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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.

Study objective Human immunity has been found to have two major components, cellular and humoral immunity. T-helper type 1 (Th1) pathway favors cellular immunity and Th2 pathway favors humoral immunity. Early determination toward Th1 and Th2 cells in the immune response is dependent on the balance between interleukin-12 (IL-12), which favors Th1 responses, and IL-4, which favors Th2 responses. IL-2 and interferon-γ (IFN-γ) are produced in the Th1 pathway, and IL-4 and IL-10 are produced in the Th2 pathway. Lack of cellular immunity, IL-2, and IFN-γ had been reported in malignant pleural effusions. However, to our knowledge, there are no previous reports on other cytokine components involving Th1 or Th2 pathway. The present study was designed to answer these questions. Design Cytokine levels in peripheral blood and pleural fluid of 21 patients with malignant pleural effusion, including IL-4, IL-10, and IL-12, were analyzed with enzyme-linked immunosorbent assays. Lymphocyte subpopulations of peripheral blood and pleural effusion were also studied by using flow cytometry. Measurements and results The results showed a significant increase in IL-10 level as compared with blood samples. IL-4 and IL-12 were below minimal detectable concentrations both in the blood and the effusion. The ratio of pleural helper T cells was significantly higher than in the blood (p=0.0002). The ratio of pleural natural killer (NK) cells was significantly lower than in the blood (p=0.0001). The ratio of pleural suppressor T cells was lower than blood with borderline significance (p=0.0522). No significant change in B-lymphocyte ratio between blood and pleural effusion was found (p=0.2471). There was no correlation between difference in IL-10 level and lymphocyte subpopulation of pleural effusion and blood samples. Conclusions Helper T-cell subpopulations were increased while NK and suppressor T-cell subpopulations were decreased in malignant pleural effusions. The decrease in NK cell subpopulations with elevated IL-10 and minimal IL-12 concentration in neoplastic pleural effusion would suggest the usage of IL-12 or antibody of IL-10 to improve local cellular immunity. Further study is needed.

Treatment of mice with hepatic carcinogens, including CCl4, has been shown to rapidly enhance the transcription of endogenous murine leukemia virus—related proviral sequences in the liver. To understand the mechanism for this transcriptional stimulation, we used nuclear protein preparations from mouse livers to perform DNase I protection analyses and identified nuclear protein binding on approximately 20 individual sequences within the regulatory regions of the long terminal repeat (LTR) of a polytropic-class endogenous provirus clone. From 3 to 144 h after treatment with CCl4, the livers of FVB/N mice were analyzed for specific nuclear protein binding to the LTR DNA. Three to nine hours after CCI4 treatment, decreased protection was seen at potential regulatory cis-elements throughout the LTR, including specific sites within the putative negative regulatory element (located 5′ of the consensus enhancer sequences) and the 3′ terminal portion of the polytropic class-specific enhancer-like inserted sequence element and around the CCAA(C/T) box in the promoter region. In addition, by 3–6 h after treatment, a transient increase in protection activity for the transcription initiation site occurred. The loss of cis-element protection expanded to other binding sites and became most marked by 48 h after treatment. As the regenerating liver recovered, the nuclear protein binding activities for these LTR sequences also recovered, but protection at the TATAA and transcription initiation sites remained deprotected at 144 h after treatment. Nuclear protein protection of other sites, particularly in the conserved LTR enhancer sequences, was minimally affected by CCl4 treatment. Three nuclear protein binding sites that showed rapid CCI4-induced kinetic changes were homologous to the consensus sequence for the binding of the transcription factor families MEF-2, HNF-1, and C/EBP. The complex kinetic changes in factors that may contribute to the rapid and transient induction of endogenous retroviral gene expression in the liver after CCI4 exposure are discussed.

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.

Fv-1 gene-specific resistance to ecotropic murine retroviruses can be transferred to permissive cells with RNA prepared from restrictive cells. The active RNA has a sedimentation coefficient of 20-22S at low ionic strength and multiple regions of activity (5-6S, 10-12S, 24-27S) at high ionic strength. The maximum level of resistance transfer cannot be enhanced with noninhibitory carrier RNA or facilitators of RNA uptake, and the maximum inhibition (40-60%) is independent of the MOI. The low efficiency of resistance transfer is, therefore, probably the result of a fraction of cells resistant to RNA uptake.

RFM/Un mice express an endogenous type C retrovirus throughout their life span in many tissues; primary or established embryo fibroblast cell cultures do not express a virus but can be induced by exposure to 5-iodo-2'-deoxyuridine. All of our sources yielded a single ecotropic virus (RFV) which appeared to be related more closely to the endogenous N-tropic virus (WN1802N) of BALB/c mice than to Gross leukemia virus on the basis of two-dimensional gel electropherograms of virion proteins. No xenotropic or recombinant viruses were isolated by cocultivation techniques. RFV is N-tropic, and RFM/Un cells possess the Fv-1n allele, as indicated by restriction of B-tropic virus and susceptibility to Gross strain N-tropic virus. However, RFM cells are highly resistant to RFV and other endogenous N-tropic viruses. This resistance is expressed by two-hit titration kinetics and by inhibition of viral linear duplex DNA formation. This is similar to the effects of the Fv-1 locus, but preliminary work has shown no apparent genetic linkage between the two restrictions. The relative strength of the restriction, the presence of a single class of ecotropic virus, and the absence of recombinant viruses suggest that in RFM mice virus is expressed only in cells in which it is induced and not by cell-to-cell transmission.

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.

We molecularly cloned unintegrated viral DNA of the BALB/c endogenous N-tropic and B-tropic murine leukemia retroviruses and in vitro passaged N-tropic Gross (passage A) murine leukemia retroviruses. Recombinant genomes were constructed in vitro by exchanging homologous restriction enzyme fragments from N- or B-tropic parents and subsequent recloning. Infectious virus was recovered after transfection of these recombinant genomes into NIH-3T3 cells and cocultivation with the Fv-1 nonrestrictive SC-1 cells. XC plaque assays of recombinant virus progeny on Fv-ln and Fv-lb cells indicated that the Fv-l host range was determined by sequences located between the BamHI site in the p30 region of the gag gene (1.6 kilobase pairs from the left end of the map) and the HindIII site located in the pol gene (2.9 kilobase pairs from the left end of the map).

The decline in phytohemagglutinin (PHA) responsiveness of spleen cells from aged mice was studied in an effort to determine the nature of the depressed PHA reactivity. The reduced response of DNA synthesis (thymidine incorporation) to PHA stimulation could not be attributed to a decreased viability of spleen lymphocytes from old mice in in vitro culture conditions, to different PHA dose requirements, or to longer periods of culture time for old mouse spleen cells to reach maximal activity. Culture of cells for 24–48 h prior to addition of PHA gave patterns of thymidine incorporation similar to those of freshly prepared spleen cells. The elimination of red blood cells by a brief hypotonic shock significantly increased thymidine incorporation in spleen lymphocytes from both young and old mice.

Specific antisera were prepared against the Bowman-Birk trypsin inhibitor and four other trypsin inhibitors of low molecular weight isolated from soybeans (Glycine max L. cv. Tracy). These antisera were used to detect the presence and amount of the inhibitors in: (a) seeds and protein extracts of soybean meal; (b) seedlings; and (c) the water surrounding the seeds and roots of seedlings. Lectin activities in seeds, seedlings, and water were also determined at the same time as the protease inhibitor activities. By competitive inhibition of immunoprecipitation, the combined five low molecular weight protease inhibitors were found to constitute the following percentages of proteins (w/w): 6.3% in defatted soybean meal; 8.1% of the protein extracted from the meal by a buffer of pH 8.6; 8.3, 14.7, 15.2, 16.1, 17.2, and 18.9% of the protein in a lyophilisate of water in which seeds were incubated for 4, 8, 12, 16, 20, and 24 hours, respectively; 8.2% in a lyophilisate of water in which roots of seedlings grew for 20 days; 1.5% in cotyledons; and less than 0.1% in epicotyls, hypocotyls, and roots of 12-day-old seedlings. Hemagglutination activities, expressed as the lowest amount of protein required to give a positive agglutination of 0.2 ml of 2% rabbit red blood cells, were as follows: purified soybean lectin, 0.08 μg; lyophilisate of water in which seeds were incubated for 4, 8, 12, 16, 20, and 24 hours, 10, 2.5, 5, 5, and 2.5 μg, respectively; lyophilisate of water in which roots grew for 20 days, 5 μg; 12-day-old cotyledons, roots, epicotyls, and hypocotyls, 12.5, 100, >1,000, and >500 μg, respectively. The results indicate that a large amount of protease inhibitors as well as lectins are released from seeds during the first 8 hours of imbibition. Neither lima bean trypsin inhibitor (mol wt, 10,000) nor Kunitz soybean trypsin inhibitor (mol wt, 21,500) showed competitive inhibition in tests with antisera against low molecular weight soybean protease inhibitors.

We molecularly cloned and analyzed an N-tropic ecotropic retrovirus induced with iododeoxyuridine from RFM/Un mouse cell cultures. Based on the restriction map, the RFM/Un virus appears to be indistinguishable from other induced N-tropic retroviruses. A nucleotide sequence analysis of the long terminal repeat of an infectious clone revealed structural features characteristic of murine type C retrovirus long terminal repeats. The U3 region of the RFM/Un virus long terminal repeat, however, contained no short sequence duplication or insertion found in other murine leukemia virus isolates.

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.

Resume Le RNA genomique 35S des retrovirus de type C est present au niveau des polysomes 4 heures apres infection dans des lignees cellulaires non differenciees et differenciees derivees du teratocarcinome de la souris. Une proteine specifique virale de 65.000 daltons est alors produite dans les cellules infectees. Ces resultats montrent que le genome viral fonctionne comme RNA messager pour la synthese du precurseur des proteines gag Pr65 a une etape precoce du cycle infectieux des retrovirus ecotropiques murins.

Nucleotide sequences of the region that corresponds to the site of tRNA primer binding for a functional retrovirus were determined in five murine leukemia virus-related sequence clones from mouse chromosomal DNA, which contain a unique 170 to 200-base-pair additional internal segment in the long terminal repeats. The 3'-terminal 18-nucleotide sequence of a major glutamine tRNA isoacceptor was found to match well with the putative primer binding site: 18 of 18 in three clones, 17 of 18 in one clone, and 16 of 18 in one clone. This implies that most of these endogenous proviral sequences of the mouse genome, if replicated as retroviruses, will be different from ecotropic murine leukemia viruses and most mammalian type C retroviruses in using glutamine tRNA, rather than proline tRNA, as a primer.

Histidyl transfer ribonucleic acid (tRNA) from 17 day mouse placenta was fractionated into six components by reversed-phase plaskon chromatography. Codon specificity of three components (tRNA2His, tRNA4His and tRNA6His) was studied by binding to mouse liver ribosomes in the presence of poly(AC) and poly(ACU). All three tRNAs respond equally to each polymer indicating a dual in vitro recognition of CAC and CAU. At present the data do not support changes in coding as a rationale for the quantitative variation shown by placental His-tRNAs during pregnancy.

A recombinant DNA clone, named AL10, that contains murine leukemia virus (MuLV) related sequences was isolated from BALB/c mouse chromosomal DNA and examined in detail. Restriction endonuclease mapping revealed that the 10.5 kbp EcoRI insert consists of a 3.6 kbp left flanking cellular DNA region and a 6.9 kbp MuLV-related region that has a typical proviral LTR-gag-pol-env structure up to the EcoRI site in the env gene region. Comparison of the AL10 map with ecotropic and xenotropic virus isolates revealed many common restriction sites in the LTR and pol gene regions, but much fewer in the leader and gag regions. A stretch of 1,700 nucleotides containing the cellprovirus junctional region was sequenced and revealed transcriptional consensus signals and other structural features characteristic of MuLV LTRs, as well as two distinctive features: (a) a sequence of approximately 170 bp with direct and inverted terminal repeats not seen in infectious MuLV LTRs was identified in the U3 region between the "enhancer" region and the "CAT" box. This novel segment or its homologous sequences appear to be present in most of the endogenous MuLV-related LTRs and in other chromosomal locations of the mouse (b) The tRNA primer binding site is not complementary to proline tRNA, the primer for all known MuLVs, but is a 17/18 match with rat glutamine tRNA. The integration site of AL10 provirus was in a unique DNA region but contained an "Alu"-like short interdispersed repeat in the 5' adjacent cellular region. The AL10 proviral integration found in BALB/c was also apparent in RFM, AKR and SENCAR mouse cells but not in cells of NFS/N, C3H, HRS/J, SC-1, and a California Lake Casitas wild mouse.

The DNA polymerase activities in the spleens of young adult (3–8 months) and senescent (24–30 months) female BALB/c mice have been examined. Comparisons were made by direct assay of activated DNA-dependent and (rA) n · (dT) n - dependent activities in extracts from cytoplasmic and nuclear fractions and also by sucrose gradient sedimentation analysis of the two major forms of DNA polymerases in the subcellular fractions. The high molecular weight DNA polymerase activity was equivalent in the spleens of young and old BALB/c mice. The low molecular weight DNA polymerase activity was decreased in the spleens of old BALB/c mice. This decreased activity was reflected most markedly in the nuclear fraction of the spleens. Mixing experiments did not indicate the presence of inhibitors in the nuclear extract from the old mouse spleens. Stepwise extractions of isolated nuclei with increasing NaCl concentrations showed consistent results without revealing any difference in extractability of the low molecular weight DNA polymerase from the old mouse chromatin. Isolated low molecular weight DNA polymerase preparations from the young and old BALB/c mouse spleens are similar in chromatographic migration, sucrose gradient sedimentation, heat lability in vitro and K m of substrates. The low molecular weight DNA polymerase activity was not found to decline in livers and kidneys of 24 to 30-month-old BALB/c mice, nor in spleens of 30 to 34-month-old BC3F 1 mice.

Different mouse plasma cell tumors have been shown to produce different homogenous species of myeloma immunoglobulin in large amounts and are currently thought to consist of monoclonal cells ( Potter et al. , 1965 ; Lennox and Cohn, 1967 ). We have previously observed multiple isoaccepting tRNA's in a tumor of this type, MOPC 31C ( Yang and Novelli, 1968a ). In the present work, comparative studies of isoaccepting tRNA's by co-chromatography on a reversed-phase chromatographic column have been performed on cell lines of transplantable mouse plasma cell tumors that produce different kinds of myeloma immunoglobulin. Similar chromatographic patterns were observed for most of the aminoacyl-tRNA's examined in these tumors. A marked difference, however, was found between the seryl-tRNA's of an immunoglobulin-A producer (MPC 62) and those of immunoglobulin-G producers (MPC 47 and MOPC 31C).

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).

Characteristic long terminal repeats (LTR) of approximately 700 and 750 by were found, respectively, in the two classes (polytropic and modified polytropic) of murine leukemia virus (MuLV)-related nonecotropic nonxenotropic proviral sequences in eight individual molecular clones of RFM/Un mouse chromosomal DNA fragments. Three proviral clones, two polytropic and one modified polytropic, contained sequence deletions in the viral structural genes. Nucleotide sequence analysis revealed that 7-bp direct repeats occur at both ends of deleted sequences in intact structures and one of the repeats remains in genomes with the deletion. Specifically, the deleted sequences were a 1487-bp- gag-pol sequence with ACTGCCC repeat, a 113-bp mid- pol sequence with CAGGCAA repeat, and a 1811-bp env sequence with GGTCCAG repeat. The same specific sequence deletions were found in both classes of MuLV-related proviral structures. Examination of chromosomal DNA from eight inbred laboratory mouse strains and six wild mouse spacies showed that a minor population of proviruses with these specific deletions were present in Mus musculus and Mus spretus , all of which contain prominent 700-bp LTR polytropic proviral structures. The 750-bp LTR modified polytropic proviral structures were phylogenetically more restricted, being equally predominant in Mus musculus domesticus mice, but minor to undetectable in Mus spretus subspecies, and absent in other wild mouse populations.

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.

Using 125I-labeled red kidney bean phytohemagglutinin (125I-PHA), we have found that spleen cells from old BC3F1 mice bind this plant mitogen equally well, if not better, than spleen cells from young BC3F1 mice, although PHA-induced blastogenesis of spleen cells from old mice is sharply reduced. Analyses demonstrated that there is neither significant alteration of binding affinity nor decreased total number of membrane receptor sites for PHA in senescing mouse spleen cells. The amount of PHA which was initially bound to spleen cells in serum-free medium appeared to be insufficient for a subsequent full stimulation of blastogenesis ([3H]thymidine incorporation) in either young or old mouse spleen cells; when washed free of unbound extracellular PHA and upon cultivation in serum-containing culture medium, spleen cells rapidly released more than 90% of the bound PHA. Also, temperatures which change cell membrane morphology played a significant role in the binding and retention of PHA. However, no difference was observed between young and old mouse spleen cells in all these phenomena of PHA-cell membrane interaction.

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.

Publisher Summary This chapter discusses the restriction of murine leukemia viruses by Fv-1—that is, a model for studying host genetic control of retroviral gene movement and leukemogenesis. The Fv-1 locus is known as a genetically determined host-resistant factor to murine viral leukemogenesis. With the development of an in vitro cell culture system to define the host range of different murine leukemia virus isolates, this genetic factor was found to have two kinds of alleles, Fv-1n and Fv-lb, which specify resistance to B-tropic and N-tropic viruses, respectively, in a dominant fashion. The locus has been mapped adjacent to the Gpd gene in chromosome 4 of the mouse. It is significant that all mouse strains, wild mice included, have an Fv-1 locus and that all naturally occurring or chemically induced endogenous ecotropic murine leukemia viruses are susceptible to restriction by one allele or another. The chapter summarizes the basic facts that are important for further investigation of the molecular mechanism of Fv-1 restriction.

Publisher Summary This chapter discusses the analyses of molecular clones of an endogenous ecotropic virus of BALB/c mice, WN1802N, to identify possible changes that occur in the long terminal repeat (LTR) region. In these analyses, covalently-closed circular DNA intermediates were isolated from cell cultures 48 hours post-infection and cloned by constructing Hin dIII recombinants with Charon 9 bacteriophage. The DNA of 15 independent isolates was first analyzed by Hin dIII restriction and endonuclease digestion, agarose gel electrophoresis, and Southern blot hybridization. Seven of the clones (λWN7, λWN12, λWN26, λWN30, λWN36, and λWN41) have an insert size that corresponds approximately to the complete MuLV genome containing two LTRs. There are four different size LTRs in this population of clones. The most abundant size class is 520 bp and it is probable that this is the size of the endogenous provirus LTR. Seven of these are infectious. There are three isolates with a 600-bp LTR, one of which is infectious, one with 570 bp, and one with 670 bp. Neither of these last two are infectious, but it is yet to be determined whether this is related to the LTR.

Soluble extracts of mouse cells with Fv-1 n or Fv-1 b gene alleles specifically and reciprocally inhibit infection of B- or N-tropic mouse leukemia viruses in permissive cell cultures. NB-tropic virus infection was not inhibited by either cell extract. Extracts from Fv-1 - cells did not inhibit infection by the three virus host-range types, but N- or B-tropic virus infection of Fv-1 - cells was inhibited by extracts of the nonpermissive cells, and Fv-1 nb cell extracts inhibited both viruses. The maximum degree of inhibition was 50-80% as determined by immunofluorescent or plaque assays, with extracts containing up to 500 μg/ml of nonpermissive cell protein. The inhibitor(s) is relatively unstable since activity is lost after 2 hr at 37° or 30 min at 56°. The inhibitor(s) was most effective if added 2 hr before or within 2 hr after infection, did not react with the virus directly, inhibit virus attachment, or inhibit the normal cell functions tested. These results indicate that nonpermissive mouse cells contain a product, possibly determined by the Fv-1 gene, which inhibits some early postpenetration event(s) in leukemia virus infection.

Publisher Summary Radiogenic myeloid leukemia in RFM/Un mice is a potentially valuable model system to study specific chromosomal rearrangements of retrovirus genes. One of the key aspects of this system is that infection by an endogenous virus is strongly restricted; therefore, an extracellular spread of the virus is not likely to play a role in the formation of additional integrated viral genomes, as is the case in other well-studied murine retrovirus models. The role RFV plays in radiogenic myeloid leukemia is uncertain. As with other endogenous ecotropic retroviruses, the virus does not transform cells in culture and does not appear to accelerate leukemogenesis in vivo . A downstream promotion model for leukemogeneis by avian leukosis virus does provide an attractive hypothesis for the involvement of RFV in the myeloid leukemia. Studies indicate that there is a single locus for the inducible ecotropic provirus that resides on chromosome 5. The myeloid leukemic cell line clearly has additional copies of the ecotropic provirus.

Unique 4S RNA species from AKR mouse embryo cells hybridize with AKR murine leukemia virus and avian myeloblastosis virus 35S RNAs in vitro . Analyses by reversed-phase column chromatography indicate that the major 4S species that hybridize with the two viral RNAs are probably the same. A 4S RNA species with similar chromatographic properties is a major component of the AKR viral 4S RNA which associates with the viral 70S RNA in vivo .

A standardized bioassay for transfer of Fv-1 gene-specific resistance to N-tropic and B-tropic murine retroviruses was developed using X plaque reduction in SC-1 (Fv-1-) cells inoculated with virus. Testing of subcellular fractions of restrictive cells showed that the resistance transfer activity was present in the cytoplasmic (microsomal and cytosol) fractions. The activity of the cytoplasmic extract was destroyed by treatment with ribonuclease, but not with deoxyribonuclease or proteases. RNA prepared by phenol-chloroform extraction of mouse tissues, including embryos and livers of weanling mice, transferred Fv-1 locus-specific resistance into DEAE-dextran-treated SC-1 cells. The activity of isolated RNA preparations against virus of the appropriate host-range type has been demonstrated to correspond to the Fv-1 genotypes of the cell sources. The specific transfer of resistance with cellular RNA was effective within a 5- to 6-h period from 2 h before to 4 to 5 after virus infection. Sucrose gradient centrifugation of the RNA showed that the activity sedimented as a broad peak, with an apparent maximum in the 22S region. Affinity chromatography of whole-cell RNA on polyuridylic acid-Sepharose tended to separate more activity into the polyadenylic acid RNA fraction than the non-polyadenylic acid RNA fraction. Except for the reciprocal inhibitory activity for the two host-range virus types, the RNAs of Fv-1n and Fv-1b specificities showed similar properties in all aspects studied.

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.

Flow microfluorometric assay with a Cytofluorograf model 4801 in combination with immunofluorescence was applied to the quantitative assay of cells exogenously infected with mouse leukemia viruses and to the chemical induction of virus in AKR cells. The Cytofluorograf provides sensitivity equal to the visual method and is capable of assaying up to 5000 cells/sec with specificity equivalent to that of the direct visual immunofluorescence assay, thereby providing a large, statistically valid sampling in a fraction of the time required by visual counting. Flow microfluorometry also provides a method of quantitatively resolving fluorescent cell populations on the basis of relative size and degree of fluorescence.

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.

We analyzed 15 recombinant DNA clones of the unintegrated closed circular DNA intermediate of the BALB/c endogenous ecotropic murine leukemia virus WN1802N. Thirteen of these clones had an insert which corresponded to the complete murine leukemia virus genome. Of these, six contained a single long terminal repeat (LTR) and seven contained two LTRs. The viral genomes in nine clones had an LTR of 520 base pairs (bp), one had an LTR of 570 bp, three had an LTR of 600 bp, and one had an LTR of 670 bp. Restriction endonuclease analysis demonstrated that the size variability resides in the U3 region. Seven of eight clones which yielded infectious virus by DNA transfection had the 520-bp LTR, and the other had a 600-bp LTR. More detailed examination of plasmid subclones of three isolates with different-sized LTRs revealed that the approximate position which varies in the U3 region corresponds to the 72-bp repeat region of Moloney sarcoma virus. Possible consequences of these variations are discussed.

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.