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.     

A rapid,sensitive assay for starch phosphorylase and ADPglucose pyrophosphorylase

Starch phosphorylase (EC 2.4.1.1) and ADPglucose pyrophosphorylase (EC 2.7.7.27) activities can be measured very accurately and quickly using a recording pH meter. Activities less than 0.28 nmol/min are readily measured.     

Demonstration of tubulin, actin and alpha-actinin by immunofluorescence in the microtubule-microfilament complex of the cytopharyngeal basket of the ciliate Pseudomicrothorax dubius

Studies were undertaken to identify cell surface markers specific for different phases of the cell cycle. Antisera were prepared in rabbits against membrane protein preparations from synchronized BW 5147 cells, an AKR mouse T-lymphoma cell line, in the G1, S, G2 or M phases of the cell cycle. These antisera were used to precipitate radioiodinated surface proteins from synchronized cells in the different phases. The immunoprecipitates were quantitatively analyzed by sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Cells in S phase had significantly higher concentrations of proteins weighing 70 × 10³ and 165 × 10³ D than cells in G1 or G2 phase. The other major labeled surface components did not vary. These results were confirmed by quantitative absorption of the antisera with synchronized cells. Comparative analysis of the antisera showed that the 165 × 10³ D peak contained at least two antigens, one recognized by both a-G1 and a-S and the other by a-G1 only. Though cells in S phase had large quantities of the 70 × 10³ D protein, intact and SDS-solubilized membrane preparations from S phase could not elicit in rabbits any antibody against that protein. These antisera did, however, have good antibody titers to the other major protein peaks and the antisera developed against cells in G1, G2 or M had good anti-70 × 10³ activity. The results suggest a qualitative molecular change in the 70 × 10³ protein during S phase.     

The glycoproteins of Dictyostelium discoideum. Changes during development.

The glycoproteins of D. discoideum have been analyzed by direct binding of radio-iodinated lectins to SDS gels of the successive developmental stages. Compared with the total pattern of proteins, many changes are found in the glycoproteins during development. WGA reacts with few gel bands from the vegetative cells and most of these, including a very intense band at the top of the gel, are lost during the first few hours of development. Approximately half-way through the developmental cycle at least 14 new glycoproteins reacting with WGA begin to appear and progressively accumulate. In contrast, ConA labels many glycoproteins over the complete molecular weight range and most are unaffected during development. Lectins which bind fucose label a single component at the top of the gel of vegetative cells and this decreases rapidly as development begins. No other reactive gel bands are revealed by fucose-binding lectins until the final stages of spore and stalk formation, when four high molecular weight glycoproteins are detected. Lectins specific for terminal galactose residues and for N-acetyl-galactosamine, including the intrinsic lectins produced by D. discoideum during its development, failed to reveal any reactive glycoproteins.     

Accumulation of alpha-mannosidase-1 in Dictyostelium discoideum requires many developmentally essential genes

alpha-Mannosidase-1, one of the earliest known developmentally controlled gene products in the cellular slime mold Dictyostelium discoideum, accumulates intracellularly during both axenic growth and development. The accumulation of alpha-mannosidase-1 activity prematurely ceases in all of 125 randomly isolated aggregation-deficient mutants at discrete times in development resulting in significantly reduced levels of cellular enzyme activity. This suggests that, unlike other developmentally controlled enzymes in this organism, the continued accumulation of alpha-mannosidase-1 activity is controlled by a large number of genes essential for early development. alpha-Mannosidase-1 misregulation and the aggregation-deficient phenotype are caused by the same mutation since (1) morphological revertants exhibit a coreversion to both fruiting ability and wild-type alpha-mannosidase-1 accumulation and (2) normal enzyme accumulation depends on the ability to aggregate and ultimately fruit in a conditional aggregation-deficient mutant. This type of regulation does not appear to be due to differences in enzyme secretion or changes in the overall rate of total protein synthesis. Aggregation-deficient mutants continue to synthesize protein beyond the time in development at which alpha-mannosidase-1 accumulation ceases. Our studies indicate that most of the 50-125 genes required for aggregation in Dictyostelium are also required for the normal accumulation of alpha-mannosidase-1 activity.     

Compositional heterogeneity of protochlorophyllide ester in etiolated leaves of higher plants

The formation and degradation of protochlorophyllide esters, i.e., protochlorophylls, were studied in etiolated leaves of kidney bean in relation to their aging. By the sensitive analysis of the pigments using high-performance liquid chromatography, the presence of four protochlorophylls esterified with phytol, tetrahydrogeranylgeraniol (THGG), dihydrogeranylgeraniol (DHGG), and geranylgeraniol (GG) was detected in kidney bean grown in the dark. Similar components were also observed in the etiolated seedlings of cucumber, sunflower, and corn. The content of each protochlorophyll species changed with the plant species and age of plants. In the case of kidney bean, the content of protochlorophyll phytol reached a maximal level at 9 days, then decreased rapidly during the subsequent development, in spite of the total protochlorophyll content remaining unchanged. In contrast to the degradation of protochlorophyll phytol, the other three protochlorophylls esterified with THGG, DHGG, and GG accumulated. These results may indicate that (i) protochlorophyll phytol is formed from the first esterified protochlorophyll GG through the next three hydrogenation steps as in the case of chlorophyll a phytol formation; (ii) the esterification reaction stops at 9 days and then reaction proceeds in sequence in the reverse direction, leading to the dehydrogenation of the alcohol moiety of protochlorophyll phytol to protochlorophylls THGG, DHGG, and GG.     

Effect of extracellular Ca2+ on the morphogenesis of Dictyostelium discoideum.

Effect of extracellular Ca²⁺ on the morphogenesis of the cellular slime mold Dictyostelium discoideum was examined on agar plate. The concentration of Ca²⁺ in agar plate was controlled by keeping the concentration of a chelating reagent EGTA constant and varying the concentration of total calcium. From experiments in which EGTA concentration was kept at 2.0 × 10^?3 M, it was found that by decreasing Ca²⁺ concentration the morphogenesis was modified so that development of the aggregating amebae into fruiting bodies was accelerated and the period of migrating slugs was shortened. Below 1.0 × 10^?3 M of Ca²⁺ concentration, the total number of aggregates initially increased with decreasing Ca²⁺ concentration, reached a maximum at about 3.0 × 10^?7 M of Ca²⁺ concentration and hereafter decreased with decreasing Ca²⁺ concentration. The number of mature fruiting bodies obtained at 36 h period after starvation depends on Ca²⁺ concentration and the total number of aggregates. The cell aggregation initiated at the same time period after starvation even at an extreme case of 1.0 × 10^?8 M of Ca²⁺ concentration as under enough Ca²⁺ supply, while the formation of mature fruiting body was seriously inhibited. These observation suggested that the cAMP-mediated cell aggregation in D. discoideum is a Ca²⁺-independent phenomena, although extracellular Ca²⁺ is necessary for the normal development of the aggregated amebae.     

ADP phosphorylation and glutamate oxidation in mitochondria isolated from Dictyostelium discoideum amoebae

Mitochondria have been isolated from $\text{D.}\text{discoideum}$ amoebae in which respiration is coupled to ADP phosphorylation. P:O ratios and respiratory control ratios have been obtained for a number of metabolites. In rat liver mitochondria, glutamate is oxidized almost exclusively by a respiration-dependent cyclic transamination pathway, in which glutamate is converted to aspartate. When $\text{D.}\text{discoideum}$ amoebae are incubated with glutamate alone, aspartate does not accumulate appreciably. Furthermore, when the mitochondria are incubated with glutamate plus malonate at a concentration sufficient to inhibit respiration, their utilization of glutamate is depressed only slightly. Thus, it appears that glutamate oxidation within the mitochondria of $\text{D.}\text{discoideum}$ amoebae does not, for the most part, proceed by the cyclic transamination pathway.     

Effect of stimulus strength and adaptation on the thermotactic response of Dictyostelium discoideum pseudoplasmodia

The thermotactic responses of Dictyostelium discoideum strain HL50 and mutants derived from this strain have been characterized by curves of stimulus-strength vs response. With gradient midpoint temperatures of 16 and 24 °C, these curves are typical of those of a single response, i.e., the strength of the response increases with increasing stimulus strength until at some strength the response saturates. However, with a gradient midpoint temperature close to the transition from negative to positive thermotaxis, the sign of the thermotactic response depends on gradient strength. These observations support the hypothesis that the transduction pathways for positive and negative thermotaxis act concurrently and contain separable elements. An investigation of the adaptation of thermotaxis indicated that the stimulus-strength-dependence and midpoint-temperature-dependence of both thermosensory responses was altered by shifting the growth and development temperature.     

Analysis of collagen cyanogen bromide peptides using electrophoresis in continuous concave gradient polyacrylamide gels.

A rapid and simple spectrophotometric method is described for the estimation of microgram quantities of glycosaminoglycans following the formation of soluble complexes with alcian blue dye. The method is based on the different absorption spectra of the dye and dye-glycosaminoglycan complexes. No heating, centrifugation, lengthy equilibration, or sophisticated instrumentation which hamper other methods are required. Samples are mixed with freshly prepared dye solution and absorbance readings at 480 nm are compared to an appropriate standard curve. Albumin and individual monosaccharides do not interfere with the assay but high concentrations of chloride ion do. The method is suitable for the estimation of total glycosaminoglycan levels in biological fluids such as urine and blood.     

Interchain heterogeneity in 2-hydroxyethylglutamine-l-valine random copolymers

Solid state circular dichroism (c.d.) and infrared (i.r.) studies of water soluble and insoluble fractions of poly(hydroxyethylglutamine-valine) random copolymers, prepared from parent γ-benzyl l-glutamate valine copolymers, show that interchain conformational heterogeneity with interchain compositional heterogeneity is present when the respective N-carboxyanhydrides are copolymerized in dioxan or benzene/methylene chloride. Use of previously determined reactivity ratios for the aforementioned copolymer systems permits the determination of the variation of the average copolymer composition, $\text{f}$_G, with conversion. The experimentally determined average copolymer composition.$\text{f}$_G for the use of the respective reactivity ratios and the copolymer hydroxyethylglutamine, valine are predicted by the use of the respective reactivity ratios and the copolymer composition equation. As the valine content of the copolymer chains in the fractions increases, the expected increase in β-sheet contribution is seen. Comparison of the experimentally determined solid state c.d. spectra with Greenfield and Fasman's computer generated c.d. spectra for varying amounts of α-helix, β-sheet and random structures, shows that the water insoluble fractions with their increased valine contents have a greater contribution of β-sheet structure than the respective soluble fractions.     

Stable heterogeneity of mitochondrial DNA in grande and petite strains of S. cerevisiae.

Several instances of mitochondrial DNA heterogeneity in grande and petite strains of Saccharomyces cerevisiae were examined. We have detected heterogeneity in the mtDNA from some of the progeny strains of a cross between two grande strains (D273-10B, MH41-7B) which differ in genome size and restriction cleavage pattern of their mtDNA. The progeny strains transmit restriction fragments characteristic of both parental strains from homologous regions of the mitochondrial genome, and this sequence heterogeneity is not eliminated by additional subcloning. Sequence diversity is more common in the mtDNA of petite than of grande strains of yeast. We have examined subclones of one petite strain to identify the origin of this variability. Many of the submolar restriction fragments persist in independent subclones of this petite after 15 and 30 cell divisions; some submolar fragments disappear, and some new fragments appear. We conclude that the observed sequence heterogeneity is due to molecular heterogeneity, i.e., to differences in the multiple copies of the petite mitochondrial genome, as well as to clonal heterogeneity. It is likely that tandem repeats on the same mtDNA molecule also differ, i.e., that there is intramolecular heterogeneity, and that this accounts for the stability of the heterogeneity. Continuing deletion is probably responsible for the appearance of “new” fragments in petite subclones.     

Transfer of cell-mediated immunity with cell-free leukocyte extracts: II. Demonstration of antigen-specific and nonspecific components

Transfer of cell-mediated immunity was achieved with dialyzable cell-free extracts from lymphoid cells of mice primed to the contact sensitizing agent, 2,4-dinitrofluorobenzene (DNFB). The biological activity of the extract (Transfer Factor, TF) was analyzed in vivo by the ear thickness assay and in vitro by the macrophage migration inhibition (MMI) test and lymphocyte transformation using the soluble analog, sodium 2,4-dinitrobenzenesulfonate. Consistently positive responses occurred 20 hr following a single intravenous injection of 5 × 10⁷ lymphocyte equivalents per recipient. The most potent source of TF (memory TF) was lymph node cells obtained 30 days after primary exposure to DNFB. By contrast TF prepared at the peak of the response to DNFB was less potent which was shown to be due to the presence in it of a suppressor factor. Memory TF elicited macrophage inhibition factor production in naive lymph node cells whereas positive responses were only obtained in the ear thickness and lymphocyte transformation assays provided recipients had undergone prior subliminal sensitization. Specificity of TF was tested using picryl chloride and oxazolone as control antigens. Results from the MMI and ear thickness assays were consistent with the presence in Transfer Factor of an antigen-specific component. Its effects, however, on the proliferative response to antigen lacked specificity and depended on prior sensitization of recipients, rather than donors, to the inducing antigen. The target of the specific component was considered to be an Ly-1⁺, Ia^?, $\text{Ly}\text{2}\text{3}{}^{\mathrm{?}}$ T cell since MIF production and in vivo delayed hypersensitivity are known to be mediated by a T cell bearing this phenotype. Taken together these findings emphasize the value of using a battery of tests of cell-mediated immune function when studying soluble mediators such as Transfer Factor and suggest that the current system is a valid experimental model for analysis of the Transfer Factor phenomenon.     

The production and storage of Nerve Growth Factor in vivo by tissues of the mouse,rat, guinea pig,hamster, and gerbil

The Nerve Growth Factor (NGF) content in vivo of tissues from the mouse and rat at various stages of development from 3 days embryonic gestation to the attainment of full maturity has been determined using the standard biological assay. A less extensive survey has also been made of tissues from the guinea pig, hamster, and gerbil. With the exception of the well-documented high levels of NGF in the mouse submaxillary glands, none of the organs examined contained detectable NGF. These results, which are consistent with those previously reported using the biological assay, stand in contrast to the high levels of NGF detected in virtually all tissues by some published radioimmunoassays. It is likely that the discrepancies are due to the use in the radioimmunoassays of antisera containing antibodies to proteins other than NGF, and to the inability of one-site radioimmunoassays to distinguish between the presence of NGF and that of agents capable of binding NGF. The apparent lack of widespread NGF production in vivo contrasts with the ability of many tissues to synthesize the protein in vitro. This may imply that physiologically significant levels of NGF are below the limits of sensitivity of the assay systems presently available, that NGF synthesis in vivo occurs only during a very restricted period of development, or that the presence of a normal innervation pattern influences NGF production.     

Protein kinase assay on isoelectric focusing gels: evaluation of its quantitative potentials.

The conditions of the Hirsch-Rosen assay (1974, Anal. Biochem.60, 389–394) for protein kinase activity oncomplete isoelectric focusing gels have been analyzed and further developed with the consequence that the test can be easily adapted to other protein kinases in a quantitative manner. Special attention was given (i) to the breakdown of the pH gradient in relation to gel size and Ampholine concentration in order to achieve optimal pH ranges for the assay, (ii) to the introduction of histone as a substrate besides protamine, and (iii) to the distribution of the particular substrates and products throughout the gel. The results of the protein kinase assay on gels were shown to be linear for at least 1 h, and to be dependent on the amount of ATP and on the amount of protein kinase applied, thereby fulfilling the requirements necessary to yield quantitative data.     

Direct visualization, by beta-galactosidase histochemistry, of differentiated normal cells derived from malignant teratocarcinoma in allophenic mice.

Mice obtained from blastocysts injected with malignant teratocarcinoma stem cells may comprise tumor-derived cells in their tissues. Evidence for their presence has hitherto been indirect, i.e., through detection of tissue-specific products of the tumor genotype or of strain-specific enzyme variants in tissue homogenates from healthy mice. Direct visualization and identification of the tumor-derived cells would permit their normalcy and their state of differentiation to be assessed. For this purpose, a histochemical marker is required. The marker chosen was β-galactosidase (BGS), which allows high- vs low-activity cell strains to be distinguished in situ by their differences in staining intensity. BGS has previously been employed for such visualization only in brain [Dewey, M., Gervais, A., and Mintz, B. (1976). Develop. Biol.50, 68–81] and has here been shown to be applicable to other tissues, including kidney, pancreas, and salivary gland. Two unexpected results concerning the marker itself were obtained and affected its application to histochemical comparisons: BGS activity in some tissues of some inbred strains was not concordant with that of brain, on which the existing genotypic classification is based; and some cell types within a tissue varied independently in BGS levels among strains (e.g., exocrine vs endocrine pancreas). BGS visualization clearly disclosed the presence of large numbers of fully differentiated normal cells of the teratocarcinoma strain in tissues, including the Purkinje layer of the cerebellum, the kidney tubules, and the exocrine pancreas of experimental animals. In one individual, the relevant brain region was almost entirely derived from the teratocarcinoma. Yet all tissues were indistinguishable in structure and differentiation from adult controls, and showed no malignant growth. The pattern of cell-strain distribution, which was fine-grained in the brain and patchy in the other tissues named, also resembled that of ordinary allophenic mice produced from blastomere aggregates of two strains. Thus, teratocarcinoma stem cells are here seen to undergo normal histogenesis after they are successfully incorporated into a developing host embryo.     

Activation of liver tryptophan oxygenase by hydrocortisone, hematin and tryptophan in streptozotocin-diabetic rats

This study compared changes in liver tryptophan oxygenase (TPO) activity in response to hydrocortisone, hematin and tryptophan administration to non-diabetic and diabetic (streptozotocin) rats. Hydrocortisone caused similar increases in apoenzyme (inactive), holoenzyme (heme-saturated) and total (holoenzyme + apoenzyme) TPO activities in non-diabetic and diabetic rats. The ability of hematin to increase total TPO activity was significantly less in diabetic rats. The largest differences between diabetic and non-diabetic rats were found with tryptophan which increased total TPO and holoenzyme activities 300% and 650% respectively in non-diabetic rats. However, tryptophan increased both apoenzyme (unchanged in non-diabetic rats) and holoenzyme activities by 300% in diabetic rats. These results indicate that in the diabetic state, the TPO-heme conjugation process is impaired, especially substrate mediated TPO-heme saturation.