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The human BK polyomavirus (BKPyV) is latent in the kidneys of most adults, but can be reactivated in immunosuppressed states, such as following renal transplantation. If left unchecked, BK polyomavirus nephropathy (PyVAN) and possible graft loss may result from viral destruction of tubular epithelial cells and interstitial fibrosis. When coupled with regular post-transplant screening, immunosuppression reduction has been effective in limiting BKPyV viremia and the development of PyVAN. Antiviral drugs that are safe and effective in combating BKPyV have not been identified but would be a benefit in complementing or replacing immunosuppression reduction. The present study explores inhibition of the host DNA damage response (DDR) as an antiviral strategy. Immunohistochemical and immunofluorescent analyses of PyVAN biopsies provide evidence for stimulation of a DDR in vivo. DDR pathways were also stimulated in vitro following BKPyV infection of low-passage human renal proximal tubule epithelial cells. The role of Chk1, a protein kinase known to be involved in the replication stress-induced DDR, was examined by inhibition with the small molecule LY2603618 and by siRNA-mediated knockdown. Inhibition of Chk1 resulted in decreased replication of BKPyV DNA and viral spread. Activation of mitotic pathways was associated with the reduction in BKPyV replication. Chk1 inhibitors that are found to be safe and effective in clinical trials for cancer should also be evaluated for antiviral activity against BKPyV.

Many differentiated plant cells can dedifferentiate into stem cells, reflecting the remarkable developmental plasticity of plants. In the moss Physcomitrella patens, cells at the wound margin of detached leaves become reprogrammed into stem cells. Here, we report that two paralogous P. patens WUSCHEL-related homeobox 13-like (PpWOX13L) genes, homologs of stem cell regulators in flowering plants, are transiently upregulated and required for the initiation of cell growth during stem cell formation. Concordantly, Δppwox13l deletion mutants fail to upregulate genes encoding homologs of cell wall loosening factors during this process. During the moss life cycle, most of the Δppwox13l mutant zygotes fail to expand and initiate an apical stem cell to form the embryo. Our data show that PpWOX13L genes are required for the initiation of cell growth specifically during stem cell formation, in analogy to WOX stem cell functions in seed plants, but using a different cellular mechanism.

An indazole based series of glucocorticoid receptor agonists is reported. The SAR exploration of this scaffold yielded compounds with nanomolar affinity for the glucocorticoid receptor with indications of selectivity for the preferred transrepression mechanism; in vivo efficacy was observed in the mouse LPS induced TNFα model for compound 28.

The introduction of an aryl ring onto the 4-position of the C-6 benzyl amino group of the Cdk inhibitor roscovitine (2), maintained the potent Cdk inhibition demonstrated by roscovitine (2) as well as greatly improving the antiproliferative activity. A series of C-6 biarylmethylamino derivatives was prepared addressing modifications on the C-6 biaryl rings, N-9 and C-2 positions to provide compounds that displayed potent cytotoxic activity against tumor cell lines. In particular, derivative 21h demonstrated a >750-fold improvement in the growth inhibition of HeLa cells compared to roscovitine (2).

The introduction of an aryl ring onto the 4-position of the C-6 benzyl amino group of the Cdk inhibitor roscovitine (2), maintained the potent Cdk inhibition demonstrated by roscovitine (2) as well as greatly improving the antiproliferative activity. A series of C-6 biarylmethylamino derivatives was prepared addressing modifications on the C-6 biaryl rings, N-9 and C-2 positions to provide compounds that displayed potent cytotoxic activity against tumor cell lines. In particular, derivative 21h demonstrated a750-fold improvement in the growth inhibition of HeLa cells compared to roscovitine (2).

Cyclosporin A (CsA) was converted into cyclosporin methyl vinyl ketone (Cs-MVK) by either a biocatalytic method utilizing 1-hydroxybenzotriazole-mediated laccase oxidation or by a chemical oxidation using t-butyl hydroperoxide and potassium periodate as co-oxidants. Cs-MVK is a novel, versatile synthetic intermediate that can be used for the preparation of many novel cyclosporin analogues possessing therapeutic potential as immunosuppressive agents.

Background Simian Virus 40 (SV40) infection of growth-arrested monkey kidney cells stimulates S phase entry and the continued synthesis of both viral and cellular DNA. Infected cells can attain total DNA contents as high as DNA Index, DI = 5.0–6.0 (10–12C), with host cell DNA representing 70–80% of the total. In this study, SV40-infected and uninfected control cells were compared to determine whether continued DNA replication beyond DI = 2.0 was associated with rebinding of the minichromosome maintenance (MCM) hexamer, the putative replicative helicase, to chromatin. Method Laser scanning cytometry was used to measure the total expression per cell and the chromatin/matrix-association of two MCM subunits in relation to DNA content. Results MCM2 and MCM3 proteins that were associated with the chromatin/matrix fraction in G1 phase of both uninfected and SV40-infected cells were gradually released during progression through S phase. However, in SV40-infected cells that progressed beyond DI = 2.0, chromatin/matrix-associated MCM2 and MCM3 remained at the low levels observed at the end of S phase. Rereplication was not preceded by an obvious rebinding of MCM proteins to chromatin, as was observed in G1 phase. Conclusions The rereplication of host cell DNA in the absence of the reassociation of MCM proteins with chromatin indicates that SV40 infection induces a novel mechanism of licensing cellular DNA replication. © 2005 International Society for Analytical Cytology

The infection of monkey kidney (CV-1) cells with simian virus 40 (SV40) stimulates the cells into successive rounds of DNA synthesis without an intervening mitosis, leading to the acquisition of a >G2 DNA content. To elucidate the role of small t antigen in cell cycle progression and in viral replication during infection, studies were performed using an SV40 mutant (dl888) that lacks the ability to produce small t. Initially dl888-infected cells move through the first S phase at roughly the same rate as wild-type infected cells. Upon reaching G2, however, the dl888-infected cells progressed to >G2 at a reduced rate relative to wild-type. The slower rate of entry into >G2 of dl888-infected cells is associated with a decrease in total pRb and an increase in the ratio of hypophosphorylated to hyperphosphorylated pRb. The expression of cyclin D1 and p27kip1 were elevated in dl888-infected cells compared to wild-type-infected CV-1 cells. Taken together, these results indicate that small t antigen plays a role in stimulating entry into >G2 in SV40-infected CV-1 cells, possibly by affecting the regulation of key cell cycle proteins.

Simian virus 40 (SV40) infection of quiescent monkey kidney cells stimulates two successive rounds of cellular DNA synthesis without an intervening mitosis. This uncoupling of S phase and mitosis indicates that SV40 modulates pathways regulating the G2-to-M phase transition. To examine the integrity of mitotic initiation pathways in infected cells that have bypassed mitosis, SV40-infected CV-1 cells were treated with okadaic acid (OA), a known inducer of premature mitosis in other cell types. OA treatment triggered the appearance of the mitotic marker MPM-2 in SV40-infected CV-1 cells progressing through either the first (diploid) or second (tetraploid) S phases. These results demonstrate that a subset of mitotic pathways are intact but inactive in SV40-infected cells that have bypassed mitosis and initiated tetraploid S phase.

Multicellular organisms possess pluripotent stem cells to form new organs, replenish the daily loss of cells, or regenerate organs after injury. Stem cells are maintained in specific environments, the stem cell niches, that provide signals to block differentiation. In plants, stem cell niches are situated in the shoot, root, and vascular meristems—self-perpetuating units of organ formation. Plants' lifelong activity—which, as in the case of trees, can extend over more than a thousand years—requires that a robust regulatory network keep the balance between pluripotent stem cells and differentiating descendants. In this review, we focus on current models in plant stem cell research elaborated during the past two decades, mainly in the model plant Arabidopsis thaliana. We address the roles of mobile signals on transcriptional modules involved in balancing cell fates. In addition, we discuss shared features of and differences between the distinct stem cell niches of Arabidopsis.

Simian virus 40 (SV40) infection stimulates confluent cultures of monkey kidney cells into successive rounds of cellular DNA synthesis without intervening mitosis. As an initial step in defining the mechanisms responsible for viral inhibition of mitosis, M-phase-promoting factor (MPF) was examined in SV40-infected CV-1 cells passing from G2 phase into a second S phase. MPF is a serine-threonine protein kinase that is essential for mitosis in eukaryotic cells. In SV40-infected cells exiting G2 phase, there was a reduced amount of MPF-associated H1 kinase activity relative to that of uninfected cells passing through mitosis. Both subunits of MPF, cyclin B and the p34cdc2 catalytic subunit, were present and in a complex in infected cells. In uninfected cultures, passage through mitosis was associated with the dephosphorylation of the p34cdc2 subunit, which is characteristic of MPF activation. In contrast, the p34cdc2 subunit remained in the tyrosine-phosphorylated, inactive form in SV40-infected cells passing from G2 phase into a second S phase. These results suggest that although the MPF complex is assembled and modified normally, SV40 interferes with pathways leading to MPF activation.

Simian virus 40 (SV40) large T antigen (T), a potent dominant oncogene product, forms a specific complex with the human retinoblastoma protein (pRb), a cellular growth suppressor. We have used a recombinant pRb fusion protein (GST-Rb) in combination with extracts from a line of SV40-transformed human lung cells (WI-26 VA4) to develop a simple, non-radioactive assay to rapidly screen for competitive inhibitors of T/pRb binding. We illustrate the use of the assay by demonstrating that several short peptides containing the signature sequence, Leu-X-Cys-X-Glu, can inhibit T/pRb complex formation. In contrast, peptides containing the related motif, Leu-X-Glu-X-Glu, including two peptides derived from the transcription factor E2F, are inactive in this assay. These results show that Glu cannot substitute for Cys in the Leu-X-Cys-X-Glu motif. This assay will facilitate the identification of agents that are inhibitors of T/pRb complex formation and that might exert effects on cellular growth regulation.

The retinoblastoma gene product (Rb), a cellular growth suppressor, complexes with viral and cellular proteins that contain a specific binding domain incorporating three invariant residues: Leu-X-Cys-X-Glu, where X denotes a nonconserved residue. Hydrophobic and electrostatic properties are strongly conserved in this segment even though the nonconserved amino acids vary considerably from one Rb-binding protein to another. In this report, we present a diagnostic computer pattern for a high-affinity Rb-binding domain featuring the three conserved residues as well as the conserved physico-chemical properties. Although the pattern encompasses only 10 residues (with only 4 of these explicitly defined), it exhibits 100% sensitivity and 99.95% specificity in database searches. This implies that a certain pattern of structural and physico-chemical properties encoded by this short sequence is sufficient to govern specific Rb binding. We also present evidence that the secondary structural conformation through this region is important for effective Rb binding.

Flow Cytometry (FC) is a popular method for the simultaneous quantitation of multiple specific proteins and other molecules in each cell of a single cell suspension (1). Laser Scanning Cytometry (LSC) is a closely related technology that provides many of the benefits of FC and offers additional features that allow morphological and biochemical analysis of a cell population (2,3). In both methods, quantitation of endogenous cellular proteins generally requires antibodies against the protein of interest. These antibodies must be either directly labeled with a fluorochrome or detected by a fluorescently-labeled secondary antibody. Using fluorescently-labeled antibodies in combination with fluorescent dyes that quantitatively bind to DNA, both FC and LSC have been valuable in measuring the quantities of specific proteins in relation to cell cycle position (4). In contrast to FC, where a single cell suspension is passed through a fixed-position laser beam, LSC analyzes cells fixed to a surface such as a microscope slide or cover slip. Since the position of the cell on the slide is part of the recorded data, it is possible to visualize the cell after the initial recording event or to subsequently stain the cell with another fluorescent probe and make a second measurement. LSC also allows analysis of cells following biochemical treatments that are incompatible with analysis by FC. In this chapter we describe the value of LSC in analyzing chromatin-associated proteins. Specifically, we describe an in situ fractionation procedure that allows measurement of total and chromatin-associated MCM2, a DNA helicase subunit, as a function of cell cycle position.

Quantitation of specific proteins in complex mixtures is simplified by the use of antibodies directed against the protein of interest. If the specific protein is differentially expressed within a population of cells, quantitation of the protein in cell lysates by immunoblotting will provide an average quantity of the protein per cell. As a result, when lysates of different cell populations are compared, large changes in the amount of a protein in a small percentage of cells cannot be distinguished from small changes in the amount of a protein in a large percentage of cells. Flow cytometric analysis solves this problem by providing a means to measure the amount of a specific protein within each individual cell in a population. A fluorescently labeled probe, usually an antibody, is required for detection of the protein. A single cell suspension is reacted with an antibody and passed through a flow cytometer, which focuses the cell suspension into a stream that intersects a laser beam. As each cell passes through the beam, the fluorescent probe in each cell is excited and photomultiplier tubes register the degree of fluorescence (1). Multivariate analysis, the simultaneous measurement of multiple parameters, is a powerful feature of flow cytometry. When determining the quantity of a specific protein in a cell, the quantity of other molecules in the same cell can also be measured by using additional fluors specifically targeted to other molecules. Examples of second molecules measured simultaneously include DNA (2–4) or additional proteins (5). In addition, measurement of light scatter can provide information about cell size and granularity. Most flow cytometers are capable of measuring six colors plus light scatter, but specialized flow cytometers that are capable of measuring as many as eighteen colors are now available.

A series of A-ring and E-ring analogues of the natural product luotonin A, a known topoisomerase I poison, was evaluated for growth inhibition in human carcinoma and leukemia cell lines. Rational design of structures was based on analogues of the related alkaloid camptothecin, which has been demonstrated to exert cytotoxic effects by the same mechanism of action. When compared to luotonin A, several compounds exhibited an improved topoisomerase I-dependent growth inhibition of a human leukemia cell line.

Lytic infection of African green monkey kidney (CV-1) cells by simian virus 40 (SV40) is characterized by stimulation of DNA synthesis leading to bypass of mitosis and replication of cellular and viral DNA beyond a 4C DNA content. To define mechanisms underlying the absence of mitosis, the expression levels of upstream regulatory molecules of mitosis-promoting factor (MPF) were compared in parallel synchronized cultures of SV40-infected and uninfected CV-1 cells. The DNA replication/damage checkpoint kinase Chk1 was phosphorylated in both uninfected and SV40-infected cultures arrested at G 1 /S by mimosine, consistent with checkpoint activation. Following release of uninfected cultures from G 1 /S, Chk1 phosphorylation was lost even though Chk1 protein levels were retained. In contrast, G 1 /S-released SV40-infected cultures exhibited dephosphorylation of Chk1 in S phase, followed by an increase in Chk1 phosphorylation coinciding with entry of infected cells into >G 2 . Inhibitors of Chk1, UCN-01 and caffeine, induced mitosis and abnormal nuclear condensation and increased the protein kinase activity of MPF in SV40-infected CV-1 cells. These results demonstrate that SV40 lytic infection triggers components of a DNA damage checkpoint pathway. In addition, chemical inhibition of Chk1 activity suggests that Chk1 contributes to the absence of mitosis during SV40 lytic infection.

Livers from wild-type and p53-deficient mice were analyzed for the expression of cell-cycle regulatory proteins in an attempt to determine the mechanism for the increased proliferation of liver cells in p53-deficient miceassociated with enhanced susceptibility to aflatoxin-induced liver cancer. The most striking difference found was a significant reduction of the cyclin-dependent kinase inhibitor p27 k i p 1 in the livers of 3-mo-old p53-/- mice, whereas only small changes were found in the expression of cyclins, cyclin-dependent kinases, and the inhibitors p21 c i p 1 and p16 i n k 4 a . Relative to wild-type liver, the amounts of p27 k i p 1 mRNA were reduced at both 1 and 3 mo, whereas the levels of p27 k i p 1 protein were decreased only at 3 mo. These results identify an uncharacterized link between the expression of p53 and p27 k i p 1 that may involve both transcriptional and post-transcriptional regulation and allow hepatocytes to continue to proliferate after 3 wk of age. We postulate that this increased proliferation leads to increased susceptibility to aflatoxin-induced hepatocarcinogenesis.

Viruses are obligate intracellular parasites that require the host cell replication, transcription, and translation machinery for reproduction. Each viral group provides a unique series of viral-cellular interactions. Studies have provided insight not only into viral replication and control of host functions, but also into cellular functions such as eukaryotic replication, transcription, and translation as well as the regulation of these events. This unit presents a protocol for flow cytometric monitoring of viral infection and quantitating viral-cellular events. The availability of monoclonal and/or polyclonal antibodies directed to both viral and cellular proteins offers the ability to assay a specific molecule in the intact fixed cell and the opportunity to correlate viral events with cellular processes such as progression through the cell cycle.

The infection of permissive monkey kidney cells (CV-1) with simian virus 40 induces G1 growth-arrested cells into the cell cycle. After completion of the first S phase and movement into G2, mitosis was blocked and the cells entered another DNA synthesis cycle (second S phase). Growth-arrested CV-1 cells replicated significant amounts of viral DNA in the G2 phase with the majority of synthesis occurring during the second S phase. When mimosine-blocked (G1/S) infected cells were released into the cell cycle, a major portion of the viral DNA was detected in G2 with the largest accumulation in the second S phase. The total DNA produced per infected cell was 10–12 C with approximately 0.5–2 C of viral DNA replicated per cell. Therefore the majority of the DNA per cell was cellular, 4 C from the first S phase and approximately 4–6 C from the second cellular synthesis phase.

The changes in cell size and total protein were determined for G1-arrested, contact-inhibited CV-1 cells infected with Simian virus 40 (SV40). The assays used were the Biorad total protein assays (Bradford and DC protein assays) on a standard number of cells, total protein as assayed by fluorescein isothiocyanate (FITC) and SR101 by flow cytometry, orthoganol (90°) light scatter by flow cytometry, and direct microscopic measurement with an ocular micrometer. Uninfected CV-1 cells and two cell lines with variations in DNA content (diploid vs. tetraploid) were used as controls for the studies presented. The results demonstrated a 40–60% increase in total protein at 32 to 42 h postinfection. These increases were similar to values obtained as control cells progress through the cell cycle. At later times postinfection (>42 h), total protein decreased due to cellular changes resulting from viral replication and cell death.

Previous experiments with the simian virus 40 mutant tsA357R-K (tsA30) demonstrated a T-antigen function that is required for production of cells with a greater-than-G2-phase DNA content. In this study, temperature shift experiments indicated that the temperature-sensitive function of tsA357R-K, which is necessary for entry into the greater-than-G2 phase, is not required in G1 or S but must be supplied in the G2 phase.

These studies examined cell cycle progression and quantitative changes in T-antigen following infection by SV40. Single cells were assayed by multiparameter flow cytometric analysis (FCM) for DNA content and T-antigen expression. Conditions were used which permitted permissive, semi-permissive, and non-permissive cells to be monitored through two rounds of DNA synthesis induced by SV40. The permissive cells included the monkey kidney cell lines; CV-1, Vero and BSC-1 and the COS-1 and COS-7 which are CV-1 cells transformed with an origin defective SV40. The non-permissive cell strains included mouse embryo fibroblasts, Chinese hamster fibroblasts, and IMR-90, a human diploid fibroblast. Cell types differed in the maximal amount of T-antigen expressed per cell. Additionally, all cell types expressed a limited quantity of T-antigen for each cell cycle phase and the quantity increased in each successive phase. The level in each phase was increased only two-fold when 100 times more virus was used. Thus, for an infected population the quantity of T-antigen was dependent on cell cycle distribution. High levels of T-antigen were not required for permissive infection; however, permissive cells were distinguished from non-permissive cells by the G2 levels. Permissive G2 cells had more than double the T-antigen content expressed in G1, while nonpermissive G2 cells had less than a two-fold increase over G1 levels. The appearance of cells with tetraploid DNA content and the failure to undergo mitosis correlated to the higher T-antigen levels in the G2 of the permissive cells. Two other strains of SV40, 776, and VA45 exhibit similar values for T-antigen expression and movement into tetraploid DNA content. This study establishes the levels of T-antigen correlated to the cell cycle and cell type.

L-DOPA had no effect on the endogenous phosphorylation of proteins after extraction with 1% Triton X-100 from hamster melanoma. When proteins were purified further by wheat germ-agglutinin chromatography, however, a dramatic and dose-dependent inhibitory effect of DOPA on glycoprotein phosphorylation was observed in the presence of Mn +2.

Infection of quiescent CV-1 cells with simian virus 40 mutant tsA30 at 37 degrees C resulted in the induction of two rounds of cellular DNA synthesis in T-antigen-positive cells, as previously described for wild-type simian virus 40. Following infection with tsA30 at 40.5 degrees C, T-antigen-positive cells were induced into S phase and reached a diploid G2 DNA content; however, a second S phase was not initiated. The failure of tsA30-infected CV-1 cells to enter tetraploid S phase at 40.5 degrees C identifies a T-antigen function, distinct from T-antigen functions responsible for stimulation of cell DNA synthesis, which is required for initiation of a second round of DNA synthesis without mitosis.

Testicular teratomas can be experimentally induced in some strains of mice by grafting 12·5-day male genital ridges to the testes of adults. The grafts develop into testes and most of them have teratomas. The cells of 12·5-day foetal gonads were dissociated and the germ cells and somatic cells were separated. When germ cells were reaggregated with somatic cells and implanted in adult testes, they formed seminiferous tubules with teratomas. The somatic cell populations were contaminated with about 1 % germ cells, and when they were implanted in adult testes, they formed testes with a comparatively low incidence of teratomas. When germ cells of a highly susceptible strain were combined with somatic cells from a resistant strain, they formed chimaeric testes with a high incidence of teratomas. When germ cells from a resistant strain were combined with somatic cells from a susceptible strain they formed chimaeric gonads and the incidence of teratomas was low. This indicates that at 12·5 days the genotype of the germ cells is responsible for susceptibility. When germ cells from older foetal gonads were combined with somatic cells of 12·5-day gonads, the incidence of tera-tomas was low. This showed that 12·5-day somatic cells cannot ‘rejuvenate’ older germ cells in a way to regain their susceptibility. When 12·5-day germ cells of highly susceptible strains were combined with older somatic cells the incidence of tumours was low indicating that the age of the somatic cells influences susceptibility to teratocarcinogenesis.

Three casein kinase activities have been resolved by column chromatography of HeLa cell nuclear extracts. In addition to casein kinases NI and NII, which have been described in other cell types, HeLa nuclei contain a third casein kinase activity which we have named NIII. NIII is a cyclic nucleotide-independent casein kinase which uses either Mg2+ or Mn2+ as a divalent cation, but is inhibited by increasing NaCl concentrations in the presence of Mg2+ and has optimal activity at 50 mM NaCl in the presence of Mn2+. In Mg2+, NIII uses only ATP as a phosphate donor, but in Mn2+ NIII transfers phosphate from either ATP or GTP. NIII phosphorylates the serine and threonine residues of casein, but does not phosphorylate phosvitin or calf thymus histones.

Murine embryonal carcinoma (EC) cells do not express virus-specific T antigen following infection with SV 40 . To investigate this block in T antigen expression, the state of the viral DNA in SV 40 -infected EC cells was examined. When assayed by filter hybridization and transfection, SV 40 DNA was present in 247DESC12 EC cells at early times after infection, but was lost with continued growth of the infected EC cell cultures. Although PCC4azal EC cells were T antigen negative following infection with SV 40 , T antigen was expressed in a small percentage of differentiated cells when infected with SV 40 following DMA treatment. In addition, T Ag was not expressed in PCC4azal EC cells infected with SV 40 and subsequently differentiated with DMA.

The stem cell of the murine teratocarcinoma is refractory to infection with Simian virus 40 and polyoma. Utilizing various procedures, we attempted to alter this block to infection by modifying the infection procedure. Multiple infections with high-titer SV40 and pretreatment of cells with DEAE-dextran or the carcinogen 4-nitroquinoline 1-oxide did not induce embryonal carcinoma cells to produce T-antigen. Co-infection with adenovirus 5, which infects the embryonal carcinoma, and SV40 did not induce the expression of SV40 T antigen. Therefore, these procedures did not overcome the block to virus infection. The assay for the SV40 T antigen was immunofluorescence; however, the immunoprecipitation technique did not detect T antigen in the infected embryonal carcinoma cells. Finally, the viral DNA present in the embryonal carcinoma was examined for its ability to replicate. These studies showed that viral DNA was not replicating as assayed by the viral DNA's sensitivity to UV irradiation when replicating in the presence of 5-bromodeoxyuridine.

Testicular teratomas can be induced experimentally by grafting genital ridges from male mouse fetuses to the testes of adults. A high incidence of teratomas occurs in genital ridges grafted to scrotal testes, but not in genital ridges grafted to testes maintained at body temperature. Genital ridges were cultured at 32 degrees C or 37 degrees C prior to grafting to the testes to determine the effect of temperature on the incidence of teratomas. Genital ridges cultured at 32 degrees C for two or three days produced a high incidence of teratomas when grafted to the testes, in contrast to genital ridges cultured at 37 degrees C for two or three days which produced a low incidence. Histologically, genital ridges cultured at 32 degrees C contained disorganized testicular tubules and were retarded in development. Genital ridges continued to develop in vitro at 37 degrees C, but were histologically different from genital ridges maturing in the fetus. Genital ridges cultured at 32 degrees C for 10 to 12 days did not develop teratomas in vitro or after grafting to the testes. Further characterization of temperature effects in vitro may lead to a better understanding of teratocarcinogenesis in vivo.