bovine serum albumin. Nucleotide-specific antibodies, isolated by affinity chromatography on nucleotide-agarose columns, were at least 95 % pure 7-S γ-globulin. The antibodies precipitated RNA, homologous nucleotide-rabbit albumin conjugates, and to a lesser extent, heterologous conjugates. Antibodies further purified by passage through an affinity column of heterologous nucleotides showed specificity for the single homologous nucleotide. Equilibrium dialysis measurements gave dissociation constants for the antibody-homologous nucleotide complex of about 10 −5 M, while binding of a heterologous nucleotide was insufficient to be measured. The 5′-phosphate group, the base, and the ribose derivative in the conjugate were all elements of structure recognized by the antibodies. Hydrolysis of antibodies with insolubilized papain produced monovalent Fab fragments with dissociation constants essentially as determined for intact antibodies. Antibodies were found to compete with pancreatic ribonuclease for dinucleoside phosphate substrates in a manner consistent with the binding results. Anti-nucleotide Fab fragments inhibited the nuclease hydrolysis of f2 RNA, but no directed cleavage to give altered RNA fragmentation patterns was observed.

Nucleotides at the 5′-linked ends of bromegrass mosaic virus RNA and its fragments

Abstract The RNA of bromegrass mosaic virus (BMV-RNA) was isolated as three components (L, M, and S, of about 1.0, 0.7 and 0.3·106 mol. wt.) and studied by terminal labeling. Periodate oxidation of the 5′-linked terminus followed by [14C]-dimedone treatment or by reduction with NaB3H4 showed Components L and M to each contain one free 5′-linked end per molecule. Chromatography of ribonuclease T1 hydrolysates of NaB3H4 labeled Components L or M indicated that the two RNA fractions contain the same terminal oligonucleotide; purification and analysis of each oligomer confirmed their identity and permitted tentative identification of the terminal sequence as ... GpCpCpCpA. Component S incorporated less than equimolar quantities of dimedone or NaBH4 after oxidation, and chromatography of 3H terminally labeled material showed some terminal heterogeneity but also much of the same terminal oligonucleotide identified above. Phosphomonoesterase treatment of Component S produced a variety of new periodate-sensitive ends. It was concluded that intact viral RNA (Component L) may be cleaved in a sensitive region (but not at a single internucleotide linkage) to form molecules of Component M which carries the 5′-linked terminus and Component S which usually bears a 3′-phosphate end. Component M may then be further cleaved to form two molecules of Component S, one carrying the original 5′-linked end and one usually terminating in a 3′-phosphate.

Terminal nucleotides of avian myeloblastosis virus RNA and of ribosomal RNA from chicken leukemic myeloblasts

Abstract The 18- and 28-S ribosomal RNA components from chicken leukemic myeloblasts have been purified and separately analyzed for terminal nucleotides. Alkaline hydrolysates of 18-S rRNA contain, per mole RNA, about 1 mole of cytidine 3′(2′),5′-diphosphate and 1 mole of adenosine. Similar analysis of the 28-S component gives only guanosine 3′(2′),5′-diphosphate and uridine. Fractionation of nuclease hydrolysates of each RNA by a “diagonal” electrophoretic method (which exploits the absence of monoesterified phosphate on the 3′-terminal oligonucleotide) permits isolation of a terminal fragment from each. Analysis of these fragments indicates that the terminal nucleotide sequence of myeloblast 18-S rRNA is -GC[(AU) 2 C 2 U 2 ]A OH and that of the 28-S rRNA is -PyAGGU OH . Analysis of the nucleosides released upon alkaline hydrolysis of avian myeloblastosis virus RNA show the 3′-terminus to be predominantly adenosine.

Localization of Eosinophil–Derived Neurotoxin and Eosinophil Cationic Protein in Neutrophilic Leukocytes

Eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) are generally regarded as eosinophil-specific proteins. We tested whether EDN and ECP are present in mature neutrophils. By indirect immunofluorescence, both eosinophils and neutrophils stained with antibodies to EDN and ECP. Lysates of purified (F0.1% eosinophil contamination) neutrophils contained EDN, 112 6 4 ng/10 6 cells, and ECP, 163 6 2 ng/10 6 cells, whereas eosinophil major basic pro- tein (MBP) was not detectable. Electron micro- scopic examination of immunogold-labeled buffy coat cells stained with EDN antibody showed that EDN is localized to neutrophil granules. Finally, EDN mRNA was detected in lysates of highly purified neutrophils (0.001% eosinophil contami- nation) by the reverse transcription-polymerase chain reaction. We conclude that proteins that are either identical to or immunologically cross- reactive with EDN and ECP are present in neutro- phils and that EDN is synthesized and localized to neutrophil granules. Thus, caution must be exer- cised in interpreting the presence of EDN and ECP as specific markers of eosinophil-associated inflam- mation in human disease. J. Leukoc. Biol. 63: 715-722; 1998.

[34] Antibody probes of ribosomal RNA☆

Publisher Summary This chapter focuses on the RNA structure in the ribosome with an emphasis on immunoelectron microscopy. The chapter has used antibody probes to naturally occurring modified nucleotides, to reagents used in the site-specific modification of ribosomal RNA, and to chemically synthesize modified oligo deoxynucleotide that complement and bind specific ribosomal RNA sequences. The chapter discusses the detail methods for the production and characterization of these antibodies and for their use in the study of ribosome structure. There are significant difficulties in probing the structure of the RNA within the ribosome; the secondary structure of the RNA is extensive, and a considerable portion of each sequence is likely to be buried within the subunits and unavailable to probes. A limited number of natural targets—modified nucleotides or chemically unique sites—occur in each subunit, but most of the RNA is lacking in distinct markers.

Bovine ribonucleases: Identification of tissue-specific enzymes by immunological methods

Abstract 1. 1. Antibodies directed against bovine brain or bovine pancreatic ribonucleases (EC 3.1.4.22, ribonucleate 3′-pyrimidino-oligonucletidohydrolase) have been shown to be highly specific and sensitive in enzyme quantitation. In competitive binding radioimmunoassays, cross-reactivity with the heterologous ribonucleasesis less than 0.1% and sensitivity for detection of the homologous enzyme is 1–10 ng per assay, comparable to enzymatic analytic l procedures. 2. 2. Immunological analysis of each enzyme in crude extracts of brain and pancreas accurately measures the level of each enzyme. Radioimmunoassay also shows materials that cross-react with each antibody preparation in extracts of liver, kidney, and seminal vesicles, but in most tissues the level of immunological cross-reactivity is not sufficient to account for all enzymatic activity. Instead, these tissues have one or more RNases which are structurally different from either the brain or pancreatic enzymes. 3. 3. Gel filtration of extracts usually separates pancreatic-like and brain-like immunological cross- reactivity. Ribonuclease(s) which are not closely related to either the brain or pancreatic enzymes are sometimes apparent in the elution patterns. 4. 4. Cross-reactivaty of anti-bovine brain ribonuclease with extracts of pig, rat, chicken, or turtle brain is very low. It is suggested that structural evolution of the brain ribonucleases may have been rapid, as previously suggested for pancreatic ribonucleases.

Incorporation of Dinitrophenyl Protein L23 into Totally Reconstituted Escherichia coli 50 S Ribosomal Subunits and Its Localization at Two Sites by Immune Electron Microscopy

Escherichia coli ribosomal protein L23 was derivatized with [3H]2,4-dinitrofluorobenzene both at the N terminus and at internal lysines. Dinitrophenyl-L23 (DNP-L23) was taken up into 50 S subunits from a reconstitution mixture containing rRNA and total 50 S protein depleted in L23. Unmodified L23 competed with DNP-L23 for uptake, indicating that each protein form bound in an identical or similar position within the subunit. Modified L23, incorporated at a level of 0.7 or 0.4 DNP groups per 50 S, was localized by electron microscopy of subunits complexed with antibodies to dinitrophenol. Antibodies were seen at two major sites with almost equal frequency. One site is beside the central protuberance, in a region previously identified as the peptidyltransferase center. The second location is at the base of the subunit, in the area of the exit site from which the growing peptide leaves the ribosome. Models derived from image reconstruction show hollows or canyons in the subunit and a tunnel that links the transferase and exit sites. Our results indicate that L23 is at the subunit interior, with separate elements of the protein at the subunit surface at or near both ends of this tunnel.