Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies

Protein G is an immunoglobulin (IgG)-binding bacterial cell wall protein recently isolated from group G streptococci. We have investigated the avidity of protein G for various monoclonal and polyclonal Ig of the IgG class, and compared it with the binding properties of protein A, the staphylococcal Fc-binding protein. Radiolabeled Ig were mixed with Sepharose-coupled protein G or protein A, and the amounts of radioactivity bound to the matrix-coupled bacterial proteins were determined. The avidity was found to be greater for protein G than for protein A for all examined Ig. Protein G bound all tested monoclonal IgG from mouse IgG1, IgG2a, and IgG3, and rat IgG2a, IgG2b, and IgG2c. In addition, polyclonal IgG from man, cow, rabbit, goat, rat, and mouse bound to protein G, whereas chicken IgG did not. The binding property of protein G was additionally exploited in the Western blot assay, in which iodine-labeled protein G was used successfully for the detection of a rat monoclonal antibody against ovalbumin, and for the detection of rabbit and goat polyclonal whole antisera against human urinary proteins. In these experimental situations, protein G was found to be a powerful reagent for the detection of IgG, and consequently the antigen against which these antibodies are directed.     

Partial expression of catecholaminergic traits in cholinergic chick ciliary ganglia: Studies in vivo and in vitro

We have previously demonstrated that at embryonic Day (E) 8, some cells of the chick ciliary ganglion (CG) contain the catecholaminergic (CA) enzyme tyrosine hydroxylase (TH), but not phenylethanolamine-N-methyltransferase (PNMT); and that in culture essentially all cells express both enzymes. In the present study, we sought to determine, first, whether the expression of adrenergic traits in the CG in vivo is transient or permanent in the CG. To do so, CGs were removed from E5 to postnatal Day 5, fixed, and processed for the immunocytochemical localization of the CA enzymes: TH, L-amino acid decarboxylase (AADC), and PNMT. At all stages examined, some CG neurons expressed TH immunoreactivity (TH-IR) and all contained AADC-IR. However, none stained with PNMT antibodies, indicating that these cells stably express some, but not all, of the CA enzymes. Second, we examined whether CG neurons in culture expressed other CA markers. CG neurons did not contain detectable levels of TH enzyme activity nor did they transport and store exogenously supplied monoamines. These results indicate that some but not all traits necessary for adrenergic function are present in CG neurons in vitro. Third, we sought to establish whether CA expression in CG neurons is affected by modification in culture conditions. Cultures of CG neurons continued to express TH-IR even when grown in the presence of either 50% HCM or 20 mM KCl for 5 days. Finally, the expression of the cholinergic enzyme, choline acetyltransferase (CAT) was assessed in CG cultures by biochemical assay. CAT activity increased five-fold between 5 and 17 days in vitro, irrespective of the presence of TH-IR in 100% of the CG neurons of sister cultures. These data suggest that at least a subpopulation of CG neurons express both TH and CAT in culture. We conclude that the postmitotic neurons of the CG are able to express some but not all of the traits characteristic of a CA phenotype while maintaining cholinergic expression. These findings suggest that (1) the appearance of the full complement of adrenergic properties is not coordinated and may be regulated by different environmental cues and (2) parasympathetic neurons can express both adrenergic and cholinergic traits simultaneously.     

c-Ha-ras-1 proto-oncogene amplification and overexpression during the limited replicative life span of normal human fibroblasts

The cellular proto-oncogene c-Ha-ras-1 undergoes up to 4-fold amplification during the limited replicative life span of normal human diploid fibroblasts in vitro. Levels of c-Ha-ras-1 messenger RNA and its p21 protein product are correspondingly elevated. Cellular proto-oncogene amplification and overexpression, although frequently associated with tumorigenesis, may thus occur during normal cellular growth.     

Streptococcal Fc receptors. II. Comparison of the reactivity of a receptor from a group C streptococcus with staphylococcal protein A

The reactivity of a soluble Fc receptor from a group C streptococcus ( FcRc ) was compared antigenically and functionally with the staphylococcal Fc receptor, protein A. Protein A and FcRc were found to inhibit each others' binding to the Fc region of human IgG, indicating that they bind to sites that are in close proximity on the Fc region of human IgG. The two bacterial Fc receptors were antigenically unrelated. Differences were observed in the species and subclass reactivity of the two receptors. The patterns of binding of protein A and FcRc under various conditions suggested that these receptors reacted with distinct regions on the Fc region of immunoglobulins. FcRc bound more efficiently to goat, sheep, and cow IgG, protein A bound more efficiently to dog IgG, and neither receptor bound to rat IgG. Differences were also observed in the reactivity towards human IgG subclasses. The FcRc bound to all samples of the four human IgG subclass standards. Protein A bound to IgG1, IgG2, and IgG4, and to one of two IgG3 myeloma proteins tested. The reactivity of our soluble FcRc corresponds to a type III streptococcal Fc receptor classified by the reactivity of intact bacteria.     

Streptococcal Fc receptors. I. Isolation and partial characterization of the receptor from a group C streptococcus

A receptor for the Fc region of immunoglobulin G was extracted from a group C streptococcus, purified and physicochemically characterized. The Fc receptor was extracted in high yield by lysis of the bacteria after infection with bacteriophage. The soluble receptor was purified to functional homogeneity by sequential chromatography on cellulose phosphate, DEAE, and selective elution from a column of immobilized human IgG. Four hundred micrograms of the functionally pure protein was obtained per gram (wet weight) of bacteria extracted. The affinity-purified receptor was functionally homogeneous in binding to the Fc region of human IgG; however, the product was heterogeneous on both non-denaturing and SDS polyacrylamide gels. Four major protein bands were observed, with the predominant form of the Fc receptor having an m.w. of 64,000 daltons. Antibody prepared against the major Fc receptor protein ( FcRc -II) was capable of reacting with all the fractions and completely inhibiting functional activity. The results of the competitive binding studies suggest that the purified Fc receptor behaves as a single receptor, and that the differences in charge and size were probably due to covalently bound cell wall constituents.     

A further study on the dietary-regulated biosynthesis of high-sulphur wool proteins

When the diet of sheep is supplemented by the infusion of sulphur-containing amino acids or casein into the abomasum, the newly synthesized wool shows characteristic changes in its amino acid composition, with significant increases in cystine, proline and serine and decreases in aspartic acid and phenylalanine. This modification seems to be due entirely to an alteration in the overall composition of the high-sulphur proteins and to an increase in their proportion in the fibre. These variations are not the result of a change in the composition of individual proteins, but are due to alterations in their relative proportions and to the initiation of the synthesis of `new' proteins, many of which are extremely rich in cystine. It is suggested that the heterogeneity of the high-sulphur proteins may be due, in part, to similar changes in composition caused by natural variations in the nutrition of sheep.     

Cytologic and ultrastructural findings of a peculiar alteration in cervical cells from patients with human papillomavirus infections

Inclusion bodies were previously found in Papanicolaou smears of patients infected by the human papillomavirus (HPV). To ascertain their origin, 12 biopsies from patients with colposcopic evidence of papillomatous lesions were studied by cytology and electron microscopy. In several instances, koilocytotic atypias and electron-dense masses included within epithelial cells were observed. Some of these epithelial cells appeared to be surrounded by other cells in a sort of concentric arrangement. The electron-dense masses were composed of intermediate filaments, vacuoles and electron-dense material. They seemed to be dyskeratotic cells. They can be compared with apoptotic bodies and may be related to a disturbance of the involucrin expression caused by the HPV infection.     

Sexual and site differences in calcium consumption by the Malabar Giant Squirrel Ratufa indica

Summary The calcium ingestion of Malabar Giant Squirrels Ratufa indica (Sciuridae) was examined at two sites, Magod and Bhimashankar, in western India. In females at Magod a positive correlation was found between rates of calcium ingestion from food resources and the contribution of those resources to the daily diet. This relationship was true for all females at Magod irre-spective of their reproductive condition. This relationship was neither significant for males at the same site nor for both sexes at Bhimashankar. A differential requirement for calcium between the sexes and the occurrence of higher rates of calcium ingestion or the probable presence of more easily digestible calcium at Bhimashankar are postulated to explain the observed phenomenon. Mature leaves and bark appear to be reliable sources of calcium at these sites.     

Band-specific localization of the microsatellite at D13S71 by microdissection and enzymatic amplification.

Microsatellite DNA consists of tandemly repeated simple DNA sequence motifs, the number of these repeats being polymorphic. These recently described polymorphisms are ubiquitously distributed throughout the human genome and are highly informative, making them ideal markers for linkage analysis. Physical localization of these microsatellites is an important prerequisite for aligning physical and genetic maps. We have physically mapped the microsatellite at D13S71, which has previously been assigned to chromosome 13. Band-specific mapping of D13S71 to the distal part of band 13q32, near 13q33, was achieved by microdissection of GTG-banded chromosomes and subsequent enzymatic amplification with a heminested PCR approach. Analysis of a panel of somatic cell hybrids confirmed this localization. The technique presented may also be useful in a variety of complex mapping situations and whenever the precise localization of very small (as small as 70 bp) DNA probes is necessary.