Gelatin Pellets Research Articles

Comparison of Gelatin Flavors for Oral Dosing of C57BL/6J and FVB/N Mice.

Precise oral dosing in rodents is usually achieved by intragastric gavage. If performed incorrectly due to technical difficulties, inexperience, or animal resistance, oral gavage may have animal welfare implications such as esophageal and gastric rupture and aspiration. The stress that is induced by this procedure can also lead to confounding results. In several animal models, drug vehicles must be sugar-free, deliver drugs in a specific formulation, and sometimes supply water. Gelatin has all of these properties. The current study aimed to evaluate the use of gelatin vehicles with different sensory features as an alternative to oral gavage. We investigated the time taken by 2 different inbred mouse strains, FVB/N and C57BL/6J, to ingest sugar-free gelatin pellets of varying flavors. Results showed that FVB/N mice took more time to eat the unflavored, strawberry and diet-flavored gelatin pellets than did C57BL/6J mice. Both strains showed low preference for lemon flavor, with the same ingestion times after the second day. This study showed that the C57BL/6J mice are more likely to eat gelatin than are FVB/N mice, and that the 2 strains of mice show a lower preference for lemon flavoring as compared with other flavors. This method of voluntarily oral administration offers an alternative to gavage for studies that use oral dosing studies.

Mangiferin protects against adverse skeletal muscle changes and enhances muscle oxidative capacity in obese rats.

Obesity-related skeletal muscle changes include muscle atrophy, slow-to-fast fiber-type transformation, and impaired mitochondrial oxidative capacity. These changes relate with increased risk of insulin resistance. Mangiferin, the major component of the plant Mangifera indica, is a well-known anti-inflammatory, anti-diabetic, and antihyperlipidemic agent. This study tested the hypothesis that mangiferin treatment counteracts obesity-induced fiber atrophy and slow-to-fast fiber transition, and favors an oxidative phenotype in skeletal muscle of obese rats. Obese Zucker rats were fed gelatin pellets with (15 mg/kg BW/day) or without (placebo group) mangiferin for 8 weeks. Lean Zucker rats received the same gelatin pellets without mangiferin and served as non-obese and non-diabetic controls. Lesser diameter, fiber composition, and histochemical succinic dehydrogenase activity (an oxidative marker) of myosin-based fiber-types were assessed in soleus and tibialis cranialis muscles. A multivariate discriminant analysis encompassing all fiber-type features indicated that obese rats treated with mangiferin displayed skeletal muscle phenotypes significantly different compared with both lean and obese control rats. Mangiferin significantly decreased inflammatory cytokines, preserved skeletal muscle mass, fiber cross-sectional size, and fiber-type composition, and enhanced muscle fiber oxidative capacity. These data demonstrate that mangiferin attenuated adverse skeletal muscle changes in obese rats.

Subcapsular Hematoma of the Kidney Secondary to Paintball Pellet Injuries

Since the paintball game was introduced to the United States more than two decades ago, the popularity of this recreational sport has grown significantly. It is estimated that, as of 2002, more than 12 million people in over 6000 paintball sites have participated in this simulated combat sport (1). With the rising popularity of this recreational sport supported in part by a rapid industry growth, there has been a steady rise in the incidence of paintball-related injuries (1). Because a paintball firearm is capable of firing paint-filled gelatin pellets at a high velocity, this recreational weaponry can cause significant blunt injury to bodily organs upon impact.

Behavioral response to selected feed attractants and stimulants in Pacific white shrimp, Litopenaeus vannamei

Nine commercial feeding attractants and stimulants for Litopenaeus vannamei were evaluated by observation of behavioral responses in animals allotted in one Y-maze aquarium apparatus. In the validation phase, fishmeal–Brazilian origin (FMBO); fishmeal–Peruvian origin (FMPO); blood meal (BM), meat and bone meal (MBM), squid meal (SM), fish oil (FO) and fish solubles (FS) were evaluated. There was also a control without stimulatory raw material. The tested materials were included at 3% in neutral gelatin pellets (wet basis). In each behavioral observation, two different ingredients were offered at the same time in equal quantities, being allotted in the end of each chamber's arm. In Phase II after system validation, the following commercial attractants were tested: 80% crude protein (CP) vegetable dried biomass (VDB 80), 68% CP vegetable dried biomass + glutamate + betaine (VDB 68), complex of amino acids (alanine, valine, glycine, proline, serine, histidine, glutamic acid, tyrosine and betaine) with enzymatically digested bivalve mollusk (CAA), condensed fish soluble protein (CFSP), squid liver meal (SLM), betaine (Bet), dried fish solubles–low biogenic amines (DFS LB), dried fish solubles–high biogenic amines (DFS HB) and whole squid protein hydrolysate (WSPH). Attractants were used at a 3% level wet basis with neutral gelatin, without any additional ingredient source available. The best four commercial attractants from this phase (CAA, CFSP, SLM and WSPH) were compared under 0.5% and 1.0% levels. In Phase I of the study, a higher percentage of choices were observed for FMPO and FMBO. BM and FO were the least chosen ingredients. In Phase II, the worst results were observed for Bet, DFS HB and, mainly, for VDB 80 and VDB 68. When two-by-two comparisons were performed, results suggested that CFSP and CAA were the best commercial attractants tested. In the last phase, both CFSP and CAA at 1.0% level were significantly more chosen by shrimp than CFSP (0.5%), SLM (0.5 or 1.0%) or WSPH (0.5 or 1.0%). At both 0.5% and 1.0% levels, shrimp spent a similar amount of time feeding on CFSP and CAA. However, only CAA was statistically better than SLM and WSPH together. Further work is required to better elucidate the chemical drivers of chemostimulation for L. vannamei for each one of the attractants tested.

Protein Kinase Cζ Is Up-regulated in Osteoarthritic Cartilage and Is Required for Activation of NF-κB by Tumor Necrosis Factor and Interleukin-1 in Articular Chondrocytes

Protein kinase Czeta (PKCzeta) is an intracellular serine/threonine protein kinase that has been implicated in the signaling pathways for certain inflammatory cytokines, including interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha), in some cell types. A study of gene expression in articular chondrocytes from osteoarthritis (OA) patients revealed that PKCzeta is transcriptionally up-regulated in human OA articular cartilage clinical samples. This finding led to the hypothesis that PKCzeta may be an important signaling component of cytokine-mediated cartilage matrix destruction in articular chondrocytes, believed to be an underlying factor in the pathophysiology of OA. IL-1 treatment of chondrocytes in culture resulted in rapidly increased phosphorylation of PKCzeta, implicating PKCzeta activation in the signaling pathway. Chondrocyte cell-based assays were used to evaluate the contribution of PKCzeta activity in NF-kappaB activation and extracellular matrix degradation mediated by IL-1, TNF, or sphingomyelinase. In primary chondrocytes, IL-1 and TNF-alpha caused an increase in NF-kappaB activity resulting in induction of aggrecanase-1 and aggrecanase-2 expression, with consequent increased proteoglycan degradation. This effect was blocked by the pan-specific PKC inhibitors RO 31-8220 and bisindolylmaleimide I, partially blocked by Gö 6976, and was unaffected by the PKCzeta-sparing inhibitor calphostin C. A cell-permeable PKCzeta pseudosubstrate peptide inhibitor was capable of blocking TNFand IL-1-mediated NF-kappaB activation and proteoglycan degradation in chondrocyte pellet cultures. In addition, overexpression of a dominant negative PKCzeta protein effectively prevented cytokine-mediated NF-kappaB activation in primary chondrocytes. These data implicate PKCzeta as a necessary component of the IL-1 and TNF signaling pathways in chondrocytes that result in catabolic destruction of extracellular matrix proteins in osteoarthritic cartilage.

Modulation of repulsive forces between neurofilaments by sidearm phosphorylation

Recent studies have advanced the notion that the axonal organization of neurofilaments (NFs) is based on mutual steric repulsion between the unstructured “sidearm” domains of adjacent NFs. Here, we present experimental evidence that these repulsive forces are modulated by the degree of sidearm phosphorylation. When NFs are sedimented into a gelatinous pellet, pellet volume falls with increasing ionic strength and enzymatic dephosphorylation; sedimentation of phosphorylated NFs in the presence of divalent cations also dramatically reduces pellet volume. Further, atomic force microscopy imaging of isolated mammalian NFs reveals robust exclusion of colloidal particles from the NF backbone that is reduced at high ionic strength and attenuated when the filaments are enzymatically dephosphorylated. Phosphate–phosphate repulsion on the NF sidearm appears to modulate NF excluded volume in a graded fashion, thereby controlling axonal NF organization through interfilament forces.

Cross-Linking the Protein Precursor of Marine Mussel Adhesives: Bulk Measurements and Reagents for Curing

Marine mussels affix themselves to surfaces by use of a highly cross-linked, protein-based adhesive. Metal levels (e.g., Fe, Zn, Cu, Mn) of the cured glue are significantly concentrated relative to surrounding waters. Specific details on the reagents used by mussels to induce protein cross-linking are not known at this time. To provide insight on the cross-linking agents and reactions taking place while curing mussel glues, we performed a study in which various compounds were tested for the ability to bring about protein curing. A precursor to adhesion, with proteins containing the unusual amino acid 3,4-dihydroxyphenylalanine, was extracted from mussel feet. Potential cross-linking agents were mixed with this gelatinous pellet. The compressibility and shear properties of the resulting material were investigated by use of a penetration test. The reagents examined included simple metal ions (e.g., Na+, Zn2+), oxidizing transition metals (e.g., Fe3+, Cr2O7(2-)), nonmetallic oxidants (e.g., H2O2,IO4-), and oxidizing enzymes (e.g., tyrosinase). We found that protein curing was brought about by simple oxidants and transition metal ions. The results show that optimal curing occurs when the reagent is an oxidizing metal ion (e.g., MnO4-, Fe3+). We conclude that marine mussels are likely to employ Mn3+ and Fe3+ for protein cross-linking and adhesive synthesis.

Immobilization of an L-aminoacylase-producing strain of Aspergillus oryzae into gelatin pellets and its application in the resolution of D,L-methionine.

The conditions for immobilization of an l-aminoacylase-producing strain of Aspergillus oryzae in gelatin and the enzymic characteristics of the immobilized pellets were studied. The optimal concentrations of gelatin, glutaraldehyde and ethyldiamine and time of immobilization were determined. Scanning electron micrographs reveal the cross-linked structure differences between the native and immobilized pellets. Optimum pH and temperature of the native and immobilized pellets were determined. Effects of ionic strength and substrate concentration on relative activity of the native and immobilized pellets were investigated in detail. The immobilized pellets were more stable over broader temperature and pH ranges. In addition, the immobilized pellets showed stable activity under operational and storage conditions. The immobilized pellets lost about 20% of their initial activity after five cycles of reuse. The results reported in this paper show the potential for using the immobilized A. oryzae pellets to resolve d,l-methionine.

Effects of topical Catalase application on dental pulp tissue: a histopathological evaluation

Objectives: recently, the role of antioxidant enzymes in wound healing and their therapeutic potential has been a subject of interest in medical sciences. However there is restricted knowledge on the results of topical application, optimal therapeutic dosage and carrier types. The purpose of this study was to investigate the wound healing response of dental pulp after direct pulp capping with an antioxidant enzyme; Catalase (C-40). Methods: three mongrel dogs were used in this study. A total of 32 perforated Cl V preparations were treated with either 0.8 mg ( n=16) or 1.6 mg ( n=16) Catalase (C-40) which was incorporated in gelatin carriers whereas 16 control teeth had carriers without Catalase. One millimeter thick Teflon disks were placed over the gelatin pellets and the cavities restored with silver amalgam. Short term and long term histopathologic reactions were evaluated after 7 and 90 days. Results: there were no statistically significant differences between the inflammatory cell responses of control and either dosage groups of Catalase at the 7-day evaluation period. However, after 90 days, better tissue healing was observed in the teeth treated with Catalase, resulting in significant differences with the control group. Conclusions: it can be concluded that the results of this study encourage the topical use of antioxidant Catalase as a pulp-capping agent. However, further studies are required to determine optimal antioxidant dosages.

Recovery and Composition of Swiss Cheese Whey Lipid-Containing Fractions

Cheese whey contains 0.1-0.2% residual lipids. The present study was conducted to develop a high-speed centrifugation procedure for fractionating and recovering residual whey lipids (RWL) from commercial Swiss cheese whey (SCW) and to determine the yields and compositions of the resulting fractions. Three RWL fractions were obtained: (1) a low-density lipid-containing fraction (LDLF) at the top of the centrifuge tubes; (2) a medium-density lipid-containing fraction (MDLF) in the clear, middle zone; and (3) a high-density lipid-containing fraction (HDLF) in the small, compact, and gelatinous pellet at the bottom of the centrifuge tubes. LDLF, MDLF, and HDLF contained about 95, 5, and 0.13% of the total SCW lipids, respectively. Palmitic acid was the major fatty acid, and oleic, stearic and myristic acids were present in lower concentrations. The major phospholipid components of all three SCW lipid fractions were phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin.

Gustatory control of feeding behavior in goldfish

The present study tests the effects of gustatory quality on feeding behaviors exhibited by goldfish. Goldfish were trained to feed on gelatin pellets, then tested with pellets containing potentially aversive taste stimuli (quinine or caffeine), or mixtures of aversive and appetitive components (amino acids or food). Goldfish feeding includes: initial intake of food particles, a sorting phase consisting of rinsing and backwashing, and a final phase terminating in either rejection or ingestion. Pellets flavored with quinine or caffeine were rejected at concentrations above 10 −5 and 10 −3 M, respectively. Pellets containing a mixture of quinine and ground food or quinine and either l-alanine or l-proline were ingested more frequently than pellets flavored only with quinine. These pellets with mixed chemical components also produced extended sorting behaviors, including prolonged periods of rinsing and backwashing. The prolonged sorting of quinine-food mixtures enabled the animals to separate mechanically the food from the gelatin matrix. These results show that goldfish use chemical cues to drive the intraoral sorting and rejection/ingestion behaviors.

Nuclear assembly with lambda DNA in fractionated Xenopus egg extracts: an unexpected role for glycogen in formation of a higher order chromatin intermediate.

Crude extracts of Xenopus eggs are capable of nuclear assembly around chromatin templates or even around protein-free, naked DNA templates. Here the requirements for nuclear assembly around a naked DNA template were investigated. Extracts were separated by ultracentrifugation into cytosol, membrane, and gelatinous pellet fractions. It was found that, in addition to the cytosolic and membrane fractions, a component of the gelatinous pellet fraction was required for the assembly of functional nuclei around a naked DNA template. In the absence of this component, membrane-bound but functionally inert spheres of lambda DNA were formed. Purification of the active pellet factor unexpectedly demonstrated the component to be glycogen. The assembly of functionally active nuclei, as assayed by DNA replication and nuclear transport, required that glycogen be pre-incubated with the lambda DNA and cytosol during the period of chromatin and higher order intermediate formation, before the addition of membranes. Hydrolysis of glycogen with alpha-amylase in the extract blocked nuclear formation. Upon analysis, chromatin formed in the presence of cytosol and glycogen alone appeared highly condensed, reminiscent of the nuclear assembly intermediate described by Newport in crude extracts (Newport, J. 1987. Cell. 48:205-217). In contrast, chromatin formed from phage lambda DNA in cytosol lacking glycogen formed "fluffy chromatin-like" structures. Using sucrose gradient centrifugation, the highly condensed intermediates formed in the presence of glycogen could be isolated and were now able to serve as nuclear assembly templates in extracts lacking glycogen, arguing that the requirement for glycogen is temporally restricted to the time of intermediate formation and function. Glycogen does not act simply by inducing condensation of the chromatin, since similarly isolated mitotically condensed chromatin intermediates do not form functional nuclei. However, both mitotic and fluffy interphase chromatin intermediates formed in the absence of glycogen can be rescued to form functional nuclei when added to a second extract which contains glycogen. This study presents a novel role for a carbohydrate in nuclear assembly, a role which involves the formation of a particular chromatin intermediate. Potential models for the role of glycogen are discussed.

Formula omitted] utilization in rats following tracheal implant exposure

Open-ended rat tracheal implants (OETI) were exposed to 40 μg [ 3 H] benzo[a] pyrene ( B[a] P)- gelatin pellets and the 3H activity in the OETI, the host's tissues and excretia was determined 3–96 h after insertion of the pellets. The radioactivity in the OETI reached near peak activity by 3 h, and decreased almost 10-fold by 24 h. Most of the activity was associated with parent B[ a]P throughout the 95 h. The 3H activity in the surrounding tissue also was mostly associated with B[ a]P, but the 3H activity in the liver, kidney, blood and urine was mostly associated with water-soluble plus conjugated metabolites. In the feces, 68% of the 3H activity was in B[ a]P at 3 h, but mostly organic as well as water-soluble plus conjugated metabolites were extracted from it throughout the remaining 96 h. Forty-eight hours after insertion of the B[ a]P pellets, the feces contained almost 16% of the total 3H activity. Pre-exposure of the OETI to B[ a]P for 4 days before insertion of the [ 3H]B[ a]P pellets stimulated metabolism of B[ a]P in the tracheas approximately 2-fold, but had no significant effect on the host tissues.

Immobilization of Kluyveromyces marxianus cells containing inulinase activity in open pore gelatin matrix: 1. Preparation and enzymatic properties

Kluyveromyces marxianus cells with inulinase (2,1-β- d-fructan fructanohydrolase, EC 3.2.1.7) activity have been immobilized in open pore gelatin pellets with retention of > 90% of the original activity. The open pore gelatin pellets with entrapped yeast cells were obtained by selective leaching out of calcium alginate from the composite matrix, followed by crosslinking with glutaraldehyde. Enzymatic properties of the gelatin-entrapped cells were studied and compared with those of the free cells. The immobilization procedure did not alter the optimum pH of the enzymatic preparation; the optimum for both free and immobilized cells was pH 6.0. The optimum temperature of inulin hydrolysis was 10°C higher for immobilized cells. Activation energies for the reaction with the free and immobilized cells were calculated to be 6.35 and 2.26 kcal mol −1, respectively. K m values were 8 mM inulin for the free cells and 9.52 mM for the immobilized cells. The thermal stability of the enzyme was improved by immobilization. Free and immobilized cells showed fairly stable activities between pH 4 and 7, but free cell inulinase was more labile at pH values below 4 and above 7 compared to the immobilized form. There was no loss of enzyme activity of the immobilized cells on storage at 4°C for 30 days. Over the same period at room temperature only 6% of the original activity was lost.

Continuous ethanol production by yeast cells immobilized in open pore gelatin matrix

Open pore gelatin pellets with entrapped yeast cells were obtained by selective leaching out of Ca alginate from the composite matrix followed by crosslinking with glutaraldehyde. Saccharomyces uvarum cells immobilized in the porous carrier showed high ethanol productivities with a maximum value of 25 g/l.h when monitored in packed bed reactors at 35°C with continuous cane molasses feedstock containing 10% fermentable sugars.

Effect of blood rheology on hematocrit distribution in pulmonary sheet flow.

Using a physical model of convenient macroscopic scale with blood simulated by gelatin pellets in a silicone fluid, the red cell distribution in the pulmonary alveoli was studied. Information pertaining to the flow characteristics of blood at the bif

Relationships between electroencephalographic pattern and biochemical picture of the cobalt epileptogenic lesion after cortical superfusion with taurine

Cobalt gelatin pellets were implanted into the feline cortex pretreated with a superfusion of taurine. The following results were obtained. (i) The antiepileptic action of taurine was confirmed. Taurine superfusion remarkably delayed the onset and subsequent development of focal epileptic activity. At the end of superfusion, the taurine cortical concentration was highly increased whereas the glutamic acid level was reduced. The appearance and evolution of focal epileptic discharges was paralleled by a progressively more extensive decrease in concentration of several amino acids. A relationship between the taurine and glutamate levels and the depression of epileptic susceptibility of cortex is pointed out. In addition, the more severe alterations of cortical amino acid content in the cobalt focus in comparison with the penicillin focus under the same experimental conditions as used previously (L. Durelli et al. 1976. Exp. Neurol. 52: 30–39) are outlined. (ii) The taurine-induced prolonged suppression of focal epileptic activity was associated with epileptiform electroencephalographic abnormalities recorded in cortical regions far from the site of cobalt implantation. These extrafocal spikes occurred before the onset of the focal activity and decreased, even disappeared, simultaneously with progressive enhancement of the epileptic discharges in the cobalt area. The hypothesis is put forward that cobalt, though topically implanted, might exert an epileptogenic action diffuse to different regions of the brain. Under normal conditions the inhibition by the epileptogenic focus in the cobalt area would not allow the simultaneous development of other epileptogenic zones. The latter would, however, develop if the first zone had been previously inhibited by the local action of taurine.

Purification and properties of filamin, and actin binding protein from chicken gizzard.

Filamin, a protein recently identified in chicken gizzard (Wang, K., Ash, F., and Singer, S. J. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 4483-4486), has been purified free of other components and its molecular properties have been examined. Filamin has a sedimentation constant (S020,w) of 8.86 S and a partial specific volume of 0.734 ml/g. Sedimentation equilibrium experiments give a value of 498,000 for the molecular weight of native filamin. From these data a frictional ratio of 2.32 has been calculated. On sodium dodecyl sulfate gel electrophoresis, filamin migrates as a single protein band with an estimated molecular weight of 240,000. Filamin is a soluble protein and under a variety of conditions tested does not by itself form filaments or precipitate from solution. However, filamin binds to rabbit skeletal muscle F-actin, and the complex is readily sedimented by centrifugation to yield a gelatinous pellet containing actin and filamin. These results indicate that filamin is a dimeric protein with a moderate degree of asymmetry that binds to actin. The results also suggest that the distribution of filamin in cells is derived from its interaction with polymerized actin.

The separation and isolation of plasma membranes and mesosomal vesicles from Staphylococcus aureus.

Abstract We have separated and Isolated the plasma membranes and mesosomal vesicles of Staphylococcus aureus ATCC 6538P. Cells were grown aerobically in Difco synthetic AOAC broth, washed and resuspended in hypertonic buffer (3.45 M NaC1) containing 0.02 M MgSO4. Cell wall was removed by treatment with lytic enzyme from S. aureus, strain LS. The protoplasts were collected by centrifugation at 10,000 × g for 1 hour, resuspended in hypotonic buffer containing 0.02 M MgSO4 and lysed. The resultant plasma membranes were washed and centrifuged on a 60tr>75Z sucrose density gradient at 55,000 × g for 15 hours. Gradient patterns showed two bands of membranes. Crude mesosomes were obtained from the 10,000 × g supernatant fractions by centrifugation at 100,000 × g for 2 hours. The reddish-brown gelatinous pellet, which consisted of mesosomal vesicles and a few ribosomes, was washed and centrifuged on a 60 to 85% sucrose density gradient at 100,000 × g for 15 hours. Gradient patterns produced two bands of mesosomal...

Nascent ribosomes from HeLa cells.

In recent years several stages in the synthesis of mammalian ribosomes have been resolved. (a) The 45S precursor RNA is synthesized in the nucleolus as a single polynucleotide chain.' (b) The 45S RNA is cleaved in a number of steps to 32SRNA and 18S RNA.1-3 (c) While still in the nucleolus, the 32SRNA is further cleaved to 28S RNA,3 to which is bonded noncovalently a small, 7S, piece of RNA.4 7S RNA may be released by heat, dimethyl sulfoxide, or urea. (d) The mature 28S and 18S RNA's can be detected transiently in ribonucleoprotein particles in the nucleus.5 (e) The individual subribosomal particles pass quickly to the cytoplasm where they become incorporated into polyribosomes and subsequently equilibrate with single ribosomes.6 7 A major point of interest is the process by which ribosomal protein is assembled with ribosomal RNA, which probably occurs in the nucleolus. Since available techniques for nuclear fractionation have not yet permitted a direct approach to this problem, we have attempted to isolate from the nucleolus ribonucleoprotein particles in various stages of development toward cytoplasmic ribosomes. Two types of such particles have been identified, which we have termed ribosomal for reasons which will become evident. These particles may be similar to those described by Tamaoki and Mueller,8 and by Yoshikawa-Fukada.9 Materials and Methods.-Cells: HeLa S3 cells were grown in spinner culture in Eagle's'0 minimal essential medium (MIEM) supplemented with 7% horse serum. Solutions: RSB: 0.01 M NaCl; 0.0015 Mll MgCl2; 0.01 M Tris, pH 7.4; HSB: 0.5 M NaCl; 0.05 M MgCl2; 0.01 M Tris, pH 7.4; NEB: 0.01 M NaCl; 0.01 M EDTA; 0.01 M Tris, pH 7.4; NETS: 0.1 M NaCi; 0.001 M EDTA; 0.01 AM Tris, pH 7.4; 0.1% SDS. Radioactive labeling of cells: Cells were labeled with C14and H3-uridine as previously desCribed.6 For labeling with C'4-leucine, a culture was centrifuged at 1500 rpm for 2 min, and the resulting cell pellet was resuspended in 1/' to 1/lo the original volume of a modified Eagle's MEM, containing 7% whole serum but no added leucine. C'4-leucine was added for a given length of time, followed by dilution of the culture to the original volume in Eagle's complete MEM with serum plus ten times the normal concentration of leucilne. Cell fractionation: Nutcleoli were prepared by a modification of the method of Penman.2 Cells from a 100-ml culture were harvested by centrifugation at 1500 rpm for two min, followed by two washes with cold Earle's solution. All subsequent steps were carried out at 4°. The cells were resuspended in 1.5 ml RSB and were subsequeiitly ruptured with a I)ounce homogenizer. The nuclei were collected by centrifugation for 3 mim at 1300 rpm and were washed with another 1.5 ml RSB. Ribosomes were prepared from the pooled supernatants by Mg++ precipitation as previously described.'2 The nuclei were suspended in 2.0 ml RSB to which was added 0.3 ml of a solution containing 6.7% Tweeni 40 and 3.3% sodium deoxycholate. The suspension was briefly mixed on a Vortex mixer and centrifuged at 1500 rpm for 5 min. The gelatinous pellet of nuclei was resuspended in 2.5 ml of cold HSB containing 100 lOg D)Nase (Worthington, electrophoretically). The viscosity of this suspension was reduced markedly after vigorous pipetting for several minutes. The crude nucleoli were collected by centrifugation in the cold for 10 min at 10,000 rpm. The nucleolar pellet was resuspended in 1.5 ml of NEB containing 0.01 M dithiothreitol and gently stirred for 15 min at room temperature. After centrifugation for 10 min at 15,000 rpm the supernatant, containing the nascent ribosomal particles, was analyzed on a 15-30% w/w sucrose gradient in NEB solution.