Uptake and conversion of the antibiotic albomycin by Escherichia coli K-12.

The antibiotic albomycin is transported into cells of Escherichia coli K-12 by the same uptake system as the iron-supplying ferrichrome complex. The iron-complexing hydroxamate moieties of albomycin and ferrichrome are structurally similar. During the phase of rapid iron uptake the chelators were not found in the cells. In order to understand the antibiotic activity of albomycin, it was labeled in the hydroxamate with tritium and in the presumed antibiotically active area with radioactive sulfur. While the tritium label was not retained by the cells, part of the sulfur label was taken up and concentrated 500-fold within the cell. The sulfur was not incorporated into proteins or nucleic acids since it was recovered as a low molecular weight component. Gel filtration on Bio-Gel P-2 revealed one tritium-labeled and two sulfur-labeled cleavage products in the incubation medium. We conclude that albomycin is actively transported via its ferrichrome-like portion into the cells and that the growth-inhibitory moiety is released by hydrolysis intracellularly and remains there.     

C14-variamycin content in normal and tumorous brain tissues of white rats]

Penetration of variamycin, a new antitumor antibiotic into the normal and tumor tissues of the brain of rats with multiform glioblastoma was investigated. The content of the C14-labeled antibiotic was determined radiometrically. The radioactive label penetrated into the normal and tumor tissues of the brain during the first hours after the drug administration. The level of the radioactivity in the tumor tissue was higher than that in the normal brain tissue during the whole period of the study. The greatest deviation in the contents of the radioactive label in the tumor and normal tissues was observed 2 and 3 hours after administration of the labeled antibiotic, i. e. 4.3 and 3.6 times respectively.     

Biosynthesis of the labelled rifamycin B in the presence of different 14C- and 3H-precursors

The results of studying the effectiveness of incorporation of the label from different 14C- and 3H-precursors into the molecule of rifamicin B during its biosynthesis are presented. The regularities of the label incorporation into the antibiotic composition as dependent on the time of the precursor addition were investigated. A radiochemically pure preparation of 14C-rifamicin B with specific radioactivity of 3 mcurie/mg was obtained.     

Labeling of proteins with [35S]methionine and/or [35S]cysteine in the absence of cells

Incubation of [35S]methionine and [35S]cysteine with bovine albumin, globulin, catalase, hemoglobin, or human globulin resulted in incorporation of the 35S label into each of these proteins. Trichloroacetic acid (TCA) precipitation revealed that the percentage of label incorporated ranged from 1 to 15%. The 35S labeling was resistant to dissociation by reducing SDS-PAGE, prolonged dialysis against 4 M urea, heating, TCA precipitation, and dilution by gel filtration. The labeling effect was more efficient with [35S]cysteine than [35S]methionine. Incubation of 35S label with proteins differing in methionine and cysteine content revealed no requirement for sulfur-containing amino acids in the target protein. Protein carboxymethylation reduced but did not prevent 35S label incorporation. Amino acid analysis of labeled proteins revealed that the radioactive label was not consistently associated with an individual amino acid. Differences in the ability of various proteins to spontaneously label with these amino acids suggest caution in the interpretation of metabolic labeling experiments and the necessity for inclusion of additional controls. Alternatively, our experience indicates a potentially useful method for labeling proteins in the absence of cells.     

Nucleic Acid Precursor Incorporation by Eimeria nieschulzi (Protozoa: Apicomplexa) and Jejunal Villus Epithelium*

Autoradiography was used to investigate incorporation of tritiated adenine, adenosine, guanosine and thymidine by Eimeria nieschulzi and rat jejunal villus epithelial cells. At 2 1/2 days postinoculation, parasitized and control tissues were incubated for 20 min in oxygenated Tyrode's solution (37 C, pH 7.5) containing 30 μCi/ml of each nucleic acid precursor. Treatment of tissues with ribonuclease revealed that E. nieschulzi incorporated label from [³H]adenine primarily into RNA while that from [³H]adenosine and [³H]guanosine was present mainly in DNA. Label from [³H]thymidine was not utilized by parasites. Host villus epithelial cells incorporated label from [³H]purines primarily into RNA. Labeled cytoplasmic RNA was significantly increased in parasitized cells after incubation in [³H]adenine. Tritiated nuclear RNA and cytoplasmic RNA were significantly decreased in parasitized cells after incubation in [³H]adenosine. Incorporation of label from [³H]guanosine was similar for parasitized and control cells. A small quantity of label from each [³H]precursor was incorporated into DNA of villus epithelial cell nuclei.     

Movement of Carbon from Vegetative Cells to Heterocysts in Anabaena cylindrica

Radioactive carbon assimilated by vegetative cells of Anabaena cylindrica in the light passed via an intrafilamentous route into heterocysts in the dark. After several hours, label per heterocyst approximated label per vegetative cell. Much of the label entering heterocysts was not available for diffusional exchange back into vegetative cells.     

An autoradiographic study of the implantation of transferred mouse blastocysts

Mouse blastocysts collected on day 4 were cultured in [3H]thymidine (0.01 muCi/ml) for 24 h and transferred to the uteri of pseudopregnant recipients. Autoradiography revealed that when such blastocysts were allowed to develop for 48 h in utero, label was apparent in the nuclei of decidual cells. The experimental conditions were physiological since blastocysts developed into normal offspring when gestation was allowed to proceed in pseudopregnant recipient animals. The transfer of foetal DNA into maternal decidual cells may be of important immunological significance.     

Translocation in rhizomorphs ofArmillaria mellea

The uptake and transport of exogenously applied [U-¹⁴C]glucose and³²PO₄ by the root-like structures termed rhizomorphs of the fungusArmillaria mellea were studied. Rhizomorphs were grown in petri dishes from a defined medium, across and air space, into either defined medium or water agar. Uptake of label and transport of absorbed label from bases to tips of rhizomorphs occurred in cultures growing aerobically. Label was present in all segments sampled along the lengths of such rhizomorphs with the concentration of label generally decreasing with increasing distance from the base to which isotope has been applied. The air space eliminated background due to diffusion of isotope directly through agar. Control experiments revealed that translocated compounds do not diffuse on the surface of rhizomorphs or their cells, but instead, are carried within the cells of the rhizomorphs. Both absorption and transport of label were eliminated by glutaraldehyde fixation. Rhizomorphs living under anaerobic conditions absorbed, but did not transport, label. This suggests a mechanism of transport that is dependent upon aerobic respiration. Essentially no transport of label was observed in the reverse direction from tip to base of rhizomorphs under the experimental conditions used.     

Metabolism of Transpired Ethanol by Eastern Cottonwood (Populus deltoides Bartr.)

Ethanol has previously been shown to be present in the xylem sap of flooded and nonflooded trees. Because of the constitutive presence of alcohol dehydrogenase in the mature leaves of woody plants, we hypothesized that the leaves and shoots of trees had the ability to metabolize ethanol supplied by the transpiration stream. 1-[14C]Ethanol was supplied to excised leaves and shoots of eastern cottonwood (Populus deltoides Bartr.) in short- and long-term experiments. More than 99% of the radiolabel was incorporated into plant tissue in short-term experiments, with more than 95% of the label remaining in plant tissue after 24 h. In all experiments, less than 5% of the label was transpired as ethanol and less than 1% was emitted as CO2. In excised leaf experiments, less than 0.5% of the radiolabel escaped from the leaf. Fifty percent of the label was incorporated into the petioles of excised leaves; 56% was incorporated into the stems of excised shoots. Very little label reached the leaf mesophyll cells of excised shoots, as revealed by autoradiography. Radiolabel appeared primarily in the water- and chloroform-soluble fractions in short-term experiments, whereas in long-term experiments, label was also incorporated into protein. These results demonstrate that the leaves and stems of trees appear to have substantial ability to scavenge ethanol from the transpiration stream, allowing efficient recovery of ethanol produced elsewhere by hypoxic tissues. When labeled ethanol was supplied to excised petioles in a 5-min pulse, 41% of the label was incorporated into organic acids. Some label was also incorporated into amino acids, protein, and the chloroform-soluble fraction, with very little appearing in neutral sugars, starch, or the insoluble pellet. Labeled organic acids were separated by high performance liquid chromatography and were composed of acetate, isocitrate, [alpha]-ketoglutarate, and succinate. There was no apparent incorporation of label into phosphorylated compounds. We conclude that, in higher plants, ethanol is metabolized to acetaldehyde and then to acetate by alcohol and aldehyde dehydrogenases, and then into general metabolism.     

Effect of cerulenin on macromolecule synthesis in chemoheterotrophically and photoheterotrophically grown Rhodopseudomonas sphaeroides.

The antibiotic cerulenin causes the immediate cessation of phospholipid biosynthesis in both chemoheterotrophic and photoheterotrophic cultures of Rhodopseudomonas sphaeroides. Macromolecule biosynthesis in photoheterotrophic cells was unaffected by cerulenin for the first 2 h after antibiotic addition and then continued at a reduced rate for an additional 8 h. In contrast, macromolecule biosynthesis in chemoheterotrophic cells was severely affected by cerulenin within the first 2 h of treatment. Pulse-labeling of protein after cerulenin addition revealed that all subcellular fractions were equally affected by the action of cerulenin with chemoheterotrophic cell fractions more profoundly affected than those derived from photoheterotrophic cells. Protein insertion into the intracytoplasmic membrane of photoheterotrophic cells continued for up to 6 h after the onset of cerulenin treatment. Residual macromolecule synthesis was correlated with the presence of the photosynthetic membrane system under all conditions of growth.     

Expression of human chromosomal proteins HMG-14 and HMG-17 in Saccharomyces cerevisiae

The cDNAs coding for human chromosomal proteins HMG-14 and HMG-17 were cloned into yeast expression vector pBM150, under the control of the Gal10 promoter. Northern analysis of transformed yeast cells revealed that both cDNAs were efficiently transcribed. Western analysis indicated that the mRNAs were translated into authentic proteins. Expression of human HMG proteins in yeast cell did not produce detectable phenotypic changes, as measured by the growth rate of the yeast cells under a variety of conditions. The antibiotic resistance of the transfected cells was similar to that of control cells, suggesting that the presence of HMG did not affect the expression of actively transcribed genes. However, examination of the protein profile on two-dimensional polyacrylamide gel electrophoresis revealed differences between control and HMG-transfected cells.     

Neural repair in an insect central nervous system: cell kinetics and proliferation after selective glial disruption

Summary Autoradiographs of tritiated thymidine uptake and subsequent light- and electron-microscopical examination revealed an onset of perineurial glial cell proliferation 3 days after injury to the CNS. The number of cells labelled increased rapidly until 7 days post-lesioning. At 2 weeks, the labelled cells equalled the number of nuclei present in the perineurium. No label was seen in the subperineurial cells, possibly because of the inability of the label to penetrate into a region where localised division is taking place.Prior to the onset of thymidine uptake, the damaged nerve cord was invaded by an exogenous reactive cell. The number of these cells increased rapidly in the first 48 h, then decreased as a negative exponential, very few remaining after 7 days. We suggest that this cell type must either return to the haemocoel or transform into a functional glial cell class.The repair of the insect central nervous system can be divided into three phases which show striking similarities to vertebrate repair sequences. These include: initial invasion of the lesion by exogenous cells, subsequent proliferation of glial cells, the longer term flux of cell numbers, their distribution and the time scale of events. This suggests that the insect CNS might provide a system for examining common cellular mechanisms and events.     

DNA SYNTHESIS IN THE OOPLASM OF DROSOPHILA MELANOGASTER

Tritiated thymidine was injected into 2-day-old Drosophila melanogaster females, and tissue sections were prepared from the ovary for radioautography with both the light and electron microscopes. Besides the expected incorporation of H³-thymidine into nuclei of nurse cells and follicle cells, there was a relatively high level of incorporation of label into ooplasmic DNA. The highest level of incorporation occurred at stage 12. At the same time, the 15 nurse cell nuclei also incorporate thymidine in spite of the fact that they are breaking down and degenerating. The label in the ooplasm is not removed by extraction with DNase (although this removes nuclear label) unless extraction is preceded by a treatment with protease. Electron microscopic radioautography revealed that 36% of the silver grains resulting from decay of H³-thymidine are found over mitochondria, with a further 28% being located within 0.25 µ of these organelles. The remaining 36% of the silver grains was not found to be associated with any organelles, and it probably represents synthesis in the cytoplasm by the "storage DNA" characteristic of many eggs. It is suggested that one mechanism acting throughout the egg chamber is responsible for the synchronous synthesis of DNA in the degenerating nurse cells, in the mitochondria of the egg, and in the "storage DNA" of the ooplasm.     

Phosphorylation of Escherichia coli enolase

In vivo labeling of Escherichia coli JA200 pLC 11-8 resulted in 32P incorporation into enolase as demonstrated by immunoaffinity chromatography and electrophoresis followed by autoradiography. Complete acid hydrolysis, followed by thin layer chromatography was employed for determination of the phosphoamino acid residue. Comparison with phosphoamino acid standards resulted in the identification of a labeled residue corresponding to phosphoserine. In vitro labeling of cell extracts from glucose and acetate grown cells resulted in differential labeling of enolase. When specific radioactivities of in vivo labeled enolase were compared, 7 times more label was incorporated at late log phase in glucose grown cells than in late log acetate grown cells. At stationary phase, only 2.5 times more label was incorporated into glucose compared to acetate. When 32P-labeled enolase from glucose grown cells was subjected to treatment with potato acid phosphatase, dephosphorylation of the enzyme could be observed. Monitoring enzyme activity during the acid phosphatase treatment revealed a 70% decrease for the forward enzyme reaction, and a 3-fold increase, followed by a gradual decrease to almost zero, for the reverse enzyme reaction. Complete reversal of the changes in activity was possible by adding an aliquot of partially purified enolase kinase plus ATP.     

Genetic Integration in the Heterospecific Transformation of Haemophilus influenzae Cells by Haemophilus parainfluenzae Deoxyribonucleic Acid

The in vivo chemical linkage of Haemophilus parainfluenzae deoxyribonucleic acid (DNA) with the H. influenzae genome has been found to occur at a much higher level than is suggested by the low efficiency of the heterospecific transformation of an antibiotic resistance marker. This linkage, about 60% of the level with homospecific DNA, was found to involve alkali-stable bonding. The amount of host DNA label released (about 60%) was about the same as that released during homospecific transformation. Also, over 60% of the H. influenzae cells adsorbing H. parainfluenzae DNA could not form colonies upon plating. This lethality of the heterospecific transformation was not immediate but followed considerable metabolic activity of the host cells. These data are presented to show that the "limited-pairing" hypothesis may be only a partial explanation for the low efficiency of heterospecific transformation. Another hypothesis is presented which takes into account the lethal effect of this kind of transformation.     

Synthesis and migration of 3H-fucose-labeled glycoproteins in the retinal pigment epithelium of albino rats, as visualized by radioautography

3H-fucose was injected into the vitreous body of the eye(s) of 250-gm rats, which were then killed by means of an intracardiac perfusion with glutaraldehyde after intervals of 10 min, 1 and 4 hr, and 1 and 7 days. The eyes were removed and further fixed, and pieces of retina were processed for light and electron microscope radioautography. Light microscope radioautography showed that the pigment epithelial cells actively incorporated 3H-fucose label. The intensity of reaction peaked at 4 hr after injection of the label and then slowly declined. Quantitative electron microscope radioautography revealed that, at 10 min after 3H-fucose injection, over 70% of the label was localized to the Golgi apparatus, indicating that fucose residues are added to newly synthesized glycoproteins principally at this site. With time the proportion of label associated with the Golgi apparatus decreased, but that assigned to the infolded basal plasma membrane, the apical microvilli, and various apical lysosomes increased. These results indicate that in retinal pigment epithelial cells newly synthesized glycoproteins continuously migrate from the Golgi apparatus to lysosomes and to various regions of the plasma membrane. In this case, the membrane glycoproteins may play specific roles in receptor functions of the basal plasma membrane or phagocytic activities at the apical surface. Very little label migrated to Bruch's membrane, indicating either a very slow turnover or a paucity of fucose-containing glycoproteins at this site.     

Acetylcholine stimulates selective liberation and re-esterification of arachidonate and accumulation of inositol phosphates and glycerophosphoinositol in C62B glioma cells

Glioma C62B cells, incubated for 18 h with either an unsaturated (arachidonate or oleate) or saturated (palmitate or stearate) radioactive fatty acid, incorporated label into most species of cellular glycerolipids. Treatment of prelabeled C62B cells with 1 mM acetylcholine (ACh) resulted in an accumulation of radioactive phosphatidate irrespective of which fatty acid was used as a label. However, only in cells prelabeled with unsaturated fatty acids were increases in radioactive fatty acids observed. When exogenous radioactive arachidonate was added to C62B cells in the presence of 1 mM ACh, there was a rapid, selective, and transiently enhanced incorporation of label (several times the control) into phosphatidylinositol (PI). The ACh-enhanced incorporation into PI was not preceded by enhanced incorporation of label into sn-1,2-diacylglycerol or phosphatidate but was followed by an increased labeling of polyphosphoinositides. Similarly, incorporation of oleate into PI was enhanced by ACh. In contrast, ACh did not enhance the incorporation of label into any glycerolipids when saturated fatty acids were used. C62B cells, incubated with [2-3H]inositol for 18 h selectively incorporated label into phosphoinositides. Stimulation of [2-3H]inositol-labeled cells with 1 mM ACh in the presence of 25 mM LiCl resulted in a rapid accumulation of radioactive inositol phosphates (mono-, bis-, and trisphosphates) and glycerophosphoinositol. The accumulation of inositol trisphosphates preceded that of inositol monophosphate and glycerophosphoinositol, while the accumulation of glycerophosphoinositol paralleled the time required for the ACh-stimulated esterification of arachidonate. These results suggest that ACh stimulates activation of a phospholipase C in C62B cells and release of 1,4,5-inositol trisphosphate. There is subsequent activation of phospholipase A2, which in turn liberates arachidonate from PI. The resulting lyso PI is either rapidly reesterified with unsaturated fatty acid to resynthesize PI, or further deacylated to yield glycerophosphoinositol.     

RADIOAUTOGRAPHIC COMPARISON OF THE UPTAKE OF GALACTOSE-H3 AND GLUCOSE-H3 IN THE GOLGI REGION OF VARIOUS CELLS SECRETING GLYCOPROTEINS OR MUCOPOLYSACCHARIDES

The radioautographic distribution of the label of galactose-H³ was compared with that of glucose-H³ in a series of secretory cells of the rat. Whereas the glucose label appeared in all mucous cells, the galactose label was incorporated only into certain mucous cells. Whenever either label was incorporated, however, it was located first in the Golgi region and later in the secretion product, mucus. Several lines of evidence, including extraction of glucose label with peracetic acid—beta glucuronidase, indicated that the material synthesized in the Golgi region was glycoprotein in nature. In chondrocytes, both the galactose and the glucose label appeared first in the Golgi region and later in cartilage matrix; extraction of glucose label with hyaluronidase indicated that much of it consisted of mucopolysaccharide. In all secretory cells, the extraction of glycogen by amylase had no effect on Golgi radioactivity. Such extraction did not eliminate the scattered cytoplasmic label also seen after glucose-H³ injection, but completely eliminated that seen after galactose-H³. Consequently, the galactose-H³ label in the Golgi region stood out more clearly, and was detected in many cells: pancreas, liver, epididymis, and intestinal columnar cells. In the latter, label later appeared in the surface coat. Thus, radioautography after injection of galactose-H³, as after glucose-H³, indicates that synthesis of complex carbohydrates takes place in the Golgi region of many secretory cells.     

Immunocytochemical localization of vasopressin at its absorption by cells of rat small intestine

Morpho-physiological characteristics of the transport of cyclic nonapeptide arginine vasopressin (AVP) across the rat intestinal epithelium was studied in experiments in vitro. A partial absorption of physiologically active AVP was followed when filling the isolated intestinal lumen by hormone solution. By methods of immunoelectron and immunofluorescence confocal microscopy, using polyclonal anti-AVP antibodies, cytoplasmic localization of AVP label was shown in enterocytes. The AVP label was also observed in the intercellular space in the basal area of epithelium. No label was revealed in the intercellular junctions, and no predominant label accumulation was found in any cytoplasmic structures of the epithelial cells. The obtained results are considered as evidence for the transcellular pathway of partial AVP absorption in rat small intestine.     

Cytotoxic effect of multinucleation in HeLa cell cultures associated with the presence of Vir plasmid in Escherichia coli strains

The occurrence activity and localization of calmodulin in three heterocystous cyanobacteria of the genus Anabaena were studied. Boiled crude extracts caused a Ca2+-dependent stimulation of NAD kinase. Such a stimulation was blocked by EGTA and chlorpromazine, SDS-PAGE and Western blot analysis using antiserum against eukaryotic spinach calmodulin, revealed a polypeptide of about 17 kDa. Immunogold localization of calmodulin gave a dense gold label in both vegetative cells and heterocysts. The label was mainly confined to the centroplasm in vegetative cells, while it was evenly distributed in the cytoplasm of mature heterocysts.