The kinetic significance of cell size : I. Variation of cell cycle parameters with size measured at mitosis

Using a cell line of human lymphoid cells, the kinetic significance of cell size measured at mitosis has been explored using fraction of labelled mitoses data. It was found that smaller cells tend to have progressively longer generation times. The principal mechanism for this generation time dilation is a progressively protracted G 1 duration as cell size decreases. There is a concomitant, but much slighter increase in S phase duration. G 2 duration remains essentially constant irrespective of cell size.     

CSF distribution of morphine, methadone and sucrose after intrathecal injection

The lumbar to cisternal CSF distribution of morphine and methadone were compared to C-14 sucrose, a standard marker of CSF bulk flow, after lumbar subarachnoid injections in a sheep preparation. Morphine appeared and peaked simultaneously with C-14 sucrose in cisternal CSF at 90 to 190 minutes. The mean peak cisternal CSF morphine concentrations were sustained for 30-40 minutes, and averaged 148 ng/ml, representing 0.3% of the administered dose. Methadone was not detectable in cisternal CSF up to 240-300 minutes after lumbar subarachnoid administration. The C-14 sucrose/morphine ratio was increased an average of 6.7 times in cisternal CSF as compared to the ratio of the two compounds injected into the lumbar subarachnoid space. These studies demonstrate that morphine, a hydrophilic opioid, given intrathecally moves rostrally and appears in cisternal CSF by bulk flow. Furthermore the rostral redistribution of morphine is associated with the clearance of morphine from CSF. Methadone, a lipophilic opioid, appears to be completely cleared from CSF before it reaches the cisterna magna. These pharmacokinetic studies support a contribution of supraspinal sites to the analgesic and adverse effects produced by morphine given by spinal routes of administration. In contrast methadone appears to exert its effects predominantly at spinal sites.     

Opiates and enkephalins: a common binding site mediates their analgesic actions in rats

³H-Labelled opiate and enkephalin ligands appear to bind with highest affinity to a single site responsible for their analgesic properties. Administered $\text{in vivo}$, naloxazone, an irreversible opiate, selectively inhibits for over 24 hours the high affinity binding of ³H-labelled mu, and kappa opiates and enkephalins. This inhibition of binding gradually resolves over 3 days, perhaps correlating with receptor turnover. Naloxazone treatment also abolishes morphine, D-ala²-met⁵-enkephalinamide and betah-endorphin analgesia. Although morphine and D-ala²-met⁵-enkephalinamide bind with similar potencies to the high affinity site, morphine's potency for the low affinity D-ala²-met⁵-enkephalinamide site is far less than the enkephalin analog. These results imply that all ³H-ligands examined bind with highest affinity to a mu-like receptor while low affinity D-ala²-met⁵-enkephalinamide binding, with a K_D of 6 nM, represents a delta-like receptor.     

The binding to rat brain homogenates of Mr2034, a universal opiate

Mr2034 has been proposed as a kappa opiate. While Mr2034 inhibited the binding of the kappa opiate 3H-ethylketocyclazocine better than unlabeled ethylketocyclazocine, it also displaced the binding of 3H-dihydromorphine and 3H-SKF 10047 more potently than morphine and SKF 10047, respectively. 3H-D-ala2-D-leu5-enkephalin was displaced equally well by Mr2034 and D-ala2-D-leu5-enkephalin. Saturation studies of 3H-Mr2034 binding demonstrated curvilinear Scatchard plots which could be dissected into two components by computer: KD1 0.06 nM, Bmax1 2.49 fmoles/mg tissue; and KD2 2.4 nM, Bmax2 6.57 fmoles/mg tissue. A portion of the higher affinity (KD 0.06 nM) component was inhibited by naloxonazine treatment in vitro (50 nM), suggesting that 3H-Mr2034 bound with very high affinity to mu1 sites. Displacement of 3H-Mr2034 binding by opioids was multiphasic, again implying that 3H-Mr2034 was binding to more than one class, of site. In view of its similar potency in inhibiting mu (3H-dihydromorphine), kappa (3H-ethylketocycla-zocine), sigma (3H-SKF 10047) and delta (3H-D-ala2-D-leu5-enkephalin) opioids Mr2034 might be considered a universal opiate.     

The actions of naloxazone on the binding and analgesic properties of morphiceptin (NH2Tyr-Pro-Phe-Pro-CONH2), a selective mu-receptor ligand

Active in both binding and biological assays, morphiceptin (NH₂ Tyr-Pro-Phe-Pro-CONH₂), a potent opioid peptide derivative of β-casamorphine, binds specifically and selectively to mu or morphine-type receptors with little affinity for delta sites. Displacement studies of a variety of ³H-labeled opiates and enkephalins show biphasic curves. Naloxazone, which blocks irreversibly and selectively high affinity opiate and enkephalin binding, abolishes morphiceptin's inhibition of binding at low concentrations, suggesting that the high affinity binding of enkephalins and opiates represents a mu or morphine-type receptor. Unlike the reversible antagonist naloxone, naloxazone treatment $\text{in}$$\text{vivo}$ inhibits for over 24 hours the analgesic activity of morphiceptin like it inhibits morphine, β-endorphin and enkephalin analgesia. Together, these studies imply that opiates and enkephalins bind with highest affinity to a mu receptor which mediates their analgesic activity. The ³H-D-ala²-D-leu⁵-enkephalin binding remaining after naloxazone treatment, representing a lower affinity site (K_D 4 nM), is quite insensitive to morphiceptin inhibition and has the characteristics of a delta receptor. However, the ³H-dihydromorphine binding present after naloxazone treatment, which also represents a lower affinity site (K_D 6 nM), is far more sensitive to both morphine and morphiceptin and may represent a second morphine-like, or mu, receptor.     

Protamine-DNA association in mammalian spermatozoa

We have previously identified two subsets of basic nuclear proteins of mouse sperm: the protamines and a group of less basic proteins and, with the aid of a polyvalent antiserum, we have demonstrated their differential extractibility by NaCl in reducing solution (Rodman et al., J cell sci 53 (1982) 227) [9]. By affinity purification with isolated mouse sperm protamines we have obtained a protamine-specific fraction of that antiserum and a fraction that contains antibodies to the subset of less basic proteins. With those immunochemical probes we have shown the following The antigenic sites recognized by the protamine-specific antibodies are accessible, intranuclearly, only after the DNA has been removed by DNase I. The antibodies and DNA compete for binding sites on the protamines. DNA removal and consequent availability of the antigenic sites of the protamine molecules to the antibodies are possible only after displacement of the less basic proteins and chromatin decondensation have been induced. Immunoreactivity by the less basic proteins takes place without intervention of DNase. Those data indicate that the protamines are DNA-bound but that the less basic proteins are not or, alternatively, their putative DNA-binding sites do not coincide with their immuno-reactive sites. Those data also suggest that a function of the subset of less basic proteins may be to provide a shield for the protamine-DNA complex. The mouse protamine-affinity-bound antibodies are highly cross-reactive with protamines of other mammalian sperm suggesting that, despite considerable molecular diversity among mammalian protamines, the DNA-binding sites are conserved.     

Fluorescence-activated cell sorting of human T and B lymphocytes: I. Direct evidence that lymphocytes with a high density of membrane-bound immunoglobulin are precursors of plasmacytes

Human peripheral lymphocytes bearing either a high or a low amount of membrane-bound immunoglobulin were studied. Cells were “tagged” with fluorescein-labeled antiimmunoglobulin reagents and separated by means of a new electronic instrument, a fluorescence-activated cell sorter (FACS), into populations with either > 10⁵ or < 5 × 10³ immunoglobulin molecules per cell. Fractions of high purities were obtained. (>80% and >99.9%, respectively). In vitro, different functional properties were observed: lymphocytes with high densities of membrane-Ig gave a late proliferative response after stimulation with Pokeweed mitogen (PWM). A considerable proportion of stimulated cells developed into mature plasmacytes as detected by cytoplasmic staining. Those lymphocytes with a low density or complete absence of membrane-Ig could be stimulated by both Phytohemagglutinin (PHA) and Pokeweed mitogen, but no differentiation into plasmacytes occurred. The functions are similar to those of bone marrow-derived (B) and thymus-derived (T) lymphocytes in mice. Thus, the designation as B lymphocytes for human lymphocytes with a large quantity of membrane-bound immunoglobulin seems justified.     

Anti-VPg antibody inhibition of the poliovirus replicase reaction and production of covalent complexes of VPg-related proteins and RNA

Anti-VPg antibodies inhibited host-factor-dependent RNA synthesis by the poliovirus replicase but not oligo(U)-primed synthesis, implicating VPg in the de novo initiation of replicase products. Complexes of VPg-related polypeptide and newly made RNA could be immunoprecipitated by anti-VPg antibody from the host-factor-stimulated products of the replicase reaction. The complexes appeared to be covalently linked and involved 50 to 150 nucleotide chains of RNA that were RNAase-T1-resistant and could be largely poly(U).     

Analytical techniques for cell fractions. XIX. The cyclum: an automatic system for cyclic chromatography.

An automatic system, termed a Cyclum, is described which allows column chromatographic separations to be repeated precisely a large number of times. Provision is made for the adjustment during operation of parameters such as equilibration, wash, elution, and sample flow times and duration of fraction collection. The system is applicable to both analytical and preparative use in various types of column chromatography (e.g., affinity, gel filtration, ion-exchange), but has been especially developed for separations based on immunosorption.     

A novel receptor for secretory component on porcine mononuclear cells

Porcine mononuclear cells from peripheral blood separated by Ficoll-Hypaque gradient technique bound secretory IgA preferentially but not serum IgA or IgG after capping with anti-Ig. When the cells were incubated with free secretory component and washed, serum IgA binding was facilitated. Thus, the results indicate that a subpopulation of porcine circulating mononuclear cells bear surface receptors for secretory component (SC-receptor).     

Effect of vitamin D and its metabolites on developing myogenic cultures

Growth, protein synthesis and expression of creatine kinase (CK) by embryonic chick myogenic cells are inhibited by vitamin D and certain of its metabolites. 25-OH cholecalciferol was most active in concentrations of 10⁻⁵–10⁻⁶ M, with cholecalciferol and ergocalciferol less active in that order. Ergosterol had no activity of this sort. Inhibition of CK was most marked on the 4th and 5th day of culture and was due to suppression of the appearance of CK-MM and MB. CK-BB was not affected and CK-MB was more affected than CK-BB. Skin fibroblasts by comparison were slightly stimulated in growth at 10⁻⁶ M and much less affected at 10⁻⁵ M than the myogenic cells. It is suggested that vitamin D has a direct effect upon the muscle cell, to cause a selective diminution in the production of certain polypeptides.     

Distribution of acid hydrolases in subcellular fractions of proliferating vs non-proliferating fibroblasts

We have employed colloidal silica (Percoll) density-gradient subcellular fractionation technique to examine the distribution of lysosomal hydrolases between intermediate vesicles (primary lysosomes) and secondary lysosomes in contact-inhibited non-proliferating vs proliferating chicken embryo fibroblasts. We find that the activities of lysosomal specific enzymes from both phases of growth are distributed within two peaks; however, the relative amounts differ markedly. In normal, non-proliferating cells approx. 60% of the total activities of cathepsin B, beta-mannosidase, alpha-fucosidase, beta-galactosidase and hexosaminidase is recovered in the heavier density fraction corresponding to secondary lysosomes, while less than 9% of the enzyme activities are recovered in the light-density peak. With transformed cells, between 16 and 22% of activity for these enzymes are recovered in the lighter density intermediate vesicle fraction, when less than 40% of the enzyme activities recovered in the heavy density fraction. beta-Glucuronidase distribution was different from that of the above enzymes. First, a more even distribution between the two lysosomal fractions was found with non-proliferating normal cells (33% in heavy-density fraction and 21% in light-density fraction), whereas more than 40% of the total enzyme activity was recovered in the lighter density fraction from transformed cells. Also, the amount of cathepsin B contained in the vesicle fractions is increased severalfold relative to that of contact-inhibited normal cells. However, the apparent differences in enzyme distribution between confluent normal and transformed cells are not found when vesicles are prepared from subconfluent, actively proliferating cultures. We have also compared the Percoll density gradient patterns of membrane vesicles from proliferating and non-proliferating human fibroblasts, since most earlier studies utilized this system. Again, we find that the majority of beta-hexosaminidase activity (41%) of contact-inhibited, confluent cells is recovered in the heavier density fraction with less than 15% in the lighter density fraction. Also, the distribution of beta-hexosaminidase between the heavy density and light density vesicle fractions is altered in homogenates from exponentially growing cells, being 22% and 26% respectively. We conclude that the distribution of lysosomal hydrolases between the two vesicle populations is growth-phase dependent and is markedly heterogeneous in proliferating cells.     

Erythropoietin production in long-term cultures of human renal carcinoma cells : The role of cell population density

The present studies report the maintenance of erythropoietin (Ep) production in long-term cultures of a human renal carcinoma from a patient with erythrocytosis. The renal carcinoma cells were grown and maintained in monolayer cultures for 7 months. They were serially passaged every 2-3 weeks when the cultured cells reached confluency. Ep levels measured with a sensitive radioimmunoassay in the spent culture media of the cells in the stage of semiconfluent or confluent density were less than 20 and 30 mU/ml, respectively, throughout the period of 15 successive passages. However, when the renal carcinoma cells were maintained in culture without passage after reaching confluency, Ep levels in the spent media of these cells reproducibly showed an exponential increase to more than 300 mU/ml at the time of saturation density. The importance of cell population density in Ep production by the renal carcinoma cell cultures was further confirmed by the observation that the cultures with higher seeding density reached confluency earlier and began an exponential increase in Ep production sooner than those cultures with lower seeding density.     

The search for a digitalis substitute II milrinone (Win 47203) its action on the heart-lung preparation of the dog

Milrinone (Win 47203) is a dipyridine related to amrinone, which is about 20–50 times as effective as amrinone when assayed on cardiac contractility. In dog heart-lung preparations, milrinone in a concentration of 0.25–0.5 μM produced a near maximal positive inotropic effect on a variety of acute heart failures. This dosage produced a minimal increase in heart rate and reduced the PR interval. Large doses of milrinone did not produce cardiac irregularities and in Nifedipine heart failure with ventricular irregularities, it eliminated these irregularities. Papaverine-induced heart failure was resistant to ouabain, epinephrine and milrinone therapy. In the presence of positive inotropic amounts of papaverine or theophylline, a pentobarbital heart failure was superimposed. This heart failure responded poorly to milrinone, although it responded to both the addition of epinephrine and ouabain. It is thus possible that milrinone, papaverine and theophyline have closely related sites of action.     

Effects of ascorbate on methemoglobin reduction in intact red cells

An endpoint of 75% HbO₂/25% methemoglobin (MetHb) was approached in red cells incubated with a greater than physiologic concentration of ascorbate (10 mm). The presence of glucose (5 mm) with ascorbate shifted the endpoint to 90% HbO₂/10% MetHb while lactate (2 mm) plus pyruvate (0.1 mm) had no effect. These endpoints were approached regardless of the $\text{HbO}{}_2\text{MetHb}$ ratio at zero time. No hemoglobin degradation was observed. When red cells containing 100% MetHb at zero time were used, analysis of the initial rate of HbO₂ formation in the presence of various substrates showed synergistic interaction between ascorbate (10 mm) and glucose, additive activity with ascorbate and lactate, and less than additive activity with glucose and lactate. Incubation of red cells with a phsyiologic concentration of ascorbate (0.1 mm) resulted in no significant HbO₂ formation in the absence of other additions. When red cells were incubated with glucose and/or lactate plus pyruvate, an endpoint of about 99% HbO₂/1% MetHb was approached regardless of the HbO₂/MetHb ratio at zero time or the presence or absence of physiologic ascorbate. Physiologic ascorbate slightly but consistently increased the rate of HbO₂ formation in red cells incubated with glucose but not with lactate. HbO₂ formation was not increased by ascorbate in red cells which contained more than about 90% HbO₂ at zero time. The results indicate that excess ascorbate functions stoichiometrically driving cellular chemistry to a steady state between HbO₂ and MetHb formation whereas physiologic ascorbate functions catalytically allowing electron transport from glucose to MetHb via the hexose monophosphate shunt.     

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.     

In vitro sensitization to transplantation antigens. VI. Inhibition of sensitization

We have previously reported that C57BL/6 lymph node cells cultured with C3H/He macrophage monolayers are subsequently able to transfer specific allograft immunity to recipient C57 mice. The present communication is an investigation of the requirement for the functional integrity of cells to mediate allosensitization in vitro and to transfer allograft immunity. Our results indicate that C3H macrophage monolayers subjected to X-irradiation, actinomycin D, or antimacrophage serum pretreatment can no longer sensitize C57 lymph node cells in vitro; supernatants of C3H macrophage cultures do not substitute for monolayered cells and cannot sensitize C57 lymph node cells. The present data also indicate that the integrity of the lymph node cells is required after sensitization in vitro: X-irradiated or sonicated allosensitized lymph node cells do not enable recipient mice to accelerate C3H allograft rejection. The results of this report, therefore, suggest that intact, functionally normal cells are required to sensitize, and to transfer allosensitization.     

The connection between ionophore-mediated Ca2+-movements and intermediary metabolism in human red cells. II. Site and mode of glycolytic activation during Ca2+-loading

Human red cells (RBC) respond to moderate Ca²⁺-loading with increased ATP consumption and stimulation of glycolytic flux. 1. Ca²⁺-induced metabolite transitions at different pH-values showed a clearcut crossover at the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase ($\text{GAPDH}\text{PGK}\text{)-steps}$. 2. The behavior of glycolytic metabolites in iodoacetate-treated, GAPDH-inhibited, and in phosphoenolpyruvate-loaded RBC ruled out activation of hexokinase, phosphofructokinase and pyruvate kinase. 3. Glycolytic stimulation is linked to Ca²⁺-extrusion rate and not to the loaded Ca²⁺. 4. Adenine nucleotides and inorganic phosphate could be ruled out as the connecting link between glycolytic activation and Ca²⁺-extrusion. 5. NADH oxidation was observed at all pH-values studied when the RBC were incubated either at low or high extracellular potassium. NADH is product-inhibitor of GAPDH. The concentration (34 μM) of thermodynamically free NADH calculated from the $\text{GAPDH}\text{PGK}$ equilibrium reactants was in the inhibitory range: any decrease in NADH is therefore followed by activation of GAPDH. $\text{NAD}\text{NADH}$ ratio seems to be the connecting link between ATP consuming ion transport and ATP generation by glycolysis.     

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.