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In this study, the effectiveness of a 188Re labeled sulfur colloid with two particle size ranges was used to evaluate the effectiveness of this agent on melanoma tumors in mice in terms of animal lifespan.Two separate group of animals were used for investigating biodistribution and survival time. A total of 188 B16F10-melanoma-bearing BDF(1) mice were injected intraperitoneally with 3.7 MBq (0.1mCi)/2mL of radiolabeled sulfur colloid ten days after intraperitoneal inoculation of 5x10(5) B16F10 melanoma cells/2ml. For group 1, 30 mice were sacrificed at 1, 4, 24, 48 and 72 hours for biodistribution studies. In group 2, 158 mice were divided into 9 groups (n=16 approximately 18/groups)each receiving respectively tumor alone, tumor with normal saline, cold colloid or hot colloid with 16, 23, 31, 46, 62, or 124 MBq activity. Each of these colloid groups was further divided into two groups, one receiving smaller particle sizes (3 microm:80.4 +/-7.2%, colloid 1) and the other receiving larger particle sizes (3 microm:12.3+/-1.0%, colloid 2). The animals were checked daily until death and their survival recorded.Colloid 2 showed higher accumulation in almost all tissues, the highest accumulation organ was tumor ( approximately 40%), then spleen ( approximately 20%), stomach ( approximately 15%), diaphragm ( approximately 3%), and liver ( approximately 2%). There was a significant increase in survival time with increasing amount of the larger-particle-size colloid. Administered levels of 16-31 MBq/mouse were most efficacious and with higher amounts the survival times decreased significantly below that of the controls. There was a significant difference in the dose-response curves for the two preparations. Protection factors (1/Relative-risk) of nearly 5 were achieved using the larger colloid size, and nearly 30 using the smaller colloid size. An amount of 16-31 MBq of the colloid 2 was the optimal activity in these studies. On the one hand, the survival data agreed well with the biodistribution data, where higher accumulation was found in tumor with colloid 2.Rhenium-188 offers on-site availability, medium half-life, higher beta-particle energy of 2.12 MeV for therapy and emission of 155keV gamma photon suitable for imaging. The present study demonstrated that 188Re-sulfur colloid is an effective agent in controlling tumor cells in the abdominal cavity in animals.

Glial fibrillary acidic protein (GFAP) is an astrocytic lineage-specific intermediate filament protein, and its expression or non-expression is inversely correlated with the tumourigenecity of astrocytoma cells. To estimate the GFAP levels of astrocytes in intracranial tumour tissues, we established primary cultures from six astrocytic tumour specimens and used a double-staining flow cytometric method to detect the different levels of GFAP among these primary cultures. Although these primary cultures exhibited the same Matrigel invasiveness, their GFAP expression is inversely related to the rate of cell growth and the histologic grade of the original tumour. Phenylacetate, 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate, which are potent inducers of differentiation in various cancer cells, have been examined for their effects on these primary cultures. Cytostasis was more or less caused by these compounds in all six primary cultures, but induction of GFAP was observed only in the primary culture derived from a less malignant astrocytoma specimen having the highest intrinsic GFAP level. Interestingly, this primary culture, but not others, also exhibited increased HRG-α expression after phenylacetate or sodium butyrate treatment. Loss of the inducibility of differentiation-related gene expression could be one of the events involved in the malignant progression of astrocytomas. In addition, the chemotherapeutic agent BiCNU has a killing effect on all six primary culture cells, with LD50 less than 60nM. The underlying mechanism was through the induction of apoptosis in these primary culture cells regardless of their varying malignancies of original tumours. However, unlike colon cancer and leukaemia cells, sodium butyrate could not induce apoptosis within 4 days in these astrocytic tumour cells, indicating that the cell context of different cell types indeed determined the ability of sodium butyrate to induce apoptosis.

As one of the most intensively studied probes for imaging of the cellular proliferation, [ 18 F]FLT was investigated whether the targeting specificity of thymidine kinase 1 (TK1) dependency could be enhanced through a synergistic effect mediated by herpes simplex type 1 virus (HSV1) tk gene in terms of the TK1 or TK2 expression. 5-[ 123 I]Iodo arabinosyl uridine ([ 123 I]IaraU) was prepared in a radiochemical yield of 8% and specific activity of 21 GBq/μmol, respectively. Inhibition of the cellular uptake of these two tracers was compared by using the arabinosyl uridine analogs such as 5-iodo, 5-fluoro and 5-( E )-iodovinyl arabinosyl uridine along with 2′-fluoro-5-iodo arabinosyl uridine (FIAU). Due to potential instability of the iodo group, accumulation index of 1.6 for [ 123 I]IaraU by HSV1-TK vs. control cells could virtually be achieved at 1.5 h, but dropped to 0.2 compared to 2.0 for [ 18 F]FLT at 5 h. The results from competitive inhibition by these nucleosides against the accumulation of [ 18 F]FLT implied that FLT exerted a mixed TK1- and TK2-dependent inhibition with HSV1- tk gene transfection because of the shifting of thymidine kinase status. Taken together, the combination of [ 18 F]FLT and HSV1-TK provides a synergistic imaging potency.

Diabetes is often associated with atherothrombosis. It is unknown whether high glucose can modulate the expression of tissue factor (TF) and thrombomodulin (TM) in human aortic endothelial cells (HAECs). HAECs were treated with a lower-degree high glucose condition (LG, 11.2 mM) for 8 days and a higher-degree high glucose condition (HG, 30 mM) for 4–6 h. Methoxyphenyl tetrazolium inner salt assay, real-time polymerase chain reaction, western blot, and TF activity assay were performed. In HAECs, both LG and HG conditions were nontoxic. LG caused a 74 ± 20% decrease (P = 0.273) and HG caused a 57 ± 5% decrease in TF mRNA expression (P = 0.001). LG caused a 53 ± 13% decrease (P = 0.036) and HG caused a 75 ± 10% decrease in TF protein expression (P = 0.096). TF activity was not significantly changed by LG (127 ± 13%, P = 0.40) or HG treatments (120 ± 42%, P = 0.70). In contrast, LG caused a 153 ± 16% increase (P = 0.03) and HG caused a 211 ± 20% increase in TM mRNA expression (P = 0.005). LG caused a 131 ± 31% increase (P = 0.35) and HG caused a 140 ± 9% increase in TM protein expression (P = 0.006). Different high glucose conditions do not provide the sufficient stress required to induce TF expression in HAECs. In contrast, high glucose conditions can induce TM expression in HAECs.

Paclitaxel is a potential anti-cancer agent for several malignancies including ovary, breast, and head and neck cancers. This study investigated the kinetics of paclitaxel-induced cell cycle perturbation in two human nasopharyngeal carcinoma (NPC) cell lines, NPC-TW01 and NPC-TW04. NPC cells treated with higher concentrations (0.1 or 1 micro M) of paclitaxel showed obvious G2/M arrest and then converted to a cell population with reduced DNA content, which was detected as a sub-G2 peak in the flow cytometric histographs. If a low concentration (5 nM) of paclitaxel was used instead, transient G2/M arrest was observed in NPC cells, which subsequently converted to a sub-G1 form during the treatment period. Internucleosomal fragmentation and chromatin condensation were detectable in these sub-G1 and sub-G2 cells, suggesting that persistent or transient G2/M arrest is a prerequisite step for apoptosis elicited by varying doses of paclitaxel. The levels of cyclins A, B1, D1, E, CDK 1 (CDC 2), CDK 2 and proliferating cell nuclear antigen (PCNA) were unchanged in NPC cells following treatment with any concentration of paclitaxel; however, apoptosis-related cyclin B1-associated CDC 2 kinase was highly activated by paclitaxel even at concentrations as low as 5 nM, which is consistent with the finding that low-dose paclitaxel is also able to induce apoptosis in NPC cells. Activation of cyclin B1-associated CDC 2 kinase seems to be an important G2/M event required for paclitaxel-induced apoptosis, and this activation of cyclin B1/CDC 2 kinase could be attributed to the increased activity of CDK 7 kinase.

GL331 is a semisynthetic topoisomerase II inhibitor derived from a plant toxin podophyllotoxin. In 72-h exposure assays, LD50 values of GL331 range from 0.5 to 2 microM, which are three- to ten-fold lower than those of its homologous compound etoposide (VP-16), depending on different cancer cell lines including nasopharyngeal, hepatocellular, gastric, cervical and colon cancer types. Apoptotic DNA ladders could be detected when cancer cells were treated with GL331 for 24 h even if the Bcl-2 and Bax protein levels were not altered during the period. Besides acting as topoisomerase II inhibitors, both GL331 and VP-16 decrease the cellular protein tyrosine kinase (PTK) activities in cancer cells. The activities of protein tyrosine phosphatase (PTP) are significantly increased after GL331 treatment but are not affected by VP-16. GL331-induced internucleosomal cleavage can be efficiently prevented by two inhibitors of PTP, sodium orthovanadate and zinc chloride, but not by okadaic acid, which inhibits serine/threonine phosphatase activity. These results indicate that GL331 may induce apoptotic cell death, and that activation of protein tyrosine phosphatases may be involved in this process.

Paclitaxel is a potential cancer chemotherapeutic agent for ovary, breast, and head and neck cancers; its effects on nasopharyngeal carcinoma (NPC) have not been reported previously. This study investigated the cytotoxic mechanism of paclitaxel in two NPC cell lines, NPC-TW01 and NPC-TW04. NPC cells treated with pacli-taxel showed convoluted nuclei, condensed chromatin and decreased cellular and nuclear volume, and also exhibited genomic DNA degradation into multiple oligonucleosomal fragments, suggesting that pacli-taxel induced apoptosis in these cells. The effects of paclitaxel on apoptosis-related proteins including Bcl-2, Bax and CDC 2 were also detected. Although the levels of Bcl-2 and Bax were not changed in NPC cells following treatment with 5 nM-1 μM of paclitaxel, phosphorylation of Bcl-2 was significantly observed in the cells treated with 1 μM of paclitaxel for 12 hours. In addition, cyclin B1-associated CDC 2 kinase was highly activated in the NPC cells exposed to paclitaxel even at low (5 nM) concentration, and this result is associated with the finding that low concentration of paclitaxel is able to induce apoptosis in NPC cells.

DNA polymerase activities were studied in rabbit endometria at estrus and through the first seven days of pregnancy. Extracts of total endometrial homogenates by high salt gave two peaks of polymerase activity by sucrose gradient centrifugation; the 6-8S polymerase showed considerable increase in specific activity which reached a maximum at 5-7 days post-coitum, whereas the 3-4S polymerase showed only a slight increase after 2 days of pregnancy. A 5-6S RNA-dependent DNA polymerase activity, extracted by combined use of hypertonic salt solution and non-ionic detergent from particulate subcellular functions, appeared in the endometrium after coitus, reached a maximum at Day 3-4 and declined thereafter. This RNA-dependent DNA polymerase activity, isolated by phosphocellulose chromatography at distinct regions of salt gradient, showed preference for template-primers such as (rA)/sub n/.(dT)/sub 9/ and (rC)/sub n/.(dG)/sub 6/ but did not utilize 70S RNA of RNA tumor viruses. Attempts to demonstrate oncorna virus expression by other means have been unsuccessful with endometrium of 3 day pregnant rabbits. Interrelation between the changes of these DNA polymerase activities and the DNA replication in the endometrial cells is discussed.

A purification procedure is described for two major toxic proteins from seeds of Abrus precatorius by successive chromatography on diethylaminoethyl-Sephadex A-50, carboxymethyl-cellulose, and DEAE-cellulose. The two toxins obtained both appeared homogeneous as judged by electrophoresis and analytical ultracentrifugation. Abrin A, the more positively charged protein obtained from the DEAE-cellulose chromatography, can be crystallized as needles from ammonium sulfate solution by the free interface diffusion technique; under various similar conditions, the other fraction, abrin C, failed to yield crystals. X-ray precession photographs have indicated that abrin A crystallizes in an orthorhombic unit cell of symmetry P212121 and dimensions a = 74.9, b = 269.8, and c = 70.3 A. The amino acid composition of abrins A and C are different. Molecular weights determined by sedimentation equilibrium are 60,100 for A as compared to 63,800 for C. After A has been treated with 2-mercaptoethanol at 100° for 2 min, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gives rise to one strong (corresponding to apparent subunit molecular weight of 32,000) and two much weaker bands. This is in sharp contrast with the pattern for C, two bands of approximately equal intensity, corresponding to apparent subunit molecular weights of 33,000 and 28,000. The two proteins also behave differently in affinity chromatography on a Sepharose 4B column. Abrin C at 1 µg per ml concentration level agglutinates sheep erythrocytes, whereas abrin A shows no such activity even at 40 µg per ml concentration level. Abrin C exhibits a more toxic effect on mice than abrin A. These differences in physicochemical and biological properties for the two abrins are presented in detail.

Replication of Gross strain N-tropic type C retrovirus was markedly restricted in a pluripotential undifferentiated embryonal cell line (PCC4) of murine teratocarcinoma, whereas the same virus could cause productive infection in a myoblast-derived differentiated line (PCD1) of the same tumor origin. To investigate the restriction mechanism, we compared the initial viral DNA formation in these two cell lines. Analyses by means of a modified Hirt extraction procedure and a modified Southern gel transfer method indicated that PCC4 and PCD1 cells supported the synthesis of viral DNA intermediates after inoculation of the Gross virus. In both cells, a linear DNA duplex (from III viral DNA) appeared at 4 hr, reached a maximal level at 8-9 hr, and declined rapidly thereafter, while two closed-circular supercoiled DNA duplexes (form I viral DNA) showed their appearance, increase and decline in the 8-24 hr period. During the period from 34 to 78 hr after virus inoculation, another burst of viral DNA synthesis occurred in PCD1 cells, presumably due to secondary virus infection, while at this period both form III and form I viral DNAs became undetectable in PCC4 cells. The Hirt supernatant DNAs prepared from PCD1 and PCC4 cells 10 hr after virus inoculation were equally infectious for NIH3T3 cells in a DNA transfection, although one positive result with PCD1 cells might suggest a difference between the two cell types in this aspect. These results indicate that restriction of type C retrovirus in undifferentiated embryonal carcinoma cells occurs at a step subsequent to formation and maturation of viral DNA intermediates.

The major lectin in seeds of a soybean cultivar (Glycine max cv D68-127) has been purified to apparent homogeneity by hydroxyapatite and DEAE-cellulose chromatography. In the latter, the behavior of the lectin was similar to that of the minor isolectins previously described in other soybean cultivars. Molecular weights of 92 000 for the molecule and 23 000 for the subunits were determined by gel filtration and sodium dodecyl sulfate-gel electrophoresis; these are smaller than those previously reported for the major lectin in another soybean variety. Hemagglutination by the lectin was inhibited specifically by N-acetyl-D-galactosamine and galactose-containing sugars.

Formation of viral closed circular supercoiled DNA duplexes and production of progeny virus were both inhibited in cultured mouse cells treated with cycloheximide in the first 4 h of type C retrovirus infection. With different doses of cycloheximide to cause different degrees of inhibition, the number of viral supercoiled DNA duplexes detected in the cells at 11 h showed an apparent correlation with the amount of progeny virus produced in the 12- to 22-h period of infection. A slight accumulation of the full-genome linear duplex and an open circular duplex of viral DNA intermediate was observed in the cycloheximide-treated cells. Cycloheximide given to the cells during the time of conversion of viral DNA from linear to supercoiled duplex forms (6 to 11 h after virus inoculation) did not inhibit the conversion. These kinetic data suggest that a cycloheximide-sensitive metabolic process, probably early viral protein synthesis, is required for retrovirus replication and supercoiled viral DNA formation in the cell.

Transfer RNAs from liver and brain of young and old BC3F1 mice were compared in regard to extent of aminoacylation and cochromatographic profiles of isoaccepting tRNA species on reversed-phase columns. Homologous synthetase preparations and optimal aminoacylation conditions were employed, having been determined for each amino acid and found to be the same for those from old and young mice. Small differences were found between tRNAs from young and old mice in the extent of acceptance for arginine and tyrosine in the liver and for aspartic acid in the brain. There were no differences observed between preparations from young and old mice in any of the cochromatographic profiles for the amino acids examined in this study, which included arginyl-, aspartyl-, glutamyl-, histidyl-, leucyl-, lysyl-, phenylalanyl-, seryl-, and tyrosyl-tRNAs from liver and arginyl-, aspartyl-, histidyl-, leucyl-, lysyl-, and seryl-tRNAs from brain. Comparisons of tRNA preparations from fetal and neonatal liver with those from adult liver did reveal both qualitative and quantitative differences. These results suggest that the postulated accumulation of errors as a result of age-related alterations in the translational mechanism does not occur in tRNA or aminoacyl-tRNA synthetases of these two tissues.

A high level of RNA-directed DNA polymerase activity is found in spleens of Balb c mice infected with Rauscher murine leukemia virus. The enzyme is associated with sedimentable subcellular fractions, can be separated from other cellular DNA polymerases, and is present in useful quantity for purification purposes. Complete solubilization of the enzyme, achieved and maintained by using a combination of detergent and high salt solution, is required for a satisfactory purification both in the initial extraction procedure and in subsequent column chromatography.

The tRNA synthesized by Escherichia coli during chloramphenicol treatment has altered chromatographic properties, as analyzed by reversed-phase chromatography. The data suggest that most of the tRNAs synthesized in the presence of chloramphenicol are immature, chromatographically distinct forms, capable of being converted to mature forms upon removal of chloramphenicol. Methylation of tRNA during chloramphenicol treatment appears to be quantitatively and qualitatively normal. The tRNA synthesized during chloramphenicol treatment differs from normal tRNA mainly in that it contains 60–70% less 4-thiouridine and dihydrouridine. Preliminary experiments indicate that these two minor bases are not required for normal aminoacylation. Phenylalanyl-, valyl-, isoleucyl- and lysyl-tRNAs from untreated and chloramphenicol-treated cells are equally functional in an in vitro hemoglobin-synthesizing system.

Lectin is released from soybean seeds during water uptake. Hemagglutination activity data show that the lectin is a preferential release product within the first 8 hours of hydration. A qualitative filter-paper assay for detection of lectin released by single seeds is used to show that the release phenomenon is independent of seed viability and insensitive to azide.

Tryptophan transfer RNA from chick cells chromatographs differently in the reversed-phase column chromatographic system 5 depending upon whether it is aminoacylated or not. This property was utilized to prepare pure tryptophan tRNA. Oligonucleotide fingerprints of tryptophan tRNA purified in this manner are identical with those reported for "spot 1" RNA isolated from Rous sarcoma virus.

Selected species of 4S RNA of chick embryo cells will hybridize in vitro with 35S RNA of avian myeloblastosis virus. A major tRNA component of the hybridizable 4S RNA is tryptophan tRNA. A hybrid prepared from purified tryptophan tRAN and 35S RNA of avian myeloblastosis virus in vitro is an efficient templateprimer for DNA synthesis catalyzed by reverse transcriptase (RNA-dependent DNA polymerase).

Five protease inhibitors, I–V, in the molecular weight range 7000–8000 were purified from Tracy soybeans by ammonium sulfate precipitation, gel filtration on Sephadex G-100 and G-75, and column chromatography on DEAE-cellulose. In common with previously described trypsin inhibitors from legumes, I–V have a high content of half-cystine and lack tryptophan. By contrast with other legume inhibitors, inhibitor II contains 3 methionine residues. Isoelectric points range from 6.2 to 4.2 in order from inhibitor I to V. Molar ratios (inhibitor/enzyme) for 50% trypsin inhibition are I = 4.76, II = 1.32, III = 3.22, IV = 2.17, V = 0.97. Only V inhibits chymotrypsin significantly (molar ratio = 1.33 for 50% inhibition). The sequence of the first 16 N-terminal amino acid residues of inhibitor V is identical to that of the Bowman-Birk inhibitor; all other observations also indicate that inhibitor V and Bowman-Birk are identical. The first 20 N-terminal amino acid residues of inhibitor II show high homology to those of Bowman-Birk inhibitor, differing by 1 deletion and 5 substitutions. Immunological tests show that inhibitors I through IV are fully cross-reactive with each other but are distinct from inhibitor V.

Transfer ribonucleic acid (tRNA) profiles in human lymphocytes stimulated by various mitogens have been compared with profiles from nonstimulated cells and from leukemic cells using reversed-phase chromatography. Comparisons of (/sup 3/H)- or (/sup 11/C)uridine- or (/sup 32/P)phosphate-labeled tRNAs showed that the greatest changes in tRNA composition upon phytohemagglutinin (PHA) stimulation occurred in the first 8 h after mitogen addition. Stimulation of lymphocytes by pokeweed mitogen, anti-human immunoglobulin, or bacterial lipopolysaccharide resulted in tRNA species which showed distinct differences from each other and also from the tRNAs produced by phytohemagglutinin stimulation. Leukemic lymphocyte tRNAs showed the most extensive differences in profile when compared with chromatograms from non-neoplastic cells stimulated by a variety of mitogens. Specific isoaccepting species of tyrosyl-, aspartyl-, and phenylalanyl-tRNAs were also compared in PHA-stimulated and resting lymphocytes and no differences were found. When these same species were studied in leukemic cells, tyrosyl-tRNA profiles were shifted to elute at a lower salt concentration, while the aspartyl-tRNA profile showed a new peak not present in noncancerous cells.

Previously, in vitro recombinant DNA studies demonstrated that genetic determinants of N-tropism and B-tropism, or Fv-1-related host range properties of murine leukemia viruses, were located in a BamHI-HindIII DNA segment derived from the 5' portion of the cloned viral genome. We sequenced this segment and its immediate 5' region from cloned DNA of two BALB/c mouse C-type viruses (WN1802N and WN1802B) and found base differences at 12 positions out of the otherwise identical 1,390-base-pair sequences. Analysis of the most likely reading frame showed that 6 of the 12 base differences would result in four encoded amino acid changes, three of which occur at positions 109 (glutamine in WN1802N versus threonine in WN1802B), 110 (arginine in WN1802N versus glutamic acid in WN1802B), and 159 (glutamic acid in WN1802N versus glycine in WN1802B) of the p30 protein. The remaining one is located at position 36 (threonine in WN1802N versus isoleucine in WN1802B) of the viral polymerase protein. Significant conformational alteration of the p30 protein could be predicted from these amino acid changes.

Specific adsorption capacity of Sepharose 4B in affinity chromatography for two purified galactose-binding lectins, designated as III/sub L/ and III/sub H/, from the seed of Ricinus communis (castor bean) was measured from 7 to 24/sup 0/C. The adsorption coefficients for these two protein fractions as a function of temperature were also obtained. It was found that there is a characteristic transition of adsorption coefficient at 18/sup 0/C for both lectins. Adsorption coefficients between Sepharose 4B and these two lectins were also expressed in terms of ..delta..G, ..delta..H, and ..delta..S. It is suggested that the difference in the temperature dependence of the binding energy of these two lectins may be used for their separation at selected temperature.

Retrovirus infection of cultured murine teratocarcinoma cells depends upon the state of differentiation. We have used two cell lines derived from a teratocarcinoma of mouse, strain 129. One, an undifferentiated pluripotential cell line (PCC4), is restrictive to viral infection, while the other, a differentiated myoblast-derived cell line (PCD1), is fully permissive to virus replication. We have shown that no virus RNA expression can be found in PCC4 cells 48 h post-infection and that no nucleic acid sequences can be found in an integrated form in PCC4 cells. However, the kinetics of formation of free proviral intermediates show that the three forms (I, II and III) of free virus DNA are synthesized in both PCC4 and PCD1. Free proviral DNA disappears gradually after 24 h in PCC4 cells while all forms increase in PCD1. These results suggest that the viral multiplication restriction occurs somewhere between the proviral DNA synthesis and integration of DNA in the cellular genome.

Twenty molecular clones containing sequences homologous to the long terminal repeats (LTRs) of the endogenous ecotropic murine leukemia virus (MuLV) of the RFM/Un mouse were isolated from a library of RFM/Un mouse spleen DNA in phage λ Three of these LTRs were not associated with any viral structural genes. Nucleotide sequence analysis demonstrated that they were solitary LTRs which were flanked by 4-bp directly repeated cellular sequences and which lacked primer binding sites. Two of the three subclones were found to be identical except for their orientations in the vector pBR322. Unique-sequence regions on either side of the two nonidentical elements were used to characterize their integration sites in genomic DNA. The solitary LTRs and their flanking regions were found to be conserved in a number of inbred mouse strains, including three strains known not to harbor endogenous ecotropic MuLV-type proviruses. Comparison of cleavage by the methylation-sensitive restriction enzyme Smal and methylation-insensitive Kpn l at the characteristic LTR Sma l /Kpn l site suggested that at least one of these solitary LTRs is methylated to a lesser extent than are most endogenous proviral LTRs. These particular solitary LTRs, like endogenous proviral sequences, appear to be stably transmitted genetic elements.

It is well recognized that immunoglobulins consist of heterogeneous populations of antibody proteins which are synthesized following a complicated series of cellular events induced by antigen stimulation. Chemical studies of purified antibodies suggested that antibodies of different specificities may differ from one another in their primary protein structure (Koshland and Englberger, 1963). The mechanism by which this tremendous variety of immunoglobulins are synthesized has thus been a very interesting biological problem. Biochemical knowledge concerning transcription and translation of the genetic messages for the synthesis of these proteins is, however, extremely deficient. This is due mainly to the difficulty in obtaining for study a sufficient quantity of lymphoid tissue with a given homogeneity of antibody production.

Publisher Summary This chapter describes an application of the reversed-phase column chromatographic system (RPC); presents general procedures for preparing tRNA and aminoacyl-tRNA synthetases from the tissue and for preparing labeled aminoacyl-tRNA suitable for the chromatographic analysis; and demonstrates the analysis of iso-accepting tRNAs of an amino acid with a sample size in the range of 1 A260 unit of mixed tRNA. This method is very useful, especially in cases, where the source of material for preparing tRNA is limited, such as tissue culture cells and small animal organs. The sensitivity of this method is facilitated by the availability of radioactive amino acids with high specific activity. The isoaccepting peaks of an aminoacyl-tRNA are sufficiently well resolved in the column that quantitative comparative studies can be done by cochromatography of two samples with different labels. Because, the size of the tRNA sample is small, it becomes very important that artificial modifications of amino-tRNA should be prevented. The profiles of isoaccepting tRNAs for individual amino acids are much different between the mammalian and the bacterial sources.