Disseminated intravascular coagulopathy during experimental pneumococcal sepsis: studies in normal and asplenic rhesus monkeys.

Disseminated intravascular coagulation (DIC) was induced in both normal and asplenic rhesus monkeys by intravenous challenge with Streptococcus pneumoniae. Our observations in the infected monkeys have led us to conclude that (1) pneumococcal capsular polysaccharide (PCP), immune complexes and complement may not have primary roles in the initiation of DIC; (2) intact pneumococci may be catalysts for the development of DIC; (3) the initial event in DIC may be activation of Hageman factor; and (4) evidence of activation of Hageman factor-dependent systems is present regardless of severity of infection.     

ELISA identification ofRhizobium strains by use of enzymelabeled protein A

Protein A coupled to alkaline phosphatase has been used for indirect enzyme-linked immunosorbent assay (ELISA) to identify strains ofRhizobium in culture and nodules. This conjugate shows no background reactions and provides a highly specific and sensitive detection of rhizobial antigens. An additional advantage is the simplicity of this technique for routine rhizobial detection. Currently, enzyme-labeled protein A has been used at considerably higher dilutions than conjugated goat anti-rabbit immunoglobulin.     

Diversity and relative inaccuracy of internal images of antigen group A revealed by IEF analysis]

The diversity of a polyclonal anti-idiotypic response (Ab2) to a murine monoclonal anti-A (Ab1) was investigated after purification of two Ab2 populations. One was eluted from human polyclonal anti-A column and the other from Ab1. Analysis of the Ab specificity, as well as screening of the clonotypic distribution, were achieved after splitting Ab by IEF; this was followed by immunoblotting and probing with various anti-ABH mAb. The first population reacted with almost all the murine anti-ABH mAb, as well as with four human anti-A mAb, and consequently consisted of Ab2 beta. The second was composed of "true" Ab2 directed against Ab1. In the first population internal images mimicked either A Ag, or H Ag, or some epitopes common to both. This study demonstrates the plurality of internal images-bearing Ig molecules, some mimicking completely, and some only partially or even unfaithfully the nominal A determinant. The analysis of this idiotypic cascade proves the existence of a degeneracy of the initial restricted antigenic specificity. The consequences of such a process are discussed.     

A Method for Processing Paraffin Sections of Multilayer Cultured Tissue

A simple and rapid method is described for processing histological preparations from multilayer cultures growing in plastic Petri dishes. A covering collodion film is utilized to remove the tissue from the plastic dish and transfer it onto a paper block prior to embedding in Paraplast. To avoid any disruption by the collodion of the plasticware, the cultured tissue is first immersed in a solution of collodion and absolute alcohol (1:1) and then covered with pure collodion. All steps are carried out in the cold. This procedure allows morphological, histochemical, immunofluorescent, and autoradiographic studies to be carried out on serial sections of cultured tissue.     

Cell culture factors influencing in vitro expression of mouse mammary tumor virus

Summary Several cell culture factors were found to influence in vitro expression of mouse mammary tumor virus (MMTV) in the mouse adenocarcinoma cell line Mm5mt/c₁. Cells were propagated in a variety of commercially available cell culture media to which dexamethasone (DXM) was added as a stimulator of MMTV production. Culture seeding density, culture medium type, and glucose concentration each influenced MMTV production when expressed on a per cell basis. Maximum cell growth occurred in cultures grown in RPMI-1640 medium containing insulin. Those media which provided good cell growth were not necessarily optimal for virus expression. Addition of insulin did not stimulate MMTV synthesis although dexamethasone alone was stimulatory in all media used; however, maximum MMTV expression was obtained when dexamethasone and insulin were used in concert. Equivalent levels of MMTV-specific cell membrane antigen, MMTV-specific protein, and virus particles were produced at incubation temperatures of 32°, 34° or 37° C; however, higher levels of virus-related RNA-dependent DNA polymerase (RDDP) activity were recovered from cultures incubated at 32° and 34° C than at 37° C. Decreased levels of RDDP were attributed to enzyme thermolability at 37° C incubation. Research sponsored by the National Cancer Institute under Contract No. N01-CO-25423 with Litton Bionetics, Inc., and Contract No. N01-CP-33253 with the University of California.     

Lysosomal enzyme precursors in coated vesicles derived from the exocytic and endocytic pathways

The molecular forms of two lysosomal enzymes, cathepsin C and cathepsin D, have been examined in lysosomes and coated vesicles (CVs) of rat liver. In addition, the relative proportion of these lysosomal enzymes residing in functionally distinct CV subpopulations was quantitated. CVs contained newly synthesized precursor forms of the enzymes in contrast to lysosomes where only the mature forms were detected. Exocytic and endocytic CV subpopulations were prepared by two completely different protocols. One procedure, a density shift method, uses cholinesterase to alter the density of CVs derived from exocytic or endocytic pathways. The other relies on electrophoretic heterogeneity to accomplish the CV subfractionation. Subpopulations of CVs prepared by either procedure showed similar results, when examined for their relative proportion of cathepsin C and cathepsin D precursors. Within the starting CV preparation, exocytic CVs contained approximately 80-90% of the total steady-state levels of these enzymes while the level in the endocytic population was approximately 10-13%. The implications of these findings are discussed with regard to lysosome trafficking.     

Separation of endocytic from exocytic coated vesicles using a novel cholinesterase mediated density shift technique

Coated vesicles isolated from rat liver perfused with diisopropylfluorophosphate (DFP) to inactivate endogenous cholinesterase contained newly synthesized secretory cholinesterase after a 30 min recovery. The cholinesterase is found in coated vesicles of presumed endocytic origin following DFP treatment and perfusion for 3 min with galactosylated cholinesterase, a ligand for the asialoglycoprotein receptor. Highly enriched populations of endocytic and exocytic coated vesicles can be separated by use of a novel cholinesterase mediated density shift technique. The two coated vesicle classes have very similar polypeptide compositions but differ significantly in the ratio of cholesterol to phospholipid.