ALBERT

Notizen: The density distribution and cation composition of red blood cells from newborn puppies have been studied. The density distribution of red cells from a newborn puppy in a bovine serum albumin density gradient resembles a normal distribution with a peak density at a region less than that found for adult dog red cells. In two weeks the whole distribution shifts toward a more dense region, and a second cell peak appears so that the distribution becomes bimodal. This second cell peak is smaller than the original peak, and it appears at a region of lower density. In nine weeks the distribution becomes a normal one again, but the peak density corresponds to the peak density of the second cell peak which first appeared at two weeks. Evidence has been obtained to show that fetal red cells are located in the more dense cell peak and neonatal cells are in the less dense second peak. These results were obtained by labeling fetal cells with Cr51 and neonatal cells with Fe59. The analysis of the cation content of these cells shows that fetal cells contain more K and Na and have a higher K/Na ratio than adult red cells. Furthermore, neonatal cells contain considerably less cation and hemoglobin than do fetal cells. From a study of the cation and hemoglobin content of red cells appearing in various density fractions it is concluded that fetal cells lose K and Na during the first two weeks after birth. Thus, the change in the density disribution of the erythrocytes is thought to be due to two factors: (1) An increase in the density of fetal cells due to the loss of K and Na and, hence, water during the first two weeks after birth, and (2) the entry of less dense neonatal cells into the circulation.

Notizen: A method is described for measuring lymphopoiesis that enables the production of recirculating and non-recirculating small lymphocytes to be estimated simultaneously. Using this technique, experiments were undertaken to determine whether the production of recirculating cells is influenced by the number present in the recirculating lymphocyte pool. The results suggest that neither a massive lymphocyte transfusion nor depletion of the pool by whole body irradiation or chronic lymph drainage affect the rate at which recirculating small lymphocytes are generated.

Notizen: In extracts of normal and Lesch-Nyhan (LN) heterozygous skin fibroblast monolayer cultures, hypoxanthine-guanine (H-G) and adenine (A) phosphoribosyltransferase (PRT) activities are correlated. These activities vary concomitantly during the life cycle of a culture: Peak activities occur during exponential growth. The ratio of H-GPRT to APRT activity can manifest heterozygosity at the LN locus when H-GPRT activity, per se, is close to and unreliable different from the normal range.If 2 or 8 × 104 cells are planted per 35 mm petri dish, a strain with clearly heterozygous levels of H-GPRT activity in its cell extract may not reveal its genotype by simultaneous measurement of adenine and hypoxanthine incorporation into its acid-insoluble fraction one day after subculture. In contrast, a 1:1 coculture of normal and mutant cells yields the ratio of adenine to hypoxanthine incorporation expected from the behavior of each cell type separately. In heterozygous cultures at the higher population density, the incorporation of hypoxanthine relative to adenine is 28% greater than in those at the lower population density. A 1:1 mixture of normal and mutant cells exhibited a 20% increase in the relative incorporation of hypoxanthine over the same four fold increase in population density. These observations appear to provide a quantitative expression of the phenomenon of metabolic cooperation between contacting H-GPRT-negative and H-GPRT-positive cells.

Notizen: In glucose exchange transport into red blood cells the rate of glucose uptake showed two pH dependent maxima, with the larger at approximately pH 7.5 and the smaller one at pH 3.In the studied pH range the relation between the rate of glucose uptake and the substrate concentration followed Michaelis-Menten kinetics. While the maximal velocity (V) reflected the pH changes of the media, the Michaelis constant (Km) remained constant.The dissociation constants of the groups of the free carrier and the carrier-glucose complex were the same. The pK of the acidic group was 5.2 and of the basic group 9.5.Glucose was not bound to groups of the carrier which dissociated protons in the pH range of three to nine.By rearranging the equation for the pH dependence of the relative influx a more definitive graphic determination of the pK values was produced.

Notizen: Chinese hamster mitotic cells (95% in mitosis with 85% in metaphase and 10% in anaphase) were shaken loose from monolayer asynchronous cultures and stored at 1°C for up to 28 hours. During this period, the mitotic index did not decrease and the cells remained cytologically normal. However, over a four-hour period, metaphase cells located within 1.8 minutes of anaphase, 10% of the metaphase population, were able to move into anaphase; this point of 1.8 minutes corresponds in time to that reported for the spindle activation marker. When the cells were warmed to 37°C, they were delayed in entering G1 and S (35 and 70 minutes, respectively, after a 4-hour treatment). This delay of 70 minutes was maintained for three cell cycles, during which a high degree of synchrony was maintained. Cold treatment for 12 hours produced delays into G1 and S of 50 and 110 minutes, respectively. A fraction of the metaphase cells (12 or 50% after treatments of 4 or 12 hours, respectively) evidenced chromosomal aggregation, were unable to complete cytokinesis, and appeared in the next division as tetraploid cells. These tetraploid cells were unable to survive and produce macroscopic colonies. It is concluded that this decrease in viability is caused by irreversible effects on the spindle and/or centriolar components in metaphase cells located prior to the spindle activation marker.

Notizen: The ADP-stimulated (State 3) respiration of myocardial mitochondria with glutamate-malate, glutamate-pyruvate, palmitylcarnitine and β-hydroxybutyrate as substrates declined in rats after the age of 20 months. There was no significant decline in pyruvate-malate, α-oxoglutarate, palmityl-CoA, succinate and ascorbate cytochrome c oxidation. Skeletal muscle mitochondria from senescent animals showed a similar decline in glutamate-malate oxidation but not in palmityl-CoA, palmitylcarnitine, succinate and ascorbate-cytochrome c oxidation. The controlled oxidation with ADP-limiting (State 4) and the ADP/O ratio were not affected. The results indicate an alteration in the subtle regulatory capacity for mitochondrial oxidation in senescent rats. It is suggested that the alteration may be in certain anion transport and associated functions across the mitochondrial membrane or dehydrogenase activity.

Notizen: Self-diffusion coefficient measurements of water in untreated ovarian eggs of Rana pipiens using nuclear magnetic resonance indicate that cytoplasmic water has reduced translational mobility compared with pure water. Using a simple two-state model, we find that ∼67% is “relatively immobile.” Consideration of the nuclear magnetic resonance spin-lattice and spin-spin relaxation times indicates that the decreased mobility can largely be ascribed to hydration. Our value for the self-diffusion coefficient (6.8 × 10-6 cm2/sec) is lower than those reported by other investigators using isotopic water exchange techniques on frog eggs chemically treated to remove the membrane. However, the results reported here are in agreement with unpublished data on untreated frog eggs implying that chemical treatment has modified the cytoplasm in some manner.

Notizen: The levels of carbohydrates were measured in cells before and after transformation with DNA and RNA-containing oncogenic virus. The most consistent finding was a markedly lower content of L-fucose in all transformed cells relative to their normal counterpart. The cell pellet from a line of Chinese hamster cells transformed with polyoma or SV-40 viruses showed a marked decrease in the level of carbohydrates. However, when the carbohydrate levels were calculated for the whole system (cells plus wash) the transformed cells except for L-fucose had almost the same amount of carbohydrate as the controls. The levels of various carbohydrates in cells transformed by Rous sarcoma virus were the same or were elevated above the controls. Here consideration of the washes did not alter the results. Possible explanations for the results and causes of error in these comparative studies are discussed.

Notizen: The response of unfertilized sea urchin eggs stimulated with continuous D.C. was studied using cinematographic techniques. As reported by previous workers, the initiation of the response was anodal in polarity and followed a typical strength-duration curve. However, the anodal response may be swelling and/or contraction, depending on the amplitude of the applied stimulus, i.e., the magnitude of the contraction is directly related to the applied stimulus and is a graded response. It is suggested that the phenomena of anodal swelling and anodal contraction represent a continuum of responses based on ion exchange.

Notizen: Hemopoietic colony formation in agar occurred spontaneously in mass cultures of marrow cells obtained from a number of species (guinea pig, rat, lamb, rabbit, pig, calf, human and Rhesus monkey). This contrasted with the observation that colony formation by mouse bone marrow exhibited an absolute requirement for an exogenous source of a colony stimulating factor. Analysis of spontaneous colony formation in Rhesus monkey marrow cultures revealed the presence of a cell type in hemopoietic tissue, capable of elaborating colony stimulating factor when used to condition media or as feeder layers. Equilibrium density gradient centrifugation separated colony stimulating cells from in vitro colony forming cells in monkey bone marrow. Separation studies on spleen, blood and marrow characterized the stimulating cells as of intermediate density, depleted or absent in fractions enriched for cells of the granulocytic series and localized in regions containing lymphocytes and monocytes. Adherence column separation of peripheral blood leukocytes showed the stimulating cells to be actively adherent, unlike the majority of lymphocytes, and combined adherence column and density separation indicated that stimulating cells were present in hemopoietic tissue within the population of adherent lymphocytes or monocytes.

Notizen: The study of the cytological evolution of the foetal thymus enables us to distinguish an early foetal period (up to 20 weeks) from a late foetal period. The cytological maturation is achieved at 20 weeks of gestation. The thymus in the late foetal phase is comparable to that of the child.The study of the lymphopoietic function shows a higher activity in the foetal thymus up to 20-22 weeks, lymphopoiesis is also slightly increased, in the thymus of the child up to the age of three years. The study of the contribution of the various cell types to the cellularity of the thymus, emphasizes the importance of the medium lymphocytes in the early foetal period. The small lymphocytes though to a lesser extent, are also capable of division in the foetal thymus and in the infantile thymus. Incorporation of uridine is also increased in the early foetal thymus.Cytological composition and lymphopoiesis of the foetal thymus and lymph node, have been compared.

Notizen: We have isolated several cell lines which are temperature-sensitive for growth. One of these appears to be temperature-sensitive for cytokinesis. It was isolated from a Syrian hamster cell line by selecting cells which were not killed by 1 μg/ml cytosine arabinoside at 39° but which grew normally at 31°. It shows an increased proportion of binucleate cells when shifted to the non-permissive temperature and time-lapse photomicroscopy shows that a high proportion of attempted mitoses fail at 39°, apparently at the stage of cytokinesis. The cells which have failed to complete mitosis reattach to the plate and have two normal-size nuclei but otherwise behave normally.

Notizen: A reduced rate of transport of 2-amino-isobutyric acid (AIB), was found to be associated with M and early G1 periods in synchronized populations of CHO and L cells. A doubling of the transport rate occurred with progress of CHO cells further into G1, and the specific transport activity remained constant during the rest of the cycle, The rate of uridine and thymidine transport also doubled at this point in the cell cycle. There was no change in Km values. The rise in transport rate was blocked by cycloheximide, or when the cells were prevented from leaving metaphase by colcemid.

Notizen: Chinese hamster ovary cells with a deficiency in Serine Hydroxymethylase which produces a specific glycine auxotrophy (gly- A) were fused with human cells from a variety of sources and the resulting hybrids analyzed for human gene linkage. Of 102 hybrid clones examined 65 possessed both glyA and lactic dehydrogenase B markers, 35 possessed neither marker. Two clones were found with altered glycine responses which were not linked to LDH-B. The data indicate linkage between genes responsible for serine hydroxymethylase activity and lactic dehydrogenase B. Evidence for absence of linkage between these and a variety of other genes is also presented.

Notizen: Although it is well known that thymus function changes with age, it is not known whether these changes are associated with specific thymocyte populations. Since one criterion of specificity is cell size, we studied the size distribution of thymocytes from mice 0.5 days to 30.5 months of age. Body weight, thymus weight, and thymocyte yield were also measured. The mean cell volume of thymocytes from 8.5 to 13 week old mice was 326 μ3, with two detectable subpopulations. Mean thymocyte size was found to change with age. During the first postnatal week, the mean cell volume of the whole thymocyte population increased from 200 to 350 μ3, and the percentage of large cells increased greatly and constituted 90% of the whole population at four days of age. A rather slow decline in mean cell volume with some fluctuation occurred throughout the remaining life span, and at 30.5 months the mean had dropped to about 190 μ3. We suggest on the basis of these data that large thymocytes are involved in the contribution of the thymus to early postnatal development of the immune system and that the age-related functional capacity of the thymus is related to the size of the thymocyte population.

Notizen: The sedimentation behaviour of single human red blood cells fixed with glutaraldehyde at pH 7.4 and 6.4 was studied and compared to resiflts previously reported for normal fresh cells. The cells fixed at pH 7.4 were observed to have normal shapes while those fixed at pH 6.4 were more spherical and less disc-like. Fixation of glutaraldehyde removed “membrane flicker,” resulting in increased stability as indicated by a decrease in the number of orientation changes per minute from 2.93 ± 0.16 (SEM) to 1.74 ± 0.10 (SEM). An orientation change was defined as a change of 45° in any direction. Fixation also increased the edge-to-flat time preference ratio from 2.5 to 4.7, and increased the sedimentation velocities in all three orientations, despite a measured 2.5% decrease in mean cell density.Fixation of cells at pH 6.4 showed that the decrease in stability and preference for the on-edge orientation was associated with an increase in the sphericity of the cell.

Notizen: Improvements have been made in methods of preparation of single cell suspensions from mouse testes for separation by sedimentation at unit gravity. Mechanical means of preparation specifically damage spermatogonia, young primary spermatocytes, and spermatids at various stages of development, resulting in apparent sedimentation velocities which correspond closely to those of free nuclei. Trypsin has been used to release these cells from the seminiferous tubules allowing measurement of the sedimentation properties of the intact cell. The exclusion of trypan blue from the cells, resistance to trypsin, and the persistence in vitro of the highly differentiated forms of macromolecular synthesis demonstrate the integrity of the cells in the suspensions prepared with trypsin. Differentiation pathways in terms of sedimentation velocities of the cells and nuclei have been constructed and are in agreement with morphological changes.

Notizen: The calcitonin (SCT) from salmon ultimobranchial bodies which (like mammalian calcitonins) lowers the plasma calcium concentration in mammals can also affect cyclic AMP (cyclic adenosine 3′,5′-monophosphate) metabolism and proliferation of lymphoblasts in normal and prostaglandin E1 (PGE1)-treated rat thymocyte populations in three different ways. In the first case, low concentrations (0.5-5.0 ng per milliliter) of SCT lower (by a calcium-mediated process) the ability of PGE1 to transiently increase cyclic AMP synthesis, but the reduced surge of cyclic AMP production is still ample to stimulate lymphoblasts in the cell population to initiate deoxyribonucleic acid (DNA) synthesis. Secondly, these low SCT concentrations affect the eventual progression of the PGE1-stimulated, DNA-synthesizing lymphoblasts into mitosis by a calcium-mediated process. Depending on the extracellular calcium concentration and the magnitude of the initial increment in the intracellular cyclic AMP content, SCT can either promote or inhibit the progression of the stimulated cells into mitosis. SCT's third action is a rapid (within 5 minutes), calcium-independent elevation of the cellular cyclic AMP content in otherwise untreated thymic lymphocyte populations exposed to a very high concentration (100 ng per milliliter) of the hormone. This early, transient rise in the cyclic AMP level is followed by a calcium-dependent increase in lymphoblast proliferation. An attempt is made to interrelate and explain the different actions of SCT on cyclic AMP metabolism and mitogenesis.

Notizen: Colonies of elongated fibroblast-like cells (stellate colonies) developed in agar cultures of mouse pleural cavity cells mixed with whole blood. Cultures of pleural cells alone developed only abortive clusters of round cells. The frequency of colony-forming cells in the pleural cavity was highest in neonatal mice (200/105 cells) and fell progressively with aging. Stellate colony-forming cells were not in cell cycle but were radiosensitive. In adult mice, only occasional colony-forming cells were detected in peritoneal cavity, thymic, spleen, lymph node or bone marrow cell populations. Stellate colony formation was not stimulated by the granulopoietic regulator, colony stimulating factor. The active component in whole blood required for stellate colony formation was present in plasma but not serum or washed red or white cells.

Notizen: The cellular response to an intraperitoneal injection of antigen (tetanus toxoid) was studied in reconstituted animals in order to determine the mechanism of control of eosinophil granulocytopoiesis. Antigen treatment of the marrow cell donors did not consistently increase the number of spleen and bone marrow colonies in recipient animals or change the percentage of eosinophil or other hemopoietic colony types. Antigen pre-treatment of the irradiated recipients increased the percentage of eosinophil-containing colonies in the spleen and femoral bone marrow without significantly changing the total number of either spleen or marrow colonies. Antigen treatment of both the bone marrow cell donor and recipient produced a further increase in the percentage of eosinophil-containing colonies in the marrow cavity, but not in the spleen. Antigen treatment of the irradiated recipient increased the number of eosinophilic cells (but not the total number of cells) in both the peritoneal cavity and the bone marrow. Antigen treatment of both the marrow donor and recipient produced a further increase in the number of eosinophilic cells in the peritoneal cavity, but not in a single femur. Since antigen treatment of the marrow recipient, or recipient and donor, but not of the marrow donor alone, results in increased eosinophilic cell and colony numbers, the effect of antigen appears to be mediated through some host factor(s), perhaps the eosinophilic hemopoietic inducing microenvironment (HIM), rather than directly on the hemopoietic stem cells.

Notizen: Calcium-influx associated with the action potential was studied in squid giant axons using an EDTA-free preparation of aequorin as a probe. Associated with an individual action potential there was a transient increase in aequorin luminescence and the time course of this increase was examined on a kinetic basis. The luminescent response associated with a train of action potentials was compared with that expected on the basis of superposition of individual responses. The analyses of luminescence curves produced by long trains of action potentials were complicated by failure of superposition. A long pulse of inward current was found to produce a very large enhancement of luminescence.

Notizen: Activities of two membrane associated enzymes, viz. 5′-nucleo-tidase (EC 3.1.3.5) and alkaline phosphatase (EC 3.1.3.1) are known to be altered by glucocorticoid treatment of cells in culture. In certain HeLa cell lines, their activity is elevated, while in others, it is decreased. In order to confirm the data obtained previously with cell homogenates, cells were fractionated essentially as described by Bosmann, Hagopian and Eylar ('68) after 96 hours exposure to 0.5 μg/ml prednisolone. This treatment resulted in changes both in the specific activity and in the distribution of the two enzymes among the isolated membrane fractions. The alterations in specific activity in the individual membrane fractions reflected changes previously observed in preparations from whole cells. A shift in distribution of total enzymatic activity to fractions of higher density occurred in both cell lines in response to the steroid. These results may be indicative of cellular membrane alterations as a consequence of steroid treatment.

Notizen: The effects of cytochalasin B or low concentrations of adenosine 3′,5′-monophosphate (cyclic AMP) were tested on melanophores in hanging drop preparations of neural fold explants from Xenopus laevis embryos in Barths' solution. After one week in culture, the melanophores were punctate in this medium. Cyclic AMP at 5 mM consistently caused reversible morphological transformation of these cells to the stellate state, whether they were situated within an epithelial outgrowth or isolated on the surface of the coverglass. Only the isolated melanophores consistently responded to 1 mM cyclic AMP. Cytochalasin B at 1-10 μg/ml caused aggregation of melanin granules in stellate cells, but left long, narrow cell branches containing some melanosomes. Its effect was at least partially reversible and appeared to be dose dependent. At 1% concentration, dimethyl sulfoxide caused melanin dispersion.

Notizen: The effect of dibutyryl adenosine cyclic 3′:5′ monophosphate and testosterone on growth, response to serum and transport properties of Chinese hamster ovary cells was studied. There was a marked depression of growth in the presence of both compounds. A change of medium was sufficient to permit a partially synchronized burst of growth of the treated cells either in the presence or absence of dibutyryl adenosine cyclic 3′:5′ monophosphate plus testosterone. However, in the presence of these compounds a second round of cell division was prevented. Initiation of the cell cycle by cells exposed to dibutyryl adenosine cyclic 3′:5′ monophosphate plus testosterone displayed a greater serum requirement than untreated cells. It is concluded that serum and cyclic AMP could have antagonistic interactions in growth regulation.The treated cells had a reduced ability to accumulate amino-isobutyrate and glutamine, but no difference was observed with uridine uptake. The data suggest that a functional alteration of the membrane is induced by the exposure to dibutyrl adenosine cyclic 3′:5′ monophosphate plus testosterone.

Notizen: Medium calcium ions were essential to the development of the lymphocyte transformation response induced by PHA. Magnesium ions could not substitute for calcium ions. Calcium exerted its influence during the induction phase of the process before DNA synthesis began since its removal after the response had fully developed did not alter subsequent levels of nucleic acid synthesis. The early development of RNA synthesis was prevented by low calcium levels indicating that calcium either directly influenced the initiation of this process or some prior event(s).

Notizen: Chinese hamster cell lines have a strong tendency to maintain as modal chromosome number the euploid one characteristic of the species. By following the evolution of karyotypes in cloned subpopulation it has been possible to show that these pseudodiploid constitutions are not simply established due to a selective advantage but they are actually generated in populations where pseudodiploid classes had been absent. The possible role of the mitotic apparatus in keeping and restoring the euploid number is discussed.

Notizen: Mechanisms which determine sodium and potassium content and volume of rat thymic and human chronic lymphocytic leukemia (CLL) lymphocytes have been studied. The deleterious effect of cell isolation on monovalent cation content was proven by comparing thymus sodium and potassium concentration to that of thymocytes prepared from autologous hemithymus. In vivo distribution ratios of sodium-24 and potassium-42 between thymus water and plasma water were very similar to the distribution ratios of non-radioactive isotopes (sodium-23 and potassium-39). The similar lymphocyte: thymocyte ratio of (a) cell volume (1.48), (b) cell sodium plus potassium (1.47) and (c) cell water (1.50) demonstrated the close correlation of lymphocyte volume with monovalent cation content and water content. Steady-state CLL lymphocyte sodium (32 ± 1.9 mM) and potassium (131 ± 5.1 mM) and thymocyte sodium (31 ± 1.2 mM) and potassium (136 ± 3.9 mM) were similar; however, these steady-state levels were maintained by quantitatively different membrane functions. Radiopotassium and radiosodium uptake by thymocytes was more rapid than by CLL lymphocytes. Ouabain-sensitive potassium influx was 2.4 times greater in thymic (8.70 ± 2.28 mmoles/cm2/min × 10-8) than in CLL (3.24 ± 0.45 mmoles/cm2/min × 10-8) lymphocytes. Potassium exodus was also slower in CLL lymphocytes as compared to thymocytes. Ouabain-sensitive sodium accumulation and ouabain-insensitive sodium accumulation were also slower in CLL lymphocytes than in rat thymocytes. Half-maximal ouabain inhibition of sodium entry was 7.5 × 10-3 M in thymic and CLL lymphocytes. The inhibitory effect of ouabain on sodium and potassium transport was easily reversible. Oligomycin inhibited ouabain-sensitive potassium accumulation in both lymphocyte types. Four lines of evidence indicate the presence in the lymphocyte of a system of leaks and pumps, the latter subserved by a ouabain and oligomycin-sensitive (sodium-potassium) ATPase: (a) steady-state monovalent cation gradient (K ∼ 20:1, Na ∼ 5:1), (b) the inability to maintain normal sodium and potassium gradients at cold temperature and in the presence of ouabain, (c) the effect of ouabain and oligomycin on active potassium influx and (d) the restitution of steady-state sodium and potassium concentration after cell isolation, ouabain treatment and cold exposure. CLL lymphocytes as compared to rat thymocytes have a decreased rate of ouabain-insensitive sodium uptake and potassium exodus requiring a reduced rate of active sodium extrusion and potassium accumulation to maintain steady-state cation content. Ouabain-sensitive ATPase is difficult to locate in lymphocytes in vitro possibly because it comprises a very small proportion of membrane ATPase since magnesium activated ecto-ATPase in intact lymphocytes is 1500 to 2500 times that of the intact erythrocyte. The inhibition by ouabain of blast transformation, mitosis, amino acid accumulation and nucleic acid synthesis in vitro, may reflect the importance of ouabain-sensitive ATPase and monovalent cation transport in the function of lymphoid cells.

Notizen: Effects of ionizing radiation and of sulfhydryl reagents on the 45Ca binding of red cell membranes were studied. Corresponding effects of these agents on potassium leak from intact red cells were also determined. Essentially all the 45Ca associated with the ghosts appeared to be bound. Calcium binding could be described by assuming two independent groups of binding sites with dissociation constants of about 6 × 10-4 m and 2 × 10-4 m. The total binding capacity was about 2.5 × 10-4 moles/g ghost protein. Membrane calcium was decreased by radiation and by the two sulfhydryl reagents, p-chloromercuribenzoate (PCMB) and N-ethyl maleimide (NEM). The tightly bound calcium fraction appeared to be most affected by these agents. Changes in potassium leak evoked by varying doses of agents appeared to parallel effects on membrane calcium. These investigations suggest that the increased cation permeability observed after exposure or red cells to radiation or sulfhydryl reagents may be related to alterations in the calcium-binding properties of the cell membrane.

Notizen: Marmosets are unique in that they are “always” blood cell chimeras. When the nucleated cells from the bone marrow and from the peripheral blood of marmosets were incubated in the appropriate culture fluid they were shown capable of extensive proliferation in vitro. Two patterns of cellular proliferation, adherent and nonadherent, occurred in the same culture vessel. Repeated passage of nonadherent cells in RPMI 1640 medium supplemented with calf serum resulted in relatively long-term fluid bulk cultures showing myelocytic differentiation and megakaryocytic maturation. As myelocytic maturation became the predominant feature of cultures mitoses of the precursors diminished. About half of 41 marrow-derived cultures underwent extensive proliferation lasting about two months, as evidenced by an increasing cellularity and the presence of dividing cells. Such active growth occurred in one culture for over 120 days. The natural blood-cell chimerism of marmosets was demonstrated in vitro by cytogenetic analyses of metaphases from four relatively long term marrow cultures. The ratios of male and female cells remained either relatively stable or changed slowly with time in culture. Cells having both diploid and polyploid number of chromosomes were identified male or female, suggesting chimerism in myelocytic and megakaryocytic series. Marmoset lymph node and spleen cells proliferated as lymphoid cultures for various lengths of time up to five weeks but these cells did not differentiate into hemic cell lines. Attempts to culture human and rodent hemic tissue by the procedure used on marmoset tissue were unsuccessful.

Notizen: The effect of extracellular Pi and arsenate on Pi-transport in Ehrlich ascites tumor cells has been studied. Pi-transport can be described by Michaelis-Menten kinetics; the maximal flux equal to 44 mmoles (kg cell water)-1 hour-1 and Km equal to 3.3 × 10-4 M. Arsenate is a competitive inhibitor of Pi-transport with an inhibition constant (Ki) equal to 2.41 × 10-3 M.The data support the hypothesis that cellular Pi is regulated by the cell membrane through the mediation of a carrier system.

Notizen: The lipid composition of a highly purified preparation of rat skeletal muscle sarcoplasmic reticulum was studied. These membranes contain per mg of protein 0.50 ± 0.02 μmoles of phosphatidylcholine, 0.13 ± 0.02 μmoles of phosphatidylethanolamine and 0.07 ± 0.02 μmoles ofphosphatidylinositol. These three components account for 97.3% of total lipid phosphorus. Unlike other mammalian membranes so far studied, this preparation contains neither sphingomyelin nor phosphatidylserine. Neural lipids were also measured and it is concluded that neither cholesterol nor other neutral lipids are components of the membranes studied. The results of this study indicate therefore that the lipid profile of sarcotubular membranes is relatively simple compared with most other membranes.

Notizen: The effect of di- and trivalent cations on the membrane potential of the Ehrlich ascites tumor cell has been investigated using micro-electrode techniques. In solutions free of multivalent cations the average membrane potential for 46 cells was 8.3 ± 0.5 mv (SE). However, the potentials were not stable and decayed with a half-time of about six seconds. Addition of Ca++ decreased the rate of decay and concomitantly increased the membrane potential. The magnitude of these effects was a function of the Ca++ concentration. At the optimum concentration (2 mM), the half-time of decay was increased to 12 seconds and the membrane potential was raised to 17.8 ± 1.7 mv (SE). The related alkaline-earth cations, Sr++, Ba++ and Mg++ had similar effects on both the stability and magnitude of the membrane potential. The effect of La+++, which was qualitatively similar to that of the divalent cations, was also concentration dependent. However, 100-fold lower concentrations were adequate to achieve comparable effects. Moreover, membrane potentials were stable for up to ten minutes in La+++-containing solutions. Variations in intracellular Cl- content induced by temperature changes were paralleled by changes in membrane potentials. However, the potentials were not those expected for a simple Cl- electrode.

Notizen: The intracellular sodium and potassium concentrations and membrane transport properties for these ions were investigated in red blood cells from newborn puppies and adult dogs. At birth the intracellular concentrations of sodium and potassium are much higher than those found in adult dog red cells. During the first few weeks of life the intracellular concentrations of these ions gradually decrease until the adult level is reached. Changes in the membrane transport properties develop concurrently. The rate of active potassium influx, as measured by ouabain-sensitivity, and the pump to leak ratio are greater in red cells from newborn puppies than in those from adult animals. No ouabain-sensitive sodium efflux could be demonstrated in red cells from older puppies or adult dogs. When either puppy or adult dog red cells are depleted of ATP (by incubation at 37°C with no substrate), potassium permeability increases, and the permeability of the membrane to sodium decreases. The addition of adenosine reverses the effect of depletion.

Notizen: Uptake of 3H-thymidine into suspension cultures of mouse marrow cells was stimulated by the addition of serum-free conditioned medium harvested from cultures of mouse L-cells. Characterization of the “conditioning factor activity” (CFA) by gel filtration, ion exchange chromatography, velocity sedimentation, polyacrylamide gel electrophoresis and susceptibility to trypsin digestion indicated that the CFA detected by stimulation of 3H-thymidine uptake is the same as the CFA detected previously by its ability to stimulate colony formation by marrow cells in vitro. The 3H-thymidine uptake assay was used to investigate the kinetics of disappearance of CFA as a function of time in the presence of mouse marrow cells. The CFA recoverable from the cultures decreased rapidly during the first day, and approached background levels by the fifth day. There was no evidence of inhibitory substances in the depleted media. Even if the cultures continued to receive fresh conditioned medium at daily intervals, 3H-thymidine uptake decreased sharply after the fifth day, indicating that the marrow cells had lost their capacity to respond to CFA.

Notizen: A study of aldolases in rat hepatoma clones and subclones has revealed that they synthesize all three forms of aldolase monomers: A (the ubiquitous glycolytic isozyme), B (the form characteristic of the liver) and C, and that in vitro-in vivo passage results in a reversible modulation in aldolase A activity.Three kinds of somatic hybrids, between rat hepatoma cells and either mouse fibroblasts or rat epithelial cells, have been studied. In each case, aldolase B, found only in the hepatoma parent, was absent in the hybrid cells. The absence of aldolase B in the somatic hybrids seems not to be due to trivial factors (species differences, inactivation of all hepatoma aldolase genes, increase in ploidy or loss of chromosomes); it is concluded that extinction of this differentiated function of the hepatoma parent reflects a genetic regulatory phenomenon.

Notizen: The term “contact inhibition of cell division” was borrowed from “contact inhibition of cell movement.” We prefer the term “postconfluence inhibition of cell division” as being more operational and less mechanistically biased; it is operationally defined as a pronounced depression of the mitotic rate in a postconfluent culture which displays a stationary density despite periodic nutrient renewal, the inhibition being locally reversibly by removal of the adjacent cells.The mechanism of postconfluence inhibition is of considerable interest because of the inverse correlation between postconfluence inhibition and the tumorigenicity of a number of cell lines. Several hypotheses, involving direct cell-to-cell contacts or locally restricted diffusion gradiens, could explain postconfluence inhibition.With the goal of discriminating among these hypotheses, time-lapse films were taken of carefully regulated, perfused cultures of 3T3 mouse cells, in which the transition from rapid growth to the stationary phase was recorded. Measurements of cell-to-cell contact, local cell density, and generation times were made on an individual cell level and analyzed with the aid of a computer.We observed that all-around cell-cell contact or a high local cell density present throughout G1 often did not produce immediate inhibition of cell division. We conclude that either (i) simple visible cell-cell contacts or a high local cell density are not the direct cause of postconfluence inhibition of cell division, or (ii) their effects often do not inhibit cell division until after a delay of about one cell generation time. Such a delay may be partly responsible for the 50% overshoot past the stationary density that we observed in 3T3 cultures.

Notizen: Antisera have been prepared to clonal cultured rat astrocytic cells (C6 cells) and clonal human astrocytic cells (CHB4 cells). When cultured glial cells are grown in the presence of neuraminidase the serological activity of the cell membrane with homologous antiserum is increased extensively. A similar activation of serological activity can be effected by incubating washed harvested cells with this enzyme. Membrane bound N-acetyl-neuraminic acid is extensively reduced by either incubation. The activity responsible for promoting the enhanced serological activity co-chromatographs and co-electrophoreses with neuraminidase. Proteolytic digestion, unlike neuraminidase treatment, was unable to enhance the serological activity of cultured glial cells.

Notizen: Two kinds of extracts of peripheral blood leukocytes induced erythroblast increase and maturation in vitro in rabbit marrow cells. One extract was a secretion or exudate into plasma from intact leukocytes; the other was the supernatant after homogenizing the cells in ethanol. The extracts were less active than the intact leukocytes. The response with the two extracts together was a little greater than with either alone, but less than additive.11-Ketopregnanolone and 3′5′ cyclic AMP were active inducers. Neither increased the effects of leukocytes or their extracts.

Notizen: We have investigated the effect of varying La+3 concentrations (0.01 mM to 2.0 mM) on membrane potential and electrolyte composition of Ehrlich ascites tumor cells. La+3 concentrations less than 0.02 mM had no effect. Above 0.02 mM, La+3 induced concentration-dependent loss of electrolytes and water from the cells. At 1.0 mM the effect was maximal and resulted in an 87% reduction in cellular K+, 79% in Cl- and 21% in Na+ within 4.8 minutes. The Na+ loss occurred even in the face of an electrochemical potential gradient favoring Na+ entry. La+3 increased the recorded values of membrane potential; the magnitude of the effect was related to the external La+3 concentration, and was maximal at 1.0 mM. Studies using 140La showed that La+3 binds rapidly to the cell surface and does not enter the cells. The amount of La+3 bound to the cells was related to the external La+3 concentration by a sigmoidal curve and was maximal at about 1.0 mM. The bound La+3 could not be displaced by either added La+3 or Ca+2. Agents known to effect the integrity of the cell membrane, such as phospholipase C, neuraminidase, pronase and Hg+2 were tested for their ability to displace bound La+3. Only pronase displaced bound La+3, indicating that La+3 associates with cell protein. It is hypothesized that La+3 rapidly interacts with membrane protein causing alterations in membrane permeability and capacity to actively transport ions.

Notizen: Treatment of adult mice with rabbit anti-mouse thymocyte serum decreased the number and frequency of alloantigen-sensitive units responsible for graft-versus-host reactions and prolonged the survival of skin allografts. Whereas alloantigen-sensitive units were suppressed directly in vitro, they were not apparently suppressed directly in vivo since the fall-off of their numbers and/or function did not occur during the first day after serum injection. Treatment of prospective recipients of thymus cell grafts impaired the production of alloantigen-sensitive units by transplanted primitive progenitors. Differentiation with proliferation of alloantigen-sensitive units was less affected.Similarly, treatment of prospective recipients of thymus cell grafts with antilymphocytic serum impaired the production of specific inducer cells responsive to sheep erythrocytes by transplanted more primitive cells, presumably antigenreactive cells. Production of new precursors of anti-sheep erythrocyte hemolytic plaque-forming cells by transplanted bone marrow was not affected. Thus, antilymphocytic serum impairs the generation of immunocompetent cells of thymic origin by altering a hemopoietic inductive environment necessary for an early stage in differentiation.

Notizen: Removal of the parathyroid glands caused a marked and permanent lowering of the mitotic activity in rat bone marrow, which was followed by a pronounced decline (ca. 40%) in the size of the marrow's nucleated cell population. These changes occurred only when the hypocalcemia induced by the lack of parathyroid hormone (PTH) was maintained by feeding the animals a calcium deficient diet. Since the overall marrow hypoplasia was almost entirely due to strikingly large reductions (ca. 70%) in the size of the erythroid and lymphoid subpopulations, it is concluded that PTH and calcium are major physiological regulators of the proliferation of erythroid and lymphoid cells.

Notizen: Electrogenesis of embryonic chick skeletal muscle cells differentiated in monolayer cultures was investigated. Muscle fibers in vitro generate spike potentials similar to those of fibers in vivo. However, other responses, plateaux resembling those in heart muscle, are also elicited. These results suggest that a functional differentiation exists in cultured muscle fibers.

Notizen: Sixty to eighty per cent of the cells in a culture of human diploid fibroblasts may be stimulated from the state of density dependent inhibition of replication to active DNA synthesis and division. The maximum response is effected by 50% serum within the pH range 7.2-8.0. The proportion of cells responding depends on the concentration of serum protein in the medium which may be effectively substituted by crystalling serum albumin. There is a differential sensitivity to the stimulus of cells in the densely packed centers of whorls and in the less dense areas between the whorls. The cell response is parasynchronous and the median durations of the various phases of the cell cycle are: G1I6β Œæ® ¿ ∞ 8 hours, G2 = 6 hours and doubling time = 30 hours.The stimulatory effect of fresh medium is lost during contact with dense cultures so that it has only 50% of its initial capacity after 14 hours. It can be restored by dialysis against serum-free medium. The stimulus must be applied for at least ten hours to be effective in inducing DNA synthesis. During the latter half of ten hour induction period subsequent DNA synthesis becomes exquisitely sensitive to actinomycin D. After this time an increasing number of cells become irreversibly committed to replicate. The data are interpreted to indicate that during contact with serum proteins (including albumin) changes in the cell surface, if continued long enough, trigger a mechanism which involves the synthesis of a unique RNA species during the fifth to tenth hours. After this RNA has been synthesized the cells are then committed to DNA synthesis.

Notizen: Growth of cultured human fibroblasts in low oxygen resulted in reciprocal changes in the levels of cytochrome oxidase and several glycolytic enzymes. After five days' growth in low oxygen, cytochrome oxidase specific activity fell to 40% of the level of control cultures, while lactic dehydrogenase (LDH), aldolase, and triose phosphate dehydrogenase (TDH) levels were increased by 2- to 3-fold. These changes were accompanied by a change in the LDH isoenzyme pattern resulting from an increase in the proportion of LDH A subunits; the aldolase electropherogram was unchanged. When fibroblasts were grown for five days in medium containing chloramphenicol, cytochrome oxidase specific activity fell to 10% of control values, but LDH, aldolase and TDH specific activities and LDH and aldolase electropherograms did not differ significantly from controls. These findings are interpreted to indicate that the increased accumulation of LDH, aldolase and TDH induced by low oxygen is not mediated by the rate of accumulation of cytochrome oxidase.

Notizen: Circadian rhythms are demonstrated in the tongue of adult rats for both the mitotic index of the basal epithelium and the uptake rate of injected 3H-thymidine by the tongue tip. The animals were entrained to a light-dark cycle for four weeks prior to the experiments with the light phase extending from 0600 to 1800 hours (CST). The daily fluctuation is approximately 300% for the mitotic index and 185% for the uptake rate of 3H-thymi-dine. The highest mitotic index occurs at 1100, and the highest uptake of 3H-thymidine occurs four hours earlier at 0700. The least activity for both parameters occurs during the first part of the dark span of the light-dark cycle. Estimates of several other rhythmic parameters are determined by a computerized method.

Notizen: Addition of AMP to cultures of Tetrahymena pyriformis caused an increase in glycogen content of the cells and a small inhibition of growth. Adenine, adenosine, ADP, and ATP also increased glycogen content. Inosine and GMP were less effective; cytidine and uridine were ineffective. The increase in glycogen content was also observed in cultures supplemented with ribose, fructose, or glycerol, and when glyconeogenesis was increased by partial anaerobiosis. Adenine itself did not serve as a glycogen precursor, nor could the lipids of the cell have been the source of the carbon for the increased glycogen. The specific activity of glycogen from cultures supplemented with labelled amino acids was lower in AMP-treated cells than in controls. AMP-treatment had little effect on 14CO2 production from labelled glucose, acetate, or pyruvate, but baused a marked inhibition of the oxidation of labelled glyoxylate. It was suggested that AMP increases the rate of glyconeogenesis from precursors other than amino acids and interferes with malate synthase activity or malate transfer from peroxisomes to mitochondria.

Notizen: Suspension cultures of L-929 fibroblasts grown to densities of 6 to 10 × 106 cells/ml through daily centrifugation and resuspension in fresh media, have been maintained for periods up to five months without change in viability or cell size. DNA synthesis and mitosis in these cultures is limited to 5% of the cells per day, a fraction very nearly equal to the fraction of cells rendered nonviable, most likely during the manipulations associated with medium renewal. The kinetics of the flow of cells into the S and M periods following (a) renewal of the medium and (b) dilution of the high density cultures, suggest that the large majority of the cells are in a G0 or early G1 phase, resuming growth readily in response to decreased cell density. This is further indicated by the sequence of the marked shifts occurring in the cell volume distribution spectrum of the high density cultures after dilution. Long term, steady state regulation of growth with retention of intact viability was thus demonstrated in the case of a long established aneuploid cell line. The fact that this occurs in suspension but not in attached cultures, supports the concept that impairment of growth control in such cells affects predominantly regulatory mechanisms located at the cell surface rather than those concerned with intracellular synthesis and metabolism.

Notizen: Erythropoiesis was suppressed in CDF1 female mice using one of three different techniques: hypertransfusion, daily injections of low doses of actinomycin D, or post-hypobaric polycythemia. Spleens and bone marrows of these mice contained increased numbers of stem cells as determined by transplantation into lethally irradiated syngeneic mice. These stem cells were capable of producing both erythrocytic and granulocytic progeny. An increase in the number of multipotential stem cells or increases in the numbers of both unipotential granulocytic and erythrocytic stem cells is compatible with these observations and implies that hematopoietic stem cells may have a basic rate of division that is in part independent of stimuli to differentiate. The implications of these findings for the possible modification of antineoplastic drug-induced bone marrow toxicity are discussed.

Notizen: The electrophoretic mobilities (E.P.M.) and pH-mobility relationships of chick blastoderm cells at successive developmental stages were determined. Mobilities in 0.145 M NaCl, pH 7.2, showed a wide spread and decreased from a mean value of 1.63 μ/sec/V/cm in the prestreak blastoderm, to 1.49 in the intermediate streak and 1.34 in the head process stages. Cell surface charge density changes as detected by E.P.M. were observed in all regions of the embryo.pH-mobility relationship studies showed that two cell populations differing in their approximate isoelectric point appeared at the intermediate streak and head process stages. Neuraminidase treatment indicated that the contribution of sialic acid residues to the negatively charged groups at the cell surfaces is very small at the stages studied.

Notizen: Two isogenic acyclic strains of Paramecium multimicronucleatum, syngen 2, expressing mating types III and IV, and a cycler strain, which displays circadian alternation of the two mating types, were treated with proteolytic enzymes, or the inhibitors actinomycin D or puromycin at various stages of the mating reactive period induced by food depletion. Reactivity of acyclic animals was sensitive to proteolytic enzymes, and to both inhibitors before or after the onset of reactivity, suggesting that reactivity is attained and maintained via continuous RNA and protein synthesis, and that protein integrity is essential to the activity of the type-specific substances. A new cell cycle was not necessary for recovery from a block in reactivity.Type III animals were consistently more sensitive to inhibition by puromyin, and required longer recovery periods after all treatments except actinomycin D given after the development of reactivity. Possible explanations of these observations are considered.The daily type changes seen in cycler animals seemed to occur by replacement of one mating type substance with the other. Type-switching was neither prevented nor displaced by exposure to actinomycin D or puromycin during a period before or including a type change, even though reactivity was reduced by more than 95%, suggesting that type-switching is not directly associated with transcription or translation of the type-specific genes.

Notizen: The mitochondrial-plus-lysosomal fraction of rabbit lung parenchyma was studied by equilibrium density centrifugation in continuous sucrose density gradients (specific gravity 1.035 to 1.250). High concentrations of lysosomal marker enzymes were found both in a broad band at density 1.15-1.18, a density typical for lysosomes, and in a band at density 1.06-1.07. This light density band also had the highest specific activity of phospholipid, which thin layer and gas-liquid chromatography showed to be primarily lecithin with a high content of palmitic acid residues. Electron microscopy of material from the light density band showed a homogeneous array of particles which bear a strong resemblance to the inclusion bodies of the type II alveolar epithelial cell as seen in electron micrographs of rabbit lung tissue sections. These data suggest that the light density band is an isolation of intact type II alveolar epithelial cell inclusion bodies, which previous studies have implicated as the storage site of the phospholipid moiety of pulmonary surfactant.

Notizen: Livers from five-day chick embryos maintained as organ cultures on Eagle's minimum essential medium (MEM) develop an ultrastructure similar to more mature liver cells, except for glycogen deposits and the smooth endoplasmic reticulum (SER) normally associated with such deposits. The enzymes, glycogen synthetase and glycogen phosphorylase, failed to develop in these cells. The addition of zinc-free insulin (insulin-HCl) to MEM promoted the development of small amounts of SER in the cultured cells, as well as an increase in both glycogen synthetase and phosphorylase activities. The addition of zinc-insulin also stimulated an increase in the activities of both enzymes, and promoted the development of greater amounts of SER and the deposition of glycogen, as well. In addition, both forms of insulin not only prevented the fall of total tissue protein which occurs during organ culture on MEM, but also stimulated net protein synthesis in the explanted liver (Benzo and de la Haba, '71).

Notizen: Treatment of polymorphonuclear leukocytes with sialidase release about one-half of the total sialic acid of the cells. Such treatment affects neither phagocytic ingestion nor glycolysis. These results are in contrast to earlier data in the literature that indicate suppression of both functions mentioned upon desialation of these cells. The presence of an inhibitor of glycolysis that contaminated the crude enzyme previously used is suggested.

Notizen: The rate of synthesis of different hemoglobin (Hb) species  -  embryonic, primitive, definitive, and adult  -  in the circulating red cells of the chick embryo have been measured. On the fifth day of chick embryogenesis, there is a marked decrease in the capability of these red cells to synthesize Hb. The rates of synthesis of embryonic and primitive Hb decrease markedly compared to that of definitive and adult Hb. On the fourth day of embryogenesis nucleic acid synthesis drops sharply in these cells. These decreases in metabolic activity preceed the disappearance of the primitive erythrocyte from the chick embryo circulation. It appears that the primitive cell line is specifically shut off with respect to its metabolic activities and possibly destroyed at this time.

Notizen: A clonal strain of mammalian cells with increased resistance to acute heat shock at 46° was compared with the heat-sensitive parental line from which it was derived for possible differences that might account for this acquired resistance. Studies on synchronized populations of the two cell strains did not reveal any differential heat sensitivities in the various parts of the cell cycle (G1, S, G2) within either the sensitive or resistant populations. During thermal stress both cell types exhibited marked inhibition of ability to incorporate isotopically labeled precursors into macromolecular RNA, DNA, and protein.However, significant differences were observed between the sensitive and resistant cells in the amount of leakage of materials from the two cell types during heat stress and in their relative rates of recovery after stress. Sensitive cells pre-labeled with 3H-uridine released considerably more acid-soluble (cold, 5% TCA) label-containing materials during heat stress than did pre-labeled resistant cells. This differential release of uridine-containing materials was not paralleled by a generalized differential leakiness to other compounds. In addition, the resistant cells were found to regain the capacity to synthesize vital macromolecules sooner, and at initially faster rates, than the sensitive cells after stress.These results suggest that permeability changes causing decreased leakage of uridine-containing materials during heat stress combined with accelerated rates of recovery of synthesis of essential macromolecules after stress may be important cellular mechanisms in resistance to heat shock.

Notizen: Blood leukocytes incubated in vitro with rabbit-marrow cells induced a several-fold increase in basophilic erythroblasts and smaller increases in acidophils and reticulocytes. The main effect was nearly complete in one hour at 37°. Erythropoietin augmented the leukocyte effect; anti-erythropoietin inhibited it with or without erythropoietin. The erythroblast increase came entirely from the marrow cells; the precursor cell class has not been identified, except that division of pre-existing basophils appears to be excluded. Autologous and homologous leukocytes were about equally effective.A method is described of measuring on stained smears changes in relative concentrations of different classes of cells induced experimentally. A method of preparing highly concentrated peripheral blood leukocytes is described.

Notizen: Buoyant density gradient separation of Rhesus monkey bone marrow, spleen and blood leukocytes has demonstrated a reproducible and homogeneous light density distribution profile of cells capable of forming hemopoietic colonies in agar culture (in vitro colony forming cells  -  CFC). High resolution density gradient separation performed on a light density fraction of bone marrow produced on average a 100-fold enrichment of in vitro CFC with the most enriched fractions containing the majority of the in vitro CFC population present in the original marrow. Fractions were routinely obtained in which up to 23% of cells formed colonies and 33% were capable of proliferating to some degree upon stimulation. Tritiated thymidine suiciding showed the active proliferative status of the in vitro CFC and application of autoradiography and morphological characterisation to highly enriched density fractions has shown that the in vitro CFC in normal marrow is a transitional lymphocyte. Single cell transfer experiments have shown that in vitro CFC's formed colonies containing both granulocytes and macrophages, formally demonstrating the clonal origin of in vitro colonies and the common origin of granulocytes and macrophages.

Notizen: A multiplication-stimulating activity for chicken embryo fibroblasts has been purified approximately 6000-fold from calf serum by a three step procedure. The partially purified multiplication-stimulating activity stimulated DNA synthesis, mitosis, and growth of cultured chicken embryo fibroblasts. In addition, the active step 3 material was shown to have non-suppressible insulin-like activity and to be an inhibitor of trypsin activity.The activity appeared to be associated with a small protein and was heat stable, but sensitive to dithiothreitol and to periodate.

Notizen: Previous work from this laboratory (Rovera and Baserga, 1971) has shown that, when density-inhibited WI-38 human diploid fibroblasts are stimulated to proliferate by a change of medium, the synthesis of nuclear acidic proteins increases within 30 minutes after stimulation; several hours before DNA synthesis begins to increase. Similar results have now been obtained with density-inhibited 3T6 mouse fibroblasts, also stimulated by a change of medium.Gel electrophoretic analysis of nuclear acidic proteins in both WI-38 human diploid fibroblasts and 3T6 mouse fibroblasts stimulated to proliferate indicates that the increased synthesis of nuclear acidic proteins is limited to certain classes of proteins while other classes are totally unaffected.The increase in nuclear acidic proteins synthesis is inhibited when WI-38 cells or 3T6 cells are stimulated in the presence of 5-azacytidine (10 μg/ml), a treatment which also inhibits the subsequent stimulation of DNA synthesis.These results, confirming and extending similar findings previously reported in other models of stimulated DNA synthesis, lend further support to the hypothesis that nuclear acidic proteins may play a critical role in the control of DNA synthesis and cell division in mammalian cells.

Notizen: Fluctuations in the colony stimulating factor (CSF) content of submaxillary salivary gland, lung, kidney and spleen were studied in male C57BL mice which had been subjected to a variety of stimuli (endotoxin, x-irradiation or polyadenylic-polyuridylic acid complex) that caused elevations of the serum CSF. The temporal relationships and magnitudes of the rises in CSF were complex and differed from tissue to tissue according to the stimulus used. In the tissues examined not only was the measurable CSF content in each case found to equal or exceed the prestimulatory levels, but in some cases distinctly different forms of CSF were observed as shown by differences in the zone sedimentation, electrophoretic, and calcium phosphate binding characteristics of the material. The patterns of response to the stimuli investigated suggested that tissue injury either directly, or indirectly via the release of various cellular constituents, might mediate the release of CSF by various widely disseminated and/or differing cell types.

Notizen: We have shown previously that Ehrlich ascites tumor cells maintained at room temperature under an oxygen atmosphere lose Na+, K+ and Cl- isosmotically when exposed to La+++ (0.1 to 1.0 mM). Concomitant with these changes there is an increase in the recorded membrane potential (increasing intracellular negativity). The present studies further characterize the effect of La+++ on electrolyte distribution. Ehrlich ascites tumor cells were maintained at 0.5° C to permit Na+ gain and K+ loss. The addition of 1 mM La+++ to low temperature cells induces rapid loss of Na+, K+ and Cl-. This net loss of cellular electrolytes occurs even in cells depleted of ATP content using 2-deoxyglucose (5 mM) and rotenone (10-6 M). Analysis of the appearance of tracer 22Na in the environment of cells preloaded with the radioisotope shows that La+++-induced changes in membrane permeability or in active ion transport mechanisms are not responsible for the dramatic loss of electrolytes from experimental cells. The electrolyte loss occurs only when the cells are resuspended mechanically during the washing procedure used to prepare the cells for electrolyte determination.We conclude that the results of La+++ interaction with Ehrlich ascites tumor cells are twofold. As we have previously reported, La+++ stabilizes and causes a hyperpolarization of the membrane potential. Secondly, La+++ predisposes the cell membrane to become highly permeable when subjected to mechanical stress.

Notizen: Cell division, net Na+-K+ and amino-acid transport of cultured Ehrlich ascites is reversibly inhibited by Ouabain at a final concentration of 1 × 10-3M. A line of Ehrlich ascites cells resistant to the growth inhibiting effects of Ouabain has been developed. These cells behave similarly to Ouabain-sensitive cells in the following respects doubling time, S phase time, chromosome number, cell surface charge density, rate of incorporation of C14 Uridine and 3H-Thymidine, sensitivity to Digoxin and Digitoxin, steady state Na+, K+ levels and rate of loss of K+ and gain of Na+ in cold.Ouabain resistant cells differ from sensitive cells only with respect to the effect of ouabain on active Na+, K+ transport. Although Ouabain inhibits active Na+, K+ transport in sensitive cells it has no significant effect in resistant cells.

Notizen: Studies of human erythrocyte adhesion to glass have demonstrated consistently greater adhesion with serum-containing media than with a comparable concentration of plasma. This serum-plasma difference is explained by the adhesion-inhibiting property of plasma fibrinogen. The fibrinogen effect is probably mediated through its firm binding to glass, since no adsorption onto the red cell surface could be demonstrated.The ability of more red cells to adhere to a foreign surface after plasma coagulation (the formation of serum from plasma) may be significant in the red cell surface interactions necessary for the formation of a fibrin-red cell thrombus.

Notizen: A Coulter-orifice pulse-height analyzer system was used to measure volume spectra of mammalian cells in suspension at different times after the addition of an equal volume of water. In appropriate hypotonic medium, cultured mammalian cells rapidly increase in volume and then shrink, more slowly, approaching their initial volumes within 20 to 30 minutes at 37.5°C. The shrinking phase was found to be reversibly inhibited by ouabain and inhibited in both K+-free and Na+-free solutions; neither choline+ nor Li+ could substitute for extracellular Na+ in supporting the shrinking phenomenon but Rb+ and Cs+ were fairly good substitutes for K+.Under conditions similar to those with which the shrinking phenomenon was observed with cultured cells, it was not found with either human or mouse red blood cells.Two methods were used to determine intracellular Na+ and K+ content in osmotically shocked cells and in unshocked controls. An isotope equilibration method was employed with L5178-Y mouse lymphoblasts and a chemical determination by flame photometry was used with Ehrlich ascites tumor cells. The K+ content was significantly reduced and the Na+ content was unchanged or somewhat increased in cells which had returned to their original volumes in hypotonic medium. The K+ content was even more reduced but the Na+ content was greatly increased in cells which were osmotically shocked in the presence of ouabain.

Notizen: Electrophoretic patterns of newly synthesized proteins have been compared for hamster embryo fibroblasts in asynchronous cultures, mitotically synchronized cultures, and stationary phase cultures. Only proteins with molecular weight between 30,000 and 150,000, comprising 60-70% of the total cell proteins and excluding histones and collagen were included in the comparison. Although no significant differences could be detected between such patterns for cells at different stages of the cell cycle, significant differences were detected between patterns for cells in stationary phase and for proliferating asynchronous or synchronous cells in any stage of the cell cycle. These differences amounted to at least 5% of the newly synthesized cellular proteins. Much larger differences were detected between patterns from a nuclear fraction of proliferating and resting cells. These results indicate that normal cells in stationary phase are arrested in a state distinct from any phase of the normal cell cycle and may provide a biochemical marker for resting cells.

Notizen: Two embryonic hemoglobins are found in chick erythrocytes during early development by column chromatography on DEAE-cellulose and by electrophoresis on starch gel and gelatinized cellulose acetate. Beginning during the sixth day of incubation these are replaced by three different hemoglobins. Following hatching, chicks lose the most alkaline component, so that in the adult two hemoglobins are again found. Chromatographic fractionation on phosphonic acid cellulose also revealed an increase in the number of hemoglobins present in chicks during their development. We report the presence of at least 11 hemoglobins whose relative concentrations rise and fall during chick ontogeny. Failure to recognize the multiplicity of chicken hemoglobins is believed to be the major source of the confusion regarding this subject in the present literature.

Notizen: Sublines derived from V79 and B150 Chinese hamster cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and thymidine kinase (TK) respectively. When these strains are cultivated together without virus treatment in HAT medium, hybrid colonies with normal enzymic patterns arise in high frequency. The ratio of proliferating hybrid colonies to the minority cell inoculum in mixed cultures is approximately 1/1000. This observation has been used to examine the effect of x-irradiation pretreatment of one cell type on the frequency of hybrid formation in mixed cultures. B150 cells (TK-) were irradiated in monolayer cultures at dose levels of 200-4000r (180 KV, 15 ma). After trypsinization, the irradiated cells were mixed with unirradiated V79 cells (subline 129, HGPRT-) and cultivated in HAT medium. The per cent decline in hybrid frequency as a function of x-irradiation was compared to the relative decline in viability of x-irradiated B150 cells when assayed in cultures alone. These curves diverged markedly, with the frequency of proliferating hybrids decreasing much less rapidly with radiation dose than survival of B150 cells. By contrast, when B150 cells were subjected to acute heat shock for graded intervals and subsequently mixed with untreated V79-129 cells, both viability of B150 cells and hybrid formation declined at the same rate. Our results suggest that lethally irradiated cells can still fuse with normal cells, and may contribute functional chromosomes to the hybrid karyotype. At limiting doses of x-irradiation this technique may be useful as a means for induced segregation of marker chromosomes.

Notizen: Density dependent inhibition of cell replication is released in stationary cultures of BALB/c-3T3 fibroblasts by the potent tumor promoter for mouse skin, phorbol myristate acetate (PMA). The saturation density of these cultures, which is dependent on the serum concentration of the medium, is increased by PMA. Cell division, which can be induced by PMA or serum in stationary monolayers of BALB/c-3T3 cells, is not blocked by several inhibitors of proteolytic enzymes or the plant lectins, concanavalin A or wheat germ agglutinin. Induction of cell replication by PMA or serum does not appear to be dependent on proteolytic activity, and the membrane sites associated with this induction appear to be distinct from the agglutinin-binding sites.

Notizen: Incubation of rat liver parenchymal cell suspensions at 37° results in degradation, to acid-soluble material, of 15% of cellular RNA at 30 minutes and 40% at three hours, beyond which there is little, if any, further degradation. The RNA which remains in the acid-insoluble form in the cells up to 30 minutes appears to exist largely in the native state. However, after 30 minutes, the acidinsoluble RNA of the cells is found to be partially depolym-erised. These observations suggest the activation of an intracellular nuclease on dispersion of the liver tissue to a single cell suspension and incubation at 37°. This nuclease appears to be responsible also for the degradation, reported earlier, of exogenous RNA taken up by the cells. Activation of the nuclease is not due to depletion of pool of ATP or of other ribonucleotides from the cells, during either dispersion of the tissue or incubation at 37°. Incubation of the cells at 28°, or of liver slices at 37°, does not lead to any significant degradation to acid-soluble material, or to partial depolymerisation, of RNA. Analysis of RNA obtained from cells incubated at 37° for various periods showed that chromatography on methylated albumin-kiesulguhr (MAK) and Sephadex columns is not suitable for detecting partial depolymerisation of cellular RNA; RNA shown to be partially depolymerised by analysis on sucrose density gradient, in an analytical ultracentrifuge, and on a cellulose column, gave the normal pattern in MAK or Sephadex runs.

Notizen: The electrical response of the taste cells of the frog fungiform papillae to four fundamental taste solutions (NaCl, acetic acid, quinine-HCl and sucrose) was studied by using the intracellular recording technique. The average value of resting membrane potential was 22.5 mV, inside negative. Each of the four taste solutions applied to the tongue produced a slow depolarizing potential, the receptor potential, on which no spike potential was superimposed. The amplitude of the receptor potentials increased linearly as a function of the logarithm of the concentration of the stimulus. Amplitudes of depolarizations to a given taste stimulation varied from one cell to another even within a single taste bud. Most of the cells responded to more than two of the four basic taste solutions. Sensitivity patterns in terms of the number of effective solutions and the relative effectiveness of different kinds of solutions were variable among cells. Statistical analysis suggests that at the receptor membranes of the taste cells, the sensitivities for the four basic stimuli are independent and random.

Notizen: Increased activity of the enzyme nucleoside deaminase is found in the spleens of mice protected from lethal whole-body irradiation by transfusion of isogenic bone marrow cells. As few as 10,000 nucleated bone marrow cells produce a significant elevation in enzyme level. This increase precedes any detectable increase in hemoglobin synthesis and is temporarily associated with an increase in protein synthesis during the early stages of recovery. Hypertransfusion of the irradiated, protected mice eliminates most of the stimulus to erythroid differentiation and prevents the increase in nucleoside deaminase. The present studies indicate that this enzyme is present in a differentiated red cell precursor not found in the normal stem cell population.

Notizen: The circulating leukocyte is usually considered a mature, differentiated cell which does not normally divide. Cytophotometry of human and mouse leukocyte nuclei, however, reveals significant fluctuations in amounts of Feulgen-DNA in all leukocytes at various times of the day. When analyzed for DNA content by the diphenylamine method, the same samples demonstrate variations closely correlated with those obtained by cytophotometry. Although these data may be interpreted as suggesting, among other things, the presence of metabolic DNA, a preliminary radioautographic study of the uptake of tritiated thymidine in mouse leukocytes failed to demonstrate evidence of DNA synthesis in the entire population of cells. In this study only 6-7% of the total leukocyte population was labelled over a 24-hour period. This discrepancy, when considered in light of other uncertainties concerning the Feulgen and diphenylamine reactions, has led to reservations over the ability of either reaction to reflect the true amount of DNA in cells at all times. Rather, we consider it more likely that both procedures reveal similar incomplete fractions of leukocyte DNA at various times of the day, and that the magnitude of this fraction is, perhaps, determined by cyclic influences acting on such parameters as nuclear volume and degree of packing of the nucleohistones.

Notizen: The transmembrane potential of cells from a continuous cell line (BHK-21) has been investigated by a combination of electrophysiological and flame photometric techniques. The ratio of sodium permeability to potassium permeability (PNa/PK) determined from membrane potentials recorded at varying external potassium concentrations was 0.082; from membrane potential measurements and the intracellular sodium and potassium concentrations of cells in 6.8 mM K+ media the value was 0.075. The PNa/PK ratio was not temperature dependent. Dinitrophenol (1 mM) did not significantly alter the membrane potential of cells incubated for one hour with the inhibitor. However, iodoacetate (1 mM) and sodium fluoride (30 mM) caused a significant depolarization during a one-hour incubation. Measurements of sodium and potassium concentrations during incubation at 4°C showed a decrease in internal potassium and an increase in internal sodium accompanied by a decreased membrane potential. Ion concentrations and membrane potentials were measured in cells recovering at 37°C following 24 hours at 4°C. Membrane potentials in excess of EK during the first ten minutes of recovery may indicate electrogenic pumping.

Notizen: In both photosynthetic (Pyrodinium bahamense, Gonyaulax polyedra, Pyrocystis Iunula, P. noctiluca, P. fusiformis) and nonphotosynthetic (Noctiluca miliaris) bioluminescent dinoflagellates chemical stimulation can by-pass mechanical stimulation. The effective ions are Ca++, K+, NH4+ and H+. Other chemicals found effective are those implicated in Ca++ transport or binding. There are interspecies differences in degrees of mechanical and chemical stimulability. Photoinhibition of mechanical stimulability is the result of two effects, the first a reduction in total bioluminescence potential and the second a decrease in mechanical stimulability resulting experimentally in a decreased rate of light emission. This latter effect can be reversed with Ca++ ions. Chemicals which bind Ca++ or displace Ca++ can mimic the effects of photoinhibition. The chemical inhibition of mechanical stimulability is also reversed by Ca++ ions. A scheme is proposed which is consistent for all species examined.

Notizen: The calcium uptake and ATPase activities of isolated sarcoplasmic reticulum were studied during the first six days of chick skeletal muscle maturation in tissue culture. Statistically significant increases in these activities were observed between the second and the sixth day of maturation. Increases in oxalate-dependent calcium uptake were demonstrated at concentrations of 2.5 × 10-5 M calcium and 10-4 M calcium. Calcium-binding determinations conducted in the absence of oxalate displayed changes manifested by an increase at day 5 followed by a significant decrease at day 6. Increases in total ATPase activity during maturation paralleled the sequential increases in calcium uptake. Calcium-stimulated ATPase activity, however, did not change significantly during periods of marked increase in calcium uptake, suggesting that these activities are dissociated during development of the sarcoplasmic reticulum. These data demonstrate that calcium uptake and total ATPase activity increase during muscle maturation in tissue culture and that these activities are present prior to spontaneous contractions.

Notizen: 3T3 and SV-40 transformed 3T3 mouse fibroblasts were cultured in media with serum and antibiotics plus ammonia (NH3 z NH4+) added as NH4C1. Both cell lines cultured without added ammonia showed normal morphology and multiplication even though ammonia in the medium at the end of the culture period ranged from 35 to 48 μg/ml. Ammonia concentrations being significantly higher in media removed from cells at the end of the culture period than in media incubated identically without cells, verified that cells released substantial quantities of ammonia in addition to components of the medium which underwent spontaneous breakdown. Both cell lines showed changes in morphology and highly significant reductions in cell multiplication which increased progressively as the concentration of added ammonia on the initial day of culture was increased to 35μg/ml. Control 3T3 cultures released significantly greater quantities of ammonia per cell than control cultures of transformed cells but their multiplication was more adversely affected by added ammonia. There were downward shifts in pH of the culturing medium for both cell lines as culture age increased at all concentrations of added ammonia, However, significant reductions in cell multiplication resulted from additions of ammonia that did not produce significant changes in extracellular pH. The data show that studies upon the effects of pH of the medium on cultured cells require control of ammonia concentrations.

Notizen: Normal and virally transformed mouse cells respond differently in vitro to high concentrations of potassium. A higher potassium concentration is required to inhibit multiplication of SV40 transformed 3T3 cells to the same extent as that of normal 3T3 cells. This potassium effect correlates to specific activity of Na-K dependent ATPase in membranous fraction, normal 3T3 cells having the higher enzyme activity being more sensitive to potassium than SV40-3T3 cells which have the lower specific activity of the enzyme.Contact inhibition of growth and changes of concanavalin A binding sites which are characteristics of viral transformation influence specific activities of Na-K dependent ATPase and of adenyl cyclase. Incubation with trypsinized concanavalin A causes SV40-3T3 to show contact inhibition of growth and at the same time, higher specific activities of both enzymes than the observed in untreated cells. Cellular content of cyclic 3′,5′-AMP of contact inhibited 3T3 is higher than that in transformed SV40-3T3, but no difference is detectable in ATP content.

Notizen: Die PR-Spektren einiger Methyl-, Acetyl- und Benzoylderivate von Mannan aus Steinnuß und Tubera salep sowie der d-Mannopyranose wurden analysiert. Durch einen Vergleich der Kopplungskonstanten und der Signallagen von entsprechend substituierten Polymeren und Monomeren wird, mit Ausnahme der Konfiguration an C(1), die Struktur dieser Polysaccharide bestimmt. Es wird auf charakteristische Unterschiede der Resonanzlagen von H(1) in Polysacchariden mit α-bzw. β-glycosidischen Bindungen hingewiesen. Dieser Befund eröffnet die Möglichkeit, auch in solchen Fällen eine Entscheidung über die Art der glycosidischen Bindung zu treffen, in denen, wie im Falle der Mannoside mit äquatorialständigem H(2), der Wert der Kopplungskonstante J12 keine Aussage erlaubt.Die auffallend große Breite der Signale in den Spektren der nieder- und hochmolekularen Acetyl- und Benzoylmannane wird auf eine geringe Beweglichkeit der Kette oder einzelner Kettensegmente bei Mannanderivaten in Lösung zurückgeführt.

Notizen: L'effet de solvant sur les éthers disubstitués de formule (X—CH2—CH2)2O avec X = Cl, Br, I a été étudié. Les pourcentages des différentes formes stables ont été déterminés pour chaque solvant suivant la méthode de Lin et il est montré que ces trois halogénures évoluent vers les formes gauches quand la constante diélectrique de la solution augmente.De plus, il est montré que les couplages géminés dans le cas du dérivé iodé diminuent algébriquement quand la polarité du solvant augmente. Les auteurs pésentent une tentative de corrélation des variations des couplages géminés avec l'évolution conformationnelle.

Notizen: C-13 and F-19 NMR spectra of seventeen para-substituted fluorobenzenes were measured and the chemical shifts as well as coupling constants with respect to substituents were analysed. The chemical shifts of the fluorine, the C1 and the C2 atoms were found to depend on the total electron densities. In the case of the C3 atom, the chemical shifts seem to depend on π-electron densities rather than the total electron densities. The present calculations also indicate that the chemical shift of the C4 atom depends mainly on σ-electron densities due to the inductive effects of substituents. The strongest factor influencing the coupling constant, nJ(C—F), is also considered to be the π-electron densities on the carbon atoms. In the case of the direct couplings, 1J(C—F), the π-bond orders are important.

Notizen: 14N and 35Cl NQR spectra have been investigated for 24 organophosphorus compounds using a pulse technique. The electron populations of the nitrogen lone pair orbital and the N—P bond are calculated according to the Townes and Dailey method. The experimental data are interpreted assuming a partial double bond character of the N—P bond due to the pπ—dπ interaction and pπ—σ conjugation of the lone pair electrons of the nitrogen atoms. The effect of the different nature of substituents X on the N—P bond populations is observed in X = PRn (R2N)3-n molecules (where X is O, S, Se, or lone pair electrons and n = 0, 1, 2). It can be seen from this dependence that the effective electronegativity of the phosphorus atom is largest in selenophosphoramidates and falls in the sequence P=Se 〉 P=S 〉 P=O 〉 P.

Notizen: Praseodymium(III) tris(dipivalomethanato) [Pr(DPM)3] reduces the complex overlapping aromatic absorptions in the proton NMR spectrum of a monosubstituted naphthalene, cis-3-(1-naphthyl)-1,3,5,5-tetramethlcyclohexan-1-ol(1) to a virtually first-order pattern, whereas Eu(DPM)3 at the same and higher molar concentrations does not completely resolve the aromatic proton signals. Plots of the Pr(DPM)3-induced shifts measured in carbon tetrachloride solution versus molar equivalents of Pr(DPM)3 added may be extrapolated to provide accurate chemical shifts in the absence of added lanthanide shift reagent (LSR). The qualitative conformational conclusions from the Pr(DPM)3 shifts agree with those from the Eu(DPM)3-induced shifts, but there are detailed differences in the relative lanthanide-induced shifts (LIS) for different proton types in the molecule.

Notizen: Les déplacements chimiques du fluor de différents phényls-1 et 2 fluoro éthanols ont été mesurés; les valeurs obtenues ont été extrapolées à dilution infinie en soluté et référence interne. Les corrélations linéaires ou non, obtenues entre ces déplacements chimiques et les constantes de substituant σ donnent lieu à une discussion générale relative à la validité des relations d'enthalpie libre étendues aux données de la RMN; les limites et les possibilités de telles relations sont précisées.

Notizen: The NMR spectra of a spiro oxirane (1) derivative of codeinone, codeine (2), isocodeine (3), 6-methylcodeine (4) and 6-methylisocodeine (5) were compared. NOE and double-resonance experiments were used to confirm the conformation of 1, and the configuration about C-6 of 6-methylcodeine and 6-methylisocodeine. An interchange of the chemical shifts of the olefinic protons in 1 was noted, as compared with those in all of the other compounds. This interchange could be attributed to the bond anisotropies of the oxirane moiety.

Notizen: Die 13C-chemischen Verschiebungen von 19 para-und 5 ortho-Cyclohexadienonen werden durch 13C-Fourier-Transform-Spektroskopie ermittelt und zugeordnet. Der Substituenteneinfluß auf die chemische Verschiebung der Signale der Ringkohlenstoffatome wird diskutiert. Durch die Aufstellung von Regressionsgeraden wird gezeigt, daß die Resonanzlagen der olefinischen Ringatome und des allylischen C-Atoms im para-chinoliden Ring wechselseitig voneinander abhängen. Eine Korrelation zwischen 1H-NMR- und 13C-NMR-Daten wird am Beispiel der Cyclohexadienone überprüft.

Notizen: By double and triple nuclear magnetic resonance experiments performed at 100 MHz the stereochemistry of the epoxides of Cyclopentadiene with α-methyl-β-acetyl acrylic acid lactone, were determined to have the exo position.

Notizen: The rates of NH—COOH proton exchange between 5-amino-(1a) and 5-N-methylamino-(1b)3-[2-(5′-nitro-2′-furyl)vinyl]-1,2,4-oxadiazoles and trifluoroacetic acid (TFA) have been measured by NMR spectroscopy. The values of the first-order rate constant and thermodynamic parameters for 1a and 1b, respectively, are: kapp (sec-1) = 820 and 40 (50°C), ΔF323# (kcal/mole) = 14·7 and 16·5, ΔH# (kcal/mole) = 17·3 and 24·3 and ΔS# (e.u.) = 17 and 34. The comparison of rate constants indicates that after correction for proton equivalency proton exchange in 1a is faster than in 1b by a factor of ten. The presence of an NH2 proton resonance (1a) and an N-methyl doublet (J = 5·0 Hz) between 0 and 30° (1b) suggests that 1a and 1b are present as amines and not as imines in TFA.

Notizen: Die Konformerengleichgewichte verschiedener monosubstituierter Cyclooctane werden durch 13CMR-Tieftemperatur-Spektroskopie bestimmt. Aus den 13C-Verschiebungen kann auf die Geometrie der Konformeren geschlossen werden. Durch Vergleich berechneter und experimenteller Spektren werden für die Konformerenumwandlungen einige ΔG#-Werte erhalten.