Mass production of animal cells in nude mice with retention of cell specific markers.

Biochemical analysis of macromolecular constituents of somatic cells in culture frequently requires the production of large quantities of cells from small initial populations, often a laborious and expensive procedure. Here we show that nude mice, because of their genetic immune deficiency, provide a relatively simple and reliable means of growing large quantities of cells irrespective of the species or tissue of origin. Most established animal cell lines are capable of growing as large tumors in these athymic mice. The passage of mammalian cells in nude mice does not cause either the loss or modification of specific biochemical, chromosomal, antigenic or other cellular markers, nor the induction of malignant transformation of the host cells. Tumors formed by the injected cells grow as localized, encapsulated masses, and host cell admixture due to elements of the vascular system in the tumor is variable but not extensive. Pure cultures of the original cell type can easily be recovered from the tumors if they are derived from previously established cell lines. The use of nude mice for large scale growth of animal cells is particularly advantageous for cell lines which are not adapted to mass suspension culture for highly differentiated functional tumor cells which cannot be grown in vitro.     

Target organ regulation of substance P in sympathetic neurons in culture

The distribution of the mRNA for one of the two mouse protamines, the cysteine-rich, tyrosine-containing protamine (MP1), was examined in the polysomal and nonpolysomal compartments of total testis and purified populations of round and elongating spermatids using Northern blots. In postmitochondrial supernatants prepared from total testis, about 10-15% of MP1-mRNA sediments with the small polysomes. The nonpolysomal molecules of MP1-mRNA are homogeneous in size, about 580 bases, while the polysomal molecules are heterogeneous with a mode of about 450 bases. Digestion with RNase H and thermal chromatography on poly(U) Sepharose reveals that the difference in size of polysomal and nonpolysomal MP1-mRNA is due to a shortening of the poly(A) from about 160 to 30 bases. In round spermatids, essentially all of MP1-mRNA is 580 bases long and is in the nonpolysomal fraction. Elongating spermatids contain roughly equal proportions of the homogeneous, 580 base form in the nonpolysomal compartment, and the heterogeneous 450 base form solely in the polysomal compartment. These results indicate that mRNA for one of the mouse protamines is stored as an untranslated RNP in round spermatids, and that it is partially deadenylated when it is translated in elongating spermatids.     

The nonenzymatic glycosylation of collagen

Calf skin acid-soluble collagen, containing about 34 residues of lysine plus hydroxylysine per 100,000 dalton polypeptide chain, was treated with [¹⁴C]glucose in the presence or absence of NaCNBH₃. In 144 h, under the conditions employed, the presence of NaCNBH₃ increased the extent of glycosylation from 8 to 15% of the total residues of lysine plus hydroxylysine. The extent of glycosylation was estimated, using acid hydrolysates of the protein, by isolation and determination of reduced adducts (1-lysinohexitols) employing a system of paper chromatography followed by chromatography on an amino acid analyzer. By those means the difficulties of using specific color reactions such as that with thiobarbituric acid were obviated. Identification of the reduced adducts as forms of 1-lysinohexitol was made by comparison of that substance prepared by treatment of polylysine with [¹⁴C]glucose in the presence of NaCNBH₃. Of interest is the fact that treatment of the polymer with glucose for 144 h under conditions similar to those used for the collagen, resulted in an increase of extent of glycosylation from 3 to about 50% of the total lysine residues when NaCNBH₃ was present in the incubation medium. The greater degree of glycosylation of lysine residues in polylysine as compared with collagen (15 versus about 50%) may be ascribed to the different orders of macromolecular structure in the protein that could sequester certain of the residues from reaction with glucose. 1-Lysinohexitol was also identified in hydrolysates of neutral salt-soluble guinea pig skin collagen that had been reacted with glucose and then treated with NaB³H₄. The glycosylated collagens were fragmented by treatment with CNBr, and modified lysine residues were found to occur along the entire length of the collagen chains. The use of NaCNBH₃ in the manner described above permits measurement of both aldimine and ketoamine forms of the adducts made with glucose. The possible physiological significance of the reversibility of the ketoamine form of adduct is discussed briefly.     

The arrangement of nucleosomes in nucleoprotein complexes from polyoma virus and SV40.

Androgen-dependent and androgen-independent (autonomous), cloned, cultured cell lines of the androgen-dependent mouse mammary adenocarcinoma, Shionogi carcinoma 115, have been established. Growth of the dependent cells requires the presence of androgen, provided they are grown in suspension culture in medium containing dextran-charcoal-treated fetal calf serum. The growth rate of autonomous cells in the presence or absence of DHT is similar to that of dependent cells grown in its presence. An agar culture method has been developed that enables the proportion of dependent and autonomous cells in mixed populations to be determined. Autonomous cells appear in dependent clones, and their frequency increases with increasing time of subculture. Dependent cells from tumors preferentially in male animals, and dependent cell cytosols contain significant amounts (approximately 300 femtomoles per mg protein) of a specific androgen-binding macromolecule. Autonomous cells formed tumors equally well in both male and female mice, and autonomous cell cytosols contain very low levels (≤ 7 femtomoles per mg protein) of the specific androgen-binding macromolecule(s). These studies delineate a system which can be used to investigate the mechanism of steroid hormone-dependent and autonomous tumor growth, and the transitions between the hormone-dependent and autonomous states.     

Purification and characterization of guanylate cyclase from Caulobacter crescentus

Guanylate cyclase has been purified 60-fold from cell extracts of the bacterium $\text{Caulobacter crescentus}$. It has a molecular weight of approximately 140,000 and is dependent upon Mn²⁺ for activity. Enzymic activity is unaffected by cyclic AMP, cyclic GMP or N⁶,O^2′-dibutyryl cyclic AMP but is stimulated by N²,O^2′-dibutyryl cyclic GMP. The partially purified preparation of guanylate cyclase does not contain detectable adenylate cyclase activity.     

Effects of dibutyryl cyclic AMP on tyrosine uptake and metabolism in neuroblastoma cultures

A series of thin-layer Chromatographic (TLC) systems were employed to study the effects of dibutyryl cyclic AMP (db-cAMP) on the metabolism of ³H-tyrosine in neuroblastoma cultures. The neuroblastoma monolayer cultures incubated with radiolabelled tyrosine synthesized di-hydroxyphenylalanine (DOPA), dopamine (DA), and norepinephrine (NE), in confirmation of previous reports identifying these compounds in neuroblastoma cultures. In addition, we found evidence suggesting the presence of metabolites of DA and NE, that is, homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) together with 3-methoxy-4-hydroxymandelic acid (VMA). When these cultures were grown in the presence of db-cAMP for 3 days, tyrosine uptake was increased with a proportional increase in tyrosine hydroxylation. This effect persisted in the absence of db-cAMP, but it was not apparent with only 90 min exposure to db-cAMP. Suspension cultures showed the same baseline level of tyrosine uptake as did monolayer cultures, but the uptake in suspension cultures failed to increase with db-cAMP treatment. It is suggested that the db-cAMP induced differentiation of the neuroblastoma cells in monolayer cultures was associated with induction of a tyrosine uptake system.     

Absorption spectra of mixed monomolecular films of chlorophyll and photosynthetic electron carriers at a gas-water interface

Absorption spectra of mixed monomolecular films containing chlorophyll and endogenous redox reagents are studied at a gas-water interface. Overlapping absorption spectra are resolved by difference spectroscopy and fourth derivative analysis. Monomolecular films are formed in a Langmuir trough using a Wilhelmy film balance. A reaction between vitamin K₁ and chlorophyll is observed both in the dark and after illumination. A smaller interaction occurs between α-tocopherolquinone and chlorophyll. A light-driven reaction occurs between oxidized plastocyanin and chlorophyll, but not between reduced plastocyanin and chlorophyll. An interaction is observed between cytochrome c and chlorophyll both in the dark and after illumination. Evidence is obtained indicating that the presence and amount of aggregated species of chlorophyll are dependent on the presence of specific reagents. We suggest that redox reagents of Photosystem II and Photosystem I of photosynthesis also serve to “induce” the formation of distinct chlorophyll species.     

Progesterone antagonism of androgen-dependent marking in gerbils

The role of progesterone (P) as an androgen antagonist in Mongolian gerbil territorial marking was assessed in the present investigation. Twenty-seven mature males were observed for two consecutive weeks in a marking apparatus. On the basis of the average of the two sessions, subjects were matched and assigned to one of three groups. All animals were orchidectomized and tested three weeks after surgery. Three days after a single postoperative session a schedule of hormonal replacement was initiated. Group 1 was administered 640 μg testosterone propionate (TP). Group 2 received 640 μg TP followed immediately by 3 mg P. A third group received 640 μg TP followed by a 1 mg injection of P. Subjects were tested for marking 24 hr after the injection for a cumulative period of 6 wk.Results indicated that (a) castration drastically reduced marking; (b) TP alone and TP + 1 mg P restored the behavior; (c) TP + 3 mg P inhibited its restoration; and (d) a significant interaction was present between hormonal therapy and the 6-wk testing interval. However, all three hormonal treatments restored sebaceous gland dimensions. Results are discussed in terms of a model of hormone-gene action.     

Chemically modified porous silica gel as a bioadsorbent and a biocatalyst.

Aminopropyl silica gel was prepared from porous silica gel by reaction with γ-aminopropyltrimethoxysilane in toluene and was used for immobilizing chymotrypsin (EC 3.4.4.5) and human serum albumin. Immobilized chymotrypsin was used for the resolution of N-acetyl-dl-phenylalanine and immobilized human serum albumin was used for the purification of goat anti-human serum albumin. Epoxy silica gel, prepared by reaction of porous silica gel with γ-glycidoxypropyltriethoxysilane, was coupled with m-aminobenzamidine and the resulting matrix was used for trypsin purification.     

Expression of protease and protease-inhibitory activity in human mononuclear leukocyte cultures : Effect of conA stimulation

Trypsin-activated protease activity was observed in the supernatants of conA-treated unfractionated or monocyte-depleted cultures at 1 h of incubation and trypsin-inhibitory activity was detected at 24 h. In contrast, supernatants from wheat germ agglutinin (WGA)-treated cultures or untreated cultures exhibited trypsin-inhibitory activity at 1 h and trypsinactivated protease activity after 24 h. The expression of proteases and protease inhibitors may be early events that occur during the activation of quiescent lymphocytes to a proliferative state.     

The encapsulation process in Biomphalaria glabrata experimentally infected with the metastrongylid Angiostrongylus cantonensis: enzyme histochemistry.

Enzyme histochemistry has revealed that encapsulation reactions surrounding larvae of the nematode Angiostrongylus cantonensis 4 wk after infection of the gastropod Biomphalaria glabrata are the sites of highly localized acid phosphatase and nonspecific esterase activities. Lesser amounts of alkaline phosphatase and β-glucuronidase activities also occur within such capsules, but aminopeptidase activity cannot be demonstrated. In addition, it has been ascertained that acid phosphatase activity gradually increases as the encapsulation reaction progresses during the first to the fourth week postinfection.     

Cell density-dependent stimulation of glutamine synthetase activity in cultured mouse teratoma cells

A density dependent stimulation of glutamine synthetase (GS) activity has been observed in cultures of mouse teratoma cells. GS specific activity increased as cultures approached confluency to a level greater than 2-fold over the basal level found in sparse cultures. After confluency the GS specific activity returned to the basal level found in sparse cultures. The enzyme increase could not be attributed to age of cultures, medium or glutamine depletion, cell leakage of GS, or change in the amount of cellular protein. Dibutyryl cyclic AMP (db-cAMP) plus theophylline lowered GS specific activity both in cultured teratoma and in teratoma obtained from ascites grown tumors. The enzyme increase observed in cultured teratoma cells could be prevented by cycloheximide, and enhanced by hydrocortisone or actinomycin D.     

Axon initiation by ciliary neurons in culture

A nerve culture system for the study of axon initiation is described. A population of individual chick embryo ciliary neurons, free from contact with other cells and attached to a polyornithinecoated culture dish, is exposed to heart cell-conditioned medium (HCM). Within 30 min after the addition of HCM the majority of neurons have formed growth cones, and by 90 min more than 80% of the neurons bear at least one axon longer than 15 μm. Before the addition of HCM, ciliary neurons generate membrane ruffles and extend filopodia around the entire periphery of the rounded cell body. Axon initiation, following addition of HCM, consists of two distinctive changes in the cell surface: (1) organization of the randomly distributed surface movements into localized highly active growth cones, which then form axons; and (2) the cessation of surface movements elsewhere on the cell periphery. Heart cell-conditioned medium may induce these changes by increasing the adhesion between parts of the nerve cell surface and the substratum.     

Immunologic properties of mouse thymus cells: membrane antigen patterns associated with various cell subpopulations

The membrane antigen components of mouse thymus cells and fractions derived from BSA density gradient centrifugation were assayed by quantitative cytotoxicity tests. Two subpopulations were identified on the basis of average density and antigen patterns. The major subpopulation consisted of small lymphoid cells and comprised 80%–90% of all cells, was of high relative density and rich in θ, TL, G_IX, Ly-A, Ly-B, and Ly-C, but contained little or no H-2. The minor subpopulation was chiefly large lymphoid cells, comprised 10%–15% of cells, was of low relative density, was relatively rich in H-2 but low in θ and Ly antigens, and contained no detectable TL or G_IX. This minor subpopulation was identical in density and antigen patterns to those cells remaining in the thymus after short-term cortisone treatment or whole-body irradiation. It could also be reproduced by treating whole thymus with anti-TL or anti-θ sera. The antigenic attributes of this minor subpopulation differed from those of spleen lymphocytes only with respect to average density.     

Developmental genetics of teleosts: a biochemical analysis of lake chubsucker ontogeny

Developing embryos of the lake chubsucker, Erimyzon sucetta, were analyzed with regard to both gross morphological changes and specific enzymatic changes from the unfertilized egg stage until some 3 weeks posthatching. Total activities of three enzymes—lactate dehydrogenase, glucose-6-phosphate dehydrogenase, and isocitrate dehydrogenase—were determined throughout the course of development. Each of these different enzymes exhibited a different pattern of change during ontogeny. Electrophoretic analysis of qualitative changes in isozyme patterns was accomplished for these three enzymes and for α-amylase, glucosephosphate isomerase, mannosephosphate isomerase, creatine kinase, esterase, glutamate dehydrogenase, alkaline phosphatase, aspartate aminotransferase, malate dehydrogenase, hexose diphosphatase, phosphoglucomutase, and phosphogluconate dehydrogenase. Many of the enzyme systems investigated exhibited rich patterns of ontogenetic change, while a few remained relatively unchanged throughout the interval studied. Several of the enzymes in particular metabolic pathways exhibited coincident changes suggestive of coordinate control. The appearance of several rather “tissue-specific” isozymes was closely correlated with the morphological and functional differentiation of these particular tissues or organs.     

Behavioural ecology of mass recruitment in the army ant Neivamyrmex nigrescens

The successful rearing of the army ant Neivamyrmex nigrescens in the laboratory has enabled us to demonstrate that the pheromone trail deposited by recruiting workers is qualitatively different from the ants' exploratory trail. The recruitment trail alone can initiate as strong a mass recruitment response as can a recruiting worker that physically interacts with nestmates. The rapidity with which workers are aroused is due to secondary recruitment. Army ants are able to assemble a critical striking force before food is located, as a result of mass recruitment to new terrain. In addition to feeding behaviour, mass recruitment occurs when army ants emigrate to new nests. In both behavioural contexts, primary and secondary recruiters run more rapidly than exploring ants, and with exaggerated vertical motor patterns.     

Involvement of proteolytic acivity in early events in lymphocyte transformation by phytohemagglutinin

The stimulation by phytohemagglutinin (PHA) of DNA synthesis in cultured blood lymphocytes of guinea pig was markedly inhibited by addition of leupeptin, a well-characterized, powerful protease inhibitor of tripeptide nature. About 30 to 40 per cent inhibition was observed at 40 μg/ml of leupeptin when leupeptin was added 30 min prior to or together with PHA. Per cent inhibition by the appropriate amount of leupeptin was proportional to the amount of PHA added in the range of 0.6 to 3.0 μg of PHA at which the per cent inhibition reached maximum. This inhibitory effect of leupeptin on PHA stimulation was abolished when the lymphocytes were preincubated with PHA for more than 10 min before addition of leupeptin or preincubated with leupeptin for more than 60 min prior to PHA addition.     

Differential effects of lysosomotropic amines and polyamines on processing and biological activity of EGF

The effects of lysosomotropic amines and polyamines on rat fibroblasts were studied after the administration of epidermal growth factor (EGF) in order to determine whether the intracellular processing of EGF was important for transmission of its biological signal. Following the addition of EGF, cell cultures exhibited a dose-dependent increase in ornithine decarboxylase (ODC) activity. This increase in ODC activity was drastically reduced by both methylamine, a representative lysosomotropic amine, and putrescine, a polyamine precursor. However, inasmuch as methylamine inhibited EGF-induced DNA synthesis by greater than 50%, putrescine had no inhibitory effect. Lysosomotropic amines, but not polyamines, prevented EGF processing as evidenced by their ability to block the release of intracellular 125EGF and by their ability to inhibit the formation of the final intracellular processed product of EGF, as determined by isoelectric focusing. These data suggest that the processing of EGF is consistent with the induction of DNA synthesis and ODC activity. The cellular mechanisms involved in inhibition of ODC induction by polyamines appear to be distinct from those involved in lysosomotropic amines.