Mitogenic response to epidermal growth factor: Relationship to number,affinity, and down-regulation of EGF receptors in three murine embyro cell lines

Swiss 3T3 and C3H-M2 cells have a greater mitogenic response to epidermal growth factor (EGF) than do C3H-10T1/2 cells. The latter cell line, however, has a number of EGF receptors per cell intermediate between the two cell lines that have a more vigorous response to EGF. Scatchard analysis of binding data indicate that all three cell lines have one class of EGF receptor, with indistinguishable affinity for the ligand. When exposed to 10-nM EGF all three cell lines “down-regulate” their EGF receptors with the same time course, and to the same precentage of initial receptors.     

Primary in vitro antibody response of rabbit lymphoid cells and T-B cell collaboration in the absence of detectable mitogens

To investigate whether the antibody response and T-B-cell collaboration in vitro can be obtained in the absence of mitogens, a method of obtaining an in vitro primary anti-sheep red blood cell antibody response by rabbit spleen and lymph node cells was developed. We used Marbrook culture vessels and a specially prepared medium containing 10% autologous serum and maintained at pH 7.4–7.6. The system was shown to be devoid of any polyclonal mitogens as assessed by [³H]thymidine incorporation and by direct examination for blast cells in stained smears. The primary response increased continuously over the 5-day cultivation period and only IgM but not IgG plaque-forming cells (PFC) were detected. In over 20 experiments, the response ranged from 357 ± 17 to 4425 ± 110 PFC/10⁷ cultured cells with a median stimulation index of 52. The spleen cells required less antigen than the lymph node cells and 2-mercaptoethanol inhibited the response of the spleen cells but not that of the lymph node cells. Lymphocytes were separated into highly pure T- and B-cell populations by negative selection using antibody-coated human erythrocytes to rosette either T or B cells and Ficoll-Hypaque centrifugation to remove rosetted cells. Upon cultivation, B cells alone gave a low IgM response, whereas B cells reconstituted with T cells gave a response similar to that obtained with unseparated lymphoid cells. We concluded that: (a) optimal conditions for obtaining primary in vitro antibody responses using rabbit spleen and lymph node cells were established, (b) T-B-cell collaboration was demonstrated in the rabbit primary antibody response to sheep erythrocytes, and (c) the primary antibody response in vitro and T-B-cell collaboration may occur in the absence of detectable polyclonal mitogens.     

Preliminary observations on reproduction in the Central American agouti,Dasyprocta punctata

Two adult male and three adult female agoutis have been reproducing at the Lincoln Park Zoo since 1978. Agoutis of the species D. punctata and D. fuliginosa are capable of breeding and producing offspring in every month of the year in captivity. The minimum interbirth interval of captive punctata is 127 days, and its gestation period is apparently less than 127 calendar days. In captivity, agoutis are likely to produce multiple offspring.     

Lectin binding in skeletal muscle. Evaluation of alkaline phosphatase conjugated avidin staining procedures

Summary Cryostat sections from rat gracilis muscles were incubated with different biotinylated lectins: Con A (Concanavilin A), WGA (Wheat germ agglutinin), SBA (soybean agglutinin), GS I and GS II (Griffonia simplicifolia agglutinin), LCA (Lens culinaris agglutinin), PNA (peanut agglutinin) and PSA (Pisum sativum agglutinin). The sections were subsequently treated with alkaline phosphatase conjugated avidin. The lectin binding sites were visualized after incubation in substrate media containing: (1) 5-bromo-4-chloro indoxyl phosphate and Nitro Blue tetrazolium or copper sulphate; (2) naphthol AS-MX phosphate or naphthol AS-BI phosphate and various types of diazonium salts; (3) -naphthylphosphate and Fast Blue BB; (4) -glycerophosphate according to the method of Gomori. The results obtained with the alkaline phosphatase methods were compared with those seen with a streptavidin-horseradish peroxidase procedure. Several chromogen protocols for visualizing alkaline phosphatase activity showed differences in the ability to detect lectin binding sites. A sarcoplasmic reaction was evident for Con A, GS II, WGA, LCA, and PSA after incubation in the indoxyl phosphate medium. Sarcoplasmic reaction for GS II was also noticed after incubation with naphthol AS-MX Fast Blue BB and -glycerophosphate. The latter substrate also gave rise to a sarcoplasmic Con A reaction. With the indoxylphosphate tetrazolium salt method some muscle fibres showed a very strong intracellular reaction after incubation with Con A and GS II while the staining intensity was weak in other fibres. The same muscle fibres were stained with PAS. No sarcoplasmic reactions were observed with either naphthol phosphate media or with the diaminobenzidine peroxidase methods. Further, the staining of the muscle fibre periphery, connective tissue, and capillaries was intensified using the indoxyl method. The indoxylphosphate-tetrazolium salt method seems to be suitable for future investigations of lectin binding sites in muscle sections.     

The lysis of Gram-negative Alcaligenes eutrophus by enzymes from Cytophaga

Summary The use of Cytophaga lysing enzymes was investigated for the liberation of poly--hydroxybutyrate (PHB) granules from the Gram-negative bacterium Alcaligenes eutrophus. Complete cell lysis was approached within a 60 minute period. Contrary to previous findings for the lysis of Gram-negative bacteria, prior removal of the outer membrane was not essential for enzymic lysis. The destabilisation of the outer membrane by the removal of divalent cations resulted in no significant improvement in the disruption process.     

Levels of hypoxanthine phosphoribosyltransferase RNA in human cells

The gene for the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT) is expressed at a low level in many cells. As is the case with several other “housekeeping genes,” thorough studies of hprt gene regulation have been hampered by the low levels of its mRNA. We have used RNA/RNA hybridization in solution to determine the concentration of hprt-RNA in human cells. The sensitivity and specificity of the method have been validated, and it is shown that hprt-RNA can be accurately determined at a level of a few mRNA molecules per cell. As expected for a housekeeping gene, low and relatively constant hprt-RNA levels (0.3–0.8 pg/μg DNA) were found in primary cultures of normal amnion cells and fibroblasts, EBV-transformed lymphoblastoid cell lines, neuroblastoma, glioblastoma, and melanoma cell cultures. While resting lymphocytes were found to contain very low amounts of hprt-RNA, lymphocytes stimulated with phytohemagglutinin (PHA) showed a 10-fold increase to about 0.8–1.2 pg/μg DNA, which corresponds to 6–10 hprt-RNA molecules per cell. The level started to increase about 20 h after PHA stimulation, 5–10 h before the onset of DNA synthesis, and a steady-state level was reached after 2–3 days in culture. In PHA-stimulated lymphocytes from two brothers with inherited HPRT deficiency (LeschNyhans syndrome), the hprt-RNA level in PHA-stimulated lymphocytes was only about 25% of that in normal subjects. In T-cells selected for HPRT deficiency by growth in 6-thioguanine medium, the levels of hprt-RNA were either normal or very low, which probably reflects the different nature of the mutations involved. These results demonstrate the sensitivity of this method for determinations of low levels of RNA and clearly show induction of hprt-RNA after mitogenic stimulation of human lymphocytes.     

Application of the functional-form model to the culture of seaweeds

Selecting the most appropriate species or strains is an important first step in the development of most algal cultivation systems and is usually a tedious, time-consuming, and expensive step. The functional-form model, first developed to synthesize the adaptive significance of easily assessed thallus-form attributes relative to the productivity and survival of benthic macroalgae, is applicable to the culture of seaweeds and can expedite species or strain selection. The production ecology aspects of the model are useful particularly for applications where the desired product is not species-specific, e.g., systems in which the emphasis is on algal production, such as algal biomass farms and wastewater treatment. A thallus-form with a high surface area: volume ratio is more suited for rapid production and nutrient uptake. The utility of this model to strain selection is demonstrated with the red alga Gracilaria tikvahiae, a species that has been considered a maricultural candidate for a number of utilizations. A continuum of surface area: volume ratios for eight clones of G. tikvahiae showed that this ratio decreased as morphological complexity increased and was a good predictor of both short-term photosynthesis and long-term growth rate. Clones near opposite ends of the surface area: volume ratio spectrum had significant differences for both photosynthesis and growth. Each clone of G. tikvahiae possesses concomitant combinations of benefits as well as costs, which should be carefully evaluated for the cultivation application of interest. Knowledge of functional-form relationships in seaweeds can significantly expedite their successful cultivation.     

Glycosphingolipids in insects. Chemical structures of two variants of a glucuronic-acid-containing ceramide hexasaccharide from a pupae of Calliphora vicina (Insecta: Diptera), distinguished by a N-acetylglucosamine-bound phosphoethanolamine sidechain

The two major components of the acidic glycolipid fraction from the pupae of Calliphora vicina were isolated using high-performance liquid chromatography. The acidic moiety was identified as glucuronic acid by beta-glucuronidase cleavage and gas chromatographic analysis as the pentafluoropropionyl derivative. The structures of the carbohydrate moiety were elucidated by peracetylation, methylation, exoglycosidase cleavage, fast-atom-bombardment mass spectrometric and 1H-nuclear magnetic resonance spectroscopic analysis. The only difference between the two hexasaccharide variants was the presence, in one of them, of a between the two hexasaccharide variants was the presence, in one of them, of a phosphoethanolamine (AeP) sidechain on the third sugar of the sequence, i.e. N-acetylglucosamine. The composition of the ceramide moiety was dominated by a C20:0 fatty acid (arachidic acid) and a C14:1 sphingoid base (tetradecasphing-4-enine). The chemical structures of the two insect acidic glycosphingolipids were determined to be: GlcA(beta 1-3)Gal-(beta 1-3)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man (beta 1-4)Glc(beta 1-1)Cer; GlcA(beta 1-3)Gal(beta 1-3)GalNAc(beta 1-4)[2AeP-6]-GlcNAc(beta 1-3) Man(beta 1-4)Glc(beta 1-1)Cer. Such glucuronic-acid-containing insect glycosphingolipids have been given the generic name arthrosides, with the implied synonymity to the gangliosides.