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Three isomeric forms of a cystamine-containing chloroethylnitrosourea, N,N'-bis[N-(2-chloroethyl)-N-nitrosocarbamoyl]cystamine (CNCC), have been identified and separated by high pressure liquid chromatography. Isomer S, 3,3'-bis[N-(2-chloroethyl)-N-nitrosocarbamoyl] ethyl disulfide, was significantly less cytotoxic than isomer C, 1,1'-bis [N-(2-chloroethyl)-N-nitrosocarbamoyl] ethyl disulfide, or isomer M, 1,3'-bis[N-(2-chloroethyl)-N-nitrosocarbamoyl] ethyl disulfide, in either a human Namalva lymphoblastoid or a rat Walker 256 carcinoma cell line. Both isomers S and C inhibited DNA synthesis at a 50 microM concentration. A structural analysis of the isomeric forms suggested that bioreduction of the disulfide bond would permit both isomers to produce isocyanate moieties which would carbamoylate intracellular proteins and depress nucleic acid synthesis. The reduced cytotoxic potential of isomer S is consistent with a prolongation in the half-life of production of alkylating carbonium species that lack the capacity to cross-link macromolecules. Overall, the relative position of the NH group within each of the nitrosourea isomers appears critical to the biological properties of the drug.

The presence or absence of the Giardia lamblia double-stranded RNA virus (GLV) was surveyed among 38 axenic isolates of G. lamblia derived from both humans and animals. Of the 28 isolates lacking the virus, 19 could readily be infected by the virus. The remaining 9 isolates proved to be resistant to GLV infection even when the ratio between virus to parasite reached as high as 10(6) to 1. Evidence is also presented indicating that there are at least two "Portland 1" strains being used by the current scientific community, one containing the virus and the other lacking the virus.

A Walker 256 rat mammary carcinoma cell line (WR) resistant to bifunctional nitrogen mustards has been shown to have an approximate twofold increase in bulk glutathione-S-transferase activity compared to the parent cell line. Substrate specificity studies suggest that higher levels of Yb subunit contribute to the increased activity. By exposing WR cells to additional chlorambucil, either as a single concentration (50 micrograms/ml) or at 5 micrograms/ml for 10 days, transferase activity was further increased by up to three times the normal WR level. By using colony-forming assays, mitotic index depression, or trypan blue exclusion, the increased transferase activity could be correlated with an increase in resistance of these cells to either subsequent chlorambucil or a different bifunctional nitrogen mustard, phosphoramide mustard.

A significant number of offspring from brother-sister matings of NIH-Okamoto-Aoki spontaneously hypertensive rats (SHRs) were found to be normotensive at 20 weeks of age. Over 20% of the animals that were hypertensive at this age had mild-to-moderate unilateral hydronephrosis at the time of sacrifice. In over 90% of the rats that did not develop hypertension spontaneously, ligation of one ureter raised blood pressure above 150 mm Hg within 2 weeks. In those rats made hypertensive by obstructing one ureter and in those that developed hypertension with accompanying naturally occurring hydronephrosis, subcutaneous implants of fragmented renal medulla from unrelated normal rats decreased blood pressure to normotensive levels. In contrast, medullary implants had no significant effect in rats developing hypertension spontaneously without hydronephrosis. Renal inner medullary plasma flow was low in the obstructed kidneys of hydronephrotic hypertensive SHRs but was elevated in the kidneys of nonhydronephrotic hypertensive SHRs. The hypertension in hydronephrotic SHRs appears to be related to an impairment of the antihypertensive function of the renal medulla. Such an impairment of medullary antihypertensive function does not appear to play a significant role in the hypertension in SHRs without hydronephrosis.

Hybridomas were produced by fusing the NS1 mouse myeloma line, which does not produce mouse heavy chain Ig, with human peripheral B lymphocytes from a normal individual. Two vigorously growing colonies from this fusion were found to secrete human Ig heavy chains and were recloned. Two secondary clones, which secreted human chains, were again recloned. Among the tertiary clones, two were identified which produced intracellular human Ig chains, but did not secrete immunoglobulin. These tertiary clones were recloned, generating 6 quaternary clones which failed to produce human Ig heavy chains, and 15 quaternary clones which produced intracellular Ig chains. Hybrid clones from each successive subcloning were examined for their human chromosomal content and only those clones which were found to be individually chromosomally distinct, a total of 56 clones in all, were used to analyze the segregation of human chromosomes and human Ig heavy chain synthesis. Results of this study indicate concordant segregation of human Ig heavy chain synthesis and chromosome 14. These studies therefore confirm the previous assignment by C. M. Croce et al. (Proc. Natl. Acad. Sci. USA 1979. 76: 3416) of the genes for human Ig heavy chains to chromosome 14.

A "double-stranded" RNA was identified in the anaerobic, parasitic protozoan Trichomonas vaginalis. Electron microscopic evidence indicated linear double-stranded structure 1.5 micron in length, with no apparent hairpins or loops. Boiling in 30% dimethyl sulfoxide denatured it into single strands of 1.5 micron and shorter fragments. It consists of 23.4% G, 23.4% C, 23.0% A, and 30.3% U and melts at a transition temperature of 81.7 degrees C in 75 mM NaCl and 7.5 mM sodium citrate, pH 7.0, with 7-15% hyperchromicity. The 32P-labeled double-stranded RNA hybridized specifically with T. vaginalis DNA fragments in a single DNA band from EcoRI digest and two DNA bands from HindIII digest. Of the 33 different strains or isolates of T. vaginalis examined, all contained this double-stranded RNA. However, the only two metronidazole-resistant T. vaginalis strains examined thus far (IR78 and 85) contained no detectable double-stranded RNA, although the corresponding DNA sequence was present. DNA fragments of Escherichia coli and Giardia lamblia did not hybridize with the double-stranded RNA. But DNA fragments of a metronidazole-sensitive Tritrichomonas foetus hybridized specifically with the double-stranded RNA, even though this organism does not contain the double-stranded RNA itself.

High molecular weight DNA samples free of contaminating proteins or RNA were obtained from Tritrichomonas foetus or Trichomonas vaginalis by lysing the cells in 4 M guanidinium thiocyanate before centrifuging in CsCl density gradient and then purifying the DNA band by NACS-37 column chromatography. The bulk DNA from either organism acted as a single component in ion-exchange chromatography, agarose gel electrophoresis, CsCl density gradient centrifugation and thermal denaturation. T. foetus DNA showed a melting temperature (Tm) of 82 degrees C corresponding to a 31% GC content whereas T. vaginalis DNA melted at 84 degrees C to suggest 36% GC. Both DNA samples demonstrated 35 to 42% hyperchromicity when fully melted. Cot analysis revealed the presence of repetitive sequences in both DNAs: approximately 46.7% in T. foetus DNA and 53.3% in T. vaginalis DNA. The unique sequences of these two protozoan DNAs are of a similar size of about 2.5 X 10(7) base pairs. Agarose gel electrophoresis of restriction fragments of the two purified DNA samples gave distinct banding patterns that were characteristic of the two species of protozoan parasites.

The double-stranded RNA virus-like particles, found among several independent isolates and cloned strains of Giardia lamblia, have previously been reported to be spheres of 35 nm with a genome of 7 kilobase pairs and a major protein of 100 kDa. The virus is capable of infecting certain virus-free isolates of G. lamblia. Antisera raised in mice against the intact virus did not react with the double-stranded RNA, but reacted strongly with the 100 kDa protein in Western blots. Preincubation of the virus with antisera abolished viral infectivity, whereas the antisera against double-stranded RNA showed only a weak blocking effect. Inclusion of the antiviral sera in the cultures of virus-infected G. lamblia at 10(3)-fold dilution resulted in elimination of the virus from the protozoa. Apparently, the 100 kDa protein is necessary for the initiation of viral infection and possibly subsequent assembly or replication of viral progeny particles.

A partly defined medium was successfully designed for the cultivation of Tritrichomonas foetus, an anaerobic protozoan parasite of cattle. The medium consists of hypoxanthine, uracil, and thymidine as the sole precursors of nucleotides in T. foetus. Elimination of any one of the three precursors from the medium led to cessation of T. foetus growth. The information provided by this medium verifies our previous observations that T. foetus is incapable of de novo purine and pyrimidine synthesis, that hypoxanthine can be converted to AMP and GMP, that uracil is incorporated into all pyrimidine ribonucleotides including UDP-glucose--the precursor of glycogen synthesis, and that thymidine is the only precursor of TMP. The omission of folate from the medium, without affecting growth of T. foetus, also supports our previous finding that the parasite does not have functioning dihydrofolate reductase or thymidylate synthetase. The successful plating of T. foetus on agar plates incorporating the partly defined medium with near 100% plating efficiency makes it possible to isolate T. foetus mutants for further studies of purine and pyrimidine metabolism in this parasite.

The interrelationship of DNA to the nuclear matrix is integral to the organization of chromatin within the nucleus and to the DNA replication process. The influence of nitrosourea and nitrogen mustard interactions with the nuclear matrix were studied in log phase HeLa cells. Alkylation of the nuclear matrix by chlorozotocin (CLZ) or 1-(2-chloroethyl-3-cyclohexyl)-1-nitrosourea (CCNU) was 1.58 and 1.27 pmoles drug/micrograms protein, respectively, whereas carbamoylation by CCNU was 32.5 pmoles/micrograms. These constituted approximately 30% of the total (nuclear) drug modifications. The structural matricin fibrillar components of the matrix were alkylated and carbamoylated twice as much as the ribonuclear protein elements (RNP). However, when alkylations are measured per microgram of protein, the ratio of covalently bound drug to RNP:matricin was 1.2 for both CLZ and CCNU. The RNP:matricin carbamoylation ratio for CCNU was 0.9. The importance of DNA and matrix protein alkylations to the process of reassociation was studied. Under control conditions, in vitro, approximately 80% of the DNA was associated with the matrix at a protein:DNA ratio (micrograms for micrograms) of 50:1. Direct alkylation or carbamoylation of the matrix proteins did not affect these DNA-protein interactions. However, using in vitro alkylated DNA (1 alkylation/10(2) base pairs), there was a 60% reduction of the alkylated nucleic acid bound to the matrix at the same protein: DNA ratio. The reduced binding of DNA to matrix may be a function of interference with the DNA recognition sites by alkylation of specific bases. The interference of DNA-matrix association by DNA alkylation may contribute to the cytotoxic activity of these antineoplastic agents.

The dsRNA virus which infects some strains of Giardia lamblia has been purified and characterized with respect to its effect on growth of the parasite. Extensive purification of the virus from G. lamblia growth medium was accomplished by Millipore filtration and two successive CsCl gradient centrifugations. The purified virus possessed a single major protein species of 100,000 molecular weight. Effects of the extensively purified virus on growth of the virus-free parasite were studied. A cloned WB strain, sensitive to the viral infection, and a cloned E-9/M strain, resistant to the infection, were studied. With the WB strain, infection can occur at a ratio as low as 10 viral particles per organism. As the virus to parasite ratio increased, the rate of growth of the parasite decreased and the percentage of parasites not adhering to the culture tube wall also increased. These nonadhering cells, which differed from the nonadhering cells under normal growth conditions, were unable to divide. They contained an average number of 500,000 viral particles per cell which may be the threshold intracellular density of viral particles arresting the growth of G. lamblia. The results also suggest that the specific consequence of viral infection, even at extremely high multiplicity of infection, is not lysis of G. lamblia trophozoites but cessation of growth.

A Walker 256 rat carcinoma cell line (WR) with acquired resistance to nitrogen mustards has been found to lack cross-resistance to nitrosoureas. Although total cellular glutathione pools were similar in the parent (WS) and resistant cell lines (WS, 2.5 X 10(-6); WR, 2.0 X 10(-6) mol/mg protein), glutathione reductase activity was 3.98 in WR compared to 8.67 nmol reduced nicotinamide adenine dinucleotide phosphate oxidized per microgram protein per min in WS cells. Treatment of cells with a carbamoylating nitrosourea, N,N'-bis(trans-4-hydroxycyclohexyl)-N'-nitrosourea, produced a dose-dependent inhibition of glutathione reductase and depletion of thiols in both cell lines. The drug caused no direct DNA strand breakage, but a differential mitotic spindle-chromosome stain showed that spindle formation was inhibited in WR cells at N,N'-bis(trans-4-hydroxycyclohexyl)-N'-nitrosourea concentrations of greater than 50 microM. In WS cells, mitotic figures were still visible at 100 microM. Chromosomal damage was expressed in both cell lines at concentrations of 25 microM. The number and extent of these aberrations were greater in WR than WS. Observed karyotypic abnormalities included polyploidy, chromosome decondensation, and endoreduplication. In interphase cells, transmission electron microscopy showed that the most prevalent drug-induced lesions included (a) disappearance of plasma membrane filopodia, (b) appearance of membrane blebbing, and (c) development of irregular crescent-shaped nuclei. These morphological and cytogenetic changes correlate with cytotoxic responses of these cell lines to N,N'-bis(trans-4-hydroxycyclohexyl)-N'-nitrosourea and would be consistent with drug-induced inhibition of glutathione reductase.

Estramustine is cytotoxic in HeLa and Walker 256 carcinoma cells (with or without acquired resistance to nitrogen mustards) at concentrations equivalent to other alkylating agents. Even at lethal estramustine levels, no damage to DNA occurs. Instead, a disproportionately high amount of intact estramustine binds hydrophobically to the structural proteins of the nucleus, the nuclear matrix. In HeLa cells, estradiol receptors are absent, and estradiol per se is not toxic. Thus, estramustine has a mechanism of action distinct from that of steroids and alkylating agents and may induce cytotoxicity through interactions with the proteins of the nuclear matrix.

A linear 5.5-kilobase double-stranded RNA, identified in many strains and isolates of the parasitic protozoan Trichomonas vaginalis in a previous study, is found largely intact in ribonuclease-treated homogenates of the parasite. It can be pelleted with membranes from the homogenate at 12,500 X g and further purified in CsCl buoyant density-gradient centrifugations. The purified sample contains the double-stranded RNA as well as one major protein with an estimated molecular mass of 85 kDa in NaDodSO4/PAGE. Electron microscopic examinations indicated the presence of icosahedral virus-like particles of 33-nm diameter in the purified preparation. The exact location of the virus in T. vaginalis is not clear, except that it is not found in the nuclear fraction and is probably membrane-bound. No free virus can be recovered from the culture medium of T. vaginalis, and no successful infection of virus-free T. vaginalis strains by purified virus has yet been accomplished. There is no viral genomic sequence identifiable in host DNA. So far as we know, it is the first time a double-stranded RNA virus has been identified in a protozoan.

The structural gene encoding human delta-aminolevulinate dehydratase has been assigned to the long arm of chromosome 9 by somatic cell hybridization techniques using murine erythroleukemia-human fibroblast somatic cell hybrids. Dimethyl sulfoxide induction of erythroid differentiation in these hybrid cells resulted in a 3 to 12-fold increase in the levels of total delta-aminolevulinate dehydratase. Human delta-aminolevulinate dehydratase was detected by an immunodiscrimination assay using polyclonal mouse anti-human delta-aminolevulinate dehydratase antibodies. Of four primary hybrid clones, each from an independent fusion, one hybrid line, XX-8, was positive for human delta-aminolevulinate dehydratase. Examination of 23 secondary, tertiary, and quaternary XX-8 subclones revealed that the expression of the human isozyme segregated with human chromosome 9q, confirming the provisional regional assignment made by classical linkage studies. One positive quaternary clone, XX-8-H21-H7-2, expressed human delta-aminolevulinate dehydratase activity and contained only human 9q13----qter. In addition, studies of tertiary and quaternary subclones from two series, XX-8-A31 and XX-8-H21-H7, indicated that murine regulatory factors increased the human as well as the murine enzymatic activity following induction of erythroid differentiation.

The effects of steroid-induced modifications of chromatin structure on the extent and sites of chloroethylnitrosourea binding to chromatin were studied using log-phase HeLa cells. The cells were exposed to 0.1 to 2.0 microM hydrocortisone for 22 hr; this resulted in depressed DNA synthesis while transcriptional activity was stimulated. Hydrocortisone had no effect upon cellular or nuclear uptake of the two nitrosoureas under study, 0.6 mM chlorozotocin or 1-(2-chloroethyl-3-cyclohexyl-1-nitrosourea). Both drugs were found to alkylate transcriptional chromatin preferentially, as demonstrated by DNase II and DNase I digestion. This alkylation was stimulated 2-fold by the same micromolar concentrations of hydrocortisone, 0.1 to 2.0 microM, which stimulated transcription. The extent of nuclear RNA alkylation, determined using RNase T2 as a probe, was found to contribute less than 20% of total chromatin alkylation and was unaffected by steroid pretreatment. Instead, the increased alkylation within these chromatin subfractions was attributed to a steroid-induced alteration of chromatin structure. Electron microscopic examination of HeLa nuclear morphology revealed a hydrocortisone-induced disaggregation of nuclear membrane-associated heterochromatin resulting in a more heterogeneous, less condensed distribution of chromatin. Such data are consistent with a relaxation of the supercoiled chromatin structure, resulting in increased transcription and increased accessibility of potential target sites for nitrosourea alkylation.

An isolate of Giardia lamblia infected with the double-stranded RNA virus (GLV) has two major species of RNA that are not present in an uninfected isolate. One of these species is the previously characterized double-stranded RNA genome of GLV (1). The second species of RNA appears to be a full length copy of one strand of the double-stranded RNA genome. This full length single-stranded RNA is not present in viral particles isolated from the growth medium. The cellular concentration of the single-stranded RNA changes during exponential and stationary phases of cell growth in a fashion consistent with a viral replicative intermediate or mRNA. The single-stranded species does not appear to be polyadenylated.

Nucleic acid samples purified from trophozoites of Giardia lamblia Portland I strain contain an ethidium-stainable band that comigrates with 7.0 kilobase DNA in agarose gel electrophoresis. The band was degradable by alkali, ribonuclease A and ribonuclease T1, but the susceptibility toward the ribonucleases decreased with increasing ionic strength, suggestive of double-stranded RNA (dsRNA). This identification was confirmed by electron micrographs of the purified samples, which showed linear double-stranded structures with an estimated average length of 1.5 micron. In crude homogenates of G. lamblia, this dsRNA was protected against added ribonuclease A but disappeared upon adding sodium dodecyl sulfate or proteinase K. Differential centrifugations suggested an association of the dsRNA with the nuclear fraction, but it was freed to the 109,000 X g pelletable fraction with increasing homogenization. The dsRNA was purified by CsCl buoyant density gradient centrifugations in a distinct band with a rho value of 1.368 g ml-1. Electron microscopy revealed spherical virus-like particles (VLP) with a diameter of 33 nm. VLP of similar shape and size were also identified in the nuclei of sectioned G. lamblia trophozoites. VLP yield a major protein with an estimated molecular weight of 66,000 in sodium dodecyl sulfate polyacrylamide gel electrophoresis. VLP are abundant in the culture media of stationary-phase G. lamblia Portland I strain and are able to infect the G. lamblia WB strain, which is free of the virus. There is no sequence homology between the dsRNA and the nuclear DNA of G. lamblia and thus no apparent integration of viral genome into host DNA.

It has previously been shown that there are extensive idiotypic cross-reactions among antiphenylarsonate antibodies of A/J mice. The present work indicates that administration, into normal, adult A/J mice, of rabbit antiidiotypic antibody directed to A/J antiphenylarsonate antibody suppresses almost completely the subsequent production of antibody of the corresponding idiotype. No effect was noted on the formation of antibodies to the protein carrier or of antiphenylarsonate antibody of a different idiotype. The data are consistent with central suppression of production of the idiotypic antibody mediated through interaction with immunoglobulin receptors on lymphocytes.

In order to clarify the cellular events that precede the onset of immunological competence in the mouse, we have characterized and quantitated the lymphoid cells of the spleen as a function of age. Our results show that T cells and B cells both appeared in the spleens of Swiss-L mice as early as the 15th-16th day of gestation. Antigen-binding cells specific for each of three different antigens were also first detected during this same 24 h interval. The B cells and three varieties of antigen-binding cells increased in number rapidly and in parallel until about 1 wk after birth. The T cells, which were more numerous than B cells at first, increased in number somewhat more slowly. Coincident with the onset of response to antigen, there was a further increase in B cell numbers and a decrease in the T cell to B cell ratio. The capacity to respond to antigen by cellular proliferation and synthesis of antibody did not arise until about 2 wk after birth although there were no quantitative changes in the total numbers of T cells, B cells, and antigen-binding cells between 1 and 2 wk of age. Some qualitative change, such as the functional maturation of an antigen-reactive cell, may be required during this interval for the onset of this immunological response. Although the numbers of antigen-binding cells present in fetuses and young animals were smaller than in adults, we have as yet been unable to detect any restriction in the variety of specificities that can be expressed in fetuses, either in the kinds of antigens bound or in the range of avidities with which a single antigen is bound.

Antisera were prepared in rabbits against anti-p-azobenzoate antibodies of an A/J and a BALB/c mouse and anti-p-azophenylarsonate antibodies of an A/J mouse. After appropriate absorption the antisera reacted with the anti-hapten antibody of the donor mouse but, by sensitive quantitative tests, not at all with other components of the hyperimmune serum or with preimmune serum of the donor mouse. The absorbed antiserum therefore appeared to be specific for idiotypic determinants. Nearly all idiotypic specificities identified in the serum of the donor were also present in the serum of other mice of the same strain, immunized against the same hapten group, but not in mice immunized with a different hapten. In each case the antibodies of the donor mouse reacted most effectively on a weight basis with antiidiotypic antiserum. Cross-reactions were observed among different strains of mice but homologous anti-bodies reacted most effectively with antiidiotypic antisera. C57/BL and DBA antisera contained very low concentrations of specificities present in the A/J and BALB/c antibody populations; antibodies of A/J and BALB/c antisera are more closely related to one another. The results indicate that idiotypic specificity may provide a genetic marker for the variable regions of immunoglobulin polypeptide chains.