Uptake and intracellular transport in rat liver of formaldehyde-treated bovine serum albumin labelled with 125I-tyramine-cellobiose

1. Endocytosis of formaldehyde-treated bovine serum albumin by rat liver sinusoidal cells has been followed by injecting rats with the protein labelled with 125I-tyramine cellobiose (125I-TCfBSA). 125I-TCfBSA is quickly taken up by the liver; the radioactivity present in the organ reaches a plateau 5-10 min after injection and is maintained for up to at least 180 min. During the first 5 min most of radioactivity remains acid-precipitable. After which, labelled acid-soluble components are produced at a constant rate for up to 30-40 min. 2. Differential centrifugation shows that radioactivity is first recovered mainly in the microsomal fraction. Within a few minutes it exhibits a distribution pattern similar to that of lysosomal enzymes, being chiefly located in the mitochondrial fractions. 3. Isopycnic centrifugation in a sucrose gradient of the microsomal fraction isolated 1 min after injection indicates a similar distribution for radioactivity and alkaline phosphodiesterase. Later, the microsomal radioactivity distribution curve is shifted towards higher densities and becomes distinct from that of the plasma-membrane enzyme. After isopycnic centrifugation in a sucrose gradient of the total mitochondrial fraction a considerable overlapping of acid-precipitable and acid-soluble radioactivity distributions is observed without significant changes with time. The same is observed in a Percoll gradient except that after a relatively long time (greater than 30 min) of injection a marked shift of radioactivity distribution towards higher densities occurs. 4. A pretreatment of rats with Triton WR 1339, a density perturbant of liver lysosomes, causes a striking shift of acid-soluble radioactivity distribution in a sucrose gradient towards lower densities while having markedly less influence on the acid-precipitable distribution. As a result, a distinction between the distribution of both kinds of radioactivity becomes clearly apparent. A preinjection of yeast invertase, modifies the acid-soluble distribution without having a significant effect on the acid-precipitable distribution up to 30 min after 125I-TCfBSA injection. 5. Glycyl-1-phenylalanine-2-naphthylamide largely releases acid-soluble radioactivity associated with the mitochondrial fraction, whatever the time after 125I-TCfBSA injection. On the other hand the proportion of acid-precipitable radioactivity present in the fraction that can be released is almost zero at 10 min after injection, and it later increases. 6. The results presented here are best explained by supposing that, after being trapped in small pinocytic vesicles, 125I-TCfBSA is quickly delivered to the endosomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of influenza virus uncoating by rimantadine hydrochloride.

In freeze-thaw lysates of MDCK cells infected with 32P-labeled influenza virus A/WSN in the presence of added RNase, acid-precipitable radioactivity diminished to about 50% of initial values within 90 min after a 1-h virus adsorption period. A similar preparation containing rimantadine at a concentration of 50 micrograms/ml exhibited only a 10% reduction in acid-precipitable radioactivity. These findings suggest that rimantadine interferes with uncoating of influenza virus in infected cells.     

GABA from putrescine is bound in macromolecular form in keratinocytes

We report for the first time that the neurotransmitter γ-aminobutyric acid (GABA) exists in macromolecular form in keratinocytes. GABA derived from putrescine (Pu) has been identified as a component of acid-precipitable material of cultured human keratinocytes. Confluent, stratified cultures of human foreskin keratinocytes exposed to [³H]-Pu for 4 hours took up about 14% of the radioactivity from the medium and 1% of the total cell-associated radioactivity was precipitable by trichloroacetic acid (TCA). Both attached and shed cells were examined by HPLC for Pu and its radioactive metabolites in TCA-insoluble and TCA-soluble fractions. GABA accounted for the major portion (54%) of the radioactivity derived from Pu in the TCA-precipitable material of attached keratinocytes. Pu and spermidine represented lesser amounts, 35% and 9% respectively, of the total TCA-precipitable radioactivity. In addition, a large portion of acid soluble radioactivity derived from Pu (63%) was GABA, whereas Pu and spermidine represented 29% and 6% respectively of the total TCA-soluble radioactivity. The exact origin of GABA in acid-precipitable material, as well as its form of attachment, is currently under investigation.     

Intracellular transport and degradation of 125I-tyramine cellobiose-labelled low-density lipoprotein endocytosed in vivo in rat liver cells studied by means of subcellular fractionation

The intracellular transport and degradation of in vivo endocytosed 125I-tyramine cellobiose-labelled low density lipoprotein (125I-TC-LDL) in rat liver cells were studied by means of subcellular fractionation in Nycodenz, sucrose and Percoll density gradients, as well as by means of analytical differential centrifugation. Initially, labelled LDL was located in endocytic vesicles of low densities. Subsequently, acid-soluble and acid-precipitable radioactivities were found in organelles with buoyant densities distinctly lower than that of the main peaks of the lysosomal marker enzymes acid phosphatase and N-acetyl-beta-glucosaminidase. These prelysosomal organelles may represent multivesicular bodies (MVBs). Finally, 6 h after injection and onwards, the acid-soluble radioactivity cosegregated completely with the two lysosomal marker enzymes, suggesting that the degradation products were in secondary lysosomes. The rate of intracellular processing of LDL was very slow compared to that of asialoglycoproteins, suggesting that LDL followed a unique intracellular pathway, that may be specific for this type of ligand.     

Incorporation of radioactivity from amino acids in the presence of cycloheximide demonstrates gluconeogenic activity in Mucor rouxii.

Mucor rouxii cells induced for gluconeogenesis incorporated radioactivity from [14C]glutamic acid into trichloroacetic acid-precipitable material in the presence of 200 micrograms of cycloheximide per ml. This metabolic capacity was repressed by hexoses and required amino acids for induction. These results suggest that the incorporation of amino acids in the presence of cycloheximide represents gluconeogenic activity with associated polysaccharide synthesis.     

Excess thymidine accelerates nascent replicon maturation without affecting the rate of DNA synthesis

The effects of different concentrations of exogenously supplied dThd on DNA replication were investigated in seedlings of Pisum sativum. Nascent DNA was labeled with either [³H]dThd or [³H]dAdo in the presence of 1·10^?6, 1·10^?5 or 1·10^?4 M unlabeled dThd. The rate of DNA synthesis was determined by measuring the kinetics of radioactivity incorporation into trichloroacetic acid-precipitable material and the size of the nascent molecules was investigated using alkaline sucrose gradients. The results obtained showed that high concentrations of exogenously supplied dThd accelerated the joining of completed nascent replicons without affecting the rate of DNA synthesis. This observation strengthens the hypothesis that the dTTP pool size is one of the factors controlling the timing of nascent replicon maturation.     

Ribonucleotides Linked to DNA of Herpes Simplex Virus Type 1

Cells of a continuous cell line derived from rabbit embryo fibroblasts were infected with herpes simplex type 1 virus (HSV-1) and maintained in the presence of either [5-(3)H]uridine or [methyl-(3)H]thymidine or (32)PO(4) (3-). Nucleocapsids were isolated from the cytoplasmic fraction, partially purified, and treated with DNase and RNase. From the pelleted nucleocapsids, DNA was extracted and purified by centrifugation in sucrose and cesium sulfate gradients. The acid-precipitable radioactivity of [5-(3)H]uridine-labeled DNA was partially susceptible to pancreatic RNase and alkaline treatment; the susceptibility to the enzyme decreased with increasing salt concentration. No drop of activity of DNA labeled with [(3)H]thymidine was observed either after RNase or alkali treatment. Base composition analysis of [5-(3)H]uridine-labeled DNA showed that the radioactivity was recovered as uracil and cytosine. In the cesium sulfate gradient, the purified [5-(3)H]uridine-labeled DNA banded at the same position as the (32)P-labeled DNA. The present data tend to suggest that ribonucleotide sequences are present in HSV DNA, that they are covalently attached to the viral DNA, and that they can form double-stranded structures.     

A gradient of diffusible substance in a monolayer of cultured cells

Summary Monolayers of hypoxanthine phosphoribosyl transferase-deficient and their corresponding wildtype cells have been placed adjacent to each other with a newly described method. Autoradiographs from such preparations after incubation with³H-hypoxanthine allow the direct visualization of gradients of incorporated radioactivity at the border between the two cell types. The gradients can be described by an exponential function, and the amount of radioactivity incorporated descreases to less than 1% at a distance of 1 mm from the wild-type cells. A possible mechanism to convert exponential gradients to linear ones over a certain concentration range is discussed.     

Utilization of d-Ribose by Veillonella

Three strains of Veillonella, representing two species, were unable to utilize carbohydrates as energy sources for growth. Ribose, however, was utilized biosynthetically by all three strains. Exponentially growing cultures removed (14)C-ribose from the growth medium and retained radioactivity throughout the growth cycle. The kinetics of removal of ribose from the growth medium was found to depend on the initial ribose concentration. Uptake by resting cells was found to require active metabolism, was greatly stimulated by the presence of an energy source, and was insensitive to the presence of other pentoses. Fractionation of cells showed that the ribose was used for synthesis of acid-precipitable material, with as much as 92% of the radioactivity being found in the nucleic acid fraction. The intracellular distribution of ribose radioactivity did not change during growth after uptake was completed.     

Properties of a D-glutamic acid-requiring mutant of Escherichia coli

Some properties of a d-glutamic acid auxotroph of Escherichia coli B were studied. The mutant cells lysed in the absence of d-glutamic acid. Murein synthesis was impaired, accompanied by accumulation of uridine-5'-diphosphate-N-acetyl-muramyl-l-alanine (UDP-MurNac-l-Ala), as was shown by incubation of the mutant cells in a cell wall medium containing l-[(14)C]alanine. After incubation of the parental strain in a cell wall medium containing l-[(14)C]glutamic acid, the acid-precipitable radioactivity was lysozyme degradable to a large extent. Radioactive UDP-MurNac-pentapeptide was isolated from the l-[(14)C]glutamic acid-labeled parental cells. After hydrolysis, the label was exclusively present in glutamic acid, the majority of which had the stereo-isomeric d-configuration. Compared to the parent the mutant incorporated less l-[(14)C]glutamic acid from the wall medium into acid-precipitable material. Lysozyme degraded a smaller percentage of the acid-precipitable material of the mutant than of that of the parent. No radioactive uridine nucleotide precursors could be isolated from the mutant under these conditions. Attempts to identify the enzymatic defect in this mutant were not successful. The activity of UDP-MurNac-l-Ala:d-glutamic acid ligase (ADP; EC 6.3.2.9) (d-glutamic acid adding enzyme) is not affected by the mutation. Possible pathways for d-glutamic acid biosynthesis in E. coli B are discussed.     

Failure of lactoperoxidase to iodinate specifically the plasma membrane of cucurbita tissue segments

An attempt has been made to use lactoperoxidase-catalyzed iodination of excised Cucurbita hypocotyl hooks to monitor the distribution of plasma membrane fragments relative to that of phytochrome in particulate fractions from this tissue. Upon fractionation, the iodinated tissue yields a 20,000g pellet which contains 58% of the trichloroacetic acid-precipitable ¹²⁵I at a specific radioactivity 12 times that of the proteins in the supernatant. On sucrose gradients, the labeled fraction has a mean isopycnic density of 1.15 g · cm⁻³. The distribution profile is distinct from that of the total particulate protein and does not coincide with either mitochondrial or endoplasmic reticulum markers. These observations satisfy operational criteria commonly accepted in other systems as indices of selective labeling of the cell surface. The sucrose gradient profiles of the phytochrome and ¹²⁵I in the 20,000g pellets are noncoincident. In the absence of more direct evidence, this is readily interpreted to indicate a lack of association of the pigment with the plasma membrane.     

Uptake and intracellular fate of gelonin,a ribosome-inactivating protein,in rat liver

Gelonin, a type 1 ribosome-inactivating protein, has been used as toxin conjugate for several therapeutic purposes. We have investigated the endocytosis of gelonin by rat liver in vivo. Subcellular distribution of [125I]gelonin was established after differential and isopycnic centrifugation. Fractions were analyzed for acid-soluble and acid-precipitable radioactivity. Results show that gelonin is rapidly cleared from the blood and within 15min reaches a peak (25% of total injected) in the liver. With time, radioactivity associated with the liver markedly decreases. Two important observations are made: (a) Radioactivity associated with all fractions, at any time point, is greater than 80% acid precipitable. (b) Even at 5min, a significant amount of intact gelonin is present in the cytosolic fraction. Our work suggests that, though gelonin is rapidly cleared from the blood, there are still intact molecules that have entered the cytosol where they could exert their toxic effect.     

Newly synthesized secretory proteins from pig pancreas are not released from a homogeneous granule compartment

The pancreatic secretion of anesthetized pigs was collected by cannulation after pulse labeling with [³H]leucine. Collection at 5 min intervals started immediately post-pulse labeling up to 85 min. The volume, the protein content and the trichloroacetic acid-precipitable radioactivity of the juice were measued. The specific radioactivity of the secertory proteins was compared to that of a zymogen granule fraction isolated from the same animal. The latter was very much higher. Caerulein stimulation for 5 min at 80 min post-pulse caused a sharp drop in the specific activity of secretory proteins in the juice, to a level lower than that of the zymogen granule content. These data support the concept of more than one pool of secretory proteins in the pancreas and are incompatible with the concept that secretory proteins derive from an homogeneous granule compartment in a functionally homogeneous population of cells. To explain our results the hypothesis of a second intracellular route for the secretory proteins is proposed.     

Synthesis of nitrogenase by isolated heterocysts

Heterocysts isolated from Anabaena variabilis incorporate [¹⁴C]leucine and [³⁵S]methionine into trichloroacetic acid-precipitable material in the light. Analysis by polyacrylamide gel electrophoresis shows that the radioactivity is present in polypeptides of discrete sizes. However, the relative proportions of different proteins synthesized by isolated heterocysts differ from the relative proportions of those proteins incorporated by the heterocysts in intact filaments. The two components of nitrogenase are among the proteins synthesized by the isolated heterocysts.     

Processing of calcitonin and epidermal growth factor after binding to receptors in human breast cancer cells (T 47D).

125I-labelled calcitonin and 125I-labelled epidermal growth factor (EGF) bound to T 47D breast cancer cells at 37 degrees C in a manner that became increasingly resistant to removal by acid pH. Bound 125I-labelled EGF became resistant to acid removal more rapidly than did bound 125I-labelled calcitonin. The shift from acid accessibility to acid inaccessibility was energy-dependent since it was not seen at 4 degrees C and was inhibited in the presence of cell metabolic inhibitors. Radioactivity removed by acid represented intact hormone as assessed by trichloroacetic acid precipitation, whereas radioactivity released spontaneously by the cells was trichloroacetic acid-soluble. Inclusion of 10 mM-NH4Cl in the incubation medium resulted in an accumulation of cell-associated radioactivity without affecting the shift to acid inaccessibility. The accumulated radioactivity was relatively more trichloroacetic acid-precipitable in comparison with that associated with control cells. These data are consistent with internalization of receptor-bound EGF and a similar though slower mechanism of processing for receptor-bound calcitonin. The predominant route of hormone release from cells seems to occur via intracellular degradation rather than dissociation from cell-surface receptors.     

An assay for the endonucleolytic clevage of RNA to large oligonucleotides

A rapid assay for endonuclease activity which cleaves high-molecular-weight RNA to acid-precipitable fragments has been developed. RNA is covalently coupled to beaded agarose under conditions that produce relatively few coupling sites. The immobilized RNA can be used qualitatively or semiquantitatively in an assay for endonuclease activity by determining the release of acid-precipitable RNA from the complex. This assay is compared to one employing separation of degraded RNA by gel electrophoresis.     

Renal uptake and degradation of trapped-label insulin

In order to study the kinetics of insulin degradation in the kidneys and liver, insulin was labelled by a trapped-label procedure and injected into rats. In contrast to conventional 125I-insulin, the trapped-label preparation allows quantitative measurements of the extent of degradation in vivo because the final degradation products do not leave the cells. One hour after injection, the amount of radioactivity in the kidneys from a trace dose of trapped-label insulin was 10 times higher that from conventionally labelled insulin; over 80% of the increase was due to low molecular weight degradation products which were retained in the kidneys. The amount of acid-precipitable radioactivity in the blood was the same for both labelled preparations, indicating that their rates of clearance were similar. In the kidney, we detected no degradation products of molecular weight intermediate between intact insulin and the end products of proteolysis. After 2 h, 33% of the injected dose remained in the kidneys and only 13% in the liver. Over 80% of the renal radioactivity was sedimentable in an isotonic density gradient, indicating that intact insulin, as well as degradation products in the cells, were enclosed within membrane-bound vesicles.     

DNA-Dependent RNA Polymerase Activity Associated with Subviral Particles of Polyhedral Cytoplasmic Dexoyribovirus

Polyhedral cytoplasmic deoxyribovirus virions contain a DNA-dependent RNA polymerase which catalyzes the incorporation of ribonucleotides into an acid-precipitable product. Treatment of virions with sodium deoxycholate and dithiothreitol resulted in the formation of subviral particles which could be separated from virions by rate zonal centrifugation in sucrose gradients. Subviral particles were RNA polymerase-positive and more active per unit mass of protein than virions. In vitro enzyme activity associated with subviral particles required addition of ribonucleotides, Mg(2+), and exogenous denatured DNA template. Optimal enzyme activity occurred over a broad pH (7.2 to 8.8) and Mg(2+) concentration (2 to 10 mumol) range. The specific activity of the RNA polymerase was maximal at 37 C. Addition of DNase or actinomycin D to the reaction mixture reduced the incorporation of [(3)H]UMP into an acid-precipitable product. The product of the reaction was sensitive to degradation by RNase but not to DNase or Pronase. These data suggest that the enzyme copies DNA into RNA.     

Biosynthesis of beta nerve growth factor in mouse submaxillary glands

The biosynthesis of beta nerve growth factor (betaNGF) was studied in isolated mouse submaxillary glands incubated with L-[35S]cystine. Sodium dodecyl sulfate gels of anti-betaNGF immunoprecipitates from labeled gland homogenates showed a single major peak of radioactivity, which comigrated with purified betaNGF. This species was nearly completely precipitated by the addition of equivalent amounts of anti-betaNGF, but was absent from immunoprecipitates obtained by the addition of ferritin plus anti-ferritin. The cystine-containing tryptic peptides of the labeled species appeared identical with those of purified betaNGF. In submaxillary glands from adult male mice, labeling of betaNGF represented approximately 0.2% of the trichloroacetic acid-precipitable radioactivity. Castration reduced this value to one-third, while testosterone treatment of castrated animals restored the relative betaNGF synthesis to normal or more. No betaNGF synthesis could be detected in glands from female animals. Several tissues were examined for their ability to synthesize betaNGF in culture. Only submaxillary gland incorporated detectable amounts of radioactivity into betaNGF. Labeling of betaNGF could also be obtained by direct injection of isotope into the submaxillary gland in vivo. The results are discussed in terms of the integration of betaNGF synthesis into neuronal development and maintenance.     

A semiautomated system for the production and analysis of sucrose density gradients

A semiautomated system permitting considerable accuracy, speed and reproducibility in the making and fractionation of sucrose density gradients is described. The system consists of a modified Beckman gradient forming device which makes six gradients simultaneously and delivers them into six 12.5 ml polyallomer centrifuge tubes in such a manner that new material is continuously added to the meniscus of the gradient. The gradients are fractionated three at a time and up to 100 fractions per gradient can be collected automatically directly into scintillation vials with a choice of drop counting or time mode with rinse and automatic addition of scintillation fluid to each vial. The system can process up to six gradients per hour but centrifugation time is usually the limiting factor. With neutral sucrose gradients, sharp, reproducible, monodisperse peaks containing up to 100% of the gradient radioactivity are usually obtained but a smaller monodisperse peak containing as little as 3.5% of the gradient radioactivity can be detected under conditions where some pairs of molecules might tangle or dimerize. The resolution and reproducibility of this system when used with neutral sucrose gradients is at least the equal if not superior to that commonly claimed for alkaline sucrose gradients.