Comparison of four new cell lines from patients with adenocarcinoma of the ovary.

Permanent human tumor cell lines COLO 110, COLO 316, COLO 319, and COLO 330 were established from four patients with serous cystadenocarcinoma of the ovary. COLO 110 was derived from primary tumor tissue; COLO 316, COLO 319, and COLO 330 were derived from cells in malignant effusions. COLO 110 and COLO 316 grew as monolayers of epithelioid cells in culture; COLO 319 and COLO 330 grew as vermiform, floating colonies of epithelioid cells in culture. Epithelial-like morphology was confirmed by transmission electron microscopy. All four cell lines had marker chromosomes and double minute chromosomes. Giemsa banding revealed chromosomes 1, 3, 6, and 7 were involved in markers in all four lines, and chromosomes 2, 4, 5, 9, 11, and 15 were involved in markers in three of the cell lines. Marker chromosomes with possible homogeneous staining regions were observed in COLO 319. Estrone was elaborated by three of the lines, but neither chorionic gonadotropin, carcinoembryonic antigen, nor estrogen or progesterone receptor proteins were detected. Each cell line demonstrated a distinctive isozyme phenotype. These cell lines are maintained in active culture and in a cell bank for distribution to other investigators.

Transcription factor C/EBP-ß induces tumor-suppressor phosphatase PHLPP2 through repression of the miR-17-92 cluster in differentiating AML cells.

PHLPP2, a member of the PH-domain leucine-rich repeat protein phosphatase (PHLPP) family, which targets oncogenic kinases, has been actively investigated as a tumor suppressor in solid tumors. Little is known, however, regarding its regulation in hematological malignancies. We observed that PHLPP2 protein expression, but not its mRNA, was suppressed in late differentiation stage acute myeloid leukemia (AML) subtypes. MicroRNAs (miR or miRNAs) from the miR-17-92 cluster, oncomir-1, were shown to inhibit PHLPP2 expression and these miRNAs were highly expressed in AML cells that lacked PHLPP2 protein. Studies showed that miR-17-92 cluster regulation was, surprisingly, independent of transcription factors c-MYC and E2F in these cells; instead all-trans-retinoic acid (ATRA), a drug used for terminally differentiating AML subtypes, markedly suppressed miR-17-92 expression and increased PHLPP2 protein levels and phosphatase activity. Finally, we demonstrate that the effect of ATRA on miR-17-92 expression is mediated through its target, transcription factor C/EBPß, which interacts with the intronic promoter of the miR-17-92 gene to inhibit transactivation of the cluster. These studies reveal a novel mechanism for upregulation of the phosphatase activity of PHLPP2 through C/EBPß-mediated repression of the miR-17-92 cluster in terminally differentiating myeloid cells.

Viruses of the extremely thermophilic archaeon Sulfolobus.

Viruses of Sulfolobus are highly unusual in their morphology, and genome structure and sequence. Certain characteristics of the replication strategies of these viruses and the virus-host interactions suggest relationships with eukaryal and bacterial viruses, and the primeval existence of common ancestors. Moreover, studying these viruses led to the discovery of archaeal promoters and has provided tools for the development of the molecular genetics of these organisms. The Sulfolobus viruses contain unique regulatory features and structures that undoubtedly hold surprises for researchers in the future.

The major dimerization determinants of the nitrogen regulatory protein NTRC from enteric bacteria lie in its carboxy-terminal domain.

The NTRC protein (nitrogen regulatory protein C) of enteric bacteria is an enhancer-binding protein that activates transcription by the sigma54-holoenzyme form of RNA polymerase. NTRC is a homodimeric protein that binds to a dyad-symmetrical site in DNA. To activate transcription NTRC must be phosphorylated and must form an appropriate oligomeric species at an enhancer. In order to study subunit exchange between NTRC dimers, we constructed a fusion of the maltose-binding protein (MBP) to the amino-terminal end of NTRC (MBP-NTRC) and visualized the formation of heterodimers between MBP-NTRC and wild-type NTRC by a gel-mobility shift assay for DNA-binding. When MBP-NTRC is mixed with wild-type NTRC at 37 degrees C, subunit exchange occurs rapidly. The apparent half-life for dissociation of homodimers of NTRC is two to three minutes at 37 degrees C and is not changed by phosphorylation. The isolated carboxy-terminal domain of NTRC (91 amino acid residues) forms heterodimers with both wild-type NTRC and MBP-NTRC, indicating that the C-terminal domain is sufficient for dimerization. The apparent rate of dissociation of homodimers of the C-terminal domain is essentially the same as that of full-length NTRC, indicating that the major dimerization determinants of the protein lie in its C-terminal domain. Congruent with this, a truncated form of NTRC from which the last 58 amino acid residues were removed is a monomer in solution. Moreover, truncated forms of NTRC from which the last 16 or 26 amino acid residues were removed are predominantly monomeric in solution, as is a mutant form with the amino acid substitution A410E in its C-terminal domain. Monomerization of the above mutant forms of NTRC can be rationalized on the basis of homology between the C-terminal region of NTRC and a 50 amino acid residue region of the factor for inversion stimulation (FIS) protein.

Structural and functional analyses of activating amino acid substitutions in the receiver domain of NtrC: evidence for an activating surface.

The bacterial enhancer-binding protein NtrC activates transcription when phosphorylated on aspartate 54 in its amino (N)-terminal regulatory domain or when altered by constitutively activating amino acid substitutions. The N-terminal domain of NtrC, which acts positively on the remainder of the protein, is homologous to a large family of signal transduction domains called receiver domains. Phosphorylation of an aspartate residue in a receiver domain modulates the function of a downstream target, but the accompanying structural changes are not clear. In the present work we examine structural and functional differences between the wild-type receiver domain of NtrC and mutant forms carrying constitutively activating substitutions. Combinations of such substitutions resulted in both increased structural changes in the N-terminal domain, monitored by NMR chemical shift differences, and increased transcriptional activation by the full-length protein. Structural changes caused by substitutions outside the active site (D86N and A89T) were not only local but extended over a substantial portion of the N-terminal domain including the region from alpha-helix 3 to beta-strand 5 ("3445 face") and propagating to the active site. Interestingly, the activating substitution of glutamate for aspartate at the site of phosphorylation (D54E) also triggered structural changes in the 3445 face. Thus, the active site and the 3445 face appear to interact. Implications with respect to how phosphorylation may affect the structure of receiver domains and how structural changes may be communicated to the remainder of NtrC are discussed.

Genetic requirements for the function of the archaeal virus SSV1 in Sulfolobus solfataricus: construction and testing of viral shuttle vectors.

Directed open reading frame (ORF) disruption and a serial selection technique in Escherichia coli and the extremely thermophilic archaeon Sulfolobus solfataricus allowed the identification of otherwise cryptic crucial and noncrucial viral open reading frames in the genome of the archaeal virus SSV1. It showed that the 15. 5-kbp viral genome can incorporate a 2.96-kbp insertion without loss of viral function and package this DNA properly into infectious virus particles. The selection technique, based on the preferential binding of ethidium bromide to relaxed DNA and the resulting inhibition of endonuclease cleavage to generate a pool of mostly singly cut molecules, should be generally applicable. A fully functional viral shuttle vector for S. solfataricus and E. coli was made. This vector spreads efficiently through infected cultures of S. solfataricus, its replication is induced by UV irradiation, it forms infectious virus particles, and it is stable at high copy number in both S. solfataricus and E. coli. The classification of otherwise unidentifiable ORFs in SSV1 facilitates genetic analysis of this virus, and the shuttle vector should be useful for the development of genetic systems for Crenarchaeota.

Identification, cloning, and characterization of a major cat flea salivary allergen (Cte f 1).

An 18 kDa protein isolated from saliva of the cat flea, Ctenocephalides felis, elicits a positive intradermal skin test (IDST) in 100 and 80% of experimental and clinical flea allergic dogs, respectively. Using solid-phase enzyme-linked immuno assay (ELISA), this protein detected IgE in 100 and 80% of experimental and clinical flea allergic dogs, respectively. A cDNA (pFSI) encoding a full-length Cte f 1 protein was isolated from a C. felis salivary gland cDNA library, using a combination of PCR and hybridization screening. This cDNA is 658 bp in length, and contains an open reading frame of 528 bp. The open reading frame encodes a protein of 176 amino acids, consisting of an 18 amino acid signal sequence and a 158 amino acid mature protein. The calculated molecular weight and pI of the mature protein are 18106 Da and 9.3, respectively. The protein, named Cte f 1, is the first novel major allergen described for canine flea allergy. Recombinant Cte f 1 (rCte f 1) was expressed in Escherichia coli, Pichia pastoris and baculovirus infected Trichoplusia ni cells. Approximately, 90% of the rCte f 1 expressed in E. coli accumulated in insoluble inclusion bodies, which could be refolded to a soluble mixture of disulfide isomers with partial IgE binding activity. Small quantities of an apparently correctly refolded form of rCte f 1, which had IgE binding activity equal to the native antigen, was isolated from the soluble fraction of E. coli cells. However, P. pastoris and baculovirus infected insect cells expressed and secreted a fully processed, correctly refolded and fully active form of rCte f 1. Mass spectrometry analysis of the active forms of rCte f 1confirmed that eight intact disulfide bonds were present, matching the number observed in the native allergen. The relative ability of rCte f 1 to bind IgE in the serum of flea allergic animals, produced in these three expression systems, matched that of the native allergen. Competition ELISA demonstrated that approximately 90% of the specific IgE binding to native Cte f 1 could be blocked by the different forms of rCte f 1.

An [35S]methionine cytotoxicity assay requiring small numbers of effector and target cells.

This communication describes an assay to detect polyclonal cytotoxic activity of small numbers of effector cells. 200 tumor target cells, radiolabelled with 10,000-20,000 cpm [35S]methionine, are incubated with proportionately few effector lymphocytes in microtiter wells. The targets are sufficiently well labelled to permit detection of cytolysis caused by greater than or equal to 1000 cytotoxic cells, at effector:target ratios as low as 5:1. This method permits the rapid assay of limited numbers of effector cells, avoids lengthy culture of effector cells in order to acquire sufficient numbers, and is useful for limiting dilution analysis of cytotoxic T lymphocytes or their precursors.

Estrogen receptor proteins in diverse human tumors.

One hundred three diverse benign and malignant human tissues have been assayed for estrogen receptor proteins. Receptors were detected in many endocrine and nonendocrine tumors. Tissues with estrogen receptor activity included four of five male breast carcinomas, 11 of 14 malignant melanomas, four of eight colon carcinomas, five of seven renal carcinomas, and various sarcomas and benign and normal tissues. Some tumors also had progesterone, androgen, and/or glucocorticoid receptors. These results suggest the use of hormones and hormone antagonists for therapy of a broad range of human cancer. Clinicians of diverse expertise should be aware of, and responsive to, potential endocrinological involvement in many dissimilar disease states.

Characterization and cloning of a major high molecular weight house dust mite allergen (Der f 15) for dogs.

Although house dust mites (HDM(s)) are important elicitors of canine allergy, the low molecular weight molecules defined as major allergens for humans do not appear to be major allergens for dogs. Western blotting of Dermatophagoides farinae (D. farinae) extracts with sera from sensitized dogs showed that the majority of animals had IgE antibodies specific for two proteins of apparent molecular weights of 98 and 109kDa (98/109kDa). The N-terminal sequences of these two proteins were identical, suggesting they were very closely related, and sequencing of internal peptides showed the protein(s) to have homology with insect chitinases. A purified preparation of 98/109kDa proteins elicited positive intradermal skin tests (IDST(s)) in a group of well-characterized atopic dogs sensitized to D. farinae, but not in normal dogs. A rabbit polyclonal antiserum raised against the purified proteins was used to immunoscreen a D. farinae cDNA library. The mature coding region of the isolated chitinase cDNA predicts a protein of 63.2kDa; sequence analysis and glycan detection blotting suggest that the molecule is extensively O-glycosylated. Monoclonal antibodies made against the purified native protein were used to localize the chitinase in sections of whole D. farinae mites. The protein displayed an intracellular distribution in the proventriculus and intestine of the mite, suggesting that it has a digestive, rather than a moulting-related, function. The high prevalence of IgE antibodies to this antigen in canine atopic dermatitis makes it a major HDM allergen for dogs, and the protein has been formally designated Der f 15.

Measurement of canine IgE using the alpha chain of the human high affinity IgE receptor.

In vitro assays for allergen specific immunoglobulin E (IgE) are a convenient and reproducible alternative to intradermal skin testing in dogs. Such tests may be used to support a diagnosis of atopic dermatitis and to define appropriate allergens for immunotherapy. Current in vitro assays rely upon monoclonal or polyclonal antibodies as IgE detection reagents. However, in sera where allergen-specific IgG occurs in great excess, any IgE:IgG cross-reactivity of the detection reagent may result in lowered assay specificity. Therefore, we have developed an assay for canine IgE which uses a recombinant form of the extracellular part of the alpha chain of the human high affinity IgE receptor (FcvarepsilonRIalpha). Biotinylated FcvarepsilonRIalpha shows no significant binding to purified canine IgG, and recognizes a heat labile antibody in serum, with a detection limit of 73-146pg/ml. Comparison of assay signals using the labeled FcvarepsilonRIalpha and a highly specific anti-canine IgE monoclonal antibody (MAb) shows good agreement. The FcvarepsilonRIalpha is therefore a sensitive and specific alternative to polyclonal or monoclonal antibodies for canine serum IgE measurement.

Recombinant canine IL-13 receptor alpha2-Fc fusion protein inhibits canine allergen-specific-IgE production in vitro by peripheral blood mononuclear cells from allergic dogs.

Human IL-13, like IL-4, is involved in the regulation of B-cell development, IgE synthesis and allergic responses. However, because IL-13 does not affect either murine Ig class switching or IgE production in vitro, the use of murine models to study the role of IL-13 in IgE-mediated diseases has been limited. In this communication, we report that recombinant protein of canine IL-13 (rcaIL-13) stimulates production of allergen-specific-IgE in vitro by peripheral blood mononuclear cells (PBMC) from flea allergen-sensitized dogs, and that this stimulation activity is specifically inhibited by recombinant protein of canine IL-13Ralpha2 and Fc fragment of canine IgG heavy chain (rcaIL-13Ralpha2-Fc). The data suggest that the regulatory effects of IL-13 on IgE production in canine PBMC are similar to those reported in humans. Thus, canine IL-13 may be a central mediator of allergic diseases in dogs, and allergic dogs may be excellent models for research on IgE-mediated diseases in humans.

Pilot study to assess the effects of early flea exposure on the development of flea hypersensitivity in cats.

This pilot study was to determine if early oral flea exposure reduces the incidence of flea allergy dermatitis (FAD) in cats. Eighteen kittens, assigned to three groups, received no flea exposure, oral flea exposure or flea infestation for 12 weeks. Then all the kittens were exposed continually to fleas for 31 weeks. Sensitization was monitored using intradermal testing (IDT), in vitro measurement of anti-flea saliva immunoglobulin E (IgE) and development of FAD. There was no statistically significant difference between groups in IDT reactions, in vitro data or clinical scores. The development of FAD was not associated with the presence of anti-flea saliva IgE. However, the development of a delayed reaction to flea bite was associated with symptoms after flea exposure. Although not statistically significant, the FAD scores in the oral group were lower than in the controls. Further studies are required to determine the role of oral flea exposure in the development of FAD in cats.

Microbial biogeochemistry of Boiling Springs Lake: a physically dynamic, oligotrophic, low-pH geothermal ecosystem.

Boiling Springs Lake (BSL) in Lassen Volcanic National Park, California, is North America's largest hot spring, but little is known about the physical, chemical, and biological features of the system. Using a remotely operated vessel, we characterized the bathymetry and near-surface temperatures at sub-meter resolution. The majority of the 1.2 ha, pH 2.2 lake is 10 m deep and 50-52 °C, but temperatures reach 93 °C locally. We extracted DNA from water and sediments collected from warm (52 °C) and hot (73-83 °C) sites separated by 180 m. Gene clone libraries and functional gene microarray (GeoChip 3.0) were used to investigate the BSL community, and uptake of radiolabeled carbon sources was used to assess the relative importance of heterotrophic vs. autotrophic production. Microbial assemblages are similar in both sites despite the strong temperature differential, supporting observations of a dynamic, convectively mixed system. Bacteria in the Actinobacteria and Aquificales phyla are abundant in the water column, and Archaea distantly related to known taxa are abundant in sediments. The functional potential appears similar across a 5-year time span, indicating a stable community with little inter-annual variation, despite the documented seasonal temperature cycle. BSL water-derived DNA contains genes for complete C, N, and S cycles, and low hybridization to probes for N and S oxidation suggests that reductive processes dominate. Many of the detected genes for these processes were from uncultivated bacteria, suggesting novel organisms are responsible for key ecosystem services. Selection imposed by low nutrients, low pH, and high temperature appear to result in low diversity and evenness of genes for key functions involved in C, N, and S cycling. Conversely, organic degradation genes appear to be functionally redundant, and the rapid assimilation of radiolabeled organic carbon into BSL cells suggests the importance of allochthonous C fueling heterotrophic production in the BSL C cycle.

A spleen-derived maturational factor allows immature thymocytes, prepared as cells bearing low amounts of surface sialic acid, to become cytotoxic T cells.

A population of immature mouse thymocytes bears low levels of surface sialic acid and can be separated from the more mature high sialic acid-bearing thymocytes by selective agglutination with the sialic acid-specific lectin, lobster agglutinin 1. These immature thymocytes do not proliferate in response to concanavalin A (Con A). They do not produce interleukin 2 (IL-2), do not provide T cell help to B cells for an in vitro antibody response, and as shown here, do not become cytotoxic T lymphocytes when polyclonally stimulated with Con A + IL-2. We describe here a spleen-derived maturational factor which stimulates these immature thymocytes, in the presence of Con A and IL-2, to become cytotoxic T lymphocytes. The maturational factor is a protein secreted by Con A-stimulated mouse or rat spleen cells; it is apparently neither interleukin 1, IL-2, interleukin 3, gamma-interferon, nor combinations of these cytokines, because these materials do not replace the maturational factor. The active material in Con A-stimulated mouse spleen cell supernatant was recovered from a G-75 column in the 33,000-48,000 m.w. range. These experiments suggest that within the lobster agglutinin 1-negative thymocyte population there are cells which can mature under the influence of a spleen-derived factor. It is possible that these cells represent the small subpopulation of immature cells destined to become immunocompetent peripheral T cells. On the other hand, the factor may be rescuing cells destined to die in the thymus.

Endogenously secreted IL-4 is required for mouse thymocytes to become cytotoxic. Human, but not mouse, IL-2 induces a functionally immature thymic subset to secrete IL-4 and become CTL.

Experiments described here demonstrate that the differentiation of mouse thymocytes into cytotoxic T lymphocytes (CTL) requires interleukin-4 (IL-4). To reach this conclusion, we took advantage of our discovery that human and mouse IL-2 have very different effects on the development of CTL from a functionally immature subset of thymocytes. The lobster agglutinin 1 (LAg1)-negative subpopulation of thymocytes proliferated when cultured with concanavalin A (Con A)+ human or mouse IL-2, but these cells became CTL only when cultured with Con A+ human IL-2. Furthermore, Con A+ human IL-2, but not mouse IL-2, stimulated IL-4 production by cells within this population. Con A-induced cytotoxicity by mature LAg1-positive thymocytes and normal thymocytes was also accompanied by secretion of IL-4. The anti-IL-4 mAb 11B11 inhibited induction of cytotoxicity by all thymocyte populations tested. Taken together these experiments indicate that stimuli which induce cytotoxicity by mouse thymocytes also induce the secretion of IL-4, which is necessary for the differentiation of thymocyte CTL precursors into CTL.

B cell stimulatory factor 1 induces lobster agglutinin 1-separated mouse thymocytes to express cytotoxic T lymphocyte activity.

Mouse thymocytes low in surface sialic acid were prepared by using the lectin lobster agglutinin 1 (LAg1). These LAg1-thymocytes do not become CTL when incubated with Con A or Con A plus mouse rIL-2, whereas unseparated thymocytes and thymocytes with high levels of surface sialic acid develop good levels of polyclonal CTL activity under these conditions. However, LAg1- thymocytes developed high levels of CTL activity when incubated with B cell stimulatory factor-1 (BSF-1), provided as the supernatant of the rBSF-1-secreting T cell hybridoma D9-C1.12.17. Affinity-purified BSF-1 from D9-C1 supernatant and rBSF-1 also stimulated these cells to become CTL, but they were not as active as the D9-C1 supernatant. The ability of D9-C1 supernatant and of affinity-purified BSF-1 to induce CTL activity was inhibited by the anti-BSF-1 mAb 11B11. Moreover, this mAb inhibited the ability of 24-h Con A-stimulated spleen cell supernatant to induce these cells to express CTL activity. 11B11 also inhibited LAg1+ thymocytes from becoming CTL when stimulated with Con A alone. These experiments suggest that BSF-1 is required for LAg1- and LAg1+ thymocytes to become CTL.

Distribution of endogenous type B and type D sheep retrovirus sequences in ungulates and other mammals.

The jaagsiekte sheep retrovirus (JSRV), which appears to be a type B/D retrovirus chimera, has been incriminated as the cause of ovine pulmonary carcinoma. Recent studies suggest that the sequences related to this virus are found in the genomes of normal sheep and goats. To learn whether there are breeds of sheep that lack the endogenous viral sequences and to study their distribution among other groups of mammals, we surveyed several domestic sheep and goat breeds, other ungulates, and various mammal groups for sequences related to JSRV. Probes prepared from the envelope (SU) region of JSRV and the capsid (CA) region of a Peruvian type D virus related to JSRV were used in Southern blot hybridization with genomic DNA followed by low- and high-stringency washes. Fifteen to 20 CA and SU bands were found in all members of the 13 breeds of domestic sheep and 6 breeds of goats tested. There were similar findings in 6 wild Ovis and Capra genera. Within 22 other genera of Bovidae including domestic cattle, and 7 other families of Artiodactyla including Cervidae, there were usually a few CA or SU bands at low stringency and rare bands at high stringency. Among 16 phylogenetically distant genera, there were generally fewer bands hybridizing with either probe. These results reveal wide-spread phylogenetic distribution of endogenous type B and type D retroviral sequences related to JSRV among mammals and argue for further investigation of their potential role in disease.

pING family of conjugative plasmids from the extremely thermophilic archaeon Sulfolobus islandicus: insights into recombination and conjugation in Crenarchaeota.

A novel family of conjugative plasmids from Sulfolobus comprising the active variants pING1, -4, and -6 and the functionally defective variants pING2 and -3, which require the help of an active variant for spreading, has been extensively characterized both functionally and molecularly. In view of the sparse similarity between bacterial and archaeal conjugation and the lack of a practical genetic system for Sulfolobus, we compared the functions and sequences of these variants and the previously described archaeal conjugative plasmid pNOB8 in order to identify open reading frames (ORFs) and DNA sequences that are involved in conjugative transfer and maintenance of these plasmids in Sulfolobus. The variants pING4 and -6 are reproducibly derived from pING1 in vivo by successive transpositions of an element from the Sulfolobus genome. The small defective but mobile variants pING2 and -3, which both lack a cluster of highly conserved ORFs probably involved in plasmid transfer, were shown to be formed in vivo by recombinative deletion of the larger part of the genomes of pING4 and pING6, respectively. The efficient occurrence of these recombination processes is further evidence for the striking plasticity of the Sulfolobus genome.

The exogenous form of Jaagsiekte retrovirus is specifically associated with a contagious lung cancer of sheep.

Sheep pulmonary adenomatosis ([SPA] ovine pulmonary carcinoma) is a transmissible lung cancer of sheep that has been associated etiologically with a type D- and B-related retrovirus (jaagsiekte retrovirus (JSRV]). To date it has been impossible to cultivate JSRV in vitro and therefore to demonstrate the etiology of SPA by a classical approach. In addition, the presence of 15 to 20 copies of endogenous JSRV-related sequences (enJSRV) has hampered studies at the molecular level. The aim of this study was to investigate whether the expression of exogenous JSRV was specifically associated with neoplasia in SPA-affected animals. Initially, we found that enJSRVs were transcribed in a wide variety of normal sheep tissues. Then, by sequencing part of the gag gene of enJSRV we established a ScaI restriction site in gag as a molecular marker for the exogenous form of JSRV. Restriction enzyme digestion of PCR products obtained from the amplification of cDNA from a total of 65 tissues collected from SPA-affected and unaffected control sheep revealed that the exogenous form of JSRV was exclusively and consistently present in tumor tissues and lung secretions of the affected animals. In addition, exogenous JSRV provirus was detected only in DNA from SPA tumors and not from nontumor tissues of the same animals. This study has demonstrated clearly that the exogenous form of JSRV is specifically associated with SPA tumors.