An improved detection system applied to the study of serum lipoproteins after single-step density gradient ultracentrifugation

Starting from a single-spin ultracentrifugation procedure described previously (Foreman et al., J. Lipid Res. 18, 759, 1977), we have improved the system for detection of the fractions eluted from the gradient by monitoring them continuously at 280 nm. A graphic display readily permits assessment of the distribution of the lipoproteins and their quantification with the aid of a computer program. By the use of appropriate factors, one can convert absorbance readings into actual lipoprotein values which correspond well (±7% for low-density lipoproteins and ±5% for high-density lipoproteins) to those obtained by means of chemical analyses. The examples provided indicate the versatility of the method and its sensitivity (down to 0.1 ml of serum).     

Age-related motor and catecholomine alterations in mice on levodopa supplemented diet

Old mice reared on regular diet show reduced motor activity, decreased basal adenylate cyclase, and increased MAO activities compared to adults. Brain DDC and COMT activities, DA, NE levels and DA-stimulated adenylate cyclase remained unchanged. By contrast, mice fed levodopa for life did not develop decreased motor activity with aging, lived about 50% longer, had slightly elevated whole brain DA and NE levels and failed to develop the expected rise in MAO activity with aging. Levodopa did not alter the number of dopaminergic and muscarinic cholinergic receptors or the adenylate cyclase activity in the striatum during aging. On levodopa, hepatic and renal DA, dopa, and HVA increased but the latter two returned to basal levels by mid life. In liver, DDC was unchanged but MAO tended to be higher in levodopa-fed mice. Thus, motor impairment is an age-related phenomenon in mice associated with selective alterations in brain dopaminergic systems, which may be prevented by dietary levodopa. Extracerebral tissues, through possibly adaptive metabolic mechanisms, play a significant role in regulating brain catecholamines during chronic administration of large doses of levodopa.     

Fucose in the surface deposits of axenic and field grown roots ofZea mays L.

Summary The location of materials containing terminal fucose residues on the surface of axenic and field grown roots of corn has been determined.Binding patterns of FITC-labelled,Lotus purpureus Moench lectin indicate the presence of the fucose residues in the cell walls and mucilage of the peripheral region of the root cap. During development, fucose residues also appear in the outer periclinal walls and overlying mucilage of columnar epidermal cells. Surface material rich in these residues persists between the mature root hairs but is not found on their surface. Fucose-rich mucilage is present on the exposed surface of aerial roots and at the point where they enter the soil. No lectin binding residues are indicated elsewhere in the roots.     

Changes in plasma-membrane-associated filaments during endocytosis and exocytosis in polymorphonuclear leukocytes

We examined the filaments associated with the cytoplasmic surface of the plasma membrane in rabbit exudate PMNs during phagocytosis of particles, or during “frustrated phagocytosis” with exocytosis of storage granules. Cells were plated onto yeast particles glued to coverslips with polylysine or onto coverslips coated with sheets of heat-agglutinated IgG. After periods ranging from 1 to 15 min, we disrupted the cells by a jet of salt solution and exposed their inner membranes. These broken cells were fixed immediately and processed for SEM. Whole cells were also prepared for SEM or TEM. At the site of PMN adherence to an opsonized yeast particle, a network of globular centers and thin, branched filaments appears on the cytoplasmic surface of the plasma membrane, while the outstretching lamellipodia contain a mesh of such filaments but no globular centers. Within 1 to 2 minutes, these structures disappear from the invaginating portion of the developing vacuole, and the cell's storage granules fuse with the barren membrane regions. These activities occur in rapid sequence over the vacuolar membrane after the first contact, until the phagocytosed particle is wholly encircled by a smooth, loose membrane, separated from the cell surface. A comparable filament pattern or complex was seen during “frustrated phagocytosis” on IgG sheets. At times between 1 and 5 min after plating, the cytoplasmic surfaces of these adherent membranes contain denuded central regions and peripheral nets of globular centers with radiating, thin, branched filaments. Granules apparently fuse with the bare areas. Thus we have obtained evidence of filament association with the plasma membrane at sites of adherence (to phagocytosable or nonphagocytosable surfaces) and have traced the subsequent disappearance of the filaments with degranulation.     

Antigenic and Immunogenic Characteristics of Nuclear and Membrane-Associated Simian Virus 40 Tumor Antigen

Antisera were prepared in syngeneic hosts against subcellular fractions of simian virus 40 (SV40)-transformed cells (MoalphaPM, MoalphaNuc), glutaraldehydefixed SV40-transformed cells (HaalphaH-50-G, MoalphaVLM-G), and electrophoretically purified denatured SV40 tumor antigen (T-ag) (RaalphaT). Immune sera were also collected from animals bearing tumors induced by SV40-transformed cells (HaalphaT, MoalphaT, HAF) and from SV40-immunized animals that had rejected a transplant of SV40-transformed cells (HaalphaS, MoalphaS). Immunological reagents prepared against cell surface (MoalphaPM, HaalphaS, MoalphaS, HaalphaH-50-G, MoalphaVLM-G) reacted exclusively with the surface of SV40-transformed cells by indirect immunofluorescence or protein A surface antigen radioimmunoassay. Immunological reagents prepared against the nuclear fraction (MoalphaNuc) or whole-cell determinants (HaalphaT, MoalphaT, HAF, RaalphaT) reacted with both the nuclei and surface of SV40-transformed or -infected cells. All reagents were capable of immunoprecipitating 96,000-molecular weight large T-ag from solubilized whole cell extracts of SV40-transformed cells. The exclusive surface reactivity of HaalphaS exhibited in immunofluorescence tests was abolished by solubilization of subcellular fractions, which then allowed immunoprecipitation of T-ag by HaalphaS from both nuclear and plasma membrane preparations. Specificity was established by the fact that all T-reactive reagents failed to react in serological tests against chemically transformed mouse cells, and sera from mice bearing transplants chemically transformed mouse cells (MoalphaDMBA-2) failed to react with SV40-transformed mouse or hamster cells. Reagents demonstrating positive surface immunofluorescence and protein A radioimmunoassay reactions against SV40-transformed cells were capable of blocking the surface binding of RaalphaT to SV40-transformed cells in a double-antibody surface antigen radioimmunoassay. This blocking ability demonstrated directly that a component specificity of each surface-reactive reagent is directed against SV40 T-ag. A model is presented which postulates that the differential detection of T-ag by the various serological reagents is a reflection of immunogenic and antigenic differences between T-ag polypeptides localized in nuclei and plasma membranes.     

Immunogenicity of outer membrane derivatives ofHaemophilus influenzae type b

We prepared outer membrane derivatives ofHaemophilus influenzae type b to determine whether the residual capsular and noncapsular surface components are immunogenic and protective. These fragments consist primarily of six major proteins and lipopolysaccharide. By transmission electron microscopy, they appeared as small, membrane-like fragments or larger, cellshaped double-track ghosts. Rabbits immunized with ghosts responded with increases in serum anticapsular antibody and bactericidal activity. Antisera absorbed with capsular antigen to remove anticapsular antibody remained bactericidal and passively protected infant rats. These data suggest that antibodies to noncapsular surface antigens are protective, and that outer membrane derivatives retain some of the constituents responsible for stimulating immunity.     

Controlling the exotic diseases: 2. Nursing management.

Advance planning can facilitate the care of a patient with an exotic disease who is admitted to a hospital that lacks facilities for high-security isolation. The Department of National Health and Welfare contingency plan for dealing with such patients lacks specific information in a number of areas of medical care, as described in this paper. Consideration must be given to the number of personnel trained and readied for employment, the criteria for selection and special preparation. The protective clothing generally used for hospital isolation procedures is inadequate. Several types of special clothing, including a respirator, are available for total protection of personnel; the clothing may be uncomfortable when worn for long periods, and does restrict movement, vision and communication. All persons entering the isolation suite must change into fully protective clothing, and double layers of clothing are required for direct patient care. All personnel must shower and change before leaving the isolation suite. Suitable facilities for dressing and showering, together with entry and exit routines, must be considered. Hand washing, daily cleaning procedures and disposal of liquid and solid wastes all require special procedures. The social and psychologic problems of patients and their families must also be considered. Preplanning is required to decrease the risks involved in monitoring vital signs and implementing emergency procedures requiring contact with the patient''s blood.     

The role of RNA primer in discontinuous DNA replication in isolated nuclei from HeLa cells

RNA-primed discontinuous DNA synthesis was studied in an in vitro system consisting of washed nuclei from synchronized S-phase HeLa cells. A new technique proved useful for the purification of short nascent fragments of DNA (Okazaki fragments). Mercurated dCTP was substituted for dCTP in the DNA synthesis reaction. Short nascent pieces (4–6 S) of mercurated DNA were found to bind preferentially to sulfhydryl-agarose, and could be eluted with mercaptoethanol. The isolated fragments were assayed for the presence of covalently linked RNA by the spleen exonuclease method described by Kurosawa et al. (Kurosawa, Y., Ogawa, T., Hirose, S., Okazaki, T. and Okazaki, R. (1975) J. Mol. Biol. 96, 653–664). Following a 30 s incubation with [³H]TTP in the absence of added ribonucleotides, approximately 20% of the nascent strands synthesized in washed nuclear preparations had RNA attached. These RNA primers either preexisted in the nuclei or were formed from endogenous ribonucleotides. The 5′ ends of the primers appeared to be largely in a phosphorylated state. In the absence of added ribonucleotides, these RNA-DNA linkages disappeared within 2 min, whereas if ribonucleotides were added, the number of RNA primers increased to 40% and remained at this level for greater than 2 min. To obtain maximal levels of RNA primer, the addition of all three of the ribonucleotides, rCTP, rGTP and rUTP (0.1 mM), as well as high levels of rATP (5 mM) was required. Addition of ribonucleotides also markedly enhanced the amount of nascent DNA fragments synthesized. However, in the absence of added ribonucleotides, after RNA primers had disappeared, nascent DNA fragments were still initiated at a significant rate. These results suggest that RNA primers play an important role in the initiation of Okazaki fragments but that synthesis can also be initiated by alternative mechanisms. An important role for ATP in RNA primer synthesis is suggested.