Sea urchin hyalin: appearance and function in development

Embryonic chicken sensory cells from dorsal root ganglia and a clonal line of pheochromocytoma cells (PC-12) extended neuronal-like processes within 24 hr of seeding on a naturally produced, basement membrane-like extracellular matrix (ECM) in the absence of nerve growth factor (NGF). Plating on ECM also induced a rapid cell attachment and flattening of these cells and supported the survival of embryonic sensory cells in primary cultures. Unlike the effect of NGF on PC-12 cells, the ECM-induced morphological differentiation was transient and led to disintegration and degeneration of processes bearing PC-12 cells. The ECM-induced morphological differentiation was not inhibited by anti-NGF antibodies, and the cells retained their ability to bind and internalize NGF in a manner similar to that observed on plastic. PC-12 cell attachment and flattening occurred on dishes coated with collagen type IV in a way similar to that observed on ECM, but precoating the dishes with fibronectin had no effect. Extension of cell processes was not induced by either substrate. Morphological differentiation but not the induction of cell adhesion and flattening was inhibited by either prefixation with glutaraldehyde, oxidation with periodate, or preexposure to concanavalin A of the ECM, suggesting that the ECM and in particular its sugar moieties play an active role in the induction of neurite outgrowth. It is suggested that close contact with the ECM provides chemical or mechanical cues that permit contactmediated elongation and directed growth of both embryonic and regenerating nerve fibers.     

Properties of the genome in normal and SV40 transformed WI38 human diploid fibroblasts. I. Composition and metabolism of nonhistone chromosomal proteins.

The composition and metabolism of nonhistone chromosomal proteins associated with the genome of normal and SV40 transformed WI38 human diploid fibroblasts were examined. Evidence is presented which indicates variations in the relative protein content, specific activity and phosphorylation of several defined molecular weight classes of these chromosomal polypeptides. In addition, it is shown that blocking DNA replication with cytosine arabinoside does not inhibit the incorporation of ³H-leucine into nonhistone chromosomal proteins of SV40 transformed cells. The possibility that the nonhistone chromosomal proteins of human diploid fibroblasts transformed by SV40 virus may be involved in the regulation of viral induced alterations in gene expression is discussed.     

Interaction of far- and near-ultraviolet radiation. The occurrence of photo-augmentation and photo-recovery in cultured mammalian cells

Guided by the phenomena of photo-augmentation and photo-recovery, which have been described with respect to the induction of erythema in human skin, experiments were undertaken with cultured mammalian cells to study whether irradiation with far- and near-ultraviolet radiation results in an interaction at the cellular level with respect to cell survival and induction of mutations. Evidence was found for both photo-augmentation and photo-recovery. Photo-augmentation (more than an additive effect) was observed for cell survival when the long-wave ultraviolet irradiation (UVA) preceded the short-wave ultraviolet irradiation (UVB). Photo-recovery (less than an additive effect) was observed for cell survival if the UVA was given after or simultaneously with the UVB. The latter effect, however, was strongly influenced by dose: doses of UVA higher than 20 000 J/m2 no longer lead to photo-recovery in cell survival. For mutation induction, reduction in mutant frequency appears indicated for both combinations of UVA and UVB and for high and low doses of UVA.     

Accumulation of uridine diphosphoglucose pyrophosphorylase in Dictyostelium discoideum via preferential synthesis

At the end of the exponential growth phase, the enzyme UDP-glueose pyrophosphorylase is present in the vegetative cells of Dictyostelium discoideum NC4 (haploid) at a low level (about 0.05% of total protein). During the initial stages of fruiting body construction, while the cells are entering into multicellular aggregates, the enzyme level remains constant, but increases dramatically thereafter reaching a peak (about 0.5% of total protein) at the end of fruiting body construction, and then partially decreasing. Previous studies have shown that both the accumulation and disappearance are keyed to the flow of morphogenetic events.In this study, cells were labeled with amino acids for different periods throughout the sequence. The enzyme was quantitatively immune-precipitated from crude cell extracts, the precipitate was washed and redissolved, and the enzyme protein separated by acrylamide gel electrophoresis in order to estimate the differential incorporation ratio, i.e. $\text{disints}\text{min in enzyme protein per 10}{}^8\text{cells}\text{disints}\text{min in total protein per 10}{}^8\text{cells}\text{× 100}$ for each labeling period. During the initial stages, when the enzyme level remained relatively constant, this ratio was about 0.03 to 0.04%. As the enzyme began to accumulate it rose progressively, attaining levels of 0.6 to 0.8% toward the end of fruiting body construction before declining. The data are not consistent with the theory of Gustafson and Wright (1973) that differential turnover controls the level of this enzyme during the development of D. discoideum. They are consistent with the conclusion that directed changes in the differential rate of synthesis of UDP-glucose pyrophosphorylase is the controlling element.The estimates of enzyme content are based on a value for the specific enzyme activity of 100,000 units/mg enzyme, which had been determined previously using samples of the enzyme purified to apparent physical homogeneity. This figure has been confirmed in the present study by quantitative immuneprecipitation of the enzyme from crude extracts of homogeneously labeled cells. The method can be generally used to determine if a specific biological activity estimate obtained with a purified protein is consistent with its activity when measured before or during purification.     

2'-deoxyadenosine-3'-monophosphate: A naturally occurring inhibitor of adenylate cyclase in amphibian and mammalian cells

A naturally occurring inhibitor of adenylate cyclase has been found in various rat tissues and toad erythrocytes. The compound containing this inhibitory activity has been isolated from several cell types. Physical, chemical, and enzymatic analyses define this inhibitor as 2'-deoxyadenosine-3'-monophosphate (2'-deoxy-3'-AMP). Measurements of the effect of various nucleoside monophosphates on adenylate cyclase activity reveal that the inhibitory activity is specific for 2'-deoxy-3'-AMP. In addition, the metabolism of this inhibitor may be relatively specific since only aminophylline and cAMP appear to inhibit the breakdown of 2'-dexy-3'-AMP by liver microsomes. The presence of this inhibitor suggests the possibility that deoxynucleotides may play an important role in the action of hormones and the regulation of intracellular metabolism.     

Model of calcium movements in the mammalian myocardium: interval-strength relationship

A model is proposed to describe the interval-strength relationship in mammalian cardiac muscle in terms of "discrete" calcium movements associated with each cycle. The sarcoplasmic reticulum is assumed to be comprised of three functional sub-compartments: (1) The "main calcium store" which contains most of the calcium (predominantly bound) and is considered, due to its large buffering capacity, to account for the "long-term memory" lasting 7-10 beats. (2) The "releasable terminal" which contains the calcium readily available for release (all or most of it free) and accounts for the "short-term memory" which affects the subsequent beat. (3) The longitudinal network of the SR recirculating the myofibrillar calcium to the "main calcium store". The total content of calcium in the main store is determined by the transsarcolemmal influx and efflux. While influx occurs only during depolarization, efflux occurs during the whole cardiac cycle. The amount of free calcium in the main store is determined by an equilibrium equation. The release of calcium from the "releasable terminal" is governed by a "concentration-dependent" mechanism. This implies that when the concentration in the "releasable terminal" increases, the fraction released increases and the residual calcium left for the subsequent contraction decreases. The model predicts the following interval-strength relationships: steady state peak tension; changes from one steady rate to another; restitution curves; post-stimulation potentiation; paired stimulation; premature beats; post-extrasystolic potentiation following interpolated, basal or complimentary interval.     

Transfer of transposable drug-resistance elements Tn5, Tn7, and Tn76 to Azotobacter beijerinckii: use of plasmid RP4::Tn76 as a suicide vector

Transposable elements Tn5, Tn7, and Tn76 were transferred to Azotobacter beijerinckii. Evidence was obtained for the transposition of Tn5 but cells of the majority of presumptive transposition isolates had abnormal morphologies and rapidly lost viability when subcultured. Data are presented that indicate that plasmid RP4::Tn76 behaves as a suicide vector upon transfer to this host, allowing the isolation of A. beijerinckii::Tn76 isolates at a high frequency. Nitrogen-fixing mutants and leucine and adenine auxotrophs were isolated from cultures in which the transposition of Tn76 occurred.     

Induction and amplification of T-lymphocyte proliferative responses to periodate and soybean agglutinin by human adult vascular endothelial cells

Human mononuclear phagocyte (M phi) populations were compared to adult human endothelial cells (HEC) for their respective abilities to influence the proliferative responses of purified human T lymphocytes to the mitogenic agents Na-m-periodate (IO-4), soybean agglutinin (SBA), or allogeneic cells. HEC and M phi were both capable of inducing proliferative responses of allogeneic T lymphocytes in mixed-lymphocyte culture. Under low cell density culture conditions, purified T-lymphocyte proliferative responses to IO-4 or SBA could be restored by addition of syngeneic M phi or HEC. At higher cell density culture conditions, proliferation of T cells to IO-4 could be amplified more by HEC than M phi. T-lymphocyte proliferative responses to SBA were amplified by addition of HEC but were suppressed by addition of M phi. These findings indicate that human adult HEC are unique and potent accessory cells for T lymphocytes. Furthermore, these findings demonstrate that accessory cell functions of HEC can be discriminated from those of M phi.     

The integrated and free states of Streptomyces griseus plasmid pSG1

A 16.6-kb plasmid-pSG1-was isolated from Streptomyces griseus following transformation of protoplasts with unrelated plasmids. Southern hybridization experiments with radioactive probes prepared from pSG1 fragments and immobilized S. griseus DNA fragments indicated that the plasmid was present in the progenitor strain, in an integrated state. In the pSG1+ isolates plasmid sequences existed both as integrated sequences and as free plasmids. The integrated state of maintenance persisted in strains which have been cured of the free plasmid. The junction site on the plasmid was located on a 0.5-kb EcoRI-SalI fragment. The chromosomal integration site was demonstrated to be the same in all strains derived from S. griseus NRRL3851. The occurrence of both states of plasmid maintenance in the same clones indicates that an integrated pSG1 sequence does not interfere with free plasmid replication and partition. It suggests that the establishment of the free state may involve a replicative excision of pSG1 from the S. griseus chromosome.     

Studies on the amino groups of myosin ATPase. Trinitrophenylation of reactive lysyl residue in ventricular and atrial myosins

Myosin and heavy meromyosin from ventricular, atrial, and skeletal muscle were purified and trinitrophenylated by 2,4,6-trinitrobenzene sulfonate. The trinitrophenylation reaction followed a complex kinetics consisting of a fast and slow reaction in all preparations studied. Reactive lysine residues were trinitrophenylated during the fast reaction with a concomitant decrease in K⁺ (EDTA)-activated ATPase and an increase in Mg²⁺-stimulated ATPase activities of myosin. The extent of increase in Mg²⁺-mediated ATPase was the highest with skeletal and the lowest with atrial myosin. The trinitrophenylation of the less reactive lysyl residues continued during the slow reaction. The rate constants of the reactions and the number of reactive lysine residues were evaluated by computer analyses of the trinitrophenylation curves. Two reactive lysine residues were found in skeletal and ventricular myosins while their number in atrial myosin was somewhat lower. The rate of trinitrophenylation in skeletal muscle myosin or heavy meromyosin was always higher than in the two cardiac myosin isozymes. Addition of KCl increased the trinitrophenylation of both highly reactive and slowly reactive lysyl residues in all of the three heavy meromyosins, however, the effect was more profound with cardiac heavy meromyosins. Addition of MgADP induced spectral changes in trinitrophenylated skeletal but not in cardiac myosins. Similar changes occurred in skeletal and to a lesser degree in ventricular heavy meromyosin, but no definite spectral changes were observed in atrial heavy meromyosin. The findings suggest that structural differences exist around the reactive lysyl residue in the head portion of the three myosins.     

Insulin-like growth factors, IGF-1, IGF-2 and somatomedin C trigger cell proliferation in mammalian epithelial cells cultured in a serum-free medium

A single insulin-like growth factor which constitutes part of a defined serum-free medium is sufficient to stimulate DNA synthesis and mitosis in mammalian lens epithelial cells. Rabbit lenses were cultured in KEI-4, a medium which mimics rabbit aqueous humor, or in KEI-4 containing insulin growth factor I (IGF I), insulin growth factor II (IGF II) or somatomedin C. The magnitude of DNA synthesis and mitosis was evaluated on whole mount preparations of the epithelium at various times of culture. IGF I and II, the most highly purified of the insulin-like growth factors, and somatomedin C were equipotent lens mitogens, were active at the ng level, were more mitogenic toward lens epithelial cells than insulin, and initiated cell proliferation throughout the normally amitotic central region of the lens epithelium. The time course of the mitotic response elicited by the insulin-like growth factors was identical to that noted in lenses cultured in medium supplemented with serum or insulin. The present results, coupled with those of other investigators, suggest that insulin-like factors may regulate cell division in the mammalian lens in vivo.     

Prostacycin production by arterialized autogenous venous grafts in dogs

Arteries are capable of producing significantly larger quantities of protacyclin than are veins. To test the hypothesis, whether prostacyclin production by the vessel wall is related to blood pressure and flow, we measured the amounts of PGI₂ released and synthesized by venous segments transplanted for 6 weeks into the arterial circulation. These results were compared with the production of prostacyclin by normal veins and arteries. In 20 dogs a segment of jugular vein was interposed into the carotid system; a sham dissection was done on the opposite side. “Arterialized” vein grafts showed prominent intima lined by endothelium, medial smooth muscle cell proliferation and fibrotic proliferation in adventitia. Spontaneous and arachidonic acid- stimulated prostacyclin production (measured by radioimmunoassay for 6-keto-PGF_1α) was not significantly different between arterialized venous autografts and jugular veins. Significantly larger amounts of prostacyclin were synthesized by the carotid artery. Thus, histologic changes and rheologic effects occurring in vein grafts transposed to the arterial site do not affect prostacyclin production.     

Cell polarity in precartilage mouse limb mesenchyme cells

The patterns of orientation of individual mesenchyme cells have been evaluated in the hindlimb of the mouse embryo during the period of transition from early aggregation (Day 12) to cartilage formation (Day 13). Orientation was measured by determining the angular relationship between the Golgi-nucleus axis of each cell relative to either the longitudinal limb axis or the center of the cartilaginous aggregate. Patterns were assessed qualitatively and quantitatively in horizontal, vertical, and transverse sections of the proximal, middle, and distal precartilage mesenchyme. These analyses showed that the mesenchyme cells are oriented predominantly toward the longitudinal axes of both the early (Day 12) and late (Day 13) aggregates.     

Separation of mitogen-induced suppressor cells of human antibody-producing cells

Human antibody-forming cells were demonstrated by a plaque in agar technique following in vitro stimulation with either pokeweed mitogen or Cowan I strain of protein A-positive Staphylococcus aureus bacteria. We evaluated the effects on this antibody formation caused by the addition of cells which had been stimulated with PH A or Con A. Both Con A and PHA cells harvested after 3 days showed strong inhibition of pokeweed-induced plaque formation. The majority of the suppression could be accounted for by a blast fraction separated on 1g sedimentation gradients from the Con A or PHA cultures. Small cells from such cultures showed inhibition of PFC when added at high ratios (1:2), but this suppressive activity diluted out much more rapidly than that of the blast cells. No helper activity was noted with either small cells or blasts. Our studies indicate a T-cell blast as the suppressive fraction in Con A- or PHA-stimulated human lymphoid cells. While this T-cell suppression applies to T-dependent responses such as antibody stimulation with pokeweed mitogen, it does not have a substantial effect on Cowan I-induced plaque-forming responses. The finding that Cowan I-induced plaques could not be inhibited by Con A or PHA blasts indicates the T independence of this response.     

A partial characterization of suppressor cells in rat fetal liver cells

Rat fetal liver cells (FLC) obtained at 18–20 days gestation suppressed mixed lymphocyte reactions(MLR) of adult lymph node cells. The suppression was not strain specific: both syngeneic and allogeneic FLC were capable of suppressing the MLR. The same suppressor activity was observed with fetal spleen cells but not with fetal thymus cells. Removal of phagocytic cells from FLC failed to inhibit the suppressor activity. The suppressor cells were separated into two different types by BSA density gradient: one is radiosensitive, the other radioresistant. A stronger suppressor activity was observed in radiosensitive cells. The suppressor cells belonged to the fraction agglutinated with peanut agglutinin. The data suggest that the suppressor cells in rat FLC may be a proliferating blastoid-type cell rather than mature lymphocytes or mature macrophages.     

Proteins synthesized in lymphoid cells early after activation by phytohemagglutinin

A double-isotope labeling approach has been employed in an attempt to identify the proteins synthesized by lymphocytes early after stimulation by phytohemagglutinin (PHA). The earliest effect of PHA, within the first hour, was the induction of large aggregates of cellular proteins, which were not dissociated by 1% sodium dodecyl sulfate (SDS) in the absence of β-mercaptoethanol. These aggregates were composed of proteins of molecular weight approximately 70,000, but they did not include PHA. The aggregates were made up of preexisting as well as newly synthesized cellular proteins. Subsequently, within the first 2 hr after the addition of PHA, there was a nonspecific stimulation of protein synthesis. This was followed by the preferential synthesis of several classes of proteins including at least one group of nuclear proteins. The structural changes described here are among the earliest events known to occur within the lymphoid cell after its interaction with PHA.     

Lateral mobility of negative charge sites at the surface of sheep erythrocytes

Neuraminidase-sensitive negative charge sites on sheep red blood cells (SRBCs) are uniformly distributed over the cell surface, as in other species. Unlike most other species in which charge sites are immobile, however, in intact RBCs interaction of SRBCs with cationized ferritin (CF) induces clustering (i.e. patching) of CF-binding sites. This clustering is both time- and temperature-dependent. The clustering of negative charge sites results in the formation of domains up to 1 000 Å across which are totally free of CF binding sites. The unusually high degree of lateral mobility of ionogenic groups at the SRBC surface may contribute to the species specificity of E rosetting.     

Protein synthesis in dorsal and ventral regions of Xenopus laevis embryos in relation to dorsal and ventral differentiation

Two-dimensional gel electrophoresis has been used to analyze protein synthesis in dorsal and ventral regions in embryonic stages of Xenopus laevis. Proteins specific either to dorsal or to ventral regions are synthesized for the first time at gastrulation, concomitant with morphological differentiation. The reliability of these proteins as markers of dorsal and ventral differentiation was tested by examining their synthesis in Uv-irradiated embryos, which have severely reduced capacity for dorsal development, reflected in reduced levels of the neuromuscular-specific enzyme acetylcholinesterase, but which continue to synthesize the great majority of proteins at normal rates. Synthesis of dorsal indicator proteins should be reduced or absent in these embryos, whereas ventral indicators should be synthesized at least to the same extent as in control embryos. Some of the putative dorsal and ventral indicators failed this test, but the majority were confirmed as reliable markers of dorsal and ventral differentiation, thus providing a connection between morphology and gene expression in the establishment of the dorsal-ventral axis in X. laevis.     

The display of microtubules in transformed cells.

Monospecific tubulin antibodies have been used in indirect immunofluorescence microscopy on a variety of well characterized, transformed cell lines grown in tissue culture. Networks of colcemid-sensitive fibers are seen in SV40-transformed 3T3 cells, SV40-transformed rat embryo cells, HeLa cells and other transformed cell lines. In each case, greater than 90% of the cells contain visible microtubular networks, and where individual microtubules can be distinguished, they run for long distances. Documentation of these metworks is more difficult in transformed than in normal cells, because transformed cells are in general more rounded and have less well spread cytoplasm. In addition, the microtubular networks can be readily visualized in "cytoskeletons" of both normal and transformed cells, obtained by treatment of cells with nonionic detergents in a buffer which stabilizes microtubules in vitro. Addition of calcium to this buffer results in in situ fragmentation and destruction of the microtubular network. In view of these results, we conclude that transformed cells contain significant numbers of microtubules, and that in transformed cells, as in normal cells, microtubules are arranged in networks.