Virus-Receptor Interaction in an Adenovirus System

HeLa or KB cells each contain around 10⁴ receptor sites for adenovirus type 2. These are inactivated by treatment of live cells with subtilisin. The receptor activity of the enzyme-treated cells is regained after 4 to 8 hr of incubation in complete medium. A technique that utilizes the difference in buoyant density between free virus and virus-receptor complex was developed to demonstrate receptor activity. Cellular fractionation revealed that receptors were confined mainly to the plasma membrane fraction and that negligible receptor activity could be demonstrated in enzyme-treated cells. Subtilisin probably did not penetrate the cell membrane; thus, the receptors are limited to the cell surface. Purified fiber of the virion completely prevents attachment of adenovirus types 2 and 5 to receptor sites at a ratio of 10⁵ protein molecules per cell. Adsorption studies indicate that 10⁵ to 10⁶ receptor sites are available for the structural protein. The fiber does not affect attachment of poliovirus type 1.     

BIOCHEMTCAL CHANGES IN THE BRAIN OF EXPERIMENTAL ANIMALS IN RESPONSE TO ELEVATED PLASMA HOMOCYSTINE AND METHIONINE

The effects of high plasma concentrations of homocystine and methionine on the free amino acids of brain have been examined. Incorporation of the label from [³⁵S]methionine into the free amino acid pools of rabbit brain was enhanced in response to high plasma homocystine or high plasma homocystine and mcthionine. Under comparable conditions a marked decrease in the incorporation of the label from [¹⁴C]glycine into the free pool was observed. The corresponding incorporation of ³⁵S and ¹⁴C into brain proteins parallelled the results obtained with incorporation into the free pools of amino acids. Amino acid analyses of the free amino acid pools of rabbit brain revealed a general decrease in the concentration of amino acids in response to high plasma homocystine or high plasma homocystine and methionine. Inhibition of protein synthesis which occurs under the above experimental conditions is a general phenomenon. myelin and other brain fractions being equally affected. The decrease in concentration of brain amino acids also results in a diminution in concentration of the neurotransmitters GABA, dopamine and noradrenaline. The possible relationship of the observed changes to homocystinuria is discussed.     

Evidence for a 5' leads to 3' direction of hydrolysis by a 5' mononucleotide-producing exoribonuclease from Saccharomyces cerevisiae.

[³H]rRNA labeled at the 5′ terminus with ³²P and [³H]rRNA labeled at the 3′ end with [¹⁴C] (pA)_n have been degraded at 0° with a highly purified exoribonuclease from $\text{Saccharomyces cerevisiae}$. The results show that with the [³²P, ³H] substrate, the ³²P label is rendered acid-soluble at a much faster rate than the ³H label. Both acid-soluble labels are found in 5′ mononucleotide. With the [¹⁴C, ³H]rRNA, the ³H label is hydrolyzed at a faster rate than the ¹⁴C label. The exoribonuclease hydrolyzes in the 5′ → 3′ direction.     

MEASUREMENT OF RATES OF PHAGOCYTOSIS : The Use of Cellular Monolayers

A method has been developed for measuring the rate of phagocytosis rather than the quantity of particles ingested per cell when the process is virtually complete. The method, which is simpler and more rapid than those described previously, utilizes cellular monolayers, radioactive particles, and short incubation times. Under the conditions described, the rate of uptake of particles by either guinea-pig peritoneal or human blood leukocytes was proportional to both cell concentration and the time of incubation, and was independent of changes in the concentration of particles during the measurement. The particles were retained by the cells for at least 90 min. The most suitable particles so far used have been ³²P-labeled Salmonella typhimurium, and acetyl-¹⁴C- or methyl-¹⁴C-labeled starch particles. The oxidation of ¹⁴C-labeled glucose has been studied under the same conditions that were used for the assays of phagocytosis: the greatest increase in formation of ¹⁴CO₂ from glucose-1-¹⁴C occurred a few minutes after the most rapid period of phagocytosis.     

Affinity labeling of specific regions of 23 S RNA by reaction of N-bromoacetyl-phenylalanyl-transfer RNA with Escherichia coli ribosomes

Reaction of the affinity-labeling reagent N-bromoacetyl-[¹⁴C]phenylalanyl-tRNA with Escherichia coli ribosomes results in covalent labeling of 23 S ribosomal RNA in addition to the previously reported labeling of ribosomal proteins. The reaction with the 23 S RNA is absolutely dependent on the presence of messenger RNA. Covalent attachment of the affinity label to 23 S RNA was demonstrated by its integrity in strongly dissociating solvents, and the conversion of the labeled material to small oligonucleotides by ribonuclease treatment. After digestion of labeled 23 S RNA with T₁ ribonuclease, the radioactivity is found mainly in two oligonucleotide fragments. These results support models in which both ribosomal RNA and ribosomal protein contribute to the structure of the region of the ribosome surrounding the peptidyl transferase center.     

The effect of sterols on the metabolism of Pythium species

Summary The growth of several Pythium species is increased between 65 and 100% if cholesterol is added to the growth medium. The optimum concentration is 15 mcg per ml. Mycelium of Pythium ultimum, in which cholesterol is present, incorporates glucose-U-¹⁴C and releases ¹⁴CO₂ at a faster rate than the corresponding sterol free mycelium. In sterol containing cells, more ¹⁴CO₂ is produced from a given amount of absorbed glucose-U-¹⁴C than in sterol free cells, there is thus in sterol containing hyphae a higher level of energy production. This condition can account for the increase in growth due to cholesterol. Only if sterols are present in the cellular membranes of Pythium species is the optimum synthetic capacity reached.     

Studies on Cassava, Manihot utilissima. II. Biosynthesis of Asparagine-14C from 14C-labelled Hydrogen Cyanide and its Relations with Cyanogenesis

Seeds and seedlings of Manihot utilissima were analysed for cyanogenic glycosides und free amino acids, with special reference to valine and isoleucine which serve as precursors of the aglycone moieties of linamarin and lotaustralin. Seeds contained traces of valine and isoleucine but no glycosides, whereas seedlings contained high concentrations of these amino acids and glycosides. Illumination of seedlings led to a steep increase in the concentration of glycosides followed by a decrease without excretion of detectable HCN. Seeds accumulated asparagine, while seedlings accumulated both asparagine and glutamine in the storage and transport of nitrogen. Seedlings incorporated 13.2 per cent of label from valine-¹⁴C(U) and 2.4 per cent of label from isoleucine-¹⁴C(U)into linamarin and lotaustralin, respectively. In both cases, appreciable amounts of label were also incorporated into asparagine. 49 per cent of label from H¹⁴CN was incorporated inio asparagine in which ca. 98 per cent of total radioactivity was located in the amide-carbon atom. The different patterns of labelling which occurred during the assimilation of H¹⁴CN and ¹⁴CO₂ showed that cyanide metabolism did not proceed via CO₂, and that M. utilissima contains an efficient enzyme-system which catalyses the conversion on high concentrations of HCN into asparagine, which subsequently enters different metabolic pools involved with respiration, protein and carbohydrate syntheses. Cyanogenesis in M. utilissima appears lo be directly influenced by available pools of valine and isoleucine, and the metabolism of HCN released from linamarin and lotaustralin by the action of linamarase may be directly related to respiratory and synthetic processes by way of the incorporation of HCN as a unit into asparagine.     

Differential regulation of the N-methyl transferases responsible for phosphatidylcholine synthesis in Saccharomyces cerevisiae

The enzymes catalyzing the conversion of phosphatidylethanolamine to phosphatidylcholine were assayed by measuring the incorporation of label from [¹⁴C-CH₃]-S-adenosyl-methionine into the endogenous phospholipids of particulate, cell-free preparations from S. cerevisiae grown in the presence of N-methylethanolamine, N,N-dimethylethanolamine, or choline. The results indicate that each base in the growth medium results in reduced levels of all the N-methyltransferase activity involved in the formation of the phosphatidyl ester of the given base. By following the conversion of exogenous [³²P]-phosphatidyldimethylethanolamine to [³²P]-phosphatidylcholine it has been shown that the activity of the third methyl transfer is 90% lower in particles prepared from choline grown cells than in particles prepared from cells grown without choline. The results suggest that there are at least two enzymes involved in the conversion of phosphatidylethanolamine to phosphatidylcholine and that their levels can be regulated individually.Supplementing the growth medium with any of the three methylated aminoethanols results in markedly increased cellular levels of their corresponding phosphatidyl esters and decreased levels of the precursor phosphatidyl esters. The fatty acid composition of phosphatidylcholine also changes when the medium is supplemented with choline suggesting that the proportions of the molecular species of this phosphatide depends on whether synthesis is via methylation of phosphatidylethanolamino or from the supplemented aminoethanol.     

Labeling of adenovirus particles with PARACEST agents

Recombinant adenovirus type 5 particles (AdCMVLuc) were labeled with two different bifunctional ligands capable of forming stable complexes with paramagnetic lanthanide ions. The number of covalently attached ligands varied between 630 and 1960 per adenovirus particle depending upon the chemical reactivity of the bifunctional ligand (NHS ester versus isothiocyanide), the amount of excess ligand added, and the reaction time. The bioactivity of each labeled adenovirus derivative, as measured by the ability of the virus to infect cells and express luciferase, was shown to be highly dependent upon the number of covalently attached ligands. This indicates that certain amino groups, likely on the surface of the adenovirus fiber protein where cell binding is known to occur, are critical for viral attachment and infection. Addition of (177)Lu3+ to chemically modified versus control viruses demonstrated a significant amount of nonspecific binding of (177)Lu3+ to the virus particles that could not be sequestered by addition of excess DTPA. Thus, it became necessary to implement a prelabeling strategy for conjugation of preformed lanthanide ligand chelates to adenovirus particles. Using preformed Tm3+- L2, a large number of chelates having chemical exchange saturation transfer (CEST) properties were attached to the surface residues of AdCMVLuc without nonspecific binding of metal ions elsewhere on the virus particle. The potential of such conjugates to act as PARACEST imaging agents was tested using an on-resonance WALTZ sequence for CEST activation. A 12% decrease in bulk water signal intensity was observed relative to controls. This demonstrates that viral particles labeled with PARACEST-type imaging agents can potentially serve as targeted agents for molecular imaging.     

The photobinding of 5,7-dimethoxycoumarin to adenovirus type-2 DNA. A method for in vitro mutagenesis

The photobinding of 5,7-dimethoxycoumarin to isolated adenovirus-type 2 DNA has been investigated with respect to the influence of the ionic environment, and varying molar ratios of DNA_(p): 5,7-dimethoxycoumarin. In particular, the ultraviolet radiation-induced covalent addition of 5,7-dimethoxycoumarin to adenovirus DNA was increased by reducing the concentration of Na⁺. The maximum photobinding of 5,7-[³H]dimethoxycoumarin to adenovirus DNA under the given ionic condition was one 5,7-dimethoxycoumarin per 101 nucleotides. Moreover, restriction enzyme analysis of the 5,7-dimethoxycoumarin-DNA photoadduct versus unmodified viral DNA, suggested that the sequence d(A-T) is the preferential site for intercalation and subsequent photobinding of 5,7-dimethoxycoumarin. This susceptibility of d(A-T) sequences to 5,7-dimethoxycoumarin interaction has a corresponding influence on the survival of adenovirus because of the A-T-rich sequences that occur in some of the early gene regions of the adenovirus genome. Specifically, 5,7-dimethoxycoumarin per 800 nucleotides in adenovirus DNA reduced the surviving fraction of adenovirus to a value of 0.1 after DNA infectivity (transfection) into human 293 cells. Results suggest that 5,7-dimethoxycoumarin may be used for generating a limited ‘library’ of mutations in each of the five early gene regions of the adenovirus genome.     

Distribution of [14C] Dichlorophenoxyacetic Acid in Cultured Zygotic Embryos of Zea mays L.

The uptake of 2,4-dichlorophenoxyacetic acid (2,4-D), necessary for the in vitro induction of callus formation and somatic embryogenesis in cultured immature maize embryos, was quantified after culture on nutrient medium with [¹⁴C]2,4-D. The identity of the ¹⁴C label in the embryos was determined by high performance liquid chromatography (HPLC), and its distribution within embryos was visualized on sections of plastic embedded material. Quantification of the ¹⁴C label after a pulse label of 16 h showed a hundredfold accumulation of 2,4-D in the embryos with respect to the initial medium concentration. During tissue processing for in situ detection of ¹⁴C, however, up to 70% of the label disappeared because of the embedding process. The best structural preservation was obtained after ethanol-mediated infiltration of Technovit 7100. Water-mediated infiltration of Technovit 7100 gave the highest retention of ¹⁴C. HPLC analysis showed that more than 95% of the residual ¹⁴C label found in embryos was still 2,4-D. Autoradiography showed that the embryogenic inbred line A188 contained ¹⁴C label in distinct regions of the scutellum, coleoptile, and suspensor. The nonembryogenic inbred line A632 contained more label after 16 h of culture in a different distribution compared with A188. Subculture of the embryos for 24 and 72 h and histologic analysis showed that cell proliferation and callus formation were restricted to specific regions of the embryo in both inbred lines. The pattern of 2,4-D distribution did not codistribute with regions of proliferation, indicating that 2,4-D is not the only trigger for proliferation. Received August 18, 1997; accepted February 12, 1998     

Long Distance Transport in Macrocystis integrifolia: II. Tracer Experiments with C and P

Discs from mature regions of Macrocystis blades picked up significantly more [³²P]phosphate from the ambient medium than similar discs from young meristematic regions, and this uptake was higher in light than in darkness. Double-labeling experiments with NaH¹⁴CO₃ and [³²P]phosphate, using intact fronds as well as cut frond segments, indicated that ³²P was translocated from mature blades to sink regions at velocities of 25 to 45 centimeters per hour, velocities comparable to ¹⁴C translocation velocity in the same material. There was a slight delay in transport of ³²P which may be due to a delay in loading or to a high metabolism of ³²P in the transporting channels. Histoautoradiography of stipe segments in the translocation pathway indicated that transport of label occurred in the peripheral parts of medulla. An analysis of ³²P-labeled compounds in the fed blade and in the sieve tube sap, collected from basal cut ends of stipes, indicated major differences in labeling patterns. In the blade, a high proportion of ³²P was recovered as inorganic phosphate and relatively small amounts were found in hexose mono- and diphosphates, UDPG and ATP. In the sieve tube sap, however, only a small amount of ³²P was present as inorganic phosphate, a large proportion was found in hexose mono- and diphosphates, and appreciable amounts were present in ATP and UDPG.     

The use of 14C-labeled bacteria as a tracer of ingestion and metabolism of bacterial biomass by microbial grazers

A method for radiolabeling marine bacteria with d-[U-¹⁴C] glucose and a radiotracer method for measuring ingestion and metabolism of bacterial biomass by ciliated protozoa and other microzooplankton are presented. Problems associated with using live bacterial tracers, i.e., label retention, label recycling, tracer cell size and morphology, and intracellular distribution of label are evaluated.Bacterioplankton assemblages collected from field samples incorporated and retained label as efficiently as coastal isolates which were selected for glucose incorporation. Under grazing experimental conditions, labeled bacteria retained a high proportion of the label (hourly net loss = 1.2%). Bacterial recycling of released dissolved organic ¹⁴C (DO¹⁴C) was 0–2.2% of total ¹⁴C per h. Addition of labeled assemblages to nearshore water samples did not detectably alter mean cell size or size frequency distribution of the attendant bacterioplankton assemblages.In grazing experiments with cultured ciliates (Euplotes sp. and Uronema sp.), radioassay parameters varied as direct functions of predator and prey concentrations. Microautoradiographic analysis corroborated that ¹⁴C incorporation measured in the radioassay by filtration techniques primarily represented ingested bacterial biomass and that problems associated with attached and adsorbed labeled bacteria were minimized. Grazing experiments conducted with bacteria labeled with [U-¹⁴C]glucose yielded ingestion rates comparable to bacteria labeled with [U-¹⁴C]thymidine and additionally provided respiration and exudation rates.     

Amino acid metabolism in the pedicel-placenta-chalazal region of the developing maize kernel

The pedicel-placenta-chalazal (PPCh) region is a heterogeneous maternal tissue located at the base of the endosperm. Its active role in the transport and metabolism of nitrogen compounds supplied to the endosperm has been shown by incorporation of labelled compounds, as well as by analysis of appropriate enzyme activities and the free amino acid pool. Within 30 min incubation, 60–70% of the label given in [¹⁴C]aspartate or [¹⁴C]glutamate was recovered in the organic acid fraction of the PPCh. Although the dry matter and protein content of the PPCh region was practically unchanged between days 15 and 30 after pollination, glutamate transaminase, alanine transaminase, NADP-malic enzyme and NAD-malate dehydrogenase were increased substantially. The PPCh region contains glutamine at a significantly higher concentration and alanine at a lower concentration than the endosperm. In contrast to glutamate synthase, the activity of glutamine synthetase per endosperm and per mg protein is significantly higher in the PPCh region. This implies involvement of these tissues in the transport of nitrogen compounds, mainly in the form of glutamine, as well as possible compartmentation of the glutamine-glutamate cycle in the developing maize kernel.     

Biospecific nanoparticles for multiplex phosphorescence analysis (PHOSPHAN)

We have developed a technology for the production of polymeric nanoparticles containing the incorporated phosphorescent label (europium ions–naphthoyltrifluoroacetone complexes) and streptavidin that is covalently bound on the surface. The aggregation-stable biospecific nanoparticles (40–60 nm in diameter) include up to 2000 molecular tags/particle and retain biological activity and stable phosphorescence for at least 20 months. They can be used in phosphorescence analysis (PHOSPHANTM)-based biochip technology as an effective detector system to record phosphorescence from microzones (microarrays) printed on the well bottoms of standard polystyrene microplates. The creation of a dense monolayer on the surface of a microzone requires up to 10⁸ particles/microarray, or 10⁹ particles/mm² of area; this is in good agreement with theoretical estimates. The detection limit is as low as 300–400 phosphorescent nanoparticles per a microzone with an area of ~0.1 mm². It has been demonstrated in the model of thyroid stimulating hormone (TSH) detection in filter paper dried blood that the newly developed detector system is five times more sensitive than the conventional methods of multiplex PHOSPHAN (with Pt-coproporphyrin phosphorescent label) and lanthanide immune fluoroassay (with fluorescent Eu³⁺ chelate complexes registered in the enhancement solution). The sensitivity of phosphorescent nanoparticle-based detector system is as low as 6.8 × 10⁵ molecules/1.5 μL sample, which corresponds to a TSH concentration of 1.5 × 10^–14 M.     

C radiolabeling of proteins to monitor biodistribution of ingested proteins

The economical preparation of microgram quantities of ¹⁴C-labeled proteins by in vacuo methylation with methyl iodide is described. The ¹⁴C radiolabeling was achieved by the covalent attachment of [¹⁴C]methyl groups onto amino and imidazole groups by reaction in vacuo with [¹⁴C]methyl iodide. The method was tested by investigating the biodistribution of ¹⁴C in rats that were fed ¹⁴C-labeled human soluble cluster of differentiation 14 (CD14) protein, a receptor for bacterial lipopolysaccharide. Two other control proteins, bovine serum albumin (BSA) and casein, were also labeled with ¹⁴C and used for comparative analysis to determine the following: (i) the efficacy and cost efficiency of the in vacuo radiolabeling procedure and (ii) the extent of incorporation of the ¹⁴C label into the organs of orogastrically fed 10-day-old Sprague–Dawley rats. [¹⁴C]BSA, [¹⁴C]casein, and [¹⁴C]CD14 were individually prepared with specific radioactivities of 34,400, 18,800, and 163,000 disintegrations per minute (dpm)/μg, respectively. It was found that the accumulation of ¹⁴C label in the organs of [¹⁴C]CD14-fed rats, most notably the persistence of ¹⁴C in the stomach 480 min postgavage, was temporally and spatially distinct from [¹⁴C]BSA and [¹⁴C]casein-fed rats.     

Biosynthesis of glycosaminoglycans in the developing retina

The glycosaminoglycans of neural retinas from 5-, 7-, 10-, and 14-day chick embryos were labeled in culture with [³H]glucosamine and ³⁵SO₄, extracted, and isolated by gel filtration. The incorporation of label per retina into glycosaminoglycans increased with embryonic age, but that per cell and per unit weight of uronic acid decreased. Specific enzyme methods coupled with gel filtration and paper chromatography demonstrated that [³H]glucosamine incorporation into chondroitin sulfate increased between 5 and 14 days from 7 to 34% of the total incorporation into glycosaminoglycans. During this period, incorporation into chondroitin-4-sulfate increased relative to that into chondroitin-6-sulfate. Between 5 and 10 days, incorporation into heparan sulfate showed a relative decline from 89 to 61%. Incorporation into hyaluronic acid always represented less than 2% of the total. A twofold greater increase in galactosamine concentration than in glucosamine concentration in the glycosaminoglycan fraction between 7 and 14 days supports the conclusion that chondroitin sulfate was the most rapidly accumulating glycosaminoglycan. ECTEOLA-cellulose chromatography revealed a heterogeneity in the size and/or net charge of chondroitin sulfate and heparan sulfate. We conclude that incorporation of exogenous precursors into glycosaminoglycans in the chick retina decreases relative to cell number as differentiation progresses from a period of high mitotic activity to one of tissue specialization, and that it is accompanied by a net accumulation of glycosaminoglycan and a change in the pattern of its synthesis.     

Arginine Metabolism in Developing Soybean Cotyledons : II. Biosynthesis

Tracer kinetic experiments were performed using [ureido-¹⁴C] citrulline, [1-¹⁴C]ornithine, and isotope trapping techniques to determine if arginine is synthesized via the urea cycle in developing cotyledons of Glycine max (L.) Merrill. Excised cotyledons were injected with the ¹⁴C-solution and incubated in sealed vials containing a CO₂ trap. The free and protein amino acids were analyzed using high performance liquid chromatography and arginine-specific enzyme-linked assays. In the ¹⁴C-citrulline feeding experiment argininosuccinate was the most highly labeled compound after 5 minutes and it was the first compound to lose ¹⁴C later in the time course. Carbon-14 was also recovered in free arginine, protein arginine, and CO₂ up to 4 hours after introduction of label. All of the ¹⁴C in free and protein arginine could be accounted for in the C-6 position. Metabolism of ¹⁴C-ornithine resulted in ¹⁴C-incorporation into citrulline and free and protein arginine and the evolution of ¹⁴CO₂. Citrulline was the most highly labeled compound after 15 minutes and was the first compound to reach a steady state level of ¹⁴C. With the addition of 800 nanomoles unlabeled citrulline to the ¹⁴C-ornithine feeding solution citrulline was the only compound labeled after 5 minutes and the steady state level of ¹⁴C-citrulline increased 12-fold. The appearance of ¹⁴C in free arginine and protein arginine was also delayed. In both ¹⁴C-ornithine feedings all of the ¹⁴C in free and protein arginine could be accounted for in the C-1 position. Together, the data support the reaction sequence: ornithine → citrulline → argininosuccinate → arginine → protein arginine.     

Immobilization of lipase into mesoporous silica particles by physical adsorption

Mesoporous silica particles for immobilization of lipase from Candida rugosa were prepared by precipitation and aggregation of primary particles from highly basic sodium silicate solution but without addition of templates. The average pore size of the material was 15.8 nm, which allowed enzyme adsorption inside the pores and high enzyme loading. Specific surface area of the material was found to be 359 m²g^?1. A loading of 100 mglipasegdrysilica^?1 was obtained at initial enzyme concentration of 1.8 mgmL^?1 by physical adsorption. The FTIR spectrum showed the structural conformation of lipase to be retained after adsorption into the mesoporous silica support. Although the efficiency of the mesoporous biocatalyst was shown to be lower than that of the free enzyme, the immobilized enzyme showed enhanced thermal stability and could be desorbed with Triton X-100, indicating the hydrophobic nature of the adsorption.     

Regulation aspects of roquefortine production by free and Ca-alginate immobilized mycelia of Penicillium roqueforti

Summary Roquefortine synthesis with free and Ca-alginate immobilized Penicillium roqueforti cells was investigated under different culture conditions. Decreasing Ca-alginate concentration was related to increasing roquefortine production; free cells gave the best results. Formation of roquefortine was three times higher with mannitol and succinate than with sucrose as the carbon source; phosphate inhibited its biosynthesis in free cells by 23% to 32%. Relationships between cell density, ¹⁴C-tryptophan content of cells and roquefortine synthesis were shown. The special morphology of immobilized mycelia was demonstrated.