Subcellular Extracts Research Articles

Evaluation of the role of the pneumococcal Forssman antigen (F-polysaccharide) in the cross-serotype protection induced by pneumococcal subcellular preparations.

We tested the hypothesis that the capacity of subcellular preparations of rough pneumococci to give cross-serotype protection is due to the presence of the pneumococcal Forssman antigen (F-polysaccharide). We found by hemagglutination inhibition that the Forssman antigen is present in the subcellular extracts. However, we concluded that the Forssman antigen is not the protective immunogen in the pneumococcal subcellular preparation, since absorption with sheep erythrocytes failed to remove the protective capacity from antiserum raised against the vaccine. Other evidence mitigating against the pneumococcal Forssman antigen being the protective immunogen included the absence of a detectable hemolytic titer in protective antiserum raised against the subcellular preparation, the failure of high-titered sheep hemolysin to passively protect mice against pneumococcal infection, and the failure of purified F-polysaccharide to immunize mice against pneumococcal infection.

Nature of the cross-protective antigen in subcellular vaccines of Streptococcus pneumoniae.

Studies have been carried out to investigate the nature of the antigen present in subcellular extracts of a rough strain of Streptococcus pneumoniae A662b which has been shown to confer protection in mice against challenge with smooth, virulent organisms of the homologous and heterologous serotypes. The finding that whole, heat-killed cells were also capable of immunizing mice against challenge with organisms of heterologous serotypes suggests that the immunogen is present on the surface of the rough pneumococcal cell. Ribosomes purified by sucrose gradient centrifugation were not protective, but material recovered in the pellet retained activity. Subcellular extracts prepared from spheroplasts with a partial absence of cell wall showed decreased protective capacity, and extracts prepared from wall-deficient protoplasts were not protective. Crude cell walls evidenced cross-serotype protection, but purified walls did not protect. These results are interpreted as suggesting that the active moiety in the subcellular vaccine is present on the surface of rough pneumococci and is either a wall antigen that must be part of a larger macromolecular complex to be immunogenic, or a substance associated with the cell wall that is present in crude, but not purified, cell wall fractions.

In vitro processing of B. mori transfer RNA precursor molecules

Ribonuclease P and 3′–5′ nuclease, two enzymatic activities necessary for tRNA synthesis in E. coli, are also found in the silkgland cells of Bombyx mori. B. mori subcellular extracts containing RNAase P activity can cleave the E. coli tRNA precursor molecule endonucleolytically at the same site as the E. coli enzyme, and will also cleave in vitro all E. coli tRNA precursors (pre-tRNAs) which the bacterial enzyme recognizes. B. mori RNAase P will not cleave two E. coli RNAase P substrates that are structurally unrelated to tRNA. Pre-tRNAs from B. mori contain extra 5′ and 3′ nucleotides as judged by RNA fingerprinting and 5′ terminal phosphate analysis. Crude silkgland extracts containing both RNAase P and 3′–5′ nuclease can remove the 5′ and 3′ extra nucleotides from B. mori pre-tRNAs, whereas purified fractions containing RNAase P remove only 5′ extra nucleotides. Only large silkworm pre-tRNAs were found to be susceptible to cleavage by B. mori RNAase P. This observation and sequence analysis of intermediates of in vitro processing reactions indicate a two-step process of pre-tRNA maturation in which extra 5′ nucleotides are first removed by RNAase P and extra 3′ nucleotides are then trimmed off by a 3′–5′ nuclease.

Subcellular distribution of enkephalins and endogenous opioid activity in rat brain

The subcellular distribution of leucine- and methionine-enkephalin in rat brain was studied using a highly selective and sensitive radioimmunoassay. About 85% of the total recoverable activity of each peptide was present in crude synaptosomal and microsomal fractions which contained about 60% and 25% respectively. Total opioid activity in brain subcellular extracts was measured by competition for opiat receptor binding. It is concluded that enkephalin accounts for the majority of the opioid activity in the brain extracts. It seems unlikely that the enkephalin in microsomal fractions are exclusively associated with opiate receptors present in these fractions.

Anti-porcine antibodies in xenografted burned patients

SINCE SONG [13] AND BROMBERG [ 131 REINTRODUCED the use of porcine xenografts as a covering for burned patients there ha-s been considerable question whether the xenografts cause an immune reaction. Several observations [ 121 suggest that the xenograft does not cause an immune reaction: (a) The xenograft is not vascularized; (b) subcellular porcine skin extracts injected intradermally into human patients before and after porcine xenografting do not elicit delayed hypersensitivity; (c) and, this porcine skin extract, injected intravenously into rabbits, fails to produce precipitin antibodies against the extract. It is the purpose of this study to illustrate that by suitable assay procedures, the act of xenografting with porcine skin in burned patients elicits an immune reaction.

Murine reticulum cell neoplasms type B (Hodgkin's-like lesions) induced in BALB/c mice with field isolates of murine leukemia virus

Twenty four per cent of our specific pathogen free BALB/c female control mice and 9% of control males developed lymphoid moplasias Three fourth of the neoplasms were reticulosarcomas type B (Hodgkin's like lesions) Without change in the proportion of reticulosarcomas the tumor incidence was tripled by infecting newborn BALB/c mice with 0.05 ml 5 times concentrated subcellular extracts of reticulosarcomas, doubled by injecting 10 5 XC units of either of two leukemia type viruses recently isolated from BAIB/c mice, and tripled by grafting 10 4 cells from leukemia virus infected syngeneic secondary embryonic fibroblast cultures. Intrathymic inoculation gave the same result as subcutaneous administration 4 four log reduction tn virus given dose s.c. only lowered the tumor incidence slightly. The mean latent period was 13 months following inoculation of cell free virus preparations and 8 month after injection of infected tissue culture fibroblasts. Adult mice receiving onus did not show enhanced tumor incidence.

Protection Against Pneumococcal Infection by a Ribosomal Preparation

A subcellular extract prepared from nonencapsulated Diplococcus pneumoniae type 3 protected mice against a subsequent challenge with virulent D. pneumoniae types 1, 2, 3, and 7. Potency ratios ranged from 25 (type 3) to >1,175 (type 1). The immunity induced in mice by this preparation was best obtained by intraperitoneal inoculation followed by intravenous challenge. Mice immunized in this manner were protected up to 12 weeks and could withstand a challenge of several hundred LD(50). The protection was destroyed by treatment of the preparation with ribonuclease and was decreased by treatment with protease. The preparation consisted of 60.5% ribonucleic acid, 29.1% protein, 6.3% deoxyribonucleic acid, and 4.0% hexose. Ultracentrifugation studies indicated that this extract had five constituents which are compatible with ribosomal material.

Rat Liver Adenosine Triphosphate:Adenosine Monophosphate Phosphotransferase Activity: II. SUBCELLULAR LOCALIZATION OF ADENYLATE KINASE ISOZYMES

Abstract Adenylate kinase activity (ATP:AMP-phosphotransferase, EC 2.7.4.3) was studied in subcellular extracts of rat liver tissue. Essentially all of the activity, over 150 units per g of tissue, wet weight (1.7 units per mg of protein), was released into the cytosol when the tissue was homogenized in 0.025 m sucrose. Isotonic (0.25 m sucrose) homogenization and separation of subcellular fractions by both differential centrifugation and linear sucrose density gradient centrifugation revealed 8 units per g (0.4 units per mg) in the nuclear fraction, 84 units per g (2 units per mg) in the heavy mitochondrial fraction, 22 units per g (0.6 unit per mg) in the light mitochondrial fraction, and 30 units per g (0.5 unit per mg) in the cytosol fraction. Rat liver adenylate kinase activity was separated into four electrophoretically distinct isozymes. Isozyme I, at 3 units per g of tissue, wet weight, was observed only in the nuclear fractions. Isozyme II was found in the cytosol at 19 units per g. Isozyme III, at 110 units per g, was the predominant adenylate kinase isozyme in the rat liver; it was localized in the outer compartment of the mitochondria. Since adenylate kinase III is easily released from mitochondria, mitochondrial damage can be observed by monitoring this isozyme. Also, because of this release, the true subcellular distribution of rat liver adenylate kinase activity is probably better represented by the corresponding values for each isozyme, not the values obtained by normal subcellular extraction procedures.

Intracisternal A-type particles in murine neoplasias with and without paraprotein production.

Immature plasma cell neoplasias induced in BALB/c mice with subcellular leukaemic extracts were investigated with the electron microscope for virus‐like particles. A‐type particles located in the cisternae of the endoplasmic reticulum and in the perinuclear space were the only virus‐like particles observed. The A‐type particles average 70 mü in transverse diameter. They display two electron‐dense shells around a 30 mü wide electron‐translucent core. In most instances the particle surface is covered by a layer of granular material with moderate electron‐density. A few electron‐dense granules are noted in the core of some A‐type particles. The A‐type particles apparently are formed by inward budding from the membrane of the endoplasmic reticulum and occur whether or not the neoplasm produces paraprotein. The findings are discussed in light of our knowledge on leukaemic lesions in mice with plasma cell neoplasia.

Attempts to raise heterologous antisera against subcellular antigenic extracts.

Attempts to raise heterologous antisera against subcellular antigenic extracts.

Separation and identification of 14C-labeled glucogenic compounds by paper chromatography

Two different two-dimensional decending paper chromatographic systems are presented which permit the simultaneous separation of the following: alanine, phenylalanine, tyrosine, glutamic, glucose, fructose, pyruvic, lactic, malic, fumaric, succinic, α-ketoglutaric, 3-phosphoglyceric, hexose phosphate, and phosphoenolpyruvic. Fructose diphosphate and 2-phosphoglyceric migrate together but can be well separated from the others. Carbon-14 labeled standards were used and localization accomplished by radioautography. The chromatography described can be applied usefully to deproteinated subcellular or tissue extracts from experiments in which isotopically labeled precursors were metabolized by the pathways of glycolysis, respiration, gluconeogenesis, or the biosynthesis and degradation of some amino acids. This is so chiefly because the discrete isolations obtained with permit the accurate determination of specific activities.